Tolerogenic Compositions

Abstract

The present disclosure relates to a tolerogenic liposome-based composition and its use for treating immune disorders, the composition comprising two populations of liposomes, a first population of liposomes having a size range of 2-200 nm and a second population of liposomes having a size range of 500-2000 nm, the liposomes of the first and second populations carrying one or more antigens, and the liposome membrane of each liposome within the first and second liposome populations comprising phosphatidylserine in an amount ranging from 20-60% by weight based on the total liposome membrane composition.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of European Patent Application No. 22383126.4, filed November 23, 2022.

[0002] The present disclosure relates to the field of medicine. In particular, the present disclosure provides compositions for inducing tolerance to antigens, which are useful for the prevention and / or treatment of various immune disorders, such as autoimmune diseases. [Background technology]

[0003] Aberrant immune responses are the cause of many diseases and adverse reactions, including autoimmune diseases, allergies, transplant rejection and drug hypersensitivity.

[0004] Specifically, autoimmunity is the failure of an organism to recognize its own components as self, thereby eliciting an immune response against its own cells and tissues. Prominent examples include type 1 diabetes (T1D), lupus erythematosus, rheumatoid arthritis, multiple sclerosis (MS), Addison's disease, celiac disease, dermatomyositis, Hashimoto's thyroiditis, myasthenia gravis, pernicious anemia, reactive arthritis, Sjögren's syndrome, neuromyelitis optica, and immune thrombotic thrombocytopenic purpura.

[0005] It is estimated that 7-10% of the population in developed countries suffer from these diseases, which are often chronic, debilitating, and life-threatening. Healthcare costs associated with autoimmunity continue to grow due to the global rise in autoimmune disorders and the lack of available effective treatments.

[0006] Treatments for autoimmune diseases have traditionally been immunosuppressive, anti-inflammatory (steroids), or palliative. Non-immunotherapy, such as hormone replacement in Hashimoto's thyroiditis or type 1 diabetes, is palliative because it treats the consequences of the autoaggressive response. Dietary manipulation limits the severity of celiac disease. Steroid or NSAID treatment limits the inflammatory symptoms of many diseases. Similar limitations apply to other disorders associated with an aberrant, often excessive, immune response.

[0007] Extensive research has been devoted to developing immunomodulatory therapies to reduce or avoid unwanted immune responses. However, progress in this field has been significantly slowed due to a limited understanding of the complex details of various autoimmune diseases. Current strategies are generally based on broad-acting immunosuppressants, which are generally lifelong treatments, to maintain immunosuppression. Furthermore, the use of broad-acting immunosuppressants is associated with the risk of serious side effects, such as tumors, infections, nephrotoxicity, and metabolic disorders.

[0008] Liposomes are lipid vesicles composed of a lipid membrane surrounding an aqueous core. These vesicles are believed to have great potential as drug delivery systems for several reasons, mainly: i) they can deliver various types of active agents, hydrophilic agents can be loaded into the aqueous compartment, or hydrophobic agents can be immobilized in the membrane; and ii) they can target specific tissues, thereby enhancing therapeutic effects, reducing side effects, and increasing the bioavailability of the delivered agent. Liposome-based approaches have been proposed to effectively deliver immunosuppressants and reduce side effects in relation to autoimmune diseases. Furthermore, liposome-based approaches have been disclosed to induce tolerance in the absence of immunosuppressants, and have been somewhat successful in preventing serious autoimmune diseases (WO2015107140). The liposomes disclosed herein are not simply carriers of active agents, but are themselves part of the active agent responsible for promoting tolerance to the antigen specifically contained therein. The liposomes disclosed in WO2015107140 are relatively large in size (greater than 500 nm) and mimic apoptotic bodies that induce tolerance in dendritic cells through a mechanism similar to efferocytosis. However, despite efforts to date, there remains significant room for improvement in the treatment of autoimmune disorders and other conditions resulting from aberrant immune responses. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] International Publication No. 2015107140 Summary of the Invention [Problem to be solved by the invention]

[0010] The present inventors have surprisingly found that by combining two populations of liposomes carrying an antigen and containing at least 20% phosphatidylserine within the liposome membrane (the populations have different liposome sizes), a surprising tolerogenic effect is achieved that leads to the beneficial treatment of immune disorders. [Means for solving the problem]

[0011] Accordingly, a first aspect of the present disclosure relates to a composition comprising two populations of liposomes, wherein: the first population of liposomes has a size in the range of 2 to 200 nm; The second population of liposomes has a size in the range of 500-2000 nm; The first and second populations of liposomes carry one or more antigens; The liposome membrane of each liposome in the first and second liposome populations comprises phosphatidylserine in an amount ranging from 20 to 60% by weight relative to the total composition of the liposome membrane.

[0012] As shown in the following examples, the composition according to the first embodiment induces tolerogenic characteristics (secretion of interleukin-10 (IL-10) and transforming growth factor beta (TGF-B)) in human peripheral blood mononuclear cells (PBMCs). The following examples also show that a composition according to the first embodiment, comprising two liposome populations, one having a liposome size of 2-200 nm and the other having a size of 500-2000 nm, achieves greater efficacy in ameliorating autoimmune diseases than a liposome composition having the same characteristics except that only the 500-2000 nm population of liposomes is present.

[0013] Interestingly, the present inventors have surprisingly found that the composition according to the first aspect induces tolerance by interacting with a wide range of antigen-presenting cells. Without wishing to be bound by theory, the present inventors hypothesize that the liposome composition described herein may achieve remarkable effects by interacting with various factors involved in the process of peripheral tolerance. Considering that different abnormal autoimmune responses can be mediated by different APCs, sometimes multiple APCs, the composition of the present invention offers a significant advantage over other products in the prior art in that it has a broader spectrum of action and covers a wider range of target diseases than other products. Furthermore, the composition of the present invention can be found to achieve even greater effects because it acts through multiple mechanisms of action while inducing tolerance through various APCs.

[0014] For example, liposome populations between 2 and 200 nm in size have been shown to induce tolerance in B cells, while larger populations between 500 and 2,000 nm have been shown to induce tolerance by dendritic cells (DCs), as disclosed in International Publication No. 2015107140. The following examples demonstrate the ability of small-sized liposome populations to interact with B cells and induce tolerogenic characteristics in B cells. For example, Figure 1 specifically shows that PS-PC-Chol liposomes smaller than 200 nm interact with B cells and induce IL-10 secretion. Other examples demonstrate that liposomes in the compositions of the present invention containing the above two populations interact with and induce tolerogenic characteristics (IL-10 and TGF-B secretion) in several B cell subsets, in addition to dendritic cell IL-10 secretion (Figures 4, 5, and 6). This effect is surprising in light of International Publication No. 2015107140, which discloses that liposome sizes greater than 500 nm are essential for mimicking apoptotic cells and inducing tolerance through dendritic cells via a mechanism similar to efferocytosis. Given this prior art document, the effect of smaller liposome populations was not expected. Furthermore, given the teachings of International Publication No. 2015107140, experts would not study liposome sizes less than 500 nm because no relevant effects are predicted for liposomes with this small size. Other prior art documents also disclose the use of relatively small liposomes to enhance immune responses (Chen et al., doi:10.4049 / jimmunol.1801677). It has also been found that the liposome composition of the first embodiment can induce tolerance through hepatic sinusoidal endothelial cells (LSECs) and macrophages (Figure 4).

[0015] The tolerogenic effect of the composition defined in the first aspect of the present disclosure (hereinafter also referred to simply as the "liposome composition") not only results in effective treatment of autoimmune diseases, but also results in improved efficacy in treating autoimmune diseases when compared to liposome compositions comprising only a population of liposomes greater than 500 nm in size (Figure 2).

[0016] The liposome composition of the first embodiment has the advantage of being an antigen-specific therapy without the associated undesirable side effects. As mentioned above, most immunomodulatory approaches for the treatment of autoimmune conditions or transplant rejection involve immunosuppressants, which often result in increased susceptibility to infections and sometimes promote the development of tumors, nephrotoxicity, or metabolic disorders. In addition to conferring tolerance to the associated antigen, the liposome composition of the first embodiment does not induce any toxicity or other undesirable side effects.

[0017] The liposome composition of the first embodiment further has several advantages in terms of stability, uniformity, and ease of large-scale production.

[0018] First, the production of liposomes containing antigens, typically antigenic peptides, of the present invention can be achieved at low cost using common reagents and equipment in the pharmaceutical industry. Furthermore, product uniformity can be guaranteed, while scale-up for large-scale industrial production is affordable and dosage adjustment is easy. Furthermore, because the antigen is protected by the liposome, it is less susceptible to degradation.

[0019] Another major advantage lies in the fact that the liposome-based compositions of the present invention are defined compositions that are free of undesirable contaminants or by-products: the antigen-containing liposomes do not degrade into toxic by-products such as necrotic matter and do not induce rejection reactions as is the case with autologous or xenogeneic cell-based therapies.

[0020] Another advantage of the liposome composition disclosed herein is that it acts on two different immune tolerance mechanisms.Therefore, the effectiveness of the treatment is substantially without additional side effects.In addition, the two populations that provide dual activity can be achieved through a single preparation procedure, which is an additional advantage.

[0021] The liposome composition defined in the first aspect, due to its tolerogenic effect, provides effective prevention and treatment of disorders associated with abnormal immune responses, such as autoimmune disorders, both at the preclinical stage (i.e., the stage where an abnormal immune response has already been triggered but tissue damage and clinical symptoms are low) and at the clinical stage (i.e., the stage where tissue damage increases and clinical symptoms are evident).

[0022] In a second aspect, the present disclosure provides a liposomal composition as defined in the first aspect for use as a medicament. This aspect may also be formulated as the use of the liposomal composition as defined in the first aspect for preparing a medicament. A method of treatment is also disclosed, comprising administering a therapeutically effective amount of the liposomal composition as defined in the first aspect to a subject in need thereof.

[0023] Without wishing to be bound by theory, it is believed that the therapeutic effect of the liposome composition is achieved by tolerogenic presentation of the encapsulated antigen by antigen-presenting cells (for example, but not limited to, B cells, DCs, and macrophages) and subsequent suppression of the aberrant immune response. Thus, in a third aspect, the present disclosure provides a liposome composition as defined in the first aspect for use in inducing tolerance. This aspect may also be formulated as the use of the liposome composition as defined in the first aspect for preparing a medicament for inducing tolerance. Also disclosed is a method of inducing tolerance, comprising administering a therapeutically effective amount of the liposome composition as defined in the first aspect to a subject in need thereof.

[0024] In a fourth aspect, the present disclosure provides a liposomal composition as defined in the first aspect for use in treating a disorder associated with an abnormal immune response. This aspect may also be formulated as use of the liposomal composition as defined in the first aspect for preparing a medicament for treating a disorder associated with an abnormal immune response. Also disclosed is a method for treating a disorder associated with an abnormal immune response, comprising administering a therapeutically effective amount of a composition as defined in the first aspect to a subject in need thereof.

[0025] Finally, other aspects of the present invention provide a liposomal composition as defined above for use in immunomodulation, for use in suppressing an excessive immune response, and for use in treating a disorder associated with an aberrant immune response, wherein the liposomal composition restores tolerance to the antigen contained in the liposomal composition. [Brief explanation of the drawings]

[0026] [Figure 1] PS-PC-Chol-liposomes smaller than 200 nm interact with B cells and induce IL-10 secretion. a Percentage of NBD-positive CD19 cells and NBD-positive CD19CD1d cells from splenocytes of NOD mice incubated for 4 hours with PS-PC-Chol-NBD-Ins(h)-liposomes smaller than 200 nm. b Percentage of IL-10 expression in B cells from PBMCs after 24 hours of incubation with PS-PC-Chol-Ins(h)-liposomes smaller than 200 nm. Results are shown as mean ± SEM. [Figure 2] The PS-PC-Chol-liposome composition of the present disclosure has higher efficacy than PS-PC-Chol-liposome >500nm. Eight-week-old mice were immunized with mouse MOG peptide 35-55 (mMOG35-55) and treated intravenously with PS-PC-Chol-MOG36-55-liposome->500nm or PS-PC-Chol-MOG36-55-liposome-200 and 500 5 days after immunization. Clinical scores were assessed for 28 days. The area under the curve (AUC) difference of clinical scores was calculated to compare the beneficial effects of both liposome compositions. [Figure 3] Fluorescently labeled PS-liposomes are distributed to target organs. Histograms of relative fluorescence signal (RFU / gram of tissue) in NOD mice 1 and 6 hours after administration of PS-PC-Chol-AF750-empty-liposome-200 and 500. Mice were administered 100 μl by intravenous route (iv, n=5). Results are shown as mean ± SEM. [Figure 4] PS-PC-Chol-Liposome-200 and 500 interact with DCs, B cells, macrophages, and LSECs in vivo. The percentage of NBD-fluorescent cells in the parent gate was measured in the spleen and liver of animals treated with PS-PC-Chol-NBD-Ins(h)-Liposome-200 and 500 i.v. (1 h (n = 5-6) or 6 h (n = 5) after treatment). Cells were determined as follows: B cells, CD19+; conventional DCs (cDCs), CD11c+MHC-II+CD205+CD8a+; plasmacytoid DCs (pDCs), CD11c+MHC-II+B220+; macrophages (Mφ), F4 / 80+; Kupffer cells, F4 / 80+CD68+; and hepatic sinusoidal endothelial cells (LSECs), CD206+F4 / 80-. Data are means ± SEM; differences were observed when comparing immune subsets and time points within the same subset (*p<0.05, **p<0.01, ***p<0.001, two-way ANOVA with Tukey's multiple comparison test). [Figure 5] Liposomes of the present composition bind to B cells and induce IL-10 secretion. a) Percentage of NBD-fluorescent B cell subsets within the total subset gate after overnight culture of PS-PC-Chol-NBD-ins(h)-liposome-200 and 500 (n=3). Breg cell subsets were determined as follows: CD19+CD1dhighCD5+; B1a, CD19+CD5+CD43+; B1b, CD19+CD5-CD43+; marginal zone (MZ), CD19+CD21highCD23-. Data shown as mean ± SEM. b) Percentage of IL-10-secreting cells from the NBD-PSIns(h)-liposome-bound NBD+ B cell subset (n=3) and the non-liposome-bound NBD- B cell subset (n=3). Data shown as mean ± SEM; no differences were observed. [Figure 6]Liposomes of the composition of the present invention induce the expression of the tolerogenic cytokines IL-10 and TGF-B in B, and the expression of IL-10 in DC cells derived from PBMCs. a % IL-10 expression in NBD+ or NBD- B cells from PBMCs after 24 hours of incubation with PS-PC-Chol-NBD-liposomes-200 and 500 loaded with AChR peptide or mutated citrullinated vimentin peptide or insulin. b % TGF-b expression in NBD+ or NBD- B cells from PBMCs after 24 hours of incubation with PS-PC-Chol-NBD-liposomes-200 and 500 loaded with AChR peptide or mutated citrullinated vimentin peptide or insulin. c Percentage of IL-10 expression in NBD+ or NBD- dendritic cells from PBMCs after 24-hour incubation with PS-PC-Chol-NBD-liposome-200 and 500 loaded with AChR peptide, mutant citrullinated vimentin peptide, or insulin. d Percentage of TGF-β expression in NBD+ or NBD- dendritic cells from PBMCs after 24-hour incubation with PS-PC-Chol-NBD-liposome-200 and 500 loaded with AChR peptide, mutant citrullinated vimentin peptide, or insulin. e Percentage of pHrodo™ green-positive cells in the parent gate of various immune cell types from PBMCs after 24-hour incubation with PS-PC-Chol-MCV-200 and 500 stained with pHrodo™ green dye. f Results of IL-10 and TGF-β expression in pHrodo+ and pHrodo- B cells expressed by mean fluorescence intensity (MFI) are shown as mean ± SEM. (*p<0.05, **p<0.01, ***p<0.001, two-way ANOVA with Tukey's multiple comparison test) DETAILED DESCRIPTION OF THE INVENTION

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs at the time of filling. However, in the event of potential ambiguity, the definitions provided herein shall take precedence over any dictionary or external definitions. Furthermore, unless otherwise required by context, singular terms shall include plurals and plural terms shall include the singular.

[0028] As used herein, the indefinite articles "a" and "an" are synonymous with "at least one" or "one or more." Unless otherwise indicated, definite articles such as "the" used herein also include the plural of the noun.

[0029] Liposomes As described above, a first aspect of the present disclosure relates to a composition comprising two populations of liposomes, wherein the first population of liposomes has a size range of 2 to 200 nm and the second population of liposomes has a size range of 500 to 2000 nm. In certain embodiments, the composition of the first aspect consists essentially of two populations of liposomes, wherein the first population of liposomes has a size range of 2 to 200 nm and the second population of liposomes has a size range of 500 to 2000 nm. In another specific embodiment, the composition of the first aspect consists of two populations of liposomes, wherein the first population of liposomes has a size range of 2 to 200 nm and the second population of liposomes has a size range of 500 to 2000 nm. The liposomes of the first and second populations may carry one or more antigens. The liposome membrane of each liposome in the first and second liposome populations may contain phosphatidylserine in an amount ranging from 20 to 60% by weight relative to the total composition of the liposome membrane.

[0030] The term "liposome" refers to a self-assembled structure comprising one or more membranes formed by amphiphilic bilayers, each of which contains two monolayers containing oppositely oriented amphiphilic molecules. Amphiphilic molecules, such as amphiphilic lipids, contain a polar (hydrophilic) head group region covalently linked to one or two nonpolar (hydrophobic) chains. Energetically unfavorable contact between the hydrophobic chains and the surrounding aqueous medium induces the amphiphilic molecules to orient themselves so that their polar head groups are oriented toward the surface of the bilayer, while the hydrophobic chains are reoriented toward the interior of the bilayer. Thus, an energetically stable structure is formed that effectively shields the hydrophobic chains from contact with the aqueous environment.

[0031] Liposomes can have a single bilayer membrane (small unilamellar vesicles "SUVs" and large unilamellar vesicles "LUVs") or multiple bilayer membranes (large multilamellar vesicles "MLVs"). Liposomes can also be prepared as multivesicular vesicles "MVVs," which are liposomes that surround or enclose multiple non-concentric aqueous chambers. In contrast, MLVs have multiple concentric "onion skin" membranes, each enclosing an aqueous compartment. Given this enclosure of the aqueous volume within a protective barrier of lipid molecules, liposomes can sequester encapsulated molecules, such as peptides, from the degradative effects of factors present in the external environment, such as peptidase enzymes. The sequestered molecules can be suspended or dissolved in the aqueous compartments or associated with the liposome membrane. In general, polar, water-soluble molecules are primarily dissolved in the aqueous compartments, while less polar molecules can associate with the lipid membrane.

[0032] The liposome composition of the first embodiment comprises two populations of liposomes, the first population of liposomes having a size range of 2 to 200 nm, and the second population of liposomes having a size range of 500 to 2000 nm. Liposome size typically refers to the average diameter and can be determined by nanoparticle tracking analysis (NTA) using a Malvern Panalytical Nanosight NS300 (Whitepaper on Nanoscale Material Characterization: a Review of the Use of Nanoparticle Tracking Analysis (NTA) 2015 Malvern Instruments Limited). This method is known for determining the "hydrodynamic diameter." Therefore, in certain embodiments of the present disclosure, liposome size refers to the hydrodynamic diameter. While the size of the liposomes in the second population typically does not exceed 2000 nm, in some embodiments, the size range of this second population may be larger, e.g., 500 to about 3000, about 4000, or about 5000 nm.

[0033] In one embodiment, the first population of liposomes has a size of less than 200 nm, or less than 180 nm, or less than 160 nm, or less than 150 nm, or less than 125 nm, or less than 100 nm, or less than 75 nm, or less than 50 nm. In another embodiment, the first population of liposomes has a size of more than 2 nm, or more than 10 nm, or more than 15 nm, or more than 25 nm, or more than 50 nm, or more than 75 nm. In certain embodiments, the first population of liposomes has a size in the range of 2 to 200 nm, more particularly 10 to 200 nm.

[0034] In one embodiment, the liposomes of the second population have a size greater than 500 nm, or greater than 525 nm, or greater than 550 nm, or greater than 575 nm, or greater than 600 nm, or greater than 625 nm, or greater than 650 nm, or greater than 675 nm, or greater than 700 nm. In another embodiment, the liposomes of the second population have a size less than 5000 nm, or less than 4000 nm, or less than 3000 nm, or less than 2000 nm, or less than 1800 nm, or less than 1500 nm, or less than 1250 nm, or less than 1100 nm, or less than 1000 nm, or less than 950 nm, or less than 900 nm, or less than 850 nm, or less than 800 nm.

[0035] In one embodiment, 15-75% of the liposomes in the composition correspond to the first population, i.e., 15-75% of the liposomes in the composition have a size in the range of 2-200 nm. These percentages refer to the number of liposomes of a particular size per 100 liposomes in the composition. In a specific embodiment, 15-55% of the liposomes in the composition correspond to the first population. In an even more specific embodiment, 20-50% of the liposomes in the composition correspond to the first population. In one embodiment, 2-40% of the liposomes in the composition correspond to the second population, i.e., 2-40% of the liposomes in the composition have a size in the range of 500-2000 nm. In a specific embodiment, 2-25% of the liposomes in the composition correspond to the second population. In another even more specific embodiment, 2-15% of the liposomes in the composition correspond to the second population.

[0036] As mentioned above, liposome membrane is formed by amphiphilic molecules.Non-limiting amphiphilic molecules that can be contained in the liposome membrane of the liposome composition of the present disclosure include phospholipids; glycerophospholipids; phosphatidylserine (PS), such as 1,2-dioleoyl-phosphatidylserine, 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), 1,2-dipalmitoyl-phosphatidylserine, 1,2-dimyristoyl-phosphatidylserine, 1,2-distearoyl-phosphatidylserine, l-oleoyl-2-palmitoyl-phosphatidylserine, l-oleoyl-2- Stearoyl-phosphatidylserine, 1-palmitoyl-2-oleoyl-phosphatidylserine, and l-stearoyl-2-oleoyl-phosphatidylserine; phosphatidylcholines (PC), such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, lecithin; lysolecithin; phosphatidylethanolamines, such as dioleylphosphatidylethanolamine (DOPE); poly(ethylene glycol) copolymers. glycol) 5000-phosphatidylethanolamine; dioleyloxypropyltriethylammonium (DOTMA); cholesterol; cholesterol esters; diacylglycerol; diacylglycerol succinate; diphosphatidylglycerol (DPPG); hexanedecanol; fatty alcohols, such as polyethylene glycol (PEG); poly(ethylene glycol) 400-monostearate; polyoxyethylene-9-lauryl ether; surface-active fatty acids, such as palmitic acid or olein Included are acids; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; sorbitan fatty acid esters, such as sorbitan trioleate; phosphatidylinositol; sphingomyelin; cardiolipin; phosphatidic acid; cerebrosides; dicetyl phosphate; dipalmitoylphosphatidylglycerol; stearylamine; dodecylamine; hexadecylamine; acetyl palmitate; glycerol ricinoleate; hexadecyl sterate; isopropyl myristate, and combinations thereof.

[0037] In one embodiment, the liposome membrane may contain phospholipids, such as, but not limited to, phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidic acid (PA). The liposome membrane may also contain other lipids, such as cholesterol (CHOL). The liposome membrane may contain additional molecules that are not naturally lipids, such as proteins, carbohydrates, antibodies, or polyethylene glycol (PEG) chains. Liposomes may contain specific moieties designed to target the liposome to specific sites or target cells or to protect the liposome from harmful environments (e.g., the gastrointestinal tract). The composition of the liposome is relevant to the tolerogenic delivery of antigens. Therefore, as described above, the liposome membrane preferably contains PS in an amount ranging from 20 to 60% by weight relative to the total liposome membrane composition. The PS contained in the liposome membrane constitutes an "eat me" signal that links PS recognition by antigen-presenting cells with the outcome of tolerance induction. It is noteworthy that the liposomes of the present invention are effectively phagocytosed by antigen-presenting cells and do not require additional receptors or ligands to achieve tolerogenic delivery of antigen. However, other receptors and / or ligands may be assembled into the liposomes to improve uptake and / or tolerogenic processing.

[0038] The term "weight percent (%)" refers to the proportion of each component of the liposome membrane relative to the total weight of the liposome membrane.

[0039] "Liposome's membrane" or "liposomal membrane" refers to the entire membrane bilayer contained in a liposome.

[0040] In one embodiment, the liposome membrane comprises PS in an amount of 30-50% by weight relative to the total liposome membrane. In a further embodiment, the liposome membrane comprises PS in an amount of 30-45% by weight, or 35-45% by weight, relative to the total liposome membrane, such as 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, or 44%.

[0041] Liposomes according to the present disclosure may contain, in addition to PS, varying concentrations of other lipids. In some embodiments, the liposome membrane also contains a phospholipid with a neutral net charge. In some embodiments, the liposome membrane also contains phosphatidylethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, and / or sphingomyelin. In some embodiments, the liposome membrane also contains PC, e.g., 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments, the liposome membrane also contains a sterol lipid or a steroid. In certain embodiments, the liposome membrane also contains PC and CHOL.

[0042] In one embodiment, the liposome membrane comprises PC in an amount of 30-50% by weight relative to the total liposome membrane. In another embodiment, the liposome membrane comprises PC in an amount of 30-45% by weight relative to the total liposome membrane. In a further embodiment, the membrane PC comprises 32-42% by weight relative to the total liposome membrane, e.g., 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or 41%.

[0043] The amount of CHOL can be 10-40% by weight based on the total liposome membrane. In one embodiment, the liposome membrane contains CHOL in an amount of 12-40% by weight based on the total liposome membrane. In a further embodiment, the membrane CHOL is 15-37%, 20-35%, or 20-30% by weight based on the total liposome membrane, e.g., 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, or 29%.

[0044] To obtain liposomes with appropriate physical and chemical properties in terms of stability, permeability, and morphology, the ratios of various lipids contained in the liposome membrane must be balanced. In some embodiments, the liposome membrane comprises PS, PC, and CHOL. In some embodiments, the liposome membrane consists essentially of PS, PC, and CHOL. In some embodiments, the liposome membrane consists of PS, PC, and CHOL. In some embodiments, the molar ratio of PS:PC:CHOL is 1:(0.2-4):(0.2-5). In certain embodiments, the molar ratio of PS:PC:CHOL is 1:(0.6-1.8):(0.7-2.5). The term "ratio" is understood in its usual sense as the magnitude of quantities relative to one another. Specifically, the ratio of two quantities indicates how many times the first quantity (X) is contained in the second quantity (Y), and is expressed as X:Y. As in the above embodiment, the quantities can be expressed as a range (X-X':Y-Y'). The term "molar ratio" is used when the magnitude referred to is a molar concentration. Alternatively, the ratio can be expressed as a "weight ratio" when the magnitude referred to is a weight. Here, the molar ratio ranges are given for three specific lipids (PS, PC, and CHOL). In a more specific embodiment, the membrane contains a molar ratio of PS:PC:CHOL that is 1:(0.7-1.5):(0.9-2). In another more specific embodiment, the membrane contains a molar ratio of PS:PC:CHOL that is 1:(0.8-1.4):(1.1-1.9). In another more specific embodiment, the membrane contains a molar ratio of PS:PC:CHOL that is 1:(0.9-1.3):(1.2-1.7).

[0045] antigen As described above, the liposomes contained in the composition of the first embodiment contain an antigen. The term "antigen" refers to any substance that elicits a B cell (humoral / antibody) and / or T cell (cellular) adaptive immune response upon exposure to a host organism. The antigen may be dissolved or suspended in the aqueous compartment inside the liposome, or may be associated with the liposome membrane. An antigen is a molecule that can bind to components of the immune system, including antibodies, T cells, and B cells. A particular antigen may contain one or more epitopes or antigenic determinants. Within the meaning of the present disclosure, an antigen is an immunogenic antigen. Those skilled in the art can determine antigenic substances by methods well known in the art.

[0046] The liposome composition of the first aspect can contain one kind of antigen or multiple kinds of antigens.In certain embodiments, liposome encapsulates multiple kinds of antigens that are related to the same immunological disorder.For example, liposome can encapsulate two, three, four or five different antigens that are preferably related to the same immunological disorder.

[0047] Antigens can have a variety of chemical properties. While most antigens are peptide-based, polysaccharides, lipids, or nucleic acids can also be antigenic molecules. Furthermore, some antigens are complexes, such as complexes formed by polypeptides and other molecules selected from nucleic acids, lipids, and polysaccharides. For example, the present disclosure contemplates that liposomes contain polypeptides containing one or more antigenic sequences. When liposomes contain multiple different antigens, the present disclosure contemplates that the different antigens are independent antigenic peptide sequences. It is also contemplated that the different antigens form part of a single, separate sequence containing multiple different antigenic sequences. The antigens (antigen sequences) may be arranged contiguously within the polypeptide or separated by a non-antigenic linking sequence. The present disclosure is not limited to any particular antigen or group of antigens. The liposome compositions of the present disclosure can be prepared by containing any antigen and will induce antigen-specific immune tolerogenesis against any antigen contained therein.

[0048] In one embodiment, the antigen is selected from the group consisting of an autoantigen, a drug including a therapeutic protein, an allergen, and an alloantigen.

[0049] In one embodiment, the antigen is a peptide. In a specific embodiment, the antigen is a peptide having a size in the range of 3 to 5,000 amino acids, particularly 5 to 2,000 amino acids, or 6 to 1,000 amino acids, or 7 to 500 amino acids, or 8 to 250 amino acids, or 5 to 200 amino acids, or 8 to 100 amino acids, or 10 to 50 amino acids. In a specific embodiment, the antigen is a peptide having a size in the range of 5 to 100 amino acids.

[0050] In one embodiment of the first aspect, antigen is autoantigen.Term " autoantigen " generally refers to the normal substance, often or protein complex, that is recognized by the immune system of patients suffering from certain autoimmune diseases.These antigens should not be the target of immune system under normal conditions, but due mainly to genetic and environmental factors, these patients lose the normal immune tolerance to these antigens.

[0051] In one embodiment of the first aspect, the antigen is selected from the group consisting of type 1 diabetes (T1D), lupus erythematosus, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, reactive arthritis, psoriatic arthritis, multiple sclerosis, neuromyelitis optica, Addison's disease, celiac disease, dermatomyositis, Hashimoto's thyroiditis, myasthenia gravis, pernicious anemia, autoimmune hemolytic anemia, autoimmune neutropenia, Graves' disease, psoriasis, Sjogren's syndrome including keratoconjunctivitis sicca secondary to Sjogren's syndrome, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), alopecia areata, Crohn's disease, aphthous ulcers, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, and acute necrotizing hemorrhagic encephalopathy. It is an autoantigen associated with encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, true erythrocytic anemia, idiopathic thrombocytopenic purpura, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, sarcoidosis, primary biliary cirrhosis, posterior uveitis, and interstitial pulmonary fibrosis.

[0052] In another embodiment of the first aspect, antigen is autoantigen.Autoantigen (also called autoantigen) is known to those skilled in the art and can be found in literature.For example, the autoantigens associated with numerous autoimmune diseases are disclosed in AAgAtlas 1.0 database (doi:10.1093 / nar / gkw946.Epub 2016 Oct 19; http: / / biokb.ncpsb.org / aagatlas / ).

[0053] In certain embodiments, the autoantigen is insulin, proinsulin, protein tyrosine phosphatase (IA2, also known as islet cell antigen 512), glutamic acid decarboxylase (GAD), chromogranin and islet-glucose-6-phosphatase catalytic subunit-related protein (IGRP), peripherin (Roep BO, Peakman M. Cold Spring Harbor Perspectives). Med., 2012, vol. 2(4): a007781.oi:10.1101 / cshperspect.a007781.), myelin, myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), myelin proteolipid protein (PLP), GDP-l-fucose synthase, acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), agrin, lipoprotein-related protein 4 (LRP4), cortactin, transglutaminase, deamidated gliadin, thyroglobulin (Lernmark AJ Clin Invest., 2001, vol. 108, pp. 1091-1096), collagen (e.g., type 11 collagen), human cartilage gp 39, chromogranin A, gp130-RAPS, vimentin, citrullinated vimentin, ADAMST13, aquaporin-4, proteolipid protein, fibrillarin, nuclear protein, nucleolar protein (e.g., small nucleolar protein), histidyl-tRNA synthetase (HisRS), histidine-tRNA synthetase (HARS1), jo-1, thyroid stimulating factor receptor, histone, glycoprotein gp 70, ribosomal protein, pyruvate dehydrogenase dehydrolipoamide acetyltransferase, hair follicle antigen, human tropomyosin isoform 5, mitochondrial protein, pancreatic beta cell protein, gluten, and antigenic fragments or derivatives of any of the above.Examples of antigenic fragments include, for example, those disclosed in Table 3.

[0054] In other embodiments, the autoantigen is associated with an autoimmune disorder selected from those disclosed in Table 1. In other embodiments, the autoantigen is a polypeptide selected from the polypeptides disclosed in Table 1, and antigenic fragments of said polypeptides. In a particular embodiment, the antigen is associated with T1D. In a more particular embodiment, the antigen is selected from those disclosed in Table 2, and combinations or immunogenic fragments thereof. [Table 1] TIFF2025539844000002.tif178165 [Table 2] IGRP: glucose-6-phosphatase catalytic subunit-related protein; ZnT8: zinc transporter 8; GAD: glutamic acid decarboxylase; IA-2: insulinoma antigen 2; ICA-69: islet cell antigen-69; PDX1: pancreatic duodenal homeobox protein 1; HSP60: heat shock protein 60. [Table 3] TIFF2025539844000005.tif187165TIFF2025539844000006.tif233165TIFF2025539844000007.tif240165TIFF2025539844000008.tif229165TIFF2025539844000009.tif241165TIFF2025539844000010.tif68165X represents citrulline

[0055] In another embodiment, the antigen included in the composition of the first aspect is an allergen. An "allergen" is any substance that can cause an undesired (e.g., type 1 hypersensitivity) immune response (i.e., an allergic response or allergic reaction) in a subject. Allergens include, but are not limited to, plant allergens (e.g., pollen, ragweed allergens), insect allergens, insect sting allergens (e.g., bee sting allergens), animal allergens (e.g., pet allergens, such as animal dander or cat Fel d 1 antigen), latex allergens, mold allergens, fungal allergens, cosmetic allergens, drug allergens, food allergens, dust, insect venom, viruses, bacteria, etc. Food allergens include, but are not limited to, milk allergens, egg allergens, nut allergens (e.g., peanut allergens or tree nut allergens (e.g., walnut, cashew, etc.)), fish allergens, shellfish allergens, soy allergens, legume allergens, seed allergens, and wheat allergens. Insect sting allergens include allergens that are or are related to bee stings, hornet stings, Japanese carpenter bee stings, yellow bee stings, etc. Insect allergens also include dust mite allergens (e.g., Der P1 antigen) and cockroach allergens. Drug allergens include allergens that are or are related to antibiotics, NSAIDs, anesthetics, etc. Pollen allergens include grass allergens, tree allergens, weed allergens, flower allergens, etc.

[0056] In another embodiment, the antigen contained in the composition of the first aspect is associated with organ or tissue rejection. Examples of such antigens include, but are not limited to, antigens derived from allogeneic cells, such as antigens derived from allogeneic cell extracts, and antigens derived from other cells, such as endothelial cell antigens. Antigens also include those associated with transplantable grafts. Such antigens arise as a result of the transplantable graft or the introduction of the transplantable graft in the recipient, and may be presented for recognition by cells of the immune system, resulting in an undesired immune response in the recipient of the transplantable graft. Transplantation antigens include those associated with organ or tissue rejection or graft-versus-host disease. Transplantation antigens may be obtained from or derived from cells of biological material or information about the transplantable graft. Transplantation antigens generally include proteins, polypeptides, peptides, lipoproteins, glycolipids, polynucleotides, or are contained or expressed within cells. Information about the transplantable graft is any information about the transplantable graft that can be used to obtain or derive transplant antigens. Such information includes information about antigens predicted to be present in or on the cells of the transplantable graft, e.g., sequence information, the type or class of antigen, and / or their MHC class I, MHC class II, or B cell presentation restriction, etc. Such information may also include information about the type of transplantable graft (e.g., autograft, allograft, xenograft), the molecular and cellular composition of the graft, the body site from which the graft originates or into which the graft will be implanted (e.g., whole or partial organs, skin, bone, nerves, tendons, neurons, blood vessels, fat, cornea, etc.).

[0057] In another embodiment, the antigen included in the composition of the first aspect is a therapeutically active agent (also referred to herein as a "drug") that may generate an unwanted immune response. The therapeutically active agent may be, for example, a therapeutic protein. Therapeutic protein antigens generally include proteins, polypeptides, peptides, lipoproteins, or are contained or expressed within, by, or on a cell. Therapeutic proteins include, but are not limited to, insoluble therapeutic proteins, enzymes, enzyme cofactors, hormones, blood clotting factors, cytokines and interferons, growth factors, monoclonal and polyclonal antibodies (e.g., administered to a subject as replacement therapy), and proteins associated with Pompe disease (e.g., alglucosidase alfa, rhGAA (e.g., Myozyme and Lumizyme (Genzyme)). Therapeutic proteins also include proteins involved in the blood clotting cascade. Therapeutic proteins include, but are not limited to, Factor VIII, Factor VII, Factor IX, Factor V, von Willebrand factor, von Heldebrand factor, and the like. Factor), tissue plasminogen activator, insulin, growth hormone, erythropoietin alpha, VEGF, thrombopoietin, lysozyme, antithrombin, etc. Therapeutic proteins also include adipokines such as leptin and adiponectin. Other examples of therapeutic proteins are as follows: Fragments or derivatives of any of the therapeutic proteins disclosed herein provided as antigens are also contemplated as antigens in the compositions of the first embodiment.

[0058] Examples of therapeutic proteins used in enzyme replacement therapy of subjects with lysosomal storage diseases include, but are not limited to, imiglucerase (e.g., CEREZYME™) for the treatment of Gaucher disease, a-galactosidase A (a-gal A) (e.g., agalsidase beta, FABRYZYME™) for the treatment of Fabry disease, acid a-glucosidase (GAA) (e.g., alglucosidase alfa, LUMIZYME™, MYOZYME™) for the treatment of Pompe disease, and arylsulfatase B (e.g., laronidase, ALDURAZYME™, idursulfase, ELAPRASE™, arylsulfatase B, NAGLAZYME™) for the treatment of mucopolysaccharidoses. Other examples of enzymes include oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.

[0059] Examples of therapeutic proteins also include melatonin (N-acetyl-5-methoxytryptamine), serotonin, thyroxine (or tetraiodothyronine) (thyroid hormone), triiodothyronine (thyroid hormone), epinephrine (or adrenaline), norepinephrine (or noradrenaline), dopamine (or prolactin-inhibiting hormone), anti-Müllerian hormone (or Müllerian inhibitory factor or Müllerian inhibitory hormone), adiponectin, adrenocorticotropic hormone (or or corticotropin), angiotensinogen and angiotensin, antidiuretic hormone (or vasopressin, arginine vasopressin), atrial natriuretic peptide (or atriopeptin), calcitonin, cholecystokinin, corticotropin-releasing hormone, erythropoietin, follicle-stimulating hormone, gastrin, ghrelin, glucagon, glucagon-like peptide (GLP-1), GIP, gonadotropin-releasing hormone, growth hormone-releasing hormone, human chorionic gonadotropin , human placental lactogen, growth hormone, inhibin, insulin, insulin-like growth factor (or somatomedin), leptin, luteinizing hormone, melanocyte-stimulating hormone, orexin, oxytocin, parathyroid hormone, prolactin, relaxin, selectin, somatostatin, thrombopoietin, thyroid-stimulating hormone (or thyrotropin), thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione Hormones include dihydrotestosterone, estradiol, estrone, estriol, progesterone, calcitriol (1,25-dihydroxyvitamin D3), calcidiol (25-hydroxyvitamin D3), prostaglandins, leukotrienes, prostacyclin, thromboxane, prolactin-releasing hormone, lipotropin, brain natriuretic peptide, neuropeptide Y, histamine, endothelin, pancreatic polypeptide, renin, and enkephalins.

[0060] Examples of blood and blood clotting factors include factor I (fibrinogen), factor II (prothrombin), tissue factor, factor V (proaccelerin, unstable factor), factor VII (stable factor, proconvertin), factor VIII (antihemophilic globulin), factor IX (Christmas factor or plasma thromboplastin component), factor X (Stuart-Prower factor), factor Xa, factor XI, factor XII (Hageman factor), factor XIII (fibrin-stabilizing factor), von Willebrand factor, prekallikrein (Fletcher factor), and high molecular weight kininogen. (HMWK) (Fitzgerald factor), fibronectin, fibrin, thrombin, antithrombin III, heparin cofactor II, protein C, protein S, protein Z, protein Z-related protease inhibitor (ZPI), plasminogen, alpha 2-antiplasmin, tissue plasminogen activator (tPA), urokinase, plasminogen activator inhibitor-1 (PAI1), plasminogen activator inhibitor-2 (PAI2), cancer procoagulant, and epoetin alfa (Epogen, Procrit).

[0061] Examples of cytokines include lymphokines, interleukins and chemokines, type 1 cytokines such as IFN-γ, TGF-β, and type 2 cytokines such as IL-4, IL-10 and IL-13.

[0062] Examples of growth factors include adrenomedullin (AM), angiopoietin (Ang), cell motility stimulating factor, bone morphogenetic protein (BMP), brain-derived neurotrophic factor (BDNF), epidermal growth factor (EGF), erythropoietin (EPO), fibroblast growth factor (FGF), glial cell line-derived neurotrophic factor (GDNF), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), growth differentiation factor-9 (GDF9), hepatocyte growth factor (HGF), hepatoma-derived growth factor (HDGF), insulin-like growth factor (IGF) ), migration stimulating factor, myostatin (GDF-8), nerve growth factor (NGF) and other neurotrophins, platelet-derived growth factor (PDGF), thrombopoietin (TPO), transforming growth factor alpha (TGF-α), transforming growth factor beta (TGF-β), tumor necrosis factor alpha (TNF-α), vascular endothelial growth factor (VEGF), Wnt signaling pathway, placental growth factor (P1GF), [(fetal bovine somatotropin)] (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7.

[0063] Examples of monoclonal antibodies include abagovomab, abciximab, adalimumab, adecatumumab, afelimomab, afutuzumab, alacizumab pegol, ALD, alemtuzumab, altumomab pentetate, anatumomab mafenatox, anrukinzumab, anti-thymocyte globin (ATG), and anti-thymocyte globin (ATG). globin, Apolizumab, Arcitumomab, Aselizumab, Atlizumab (Tocilizumab), Atorolimumab, Bapineuzumab, Basiliximab, Bavituximab, Bectumomab, Belimumab, Benralizumab, Bertilimumab, Besilesomab, Bevacizumab, Biciromab, Bivatuzumab mertansine, blinatumomab, brentuximab vedotin, briakinumab, canakinumab, cantuzumab mertansine, capromab pendetide, catumaxomab, cedelizumab, certolizumab pegol, cetuximab, sitatuzumab bogatox, cixutumumab, clenoliximab, clivatuzumab tetraxetan, conatumumab, dacetuzumab, daclizumab, daratumumab, denosumab, detumomab, dorlimomab aritox, dorlixizumab, ecromeximab, eculizumab, edobacomab, edrecolomab, efalizumab, efungumab, elotuzumab, elsilimomab, enlimomab pegol, epitumomab sitaxetanCituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Felvizumab, Fezakinumab, Figitumumab, Fontolizumab, Foravirumab, Fresolimumab, Galiximab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, GCl008, Girentuximab, Glembatumumab vedotin vedotin, golimumab, gomiliximab, ibalizumab, ibritumomab tiuxetan, igovomab, imciromab, infliximab, intetumumab, inolimomab, inotuzumab ozogamicin ozogamicin, ipilimumab, iratumumab, keliximab, labetuzumab, lebrikizumab, lemaresomab, lerdelimumab, lexatumumab, ribivirumab, lintuzumab, lorvotuzumab Mertansine, Lucatumumab, Lumiliximab, Mapatuzumab, Maslimomab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Morolimumab, Motavizumab, Muromonab-CD3, Nacolomab-Tafenatoxifentafenatox, naptumomab estafenatox, natalizumab, nebacumab, necitumumab, nerelimomab, nimotuzumab, nofetumomab merpentan, ocrelizumab, odulimomab, ofatumumab, olaratumumab, omalizumab, oportuzumab monatox, oregovomab, otelixizumab, pagibaximab, palivizumab, panitumumab, panobacumab, pascolizumab, pemtumomab, pertuzumab, pexelizumab, pintumomab, priliximab, pritumumab mab), Rafivirumab, Ramucirumab, Ranibizumab, Raxibacumab, Regavirumabreslizumab, Rilotumumab, Rituximab, Robatumumab, Rontalizumab, Rovelizumab, Ruplizumab, Satumomab pendetide Pendetide, Sevirumab, Sibrotuzumab, Sifalimumab, Siltuximab, Siplizumab, Solanezumab, Sonepcizumab, Sontuzumab, Stamulumab, Sulesomab, Tacatuzumab tetraxetan, Tadocizumab, Talizumab, Tanezumab, Taplitumomab paptox, Tefibazumab, Telimomabaritox, tenatumomab, teneliximab, teplizumab, ticilimumab (tremelimumab), tigatuzumab, tocilizumab (atlizumab), toralizumab, tositumomab, trastuzumab, tremelimumab, tucotuzumab These include celmoleukin, Tuvirumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vedolizumab, Veltuzumab, Vepalimomab, Visilizumab, Volociximab, Votumumab, Zalutumumab, Zanolimumab, Ziralimumab, and Zolimomab aritox.

[0064] Examples of infusion therapies or injectable therapeutic proteins include, for example, tocilizumab (Roche / Actemra®), alpha-1 antitrypsin (Kamada / AAT), Hematide® (Affymax and Takeda, synthetic peptide), albinterferon alfa-2b (Novartis / Zalbin™), Rhucin® (Pharming Group, C1 inhibitor replacement therapy), tesamorelin (Theratechnologies / Egrifta, synthetic growth hormone-releasing factor), ocrelizumab (Genentech, Roche and Biogen), belimumab (GlaxoSmithKline / Benlysta®), pegloticase (Savient Pharmaceuticals / Krystexxa™), taliglucerase alfa (Protalix / Uplyso), agalsidase alfa (Shire / Replagal®), velaglucerase alfa (Shire). Additional therapeutic proteins useful in accordance with aspects of the present invention will be apparent to those of skill in the art, and the present invention is not limited in this regard.

[0065] In certain embodiments, the antigen contained in the liposome composition of the first aspect is a viral antigen, particularly a viral vector, such as a viral transfer vector. Viral vectors have been used to transfer therapeutic polynucleotides into cells and are increasingly used in gene therapy and RNA- or DNA-based vaccines. Unfortunately, however, re-administration of gene therapy is limited by immunogenicity, which often causes severe toxicity. Therefore, enhancing tolerance to these gene vectors is of great interest and can be achieved by using the liposome composition of the present disclosure. Non-limiting viral vectors contemplated herein include retroviral vectors, lentiviral vectors, herpes simplex virus (HSV)-based vectors, adenovirus-based vectors, adeno-associated virus (AAV)-based vectors, and AAV-adenovirus chimeric vectors. The adenoviral vector may be selected from the group consisting of adenoviral vectors of subgroup A, subgroup B, subgroup C, subgroup D, subgroup E, and subgroup F. The lentiviral vector may be selected from the group consisting of HIV, SIV, FIV, EIAV, and ovine lentiviral vectors. The adeno-associated viral vector may be selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, and AAV11 adeno-associated viral vectors. In some embodiments, the viral vector is a chimeric viral vector, such as an AAV-adenoviral vector. In certain embodiments, the antigen is a viral component, particularly a viral protein. In a specific embodiment, the antigen is an envelope protein. In a very specific embodiment, the antigen is a viral capsid component, for example, a viral capsid protein such as capsid proteins VP1, VP2, and VP3. In a preferred embodiment, when the antigen is a viral antigen, the viral antigen is selected from the group consisting of capsid proteins VP1, VP2, VP3, and combinations thereof.

[0066] As will be apparent to one of skill in the art, immunogenic fragments of any of the above antigenic proteins are also contemplated as antigens within the meaning of the present disclosure.

[0067] It is known that some oxidative and non-oxidative post-translational modifications can generate neoantigens from original antigens. Thus, in one embodiment, the antigen is a neoantigen. Examples of non-oxidative and oxidative post-translational modifications that can generate neoantigens include citrullination, glycosylation, sumoylation, neddylation, deamination, deamidation, hydroxylation, sulfation, oxidation, carbamylation, pegylation, succinylation, alkylation, sialylation, phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, arginylation, amidation, tyrosination, and lipidation. In other embodiments, the antigen may be a post-translationally modified antigen. Thus, in another embodiment, the antigen is a post-translationally modified antigen of any of the previous embodiments.

[0068] In a further embodiment, the antigen in the liposome composition of the first aspect is provided in the form of a nucleic acid encoding a peptide, polypeptide, or protein. The nucleic acid can be DNA or RNA, such as mRNA. In embodiments, the nucleic acid encoding the antigenic polypeptide is contained within an expression vector that can be transcribed when transfected into a cell. In some embodiments, the expression vector can include, inter alia, a plasmid, a viral particle, a retrovirus, or an adenovirus. For example, the antigen can be a polynucleotide encoding a viral antigen, such as a viral capsid antigenic protein, such as viral capsid protein VP1, VP2, or VP3.

[0069] Preparation of liposome compositions A variety of methods well known to those skilled in the art can be used to prepare the liposome composition of the first aspect of the present disclosure.

[0070] Antigen-carrying liposomes can be formed by directly entrapping the autoantigen during liposome formation using well-known methods such as the lipid thin film hydration method and the solvent injection method. In one embodiment, liposomes are prepared by the thin film hydration method (Bangham et al., J. Mol. Biol., 13, 238 (1965)). In another embodiment, liposomes are prepared by the solvent injection method (Pons et al., International Journal of Pharmaceutics 95 (1993) 51-56).

[0071] In one embodiment, liposomes carrying an antigen may be prepared by a process comprising: (a) preparing a lipid blend in a suitable solvent, e.g., chloroform; (b) removing the solvent, e.g., by evaporation under vacuum; and (c) hydrating the lipid blend with a suitable buffer, e.g., phosphate buffered saline, containing the antigen to obtain antigen-containing liposomes.

[0072] In another embodiment, liposomes carrying an antigen may be prepared by a process comprising: (a) preparing a lipid blend in a suitable solvent, e.g., ethanol; and (b) injecting the lipid blend into a solution containing at least one antigen and a suitable buffer, e.g., phosphate buffered saline, to obtain antigen-containing liposomes.

[0073] If the liposomes carry multiple antigens, the hydration step (c) or the injection step (b) is carried out in the presence of a buffer containing a mixture of said antigens in the desired ratio, such ratio taking into account the specific encapsulation efficiency for each antigen.

[0074] In some embodiments, the weight ratio of the total amount of lipids forming the liposome membrane to the total amount of antigen is 1000:1 to 1:1, or 500:1 to 2:1, or 350:1 to 10:1, or 250:1 to 15:1, or 210:1 to 20:1, or 190:1 to 30:1. In another specific embodiment, the weight ratio of the total amount of lipids forming the liposome membrane to the total amount of antigen is 350:1 to 10:1. In even more specific embodiments, the weight ratio of the total amount of lipids forming the liposome membrane to the total amount of antigen is 250:1 to 20:1. In even more specific embodiments, the weight ratio of the total amount of lipids forming the liposome membrane to the total amount of antigen is 210:1 to 25:1. In even more specific embodiments, the weight ratio of the total amount of lipids forming the liposome membrane to the total amount of antigen is 190:1 to 30:1.

[0075] Other methods known in the art can also be used to obtain liposomes carrying antigens.For example, some embodiments contemplate obtaining liposomes first, and then including antigens.There are well-known methods in the prior art for including compounds in liposomes (see Maurer N. et al., Expert Opin Biol Ther, 2001, vol.1(6), p.923-47; Waterhouse D. et al., Methods Enzymol., 2005; vol.391, p.40-57; Urban P. et al., Nanosc.Res.Lett., 2011, vol.6, p.620).

[0076] The resulting antigen-containing liposomes obtained as described above or by any other method known to those skilled in the art can be subjected to further purification, homogenization, and / or separation steps. In most embodiments, a purification step is applied to remove unencapsulated peptides. The purification step can be carried out, for example, by centrifugation, filtration, tangential flow filtration, dialysis, gel permeation chromatography, ion exchange chromatography, size exclusion chromatography, etc. For example, a purification step to remove unencapsulated antigens can be carried out by filtering through a 100 KDa filter. The antigen-containing liposomes can be further homogenized or separated by size. Extrusion can be used to size-homogenize liposomes, i.e., generate liposomes with a predetermined average size by passing the liposomes through a filter with specific pores under pressure. Filtration, for example, tangential flow filtration, can also be used to purify and separate liposomes according to their size, i.e., generate a population of liposomes with a desired size distribution and fewer impurities. Other methods by which liposome populations can be separated according to their size are centrifugation (eg, ultracentrifugation), size exclusion chromatography, gel permeation chromatography, and combinations thereof.

[0077] In one embodiment, the liposome composition of the first aspect is prepared by a process that includes obtaining antigen-carrying liposomes by any of the above methods and, optionally, further purification, separation, and / or enrichment steps. In certain embodiments, the process includes a purification step to remove non-encapsulated peptides. In another embodiment, the process further includes a homogenization step. In another embodiment, the process further includes a separation step. In some embodiments, the process further includes a enrichment step. For example, a portion of the composition obtained by any of the above methods may be subjected to separation to separate a liposome population, e.g., a liposome population with a size of less than 200 nm. The separate liposome population may then be added to a composition containing the two populations, thereby resulting in enrichment of the selected liposome population, e.g., a liposome population with a size of less than 200 nm. In other embodiments, extrusion, physical separation, or sonication is used to size-homogenize liposomes or to obtain a desired liposome population. The homogenized liposomes or distinct liposome populations may then be added to a liposome composition containing the population of interest defined in the first aspect, thereby enriching the composition for the particular liposome population.

[0078] The composition of the first aspect of the present disclosure may be obtained by separately preparing each of the two liposome populations defined in the first aspect and then mixing them in appropriate proportions. The composition may also be obtained by a method that results in a composition already comprising the two populations defined in the first aspect.

[0079] In one embodiment, the liposome composition of the first aspect can be prepared by a process including: (a) obtaining liposomes carrying an antigen, for example, by any of the methods described above; (b) isolating liposomes having a size of 2 to 200 nm; (c) isolating liposomes having a size of 500 to 2000 nm; and (d) mixing the liposomes having a size of 2 to 200 nm with liposomes having a size of 500 to 2000 nm.

[0080] In another embodiment, the liposome composition of the first aspect is prepared by a process comprising: (a) obtaining liposomes carrying an antigen, for example, by any of the methods described above; (b) subjecting a portion of the liposome composition obtained in (a) to a separation step to separate liposomes having a size of 2 to 200 nm; and (c) adding the separated liposomes having a size of 2 to 200 nm to the liposome composition obtained in (a) to obtain a liposome composition enriched in liposomes having a size of 2 to 200 nm.

[0081] The present disclosure contemplates liposome compositions obtainable by any of the above methods.

[0082] In certain embodiments, liposome compositions with broad size polydispersity and carrying antigens can be prepared by solvent injection, a process that involves preparing a lipid blend in an appropriate solvent, such as ethanol, and injecting the lipid blend into a solution containing at least one antigen and an appropriate buffer, such as phosphate-buffered saline, to obtain antigen-containing liposomes. Instead of vortexing or high-speed stirring, a low injection speed, a specific number of injections, and gentle shaking promote the formation of large-sized liposomes. By not applying a subsequent homogenization step, such as extrusion under pressure, and not applying any further purification steps to remove small liposomes, such as tangential flow filtration, the liposome composition exhibits a broad size distribution. To reduce the average diameter of the liposome composition, several successive extrusion cycles can be applied to the heterogeneous polydisperse composition. To achieve this, liposomes are sequentially filtered through a series of polycarbonate membranes with gradually decreasing pore diameters, thereby conveniently and reproducibly obtaining liposomes with an average size close to the membrane pore size. Furthermore, applying short-term sonication to the formulation, either directly or indirectly using a tip in a bath sonicator at room temperature, also reduces the average diameter of the liposomes. Other industrial homogenization methods for disrupting large liposomes include microfluidization, high-pressure homogenization, and shear-induced homogenization techniques. In one embodiment, the present disclosure also contemplates a liposome composition obtainable by this particular method.

[0083] formulation In one embodiment, the liposomal composition of the first aspect is a pharmaceutical composition. Accordingly, the present disclosure provides a pharmaceutical or veterinary composition comprising a therapeutically effective amount of the liposomal composition defined in the first aspect, optionally together with other suitable pharmaceutically or veterinarily acceptable excipients or carriers. In another embodiment, the present disclosure provides a pharmaceutical or veterinary composition consisting essentially of a therapeutically effective amount of the liposomal composition defined in the first aspect and a suitable pharmaceutically or veterinarily acceptable excipient or carrier. In another embodiment, the present disclosure provides a pharmaceutical or veterinary composition consisting of a therapeutically effective amount of the liposomal composition defined in the first aspect and a suitable pharmaceutically or veterinarily acceptable excipient or carrier.

[0084] As used herein, the term "therapeutically effective amount" refers to an amount of liposomes, particularly the amount of liposomes of the first and second populations of the composition of the first aspect, sufficient to prevent or alleviate to some extent one or more symptoms of the disorder being addressed when administered. The specific dose of the compound administered in accordance with the present disclosure will, of course, be determined by the specific circumstances surrounding the case, including the antigen administered, the route of administration, the specific symptoms being treated, and similar considerations. In one embodiment, a therapeutically effective amount within the meaning of the present disclosure involves reducing the level of an unwanted immune response. In another embodiment, a therapeutically effective amount involves completely preventing an unwanted immune response. In another embodiment, a therapeutically effective amount involves delaying the onset of an unwanted immune response. A therapeutically effective amount within the meaning of the present disclosure results in a tolerogenic immune response to the antigen in the subject. Achievement of any of the foregoing can be monitored by routine methods.

[0085] The present disclosure also contemplates compositions in which the liposomes carry multiple antigens, and compositions comprising different liposomes each carrying a different antigen. Preferably, all of the antigens contained in the composition of the first aspect are associated with the same immunological disorder.

[0086] In this disclosure, the term "pharmaceutically acceptable excipient or carrier" refers to a pharmaceutically acceptable material, composition, or vehicle. Each component must be pharmaceutically acceptable in the sense of being compatible with the other components of the pharmaceutical composition. It must also be suitable for use in contact with the tissues or organs of humans and animals without excessive toxicity, irritation, allergic response, antigenicity, or other problems or complications commensurate with a reasonable benefit / risk ratio. Similarly, the term "veterinarily acceptable" means suitable for use in contact with non-human animals.

[0087] The antigen-containing liposomes of the compositions of the present invention are considered to be active agents themselves, and therefore, antigen-containing liposomes of a certain size range play a role in promoting tolerance to the specifically contained antigen. Therefore, it is noteworthy that no other active agent is required to induce tolerance. Importantly, the compositions of the present invention do not need to be used with other immunomodulatory agents. In particular, the compositions do not need to contain or be used in combination with immunosuppressants. In certain embodiments, the compositions of the first aspect do not contain immunosuppressants. Otherwise, the compositions of the first aspect are described as lacking immunosuppressants.

[0088] Nevertheless, the present disclosure also contemplates combining the liposome compositions described herein with other active agents, even if this is not required to achieve the technical effect. Thus, the pharmaceutical compositions contemplated herein may contain additional active agents, such as other immunomodulatory agents, e.g., immunosuppressants. Examples of immunosuppressants include glucocorticoids (prednisolone, methylprednisone, betamethasone), cytostatics (methotrexate...), antibodies (rituximab...), drugs against immunophilins (cyclosporine, tacrolimus, sirolimus, everolimus...), interferons, TNF-binding proteins, mycophenolic acid, and small biological agents (fingolimod, myriocin).

[0089] The formulation of the compositions of the present disclosure largely depends on the route of administration. In one embodiment, the pharmaceutical composition is administered orally to a patient. Oral compositions include tablets, powders, capsules, sachets, and liquid syrups, suspensions, and elixirs, all of which can be formulated by methods well known in the art. The compositions of the present disclosure can also be administered to a patient via intravenous, intraarterial, intraperitoneal (ip), subcutaneous, intramuscular, or intradermal routes. Compositions suitable for these administration routes are also well known in the art, including solutions for injection, solutions for irrigation, powders for reconstitution of liquid injections, and pre-filled syringes. In the context of the present disclosure, the compositions disclosed herein may also be suitable for intranasal or inhalation administration, rectal administration, or topical administration, for example, in the form of creams, gels, ointments, or skin patches. Methods for preparing these formulations are well known in the art. Furthermore, the compositions of the present disclosure can be formulated as controlled-release dosage forms. Controlled-release dosage forms are known in the art and are particularly desirable for the treatment of chronic diseases or for the administration of active agents that may be toxic at high doses or that exhibit poor half-life patterns when administered to a patient.

[0090] Also disclosed herein is a kit of parts comprising: (a) a liposomal composition as defined above, optionally together with a pharmaceutically acceptable excipient or carrier; (b) optionally, a further active agent, and (c) optionally, instructions for its use.

[0091] Also disclosed herein is a container or injection device containing a liposome composition as defined above, preferably together with a pharmaceutically acceptable excipient or carrier.

[0092] therapeutic use As described above, the liposome composition of the present disclosure can be used therapeutically to promote tolerance to the antigen contained in the composition. In one embodiment, the induction of tolerance comprises B cell-mediated tolerance. In another embodiment, the induction of tolerance comprises T cell-mediated tolerance, particularly tolerance mediated through tolerogenic presentation of the antigen by dendritic cells. When referring to the medical use of the liposome composition of the present disclosure, it is intended that the composition may be a pharmaceutical composition comprising a therapeutically effective amount of the liposome composition defined in the first aspect, optionally together with other suitable pharmaceutically or veterinarily acceptable excipients or carriers.

[0093] The liposome composition of the present disclosure contains two liposome populations of different sizes, both containing an antigen a with a specific membrane composition, as broadly described above. These liposome populations comprise an active ingredient that induces tolerance and is effective in treating symptoms associated with a dysfunctional, e.g., exacerbated, immune response, without the need for additional active ingredients such as immunosuppressants. Each liposome population targets a different mechanism of immune tolerogenesis. The liposome population of 500 nm or larger induces antigen-specific tolerance through a mechanism that resembles efferocytosis and involves tolerogenic presentation of antigens by dendritic cells, whereas the liposome population of 2-200 nm can induce tolerance via B cells. This dual effect has not been previously disclosed and provides improved tolerogenic effects. Furthermore, due to this dual effect, the liposome composition disclosed herein is effective in treating immune disorders that could not be treated with previous tolerance inducers.

[0094] The liposome composition defined above is for use in immunomodulation, more particularly for suppressing excessive immune responses to specific antigens. In some embodiments, the liposome composition defined above is for use in treating disorders associated with an aberrant, mostly excessive, immune response. It is important that the tolerogenic / immunomodulatory effect of the liposome composition is antigen-specific. Thus, in certain embodiments, the liposome composition defined above is for use in treating disorders associated with an aberrant, mostly excessive, immune response, and the liposome composition restores tolerance to the antigen contained in the liposome composition.

[0095] In one embodiment, the disorder associated with an aberrant, mostly excessive, immune response is selected from autoimmune diseases, allergies, drug hypersensitivity, and transplant rejection.

[0096] In the sense of the present disclosure, the term "treatment" includes preventative treatment before the clinical onset of symptoms caused by an immune disorder, or therapeutic treatment after the clinical onset of symptoms caused by an immune disorder. In certain embodiments, the treatment is preventative treatment. In one embodiment, preventative treatment includes partial or complete prevention of an immune disorder. In one embodiment, an aberrant immune response to an autoantigen is prevented, thereby preventing the pathogenic events underlying the aberrant immune response. In another specific embodiment, the treatment includes improving, slowing, stopping, or reversing the pathological mechanisms underlying the immune disorder. In certain embodiments, the treatment includes improving, slowing, stopping, delaying, or reversing the clinical symptoms of the immune disorder.

[0097] The present disclosure contemplates treating immune conditions in patients who have an abnormal immune response and some tissue damage but who show no or few clinical symptoms of disease. This stage is often referred to as the "preclinical" stage, e.g., in T1D, transplant rejection, and many autoimmune diseases, and is referred to as prediabetes. In prediabetes, pancreatic B cells are damaged to some extent, but only some of the diagnostic criteria for diabetes are met. Diseases at this preclinical stage can be effectively treated with the liposomal compositions of the present disclosure. Thus, one embodiment relates to the treatment of autoimmune diseases during the preclinical stage. Furthermore, by administering an effective amount of the liposomal composition of the present disclosure, advanced stages of immune diseases, in which tissue damage is severe and clinical symptoms are evident, can also be effectively treated.

[0098] In certain embodiments, the disorder is T1D, lupus erythematosus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, reactive arthritis, psoriatic arthritis), multiple sclerosis (MS), neuromyelitis optica, Addison's disease, celiac disease, dermatomyositis, Hashimoto's thyroiditis, myasthenia gravis, pernicious anemia, autoimmune hemolytic anemia, autoimmune neutropenia, Graves' disease, psoriasis, Sjogren's syndrome including keratoconjunctivitis sicca secondary to Sjogren's syndrome, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), alopecia areata, cloaca, ulcerative colitis ... The autoimmune disease is selected from the group consisting of: Crohn's disease, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, true erythrocytic anemia, idiopathic thrombocytopenic purpura, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, sarcoidosis, primary biliary cirrhosis, posterior uveitis, and interstitial pulmonary fibrosis.

[0099] In certain embodiments, the present disclosure provides a liposome composition defined in the first aspect or a pharmaceutical composition thereof for use in treating MS. In another embodiment, the autoimmune disease being treated is T1D. In another embodiment, the liposome composition defined in the first aspect or a pharmaceutical composition thereof is for use in treating T1D in a prediabetic subject. In another specific embodiment, the autoimmune disease being treated is myasthenia gravis. In another specific embodiment, the autoimmune disease being treated is selected from the group consisting of rheumatoid arthritis, neuromyelitis optica, myositis or antisynthetase syndrome, thrombotic thrombocytopenic purpura, and celiac disease.

[0100] In another specific embodiment, the disorder is an allergy or allergic condition. "Allergy" or "allergic condition" includes, but is not limited to, allergic asthma, hay fever, hives, eczema, plant allergies, bee sting allergies, pet allergies, latex allergies, mold allergies, cosmetic allergies, food allergies, allergic rhinitis or coryza, topical allergic reactions, anaphylaxis, atopic dermatitis, hypersensitivity reactions, and other allergic conditions. In some embodiments, the allergy is a food allergy. Food allergies include, but are not limited to, milk allergies, egg allergies, tree nuts allergies, fish allergies, shellfish allergies, soy allergies, or wheat allergies.

[0101] Undesirable immunogenicity may also occur in response to a therapeutic drug. Thus, in another specific embodiment, the disorder is drug hypersensitivity. "Drug hypersensitivity" refers to an immune-mediated reaction to a drug. Symptoms range from mild to severe and include rash, anaphylaxis, and serum sickness. Drugs that cause hypersensitivity are not particularly limited in the sense of the present disclosure.

[0102] Increasing efforts are being made to reduce the immunogenicity of life-saving gene therapy vectors. Harmful immune responses to gene therapy vectors can often cause severe toxicity and the formation of neutralizing antibodies, which can prevent necessary re-administration. Thus, in one embodiment, the liposome composition is for use in the prevention or treatment of adverse effects caused by gene therapy. In another specific embodiment, the liposome composition is for use in the prevention or treatment of adverse effects caused by viral vectors, particularly viral transfer vectors. The present disclosure also contemplates the liposome composition disclosed herein for use in the prevention or treatment of adverse effects caused by any drug, such as a therapeutic protein.

[0103] In another specific embodiment, the disorder is transplant rejection. "Transplant rejection" occurs when transplanted tissue is rejected by the recipient's immune system, resulting in the destruction of the transplanted tissue. "Transplant" and its variants refer to the insertion of a transplant (also called a graft) into a recipient, regardless of whether the transplant is syngeneic (when the donor and recipient are genetically identical), allogeneic (when the donor and recipient are of the same species but have different genetic origins), or xenogeneic (when the donor and recipient are from different species). The term "allograft" or "allograft" or "allotransplant" refers to transplanted tissue, e.g., an organ, from a donor of a different genetic origin but the same species as the recipient. For example, an allograft can be a solid organ, particularly a kidney, but can also be a lung, heart, pancreas, liver, etc. An allograft can be any other type of tissue, such as skin, bone, muscle, vascular tissue, cartilage, etc. As will be apparent to those skilled in the art, the allografts encompassed may also be organ tissues such as kidney tissue, lung tissue, heart tissue, liver tissue, etc. As used herein, the term "transplant rejection" or "graft rejection" encompasses both acute and chronic transplant rejection and refers to the rejection of transplanted tissue by the recipient's immune system. The present disclosure contemplates any type of transplant rejection for any type of transplant. In one particular embodiment, the immune disorder is graft-versus-host disease.

[0104] The particular dose of liposomal composition administered in accordance with the present disclosure may be determined by the particular circumstances surrounding the case, including the antigen administered, the route of administration, the particular condition being treated, and similar considerations. Additionally, the clinical stage of the immune disorder being treated may also need to be considered to determine the appropriate dose of liposomal composition to be administered.

[0105] In summary, the dose of liposome composition to be administered is determined by several considerations. In some embodiments, the dose is calculated in terms of the amount of liposome per kg of body weight (mg liposome / kg body weight). In one embodiment, the dose ranges from 0.025 to 50 mg liposome / kg body weight, particularly 0.25 to 10 mg liposome / kg body weight.

[0106] Furthermore, a medical professional will determine how many doses of a pharmaceutical agent will be administered to a subject in need thereof to treat an immune disorder. In this regard, it is noteworthy that the liposome composition therapy developed by the present inventors is not permanently required. Instead, administration of the liposome composition of the present disclosure achieves long-term restoration of tolerance, resulting in effective treatment of the immune disorder. This is in contrast to known immunomodulatory or anti-inflammatory treatments for immune disorders, which are generally lifelong. A long-lasting immune tolerogenic effect can be achieved after a single administration of the liposome composition of the present disclosure, or alternatively, after no more than two to four administrations. However, a medical professional may determine that more doses are needed to treat an advanced stage of the disease or for some other reason. In one embodiment, treatment involves administering 1 to 10 doses, e.g., 2, 3, 4, 5, 6, 7, or 8 doses, of the liposome composition to a subject.

[0107] In one embodiment, the subject is in need of antigen-specific tolerance. In another embodiment, the subject has an autoimmune disease, an inflammatory disease, an allergy, graft-versus-host disease, organ or tissue rejection, or has undergone or will undergo a transplant. In another embodiment, the subject has been administered, is being administered, or will be administered a therapeutic protein that has experienced, is experiencing, is experiencing, or is predicted to experience an unwanted immune response.

[0108] As already mentioned above, the present disclosure does not exclude the combination of the liposome composition described herein with other active agents.Therefore, in one embodiment, the liposome composition for any of the above-defined uses is for use in combination with additional active agents.The liposome composition and the additional active agent can be administered in the same composition or different compositions, and in the latter case, they can be administered sequentially, simultaneously, or within a therapeutic interval.In certain embodiments, the additional active agent is another immunomodulator, for example, an immunosuppressant.

[0109] For the sake of completeness, the present specification also discloses the following numbered embodiments: 1. A composition comprising two populations of liposomes, the first population of liposomes has a size of 200 nm or less; the second population of liposomes has a size of 500 nm or greater; the first and second populations of liposomes carry one or more antigens; A composition, wherein the liposome membrane of each liposome in the first and second liposome populations comprises phosphatidylserine in an amount ranging from 20 to 60% by weight relative to the total composition of the liposome membrane.

[0110] 2. A composition consisting essentially of two populations of liposomes, the first population of liposomes has a size of 200 nm or less; the second population of liposomes has a size of 500 nm or greater; the first and second populations of liposomes carry one or more antigens; A composition, wherein the liposome membrane of each liposome in the first and second liposome populations comprises phosphatidylserine in an amount ranging from 20 to 60% by weight relative to the total composition of the liposome membrane.

[0111] 3. The first population of liposomes has a size in the range of 2 to 200 nm; the second population of liposomes has a size in the range of 500 to 2000 nm; A composition according to any one of the preceding embodiments,

[0112] 4. A composition according to any one of the preceding embodiments, wherein 15-75% of the liposomes in the composition correspond to the first population.

[0113] 5. A composition according to the preceding embodiment, wherein 15-55% of the liposomes in the composition correspond to the first population.

[0114] 6. A composition according to the preceding embodiment, wherein 20-50% of the liposomes in the composition correspond to the first population.

[0115] 7. A composition according to any one of the preceding embodiments, wherein between 2 and 40% of the liposomes in the composition correspond to the second population.

[0116] 8. A composition according to the preceding embodiment, wherein 2-25% of the liposomes in the composition correspond to the second population.

[0117] 9. A composition according to the preceding embodiment, wherein 2-15% of the liposomes in the composition correspond to the second population.

[0118] 10. A composition according to any one of the preceding embodiments, wherein the amount of phosphatidylserine in the liposome membrane is 35-45% by weight relative to the total composition of the liposome membrane.

[0119] 11. The composition according to the preceding embodiment, wherein the amount of phosphatidylserine in the liposome membrane is 35 to 45% by weight, based on the total composition of the liposome membrane, for example, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43% or 44% by weight, based on the total composition of the liposome membrane.

[0120] 12. The composition according to any one of the preceding embodiments, wherein the PS is selected from the group consisting of 1,2-dioleoyl-phosphatidylserine, 1,2-dioleoyl-sn-glycero 3-phospho-L-serine (DOPS), 1,2-dipalmitoyl-phosphatidylserine, 1,2-dimyristoyl-phosphatidylserine, 1,2-distearoyl-phosphatidylserine, l-oleoyl-2-palmitoyl-phosphatidylserine, l-oleoyl-2-stearoyl-phosphatidylserine, 1-palmitoyl-2-oleoyl-phosphatidylserine, l-stearoyl-2-oleoyl-phosphatidylserine, and combinations thereof.

[0121] 13. The composition according to any one of the preceding embodiments, wherein the liposome membrane further comprises phosphatidylcholine (PC).

[0122] 14. A composition according to the preceding embodiment, wherein the amount of PC in the liposome membrane is 20-50% by weight relative to the total composition of the liposome membrane.

[0123] 15. The composition according to the preceding embodiment, wherein the amount of phosphatidylserine in the liposome membrane is 32 to 42% by weight relative to the total composition of the liposome membrane, for example, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40% or 41% by weight relative to the total composition of the liposome membrane.

[0124] 16. The composition according to any one of embodiments 13-15, wherein the PC is selected from the group consisting of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, 1,2-dilinoleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-linoleoyl-sn-glycero-3-phosphocholine, 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine, and combinations thereof.

[0125] 17. A composition according to any one of the previous embodiments, wherein the liposome membrane further comprises a sterol lipid or a steroid, in particular cholesterol (CHOL).

[0126] 18. A composition according to the preceding embodiment, wherein the amount of CHOL in the liposome membrane is 10 to 40% by weight relative to the total composition of the liposome membrane.

[0127] 19. The composition according to the preceding embodiments, wherein the amount of CHOL in the liposome membrane is 20-35% by weight relative to the total liposome membrane composition, or 20-30% by weight relative to the total liposome membrane composition, for example, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28% or 29% by weight relative to the total liposome membrane composition.

[0128] 20. A composition according to any one of the preceding embodiments, wherein the liposome membrane comprises PS, PC and CHOL.

[0129] 21. The composition according to the preceding embodiment, wherein the liposome membrane consists essentially of PS, PC and CHOL.

[0130] 22. The composition according to any one of embodiments 1 to 21, wherein the molar ratio PS:PC:CHOL is 1:(0.2-4):(0.2-5).

[0131] 23. A composition according to the preceding embodiment, wherein the molar ratio PS:PC:CHOL is 1:(0.6-1.8):(0.7-2.5), in particular 1:(0.7-1.5):(0.9-2).

[0132] 24. A composition according to the preceding embodiment, wherein the molar ratio PS:PC:CHOL is 1:(0.8-1.4):(1.1-1.9), in particular 1:(0.9-1.3):(1.2-1.7).

[0133] 25. A composition according to any one of the preceding embodiments, wherein the first population of liposomes provides 5-30% by weight of the total liposomal PS in the composition, particularly 5-20% by weight of the total liposomal PS in the composition.

[0134] 26. A composition according to any one of the preceding embodiments, wherein the second population of liposomes provides 5-75% of the total liposomal PS in the composition, particularly 5-55% by weight of the total liposomal PS in the composition.

[0135] 27. A composition according to any one of the preceding embodiments, wherein the liposome contains one type of antigen.

[0136] 28. A composition according to any one of the preceding embodiments, wherein the liposome contains multiple types of antigens.

[0137] 29. The composition according to any one of the preceding embodiments, wherein the antigen is a peptide.

[0138] 30. The composition according to the preceding embodiment, wherein the antigenic peptide has a size of 5 to 1000 amino acids.

[0139] 31. The composition according to the preceding embodiment, wherein the antigenic peptide has a size of 5 to 200 amino acids, e.g., 15 to 100 amino acids.

[0140] 32. The composition according to any one of the preceding embodiments, wherein the antigen is an autoantigen.

[0141] 33. Autoantigens associated with type 1 diabetes (T1D), lupus erythematosus, rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, reactive arthritis, psoriatic arthritis, multiple sclerosis, neuromyelitis optica, Addison's disease, celiac disease, dermatomyositis, Hashimoto's thyroiditis, myasthenia gravis, pernicious anemia, autoimmune hemolytic anemia, autoimmune neutropenia, Graves' disease, psoriasis, Sjögren's syndrome including keratoconjunctivitis sicca secondary to Sjögren's syndrome, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), alopecia areata, Crohn's disease, aphthous ulcers, iris urinary tract infections, conjunctivitis, keratoconjunctivitis, ulcerative colitis, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, true erythrocytic anemia, idiopathic thrombocytopenic purpura, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, sarcoidosis, primary biliary cirrhosis, posterior uveitis, and interstitial pulmonary fibrosis.

[0142] 34. Autoantigens include insulin, proinsulin, protein tyrosine phosphatase (IA2), glutamic acid decarboxylase (GAD), chromogranin and islet-glucose-6-phosphatase catalytic subunit-related protein (IGRP), peripherin, myelin, myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), myelin proteolipid protein (PLP), GDP-l-fucose synthase, acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), agrin, lipoprotein-related protein 4 (LRP4), cortactin, transglutaminase, deamidated gliadin, thyroglobulin, collagen (e.g., type 11 collagen), and human cartilage protein (GP). 39, chromogranin A, gp130-RAPS, vimentin, citrullinated vimentin, ADAMST13, aquaporin-4, proteolipid protein, fibrillarin, nuclear proteins, nucleolar proteins (e.g., small nucleolar proteins), histidyl-tRNA synthetase (HisRS), histidine-tRNA synthetase (HARS1), jo-1, thyroid-stimulating factor receptor, histones, glycoprotein gp 70, ribosomal proteins, pyruvate dehydrogenase dehydrolipoamide acetyltransferase, hair follicle antigen, human tropomyosin isoform 5, mitochondrial proteins, pancreatic beta cell proteins, gluten, and antigenic fragments or derivatives of any of the above, in particular the autoantigen is selected from the group consisting of MOG, PLP, MBP, preproinsulin, insulin, peripherin, acetylcholine receptor, MUSK, citrullinated vimentin, collagen, deamidated gliadin, ADAMST13, aquaporin 4, transglutaminase, histidyl-tRNA synthetase (HisRS), and antigenic fragments or derivatives thereof, for example, antigenic fragments selected from those disclosed in Table 3.

[0143] 35. The composition according to any one of embodiments 32-33, wherein the autoantigen is selected from one of the antigens disclosed in Table 1, one of the antigens disclosed in Table 2, and an antigenic fragment or derivative of an antigen of Table 1 or Table 2.

[0144] 36. The composition according to any one of embodiments 1-31, wherein the antigen is a drug comprising a therapeutic protein.

[0145] 37. The composition according to any one of embodiments 1-31, wherein the antigen is an allergen.

[0146] 38. The composition according to any one of embodiments 1-31, wherein the antigen is an alloantigen.

[0147] 39. The composition according to any one of embodiments 1 to 31, wherein the antigen is a viral antigen, in particular a viral vector, more particularly, the antigen is a viral envelope protein or a viral capsid protein selected from, for example, VP1, VP2 or VP3.

[0148] 40. The composition according to any one of embodiments 36-39, wherein the antigen is a protein fragment, preferably an immunogenic fragment.

[0149] 41. The composition according to any one of the previous embodiments, wherein the antigen is a polynucleotide encoding an antigenic protein or a fragment thereof.

[0150] 42. A composition according to any one of the preceding embodiments, which does not contain an immunosuppressant.

[0151] 43. (a) preparing a lipid blend in a suitable solvent; and (b) injecting the lipid blend into a solution containing at least one antigen and a suitable buffer. 10. A method of preparing a liposome composition as defined in any one of the preceding embodiments, comprising:

[0152] 44. The method according to embodiment 43, wherein the lipid blend comprises PS, PC and CHOL.

[0153] 45. The method according to any one of embodiments 43-44, wherein the molar ratio PS:PC:CHOL is in the range from 1:(0.8-1.4):(1.1-1.9), in particular in the range from 1:(0.9-1.3):(1.2-1.7).

[0154] 46. ​​The method according to any one of embodiments 43-45, wherein the solvent in step (a) is ethanol.

[0155] 47. The method according to any one of embodiments 43-46, wherein the buffer in step (b) is phosphate buffered saline.

[0156] 48. The method according to any one of embodiments 43-47, wherein step (b) comprises slow injection and gentle shaking.

[0157] 49. The method according to any one of embodiments 43 to 48, further comprising a separation step selected from filtration, e.g., tangential flow filtration, centrifugation (e.g., ultracentrifugation), size extrusion chromatography, gel permeation chromatography, and combinations thereof.

[0158] 50. A liposome composition obtainable by a method defined in any one of embodiments 43 to 49.

[0159] 51. The composition according to any one of embodiments 1-42 or 50, which is a pharmaceutical composition and comprises pharmaceutically acceptable excipients and carriers.

[0160] 52. The composition according to the previous embodiment, which is for intravenous, intraarterial, intraperitoneal (ip), subcutaneous, intramuscular or intradermal administration.

[0161] 53. The composition according to any one of embodiments 51-52, further comprising an additional active ingredient.

[0162] 54. (a) a liposome composition according to any one of embodiments 1-42 or 50-53; (b) optionally, a further active ingredient; and (c) optionally, instructions for its use and Parts kit including.

[0163] 55. The composition defined in any one of embodiments 1-42 or 50-53, for use as a medicament.

[0164] 56. The composition defined in any one of embodiments 1-42 or 50-53, for use in inducing tolerance to an antigen.

[0165] 57. The composition for use according to embodiment 56, wherein the induction of tolerance comprises B cell-mediated tolerance and T cell-mediated tolerance to the antigen.

[0166] 58. The composition defined in any one of embodiments 1-42 or 50-53, for use in immunomodulation.

[0167] 59. The composition defined in any one of embodiments 1-42 or 50-53, for use in suppressing an excessive immune response.

[0168] 60. The composition defined in any one of embodiments 1-42 or 50-53, for use in treating a disorder associated with an abnormal immune response, wherein the liposomal composition restores tolerance to an antigen contained in the liposomal composition.

[0169] 61. The composition defined in any one of embodiments 1-42 or 50-53, for use in treating an autoimmune disease.

[0170] 62. T1D, lupus erythematosus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, reactive arthritis, and psoriatic arthritis), multiple sclerosis, neuromyelitis optica, Addison's disease, celiac disease, dermatomyositis, Hashimoto's thyroiditis, myasthenia gravis, pernicious anemia, autoimmune hemolytic anemia, autoimmune neutropenia, Graves' disease, psoriasis, Sjogren's syndrome, including keratoconjunctivitis sicca secondary to Sjogren's syndrome, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), alopecia areata, Crohn's disease, aphthous ulcers, iritis, and conjunctivitis. an autoimmune disease selected from the group consisting of: ulcerative colitis, urinary tract infections, keratoconjunctivitis, ulcerative colitis, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, true erythrocytic anemia, idiopathic thrombocytopenic purpura, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, sarcoidosis, primary biliary cirrhosis, posterior uveitis, and interstitial pulmonary fibrosis;

[0171] 63. An autoimmune disease selected from the group consisting of T1D, multiple sclerosis, rheumatoid arthritis, neuromyelitis optica, myasthenia gravis, myositis or antisynthetase syndrome, thrombotic thrombocytopenic purpura and celiac disease, a composition for use according to the preceding embodiments.

[0172] 64. The composition defined in any one of embodiments 1-42 or 50-53, for use in treating allergies.

[0173] 65. The composition defined in any one of embodiments 1-42 or 50-53, for use in treating a hypersensitivity reaction, e.g., drug hypersensitivity.

[0174] 66. The composition defined in any one of embodiments 1-42 or 50-53, for use in treating transplant rejection.

[0175] 67. The composition defined in the preceding embodiment, wherein the transplant rejection is graft-versus-host disease.

[0176] 68. The composition defined in any one of embodiments 1-42 or 50-53, for use in treating adverse immune effects caused by gene therapy.

[0177] 69. A composition according to any one of embodiments 1-42 or 50-53, for use in combination with a further active ingredient.

[0178] 70. A composition for combination use according to the preceding embodiments, wherein the composition defined in any one of embodiments 1 to 42 or 50 to 53 and the further active ingredient are administered sequentially, simultaneously or within a therapeutic interval.

[0179] 71. A composition for the combination according to any one of embodiments 69-70, wherein the further active ingredient is not an immunosuppressant.

[0180] 72. A composition for use according to any one of embodiments 55 to 71, wherein the dose of the liposome composition is in the range of 0.25 to 50 mg liposomes / Kg body weight.

[0181] 73. A composition for use according to any one of embodiments 60 to 72, wherein the treatment is a prophylactic treatment.

[0182] Throughout the specification and claims, the word "comprise" and variations of this word are not intended to exclude other technical features, additives, ingredients, or steps. Furthermore, the word "comprise" encompasses the cases of "consisting of" and "consisting essentially of." Additional objects, advantages, and features of the present invention will become apparent to those skilled in the art upon examination of this specification or may be learned by practice of the present invention. The following examples and drawings are provided by way of illustration and are not intended to limit the present invention. Furthermore, the present invention covers all possible combinations of the specific preferred embodiments described herein. [Example]

[0183] 1. Materials and Methods 1.1. Liposome production Antigenic peptides (antigens) were selected based on their reported relevance in the development of autoimmune diseases. Peptides (human mutated citrullinated vimentin (MCV) (SEQ ID NO: 44) and MOG 35-55 (SEQ ID NO: 4) and human AChR 146-162 (SEQ ID NO: 57)) were purchased with a purity of over 95% from the Peptide Synthesis Facility (Department of Experimental and Health Sciences, Pompeu Fabra University, Spain). Human insulin (CAS reference number: 11061-68-0; Ins(h)) was purchased from Sigma-Aldrich (USA). They were resuspended at 0.5 mg / ml in phosphate-buffered saline (DPBS, Fisher Scientific, Spain). Liposomes were manufactured under GMP-like conditions in Ahead Therapeutic's clean room (Arboc, Spain). Liposomes were composed of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine (sodium salt) (DOPS, Lipoid GmbH, Germany), 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC, Lipoid), and cholesterol (CH, Sigma-Aldrich).

[0184] Liposomes were prepared by the solvent injection method. DMPC, DOPS, and cholesterol were dissolved in ethanol (EtOH, Sigma-Aldrich) and rapidly injected into a DPBS solution containing the required antigen, with a final lipid concentration of 30 mM. The liposome emulsion was extruded twice through a 1 μm pore-size polycarbonate membrane using a Lipex Thermobarrel Extruder (Evonik, Canada) and sonicated for 10 minutes in an ultrasonic bath sonicator (Sonorex RK100, Bandelin) to obtain the liposome compositions of the present invention (abbreviated as PS-PC-Chol-liposome-200 and 500).

[0185] Sub-200 nm PS-PC-Chol-liposomes (abbreviated as PS-PC-Chol-liposome-<200) were obtained by multiple extrusions through membranes with 200 nm or 100 nm pore sizes.

[0186] PS-PC-Chol-liposomes with diameters greater than 500 nm (abbreviated as PS-PC-Chol-liposome->500) were prepared as described in WO2015107140.

[0187] Fluorescent liposomes were prepared using either 3-hexanoyl-nitrobenzoxadiazole cholesterol (NBD, Cayman Chemical, USA) or Alexa Fluor 750 dye (ThermoFisher Scientific, USA). DOPE-AF750 dye was obtained by incubating 4 mg of 1,2-dioleoyl-sn-glycerophosphoethanolamine (DOPE) with 0.11 μL of TEA in a 0.5 mg / mL ethanol solution of Alexa Fluor 750. The solution was stirred for 5 hours. 0.15% molar of either 3-hexanoyl-NBD cholesterol or DOPE-AF750 dye was added to an ethanol solution of DOPS, DMPC, and cholesterol, followed by injection into DPBS solution.

[0188] Particle size was measured using nanotracking analysis (NTA) using a Malvern Panalytical Nanosight NS300, and ζ-potential was measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern, Instruments Ltd, UK). Peptide encapsulation efficiency (EE) was indirectly calculated by the formula: EE (%) = [(total peptide - free peptide) / total peptide] × 100, where total peptide is the total peptide concentration in the formulation and free peptide is the concentration of unencapsulated peptide. To measure free peptide, the liposome emulsion was centrifuged at 3700 g for 20 minutes at room temperature, and the concentration of unencapsulated peptide in the supernatant was assessed using a PIERCE BCA Protein Assay Kit (Thermo Fisher Scientific Inc., USA).

[0189] Unencapsulated peptide was removed from the liposome suspension using 100 kDa Spectra / Por 7 dialysis tubing (SPECTRUM; 131420). The suspension was dialyzed against 50–60 volumes of DPBS Ca / Mg with gentle agitation at room temperature for 24 h with one change of dialysis bath.

[0190] 1.2.Mouse Wild-type non-obese diabetic (NOD) mice were purchased from Jackson Laboratory (USA) and housed under specific pathogen-free (SPF) conditions at the Center for Comparative Medicine and Bioimage (Badalona, ​​Spain). This mouse strain spontaneously develops autoimmune diabetes after 12 weeks of age. Eight-week-old C57BL / 6J female mice purchased from Envigo Laboratories (Milan, Italy) were used for experimental autoimmune encephalomyelitis (EAE) experiments.

[0191] All mice were housed in a temperature- and humidity-controlled facility, provided with food and water ad libitum, and subjected to a 12-hour dark / light cycle. All experiments using animal models were conducted in accordance with animal facility rules and regulations, in full compliance with the recommendations set forth in the Declaration of Helsinki for Animal Research and the National Institutes of Health's Principles of Laboratory Animal Care. Protocols were approved by the government animal ethics committees of participating institutions.

[0192] 1.3. Liposome interaction with mouse B cells (spleen cells) Spleens were harvested from 12-week-old non-obese diabetic (NOD) mice (Jackson Laboratory, Bar Harbor, ME, USA) and mechanically disrupted. The cell suspension was hemolyzed and washed twice, after which cells were counted by flow cytometry using 7aad (BD Biosciences, San Jose, CA, USA). After 10 min, 6Splenocytes at 100 cells / ml were cultured in RPMI-1640 medium (Biowest, Nuaille, France) plus 10% fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA), 100 IU / ml penicillin (Normon SA, Madrid, Spain), and 100 μg / ml streptomycin (Laboratorio Reig Jofre, Sant Joan Despi, Spain) and incubated unstimulated, with 1 mM PS-PC-Chol-Insh-liposomes-<200 (non-fluorescent), or with 1 mM PS-PC-Chol-NBD-Insh-liposomes-<200 (fluorescent) for 4 h at 37°C and 5% CO. After incubation, cells were harvested, washed, and stained with 7aad, B220 BV510, CD19 APC-R700 (BD Biosciences), and CD1d PECy7 (BioLegend, San Diego, CA) for 20 minutes at 4°C. After washing, cells were analyzed for positivity for NBD by flow cytometry using a FACS Fortessa (BD Biosciences), and data were analyzed using FlowJo software (Tree Star, Ashland, OR, USA). Experiments were performed in duplicate and triplicate.

[0193] 1.4.EAE induction and clinical follow-up. 100 μg of mouse MOG peptide 35-55 (MOG) emulsified in 100 μl of Freund's complete adjuvant (Sigma Chemicals) containing 4 mg / ml Mycobacterium tuberculosis H37RA (Difco Laboratories, Franklin Lakes, NJ, USA) was used. 35-55Anesthetized mice were immunized by subcutaneous injection of 100 μl of phosphate-buffered saline (PBS) containing 100 μl of 100 μg of pertussis toxin (Proteomics Section, Universitat Pompeu Fabra, Barcelona, ​​Spain). On days 0 and 2 post-immunization (pi), mice were intravenously injected with 250 ng of pertussis toxin (Sigma Chemicals). Mice were weighed and examined daily for neurological signs using the following criteria: 0 = no clinical signs; 0.5 = partial loss of tail tone over two consecutive days; 1 = total tail paralysis; 2 = mild paraparesis of one or both hind limbs; 2.5 = severe paraparesis or paraplegia; 3 = mild tetraparesis; 4 = tetraparesis (severe in hind limbs); 4.5 = severe tetraparesis; 5 = tetraparesis; and 6 = death (Gutierrez et al., Mol Neurobiol. 2017). All presented data follow the proposed guidelines for publication of EAE (Baker D. et al., J. Neuroimmunol. 2012). Weight loss was calculated as the percentage change in daily weight compared to the initial weight on the day of immunization. A score of 5 and a weight loss of >30% were defined as endpoint criteria to minimize suffering and ensure animal welfare. Clinical scores were monitored for 28 days.

[0194] Liposome biodistribution in NOD mice For in vivo liposome tracking, near-infrared (NIR, 0.7-1.7 μm) fluorescence imaging was performed using a Pearl Impulse imaging system (LI-COR, USA). Prediabetic (<12 weeks old) NOD mice were treated with empty fluorescent DOPE-AF750-PS-liposomes administered intravenously (i.v.) in a single 100 μl dose. In vivo imaging was performed 1 h, 6 h, and 24 h after injection. Axillary lymph nodes (LNs), bladder, brain, heart, hindlimb bones, kidneys, liver, lungs, mediastinal LNs (MDLNs), pancreas, perigonadal adipose tissue (PAT), pancreatic LNs (PLNs), salivary glands, spleen, stomach, and thymus were harvested, washed with DPBS, and finally imaged ex vivo using a Pearl Impulse system (LI-COR). Background fluorescence was subtracted to normalize fluorescence values. Fluorescence signals were expressed as relative fluorescence units (RFU) per gram of tissue for each organ.

[0195] 1.6. In vivo entrapment of fluorescent liposomes in NOD mice To characterize in vivo liposome interactions with B cells, dendritic cells, and other APCs, such as macrophages and LSECs, prediabetic NOD mice were treated with fluorescently labeled empty NBD-PS-liposomes or NBD-PSIns(h)-liposomes administered intravenously in a single 100 μl dose. Spleens were harvested 1 and 6 h after injection. Splenocytes obtained after mechanical disruption and erythrocyte lysis were then labeled with monoclonal antibodies for 20 min at 4°C. The antibody panel used in this experiment was CD11c BV786, B220 / CD45R BV510, CD205 / DEC-205 BV421, CD8a PECy7, MHC-II / MHC-IA[d] PE, and CD19 APCCy7. Dead cells were excluded using Fixable Viability Stain 575V (BD Biosciences, USA). Cells were acquired using flow cytometry (FACS LSR Fortessa, BD Biosciences), and corresponding fluorescence minus one (FMO) staining was used as a control. Data were analyzed using FlowJo software (Tree Star, OR, USA). [Table 4] DC, dendritic cell.

[0196] 1.7. In vitro liposome capture evaluation by flow cytometry in NOD splenocytes Splenocytes from female NOD mice were obtained after mechanical disruption and red blood cell lysis, and then cultured in 96-well round-bottom plates at 3 × 10 s for 1 hour in 200 μl of RPMI 1640 (Lonza, Switzerland) supplemented with 10% heat-inactivated fetal bovine serum (Gibco, USA), 2 mM L-glutamine (Corning, USA), 1 mM sodium pyruvate (Lonza), 50 μM 2β-mercaptoethanol (Sigma-Aldrich), 100 U penicillin potassium (Lonza), and 100 μg streptomycin sulfate (Lonza). 5Cells were cultured at 1000 cells / well. They were incubated with 1 mM NBD-PS-liposomes or NBD-PSIns(h)-liposomes at 37°C and 5% CO2 for 2 hours and then maintained overnight. IL-10 secretion was detected using the APC Mouse IL-10 Secretion Assay Detection Kit (Miltenyi Biotech, Germany) according to the manufacturer's instructions. + Cells were stained and then reincubated with 1 mM NBD-PS-liposomes or NBD-PSIns(h)-liposomes for 1 hour to enhance NBD marking. B cell subsets were identified by monoclonal antibody immunophenotyping using a panel designed as follows: (1) CD19 BV510, CD5 BV421, CD1d PECy7, CD43 PerCP-Cy5.5, LAG3 APC; (2) CD19 BV510, CD21 APCCy7, CD23 BV421. Cells were acquired using flow cytometry (FACS LSR Fortessa, BD Biosciences), and the corresponding FMO staining was used as a control. Data analysis was performed using FlowJo software (Tree Star).

[0197] Analysis of IL-10 and TGF-β in PBMCs Twenty milliliters of peripheral blood from healthy adult donors was obtained by venipuncture into heparin tubes (BD Biosciences). The blood was diluted 1:1 with phosphate-buffered saline and subjected to density gradient centrifugation using Ficoll (GE Healthcare Life Sciences, Marlborough, MA, USA) to obtain peripheral blood mononuclear cells (PBMCs). Cells were washed and resuspended in X-VIVO 15 medium (Lonza, Basel, Switzerland) supplemented with 2% male AB human serum (Biowest), 100 IU / ml penicillin (Normon SA, Madrid, Spain), and 100 μg / ml streptomycin (Laboratorio Reig Jofre). After counting and viability assessment using 7aad (BD Biosciences) by flow cytometry, cells were cultured with various liposome compositions for 24 hours at 37°C and 5% CO2. After incubation, cells were harvested, washed, and stained with CD19 BV785, CD14 BV711, CD3 BV650, CD4 BV570, CD8 APCCy7, CD68 BV421 (BioLegend), TGF-β / LAP PE (Miltenyi Biotec), CD11c PE Cy7, and 7aad (BD Biosciences). IL-10 secretion was analyzed using the IL-10 Secretion Assay-Detection Kit, human (Miltenyi Biotec) according to the manufacturer's instructions. PBMC subsets, NBD positivity, IL-10 secretion, and TGF-β expression were assessed using a FACS Fortessa (BD Biosciences), and data were analyzed using FlowJo software (Tree Star). Experiments were performed in duplicate and triplicate.

[0198] 1.9.Statistical analysis Statistical analysis was performed using Prism 9.0 software (GraphPad Software Inc., San Diego, CA). For comparisons between unpaired data, a parametric two-tailed Student's t-test (Gaussian distribution) or a nonparametric Mann-Whitney test was used. For comparisons between multiple groups, a one-way ANOVA (Gaussian distribution) or a two-way ANOVA with corresponding multiple comparison tests was performed. A P value of <0.05 was considered significant.

[0199] 2.Results 2.1. PS-PC-Chol liposomes smaller than 200 nm interact with B cells and induce IL-10 secretion. PS-PC-Chol-NBD-Ins(h)-liposome-<200 was prepared by ethanol injection and extrusion 10 times through a 200 nm pore membrane, resulting in a composition with 98% liposomes with a diameter of less than 200 nm as determined by NTA and a zeta potential of -20.0 mV. These liposomes were incubated with splenocytes for 4 hours, and the percentage of interacting B cells (CD19+) was assessed. Surprisingly, 6.2% of CD19+ cells were found to interact with PS-PC-Chol-NBD-Ins(h)-liposome-<200; even more significantly, this percentage increased to 28% when the interaction in the putative Breg compartment, CD19+CD1dhi, was assessed (Figure 1).

[0200] To determine whether this interaction has biological significance with respect to tolerance induction, we analyzed the expression of the tolerogenic cytokine IL-10 in B cells from PBMC samples after 24 h of incubation with PS-PC-Chol-Ins(h)-liposomes-<200 (10–200 nm: 97.5%; >500 nm: 0%; zeta potential: -20.0 mV). As shown in the figure, PS-PC-Chol-Insulin-liposomes-<200 induced ex vivo expression of IL-10 in human B cells, as a 30% increase in the expression of this cytokine in B cells was observed after incubation with these liposomes.

[0201] 2.2. The PS-PC-Chol-liposome composition of the present invention has higher efficacy than PS-PC-Chol-liposomes >500 nm Induce EAE by MOG immunization as described above, and administer 50 μL of PS-PC-Chol-MOG via injection with a 1 mL syringe and a 30 G needle using the intravenous route on day 5 post-immunization. 36-55 - Liposomes-200 and 500 (10–200 nm 43.3%; >500 nm 3.9%; ζ potential −23.9 mV), PS-PC-Chol-MOG 36-55 Mice were treated with PS-PC-cholesterol-empty liposomes->500 (10-200 nm 0%; >500 nm 95.2%; ζ potential -24.7 mV), PS-PC-cholesterol-empty liposomes-200 and 500 (10-200 nm 26%; >500 nm 8.2%; ζ potential -22.7 mV), or PS-PC-cholesterol-empty liposomes->500 (10-200 nm 0%; >500 nm 93.1%; ζ potential -23.2 mV), and clinical scores were monitored for 28 days.

[0202] The area under the curve (AUC) difference of clinical scores was calculated to compare the beneficial effects of both liposome compositions. The AUC score of each MOG-loaded liposome-treated mouse was subtracted from the AUC score of each control mouse (empty group). A positive AUC difference means that the AUC score of the empty liposome-treated mouse was higher than that of the MOG-loaded liposome-treated mouse. This means that the MOG-loaded liposome-treated mouse exhibited a milder clinical course of EAE.

[0203] As shown in Figure 2, PS-PC-Chol-MOG 36-55 -Liposome-200 and 500 are superior to PS-PC-Chol-MOG in controlling disease progression and clinical symptoms. 36-55 -Liposomes->500 show higher efficacy.

[0204] 2.3. Fluorescently labeled PS-PC-Chol-liposome-200 and 500 are distributed to target organs and taken up by phagocytes in NOD mice We used AF750-labeled PS-PC-Chol-empty liposomes-200 and 500 to examine the biodistribution of PS-PC-Chol-liposomes-200 and 500. Therefore, PS-PC-Chol-AF750-empty liposomes-200 and 500 (10-200 nm: 45.1%; >500 nm: 4.7%; ζ potential: -24.7 mV) were administered via the i.v. route, and the fluorescence signal in each organ was evaluated 1, 6, and 24 h after administration (Figure 3). The i.v. route showed a peak value 1 h after injection, and the signal negligibly decreased over time until the 24-h checkpoint. As shown in the figure, when PS-PC-Chol-AF750-empty liposomes-200 and 500 were administered i.v., fluorescence was specifically concentrated in the liver, lungs, MDLN, spleen, and stomach.

[0205] To confirm which immune cells interacted with the inventive composition in vivo, NOD mice were administered a single intravenous dose of PS-PC-Chol-NBD-insulin-liposomes-200 and 500 (10-200 nm: 37.1%; >500 nm: 16.3%; zeta potential: -24.3 mV). Spleens and livers were harvested 1 and 6 hours after administration. Splenocyte viability was 98.8 ± 0.23 (mean ± SEM, data not shown). In the spleen, conventional DCs (cDCs) (8.6 ± 2.1%) showed NBD signal, and 1.4 ± 0.5% of B cells were also positive for NBD staining, indicating that the inventive composition directly interacts with these two immunological compartments. In addition to confirming interactions with DCs and B cells, among the immune cells tested, macrophages showed the highest percentage of interaction with liposomes, which is related to the parent population. Notably, LSECs were also found to interact with liposomes (Figure 4).

[0206] 2.4. PS-liposomes interact with Breg cell subsets and promote IL-10 secretion. Because B cells play an important role as antigen-presenting cells (APCs), we investigated the tolerogenic effects of PS-PC-Chol-Ag-liposome-200 and 500 on B cells. To this end, splenocytes from NOD mice were co-cultured with PS-PC-Chol-NBD-insulin-liposome-200 and 500 (10-200 nm: 36.4%; >500 nm: 18%; zeta potential: -25 mV). Of all B lymphocytes, 34.8 ± 2.0% showed NBD fluorescence. When examining NBD fluorescence in various B cell subsets with the described regulatory capacities, CD5 + CD1d high The cells (CD5 + CD1d high The NBD signal was 48.1 ± 2.3% (relative to the entire subset), + B1a B lymphocytes represented 58.7±1.4% of all B1a B lymphocytes, and NBD + B1b B lymphocytes represented 24.8±1.5% of all B1b B lymphocytes, and NBD + Marginal zone (MZ) B cells represented 67.5±0.4% of all MZ B cells (Figure 5). To demonstrate their regulatory function after treatment, we found positive IL-10 secreting cells in each subset: 13.1±1.4% CD5 + CD1d high NBD + cells, 16.9±1.9% B1a NBD + cells, 1.5±0.8% B1b NBD + cells, and 13.4 ± 0.9% MZ NBD + Cells (Figure 5). IL-10 secretion showed a biological trend toward increased secretion in NBD-fluorescent B cells when compared to non-fluorescent B cells for each subset.

[0207] Liposomes of the composition of the present invention induce the secretion of IL-10 and TGF-β in PBMC-derived B cells and dendritic cells.

[0208] To confirm the tolerance-inducing ability of the compositions of the present invention in human cells, 100 ng / ml lipopolysaccharide, 1 mM PS-PC-Chol-NBD-Ins(h)-liposome-200 and 500 (10-200 nm 37.0%; >500 nm 2.8%; ζ potential -22.3 mV), 1 mM PS-PC-Chol-NBD-MCV-liposome-200 and 500 (10-200 nm 39.3%; >500 nm 3.6%; ζ potential -24.7 mV), or 1 mM PS-PC-Chol-NBD-AChR-liposome-200 and 500 (10-200 nm 18.1%; >500 nm PBMCs from healthy donors were incubated for 24 hours in the presence or absence of PS-PC-Chol-MCV-200 and 500 (10-200 nm 28.6%; >500 nm 9.4%; ζ potential -23.7 mV) stained with pHrodo™ green dye for labeling amines according to the manufacturer's instructions, and the expression of the tolerance-associated cytokines IL-10 and TGF-β was analyzed in NBD-positive and NBD-negative subsets of B cells and DCs. Expression of these two cytokines was also assessed in pHrodo-positive and pHrodo-negative subsets of B cells.

[0209] As shown in the figure, interaction with differently loaded liposomes induces a clear increase in IL-10 and TGF-β expression in the CD19+ compartment, whereas DCs that capture liposomes express higher levels of IL-10 than DCs that do not phagocytose liposomes, but no difference in TGF-β expression is observed. Furthermore, pHrodo staining, which is pH-sensitive and indicates direct phagocytosis of cells, confirmed that the modulation of B cells by liposome treatment is due to liposome phagocytosis rather than indirect membrane interaction. These data demonstrate that the mechanism of action for inducing tolerance via B cells and DCs is common, regardless of the antigen loading in PS-liposomes.

[0210] Citation list Patent documents: - International Publication No. WO 2015 / 107140 Non-Patent Literature: - Chen et al, J Immunol 2020, 204(2): 335 - 347. https: / / doi.org / 10.4049 / jimmunol.1801677 - Roep BO, Peakman M. Cold Spring Harb Perspect Med, 2012, vol.2(4): a007781. doi:10.1101 / cshperspect.a007781 - Lernmark A. J. Clin Invest, 2001, vol.108, p.1091 - 1096 - Bangham et al., J. Mol. Biol., 13, 238(1965) - Pons et al. International Journal of Pharmaceutics 95(1993)51 - 56) - Maurer N. et al., Expert Opin Biol Ther, '2001, vol.1(6), p.923 - 47 - Waterhouse D. N. et al., Methods Enzymol., 2005; vol.391, p.40 - 57 - Urban P. et al., Nanosc. Res. Lett., 2011, vol.6, p.620 [[ID=2​​​-Whitepaper on Nanoscale Material Characterization:a Review of the use of Nanoparticle Tracking Analysis(NTA),2015 Malvern Instruments Limited

Claims

1. 1. A composition comprising two populations of liposomes, the first population of liposomes has a size in the range of 2 to 200 nm; the second population of liposomes has a size in the range of 500 to 2000 nm; - the liposomes of the first and second populations carry one or more antigens; the liposome membrane of each liposome in the first and second liposome populations comprises phosphatidylserine in an amount ranging from 20 to 60% by weight relative to the total composition of said liposome membrane; composition.

2. 10. The composition of claim 1, wherein 15-75% of the liposomes in the composition correspond to the first population and 2-40% of the liposomes in the composition correspond to the second population.

3. The composition according to any one of claims 1 to 2, wherein the amount of phosphatidylserine in the liposome membrane is 35 to 45% by weight based on the total composition of the liposome membrane.

4. The composition according to any one of claims 1 to 3, wherein the liposome membrane further comprises phosphatidylcholine (PC) and cholesterol (CHOL).

5. 5. The composition of claim 4, wherein the liposome membrane comprises PS, PC, and CHOL in a molar ratio of PS:PC:CHOL comprised of 1:(0.6-1.8):(0.7-2.5).

6. The composition of any one of claims 1 to 5, wherein the antigen is a peptide having 5 to 200 amino acids.

7. The composition of any one of claims 1 to 6, wherein the antigen is selected from the group consisting of an autoantigen, a drug comprising a therapeutic protein, a viral vector comprising a viral capsid protein, an allergen, and an alloantigen.

8. The antigen is an autoantigen associated with an autoimmune disease, and in particular, the autoantigen is selected from the group consisting of insulin, proinsulin, protein tyrosine phosphatase (IA2), glutamic acid decarboxylase (GAD), chromogranin and islet-glucose-6-phosphatase catalytic subunit-related protein (IGRP), peripherin, myelin, myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP), myelin proteolipid protein (PLP), GDP-1-fucose synthase, acetylcholine receptor (AChR), muscle-specific tyrosine kinase (MuSK), agrin, lipoprotein-related protein 4 (LRP4), cortactin, transglutaminase, deamidated gliadin, thyroglobulin, collagen (e.g., type 11 collagen), human cartilage gp, 39, chromogranin A, gp130-RAPS, vimentin, citrullinated vimentin, ADAMST13, aquaporin-4, proteolipid protein, fibrillarin, nuclear protein, nucleolar protein (e.g., small nucleolar protein), histidyl-tRNA synthetase (HisRS), histidine-tRNA synthetase (HARS1), jo-1, thyroid-stimulating factor receptor, histone, glycoprotein gp 70, ribosomal protein, pyruvate dehydrogenase dehydrolipoamide acetyltransferase, hair follicle antigen, human tropomyosin isoform 5, mitochondrial protein, pancreatic beta cell protein, gluten, and an antigenic fragment or derivative of any of the foregoing, and an immunogenic fragment or derivative of any of the foregoing.

9. The composition according to claim 7, wherein the antigen is a viral capsid protein, in particular selected from VP1, VP2 and VP3.

10. The composition according to any one of claims 1 to 9, which does not contain an immunosuppressant.

11. The composition of any one of claims 1 to 10, which is a pharmaceutical composition and comprises pharmaceutically acceptable excipients and carriers.

12. A composition according to any one of claims 1 to 11 for use as a medicament.

13. 12. The composition of any one of claims 1 to 11 for use in inducing tolerance to said antigen, in particular said induction of tolerance comprising B cell-mediated tolerance and T cell-mediated tolerance to said antigen.

14. 12. The composition of any one of claims 1 to 11 for use in the treatment of a condition selected from autoimmune diseases, allergies, drug hypersensitivity, transplant rejection, adverse immune effects caused by gene therapy.

15. The condition is selected from the group consisting of T1D, lupus erythematosus, arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, reactive arthritis, and psoriatic arthritis), multiple sclerosis, neuromyelitis optica, Addison's disease, celiac disease, dermatomyositis, Hashimoto's thyroiditis, myasthenia gravis, pernicious anemia, autoimmune hemolytic anemia, autoimmune neutropenia, Graves' disease, psoriasis, Sjogren's syndrome, including keratoconjunctivitis sicca secondary to Sjogren's syndrome, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), alopecia areata, Crohn's disease, aphthous ulcers, iritis, 15. The composition for use according to claim 14, wherein the autoimmune disease is selected from the group consisting of conjunctivitis, keratoconjunctivitis, ulcerative colitis, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, true erythrocytic anemia, idiopathic thrombocytopenic purpura, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic sprue, lichen planus, sarcoidosis, primary biliary cirrhosis, posterior uveitis, and interstitial pulmonary fibrosis.

16. The composition according to any one of claims 1 to 11 or the composition for use according to any one of claims 12 to 15, wherein the size of the liposomes is determined by nanoparticle tracking analysis (NTA).

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