Pharmaceutical composition comprising nanoparticles for the targeted delivery of antigens

Abstract

The present disclosure provides pharmaceutical compositions comprising nanoparticles, wherein the nanoparticles each comprise a micelle comprising an amphiphilic polymer, and at least one peptide. The present disclosure further provides methods of treating an autoimmune disease (e.g., multiple sclerosis, type 1 diabetes) in a human subject in need thereof comprising administering the pharmaceutical compositions provided herein.

Description

Attorney Docket No. 14779-010-228PHARMACEUTICAL COMPOSITION COMPRISING NANOPARTICLES FOR THE TARGETED DELIVERY OF ANTIGENS

[0001] This application claims the benefit of priority to U.S. Serial No. 63 / 687,671, filed August 27, 2024, which is incorporated herein by reference in its entirety.

[0002] This application contains an electronic Sequence Listing which has been submitted in XML file format with this application, the entire content of which is incorporated by reference herein in its entirety. The Sequence Listing XML file submitted with this application is entitled “14779-010-228_SEQLISTING.xml”, was created on August 20, 2025, and is 15,259 bytes in size.1. FIELD

[0003] The present disclosure provides pharmaceutical compositions comprising nanoparticles, wherein the nanoparticles each comprise a micelle comprising an amphiphilic polymer, and at least one peptide. The present disclosure further provides methods of treating an autoimmune disease (e.g., multiple sclerosis, type 1 diabetes) in a human subject in need thereof comprising administering the pharmaceutical compositions provided herein.2. BACKGROUND

[0004] Autoimmune diseases (AD), such as multiple sclerosis (MS) and type 1 diabetes (T1D), are chronic disorders characterized by aberrant inflammatory responses of the adaptive immune system to self-antigens, resulting in cellular and tissue damage. There is a steadily increasing global prevalence of AD, affecting between 3% and 10% of the general population. These conditions not only lead to high morbidity and mortality, but also represent a major burden on global healthcare systems.

[0005] The available treatments for AD are mainly based on systemic immunosuppression, which can cause severe side effects, not only limited to infections. In contrast to general immunosuppression, the ideal approach for treating AD is to restore immune tolerance toward the respective self-antigens that are mistakenly targeted by the immune system. Autoantigenspecific immunotherapies that exclusively target disease-specific immunopathologies, leaving the patient's general immune status untouched, are considered an unmet medical need.3. SUMMARYAttorney Docket No. 14779-010-228

[0006] In one aspect, the present disclosure provides a pharmaceutical composition comprising nanoparticles. In certain embodiments, the present disclosure provides a pharmaceutical composition comprising nanoparticles, wherein each of the nanoparticles comprise: (a) an amphiphilic polymer, and (b) at least one peptide, wherein the pharmaceutical composition comprises peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.

[0007] In certain embodiments, the at least one peptide is associated with the outside of at least one nanoparticle.

[0008] In certain embodiments, each of the at least one nanoparticles comprise at least one peptide comprising the amino acid sequence of SEQ ID NO: 12.

[0009] In certain embodiments, each of the at least one nanoparticles comprise at least one peptide comprising one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11. In certain embodiments, the pharmaceutical composition comprises only peptides comprising the amino acid sequence of SEQ ID NOs: 7, 8, 9, 10, and 11.

[0010] In certain embodiments, the pharmaceutical composition disclosed herein comprises at least 5 different types of nanoparticles, wherein the different types of nanoparticles differ among each other in the peptide sequence, and wherein a first type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 7, a second type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 8, a third type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 9, a fourth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 10, and a fifth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, each type of nanoparticle is present in equimolar amounts.

[0011] In certain embodiments, the amphiphilic polymer has a number average molecular weight (Mn) of 20,000 g / mol or less, 10,000 g / mol or less, or ranging between 6,000 g / mol to 1,000 g / mol.

[0012] In certain embodiments, the amphiphilic polymer comprises the following building blockAttorney Docket No. 14779-010-228wherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a C4 to C22 alkyl group, preferably C8 to C20 alkyl group.

[0013] In certain embodiments, 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). In certain embodiments, the amphiphilic polymer is poly(maleic acid-alt- 1 -octadecene). In certain embodiments, the number average molecular weight of the amphiphilic polymer is from 6,000 to 1,000 g / mol.

[0014] In certain embodiments, each peptide is covalently linked to the outside of at least one nanoparticle or non-covalently associated.

[0015] In certain embodiments, the pharmaceutical composition of the preset disclosure further comprises an aqueous buffer, wherein the aqueous buffer comprises at least one sugar, at least one primary amine, and at least one amino acid. In certain embodiments, the buffer contains D-mannitol, Tris(hydroxymethyl)aminomethane, and L-lactic acid.

[0016] In certain embodiments, each nanoparticle optionally comprises a iron oxide core and an amphiphilic polymer comprising poly(maleic acid-alt- 1 -octadecene) having a number average molecular weight of 6,000 to 1,000 g / mol.

[0017] In certain embodiments, the pharmaceutical composition provided in the present disclosure can be of use for treatment of an autoimmune disease. In certain embodiments, the autoimmune disease is type 1 diabetes (T1D). In certain embodiments, the autoimmune disease is multiple sclerosis (MS).

[0018] In another aspect, the present disclosure provides a nanoparticle wherein the nanoparticle comprises (a) an amphiphilic polymer, and (b) at least one peptide, wherein the at least one peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.

[0019] In certain embodiments, the at least one peptide is associated with the outside of the nanoparticle.Attorney Docket No. 14779-010-228

[0020] In certain embodiments, the nanoparticle comprises at least one peptide comprising the amino acid sequence of SEQ ID NO: 12.

[0021] In certain embodiments, the nanoparticle comprises at least one peptide comprising one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11. In certain embodiments, the nanoparticle comprises only peptides comprising the amino acid sequence of SEQ ID NOs: 7, 8, 9, 10, or 11.

[0022] In certain embodiments, the nanoparticle of the present disclosure is (a) a first type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 7; (b) a second type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 8, (c) a third type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 9, (d) a fourth type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 10, or (e) a fifth type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 11.

[0023] In certain embodiments, the amphiphilic polymer has a number average molecular weight (Mn) of 20,000 g / mol or less, 10,000 g / mol or less, or ranging between 6,000 g / mol to 1,000 g / mol. In certain embodiments, the amphiphilic polymer comprises the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a C4 to C22 alkyl group, preferably C8 to C20 alkyl group.

[0024] In certain embodiments, 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). In certain embodiments, the amphiphilic polymer is poly(maleic acid-alt- 1 -octadecene). In certain embodiments, the number average molecular weight of the amphiphilic polymer is from 6,000 to 1,000 g / mol.

[0025] In certain embodiments, least one peptide is covalently linked to the outside of the nanoparticle or non-covalently associated.Attorney Docket No. 14779-010-228

[0026] In certain embodiments, the nanoparticle optionally comprises a iron oxide core and an amphiphilic polymer comprising poly(maleic acid-alt- 1 -octadecene) having a number average molecular weight of 6,000 to 1,000 g / mol.

[0027] In certain embodiments, the nanoparticle of the present disclosure can be used in a pharmaceutical composition wherein the pharmaceutical composition further comprises an aqueous buffer, wherein the aqueous buffer comprises at least one sugar, at least one primary amine, and at least one amino acid. In certain embodiments, the buffer contains D-mannitol, Tris(hydroxymethyl)aminomethane, and L-lactic acid.

[0028] In certain embodiments, the pharmaceutical composition comprises a first type of nanoparticle, a second type of nanoparticle, a third type of nanoparticle, a fourth type of nanoparticle, and a fifth type of nanoparticle, wherein each type of nanoparticle is present in equimolar amounts.

[0029] In certain embodiments, the nanoparticle of the present disclosure, and / or a pharmaceutical composition comprising the nanoparticle of the present disclosure, can be of use for treatment of an autoimmune disease. In certain embodiments, the autoimmune disease is type 1 diabetes (T1D). In certain embodiments, the autoimmune disease is multiple sclerosis (MS).

[0030] In another aspect, the present disclosure provides a composition comprising at least one nanoparticle, wherein the at least one nanoparticle comprises (a) an amphiphilic polymer, and (b) at least one peptide, wherein the peptides comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.

[0031] In certain embodiments, the at least one peptide is associated with the outside of the at least one nanoparticle.

[0032] In certain embodiments, the at least one nanoparticle comprises at least one peptide comprising the amino acid sequence of SEQ ID NO: 12.

[0033] In certain embodiments, the at least one nanoparticle comprises at least one peptide comprising one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11. In certain embodiments, the composition of the present disclosure comprises only peptides comprising the amino acid sequence of SEQ ID NOs: 7, 8, 9, 10, and 11.

[0034] In certain embodiments, the composition of the present disclosure comprises at least 5 different types of nanoparticle, wherein the different types of nanoparticle differ among eachAttorney Docket No. 14779-010-228 other in the peptide sequence, and wherein a first type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 7, a second type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 8, a third type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 9, a fourth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 10, and a fifth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, each type of nanoparticle is present in equimolar amounts.

[0035] In certain embodiments, the amphiphilic polymer has a number average molecular weight (Mn) of 20,000 g / mol or less, 10,000 g / mol or less, or ranging between 6,000 g / mol to 1,000 g / mol.

[0036] In certain embodiments, the amphiphilic polymer comprises the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a C4 to C22 alkyl group, preferably C8 to C20 alkyl group.

[0037] In certain embodiments, 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). In certain embodiments, the amphiphilic polymer is poly(maleic acid-alt- 1 -octadecene). In certain embodiments, the number average molecular weight of the amphiphilic polymer is from 6,000 to 1,000 g / mol.

[0038] In certain embodiments, each peptide is covalently linked to the outside of the at least one nanoparticle or non-covalently associated.

[0039] In certain embodiments, composition of the present disclosure further comprises an aqueous buffer, wherein the aqueous buffer comprises at least one sugar, at least one primary amine, and at least one amino acid. In certain embodiments, the buffer contains D-mannitol, Tris(hydroxymethyl)aminomethane, and L-lactic acid.Attorney Docket No. 14779-010-228

[0040] In certain embodiments, each nanoparticle optionally comprises a iron oxide core and an amphiphilic polymer comprising poly(maleic acid-alt- 1 -octadecene) having a number average molecular weight of 6,000 to 1,000 g / mol.

[0041] In certain embodiments, the composition of the present disclosure can be of use for treatment of an autoimmune disease. In certain embodiments, the autoimmune disease is type 1 diabetes (T1D). In certain embodiments, the autoimmune disease is multiple sclerosis (MS).

[0042] In another aspect, the present disclosure provides methods of making the nanoparticles and / or compositions disclosed herein. In another aspect, the present disclosure provides kits comprising the nanoparticles and / or compositions disclosed herein.4. BRIEF DESCRIPTION OF THE FIGURES

[0043] FIGS. 1A-1C depict uniform size distribution displayed by Topas Particle Conjugates (TPCs). FIG. 1A shows a schematic of the production process of TPCs. FIG. IB shows results of a representative dynamic light scattering (DLS) demonstrating size distribution of TPCs, average size (nm), and polydispersity index. FIG. 1C shows a representative transmission electron microscopy (TEM) image of TPCs.

[0044] FIGS. 2A-2E depict TPCs accumulation primarily in the liver, predominantly within LSECs. FIGS. 2A and 2B show organ distribution of TPCs ten minutes after the intravenous administration of TPC-gliadin-Cy5. The fluorescence intensity in different organ lysates was analyzed (FIG. 2A) and intravital microscopy of the liver was performed (FIG. 2B). FIG. 2C shows representative images of the liver of C57BL / 6 mice examined under intravital microscopy before and after receiving i.v. injection with QD-TPC-Ova323-339-Cy5. Excitation of QD at 488 nm with Cy5 emission at 680 nm indicated the presence of FRET. Representative images were taken at baseline (upper panels), 1 minute post-injection (middle panels), and 1 hour postinjection (lower panels). FIG. 2D shows the percentage of each nonparenchymal liver cell type among Cy5+ cells (upper panel) and the percentage of Cy5+ cells within each nonparenchymal liver cell type (lower panel) as analyzed by flow cytometry ten minutes after the intravenous administration of TPC-gliadin-Cy5. FIG. 2E shows representative images detecting TPC-like particles (i.e., former version of TPCs) within LSEC endosomes by electron microscopy of minipig liver. CNS: central nervous system; iLN: inguinal lymph nodes; KC: Kupffer cells; DC: dendritic cells; MoMF: monocyte-derived macrophages; PMN: polymorphonuclear neutrophils. Graphs show means ± SEM of five mice. Data are representative of 1-2 experiments.Attorney Docket No. 14779-010-228

[0045] FIGS. 3A-3L depict graphs showing that TPCs alleviated DTH responses and reduced the overall expression of proinflammatory cytokines and chemokines. BALB / C mice were injected i.v. with TP or TPC-OVA323-3397 days (d) and 1 d before immunization with OVA323 -339 peptides plus complete Freund’s adjuvant (CFA) (N = 10- 11 per group). FIG. 3A shows a schematic of the experimental setup. FIGS. 3B-3D show DTH responses determined 24 hours (h) after intradermal (i.d.) injections of ears with the priming peptides, using three different parameters: Ear swelling (FIG. 3B); ear swelling in response to PBS in the contralateral ear of the animals subtracted from each measure of ear swelling (A Ear swelling; FIG. 3C); and weight of a biopsy from the OVA323-339 peptide-challenged ear minus weight of the contralateral control with PBS (A Ear weight; FIG. 3D). The contralateral ear served as an internal control to minimize the interindividual variability. FIGS. 3E-3L show relative expression of proinflammatory cytokines and chemokines of the ear pinnae from OVA323-339 peptide-primed versus unprimed animals, shown as fold changes: IFNy (FIG. 3E), TNFa (FIG. 3F), IL-2 (FIG. 3G), IL-1 (FIG. 3H), GM-CSF (FIG. 31), IL-6 (FIG. 3 J), CCL2 (FIG. 3K), and CCL5 (FIG. 3L). Graphs show means ± SEM. Data are representative of 1-2 experiments. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. Mann-Whitney test for the differences in DTH responses and relative gene expression between TP and TPC groups.

[0046] FIGS. 4A-4F depict TPCs that reduced onsets of hyperglycemia in NOD mice and increased insulin-specific Tregs. FIG. 4A shows a schematic of the experimental setup. FIG. 4B shows the frequency of diabetes-free mice (“survivors”) per week of experiment. Arrows indicate timepoints of the injections. FIG. 4C shows blood sugar levels in mice treated with TP (left panel) or TPM-T1D (right panel) throughout the experiment. The horizontal black line indicates the cutoff for hyperglycemia. FIG. 4D shows representative plots of flow cytometry for IAg7-restricted control (upper panel, left) and InsB:9-23 tetramers (upper panel, right), correlation of tetramer staining with CD3 staining as quality control (lower panel, left) and gating on Foxp3+Tregs among insulin-specific CD4+T cells (lower panel, right). Ttet: tetramer positive T cells. FIG. 4E shows the frequency of insulin-specific T cells among total CD4+T cells in the spleen of mice treated with TP or TPM-T1D. FIG. 4F shows the frequency of Foxp3+Tregs among insulin-specific T cells in the spleen of mice treated with TP or TPM-T1D. Graphs show means ± SEM of 13-15 mice per group. Data are representative of two experiments, ns: not significant; *p < 0.05; **p < 0.01. Log-rank test for the survival analysisAttorney Docket No. 14779-010-228 of diabetes-free animals; Mann- Whitney test for the differences in frequency of T cell subsets between animals treated with TP and TPM-T1D.

[0047] FIGS. 5A-5F depict TPCs that prevented or alleviated EAE with significant reduction in disease severity and demyelination in spinal cords. EAE was induced in C57B / 6 mice through immunization with MOG35-55 peptide / CFA plus i.p. injections of pertussis toxin. TP or TPC-MOG35-55 was administered either one day before immunization (prophylactic treatment) or at the disease onset (therapeutic treatment). The animals were observed daily until day 35. At the end of follow-up, spinal cords were collected for histological analysis. FIGS. 5A-5C depict the prophylactic treatment setting where FIG. 5A is a schematic of the study design, FIG. 5B shows the mean clinical score, and FIG. 5C shows the demyelination score. FIGS. 5D-5F depict the therapeutic treatment setting where FIG. 5D is a schematic of the study design, FIG. 5E shows the mean clinical score, and FIG. 5F shows the demyelination score. FIG. 5G shows the preventive treatment of EAE with TPC-MOG35 55 with concurrent prednisone in different doses. Graphs show means ± SEM of 6-12 animals per group. Data are representative of two experiments. **p < 0.01; ***p < 0.001 by Mann- Whitney test for the differences in demyelination score between TP and TPC groups.

[0048] FIGS. 6A-6J depict TPCs that induced anergic cells and enhanced expression of co- inhibitory receptors. FIGS. 6A-6D show lymphocytes isolated from spinal cords (i.e., CNS) of the animals in EAE therapeutic setting (see FIG. 5D). Absolute CNS CD4+T cell count is shown in FIG. 6A. The frequency of CNS TH17 (left panel) and GM-CSF-producing T cells (right panel) are shown in FIG. 6B. A representative flow cytometry plot of CD73hlFR4hlsubpopulation in naive and effector T cells (left panel) and the frequency of anergic CD4+T cells in CNS (right panel) are shown in FIG. 6C. The frequency of CNS Tregs is shown in FIG. 6D. FIGS. 6E-6J show TCRMOGT cells that were adoptively transferred into wild-type mice, followed by treatment with TPC-MOG35 55 and immunization with MOG35-55 peptides. Organs were harvested seven days post-immunization. FIG. 6E is a schematic of the study design. The frequency of splenic TCRMOGT cells (left panel) and their mean fluorescence intensity (MFI) of Ki67 (right panel) are shown in FIG. 6F. The frequency of HGIT+cells (left panel) and LAG- 3+cells (middle panel) among TCRMOGT cells and MFI of PD-1 of TCRMOGT cells in spleen (right panel) are shown in FIG. 6G. The frequency of anergic TCRMOGT cells in spleen is shown in FIG. 6H. The frequency of splenic TCRMOGTregs is shown in FIG. 61. TheAttorney Docket No. 14779-010-228 frequency of TIGIT+cells (left panel) and MFI of CTLA-4 (middle panel) and PD-1 (right panel) among TCRMOGTregs in spleen are shown in FIG. 6 J. CNS, central nervous system. Graphs show means ± SEM of six animals per group. Data are derived from two independent experiments. *p < 0.05; **p < 0.01 by Mann- Whitney test.

[0049] FIG. 7 depicts a schematic of florescence resonance energy transfer. FRET is a phenomenon where a donor fluorophore transfers electronic excitations to an acceptor, mediated by electronic dipole-dipole coupling. As shown here, the quantum dot (QD) serves as a donor and Cy5-labelled peptides as an acceptor. When Cy5-labelled peptides remain coupled to QD, Cy5 emission is detected following QD excitation. However, when Cy5-labelled peptides dissociate from QD due to intracellular cleavage, QD emission is detected.

[0050] FIGS. 8A and 8B depict representative images demonstrating that the FRET effect existed only between QD and coupled Cy5-labelled Ova peptides. The liver of C57BL / 6 mice was examined under intravital microscopy 1 h after intravenous administration of various nanoparticles, specified respectively in the figure. FIG. 8A demonstrates no FRET effect between TPC (iron core) and coupled Cy5-labelled Ova peptides where (upper panel) 488 nm excitation did not induce QD emission, or via FRET, Cy5 emission and (lower panel) 638 nm excitation led to Cy5 emission. FIG. 8B demonstrates no FRET effect between QD and uncoupled Cy5-labelled Ova peptides where (upper panel) 488 nm excitation induced QD emission, but no Cy5 emission and (lower panel) 488 nm and 638 nm excitation induced QD and Cy5 emission, respectively.

[0051] FIG. 9 depicts a representative flow cytometry gating strategy for liver non- parenchymal cells. DC, dendritic cells; KC, Kupffer cells; LSEC, liver sinusoidal endothelial cells; MoMF, monocyte-derived macrophages; PMN, polymorphonuclear neutrophils.

[0052] FIGS. 10A-10F depict antigen presentation of TPCs by APCs that resulted in antigen-specific T cell activation and Treg induction. In FIG. 10A, lymphocytes were isolated from spleen and lymph nodes of 2D2 mice and stimulated in vitro for 72 h with TPC-MOG35 55 or soluble MOG35 55 peptides in various concentrations. The level of IFNy in the supernatant was measured by ELISA (FIG. 10A). In FIGS. 10B-10F, TCRMOGT cells were labelled with Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) and adoptively transferred into wildtype mice, followed by treatment with TP or TPC-MOG35-55 (N = 4 per group). Organs were harvested two days after treatment. FIG. 10B is a schematic of the study design. FIG. 10CAttorney Docket No. 14779-010-228 shows representative CFSE intensity histograms of TCRMOGT cells recovered from different organs (spleen, left panel; lymph nodes, middle panel; and liver, right panel) of animals treated with either TP (black) or TPC (grey). Proliferation (i.e., CFSE dilution) (FIG. 10D) and frequency (FIG. 10E) of TCRMOGT cells in different organs is shown. The frequency of TCRMOGTreg cells in different organs is shown in FIG. 10F. Graphs show means ± SEM. *p < 0.05 by Mann-Whitney test.

[0053] FIGS. 11A-11F depict that TPCs significantly attenuated disease scores in both prophylactic and therapeutic treatment. FIGS. 11 A-l 1 C demonstrate prophylactic treatment where FIG. 11A shows end score, FIG. 11B shows maximum score, and FIG. 11C shows cumulative score. FIGS. 1 ID-1 IF demonstrate therapeutic treatment where FIG. 11D shows end score, FIG. HE shows maximum score, and FIG. llFshows cumulative score. Graphs show means ± SEM of 12 animals per group. Data are representative of two experiments. ****p < 0.0001 by Mann- Whitney test for the differences in disease score between TP and TPC groups.

[0054] FIGS. 12A-12D depict that TPCs alleviated demyelination in spinal cords of TPC- treated animals. Shown are representative images of immunohistochemical staining of spinal cords using anti-MBP (myelin basic protein) antibody. Demyelination was observed as clearly visible unstained areas in white matter tracts and is associated with the presence of large vacuoles. FIGS. 12A and 12B show tissues collected from a prophylactic treatment setting. One representative image of the spinal cord slice from mice treated with TP (FIG. 12A) and with TPC-MOG35-55 (FIG. 12B) are shown. FIGS. 12C and 12D show tissues collected from a therapeutic treatment setting. One representative image of the spinal cord slice from mice treated with TP (FIG. 12C) and with TPC-MOG35 55 (FIG. 12D) are shown. N = 6 per group. Data are representative of two experiments.

[0055] FIG. 13 depicts a representative flow cytometry gating strategy for cytokine producing T cells.5. DETAILED DESCRIPTION

[0056] Provided herein is a pharmaceutical composition comprising nanoparticles, methods of preparing nanoparticles, and methods using the pharmaceutical composition comprising nanoparticles. In certain embodiments, the nanoparticles provided herein (see Section 5.1) each comprise a micelle comprising an amphiphilic polymer, as described in Sections 5.1.1 and 5.1.2,Attorney Docket No. 14779-010-228 respectively. In certain embodiments, the nanoparticles provided herein each comprise a solid hydrophobic core (see Section 5.1.3) which can be partially coated by a micelle. In certain embodiments, the nanoparticles provided herein each comprise at least one peptide. The peptides suitable for use with a nanoparticle, and a pharmaceutical composition comprising thereof, are described in Section 5.1.4 herein. The present disclosure also provides methods of preparing nanoparticles (see Section 5.2). Pharmaceutical compositions provided by the present disclosure are described in Section 5.3 and methods of using a pharmaceutical composition of the present disclosure are described at Section 5.5 herein.5.1 Nanoparticles

[0057] According to the present disclosure, the term “nanoparticle” is used interchangeably with “nanoscale particle”. Such particles have a diameter of 1 to 999 nm, preferably, of 2 to 600 nm, 5 to 500 nm, 10 to 300 nm, 30 to 100 nm or 40 to 50 nm.

[0058] In the context of the present disclosure, a nanoparticle is a structure formed by at least a micelle and at least one peptide which is associated to the micelle. In certain embodiments, a peptide disclosed herein can either be associated to the outside of the micelle or encapsulated inside the micelle. In certain embodiments, a nanoparticle disclosed herein can further comprise a solid hydrophobic core which is at least partially coated by the micelle or does not comprise a solid hydrophobic core. In some embodiments, the nanoparticle provided herein comprises a solid inorganic core. In certain embodiments, the nanoparticle provided herein does not comprise a solid inorganic core.

[0059] The nanoparticles in the pharmaceutical composition of the present disclosure can additionally comprise a moiety, e.g., a carbohydrate or a protein targeting them, or enhancing targeting to specific cells such as liver sinusoidal endothelial cells and / or Kupffer cells. Such moiety could, e.g., enhance or accelerate uptake from the circulation via receptor mediated endocytosis. Examples of suitable modifications are carbohydrates such as mannose.

[0060] Without being bound by theory, a nanoparticle provided herein is capable of being internalized by liver sinusoidal endothelial cells (LSECs) after administration to a subject or in an in vitro model. In some embodiments, the degree or efficiency of internalization of the nanoparticle by LSECs is determined according to an Internalization Assay (see e.g., Gottstein C et al., ACS Nano. 2013 Jun 25;7(6):4933-45; Rennick JJ et al., Nat Nanotechnol. 2021 Mar;16(3):266-276). Without wishing to be bound by theory, nanoparticles having aAttorney Docket No. 14779-010-228 composition according to Section 5.1 such that the hydrodynamic diameter of the nanoparticle is less than about 60 nm are capable of being internalized by LSECs.

[0061] In some embodiments, the nanoparticle is capable of being processed after internalization by LSECs to release one or more of the peptides associated or covalently linked to the nanoparticle. In some embodiments, the nanoparticle is capable of being processed after internalization by LSECs via hydrolysis of the amide bonds covalently linking the N-termini of one or more peptides to the nanoparticle. In some embodiments, the nanoparticle is capable of being processed after internalization by LSECs via hydrolysis of the ester or amide bonds covalently linking the C-termini of one or more peptides to the nanoparticle.

[0062] In some embodiments, the nanoparticle is capable of suppressing a specific immune response. In some embodiments, the nanoparticle is capable of suppressing a specific immune response to a peptide described in Section 5.1.4 or to a protein comprising the amino acid sequence of a peptide described in Section 5.1.4.

[0063] In certain embodiments, the nanoparticle, by virtue of the polymer forming the micelle, can be negatively charged or uncharged. In some embodiments, the nanoparticle is negatively charged at a pH of 6 to 7 (pH during measurement). In some embodiments, the polymer coating can comprise acid, e.g., carboxylic acid groups, leading to a negative charge of the nanoparticle.

[0064] In certain embodiments, the nanoparticle provided herein has a hydrodynamic diameter (z-average) in the range of 10 to 100 nm as determined by Dynamic Light Scattering (DLS). In some embodiments, the nanoparticle has a hydrodynamic diameter in the range of 10 to 60 nm as determined by DLS. In some embodiments, the nanoparticle has a hydrodynamic diameter in the range of 10 to 50 nm as determined by DLS. In some embodiments, the nanoparticle has a hydrodynamic diameter in the range of 10 to 40 nm as determined by DLS. In some embodiments, the nanoparticle has a hydrodynamic diameter in the range of 10 to 30 nm as determined by DLS. In some embodiments, the nanoparticle has a hydrodynamic diameter in the range of 20 to 30 nm as determined by DLS.

[0065] In certain embodiments, the nanoparticle provided herein has a zeta potential between about -20 and -50 mV. In certain embodiments, the nanoparticle provided herein has a zeta potential between about -25 and -45 mV. In certain embodiments, the nanoparticle provided herein has a zeta potential between about -28 and -42 mV. In certain embodiments, the zetaAttorney Docket No. 14779-010-228 potential of the nanoparticle is measured at a solution pH of 6 to 7. In certain embodiments, the zeta potential of the nanoparticle is measured using a Malvern Zetasizer Nano ZS instrument.

[0066] In certain embodiments, the nanoparticles disclosed herein can have a polydispersity index below 0.50, preferably between 0.05 and 0.45, more preferably between 0.10 and 0.40, as measured by dynamic light scattering (DLS).

[0067] The determination of the hydrodynamic diameter and the poly dispersity index is carried out using electrophoretic light scattering analysis methods, preferably a Malvern Zetasizer. In one embodiment, the method for determining the hydrodynamic diameter and the poly dispersity index is carried out using electrophoretic light scattering, disposable polystyrene cuvettes, Zetasizer Software 7.12, milli-Q water. The nanosphere size standards of 20 nm and 100 nm (NIST certified or equivalent) are diluted in an aqueous 0.9% sodium chloride solution and the test samples are diluted in water. All aqueous reagents are filtered through 0.22 pm membrane prior to use. In the most preferred embodiment, the method for determining the hydrodynamic diameter and the poly dispersity index is carried out using electrophoretic light scattering in combination with the following analysis conditions (Table 1):Table 1: Overview of the Analysis Conditions

[0068] The evaluation of the data is based on mean diameter (Z-Average, nm by intensity), which is a parameter also known in DLS as the cumulants mean and Polydispersity index (PDI), which is used as a measure of the size distribution.

[0069] In certain embodiments, the nanoparticles in the pharmaceutical composition of the present disclosure can have a total polymer content of between about 0.1 mg / mL to about 5 mg / mL, preferably between 0.5 mg / mL to 4 mg / mL, more preferably of 1 mg / mL to 3 mg / mL.Attorney Docket No. 14779-010-228The total polymer content is determined by GPC. For measuring the total polymer content, the peptides are hydrolyzed, and the particles are destroyed (e.g., using a 6 M HC1 solution). The polymer is extracted after addition of EDTA. After evaporation of solvent, the residue is redissolved and the polymer content is determined by GPC.

[0070] The determination of total polymer content is preferably carried out using the following reagents and reference standards: water (HPLC grade), acetonitrile (HPLC grade), tetrahydrofuran with BHT (THF-HPLC grade), acetic acid 100% (analytical grade), hydrochloric acid 37% (analytical grade), ethylenediaminetetraacetic acid disodium salt dihydrate (analytical grade), ethyl acetate (analytical grade), sodium hydroxide (analytical grade) and poly(maleic acid-alt- 1 -octadecene) as reference material. The chromatographic conditions for the determination of total polymer content are as follows (Table 2):Table 2: Chromatographic Conditions for Determination of Total Polymer Content

[0071] The nanoparticles in the pharmaceutical composition disclosed herein comprise a high amount of peptides. In certain embodiments, the pharmaceutical composition can have a total peptide content of more than 0.1 mM, preferably between about 0.2 mM to about 1 mM, more preferably of 0.3 mM to 0.8 mM. In certain embodiments, the total peptide content can be measured by hydrolyzing the peptides with HC1 and derivatizing the resulting amino acids with ortho-phthaldehyde prior to HPLC analysis. In certain embodiments, the samples can be analyzed by standard addition with amino acid standard solution.Attorney Docket No. 14779-010-228

[0072] Nanoparticles can be prepared by any method known in the art. Specifically, nanoparticles as described above can be prepared using a poly (maleic acid-alt- 1 -octadecene), a poly(maleic acid-alt- 1 -tetradecene) or a poly(maleic acid-alt- 1 -dodecene) polymer. Non-limiting methods for preparing nanoparticles include those described in International Patent Application Publication Nos.: WO 2013 / 072051 and WO 2021 / 165227, each of which is incorporated herein by reference in its entirety. In certain embodiments, the nanoparticles are prepared according to the methods described in the Examples, infra. It should be understood that these methods are merely exemplary and that the nanoparticles provided herein can be prepared according to other methods.

[0073] The different components of the nanoparticles used in the pharmaceutical composition disclosed herein are described in more detail in the following sections (e.g., Sections 5.1.1 - 5.1.4).5.1.1 Micelles

[0074] In the context of the present disclosure, the term “micelle” relates 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, sequestering the hydrophobic “tail” regions of the amphiphilic molecules on the inside of the micelle, thus providing a dissolution like distribution behavior of the nanoparticles in aqueous liquids, i.e., render the nanoparticles water-soluble. This type of micelle is also known as a normal phase micelle (or oil-in-water micelle).

[0075] In certain embodiments, the micelle disclosed herein can be formed by one, but also by more than 1, e.g., 2, 3 or 4 amphiphilic polymeric molecules. In certain embodiments, the micelle disclosed herein can be formed by the same or by different amphiphilic polymeric molecules. In general, in the context of the specification, “a” or “the” is not intended to be limiting to “one” unless specifically stated.

[0076] In a preferred embodiment, the micelle disclosed herein is formed by a single layer of amphiphilic polymers. Such a micelle can be structurally distinct from a bilayer or a liposome formed by an amphiphilic polymer. In this case the structures are not, or not to a significant percentage (e.g., not more than 10%, more than 5%, or preferably, more than 1%), comprised in the nanoparticle of the present disclosure.Attorney Docket No. 14779-010-228

[0077] In certain embodiments, the amphiphilic polymer is used to produce at least 70%, preferably at least 90% of the micelle disclosed herein. In a preferred embodiment, the micelle disclosed herein consists of the amphiphilic polymer.

[0078] In certain embodiments, the nanoparticles disclosed herein do not comprise a solid hydrophobic core. In certain embodiments, the nanoparticles disclosed herein comprise the micelle and a solid hydrophobic core.

[0079] In certain embodiments, the micelle disclosed herein can be co-stabilized with further components such as fatty acids or phosphatidylcholines. In some embodiments, preferred fatty acids are stearic acid or oleic acid. A preferred phosphatidylcholine is selected from Lipoid SI 00, Lipoid S PC3 and DSPC. In some embodiments, cholesterol can also be used as a costabilizer.5.1.2 Amphiphilic Polymers

[0080] The present disclosure relates, in part, to nanoparticles comprising an amphiphilic polymer. In certain embodiments, the amphiphilic polymer comprises: (a) a hydrophobic region; and (b) a hydrophilic region. In certain embodiments, the hydrophobic region comprises a hydrocarbyl chain comprising 4 to 30, or preferably 7 to 19, carbon atoms. In certain embodiments, the hydrophilic region of the amphiphilic polymer comprises a protonatable or deprotonatable moiety (e.g. -OH or -COOH) such that the amphiphilic polymer is capable of having a net charge (e.g. net negative charge) in solution. One of skill in the art will recognize that the degree of protonation or deprotonation of the moiety and, thus, the net charge of the amphiphilic polymer is an equilibrium process that depends on the p L of the moiety and the pH of the solution.

[0081] In certain embodiments, the amphiphilic polymer of the present disclosure generally comprises a hydrophobic region comprising a hydrophobic aliphatic chain having a length of 8 to 23, preferably 8 to 21, most preferably 16 to 18 carbon atoms. In certain embodiments, the hydrophilic region of the amphiphilic polymer can be negatively charged in an aqueous solution.

[0082] In a preferred embodiment of the present disclosure, the amphiphilic polymer spontaneously forms micelles in solution. When a solid hydrophobic core is present, the amphiphilic polymer forms micelles around the solid core, rendering the nanoparticle water- soluble.

[0083] In some embodiments, the number average molecular weight (Mn) of the amphiphilicAttorney Docket No. 14779-010-228 polymer is about 20,000 g / mol or less, about 10,000 g / mol or less, or about 6,000 g / mol or less. In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is between about 1,000 g / mol and about 20,000 g / mol, between about 2,500 g / mol and about 20,000 g / mol, between about 1,000 g / mol and about 10,000 g / mol, between about 2,500 g / mol and about 10,000 g / mol, between about 1,000 g / mol and about 6,000 g / mol, between about 2,500 g / mol and about 6,000 g / mol, between about 1,000 g / mol and about 4,000 g / mol, or between about 2,500 g / mol and about 4,000 g / mol. In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is between about 1,000 g / mol and about 6,000 g / mol. In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is between about 2,500 g / mol and about 6,000 g / mol. In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is between about 2,500 g / mol and about 4,000 g / mol.

[0084] In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is determined using gel permeation chromatography (GPC). In some embodiments, polystyrene is used as a calibration standard. In some embodiments the number average molecular weight (Mn) of the amphiphilic polymer is determined using a PL-gel mixed D column at a temperature of 40°C, a mobile phase consisting of tetrahydrofuran / acetic acid 90 / 10% (v / v), a flow rate of 1.0 ml / min, in combination with a refractive index detector at a temperature of 35°C and polystyrene as calibration standard. In some embodiments, the number average molecular weight (Mn) of the amphiphilic polymer is determined using GPC and the following measurement conditions (Table 3):Table 3: Conditions for Measuring the Number Average Molecular Weight (Mn)Attorney Docket No. 14779-010-228

[0085] In certain embodiments, the amphiphilic polymer of the present disclosure can be an alternating copolymer. In certain embodiments, an alternating copolymer is a copolymer comprising two species of monomeric units distributed in alternating sequence.

[0086] In certain embodiments, the amphiphilic polymer disclosed herein is a copolymer of maleic anhydride and at least one alkene. In certain embodiments, the alkene used in the production of the amphiphilic polymer disclosed herein can 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. In a preferred embodiment of the present disclosure, the amphiphilic polymer is a copolymer of maleic anhydride and an alkene.

[0087] In some embodiments, the amphiphilic polymer comprises a hydrophilic poly-maleic anhydride backbone and hydrophobic hydrocarbyl side chains. In some embodiments, the side chain is a linear alkyl chain. In some embodiments, the side chain comprises 5 to 31 carbon atoms. In some embodiments, the side chain comprises 8 to 20 carbon atoms. In some embodiments, the side chain comprises 10 to 18 carbon atoms. In some embodiments, the side chain comprises 16 carbon atoms.

[0088] In certain embodiments, the amphiphilic polymer disclosed herein can comprise the following building block:wherein R is a hydrocarbyl group or a substituted hydrocarbyl group. In some embodiments, R is a linear, unsubstituted hydrocarbyl group. In some embodiments, R is a C4 to C30 alkyl group. In some embodiments, R is a linear, unsubstituted C4 to C30 alkyl group. In some embodiments, R is a C7 to C19 alkyl group. In some embodiments, R is a linear, unsubstituted C7 to C19 alkyl group. In some embodiments, R is a C9 to C17 alkyl group. In some embodiments, R is a linear, unsubstituted Cgto C17 alkyl group. In some embodiments, R is a C15 alkyl group. In some embodiments, R is a C15 linear, unsubstituted alkyl group. In some embodiments, the amphiphilic polymer comprises repeating units of the building block. In some embodiments, the amphiphilic polymer comprises n repeating units of the building block, wherein n a numberAttorney Docket No. 14779-010-228 between about 6 and about 11. In some embodiments, the amphiphilic polymer consists of repeating units of the building block. In some embodiments, the amphiphilic polymer consists of n repeating units of the building block, wherein n a number between about 6 and about 11.

[0089] In certain embodiments, the amphiphilic polymer disclosed herein can consist of the building block defined above. In certain embodiments, the amphiphilic polymer disclosed herein comprises at least 50%, preferably at least 70%, most preferably more than 90% of the building block defined above.

[0090] In some embodiments, the amphiphilic polymer is selected from the group consisting of poly(maleic acid-a / z-1 -octadecene), poly(maleic acid-a / t-1 -tetradecene) and poly(maleic acid- ah- \ -dodecene). In some embodiments, the amphiphilic polymer is poly(maleic acid-a / z-1- octadecene). In some embodiments, the amphiphilic polymer is poly (maleic acid- 1 -octadecene) and the number average molecular weight of the polymer is from about 1,000 to 6,000 g / mol. In some embodiments, the amphiphilic polymer disclosed herein is selected from the group consisting of poly (maleic acid-a / z-1 -octadecene), poly(maleic acid-a / z-1 -dodecene) and poly (maleic acid-a / t-1 -tetradecene), preferably the polymer is poly(maleic acid-a / z-1 -octadecene) and the number average molecular weight of the polymer is from 5000 to 1000 g / mol.5.1.3 Solid Hydrophobic Core

[0091] In some embodiments, the nanoparticles comprise a solid hydrophobic core which is coated by the micelle. In some embodiments, the nanoparticle provided herein comprises a solid inorganic core. In certain embodiments, the nanoparticle provided herein does not comprise a solid inorganic core.

[0092] In certain embodiments, the diameter of the core disclosed herein can be 2 to 500 nm, preferably, 3 to 25 nm, more preferably, 5 to 15 nm. The diameter of the core can be determined using transmission electron microscopy (TEM) or small-angle X-ray scattering (SAXS).

[0093] Exemplary inorganic cores are iron oxide nanoparticles stabilized by oleic acid or another carboxylic acid (C14-C22, preferably, Cie-Cis), quantum dots (CdSe / CdS / ZnS stabilized, e.g., by trioctyloxinphosphinoxide), gold nanoparticles, e.g., stabilized by sulfonic compounds. Such inorganic cores by themselves are typically not stable in an aqueous solvent such as water, but embedding them in the polymeric micelles renders them water-soluble. Without being bound by any particular theory, the hydrophobic parts of the amphiphilic polymer interact with the hydrophobic core of the nanoparticle, leading to the formation of a single coating layer ofAttomey Docket No. 14779-010-228 polymer surrounding the core. In the coating process, the amphiphilic polymer can replace the hydrophobic part of the core by ligand exchange and the double layer micelle is thus formed around the core.

[0094] In certain embodiments, the polymer at least partially replaces the oleic acid on the surface of the core particle and the hydrophilic part of the polymer interacts with the surface of the iron oxide core and the hydrophobic part of the polymer interact with each other forming a double layer micelle around the iron oxide core, resulting in an iron oxide coated with polymer.

[0095] According to a preferred embodiment of the present disclosure, the core is superparamagnetic. In a specifically preferred embodiment of the present disclosure, the core is a superparamagnetic iron oxide nanoparticle (SPION), which may be stabilized by oleic acid.

[0096] In certain embodiments, the core disclosed herein preferably renders the nanoparticles of the present disclosure traceable, e.g., by their characteristics in fluorescence, electron microscopy or any other detection method.5.1.4 Peptides

[0097] In certain embodiments, the pharmaceutical composition of the present disclosure comprises at least one peptide. In certain embodiments, the pharmaceutical composition of the present disclosure comprises a nanoparticle wherein the nanoparticle comprises at least one peptide.

[0098] The present disclosure relates, in part, to nanoparticles comprising a peptide. In certain embodiments, the peptide is covalently linked to a component of the nanoparticle such that the peptide remains on the outside of the nanoparticle. In some embodiments, peptide is associated with the outside of the nanoparticle. In some embodiments, the peptide is non- covalently associated with the outside of the nanoparticle. In some embodiments, the peptide is non-covalently associated with the amphiphilic polymer. In some embodiments, the peptide is associated with the amphiphilic polymer by a linker. In some embodiments, the peptide is covalently associated with the outside of the nanoparticle. In some embodiments, the peptide is covalently linked to the amphiphilic polymer. For example, in some embodiments, the peptide can be covalently linked to amphiphilic polymer via formation of an amide bond between the N- terminus of the peptide and a carboxylate group of the amphiphilic polymer. Known methods of covalently coupling peptides include carbodiimide or succinimide coupling. In some embodiments, the peptide is covalently linked using l-Ethyl-3-(3-Attorney Docket No. 14779-010-228 dimethylaminopropyl)carbodiimide (EDC) chemistry or any other peptide coupling reagents. See, for example, Jaradat et al. , Advances in solid-phase peptide synthesis in aqueous media (ASPPS) ((Critical Review) Green Chem., 2022, 24, 6360-6372); Albericio et al., Choosing the Right Coupling Reagent for Peptides: A Twenty-Five-Year Journey, Org. Process Res. Dev. 2018, 22, 7, 760-772.

[0099] In some embodiments, the peptide of the present disclosure is 4 to 50 amino acids long. In some embodiments, the peptide is 5 to 50 amino acids long. In some embodiments, the peptide is 6 to 50 amino acids long. In some embodiments, the peptide is 7 to 50 amino acids long. In some embodiments, the peptide is 8 to 50 amino acids long. In some embodiments, the peptide is 8 to 40 amino acids long. In some embodiments, the peptide is 8 to 30 amino acids long. In some embodiments, the peptide is 8 to 20 amino acids long. In some embodiments, the peptide is 8 to 15 amino acids long. In some embodiments, the peptide is 8 to 14 amino acids long. In some embodiments, the peptide is 8 to 13 amino acids long. In some embodiments, the peptide is 8 to 12 amino acids long. In some embodiments, the peptide is 8 to 11 amino acids long. In some embodiments, the peptide is 8 to 10 amino acids long. In some embodiments, the peptide is 8 to 9 amino acids long. In some embodiments, the peptide is 8 to 40 amino acids long. In some embodiments, the peptide is 10 to 50 amino acids long. In some embodiments, the peptide is 10 to 40 amino acids long. In some embodiments, the peptide is 10 to 30 amino acids long. In some embodiments, the peptide is 10 to 20 amino acids long. In some embodiments, the peptide is 12 to 30 amino acids long. In some embodiments, the peptide is 12 to 25 amino acids long. In some embodiments, the peptide is 12 to 20 amino acids long. In some embodiments, the peptide is 12 to 19 amino acids long. In some embodiments, the peptide is 12 to 18 amino acids long. In some embodiments, the peptide is 12 to 17 amino acids long. In some embodiments, the peptide is 12 to 16 amino acids long. In some embodiments, the peptide is 12 to 15 amino acids long. In some embodiments, the peptide is 13 to 25 amino acids long. In some embodiments, the peptide is 13 to 20 amino acids long. In some embodiments, the peptide is 13 to 19 amino acids long. In some embodiments, the peptide is 13 to 18 amino acids long. In some embodiments, the peptide is 13 to 17 amino acids long. In some embodiments, the peptide is 13 to 16 amino acids long. In some embodiments, the peptide is 14 to 25 amino acids long. In some embodiments, the peptide is 14 to 20 amino acids long. In some embodiments, the peptide is 14 to 19 amino acids long. In some embodiments, the peptide is 14 to 18 amino acids long. InAttorney Docket No. 14779-010-228 some embodiments, the peptide is 14 to 17 amino acids long. In some embodiments, the peptide is 14 to 16 amino acids long. In some embodiments, the peptide is 12 to 15 amino acids long. In some embodiments, the peptide is 25 to 50 amino acids long. In some embodiments, the peptide is 30 to 50 amino acids long. In some embodiments, the peptide is 40 to 50 amino acids long. In some embodiments, the peptide is 5 amino acids long. In some embodiments, the peptide is 6 amino acids long. In some embodiments, the peptide is 7 amino acids long. In some embodiments, the peptide is 8 amino acids long. In some embodiments, the peptide is 9 amino acids long. In some embodiments, the peptide is 10 amino acids long. In some embodiments, the peptide is 11 amino acids long. In some embodiments, the peptide is 12 amino acids long. In some embodiments, the peptide is 13 amino acids long. In some embodiments, the peptide is 14 amino acids long. In some embodiments, the peptide is 15 amino acids long. In some embodiments, the peptide is 16 amino acids long. In some embodiments, the peptide is 17 amino acids long. In some embodiments, the peptide is 18 amino acids long. In some embodiments, the peptide is 19 amino acids long. In some embodiments, the peptide is 20 amino acids long. In some embodiments, the peptide is 21 amino acids long. In some embodiments, the peptide is 22 amino acids long. In some embodiments, the peptide is 23 amino acids long. In some embodiments, the peptide is 24 amino acids long. In some embodiments, the peptide is 25 amino acids long.

[0100] In some embodiments, the peptide disclosed herein comprises an MHC-I epitope. In some embodiments, the peptide comprises an MHC-II epitope. In some embodiments, the peptide comprises two or more different peptides linked to each other as a fusion polypeptide. In certain embodiments, the peptide comprises two or more different peptides comprising MHC-I epitopes linked to each other as a fusion polypeptide. In certain embodiments, the peptide comprises two or more different peptides comprising MHC-II epitopes linked to each other as a fusion polypeptide. In certain embodiments, the peptide comprises two or more different peptides comprising one or more MHC-I epitopes linked to one or more MHC-II epitopes as a fusion polypeptide.

[0101] In certain embodiments, a peptide for use in the present disclosure can comprise an amino acid sequence corresponding to an autoantigen. In certain embodiments, a peptide for use in the present disclosure can comprise an amino acid sequence corresponding to an autoantigen associated with an autoimmune disease. In certain embodiments, a peptide for use in the presentAttorney Docket No. 14779-010-228 disclosure can comprise an amino acid sequence corresponding to an autoantigen associated with type 1 diabetes (T1D). In certain embodiments, a peptide for use in the present disclosure can comprise an amino acid sequence corresponding to an islet autoantibody. In certain embodiments, a peptide for use in the present disclosure can comprise an amino acid sequence corresponding to an autoantigen associated with multiple sclerosis (MS). In certain embodiments, a peptide for use in the present disclosure can comprise an amino acid sequence corresponding to a proteolipid protein (PLP), a myelin-associated glycoprotein (MAG), or a myelin-associated basic oligodendrocytic protein (MOG). Exemplary peptides for use in the present disclosure are provided in Table 4.Table 4: Exemplary Peptides

[0102] In certain embodiments, the peptide for use in the present disclosure comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the peptide for use in the present disclosure comprises the amino acid sequence of SEQ ID NO: 1. In certain embodiments, the peptide for use in the present disclosure comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acidAttorney Docket No. 14779-010-228 sequence of SEQ ID NO: 2. In certain embodiments, the peptide for use in the present disclosure comprises the amino acid sequence of SEQ ID NO: 2. In certain embodiments, the peptide for use in the present disclosure comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the peptide for use in the present disclosure comprises the amino acid sequence of SEQ ID NO: 3. In certain embodiments, the peptide for use in the present disclosure comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the peptide for use in the present disclosure comprises the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the peptide for use in the present disclosure comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the peptide for use in the present disclosure comprises the amino acid sequence of SEQ ID NO: 5. In certain embodiments, the peptide for use in the present disclosure comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the peptide for use in the present disclosure comprises the amino acid sequence of SEQ ID NO: 6. In certain embodiments, the peptide for use in the present disclosure comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the peptide for use in the present disclosure comprises the amino acid sequence of SEQ ID NO: 7. In certain embodiments, the peptide for use in the present disclosure comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the peptide for use in the present disclosure comprises the amino acid sequence of SEQ ID NO: 8. In certain embodiments, the peptide for use in the present disclosure comprises at least 75%, atAttorney Docket No. 14779-010-228 least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the peptide for use in the present disclosure comprises the amino acid sequence of SEQ ID NO: 9. In certain embodiments, the peptide for use in the present disclosure comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the peptide for use in the present disclosure comprises the amino acid sequence of SEQ ID NO: 10. In certain embodiments, the peptide for use in the present disclosure comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the peptide for use in the present disclosure comprises the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the peptide for use in the present disclosure comprises at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO: 12. In certain embodiments, the peptide for use in the present disclosure comprises the amino acid sequence of SEQ ID NO: 12.

[0103] In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 1-12. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 1-6. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence of SEQ ID NO: 2. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence of SEQ ID NO: 3. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence of SEQ ID NO: 4. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequenceAttorney Docket No. 14779-010-228 of SEQ ID NO: 5. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence of SEQ ID NO: 6.

[0104] In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 1-5. In certain embodiments, nanoparticles of the present disclosure comprise two peptides selected from the group of amino acid sequences as set forth in SEQ ID NOs: 1-5. In certain embodiments, nanoparticles of the present disclosure comprise three peptides selected from the group of amino acid sequences as set forth in SEQ ID NOs: 1-5. In certain embodiments, nanoparticles of the present disclosure comprise four peptides selected from the group of amino acid sequences as set forth in SEQ ID NOs: 1-5. In certain embodiments, nanoparticles of the present disclosure comprise five peptides, wherein the five peptides consist of the amino acid sequences of SEQ ID NOs: 1-5.

[0105] In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 7-12. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence of SEQ ID NO: 7. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence of SEQ ID NO: 8. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence of SEQ ID NO: 9. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence of SEQ ID NO: 10. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence of SEQ ID NO: 12.

[0106] In certain embodiments, a nanoparticle of the present disclosure comprises a peptide comprising the amino acid sequence as set forth in any one of SEQ ID NOs: 7-11. In certain embodiments, nanoparticles of the present disclosure comprise two peptides selected from the group of amino acid sequences as set forth in SEQ ID NOs: 7-11. In certain embodiments, nanoparticles of the present disclosure comprise three peptides selected from the group of amino acid sequences as set forth in SEQ ID NOs: 7-11. In certain embodiments, nanoparticles of the present disclosure comprise four peptides selected from the group of amino acid sequences as set forth in SEQ ID NOs: 7-11. In certain embodiments, nanoparticles of the present disclosureAttorney Docket No. 14779-010-228 comprise five peptides, wherein the five peptides consist of the amino acid sequences of SEQ ID NOs: 7-11.

[0107] In some embodiments, the peptide disclosed herein is synthesized, recombinantly expressed, or isolated or modified from natural sources.

[0108] In certain embodiments, the peptide can be associated with the outside of the micelle or encapsulated in the inside of the micelle (in embodiments where no solid hydrophobic core is present in the nanoparticle). In certain embodiments, the peptide can be localized on the outside of the micelle or inside the micelle.

[0109] In certain embodiments, the peptide can be covalently linked to the micelle or non- covalently associated, preferably covalently linked to the micelle. In a preferred embodiment, the peptide is covalently linked to the micelle using a method of covalently coupling peptides known in the art such as carbodiimide or succinimide coupling. Preferably, the peptide is covalently linked to the micelles using l-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry.

[0110] In some embodiments, the composition of the nanoparticle is determined by standard analytical means known in the art, such as, for example, Nuclear Magnetic Resonance (NMR) or Mass Spectrometry, with or without complete or partial decomposition of the nanoparticle and separation of the individual components of the nanoparticle by chromatographic or other means. For example, the amount of peptides associated with and / or covalently linked to the nanoparticle may be determined following cleavage of the peptides from the nanoparticle, for example by hydrolysis of amide and / or ester linkages at the N- or C-terminus of peptide, respectively. In some embodiments, the composition of the nanoparticle corresponds to the average composition of a population of nanoparticles.

[0111] In certain embodiments, a nanoparticle of the present disclosure can comprise about 25 to about 300, about 50 to about 200, about 75 to about 150, or about 100 to 125 peptides per nanoparticle. In certain embodiments, a nanoparticle of the present disclosure can comprise about 25, about 50, about 75, about 100, about 125, about 150, about 175, about 200, about 225, about 250, about 275, or about 300 peptides per nanoparticle. In certain embodiments, a nanoparticle of the present disclosure can comprise about 55, about 100, about 105, about 110, about 115, about 120 about 125, or about 130 peptides per nanoparticle.5.2 Methods of Making the NanoparticlesAttomey Docket No. 14779-010-228

[0112] The nanoparticles comprising the pharmaceutical composition of the present disclosure can be produced by a method comprising the following steps: a) obtaining a hydrophobic core nanoparticle, b) obtaining an amphiphilic polymer, preferably using radical copolymerization, c) optionally purifying the amphiphilic polymer, d) mixing of the hydrophobic core nanoparticles and the amphiphilic polymer to form micelles, e) adding at least one peptide to form the nanoparticles.If the peptides are encapsulated by the micelle, step e) is performed prior to step d). In this case, the peptides are added to the amphiphilic polymer prior to micelle formation.

[0113] In certain embodiments, the hydrophobic core of step a) can be synthesized using appropriate reactants in solution. Preferably, the hydrophobic core is synthesized using metal salts and salts of carboxylic acids as reactants in the presence of organic solvents. Preferably, the reaction is conducted at elevated temperatures under oxygen restriction. In certain embodiments, where the nanoparticle of the present disclosure does not comprise a hydrophobic core, step a) of the method can be omitted.

[0114] In certain embodiments, the amphiphilic polymer used in the nanoparticles of the present disclosure can be prepared by a radical copolymerization using a radical initiator. In certain embodiments, a method of obtaining the amphiphilic polymer with a number average molecular weight (Mn) of 20,000 g / mol or less resides in synthesizing the same using a two step method, comprising a step of producing a polymer of the anhydride and a step of hydrolyzing the anhydride to obtain an acid. In some embodiments, the molecular weight of the polymer can be controlled by varying the concentrations of the reactants or the amount of radical initiator. In some embodiments, the molecular weight of the polymer can be analyzed by gel permeation chromatography. In some embodiments, the copolymerization may be conducted in an organic solvent such as 1,4 dioxane, xylene or chlorobenzene.

[0115] Many radical initiators are known in the art; they include various peroxides and azotype compounds. Non-limiting 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, t-butyl perbenzoate and perborates. Suitable azo-type compounds include 2,2'-Azobis(2-methylpropionitrile), p-Attorney Docket No. 14779-010-228 bromobenzenediazonium fluoborate, p-tolyldiazoaminobenzene, p-bromobenzenediazonium hydroxide, azomethane and phenyl — diazonium halides. In some preferred embodiments, the radical initiator is 2,2'-Azobis(2-methylpropionitrile).

[0116] In certain embodiments, the copolymerization can be conducted at elevated temperatures such as from 70°C to 120°C, preferably from 90°C to 110°C. Preferably, the copolymerization is initiated by heating the mixture to 70°C to 120°C, preferably from 90°C to 110°C.

[0117] In certain embodiments, step b) of the method disclosed herein can comprise the steps of mixing the reactants, deoxygenizing the mixture, heating the mixture and then cooling the mixture. Afterwards, the polymer may be dissolved and stirred overnight. The formed solid may be recovered, preferably using centrifugation.

[0118] In certain embodiments, step b) of the method disclosed herein can include the addition of a base to the polymer (e.g. NaOH). Preferably, the base is reacted with the polymer at elevated temperature, preferably between 50°C and 70°C, such as 60°C until almost all solids are dissolved. The resulting suspension may be acidified (e.g. pH <2). Afterwards, the reaction mixture may be extracted with an organic solvent such as ethyl acetate. The organic layer may be extracted with a sodium hydroxide solution. The aqueous solution may be again extracted with an organic solvent such as ethyl acetate and then dried to obtain the purified amphiphilic polymer.

[0119] In certain embodiments, the polymer can be further purified in step c) of the method disclosed herein. Preferably, the polymer is further purified by extracting the polymer with n- hexane or n-heptane. The extraction can be performed at concentrations of greater than 10 g / L, preferably 100 g / 1. Furthermore, an additional purification step of the amphiphilic polymer may 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 / iso-propanol. The mixtures used may contain for example 95 / 5% (v / v%) methanol / heptane, 10 / 90 (v / v%) acetonitrile / iso-propanol or 5 / 95 (v / v%) acetonitrile / iso-propanol. In a preferred embodiment, the precipitation mixture is added at temperatures of -10 to 10°C, preferably -5 to 5°C. In certain embodiments, the purity of the amphiphilic polymer after hydrolysis and workup can be measured by1H NMR.Attorney Docket No. 14779-010-228

[0120] In certain embodiments, the micelle can be formed in step d) of the method disclosed herein by forming a solution containing the amphiphilic polymer. Preferably, the micelle is formed in an aqueous solution. Co-stabilizers may be added to the amphiphilic polymer to improve micelle formation. Preferably, step d) comprises the sub-steps of solubilising the amphiphilic polymer and the core particles, removing the solvent until a thin film is formed, adding a basic aqueous solution at increased temperature and ambient pressure to form an aqueous colloidal dispersion, diluting the solution and optionally filtering it. Afterwards, several washing steps can be applied.

[0121] In certain embodiments, the peptides to be used in step e) of the method disclosed herein can be synthesized using state of the art solid phase chemistry. In certain embodiments, the synthesis of the peptides can be accomplished via Fmoc chemistry from the C to N direction using solid phase peptide synthesis (SPPS). The alpha amino group of each amino acid is protected with a fluoren-9-ylmethoxycarbonyl (Fmoc) group, while side chain functional groups are also blocked with various appropriate protective groups. In general, the SPPS consists of repeated cycles of N-terminal deprotection followed by coupling reactions. The first Fmoc- protected amino acid is coupled to the resin. Afterwards, the amine group is deprotected with a mixture of piperidine in dimethylformamide (DMF), and then coupled with the free acid of the second Fmoc-protected amino acid. The cycle is repeated until the desired sequence is obtained. The resin is washed between each step. The completion of each coupling reaction is monitored by a qualitative ninhydrin test. In the last step of the synthesis, the crude peptide-resin is successively washed with DMF and methanol, and dried. Then, the protective groups are removed from the peptide and the peptide is cleaved from the resin using trifluoroacetic acid (TFA). The obtained crude peptide is isolated by ether precipitation from the cleavage mixture. In certain embodiments, the peptide is further purified through preparative HPLC to reach purity requirements, and the counter ion TFA is replaced with chloride by using an appropriate solventbuffer system. In certain embodiments, the purified peptide is lyophilized.

[0122] In certain embodiments, the nanoparticles resulting from the method disclosed herein can be purified using intensive washing and filtration steps to remove the coupling reagent(s) and any low molecular weight components.Attorney Docket No. 14779-010-2285.3 Pharmaceutical Compositions

[0123] In certain embodiments, the pharmaceutical composition of the present disclsosure comprises a plurality of the nanoparticles disclosed herein. In certain embodiments, the pharmaceutical composition of the present disclsosure can comprise one type of nanoparticle disclosed herein. In certain embodiments, the pharmaceutical composition of the present disclsosure can comprise more than one type of nanoparticle disclosed herein. In certain embodiments, the pharmaceutical composition of the present disclsosure can comprise at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 different types of nanoparticles. In certain embodiments, the pharmaceutical composition of the present disclsosure can comprise at least 5 different types of nanoparticles. In certain embodiments, each type of nanoparticle disclosed herein comprises at least one peptide sequence, which differs from the peptide sequence or peptide sequences of the other types of nanoparticles. In certain embodiments, each type of nanoparticle disclosed herein comprises at least one peptide sequence that can be the same peptide sequence or peptide sequences of the other types of nanoparticles.

[0124] In certain embodiments, the pharmaceutical composition disclosed herein can comprise each type of nanoparticle in a concentration below 100 pM, preferably from 0.5 to 80 pM, most preferably from 1 to 50 pM. In certain embodiments, the pharmaceutical composition of the present disclosure can comprise the different types of nanoparticles in equimolar concentration. In some embodiments of the present disclosure, the pharmaceutical composition disclosed herein comprises one type of nanoparticle dispersed in an aqueous buffer. In some embodiments of the present disclosure, the pharmaceutical composition disclosed herein comprises between four and six different types of nanoparticles in equimolar concentration, preferably dispersed in an aqueous buffer. In some preferred embodiments of the present disclosure, the pharmaceutical composition disclosed herein comprises five different types of nanoparticles in equimolar concentration, preferably dispersed in an aqueous buffer.

[0125] In certain embodiments, the pharmaceutical composition of the present disclosure comprises different types of nanoparticles. Each type may comprise at least one peptide sequence, which differs from the peptide sequence or peptide sequences of the other types of nanoparticles. Thus, the nanoparticles may differ in the peptide sequence. In certain embodiments, each type of nanoparticle comprises only one single type of peptide (having one specific amino acid sequence). In certain embodiments, each nanoparticle can compriseAttorney Docket No. 14779-010-228 numerous peptides having the same amino acid sequence and the composition is obtainable by mixing different nanoparticles.

[0126] In certain embodiments, the pharmaceutical composition of the present disclosure comprises between four and six different types of nanoparticles, wherein all associated peptides of the different types of nanoparticles comprise at least one T cell epitope. In certain embodiments, the pharmaceutical composition of the present disclosure comprises between four and six different types of nanoparticles, wherein all associated peptides of the different types of nanoparticles comprise at least one antigenic peptide toward an autoantigen. In certain embodiments, the pharmaceutical composition of the present disclosure comprises between four and six different types of nanoparticles, wherein all associated peptides of the different types of nanoparticles comprise at least one antigenic peptide toward an autoantigen associated with TD1. In certain embodiments, the pharmaceutical composition of the present disclosure comprises between four and six different types of nanoparticles, wherein all associated peptides of the different types of nanoparticles comprise at least one antigenic peptide toward an autoantigen associated with MS.

[0127] In certain embodiments, the pharmaceutical composition disclosed herein comprises at least five different types of nanoparticles, wherein the different types of nanoparticles differ among each other in the peptide sequence, and wherein a first type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 1, a second type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 2, a third type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 3, a fourth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 4, and a fifth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 5. In certain embodiments, each type of nanoparticle comprises only peptides of the same peptide sequence. In certain embodiments, the pharmaceutical composition disclosed herein comprises one type of nanoparticle, wherein all the nanoparticles comprise a peptide comprising the amino acid sequence of SEQ ID NO: 6.

[0128] In certain embodiments, the pharmaceutical composition disclosed herein comprises at least five different types of nanoparticles, wherein the different types of nanoparticles differ among each other in the peptide sequence, and wherein a first type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 7, a second type of nanoparticleAttorney Docket No. 14779-010-228 comprises a peptide comprising the amino acid sequence of SEQ ID NO: 8, a third type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 9, a fourth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 10, and a fifth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 11. In certain embodiments, each type of nanoparticle comprises only peptides of the same peptide sequence. In certain embodiments, the pharmaceutical composition disclosed herein comprises one type of nanoparticle, wherein all the nanoparticles comprise a peptide comprising the amino acid sequence of SEQ ID NO: 12.

[0129] In certain embodiments, the pharmaceutical composition of the present disclosure comprises nanoparticles comprising a solid hydrophobic core, a micelle coating the core comprising an amphiphilic polymer with a number average molecular weight (Mn) of 20,000 g / mol or less, and at least one peptide. This has the additional advantage that the nanoparticles can be produced more easily and to a higher degree of purity. The inventors have surprisingly found that low molecular weight amphiphilic polymer generates fewer aggregates during coating of a solid core than a high molecular weight amphiphilic polymer. In addition, the low molecular weight amphiphilic polymer can be purified more efficiently compared to a high molecular weight amphiphilic polymer. In particular, unbound polymer can be separated more efficiently when a low molecular weight amphiphilic polymer is used in the nanoparticle of the disclosure.

[0130] In certain embodiments, all peptides covalently linked to the polymer of one nanoparticle have the same amino acid sequence and are covalently bound to the outside of a micellar structure comprising an amphiphilic polymer shell which consists of low molecular weight poly (maleic acid-alt- 1 -octadecene and a superparamagnetic iron oxide nanoparticle (SPION) core (see schematic structure illustrated in FIG. 1A).

[0131] In certain embodiments, pharmaceutical compositions disclosed herein can be formulated for administering any of the nanoparticles contemplated herein for peripheral administration, such as parenteral (e.g., subcutaneous, intravenous, intramuscular), a continuous infusion (e.g., intravenous drip, intravenous bolus, intravenous infusion), topical, nasal, or oral administration. Suitable pharmaceutically acceptable carriers and formulations comprising thereof are known the art and can be found at least in, for example, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (A. Adejare, Ed.; 23rd edition) Academic Press (2020).Attorney Docket No. 14779-010-228

[0132] In certain embodiments, the pharmaceutical composition of the present disclosure can comprise a liquid or lyophilized carrier. In certain embodiments, for administration to a human subject, the composition preferably is sterile and / or biologically compatible.

[0133] In certain embodiments, the pharmaceutical composition comprises nanoparticles in a liquid carrier. In certain embodiments, the liquid carrier is preferably water or water-based, e.g., a aqueous buffer such as phosphate buffered saline (PBS), Ringer solution, TRIS buffer or sodium chloride solution. In certain embodiments, suitable preservatives may or may not be contained within the pharmaceutical composition of the present disclosure.

[0134] In certain embodiments, the pharmaceutical composition disclosed herein comprises the nanoparticles dispersed in an aqueous buffer, wherein the buffer preferably comprises at least one sugar, at least one primary amine and / or at least one amino acid. In certain preferred embodiments, the pharmaceutical composition comprises nanoparticles dispersed in an aqueous solution of D-mannitol, Tris(hydroxymethyl)aminomethane (TRIS) and / or L-lactic acid. The use of this buffer has the advantage that the nanoparticles are very stable in this buffer and can be lyophilized later on. In certain embodiments, the nanoparticles within the pharmaceutical composition of the present disclosure are stable for at about 1-6 days, about 1-4 weeks, about 1-3 months, about 3-6 months, about 6-12 months, or more than 1 year. In certain embodiments, the nanoparticles within the pharmaceutical composition of the present disclosure are stable for at about 1-6 days, about 1-4 weeks, about 1-3 months, about 3-6 months, about 6-12 months, or more than 1 year at about at about 0°C ± 2°C, at about 4°C ± 2°C, at about 25 °C ± 5 °C, or higher than about 30°C ± 5°C.

[0135] In certain embodiments, the pharmaceutical composition disclosed herein comprises nanoparticles comprising a) a micelle comprising an amphiphilic polymer comprising the following building blockAttorney Docket No. 14779-010-228 wherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a linear alkyl group, preferably a linear Cn to C17 alkyl group, and wherein the polymer has a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, and b) at least one peptide comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-12; and c) a solid hydrophobic core which is at least partially coated by the micelle, wherein the core comprises a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver and gold.

[0136] In certain embodiments, the pharmaceutical composition disclosed herein comprises nanoparticles comprising a) a micelle comprising an amphiphilic polymer comprising the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a linear alkyl group, preferably a linear Cn to C17 alkyl group, and wherein the polymer has a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, and b) at least one peptide comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-6; and c) a solid hydrophobic core which is at least partially coated by the micelle, wherein the core comprises a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver and gold.

[0137] In certain embodiments, the pharmaceutical composition disclosed herein comprises nanoparticles comprising a) a micelle comprising an amphiphilic polymer comprising the following building blockAttorney Docket No. 14779-010-228wherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a linear alkyl group, preferably a linear Cn to C17 alkyl group, and wherein the polymer has a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, and b) at least one peptide comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 1-5; and c) a solid hydrophobic core which is at least partially coated by the micelle, wherein the core comprises a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver and gold.

[0138] In certain embodiments, the pharmaceutical composition disclosed herein comprises nanoparticles comprising a) a micelle comprising an amphiphilic polymer comprising the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a linear alkyl group, preferably a linear Cn to C17 alkyl group, and wherein the polymer has a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, and b) at least one peptide consisting of the amino acid sequence of SEQ ID NO: 6; and c) a solid hydrophobic core which is at least partially coated by the micelle, wherein the core comprises a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver and gold.

[0139] In certain embodiments, the pharmaceutical composition disclosed herein comprises nanoparticles comprising a) a micelle comprising an amphiphilic polymer comprising the following building blockAttorney Docket No. 14779-010-228wherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a linear alkyl group, preferably a linear Cn to C17 alkyl group, and wherein the polymer has a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, and b) at least one peptide comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 7-12; and c) a solid hydrophobic core which is at least partially coated by the micelle, wherein the core comprises a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver and gold.

[0140] In certain embodiments, the pharmaceutical composition disclosed herein comprises nanoparticles comprising a) a micelle comprising an amphiphilic polymer comprising the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a linear alkyl group, preferably a linear Cn to C17 alkyl group, and wherein the polymer has a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, and b) at least one peptide comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 7-11; and c) a solid hydrophobic core which is at least partially coated by the micelle, wherein the core comprises a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver and gold.

[0141] In certain embodiments, the pharmaceutical composition disclosed herein comprises nanoparticles comprisingAttorney Docket No. 14779-010-228 a) a micelle comprising an amphiphilic polymer comprising the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a linear alkyl group, preferably a linear Cn to C17 alkyl group, and wherein the polymer has a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, and b) at least one peptide consisting of the amino acid sequence of SEQ ID NO: 12; and c) a solid hydrophobic core which is at least partially coated by the micelle, wherein the core comprises a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver and gold.

[0142] In certain embodiments, the pharmaceutical composition comprises five different types of nanoparticles, wherein each nanoparticle comprises a) a micelle comprising an amphiphilic polymer comprising the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a linear alkyl group, preferably a linear Cn to C17 alkyl group, and wherein the polymer has a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, and b) at least one peptide comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1-5; and c) a solid hydrophobic core which is at least partially coated by the micelle, wherein the core comprises a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver and gold; and wherein the different types of nanoparticles differ among each other in the peptide sequence, and wherein a first type of nanoparticle comprises a peptide comprising the amino acidAttorney Docket No. 14779-010-228 sequence of SEQ ID NO: 1, a second type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 2, a third type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 3, a fourth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 4, and a fifth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 5.

[0143] In certain embodiments, the pharmaceutical composition comprises five different types of nanoparticles, wherein each nanoparticle comprises a) a micelle comprising an amphiphilic polymer comprising the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a linear alkyl group, preferably a linear Cn to C17 alkyl group, and wherein the polymer has a number average molecular weight (Mn) of 6,000 to 1,000 g / mol, and b) at least one peptide comprising any one of the amino acid sequences selected from the group consisting of SEQ ID NO: 7-11 ; and c) a solid hydrophobic core which is at least partially coated by the micelle, wherein the core comprises a traceable inorganic material selected from the group comprising iron oxide, CdSe / CdS / ZnS, silver and gold; and wherein the different types of nanoparticles differ among each other in the peptide sequence, and wherein a first type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 7, a second type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 8, a third type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 9, a fourth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 10, and a fifth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 11.5.4 Kits

[0144] In one aspect, the disclosure provides a kit comprising the nanoparticles (such as a nanoparticle according to Section 5.1) comprising a peptide (such as a peptide described inAttorney Docket No. 14779-010-228Section 5.1.4) or the pharmaceutical composition of the disclosure (such as a pharmaceutical composition according to Section 5.3) and instructions for use. In certain embodiments, the kit comprises the nanoparticles (such as a nanoparticle according to Section 5.1) or the pharmaceutical composition of the disclosure (such as a pharmaceutical composition according to Section 5.3), and instructions for use.5.5 Methods of Use

[0145] In certain embodiments, the pharmaceutical composition of the present disclosure can be used in the treatment of an autoimmune disease in a subject in need thereof. In certain embodiments, the method comprises administering a nanoparticle (such as a nanoparticle according to Section 5.1) (such as a peptide described in Section 5.1.4) and / or the pharmaceutical composition of the disclosure (such as a pharmaceutical composition according to Section 5.3) to the subject.

[0146] According to the present disclosure, the term “treating” is used to refer to the alleviation of symptoms of a particular disease in a subject, and / or improvement of an ascertainable measurement associated with a particular disease and / or disorder (e.g., an autoimmune disease). In certain embodiments, the autoimmune disease suitable for treatment with a pharmaceutical composition of the present disclosure can be type 1 diabetes (T1D), multiple sclerosis (MS), systemic sclerosis (scleroderma), polymyositis, ulcerative colitis, inflammatory bowel disease, Crohn’s disease, celiac disease, rheumatoid arthritis (RA), psoriasis, dermatomyositis, systemic lupus erythematosus (SLE), cutaneous lupus, and myasthenia gravis. In certain embodiments, the autoimmune disease suitable for treatment with a pharmaceutical composition of the present disclosure is T1D. In certain embodiments, the autoimmune disease suitable for treatment with a pharmaceutical composition of the present disclosure is MS.

[0147] In certain embodiments, the required dose and concentration for administration to the subject may be determined by the responsible medical attendant according to the facts and circumstances of the case. In certain embodiments, an exemplary dose might comprise 0.03 pmol to 0.90 pmol per patient body weight, e.g., for a human subject. In certain embodiments, the pharmaceutical composition of the present disclosure can be administered more than once. In certain embodiments, administration can be repeated, e.g., twice, three or 4 times, e.g., with, 1, 2, 3, 4, 5, 6, 7, 10 or 14 days between administrations. In certain embodiments, administrationAttorney Docket No. 14779-010-228 can also be repeated over extended periods of time, including once, twice, three times or four times a year.

[0148] In certain embodiments, the pharmaceutical composition of the present disclosure can be administered to a subject in need thereof. In some embodiments, the subject in need thereof is human. In some embodiments, the subject in need thereof is a human diagnosed as having or suspected of having an autoimmune disease.

[0149] In certain embodiments, a method of treatment provided herein can resolve at least one sign or symptom of an existing autoimmune disease of a subject. In certain embodiments, a method of treatment provided herein can at least temporarily restore the health of a subject, e.g., to restore to the health of the subject prior to the subject's development of the autoimmune disease. In certain embodiments, a method of treatment provided herein can slow the rate of progression of the autoimmune disease in a subject. In certain embodiments, a method of treatment provided herein can result in stabilization of, or prevent progression of, the autoimmune disease in a subject, e.g., can result in no functional decline in, or maintain the severity of, the disease or condition in the subject comparable to that of the subject before the method of treatment is administered according to the present disclosure.

[0150] In certain embodiments, the effect of a method of treatment provided herein may be assessed by monitoring clinical signs and symptoms of the disease to be treated (e.g., TD1, MS). In some embodiments, a method of treatment provided herein results in a change in one or more clinical laboratory measurements. Clinical laboratory measurements can be performed using standard methods in the art (e.g., ELISA, flow cytometry, RT-PCR, etc.) on a biological sample collected from a subject. Non-limiting examples of biological samples suitable for clinical laboratory measurements include urine, blood, plasma, serum, salvia, and sweat. Non-limiting examples of clinical laboratory measurements suitable for assessing the efficiency of the methods of the present disclosure include hemogram (WBC, RBC, Hemoglobin, Hematocrit, MCV, MCH, MCHC, RDW), rheumatoid factor, C-reactive protein, coagulation test, erythrocyte sedimentation test, urinalysis, renal function tests, anti-cyclic citrullinated peptide (anti-CCP) antibodies, complement tests, and ANA (antinuclear antibody) tests. An ANA test can assess the level of one or more antinuclear antibody subtypes in a sample collected from the subject. Nonlimiting examples of antinuclear antibody subtypes include anti-Ro antibodies, anti-La antibodies, anti-Sm antibodies, anti-nRNP antibodies, anti-Scl-70 antibodies, anti-dsDNAAttorney Docket No. 14779-010-228 antibodies, anti-histone antibodies, antibodies to nuclear pore complexes, anti-centromere antibodies, and anti-splOO antibodies.

[0151] In some embodiments, a clinical laboratory measurement can be used to assess the efficacy of a method disclosed herein by comparing the measured value of the clinical laboratory measurement as determined from a sample collected from a subject after treatment according to the method disclosed herein to a reference value. A reference value is (1) the measured value from a biological sample collected from one or more subjects with the same disease at the same disease severity who are not undergoing the treatment according to the method disclosed herein, (2) the value of the same subject determined at an earlier time point (i.e., “baseline”), and / or (3) a predetermined value.

[0152] In some embodiments, a method of treatment provided herein results in an improvement in one or more clinical laboratory measurements in a patient sample as compared to the reference value. In some embodiments, a method of treatment provided herein results in at least a 1%, 5%, 10%, 15%, for 20% improvement in one or more clinical laboratory measurements in a subject sample as compared to the reference value. In some embodiments, a method of treatment provided herein results in a comparable clinical laboratory measurement in a subject sample as compared to the reference value. A comparable measurement in clinical laboratory measurement can be interpreted as the treatment according to the method herein effectively preventing progression of the disease in the subject.

[0153] In some embodiments, a method of treatment provided herein can be further assessed via imaging (e.g. CAT scan, X-rays, MRI, ultrasound), physical exam, neurological exam, electromyography, lumbar puncture, eye exam, and / or evoked potentials.6. EXAMPLES

[0154] The following is a description of various methods and materials used in the studies. They are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention and are not intended to limit the scope of what the inventors regard as their invention, nor are they intended to represent that the experiments below were performed and are all of the experiments that may be performed. It is to be understood that exemplary descriptions written in the present tense were not necessarily performed, but rather that the descriptions can be performed to generate the data and the like associated with the teachings of the present invention. Efforts have been made to ensureAttorney Docket No. 14779-010-228 accuracy with respect to numbers used (e.g., amounts, percentages, etc.), but some experimental errors and deviations should be accounted for.6.1 Example 1

[0155] Autoimmune diseases (AD), such as multiple sclerosis (MS) and type 1 diabetes (T1D), are chronic disorders characterized by aberrant inflammatory responses of the adaptive immune system to self-antigens, resulting in cellular and tissue damage (Smith DA et al., Environ Health Perspect 107, 661-665 (1999)). Treating autoimmune diseases without resorting to nonspecific immunosuppression remains an unmet medical need. In the exemplary studies provided herein, a clinical stage nanoparticle-based platform technology was designed to reinstate antigen-specific immune tolerance against autoimmunity by leveraging the natural tolerogenic capacity of liver sinusoidal endothelial cells (LSECs).6.1.1 Results(a) Nanoanosized Antigen Carriers Developed for Tolerance Induction

[0156] Building on former versions of the nanosized antigen carriers to transport autoantigen-derived peptides to LSECs in vivo (Carambia A et al., J Hepatol 62, 1349-1356 (2015); Carambia A et al., Immunology 162, 452—463 (2021)), the exemplary antigen delivery system herein was developed as a platform technology for clinical application. Superparamagnetic iron oxide nanoparticles (SPIONs) were coated with low-molecular weight poly-maleic acid-alt-octadecene polymer (FIG. 1A). The polymer-coated SPIONs, termed “Topas Particles” (TPs), served as a scaffold for conjugating antigenic peptides to their surface. The peptide conjugation process utilized EDC chemistry, linking the N-terminal amine group of the peptide to the available carboxylic groups on the TP surface. Through testing a broad array of different peptides, the peptide coupling process was optimized and verified to generate TPCs. Moreover, a proprietary linker technology was implemented to adjust peptide’s net charge and solubility, thereby enabling efficient coupling of virtually any given peptide. Each TPC comprised a TP carrying one specific peptide. For simultaneous application of multiple peptides, individual TPCs were mixed to deliver “Topas Particle Mixtures” (TPMs). For formulation, TPCs and TPMs were dispersed in Mannitol / Tris / Lactate buffer, which is suitable for intravenous administration to patients.

[0157] The small size of the TPs and the high density of carboxylic groups on their surface, as shown by the negative ^-potential, enabled the achievement of high peptide loads with >100Attomey Docket No. 14779-010-228 peptide molecules per TPC. Regardless of the coupled peptides, TPCs consistently showed an overall negative ^-potential (< -30 mV).

[0158] Measures to ensure that TPCs consistently feature only minimal variation in size and retain a low poly dispersity index were implemented. Specifically, analysis using transmission electron microscopy (TEM) and dynamic light scattering (DLS) revealed that both TPs and TPCs exhibited a mean diameter (Z-average) ranging between 25 and 30 nm, with a uniform size distribution indicated by a low polydispersity index value (FIGS. IB and 1C). When exposed to serum or plasma in vitro, TPCs showed an increase in diameter (80-100 nm), indicating the formation of a protein corona, without observed aggregation.

[0159] Collectively, TPCs exhibited uniform size and surface charge independent of the coupled peptides. TPC manufacturing has been successfully established under upscaling to satisfy the creation of material for human use.(b) TPCs Showed Predominant LSEC Targeting

[0160] To investigate the organ distribution of TPCs, TPCs coupled with gliadin peptides (CD4+ T-cell epitope) fluorescently labelled with Cyanine-5 (TPC-gliadin-Cy5) were injected intravenously into wild-type mice. Among the organs assessed, the liver displayed by far the highest fluorescence intensity, whereas the spleen and kidneys showed only minimal fluorescence levels (FIG. 2A). Using intravital microscopy, fluorescence signals were specifically detected along the liver sinusoidal lining, corresponding to the anatomical location of LSECs (FIG. 2B). Moreover, to better understand intracellular peptide dissociation from TPCs within the liver, nanoparticles incorporating quantum dots instead of iron core (QD-TP), Cy5-labelled ovalbumin (Ova)323-339 peptides (Ova323-339-Cy5), TP coupled with Ova323-339-Cy5 (TPC-Ova323-339-Cy5), and QD-TPC-Ova323 -339-Cy5 were generated. Wild-type mice were injected with these nanoparticles and underwent intravital microscopy at different timepoints (FIG. 2C). Forster resonance energy transfer (FRET) indicated the proximity between the excited donor fluorophore QD and its acceptor Cy5, serving as a proxy for intact QD-TPC- Ova323-339-Cy5 (FIG. 7). FRET was used to assess uptake of intact TPC by the liver as well as subsequent peptide release. While fluorescence along the liver sinusoidal lining initially originated solely from Cy5 due to FRET, QD emission became detectable 1 hour post-injection, indicating gradual peptide dissociation. Conversely, FRET was absent upon injection of TPC- Ova323-339-Cy5 (lacking energy donors) or QD-TP plus uncoupled Ova323-339-Cy5 (lacking donor-Attorney Docket No. 14779-010-228 acceptor proximity) (FIGS. 8A and 8B). This exemplary study demonstrated that the liver was the primary target of TPCs. Among the nonparenchymal liver cells, LSECs exhibited a predominant uptake of TPCs (92.2% ± 1.64%), with >80% of LSECs showing uptake (FIG. 2D; gating strategy shown in FIG. 9). This cellular targeting specificity was further validated by electron microscopy of the minipig liver, revealing the presence of TPCs within endosomes of LSECs (FIG. 2E).

[0161] To investigate the antigen-specific response of CD4+ T cells to TPCs via antigen presentation, an in vitro stimulation assay was performed using splenocytes isolated from 2D2 transgenic mice, which expressed a T-cell receptor (TCR) specific to myelin oligodendrocyte glycoprotein (MOG)35-55 peptides. Incubating these cells with TPC-MOG35-55 resulted in antigen-specific activation, reflected by dose-dependent IFN-y secretion (FIG. 10A). In line with this, intravenous administration of TPC-MOG35-55 also induced proliferation of MOG- specific TCR transgenic CD4+ (TCRMOG) T cells recovered from different organs, approximately 80% higher than TP treatment (all p < 0.05; FIGS. 10B-10D). This resulted in a significantly higher frequency of TCRMOGT cells (all p < 0.05; FIG. 10E).(c) TPCs Induced Tolerance Across Different Animal Models

[0162] Having established LSECs as a primary target for TPC delivery, their capability of tolerance induction across various CD4+ T cell-dependent disease models was assessed. The effect of TPCs was investigated using the classical delayed-type hypersensitivity (DTH) model, frequently employed to evaluate the immunomodulatory effects of therapeutic agents on CD4+ T cell-mediated immune responses (Getts DR et al., Nat Biotechnol 30, 1217-1224 (2012); Tong J et al., Exp TherMed 15, 4441-4447 (2018)). TPC-Ova323 -339, administered intravenously seven days and one day before immunization, rendered Ova-specific CD4+ T cells unresponsive to the immunizing Ova323-339 peptides upon intradermal challenge seven days post-immunization, with an approximate 50% reduction in ear swelling and A ear swelling and nearly 75% reduction in A ear weight (all p < 0.01; FIGS. 3A-3D). This led to a significant reduction in mRNA expression of proinflammatory cytokines and chemokines in Ova-challenged ears of animals treated with TPCs compared to those treated with TPs (FIGS. 3E-3L; reduction in median fold change of IFNy: 36.48 vs. 4.10, p < 0.01; TNFa: 3.27 vs. 1.99, p < 0.01; IL-2: 5.87 vs. 1.75, p < 0.001; IL- 10: 5.95 vs. 1.95, p < 0.001; GM-CSF (granulocyte macrophage-colony stimulating factor): 5.90Attorney Docket No. 14779-010-228 vs. 1.88, p < 0.01; IL-6: 9.83 vs. 2.51, p < 0.01; CCL2: 4.27 vs. 2.3, p < 0.05; CCL5: 6.71 vs. 1.33, p < 0.0001).

[0163] TPC’s efficacy was further examined using a well-studied mouse model of T1D. In the non-obese diabetic (NOD) model, autoimmune diabetes arises spontaneously due to T cell- mediated responses to various islet autoantigens accompanied by a progressive loss of insulinproducing beta cells in the pancreas. In NOD mice, the I-Ag7MHC-II (major histocompatibility complex class-II) molecule confers genetic susceptibility to T1D, with insulin-specific CD4+T cells playing a crucial role in immunopathogenesis alongside other immune cell types (Pearson JA et al., J Autoimmun 66, 76-88 (2016); Nakayama M et al., Nature 435, 220-3 (2005)). Considering the complexity of the autoantigenic responses, a mixture of peptides derived from different autoantigens may be required to induce immune tolerance in this model. Therefore, five disease-relevant pancreatic antigens, one CD8+and four CD4+T-cell epitopes, were selected to create a mixture of TPCs called TPM-T1D (Table 5). Eight-week-old female NOD mice received seven intravenous injections of TP or TPM-T1D loaded with 50 nmol of antigen and were monitored until 24 weeks of age for diabetes incidence (FIG. 4A). Multiple administrations of TPM-T1D significantly reduced the frequency of hyperglycemia onsets compared to treatment with TP (p < 0.01 ; FIGS. 4B and 4C). An antigen-specific tolerance effect was confirmed by the detection of insulin-specific CD4+T cells in the spleen, which not only increased in numbers after treatment with TPM-T1D (p < 0.01) but also entailed a roughly 20% higher proportion of Tregs compared to TP treatment control (p < 0.05; FIGS. 4D-4F). These findings suggest that even when the disease involves multiple autoantigenic epitopes, a combination of selected peptides has the potential to induce tolerance in T1D. This treatment effect was associated with a significant accumulation of insulin-specific Tregs, possibly beyond antigen-specific suppression.

[0164] TPCs were also investigated in the experimental autoimmune encephalomyelitis (EAE) model of MS, induced by immunization with MOG35-55 peptide. A single prophylactic intravenous injection of TPC-MOG35-55 prior to disease induction significantly attenuated disease severity, whereas TP-treated mice developed clinical symptoms (FIGS. 5A and 5B; reduction in end score, maximum score, and cumulative score, all p < 0.0001; FIGS. 11A-11C). In line with the attenuated disease scores, prophylactic treatment with TPC-MOG35-55 significantly mitigated demyelination within spinal cords compared to those of TP-treated mice (p < 0.001; FIGS. 5C,Attorney Docket No. 14779-010-22812A and 12B) This study herein also explored the therapeutic potential of TPCs. Notably, a single intravenous injection of TPC-MOG35-55 at disease onset led to a significant reduction in both mean clinical scores (FIGS. 5D and 5E; reduction in end score, maximum score, and cumulative score, all p < 0.0001; FIGS. 11D-11F) and spinal cord demyelination (p < 0.01; FIGS. 5F, 12C and 12D) These findings underscored TPC’s efficacy in inducing tolerance prophylactically and therapeutically.(d) No Pharmacodynamic Interaction Between TPCs and Low-to-Medium Dose Glucocorticoids

[0165] Glucocorticoids, particularly prednisone and prednisolone, are widely used to treat various autoimmune conditions (Masamune A et al., Gut 66, 487-494 (2017)). To assess the potential impact of oral prednisone on TPCs efficacy in suppressing EAE, three doses of prednisone were administered in drinking water (2 mg / ml, 8 mg / ml, and 40 mg / ml) following prophylactic application of TP or TPC-MOG35-55. These doses were equivalent to the administration of 5 mg, 10 mg, and 50 mg / day in humans, respectively (Chan J et al., Autoimmunity 41, 405-413 (2008)). The data showed that low and medium doses of prednisone (equivalent to < 10 mg / day in humans for maintenance therapy) did not prevent mice from developing EAE in groups receiving only TPs, nor did they impair any protective efficacy of TPC-MOG35-55, which significantly inhibited the development of EAE symptoms in those groups (FIG. 5G). The results were comparable to those achieved in the group receiving TPC-MOG35- 55 without any prednisone treatment. In contrast, the high dose of prednisone eliminated the EAE clinical phenotype, thus preventing any interpretation of TPC’s tolerance effects.(e) TPCs Induced Antigen-Specific Tregs and T-Cell Anergy

[0166] To delve into the mechanism of tolerance induction, lymphocytes isolated from spinal cords on day 35 of observation in the EAE study were analyzed. While TH17 cells are considered the primary encephalitogenic T cells in EAE (Prajeeth CK et al., J Neuroinflammation 14, 204 (2017)), the RORyt-driven production of GM-CSF has been identified as crucial in autoimmune neuroinflammation (Codarri L et al., Nat Immunol 12, 560- 567 (2011)). Therapeutic administration of TPC-MOG35-55 mitigated infiltration of CD4+T cells in spinal cords by 70% (p < 0.05) and significantly reduced TH17 and GM-CSF-secreting TH cells (both p < 0.01; FIGS. 6A and 6B; gating strategy in FIG. 13). The decrease in these proinflammatory T cell subsets could derive from either their physical deletion or functional suppression, which may be associated extrinsically with induction of Treg (Reynolds BC et al.,Attorney Docket No. 14779-010-228Eur J Immunol 44, 3342-3352 (2014)), or intrinsically with anergy or exhaustion (Trefzer A et al., Cell Rep 34, 108748 (2021)). In the exemplary study herein, treatment with TPC-MOG35-55 led to a significant increase in anergic cells observed at the end of the study (p < 0.05), identified as CD3+CD4+Foxp3 CD44hiCD73hiFR4hisubset (Kalekar LA et al., Nat Immunol 17, 304-314 (2016)) (FIG. 6C). However, the frequency of Tregs remained similar between TP and TPC groups at this late timepoint (FIG. 6D).

[0167] To investigate the TPC-induced tolerance mechanisms specifically on peptidespecific CD4+ T cells at initial stages, an adoptive transfer of TCRMOGT cells (FIG. 6E) was employed. Two days after treatment with TPC-MOG35-55, there were significantly more TCRMOG Tregs in the liver of TPC-treated compared to TP -treated mice (p < 0.05; FIG. 10F). Eight days after treatment, a significant reduction in frequency of splenic TCRMOGT cells (p < 0.05) and an approximate 20% reduction in their expression of Ki67 (p < 0.01) was observed, suggestive of restricted proliferation associated with treatment (FIG. 6F). Moreover, upon TPC- treatment TCRMOGT cells displayed a phenotype of anergy and suppressed effector functions associated with a significant upregulation of TIGIT, LAG-3 and PD-1 compared to those of TP- treated mice (all p < 0.01 ; FIGS. 6G and 6H). A significant increase in TCRMOGTregs was detected in TPC-treated compared to TP-treated mice (p < 0.01 ; FIG. 61). The expression of co- inhibitory receptors on TCRMOGTregs significantly increased in TPC-treated animals, suggesting a heightened suppressive capacity of Tregs (FIG. 6J).6.1.2 Discussion of Results

[0168] Topas particles provided in the exemplary study herein were designed with the objective to induce antigen-specific tolerance in patients with AD. Compared to former nanoparticle versions (Carambia A et al., J Hepatol 62, 1349-1356 (2015); Carambia A et al., Immunology 162, 452-463 (2021)), the platform technology was optimized to achieve the required favorable physicochemical properties for selectively targeting LSECs, while expanding the possibility to conjugate the particles with an unlimited variety of different antigenic peptides and providing a formulation compatible for clinical application. Thanks to these characteristics, CD4+T cell tolerance was demonstrated across different disease models, such as DTH, T1D, and EAE, creating the rationale for moving into clinical testing. Mechanistic studies of TPCs revealed their role in promoting Tregs and inducing T cell anergy and exhaustion, leading to the deletion of proinflammatory T cell subsets.Attorney Docket No. 14779-010-228

[0169] The key characteristic of TPCs was their tailored design for selective uptake by LSECs with only negligible uptake by other nonparenchymal liver cells. This differentiates TPCs from other tolerizing particles, which exhibit either a different organ distribution pattern (McCarthy DP et al., Nanomedicine 13, 191-200 (2017); Maldonado RA et al., PNAS 112 (2015)) or, despite exploiting the hepatic tolerance, show variable degrees of different cellular target specificity within the liver, without predominant uptake by LSECs (Casey LM et al., Biomaterials 283 (2022); Ilyinskii PO et al., Front Immunol 12 (2021)). Use of the TPCs as provided herein offers significant advantages thanks to the unique features of LSECs. In fact, the predominant location of LSECs in sinusoidal blood, along with their extraordinary clearance function, facilitates targeting and rapid uptake of carriers for antigen-specific immunotherapy, such as nanoparticles sized < 100 nm (Bhandari S et al., Front Physiol 12 (2021)). Moreover, LSECs outmatch other tolerogenic cells in inducing Tregs, veto dendritic cell-induced T cell activation, and sequestrate activated CD8+T cells, all of which contribute to their extraordinary tolerogenic capacity (Knolle PA et al., Cell Mol Immunol 13, 347-353 (2016)). Notably, other conventional hepatic APCs with tolerogenic potential, such as dendritic cells, macrophages, and Kupffer cells exhibit high plasticity, displaying tolerizing functions only under homeostatic conditions (Kingham TP et al., Hepatology 45, 445-454 (2007)) but activating immune responses in the presence of inflammation (Hilligan KL et al., Cell Mol Immunol 17, 587-599 (2020); Heymann F et al., Hepatology 62, 279-291 (2015)). This may limit their role as mediators of tolerance for treatment in autoimmunity, where inflammatory dysfunction impacts on pathogenesis and maintenance of disease (Duan L et al., J Immunol Res 2019, 1-2 (2019)). Conversely, LSECs have demonstrated remarkable resilience to external inflammatory stimuli, maintaining robustness in tolerance induction (Kern M et al., Gastroenterology 138, 336-346 (2010)).

[0170] To monitor the fate of peptides bound to TPCs upon liver uptake, FRET analysis was performed to obtain spatiotemporal distribution of peptide associations inside living cells. The data confirmed that TPCs were rapidly taken up by LSECs as intact particle-peptide conjugates, followed by gradual peptide release from TPCs. This predominantly occurred in LSECs, as visualized by their characteristic anatomical location. Electron microscopy of the minipig liver revealed TPC enrichment in LSEC-derived endosomes, thanks to the contrast-enhancing properties of the TPC iron-oxide core. As endosomes represent the subcellular compartmentAttorney Docket No. 14779-010-228 where antigen processing and peptide loading onto MHC-II molecules take place (Ma W et al., Eur J Immunol 44, 650-653 (2014)), the data herein corroborate that TPC-delivered peptides can be readily processed by LSECs similarly to blood-borne antigens. These findings are of clinical relevance, because porcine and human livers share many anatomical and physiological similarities (Eberlova L et al., J Surg Res 250, 70-79 (2020)).

[0171] The physicochemical properties of TPCs such as size and charge are believed to contribute to their selective targeting and tolerogenicity. The small size facilitates efficient uptake by LSECs, aligning with the dual-cell principle of waste clearance (Bhandari S et al., Front Physiol 12 (2021)). Accordingly, particles below 200 nm were taken up by LSECs, while those above 200 nm were engulfed by macrophages. Considering the even narrower fenestrae of human LSECs, approximately 100 nm in diameter (Wisse E et al., Gene Ther 15, 1193-9 (2008)), the small size of TPCs facilitated their selective targeting to LSECs also in humans.The consistent small size of TPCs was ensured by producing particles with a controlled uniform size distribution and the absence of aggregates in plasma and serum. Additionally, the negative ^-potential of TPCs enhanced TPC’s affinity for LSECs, given that scavenger receptors on LSECs bind to diverse polyanionic molecules (Bhandari S et al., Front Physiol 12 (2021)). Furthermore, it may confer tolerogenicity to TPCs, as the negative charge of nanoparticles is commonly associated with anti-inflammatory and tolerogenic properties (Ramos GC et al., Br J Pharmacol 151, 844-850 (2007)).

[0172] The optimized coupling method described in the study herein has been complemented by a linker technology which was applied if peptides of interest lack favorable coupling properties. Adding specific amino acids to the N-terminus of such peptides facilitated efficient coupling without altering peptide binding to the restrictive MHC molecule. This technology enabled TP conjugation with an unlimited variety of disease-relevant peptides representing T- cell epitopes and offered the possibility to create a broad portfolio of potential clinical applications.

[0173] As TP conjugation was restricted to peptides only, this resulted in the immunogenic T-cell epitopes and their corresponding MHC molecules to be identified and characterized. The direct delivery of disease-relevant peptides to LSECs is beneficial, as: i) TPCs deliver considerably higher doses of relevant T-cell epitopes to LSECs compared to providing full proteins carrying only a few relevant peptides; ii) a combination of TPs coupled with selectedAttorney Docket No. 14779-010-228 peptides, even from different antigens, can be applied as TPM, thereby amplifying the tolerization effect; iii) peptides can be deliberately selected to promote T cell differentiation into tolerant states based on their affinity for TCR upon presentation by MHC (Trefzer A et al., Cell Rep 34, 108748 (2021); This S et al., Cells 10, 1530 (2021)); and iv) several AD are linked to certain MHC molecules, facilitating patient selection accordingly. Based on the above, TPC technology has the potential to treat a well-defined patient population (personalized approach), using a precise and disease-relevant tolerization strategy (targeted approach).

[0174] To assess the possibility of pharmacodynamic interactions between TPCs and glucocorticoids, the mainstay of AD treatment, the effect of the concomitant administration of TPCs and glucocorticoids was investigated in mice with MOG-induced EAE. The data provided herein show that TPC’s efficacy remained unaffected by the administration of prednisone at doses equivalent to those used in humans for maintenance therapy (Heine LK et al., Front Immunol 13 (2022)). Therefore, the mechanisms promoting TPC-mediated tolerization seem to be unaffected by the nonspecific immunosuppressive and anti-inflammatory effects of glucocorticoids, which modulate immune responses through glucocorticoid receptor-mediated gene regulation and transcription factor inhibition (Cain & Cidlowski, Nat Rev Immunol 17, 233-247 (2017)). This is of great relevance for use of TPCs in patients with AD, who often require use of glucocorticoids.

[0175] Mechanistically, TPCs initially activated MOG-specific T cells but halted their further proliferation upon encountering cognate antigens. These effector cells upregulated anergic markers and various co-inhibitory receptors. The observed phenotypes and reduced proliferative capacity of these cells align with typical features of anergy and exhaustion (Trefzer A et al., Cell Rep 34, 108748 (2021)); Kalekar LA et al., Nat Immunol 17, 304-314 (2016)), suggesting a probable link between TPC-induced tolerance and these functional impairments. Anergy typically arises from T cell activation upon TCR recognition (first signal) lacking adequate co-stimulation as the second signal (Chappert P et al., Curr Opin Immunol 22, 552-559 (2010)). As LSECs express low levels of MHC -II and co-stimulatory molecules due to the liver’s tolerogenic microenvironment (Gottwick C et al., Semin Immunopathol 44, 475-484 (2022); Knolle PA et al., Clin Exp Immunol 114, 427-433 (1998)), their interaction with antigenspecific T cells, whose specificity corresponds to the TPC-delivered peptides, might promote anergy development. Thus, TPCs may foster immune tolerance through their selective targetingAttorney Docket No. 14779-010-228 to LSECs and the resulting induction of anergy. Notably, other work shows that the anergy markers CD73 and FR4 were expressed on antigen-exhausted CD4+T cells in a dose-dependent manner, with genes expressed by antigen-exhausted cells highly enriched in anergic cells as well (Trefzer A et al., Cell Rep 34, 108748 (2021)). Thus, anergy and exhaustion share common features despite conceptual differences in T-cell priming, and essentially both are associated with quality and quantity of TCR signal strength (Trefzer A et al., Cell Rep 34, 108748 (2021); This S et al., Cells 10, 1530 (2021)). This in turn can be effectively influenced through selection of peptides coupled to TPCs to induce these dysfunctional states, thereby facilitating tolerance.

[0176] In line with the role of Tregs in preventing EAE (Carambia A et al., J Hepatol 62, 1349-1356 (2015)), data herein demonstrate that TPC treatment significantly increased Tregs within the antigen-specific compartment of CD4+T cells in the liver and spleen shortly after treatment. As it appears, antigen-specific Tregs were induced in the liver first and then circulated to the spleen, since their increase was observed in the liver before the spleen posttreatment. At a later timepoint, there was no significant increase in Tregs, but, there was a significant increase in anergic T cells and a significant reduction in the main drivers of EAE, TH17 and GM-CSF-producing TH cells, in the spinal cords of TPC-treated animals. These findings of the study herein underscore the TPC-mediated tolerization effects in suppressing disease-relevant proinflammatory cells in the target organ.

[0177] An increase in insulin-specific Tregs was also observed in the spleens of animals receiving TPM-T1D before diabetes onset together with a reduction in T1D incidence, indicating that inducing disease-relevant Tregs could slow disease progression. In line with these observations, reduced frequencies of insulin-specific Tregs were observed in the blood of children during onset of islet autoimmunity (Serr I et al., Set Transl Med 10 (2018)) and associated increased insulin-specific Tregs with slow progression to symptomatic T1D (Serr I et al., Nat Commun 7, 10991 (2016)). While clinical studies have failed to show protein- or peptide-mediated tolerance induction in adults with established T1D, the potential of antigenspecific tolerization in early T1D stages was highlighted in the Pre-POINT study, where very early treatment with daily oral insulin increased insulin-specific Tregs in at-risk individuals (Bonifacio E et al., JAMA 313, 1541 (2015)). However, while insulin dominates the autoimmune reaction in NOD mouse (Nakayama M et al., Nature 435, 220-3 (2005)), the situation is less clear in human T1D. Large cohort studies indicate that the peak incidence ofAttorney Docket No. 14779-010-228 autoantibody appearance varies by type (Ilonen J et al., Diabetes 62, 3636-3640 (2013)) and that progression to symptomatic T1D depends on autoantibody composition (Achenbach P et al., Diabetologia 56, 1615-1622 (2013)). Multiple autoantigens are likely involved in the pathogenesis of T1D. The study herein demonstrated that a broader tolerance induction approach administering several autoantigens simultaneously was more beneficial.

[0178] Overall, the mechanisms of TPCs are likely multifaceted, involving the suppressive capacity of Tregs, as well as functional and physical deletion of effector T cells through anergy and / or exhaustion. Under certain circumstances, anergic T cells could convert into Foxp3+Tregs or Foxp3 IL- 10 regulatory T cells (Kalekar LA et al., Nat Immunol 17, 304-314 (2016);Thomann AS et al., Front Immunol 12 (2021)), supporting the notion that multiple mechanisms collectively contribute to TPC-induced tolerance.

[0179] In conclusion, the exemplary study herein demonstrated that TPCs primarily target LSECs, where efficient peptide release occurs, triggering the inherent tolerogenic function of these unconventional APCs. TPC’s ability in inducing antigen-specific immune tolerance was proven in various animal models. This was achieved by conferring anergy to CD4+T cells and inducing antigen-specific Tregs. For clinical application, GMP-compliant manufacturing and upscaling was established, ensuring excellent stability of TPCs. The platform technology as provided herein represents a promising innovative treatment option for reinstating antigenspecific tolerance in AD.6.1.3 Materials and Methods(a) Study Design

[0180] This study aimed to verify the selective LSEC targeting of peptide-conjugated nanoparticles, and to test their efficacy in inducing antigen-specific tolerance in CD4+T cell- mediated immune responses together with exploring the underlying tolerance mechanisms. To this end, wild-type mice were employed for targeting studies and three different animal models for pharmacodynamics: DTH, EAE, and T1D. Apart from assessing clinical scores as measures of treatment outcome, tolerance-related parameters were also investigated using gene expression analysis, histology, and flow cytometry. Sample sizes were determined based on prior experience from similar experiments, without the use of statistical methods for predetermination. Whenever feasible, mice were age-matched and randomly allocated to experimental groups. Experiments were conducted without knowledge of treatment to ensureAttorney Docket No. 14779-010-228 blinding. In vivo experiments included biological replicates, with the number of replicates (n values) specified in the brief descriptions of the figures (see Section 4) .(b) Preparation of antigen peptide-loaded nanoparticles

[0181] The preparation of nanoparticles involved encapsulating oleic acid-stabilized SPIONs into an amphiphilic polymer, namely poly (maleic acid-a / t-1 -octadecene), similar to that previously described (Heine M et al., Beilstein Journal of Nanotechnology 5, 1432-1440 (2014)). Polymer-coated SPIONs were coupled with peptides in the presence of EDC (l-ethyl-3- (3-dimethylaminopropyl)-carbodiimide). A 150-fold excess of the relevant peptide was added, followed by incubation for 2.5 hours (h) at room temperature. Free peptide was removed using centrifugal ultrafiltration (molecular weight cut-off 100 kDa, 4200 rpm, 25°C). The hydrodynamic size of a nanoparticle was determined by DLS using Zetasizer Nano ZS (Malvern Panalytical) and confirmed after each step of the process: before coupling, after coupling, after purification, and after formulation. Subsequently, the size distribution of the starting material (i.e., TP) was further verified by transmission electron microscopy (TEM). Peptide content and coupling efficiency were determined using AccQ Tag™ derivatization method for amino acid analysis (Waters Corporation). The individual TPCs were dispersed, mixed, and diluted in D- Mannitol (5% w / v), L-Lactic Acid (6 mM), and TRIS (5 mM), also referred to as MTL buffer to obtain the respective doses.(c) Animal Studies

[0182] Animal studies including intravital microscopy, organ distribution and targeted delivery of TPC, EAE, DTH, and type 1 diabetes were approved by the respective review boards. All mice were bred and housed in specific pathogen-free conditions. The execution of animal procedures adhered strictly to both local and national guidelines and regulations.(d) Intravital Microscopy

[0183] Intravital microscopy was performed according to methods previously described (Bartelt A et al., Nat Med 17, 200-205 (2011)). Briefly, C57BL / 6 mice were anesthetized using isoflurane inhalation anesthesia. The tail vein was catheterized, skin and peritoneum were opened, and the liver was prepared and attached to a cover glass. Microscopy was performed using a Nikon Al confocal microscope equipped with a resonant scanner for image acquisition at 30 fps. For visualization of TPC targeting, Cy5-labelled nanoparticles were injected via tail vein catheter and the uptake into the liver was monitored for 15 minutes (min).Attorney Docket No. 14779-010-228(e) Organ and Liver-specific Cellular Distribution of TPCs

[0184] C57BL / 6 mice were intravenously injected with TPC coupled with Cy5-labelled gliadin peptides and sacrificed after 10 min. For organ distribution analysis, whole cardiac perfusion was performed with phosphate-buffered saline (PBS), followed by removal of the liver, spleen, kidneys, lung, CNS, and inguinal lymph nodes. Tissues were homogenized and fluorescence was measured using a Tecan microplate reader with an excitation wavelength of 633 nm and an emission wavelength of 670 nm. For analysis of targeted delivery of TPC within liver, non-parenchymal liver cells were isolated by density gradient centrifugation as described previously (Carambia A et al., Methods in Molecular Biology 1031, 101-107 (2013)). Briefly, mouse livers were perfused with 1 mg / ml collagenase type 2 (Worthington) in Gey’s balanced salt solution, mechanically dissected and then digested for 25 min at 37°C in 1 mg / ml collagenase type 2 solution. Hepatocytes were removed by centrifugation and non-parenchymal cells were recovered using a 17% Optiprep™ (Sigma-Aldrich) gradient. Cells were stained and analyzed by flow cytometry.(f) 1)111 in Response to Ova323-339

[0185] A 24-h ear swelling assay was performed to assess DTH reactions in female BALB / c mice (Janvier Labs), which were immunized subcutaneously (s.c.) with 100 pl of Ova323-339 peptides in CFA (1 mg / ml) seven days before the assay (Allen IC, Methods in Molecular Biology 1031, 101-107 (2013)). Baseline ear thickness was measured using calipers for both ears. Immediately afterwards, each mouse received an intradermal injection of Ova323-339 peptides (10 pg in 10 pl of PBS), or 10 pl of PBS alone as a vehicle control, into the bilateral ears to elicit DTH responses or serve as a control, respectively. The increase in ear thickness from baseline was determined 24 hours after peptide challenge, denoted as ear swelling (pm). A Ear swelling was calculated by subtracting the ear swelling of vehicle control from that of the peptide- challenged ear in each animal. Following this, each ear pinna underwent puncture biopsy, and the 8-mm-diameter biopsied tissue was weighed. The difference between the weight of the peptide-challenged ear and the control was determined, denoted as A ear weight (mg).(g) Chronic EAE Model

[0186] C57BL / 6 mice (Taconic Biosciences) were immunized with MOG35-55 peptide inCFA emulsion, followed by intraperitoneal (i.p.) injection of pertussis toxin as according to methods as described previously (Mendel I et al., Eur J Immunol 25, 1951-1959 (1995)).Attorney Docket No. 14779-010-228Depending on the treatment arms, the animals received either TP or TPC-MOG35-55 one day before immunization (prophylactic treatment) or at the onset of disease (therapeutic treatment). For the studies assessing the effects of TP versus TPC-MOG35-55 in the presence of glucocorticoids, prednisone was administered ad libitum in drinking water from Day -5 (five days before immunization) through Day 28 (the end of the study). Prednisone stock solution was prepared fresh daily at 3 mg / mL in 100% ethanol and was then diluted in drinking water to reach the final concentration of 2 mg / mL, 8 mg / mL, and 40 mg / mL, respectively. For the duration of treatment, all drinking water was replaced at the same time (+ / - 1 hour) each day (Gasparini SJ et al., Steroids 116, 76-82 (2016)). Individual animals were observed daily until day 35, and blinded EAE scoring was conducted on a scale of 0 to 5: 0 = asymptomatic; 1 = limp tail; 2 = hind limb weakness; 3 = hind limb paralysis; 4 = hind limb paralysis plus partial front limb paralysis; 5 = 4-limb paralysis or death. The data are presented herein as the mean clinical score. At the end of the follow-up, spinal cord and hindbrain were collected for flow cytometry analysis or histological analysis.(h) In vitro Stimulation of MOG-specific T cells

[0187] Spleen and lymph node cells (inguinal, axillary, and brachial) were isolated from TCRMOGCD45.2 mice (C57BL / 6-Tg(Tcra2D2,Tcrb2D2)lKuch / J), followed by erythrocyte depletion with ACK lysis buffer (Thermo Fisher Scientific). 5 / 105isolated cells were cultured in 200 pl of full RPMI 1640 (PAN-Biotech) supplemented with 10% heat-inactivated fetal bovine serum (Corning) at 37°C for 72 h in the presence of either MOG35-55 peptide or TPC- MOG35-55. The supernatant was collected for Quantification of IFN using ELIS / X kit (R &D Systems).(i) Adoptive Transfer of 2D2 TCR Transgenic Cells

[0188] Spleen and lymph node cells (inguinal, axillary, and brachial) were isolated from TCRMOGCD45.2 mice (C57BL / 6-Tg(Tcra2D2,Tcrb2D2)lKuch / J), followed by enrichment of CD4+ T cells through negative immunomagnetic selection (Dynabeads™ Untouched™ Mouse CD4 Cells Kit, Thermo Fisher Scientific). Subsequently, 8xl06CD4+ T cells were intravenously (i.v.) injected into C57BL / 6 mice with congenic marker CD45.1. In some experiments, the cells were labelled with CFSE (BioLegend) before adoptive transfer. The day after adoptive transfer, recipient mice were treated with either TP or TPC-MOG35-55. One day post-treatment, recipient mice were immunized s.c. with 100 mg of MOG35-55 peptide in CFA.Attorney Docket No. 14779-010-228Seven days after immunization, spleens and lymph nodes were harvested for flow cytometry analysis. TCRMOGT cells within these tissues were identified using CD45.2 and examined for a range of surface and intracellular markers.(j) The NOD Mouse Model of Type 1 Diabetes

[0189] Female NOD / ShiLtJ mice were purchased from The Jackson Laboratory at 4 weeks of age and were acclimated to the facility before the start of the experiments. At the age of 8 weeks, the animals received either TP or TPM-T1D: a mix of five TPCs, conjugated respectively with 50 nmol of diabetogenic peptides (Insulin beta chain 9-23, Proinsulin p24-33, islet-specific glucose — phosphate catalytic subunit-related protein 206-214, 2.5 hybrid insulin peptide and 6.9 hybrid insulin peptide) (see Table 5).Table 5: Diabetogenic peptides used in TPM-T1D

[0190] The animals received five weekly injections of TPs or TPM-T1D followed by two biweekly injections for a total of seven nanoparticle injections. Blood sugar levels were determined weekly until the end of the experiment when mice reached an age of 24 weeks. Mice with two consecutive blood sugar readings > 250 mg / dL were considered diabetic and euthanized. Spleens of all mice were harvested and analyzed via flow cytometry following euthanasia due to hyperglycemia or at the end of the experiment.(k) Flow Cytometry Analysis

[0191] Single cell suspension (containing immune cells) was prepared from different organs such as liver, spleen, and spinal cords, depending on the experimental design. Fc receptorAttorney Docket No. 14779-010-228 blocking with CD 16 / 32 was performed, followed by staining with fixable viability dye and various combinations of the antibodies, which are listed in Table 6.Table 6: Anti-mouse conjugated monoclonal antibodies

[0192] Before staining for cytokines, cells were cultured with phorbol myristate acetate (50 ng / ml), ionomycin (1 pg / ml), monesin (1.36 pg / ml), and brefeldin-A (1 pg / ml) for 4 h; all reagents were purchased from Sigma-Aldrich. Intracellular staining of cytokines and Foxp3 was done using Foxp3 / Transcription Factor Staining Buffer Set (eBioscience™, Thermo Fisher Scientific) per manufacturer’s protocol. Splenocytes from NOD mice were additionally stainedAttorney Docket No. 14779-010-228 with tetramer complexes composed of MHC-II Iag7 and mimotope peptides derived from Insulin-B:9-23 conjugated with a fluorescent label (sequences: HLVERLYLVCGGEG (SEQ ID NO: 13) and HLVERLYLVCGEEG (SEQ ID NO: 14); NIH Tetramer Core Facility) (Serr I et al., Nat Commun 7, 10991 (2016)). Cytometric data were acquired on a LSRFortessa™ flow cytometer (Becton-Dickinson) and analyzed using FlowJo analysis software vlO.(l) RNA Extraction from Ear Pinnae and Real-time Reverse Transcription- quantitative PCR (RT-qPCR)

[0193] The ear pinnae were stored at -80°C in RNAlater™ solution (Thermo Fisher Scientific). The thawed tissues were homogenized using a rotor-stater homogenizer, and RNA was isolated using Rneasy® Fibrous Tissue Mini Kit (Qiagen) per manufacturer’s protocol. Real-time RT-qPCR was employed to compare gene expression levels in ear pinnae. The cDNA was reverse transcribed from the extracted RNA and underwent a qPCR reaction using the QuantStudio™ qPCR System (Thermo Fisher Scientific) following the manufacturer’s protocol. The primers used were all purchased from Thermo Fisher Scientific (Table S3).(m) Histological Analysis for Encephalomyelitis and Demyelination in EAE

[0194] Spines were dissected from mice and fixed in 10% formalin. Sections with samples from cervical, thoracic, and lumbar regions of spinal cords were prepared and underwent hematoxylin and eosin (H&E) and anti-Myelin basic protein (MBP) staining, respectively. H&E-stained slides were used for count of inflammatory foci in spinal cords, while anti-MBP- stained slides were used for estimation of demyelinated area.(n) Statistical Analysis

[0195] GraphPad Prism™ (Version 10) software was employed to perform the statistical analyses. The nonparametric Mann- Whitney test was used to compare outcomes between two independent groups. For survival data (normoglycemic “survivor”), log-rank test was conducted. Comparisons were considered significant if p < 0.05.6.2 Example 2: Preparation of Precursor Nanoparticles

[0196] Superparamagnetic iron oxide crystalline cores (SPIONs) and low molecular weight poly (maleic acid-alt- 1 -octadecene) (LM-PMAcOD) can be prepared as described in International Patent Application Publication No.: WO 2021 / 165227, which is incorporated herein by reference in its entirety.

[0197] An exemplary procedure for the synthesis of precursor nanoparticles is as follows: 100 mg LM-PMAcOD are dissolved in 4 mL chloroform in a 100 mL round bottomed flask.Attorney Docket No. 14779-010-228The mixture is heated until the polymer is fully dissolved. 3.3 mL of the oleate-SPION solution as described in WO 2021 / 165227 is added to the mixture and subsequently evaporated at <10 mbar for 15 minutes at 40°C on the rotavap with 280 RPM. Then, 10 mL 5 mM NaOH is added to the mixture and stirred on the rotavap for 15 minutes at 50°C until all black solids are dissolved. The solution is diluted 8 times using 70 mL 25 mM NaOH to dissolve the entire polymer. The obtained solution is stirred on the rotavap for 15 minutes, resulting in a brown solution.

[0198] The product is filtered over a 0.45 pm and a 0.2 pm PES filter. Afterwards, the probe is purified by tangential flow filtration (TFF).6.3 Example 3: Peptides and Peptide Coupling

[0199] An exemplary procedure for the synthesis of peptides is as follows: The synthesis of the peptides is accomplished via Fmoc chemistry from the C to N direction using solid phase peptide synthesis (SPPS). The alpha amino group of each amino acid is protected with a fluoren- 9-ylmethoxycarbonyl (Fmoc) group, while side chain functional groups are also blocked with various appropriate protective groups.

[0200] In general, the SPPS consists of repeated cycles of N-terminal deprotection followed by coupling reactions. The first Fmoc protected amino acid is coupled to the resin. Afterwards, the amine group is deprotected with a mixture of piperidine in dimethylformamide (DMF) , and then coupled with the free acid of the second Fmoc-protected amino acid. The cycle is repeated until the desired sequence is obtained. The resin is washed between each step. The completion of each coupling reaction is monitored by a qualitative ninhydrin test. In the last step of the synthesis, the crude peptide-resin is successively washed with DMF and methanol, and dried. Then, the protective groups are removed from the peptide and the peptide is cleaved from the resin using trifluoroacetic acid (TFA). The obtained crude peptide is isolated by ether precipitation from the cleavage mixture.

[0201] Further, the peptide is purified through preparative HPLC to reach purity requirements, and the counter ion TFA is replaced with chloride by using an appropriate solventbuffer system. Finally, the purified peptide is lyophilized.

[0202] The peptides can have an amino acid at the N-terminus and a free acid (HC1 salt) at the C-terminus.Attorney Docket No. 14779-010-228

[0203] Characterization of the free peptides (starting materials) is performed by LC-MS. The molecular weight of the peptides is measured by multimode electrospray atmospheric pressure chemical ionization mass spectrometry.

[0204] An exemplary procedure for the coupling of peptides to precursor nanoparticles is as follows: The peptides are coupled to the surface of the precursor nanoparticles obtained in Example 11 using l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) acid / sodium tetraborate decahydrate (SBB) chemistry in boric buffer.

[0205] EDC in SBB buffer is added to the precursor nanoparticles obtained in Example 11. After 15 minutes at RT, the peptides are added and the reaction mixture is stirred for 2 hours and 15 minutes at RT. The resulting nanoparticle solution is filtered and purified by tangential flow filtration (TFF) purification.

[0206] The nanoparticles are characterized using a variety of analytical methods. Characterization of the iron oxide core is performed using TEM and SAXS on the nanoparticles dispersed in 5% (w / v) D-mannitol, 5 mM TRIS and 6 mM L-lactic acid. Characterization of particle size and distribution is performed by dynamic light scattering (DLS).

[0207] The hydrodynamic diameter (z-average) and poly dispersity index are determined using a Malvern Zetasizer Nano ZS or equivalent in unimodal mode. These measurements are performed on the nanoparticles dispersed in 5% (w / v) D-mannitol, 5 mM TRIS and 6 mM L- lactic acid.

[0208] The surface charge of the nanoparticles is analyzed by measuring the zeta potential at pH 6 to 7 (pH during measurement) using a Malvern Zetasizer Nano ZS instrument. These measurements are performed on the nanoparticles dispersed in 5% (w / v) D-mannitol, 5 mM TRIS and 6 mM L-lactic acid.

[0209] The total polymer content is determined using GPC. The peptides are hydrolyzed, and the particles destroyed in 6 M HC1. The PMAcOD is extracted with ethyl acetate after addition of EDTA. After evaporation of sol vent, the residue is re-dissolved in a THF / acetic acid mixture before analysis.

[0210] The spectroscopic properties of the nanoparticles are determined by Fourier- transform infrared spectroscopy (FUR).7. ILLUSTRATIVE EMBODIMENTS

[0211] The present disclosure provides the following non-limiting embodiments:Attorney Docket No. 14779-010-2281. A pharmaceutical composition comprising nanoparticles, wherein each of the nanoparticles comprise: a. an amphiphilic polymer, and b. at least one peptide, wherein the pharmaceutical composition comprises peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.2. The pharmaceutical composition of embodiment 1, wherein the at least one peptide is associated with the outside of at least one nanoparticle.3. The pharmaceutical composition of embodiment 1 or 2, wherein each of the at least one nanoparticles comprise at least one peptide comprising the amino acid sequence of SEQ ID NO: 12.4. The pharmaceutical composition of embodiment 1 or 2, wherein each of the at least one nanoparticles comprise at least one peptide comprising one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11.5. The pharmaceutical composition of embodiment 4, wherein the pharmaceutical composition comprises only peptides comprising the amino acid sequence of SEQ ID NOs: 7, 8, 9, 10, and 11.6. The pharmaceutical composition of embodiment 4 or 5, comprising at least 5 different types of nanoparticles, wherein the different types of nanoparticles differ among each other in the peptide sequence, and wherein a first type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 7, a second type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 8, a third type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 9, a fourth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 10, and a fifth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 11.7. The pharmaceutical composition of embodiment 6, wherein each type of nanoparticle is present in equimolar amounts.8. The pharmaceutical composition of any one of embodiments 1-7, wherein the amphiphilic polymer has a number average molecular weight (Mn) of 20,000 g / mol or less, 10,000 g / mol or less, or ranging between 6,000 g / mol to 1,000 g / mol.Attorney Docket No. 14779-010-2289. The pharmaceutical composition of any one of embodiments 1-8, wherein the amphiphilic polymer comprises the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a C4 to C22 alkyl group, preferably Cs to C20 alkyl group.10. The pharmaceutical composition of any one of embodiments 1 -9, wherein the amphiphilic polymer is selected from the group comprising poly (maleic acid-alt- 1 -octadecene), poly (maleic acid-a / t-1 -dodecene) and poly (maleic acid-alt- 1 -tetradecene).11. The pharmaceutical composition of embodiment 10, wherein the amphiphilic polymer is poly(maleic acid-alt- 1 -octadecene).12. The pharmaceutical composition of embodiment 10 or 11, wherein the number average molecular weight of the amphiphilic polymer is from 6,000 to 1,000 g / mol.13. The pharmaceutical composition of any of one of embodiments 1-12, wherein each peptide is covalently linked to the outside of at least one nanoparticle or non-covalently associated.14. The pharmaceutical composition of any one of embodiments 1-13, further comprising an aqueous buffer, wherein the aqueous buffer comprises at least one sugar, at least one primary amine, and at least one amino acid.15. The pharmaceutical composition of embodiment 14, wherein the buffer contains D-mannitol, Tris(hydroxymethyl)aminomethane, and L-lactic acid.16. The pharmaceutical composition of any one of embodiments 1-15, wherein each nanoparticle optionally comprises a iron oxide core and an amphiphilic polymer comprising poly(maleic acid-alt- 1 -octadecene) having a number average molecular weight of 6,000 to 1,000 g / mol.17. Use of the pharmaceutical composition of any one of embodiments 1-16 for treatment of an autoimmune disease.18. The use of embodiment 17, wherein the autoimmune disease is type 1 diabetes (T1D).Attorney Docket No. 14779-010-22819. The use of embodiment 17, wherein the autoimmune disease is multiple sclerosis (MS).20. A nanoparticle, wherein the nanoparticle comprises: a. an amphiphilic polymer, and b. at least one peptide, wherein the at least one peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.21. The nanoparticle of embodiment 20, wherein the at least one peptide is associated with the outside of the nanoparticle.22. The nanoparticle of embodiment 20 or 21, wherein the nanoparticle comprises at least one peptide comprising the amino acid sequence of SEQ ID NO: 12.23. The nanoparticle of embodiment 20 or 21, wherein the nanoparticle comprises at least one peptide comprising one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11.24. The nanoparticle of embodiment 23, wherein the nanoparticle comprises only peptides comprising the amino acid sequence of SEQ ID NOs: 7, 8, 9, 10, or 11.25. The nanoparticle of embodiment 23 or 24, wherein the nanoparticle is a. a first type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 7; b. a second type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 8, c. a third type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 9, d. a fourth type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 10, or e. a fifth type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 11.26. The nanoparticle of any one of embodiments 20-25, wherein the amphiphilic polymer has a number average molecular weight (Mn) of 20,000 g / mol or less, 10,000 g / mol or less, or ranging between 6,000 g / mol to 1,000 g / mol.Attorney Docket No. 14779-010-22827. The nanoparticle of any one of embodiments 20-26, wherein the amphiphilic polymer comprises the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a C4 to C22 alkyl group, preferably Cs to C20 alkyl group.28. The nanoparticle of any one of embodiments 20-27, wherein the amphiphilic polymer is selected from the group comprising poly(maleic acid-alt- 1 -octadecene), poly(maleic acid-a / z-1 -dodecene) and poly(maleic acid-alt- 1 -tetradecene).29. The nanoparticle of embodiment 28, wherein the amphiphilic polymer is poly(maleic acid-alt- 1 -octadecene).30. The nanoparticle of embodiment 28 or 29, wherein the number average molecular weight of the amphiphilic polymer is from 6,000 to 1,000 g / mol.31. The nanoparticle of any of one of embodiments 20-30, wherein at least one peptide is covalently linked to the outside of the nanoparticle or non-covalently associated.32. The nanoparticle of any of one of embodiments 20-31, wherein the nanoparticle optionally comprises a iron oxide core and an amphiphilic polymer comprising poly(maleic acid- alt- 1 -octadecene) having a number average molecular weight of 6,000 to 1,000 g / mol.33. A pharmaceutical composition comprising the nanoparticle of any one of embodiments 20-32, further comprising an aqueous buffer, wherein the aqueous buffer comprises at least one sugar, at least one primary amine, and at least one amino acid.34. The pharmaceutical composition of embodiment 33, wherein the buffer contains D-mannitol, Tris(hydroxymethyl)aminomethane, and L-lactic acid.35. The pharmaceutical composition of embodiment 33 or 34, comprising a first type of nanoparticle, a second type of nanoparticle, a third type of nanoparticle, a fourth type of nanoparticle, and a fifth type of nanoparticle, wherein each type of nanoparticle is present in equimolar amounts.36. Use of the nanoparticle of any one of embodiments 20-32, or the pharmaceutical composition of any one of embodiments 33-35 for treatment of an autoimmune disease.Attorney Docket No. 14779-010-22837. The use of embodiment 36, wherein the autoimmune disease is type 1 diabetes (T1D).38. The use of embodiment 36, wherein the autoimmune disease is multiple sclerosis (MS).39. A composition comprising at least one nanoparticle, wherein the at least one nanoparticle comprises: a. an amphiphilic polymer, and b. at least one peptide, wherein the peptides comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.40. The composition of embodiment 39, wherein the at least one peptide is associated with the outside of the at least one nanoparticle.41. The composition of embodiment 39 or 40, wherein the at least one nanoparticle comprises at least one peptide comprising the amino acid sequence of SEQ ID NO: 12.42. The composition of embodiment 39 or 40, wherein the at least one nanoparticle comprises at least one peptide comprising one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11.43. The composition of embodiment 42, wherein the composition comprises only peptides comprising the amino acid sequence of SEQ ID NOs: 7, 8, 9, 10, and 11.44. The composition of embodiment 42 or 43, comprising at least 5 different types of nanoparticle, wherein the different types of nanoparticle differ among each other in the peptide sequence, and wherein a first type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 7, a second type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 8, a third type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 9, a fourth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 10, and a fifth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 11.45. The composition of embodiment 44, wherein each type of nanoparticle is present in equimolar amounts.Attorney Docket No. 14779-010-22846. The composition of any one of embodiments 39-45, wherein the amphiphilic polymer has a number average molecular weight (Mn) of 20,000 g / mol or less, 10,000 g / mol or less, or ranging between 6,000 g / mol to 1,000 g / mol.47. The composition of any one of embodiments 39-46, wherein the amphiphilic polymer comprises the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a C4 to C22 alkyl group, preferably Cs to C20 alkyl group.48. The composition of any one of embodiments 39-47, wherein the amphiphilic polymer is selected from the group comprising poly(maleic acid-alt- 1 -octadecene), poly(maleic acid-a / z-1 -dodecene) and poly(maleic acid-alt- 1 -tetradecene).49. The composition of embodiment 48, wherein the amphiphilic polymer is poly(maleic acid-alt- 1 -octadecene).50. The composition of embodiment 48 or 49, wherein the number average molecular weight of the amphiphilic polymer is from 6,000 to 1,000 g / mol.51. The composition of any of one of embodiments 39-50, wherein each peptide is covalently linked to the outside of the at least one nanoparticle or non-covalently associated.52. The composition of any one of embodiments 39-51, further comprising an aqueous buffer, wherein the aqueous buffer comprises at least one sugar, at least one primary amine, and at least one amino acid.53. The composition of embodiment 52, wherein the buffer contains D-mannitol, Tris(hydroxymethyl)aminomethane, and L-lactic acid.54. The composition of any one of embodiments 39-53, wherein each nanoparticle optionally comprises a iron oxide core and an amphiphilic polymer comprising poly(maleic acid- alt- 1 -octadecene) having a number average molecular weight of 6,000 to 1,000 g / mol.55. Use of the composition of any one of embodiments 39-54 for treatment of an autoimmune disease.Attorney Docket No. 14779-010-22856. The use of embodiment 55, wherein the autoimmune disease is type 1 diabetes (T1D).57. The use of embodiment 55, wherein the autoimmune disease is multiple sclerosis (MS).8. EQUIVALENTS

[0212] Although the invention is described in detail with reference to specific embodiments thereof, it will be understood that variations which are functionally equivalent are within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims. Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.

[0213] All patents and publications mentioned in this specification are incorporated herein by reference in their entireties. From the foregoing description, it will be apparent that variations and modifications can be made to the invention described herein to adopt it to various uses and conditions. Such embodiments are also within the scope of the following claims.

Claims

Attorney Docket No. 14779-010-228WHAT IS CLAIMED:

1. A pharmaceutical composition comprising nanoparticles, wherein each of the nanoparticles comprise: a. an amphiphilic polymer, and b. at least one peptide, wherein the pharmaceutical composition comprises peptides comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.

2. The pharmaceutical composition of claim 1 , wherein the at least one peptide is associated with the outside of at least one nanoparticle.

3. The pharmaceutical composition of claim 1 or 2, wherein each of the at least one nanoparticles comprise at least one peptide comprising the amino acid sequence of SEQ ID NO: 12.

4. The pharmaceutical composition of claim 1 or 2, wherein each of the at least one nanoparticles comprise at least one peptide comprising one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11.

5. The pharmaceutical composition of claim 4, wherein the pharmaceutical composition comprises only peptides comprising the amino acid sequence of SEQ ID NOs: 7, 8, 9, 10, and 11.

6. The pharmaceutical composition of claim 4 or 5, comprising at least 5 different types of nanoparticles, wherein the different types of nanoparticles differ among each other in the peptide sequence, and wherein a first type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 7, a second type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 8, a third type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 9, a fourth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 10, and a fifth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 11.Attorney Docket No. 14779-010-2287. The pharmaceutical composition of claim 6, wherein each type of nanoparticle is present in equimolar amounts.

8. The pharmaceutical composition of any one of claims 1-7, wherein the amphiphilic polymer has a number average molecular weight (Mn) of 20,000 g / mol or less, 10,000 g / mol or less, or ranging between 6,000 g / mol to 1,000 g / mol.

9. The pharmaceutical composition of any one of claims 1-8, wherein the amphiphilic polymer comprises the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a C4 to C22 alkyl group, preferably Cs to C20 alkyl group.

10. The pharmaceutical composition of any one of claims 1-9, wherein the amphiphilic polymer is selected from the group comprising poly (maleic acid-alt- 1 -octadecene), poly (maleic acid-a / t-1 -dodecene) and poly (maleic acid-alt- 1 -tetradecene).

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

12. The pharmaceutical composition of claim 10 or 11, wherein the number average molecular weight of the amphiphilic polymer is from 6,000 to 1,000 g / mol.

13. The pharmaceutical composition of any of one of claims 1-12, wherein each peptide is covalently linked to the outside of at least one nanoparticle or non-covalently associated.Attorney Docket No. 14779-010-22814. The pharmaceutical composition of any one of claims 1-13, further comprising an aqueous buffer, wherein the aqueous buffer comprises at least one sugar, at least one primary amine, and at least one amino acid.

15. The pharmaceutical composition of claim 14, wherein the buffer contains D- mannitol, Tris(hydroxymethyl)aminomethane, and L-lactic acid.

16. The pharmaceutical composition of any one of claims 1-15, wherein each nanoparticle optionally comprises a iron oxide core and an amphiphilic polymer comprising poly(maleic acid-alt- 1 -octadecene) having a number average molecular weight of 6,000 to 1,000 g / mol.

17. Use of the pharmaceutical composition of any one of claims 1-16 for treatment of an autoimmune disease.

18. The use of claim 17, wherein the autoimmune disease is type 1 diabetes (T1D).

19. The use of claim 17, wherein the autoimmune disease is multiple sclerosis (MS).

20. A nanoparticle, wherein the nanoparticle comprises: a. an amphiphilic polymer, and b. at least one peptide, wherein the at least one peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.

21. The nanoparticle of claim 20, wherein the at least one peptide is associated with the outside of the nanoparticle.

22. The nanoparticle of claim 20 or 21, wherein the nanoparticle comprises at least one peptide comprising the amino acid sequence of SEQ ID NO: 12.Attorney Docket No. 14779-010-22823. The nanoparticle of claim 20 or 21, wherein the nanoparticle comprises at least one peptide comprising one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11.

24. The nanoparticle of claim 23, wherein the nanoparticle comprises only peptides comprising the amino acid sequence of SEQ ID NOs: 7, 8, 9, 10, or 11.

25. The nanoparticle of claim 23 or 24, wherein the nanoparticle is a. a first type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 7; b. a second type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 8, c. a third type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 9, d. a fourth type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 10, or e. a fifth type of nanoparticle comprising a peptide comprising the amino acid sequence of SEQ ID NO: 11.

26. The nanoparticle of any one of claims 20-25, wherein the amphiphilic polymer has a number average molecular weight (Mn) of 20,000 g / mol or less, 10,000 g / mol or less, or ranging between 6,000 g / mol to 1,000 g / mol.

27. The nanoparticle of any one of claims 20-26, wherein the amphiphilic polymer comprises the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a C4 to C22 alkyl group, preferably Cs to C20 alkyl group.Attorney Docket No. 14779-010-22828. The nanoparticle of any one of claims 20-27, wherein the amphiphilic polymer is selected from the group comprising poly (maleic acid-alt- 1 -octadecene), poly(maleic acid-a / z-1- dodecene) and poly(maleic acid-alt- 1 -tetradecene).

29. The nanoparticle of claim 28, wherein the amphiphilic polymer is poly(maleic acid-alt- 1 -octadecene).

30. The nanoparticle of claim 28 or 29, wherein the number average molecular weight of the amphiphilic polymer is from 6,000 to 1,000 g / mol.

31. The nanoparticle of any of one of claims 20-30, wherein at least one peptide is covalently linked to the outside of the nanoparticle or non-covalently associated.

32. The nanoparticle of any of one of claims 20-31, wherein the nanoparticle optionally comprises a iron oxide core and an amphiphilic polymer comprising poly(maleic acid- alt- 1 -octadecene) having a number average molecular weight of 6,000 to 1,000 g / mol.

33. A pharmaceutical composition comprising the nanoparticle of any one of claims 20-32, further comprising an aqueous buffer, wherein the aqueous buffer comprises at least one sugar, at least one primary amine, and at least one amino acid.

34. The pharmaceutical composition of claim 33, wherein the buffer contains D- mannitol, Tris(hydroxymethyl)aminomethane, and L-lactic acid.

35. The pharmaceutical composition of claim 33 or 34, comprising a first type of nanoparticle, a second type of nanoparticle, a third type of nanoparticle, a fourth type of nanoparticle, and a fifth type of nanoparticle, wherein each type of nanoparticle is present in equimolar amounts.

36. Use of the nanoparticle of any one of claims 20-32, or the pharmaceutical composition of any one of claims 33-35 for treatment of an autoimmune disease.Attorney Docket No. 14779-010-22837. The use of claim 36, wherein the autoimmune disease is type 1 diabetes (T1D).

38. The use of claim 36, wherein the autoimmune disease is multiple sclerosis (MS).

39. A composition comprising at least one nanoparticle, wherein the at least one nanoparticle comprises: a. an amphiphilic polymer, and b. at least one peptide, wherein the peptides comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, 11, and 12.

40. The composition of claim 39, wherein the at least one peptide is associated with the outside of the at least one nanoparticle.

41. The composition of claim 39 or 40, wherein the at least one nanoparticle comprises at least one peptide comprising the amino acid sequence of SEQ ID NO: 12.

42. The composition of claim 39 or 40, wherein the at least one nanoparticle comprises at least one peptide comprising one of the amino acid sequences selected from the group consisting of SEQ ID NOs: 7, 8, 9, 10, and 11.

43. The composition of claim 42, wherein the composition comprises only peptides comprising the amino acid sequence of SEQ ID NOs: 7, 8, 9, 10, and 11.

44. The composition of claim 42 or 43, comprising at least 5 different types of nanoparticle, wherein the different types of nanoparticle differ among each other in the peptide sequence, and wherein a first type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 7, a second type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 8, a third type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 9, a fourth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 10, and a fifth type of nanoparticle comprises a peptide comprising the amino acid sequence of SEQ ID NO: 11.Attorney Docket No. 14779-010-22845. The composition of claim 44, wherein each type of nanoparticle is present in equimolar amounts.

46. The composition of any one of claims 39-45, wherein the amphiphilic polymer has a number average molecular weight (Mn) of 20,000 g / mol or less, 10,000 g / mol or less, or ranging between 6,000 g / mol to 1,000 g / mol.

47. The composition of any one of claims 39-46, wherein the amphiphilic polymer comprises the following building blockwherein R is a hydrocarbyl group or a substituted hydrocarbyl group, preferably R is a C4 to C22 alkyl group, preferably Cs to C20 alkyl group.

48. The composition of any one of claims 39-47, wherein the amphiphilic polymer is selected from the group comprising poly (maleic acid-alt- 1 -octadecene), poly(maleic acid-a / z-1- dodecene) and poly(maleic acid-alt- 1 -tetradecene).

49. The composition of claim 48, wherein the amphiphilic polymer is poly(maleic acid-alt- 1 -octadecene).

50. The composition of claim 48 or 49, wherein the number average molecular weight of the amphiphilic polymer is from 6,000 to 1,000 g / mol.

51. The composition of any of one of claims 39-50, wherein each peptide is covalently linked to the outside of the at least one nanoparticle or non-covalently associated.

52. The composition of any one of claims 39-51, further comprising an aqueous buffer, wherein the aqueous buffer comprises at least one sugar, at least one primary amine, and at least one amino acid.Attorney Docket No. 14779-010-22853. The composition of claim 52, wherein the buffer contains D-mannitol, Tris(hydroxymethyl)aminomethane, and L-lactic acid.

54. The composition of any one of claims 39-53, wherein each nanoparticle optionally comprises a iron oxide core and an amphiphilic polymer comprising poly(maleic acid- alt- 1 -octadecene) having a number average molecular weight of 6,000 to 1,000 g / mol.

55. Use of the composition of any one of claims 39-54 for treatment of an autoimmune disease.

56. The use of claim 55, wherein the autoimmune disease is type 1 diabetes (T1D).

57. The use of claim 55, wherein the autoimmune disease is multiple sclerosis (MS).

Images