Tolerogenic compositions

Abstract

The present invention relates to compositions based on tolerogenic liposomes and their use for the treatment of immune disorders. The composition comprises two populations of liposomes wherein the first population of liposomes has a size of from 2 to 200 nm and the second population of liposomes has a size of from 500 to 2000 nm, the first population of liposomes and the second population of liposomes carrying one or more antigens, and the liposome membrane of each liposome in the first liposome population and the second liposome population comprises phosphatidylserine in an amount of 20% to 60% by weight relative to the total component of the liposome membrane.

Description

[0001] Cross - reference to related applications

[0002] This application claims the benefit of European Patent Application EP22383126.4, filed on November 23, 2022. Technical field

[0003] The present invention relates to the field of medicine. In particular, the present invention provides compositions for inducing tolerance to antigens, which can be used for preventing and / or treating various immune disorders, such as autoimmune diseases. Background art

[0004] Abnormal immune responses cause a variety of diseases and adverse reactions, including autoimmune diseases, allergies, transplant rejection, and drug hypersensitivity.

[0005] In particular, autoimmunity is the inability of an organism to recognize its own components as self, thus triggering an immune response against its own cells and tissues. Major examples include type 1 diabetes (T1D), systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis (MS), Addison's disease, celiac disease, dermatomyositis, Hashimoto's thyroiditis, myasthenia gravis, pernicious anemia, reactive arthritis, Sjogren syndrome, neuromyelitis optica, immune thrombocytopenic purpura.

[0006] It is estimated that 7% to 10% of the population in developed countries suffer from these diseases, which are usually chronic, debilitating, and life - threatening. With the increase in autoimmune disorders worldwide and the lack of effective treatment methods, healthcare costs related to autoimmunity continue to rise.

[0007] Traditional treatment methods for autoimmune diseases are immunosuppression, anti - inflammation (steroids), or palliative. Non - immune therapies such as hormone replacement in Hashimoto's thyroiditis or type 1 diabetes treat the consequences of the self - attacking response, so these are palliative therapies. Dietary control limits the severity of celiac disease. Steroid or NSAID treatments limit the inflammatory symptoms of various diseases. Similar limitations apply to other disorders related to abnormal (usually excessive) immune responses.

[0008] Intensive research has been devoted to developing immunomodulatory therapies to mitigate or avoid adverse immune responses. However, the limited understanding of the complex details of different autoimmune diseases has substantially slowed down the development of this field. Current strategies are usually based on broad - spectrum immunosuppressive drugs, often requiring lifelong treatment to maintain immunosuppression. In addition, the use of broad - spectrum immunosuppressants is associated with the risk of severe side effects, such as tumors, infections, nephrotoxicity, and metabolic disorders.

[0009] Liposomes are lipid vesicles composed of a lipid membrane encapsulating an aqueous core. These vesicles are considered to have great potential as drug delivery systems for several main reasons. i) They are able to deliver various active agents; hydrophilic agents can be loaded in the aqueous compartment, or hydrophobic agents can be anchored in the membrane. And ii) They can enhance the therapeutic effect and reduce side effects due to targeting specific tissues, resulting in increased bioavailability of the delivered agent. Liposome-based means have been proposed to effectively deliver immunosuppressants in the context of autoimmune diseases with reduced side effects. In addition, liposome-based means have been disclosed to induce tolerance without immunosuppressants, and there have been some successful cases in preventing hallmark autoimmune diseases (WO2015107140). The liposomes disclosed in this document are not simply carriers of active agents, but are themselves part of an active agent that plays a role in promoting tolerance to specifically included antigens. The liposomes disclosed in WO2015107140 are relatively large in size (greater than 500 nm) and mimic apoptotic bodies, inducing tolerance in dendritic cells through a mechanism similar to efferocytosis.

[0010] Despite efforts made so far, there is still much room for improvement in the treatment of autoimmune disorders and other conditions caused by abnormal immune responses. Summary of the Invention

[0011] The inventors have surprisingly found that combining two populations of liposomes that carry antigens and contain at least 20% phosphatidylserine in the liposome membrane, with the two populations being of different sizes, achieves a surprising tolerogenic effect, which translates into an advantage in treating immune diseases.

[0012] Thus, a first aspect of the present invention relates to a composition comprising two populations of liposomes, wherein:

[0013] - The first population of liposomes has a size of 2 to 200 nm,

[0014] - The second population of liposomes has a size of 500 to 2000 nm,

[0015] - The first population of liposomes and the second population of liposomes carry one or more antigens, and

[0016] - The liposome membrane of each liposome in the first population of liposomes and the second population of liposomes contains phosphatidylserine in an amount of 20 wt% to 60 wt% relative to the total composition of the liposome membrane.

[0017] As shown in the following examples, the composition of the first aspect induces a tolerogenic profile (secretion of interleukin-10 (IL-10) and transforming growth factor β (TGF-β)) in human peripheral blood mononuclear cells. The following examples also show that the composition of the first aspect comprises two liposome populations, one population of liposomes having a size of 2 to 200 nm and another population of liposomes having a size of 500 to 2000 nm, which achieves a higher efficacy in improving autoimmune diseases compared to a liposome composition having the same characteristics except for the presence of only the liposome population of 500 to 2000 nm.

[0018] Interestingly, the inventors have surprisingly found that the composition of the first aspect induces tolerance by interacting with a wide range of antigens present in cells. Without being bound by theory, the inventors hypothesize that the liposome composition described herein can interact with different components involved in the process of generating peripheral tolerance to achieve a significant effect. Considering that different abnormal autoimmune responses can be mediated by different APCs (sometimes more than one APC), the composition of the present invention offers the following important advantages over prior art products: it has a broader spectrum of action and a wider range of target diseases. In addition, while inducing tolerance through various APCs, it has been found that the composition of the present invention can act through multiple mechanisms of action, thereby achieving a greater effect.

[0019] For example, it has been found that the liposome population having a size of 2 - 200 nm is capable of inducing tolerance in B cells, while the larger-sized population of 500 - 2000 nm induces tolerance through dendritic cells (DCs) as disclosed in WO2015107140. The ability of the smaller-sized liposome population to interact with B cells and induce a tolerogenic profile in B cells was confirmed in the following examples. For example, Figure 1 it was specifically shown that PS-PC-Chol liposomes smaller than 200 nm interact with B cells and induce IL-10 secretion. Other examples show that the liposomes in the composition of the present invention containing the two populations as described above interact with some B cell subsets and induce a tolerogenic profile (IL-10 and TGF-β secretion) in these B cell subsets in addition to dendritic cell IL-10 secretion ( Figure 4 、 5and 6). WO2015107140 discloses that liposome sizes greater than 500 nm are crucial for mimicking apoptotic cells and inducing tolerance via dendritic cells through a mechanism similar to efferocytosis. In terms of WO2015107140, this effect of the present invention is surprising. In terms of this prior art document, the effect of a smaller population of liposomes would not be foreseen. Additionally, in terms of the teachings of WO2015107140, a person skilled in the art would not employ liposome sizes less than 500 nm as the relevant effects of liposomes having such a smaller size could not be foreseen. Furthermore, other prior art documents disclose the use of smaller liposomes to enhance the immune response (Chen et al., doi: 10.4049 / jimmunol.1801677). It has also been found that the liposome composition of the first aspect can induce tolerance through liver sinusoidal endothelial cells and macrophages ( Figure 4 ).

[0020] The tolerogenic effect of the composition (hereinafter also simply referred to as "liposome composition") defined in the first aspect of the present invention not only enables effective treatment of autoimmune diseases, but also provides an improved effect in treating autoimmune diseases when compared to a liposome composition containing only a population of liposomes with sizes greater than 500 nm ( Figure 2 ).

[0021] While being an antigen-specific-based therapy, the liposome composition of the first aspect has the advantage of not exhibiting relevant adverse side effects. As mentioned above, most immunomodulatory means for treating autoimmune disorders or transplant rejection involve immunosuppressants, which tend to lead to a high susceptibility to infections and sometimes also promote the occurrence of tumors, nephrotoxicity, or metabolic disorders. In addition to providing tolerance to the relevant antigen, the liposome composition of the first aspect does not cause any toxicity or other adverse side effects.

[0022] The liposome composition of the first aspect also has some advantages in terms of stability, homogeneity, and ease of large-scale production.

[0023] First, the production of this liposome containing an antigen (usually an antigenic peptide) can be achieved at low cost using conventional reagents and equipment in the pharmaceutical industry. Additionally, the homogeneity of the product can be ensured, while at the same time, the scale-up for large-scale industrial production can be afforded and the dosage can be easily fine-tuned. Furthermore, since the antigen is protected by the liposome, it is less affected by degradation.

[0024] Another significant advantage is that this liposome-based composition is a defined composition, free of adverse contaminants or by-products. The antigen-containing liposomes do not degrade into toxic by-products, such as necrosomes, and do not cause rejection reactions as in the case of autologous or allogeneic cell-based therapies.

[0025] Another advantage of the liposomal compositions disclosed herein is that they act on two different tolerance - inducing mechanisms. Thus, the efficacy is doubled and there are substantially no additional side effects. Additionally, the two populations providing dual activity can be achieved by a single preparation process, which is an additional advantage.

[0026] By virtue of its tolerance - inducing effect, the liposomal composition defined in the first aspect effectively prevents disorders associated with abnormal immune responses, such as autoimmune disorders, and effectively treats such disorders during the pre - clinical stage (i.e., the stage where an abnormal immune response has been triggered but tissue damage and clinical symptoms are mild) and the clinical stage (i.e., the stage where tissue damage is higher and clinical symptoms are significant).

[0027] In a second aspect, the present invention provides the use of the liposomal composition defined in the first aspect as a medicament. This aspect can also be expressed as the use of the liposomal composition defined in the first aspect for the preparation of a medicament. Additionally, a treatment method is disclosed, which comprises administering a therapeutically effective amount of the liposomal composition defined in the first aspect to a subject in need thereof.

[0028] Without being bound by theory, it is believed that the therapeutic effect of the liposomal composition is achieved through the tolerance - inducing presentation of the encapsulated antigen by antigen - presenting cells (such as but not limited to B cells, DCs, and macrophages) and the subsequent inhibition of the abnormal immune response. Thus, in a third aspect, the present invention provides the use of the liposomal composition defined in the first aspect for inducing tolerance. This aspect can also be expressed as the use of the liposomal composition defined in the first aspect for the preparation of a medicament for inducing tolerance. A method for inducing tolerance is also disclosed, which comprises administering a therapeutically effective amount of the liposomal composition defined in the first aspect to a subject in need thereof.

[0029] In a fourth aspect of the present invention, there is provided the use of the liposomal composition defined in the first aspect for treating disorders associated with abnormal immune responses. This aspect can also be expressed as the use of the liposomal composition defined in the first aspect for the preparation of a medicament for treating disorders associated with abnormal immune responses. A method for treating disorders associated with abnormal immune responses is also disclosed, which comprises administering a therapeutically effective amount of the composition defined in the first aspect to a subject in need thereof.

[0030] Finally, other aspects of the present invention provide the use of the liposomal composition as defined above for immunomodulation; the use of the liposomal composition as defined above for inhibiting an excessive immune response; and the use of the liposomal composition as defined above for treating disorders associated with abnormal immune responses, wherein the liposomal composition restores tolerance to the antigen contained in the liposomal composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] 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 and CD19CD1d cells from splenocytes of NOD mice incubated with PS-PC-Chol-NBD-Ins(h) liposomes smaller than 200 nm for 4 hours. b Percentage of IL-10 expression in B cells from PBMCs after incubation with PS-PC-Chol-NBD-Ins(h) liposomes smaller than 200 nm for 24 hours. Results are expressed as mean ± SEM.

[0032] Figure 2 The PS-PC-Chol liposome composition of the present disclosure has higher efficacy than PS-PC-Chol liposomes >500 nm. 8-week-old mice were immunized with mouse MOG peptide 35-55 (mMOG 35–55 ) and intravenously treated with PS-PC-Chol-MOG36-55 liposomes >500 nm or PS-PC-Chol-MOG36-55 liposomes 200&500 five days after immunization. Clinical scores were evaluated within 28 days. The area under the curve (AUC) difference of the clinical scores was calculated to compare the beneficial effects of the two liposome compositions.

[0033] Figure 3 Fluorescently labeled PS liposomes are distributed in target organs. a Histogram of relative fluorescence signals (RFU / g tissue) of PS-PC-Chol-AF750 empty liposomes 200&500 at 1 hour and 6 hours after administration in NOD mice. Mice received 100 μl via the intravenous route (i.v., n = 5). Results are expressed as mean ± SEM.

[0034] Figure 4 PS-PC-Chol liposomes 200&500 interact with DCs, B cells, macrophages, and LSECs in vivo. Percentage of NBD-fluorescent cells in the parent gate of the spleen and liver of animals treated intravenously with PS-PC-Chol-NBD-Ins(h) liposomes 200&500 at 1 hour (n = 5-6) or 6 hours (n = 5). Cells were determined as follows: B cells, CD19 + ; conventional DC (cDC), CD11c + MHC-II + CD205 + CD8a + ; plasmacytoid DC (pDC) CD11c + MHC-II + B220 + ; macrophages F4 / 80+ ; Kupffer cell F4 / 80 + CD68 + ; Liver sinusoidal endothelial cell (LSEC) CD206 + F4 / 80 - . Data are mean ± SEM; differences were found when comparing time points within the same subset and immune subsets (*p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA with Tukey's multiple comparison test).

[0035] Figure 5 Liposomes of the composition of the present invention bind to B cells and induce IL-10 secretion. a Percentage of NBD-fluorescent B cell subsets gated across the entire subset after overnight culture in PS-PC-Chol-NBD-ins(h) liposomes 200 & 500 (n = 3). Breg cell subsets were determined as follows: CD19 + CD1d high CD5 + ; B1a, CD19 + CD5 + CD43 + ; B1b, CD19 + CD5 - CD43 + ; Marginal zone (MZ), CD19 + CD21 high CD23 - . Data are presented as mean ± SEM. b Percentage of IL-10 secreting cells from NBD-PSIns(h) liposome-bound NBD + B cell subsets and from non-liposome-bound NBD - B cell subsets (n = 3). Data are presented as mean ± SEM, no differences were found.

[0036] Figure 6The liposomes of the compositions of the present invention induce the expression of the tolerance cytokines IL-10 and TGF-β in B cells from PBMCs and induce the expression of IL-10 in DC cells. a Percentage of IL-10 expression in NBD+ or NBD- B cells from PBMCs after incubation with PS-PC-Chol-NBD liposomes 200&500 loaded with AChR peptide or mutated citrullinated vimentin peptide or insulin for 24 hours. b Percentage of TGF-β expression in NBD+ or NBD- B cells from PBMCs after incubation with PS-PC-Chol-NBD liposomes 200&500 loaded with AChR peptide or mutated citrullinated vimentin peptide or insulin for 24 hours. c Percentage of IL-10 expression in NBD+ or NBD- dendritic cells from PBMCs after incubation with PS-PC-Chol-NBD liposomes 200&500 loaded with AChR peptide or mutated citrullinated vimentin peptide or insulin for 24 hours. d Percentage of TGF-β expression in NBD+ or NBD- dendritic cells from PBMCs after incubation with PS-PC-Chol-NBD liposomes 200&500 loaded with AChR peptide or mutated citrullinated vimentin peptide or insulin for 24 hours. e pHrodo TM Green-positive cells of the parental gates of different immune cell types from PBMCs after incubation with PS-PC-Chol-MCV-200&500 stained with green dye for 24 hours. f IL-10 and TGF-β expression in pHrodo+ and pHrodo- B cells expressed as mean fluorescence intensity (MFI). Results are expressed as mean ± SEM. (*p < 0.05, **p < 0.01, ***p < 0.001, two-way ANOVA with Tukey's multiple comparison test) Detailed Description

[0037] 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 filing. However, in case of any potential ambiguity, the definitions provided herein shall prevail over any dictionary definition or external definition. Furthermore, unless the context otherwise requires, singular terms shall include the plural and plural terms shall include the singular

[0038] As used herein, the indefinite articles "a" and "an" have the same meaning as "at least one" or "one or more". Unless otherwise stated, the definite articles such as "the" used herein also include the plural of the noun.

[0039] Liposome

[0040] As described above, a first aspect of the present invention relates to a composition comprising two populations of liposomes, wherein the first population of liposomes has a size of 2 nm to 200 nm and the second population of liposomes has a size of 500 nm to 2000 nm. In a particular embodiment, the composition of the first aspect consists essentially of two populations of liposomes, wherein the first population of liposomes has a size of 2 nm to 200 nm and the second population of liposomes has a size of 500 nm to 2000 nm. In another particular embodiment, the composition of the first aspect consists of two populations of liposomes, wherein the first population of liposomes has a size of 2 nm to 200 nm and the second population of liposomes has a size of 500 nm to 2000 nm. The first population of liposomes and the second population of liposomes may carry one or more than one antigen. The liposomal membrane of each liposome in the first population of liposomes and the second population of liposomes may comprise phosphatidylserine, in an amount of 20 wt% to 60 wt% relative to the total composition of the membrane of the liposome.

[0041] The term "liposome" is intended to be understood as a self-assembled structure comprising one or more membranes composed of amphiphilic bilayers, each membrane comprising two monolayers of amphiphilic molecules with opposite orientations. Amphiphilic molecules, such as amphiphilic lipids, comprise a polar (hydrophilic) head region covalently linked to one or two non-polar (hydrophobic) chains. The energetically unfavorable contact between the hydrophobic chains and the surrounding aqueous medium induces the amphiphilic molecules to arrange themselves such that their polar heads are oriented towards the surface of the bilayer, while the hydrophobic chains are reoriented towards the interior of the bilayer. Thereby forming an energetically stable structure that effectively prevents the hydrophobic chains from contacting the aqueous environment.

[0042] Liposomes can have a single bilayer membrane (small unilamellar vesicles "SUV" and large unilamellar vesicles (LUV)), or multiple bilayer membranes (multilamellar large vesicles "MLV"). Liposomes can also be prepared as multivesicular vesicles "MVV", wherein it is a liposome encapsulating or enclosing multiple non-concentric aqueous cavities. In contrast, MLV has multiple concentric "onion skin"-like membranes, each membrane enclosing an aqueous compartment. Given such encapsulation of the aqueous volume within a protective barrier of lipid molecules, liposomes are able to sequester encapsulated molecules, such as peptides, from degradation by factors such as peptidases present in the external environment. The sequestered molecules can be suspended or dissolved in the aqueous compartment or associated with the liposomal membrane. Generally, polar, water-soluble molecules will mainly dissolve in the aqueous compartment, while less polar molecules can be associated with the lipid membrane.

[0043] The liposomal composition of the first aspect comprises two liposomal populations, wherein the first liposomal population has a size of 2 nm to 200 nm and the second liposomal population has a size of 500 nm to 2000 nm. The size of the liposomes generally refers to the average diameter and can be determined by nanoparticle tracking analysis (NTA) using a Nanosight NS300 from Malvern Panalytical (Whitepaper on Nanoscale Material Characterization: a Review of the use of Nanoparticle Tracking Analysis (NTA), 2015 Malvern Instruments Limited). This method is known to measure the "hydrodynamic diameter". Thus, in a particular embodiment of the present invention, the size of the liposomes refers to the hydrodynamic diameter. The size of the liposomes in the second population generally does not exceed 2000 nm, but in some embodiments, the size range of this second population can be larger, for example, 500 nm to about 3000, 4000 or 5000 nm.

[0044] In one embodiment, the size of the liposomes in the first population is 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 size of the liposomes in the first population is greater than 2 nm, or greater than 10 nm, or greater than 15 nm, or greater than 25 nm, or greater than 50 nm, or greater than 75 nm. In a particular embodiment, the size of the liposomes in the first population is 2 to 200 nm, more preferably 10 to 200 nm.

[0045] In one embodiment, the size of the liposomes in the second population is 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 size of the liposomes in the second population is 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.

[0046] In one embodiment, 15-75% of the liposomes in the composition correspond to a first population, i.e., 15-75% of the liposomes in the composition have a size of 2 to 200 nm. These percentages refer to the number of liposomes having the specified size per 100 liposomes in the composition. In a particular embodiment, 15-55% of the liposomes in the composition correspond to the first population. In a more particular 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 a second population, i.e., 2-40% of the liposomes in the composition have a size of 500 to 2000 nm. In a particular embodiment, 2-25% of the liposomes in the composition correspond to the second population. In another more particular embodiment, 2-15% of the liposomes in the composition correspond to the second population.

[0047] As described above, the liposome membrane is formed by amphiphilic molecules. Non-limiting amphiphilic molecules that can be included in the liposome membranes of the liposome compositions of the present disclosure include phospholipids; glycerophospholipids; phosphatidylserines 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; phosphatidylcholine (PC) such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine, lecithin; lysophosphatidylcholine; phosphatidylethanolamine such as dioleoyl phosphatidylethanolamine (DOPE); poly(ethylene glycol)5000-phosphatidylethanolamine; dioleoylpropyltrimethylammonium (DOTMA); cholesterol; cholesterol esters; diacylglycerol; diacylglycerol succinate; diphosphatidylglycerol (DPPG); cetyl alcohol; fatty alcohols such as polyethylene glycol (PEG); poly(ethylene glycol)400-monostearate; polyethylene glycol-9-lauryl ether; surface-active fatty acids such as palmitic acid or oleic acid; fatty acids; fatty acid monoglycerides; fatty acid diglycerides; sorbitan fatty acid esters such as sorbitan trioleate; phosphatidylinositol; sphingomyelin; cardiolipin; phosphatidic acid; cerebroside; dicetyl phosphate; dipalmitoyl phosphatidylglycerol; stearylamine; dodecylamine; hexadecylamine; ethyl palmitate; glycerol ricinoleate; cetyl stearate; isopropyl myristate and combinations thereof.

[0048] In one embodiment, the liposome membrane can comprise, but is not limited to, phospholipids such as phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI), and phosphatidic acid (PA). The liposome membrane can also comprise other lipids such as cholesterol (CHOL). The liposome membrane can contain other molecules that are not lipids under natural conditions, such as proteins, carbohydrates, antibodies, or polyethylene glycol (PEG) chains. Liposomes can comprise specific groups that are designed to target the liposomes to specific sites or target cells, or to protect the liposomes from harsh environments (such as the gastrointestinal tract). The composition of the liposomes is related to the delivery of tolerogenic antigens. Thus, as described above, the liposome membrane preferably comprises PS in an amount of 20 to 60% by weight relative to the total composition of the liposome membrane. The PS contained in the liposome membrane constitutes an "eat me" signal, linking PS recognition by antigen-presenting cells to the outcome of tolerance induction. It is noted that the liposomes of the present invention do not require additional receptors or ligands to be effectively phagocytosed by antigen-presenting cells and achieve tolerogenic antigen delivery. However, other receptors and / or ligands can be assembled into the liposomes to improve uptake and / or tolerogenic processing.

[0049] The term "percent by weight (%)" refers to the percentage of each component of the liposome membrane relative to the total weight of the liposome membrane.

[0050] "The liposome membrane" or "liposome membrane" refers to the entire membrane bilayer contained in the liposome.

[0051] In one embodiment, the PS content of the liposome membrane is 30 to 50% by weight relative to the total liposome membrane. In a further embodiment, the PS content of the liposome membrane is 30 to 45%, or 35 to 45%, relative to the total liposome membrane, such as 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, or 44%.

[0052] In addition to PS, the liposomes of the present invention can comprise other lipids at variable concentrations. In some embodiments, the liposome membrane further comprises phospholipids with a neutral net charge. In some embodiments, the liposome membrane further comprises 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 further comprises PC, such as 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). In some embodiments, the liposome membrane further comprises sterol lipids or steroids. In particular embodiments, the liposome membrane further comprises PC and CHOL.

[0053] In one embodiment, the PC content of the liposome membrane is 30 to 50 wt% relative to the total liposome membrane. In another embodiment, the PC content of the liposome membrane is 30 to 45 wt% relative to the total liposome membrane. In a further embodiment, the PC content of the liposome membrane is 32 to 42 wt%, such as 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or 41% relative to the total liposome membrane.

[0054] The content of PC can be 10 to 40 wt% relative to the total liposome membrane. In one embodiment, the CHOL content of the liposome membrane is 12 to 40 wt% relative to the total liposome membrane. In a further embodiment, the membrane CHOL content is 15 to 37%, or 20 to 35%, or 20 to 30 wt%, such as 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, or 29% relative to the total liposome membrane.

[0055] The proportions of the different lipids contained in the liposome membrane must be balanced to obtain liposomes having suitable physical and chemical properties in terms of stability, permeability, and morphology. In some embodiments, the liposome membrane contains 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 a particular embodiment, the molar ratio of PS:PC:CHOL is 1:(0.6 - 1.8):(0.7 - 2.5). The term "proportion" is understood in its conventional meaning as the magnitude of amounts relative to each other. Specifically, the ratio of two amounts represents the multiple by which the first amount (X) is contained in the second amount (Y), and is expressed as X:Y. As described in the above embodiments, the amounts can be expressed as a range (X - X':Y - Y'). The term "molar ratio" is used when the magnitude referred to is molar concentration. Alternatively, when the magnitude referred to is weight, the proportion can be expressed as a "weight ratio". Here, ranges of the molar ratios of three specific lipids (PS, PC, and CHOL) are given. In a more particular embodiment, the molar ratio of PS:PC:CHOL contained in the membrane is 1:(0.7 - 1.5):(0.9 - 2). In another more particular embodiment, the molar ratio of PS:PC:CHOL contained in the membrane is 1:(0.8 - 1.4):(1.1 - 1.9). In another more particular embodiment, the molar ratio of PS:PC:CHOL contained in the membrane is 1:(0.9 - 1.3):(1.2 - 1.7).

[0056] Antigen

[0057] As described above, the liposomes in the composition of the first aspect contain an antigen. The term "antigen" refers to any substance that, when exposed to a host organism, elicits a B cell (humoral / antibody) and / or T cell (cellular) adaptive immune response. The antigen can be dissolved or suspended in the aqueous compartment within the liposome or 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 can contain one or more epitopes or antigenic determinants. In the context of the present invention, the antigen is an immunogenic antigen. A person skilled in the art can determine antigenic substances by methods well known in the art.

[0058] The liposome composition of the first aspect can contain one antigen or more than one antigen. In certain embodiments, the liposomes encapsulate more than one antigen associated with the same immune disorder. For example, the liposomes can encapsulate two, three, four, or five different antigens, all preferably associated with the same immune disorder.

[0059] Antigens can have different chemical properties. Most antigens are peptides, however, polysaccharides, lipids, or nucleic acids can also be antigenic molecules. Additionally, 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 liposomes containing polypeptides that comprise one or more antigenic sequences. When the liposomes contain more than one antigen, 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 discrete sequence containing more than one different antigen sequence, and the antigens (antigenic sequences) can be located continuously within the polypeptide, or alternatively, they can be separated by non-antigenic linker sequences. 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 including any antigen and can generate antigen-specific tolerance to any antigen contained therein.

[0060] In one embodiment, the antigen is selected from the group consisting of autoantigens, drugs including therapeutic proteins, allergens, and alloantigens.

[0061] In one embodiment, the antigen is a peptide. In a particular embodiment, the antigen is a peptide having a size of 3 to 5000 amino acids, particularly 5 to 2000 amino acids, or 6 to 1000 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 particular embodiment, the antigen is a peptide having a size of 5 to 100 amino acids.

[0062] In one embodiment of the first aspect, the antigen is an autoantigen. The term "autoantigen" generally refers to normal substances recognized by the immune system of patients suffering from a particular autoimmune disease, often protein complexes. These antigens should not normally be targets of the immune system, but due mainly to genetic and environmental factors, normal immune tolerance to the antigen has been lost in these patients.

[0063] In one embodiment of the first aspect, the antigen is an autoantigen 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, 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 ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell aplasia, idiopathic thrombocytopenia, immune-mediated thrombotic thrombocytopenic purpura, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic steatorrhea, lichen planus, sarcoidosis, primary biliary cirrhosis, posterior uveitis, and pulmonary interstitial fibrosis.

[0064] In another embodiment of the first aspect, the antigen is an autoantigen. Autoantigens (also referred to as self-antigens) are known to the person skilled in the art and can be found in the literature. For example, autoantigens associated with various autoimmune diseases are publicly available in the AAgAtlas 1.0 database (doi: 10.1093 / nar / gkw946, Epub 2016 Oct 19; http: / / biokb.ncpsb.org / aagatlas / ).

[0065] In particular embodiments, the autoantigen is selected from the group consisting of: insulin, proinsulin, protein tyrosine phosphatase (IA2, also known as islet cell antigen 512), glutamate decarboxylase (GAD), chromogranin, and islet - glucose - 6 - phosphatase catalytic subunit related protein (IGRP), peripherin (Roep BO, Peakman M. Cold Spring Harb Perspect Med, 2012, Vol. 2(4): a007781. doi: 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 A. J Clin Invest, 2001, Vol. 108, pp. 1091 - 1096), collagen (such as type 11 collagen), human cartilage glycoprotein 39, chromogranin A, gp130 - RAPS, vimentin, citrullinated vimentin, ADAMST13, aquaporin 4, proteolipid protein, fibrillarin, nucleoprotein, nucleolin (such as small nucleolar protein), histidyl - tRNA synthetase (HisRS), histidine - tRNA synthetase (HARS1), jo - 1, thyroid - stimulating factor receptor, histone, glycoprotein gp70, ribosomal protein, pyruvate dehydrogenase dihydrolipoamide acetyltransferase, hair follicle antigen, human tropomyosin isoform 5, mitochondrial protein, islet β - cell protein, gluten, and antigenic fragments or derivatives of any of the foregoing. Examples of antigenic fragments are those disclosed in Table 3, for example.

[0066] 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 those disclosed in Table 1 and antigenic fragments of said polypeptide. 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.

[0067] Table 1

[0068]

[0069] Table 2. T1D Autoantigens

[0070] Autoantigen Tissue distribution Preproinsulin / Proinsulin / Insulin β-cells, Thymus Amylin β-cells IGRP β-cells ZnT8 β-cells GAD65 β-cells, Adrenal, CNS, Neurons, Testis, Ovary GAD67 β-cells (weak), Neurons IA-2 β-cells IA-2β β-cells ICA-69 Islet cells, Heart, Brain Imogen 38 Islet cells PDX1 β-cells Carboxypeptidase H / E β-cells, Neuroendocrine cells, Adrenal HSP60 Widespread (mitochondria) Sulfatide Widespread Ganglioside GM2-1 Islet cells ICA69 β-cells and Neurons HSP70 Widespread Peripherin Neuroendocrine cells REG1A Islet cells Cromogranina A Neuroendocrine cells IAPP β-cells

[0071] 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 and duodenal homeobox protein 1; HSP60: Heat shock protein 60.

[0072] Table 3. Antigenic fragments

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082] X represents citrulline.

[0083] In another embodiment, the antigen contained in the composition of the first aspect is an allergen. An "allergen" is any substance that can cause an undesired (e.g., type I hypersensitivity reaction) immune response (i.e., allergic response or reaction) in a subject. Immunogens include, but are not limited to, plant allergens (e.g., pollen, ragweed allergens), insect allergens, insect bite allergens (e.g., bee sting allergens), animal allergens (e.g., pet allergens, e.g., animal dander or cat Fel d 1 antigen), latex allergens, mold allergens, fungal allergens, cosmetic allergens, drug allergens, food allergens, dust, insect venoms, viruses, bacteria, etc. Food allergens include, but are not limited to, milk allergens, egg allergens, nut allergens (e.g., peanut or tree nut allergens, etc. (e.g., walnut, cashew, etc.)), fish allergens, shellfish allergens, soy allergens, legume allergens, seed allergens, and wheat allergens. Insect bite allergens include allergens related to bee stings, wasp stings, hornet stings, yellow jacket stings, etc. Insect allergens also include house dust mite allergens (e.g., Der P1 antigen) and cockroach allergens. Drug allergens include allergens related to antibiotics, NSAIDs, anesthetics, etc. Pollen allergens include grass allergens, tree allergens, weed allergens, flower allergens, etc.

[0084] In another embodiment, the antigen contained in the composition of the first aspect is related to organ or tissue rejection. Examples of such antigens include, but are not limited to, antigens from allogeneic cells, such as antigens from allogeneic cell extracts, and antigens from other cells, such as endothelial cell antigens. The antigen also includes those related to a transplantable graft. These antigens are related to an adverse immune response in the recipient of a transplantable graft or in the body of the recipient of a transplantable graft that results from the introduction of a transplantable graft, and these antigens can be presented by cells of the immune system for recognition and can produce an adverse immune response. Graft antigens include those related to organ or tissue rejection or graft-versus-host disease. Graft antigens can be obtained or inferred from the cells of a biomaterial or information related to a transplantable graft. Graft antigens generally include proteins, polypeptides, peptides, lipoproteins, glycolipids, polynucleotides, or are contained in or expressed in cells. Information related to a transplantable graft is any information that can be used to obtain or infer graft antigens for a transplantable graft. Such information includes information about antigens expected to be present in or on the cells of a transplantable graft, such as sequence information, antigen type or class, and / or its MHC class I, MHC class II, or B cell presentation restriction. Such information can also include information about the type of transplantable graft (e.g., autograft, allograft, xenograft), the molecular and cellular composition of the graft, the graft source, or the body location where the graft is to be transplanted (e.g., whole or part of an organ, skin, bone, nerve, tendon, neuron, blood vessel, fat, cornea, etc.).

[0085] In another embodiment, the antigen contained in the composition of the first aspect is a therapeutic active agent (hereinafter also referred to as "drug") that can produce an undesired immune response. The therapeutic active agent can be, for example, a therapeutic protein. Therapeutic protein antigens generally include proteins, polypeptides, peptides, lipoproteins, or are contained in cells, expressed in cells, expressed by cells, or expressed on cells. Therapeutic proteins include, but are not limited to, infusible therapeutic proteins, enzymes, enzyme cofactors, hormones, clotting factors, cytokines and interferons, growth factors, monoclonal antibodies and polyclonal antibodies (e.g., administered to a subject as replacement therapy), and proteins associated with Pompe disease (e.g., alglucosidase α, rhGAA (e.g., Myozyme and Lumizyme of 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 Heldebrant factor, tissue plasminogen activator, insulin, growth hormone, erythropoietin α, VGEF, thrombopoietin, lysozyme, antithrombin, etc. Therapeutic proteins also include adipokines, such as leptin and adiponectin. Other examples of therapeutic proteins are described below. As the antigen in the composition of the first aspect, fragments or derivatives of any therapeutic protein provided as an antigen disclosed herein are also contemplated.

[0086] Examples of therapeutic proteins used in enzyme replacement therapy for subjects with lysosomal storage diseases include, but are not limited to, imiglucerase for the treatment of Gaucher disease (e.g., CEREZYME TM )), a-galactosidase A (a-gal A) for the treatment of Fabry disease (e.g., agalsidase β, FABRYZYME TM ), acid a-glucosidase (GAA) for the treatment of Pompe disease (e.g., alglucosidase α, LUMIZYME TM , MYOZYME TM ), arylsulfatase B for the treatment of mucopolysaccharidosis (e.g., laronidase, ALDURAZYME TM , idursulfase, ELAPRASE TM , arylsulfatase B, NAGLAZYME TM ). Examples of other enzymes include oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases.

[0087] Examples of therapeutic proteins also include hormones such as melatonin (N-acetyl-5-methoxytryptamine), serotonin, thyroxine (or tetraiodothyronine) (thyroid hormone), triiodothyronine (thyroid hormone), adrenaline (or epinephrine), norepinephrine (or noradrenaline), dopamine (or prolactin-inhibiting hormone), anti-Müllerian hormone (or Müllerian-inhibiting factor or hormone), adiponectin, adrenocorticotropic hormone (or corticotropin), angiotensinogen and angiotensin, antidiuretic hormone (or vasopressin, arginine vasopressin), atrial natriuretic peptide (or atrial peptide), 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, secretin, somatostatin, thrombopoietin, thyroid-stimulating hormone (or thyrotropin), thyrotropin-releasing hormone, cortisol, aldosterone, testosterone, dehydroepiandrosterone, androstenedione, 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 enkephalin.

[0088] Examples of blood and blood coagulation factors include factor I (fibrinogen), factor II (prothrombin), tissue factor, factor V (proaccelerin, labile 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), 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, α-2-antiplasmin, tissue-type plasminogen activator (tPA), urokinase, plasminogen activator inhibitor 1 (PAI1), plasminogen activator inhibitor-2 (PAI2), cancer procoagulant, and erythropoietin α (Epogen, Procrit).

[0089] Examples of cytokines include lymphokines, interleukins, chemokines, type 1 cytokines (such as IFN-γ, TGF-β), and type 2 cytokines (such as IL-4, IL-10, and IL-13).

[0090] Examples of growth factors include adrenomedullin (AM), angiopoietin (Ang), autocrine motility factor, bone morphogenetic proteins (BMPs), 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 α (TGF-α), transforming growth factor β (TGF-β), tumor necrosis factor-α (TNF-α), vascular endothelial growth factor (VEGF), Wnt signaling pathway, placental growth factor (P1GF), [(fetal bovine growth hormone)] (FBS), IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, and IL-7.

[0091] Examples of monoclonal antibodies include Abagovomab, Abciximab, Adalimumab, Adedcatumumab, Afelimomab, Afutuzumab, Alacizumab pegol, ALD, Alemtuzumab, Altumomab pentetate, Anatumomab mafenatox, Anrukinzumab, antithymocyte globulin, Apolizumab, Arcitumomab, Aselizumab, Atezolizumab (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, Citatuzumab bogatox, Cixutumumab, Clenoliximab, Clivatuzumabtetraxetan), Conatumumab, Dacetuzumab, Daclizumab, Daratumumab, Denosumab, Detumomab, Dorlimomabaritox, Dorlixizumab, Ecromeximab, Eculizumab, Edobacomab, Edrecolomab, Efalizumab, Efungumab, Elotuzumab, Elsilimomab, Enlimomab pegol, Epitumomabcituxetan, Epratuzumab, Erlizumab, Ertumaxomab, Etaracizumab, Exbivirumab, Fanolesomab, Faralimomab, Farletuzumab, Felvizumab, Fezakinumab, Figitumumab, Fontolizumab, Foravirumab, Fresolimumab, Galiximab, Gantenerumab, Gavilimomab, Gemtuzumab ozogamicin, GCl008, Girentuximab, Glembatumumab vedotin, Golimumab, Gomiliximab, Ibalizumab, Ibritumomab tiuxetan, Igovomab, Imciromab, Infliximab, Intetumumab, Inolimomab, Inotuzumabozogamicin), Ipilimumab, Iratumumab, Keliximab, Labetuzumab, Lebrikizumab, Lemalesomab, Lerdelimumab, Lexatumumab, Libivirumab, Lintuzumab, Lorvotuzumab mertansine, Lucatumumab, Lumiliximab, Mapatumumab, Maslimomab, Matuzumab, Mepolizumab, Metelimumab, Milatuzumab, Minretumomab, Mitumomab, Morolimumab, Motavizumab, Muromonab - CD3, Nacolomab tafenatox, Naptumomab estafenatox, Natalizumab, Nebacumab, Necitumumab, Nerelimomab, Nimotuzumab, Nofetumomab merpentan, Ocrelizumab, Odulimomab, Ofatumumab, Olaratumab, Omalizumab Moaozhu, Oportuzumabmonatox), oregovomab, otelixizumab, pagibaximab, palivizumab, panitumumab, panobacumab, pascolizumab, pemtumomab, pertuzumab, pexelizumab, pintumomab, priliximab, pritumumab, rafivirumab, ramucirumab, ranibizumab, raxibacumab, regavirumab reslizumab, rilotumumab, rituximab, robatumumab, rontalizumab, rovelizumab, ruplizumab, satumomab 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 (or atlizumab), Toralizumab, Tositumomab, Trastuzumab, Tremelimumab, Tucotuzumab celmoleukin, Tuvirumab, Urtoxazumab, Ustekinumab, Vapaliximab, Vedolizumab, Veltuzumab, Vepalimomab, Visilizumab, Volociximab, Votumumab, Zalutumumab, Zanolimumab, Ziralimumab, and Zolimomab aritox.

[0092] Examples of infusion therapies or injectable therapeutic proteins include, for example, Tocilizumab (Roche / ), α-1 antitrypsin (Kamada / AAT), (Affymax and Takeda, synthetic peptide), albinterferon alfa-2b (Novartis / Zalbin TM ), (Pharming Group, C1 inhibitor replacement therapy), Tesamorelin (Theratechnologies / Egrifta, synthetic growth hormone releasing factor), Ocrelizumab (Genentech, Roche and Biogen), Belimumab (GlaxoSmithKline / ), Pegloticase (Savient Pharmaceuticals / Krystexxa TM ), Taliglucerase alfa (Protalix / Uplyso), Agalsidase alfa (Shire / )、Velaglucerase alfa (Shire). Other therapeutic proteins relevant to aspects of the present invention will be apparent to those skilled in the art, and the present invention is not limited in this regard.

[0093] In particular embodiments, the antigen contained in the composition of the first aspect is a viral antigen, particularly a viral vector, such as a viral transfer vector. Viral vectors are used to transfer therapeutic polynucleotides into cells and are increasingly used in gene therapy and for RNA or DNA-based vaccines. Unfortunately, however, readministration of gene therapy is limited by immunogenicity, which often causes severe toxicity. Therefore, improving tolerance to these gene vectors is of great interest and can be achieved by using the liposomal compositions disclosed herein. For the purposes of this specification, non-limiting viral vectors that can be considered are retroviral vectors, lentiviral vectors, herpes simplex virus (HSV)-type vectors, adenovirus-type vectors, adeno-associated virus (AAV)-type vectors, and AAV-adenovirus chimeric vectors. Adenovirus vectors can be selected from the group consisting of subgroup A, B, C, D, E, and F adenovirus vectors. Lentiviral vectors can be selected from the group consisting of HIV, SIV, FIV, EIAV, and ovine lentiviral vectors. Adeno-associated virus vectors can be selected from the group consisting of AAV1, AAV2, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAV10, and AAV11 adeno-associated virus vectors. In certain embodiments, the viral vector is a chimeric viral vector, for example, an AAV-adenovirus vector. In particular embodiments, the antigen is a viral component, particularly a viral protein. In a particular embodiment, the antigen is an envelope protein. In very particular embodiments, the antigen is a viral capsid component, such as a viral capsid protein, such as capsid proteins VP1, VP2, and VP3. In preferred embodiments, 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.

[0094] It will be apparent to those skilled in the art that immunogenic fragments of any of the above antigenic proteins are also considered antigens within the meaning of the present invention.

[0095] It is known that some oxidative and non-oxidative post-translational modifications can generate neoantigens from the original antigen. Thus, in one embodiment, the antigen is a neoantigen. Examples of non-oxidative and oxidative post-translational modifications that can generate neoantigens are citrullination, glycosylation, sumoylation, neddylation, deamination, deacylation, hydroxylation, sulfuration, oxidation, carbamylation, polyethylene glycolylation, succinylation, alkylation, sialylation phosphorylation, ubiquitination, nitrosylation, methylation, acetylation, arginylation, amidation, tyrosination, lipidation. In other embodiments, the antigen can be a post-translationally modified antigen. Thus, in another embodiment, the antigen is a post-translationally modified antigen of any of the foregoing embodiments.

[0096] 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 an embodiment, the nucleic acid encoding the antigen polypeptide is contained in an expression vector that can be transcribed when transfected into a cell. In some embodiments, the expression vector can include a plasmid, viral particle, retrovirus or adenovirus, etc. For example, the antigen can be a polynucleotide encoding a viral antigen, such as a viral capsid antigen protein, such as viral capsid protein VP1, VP2 or VP3.

[0097] Preparation of liposome composition

[0098] The liposome composition of the first aspect of the present disclosure can be prepared using various methods well known to those skilled in the art.

[0099] Liposomes carrying the antigen can be formed by directly entrapping the self-antigen during liposome formation by well-known methods such as the lipid film hydration method and the solvent injection method. In one embodiment, the liposomes are prepared by the lipid film hydration method (Bangham et al., J. Mol. Biol., 13, 238 (1965)). In another embodiment, the liposomes are prepared by the solvent injection method (Pons et al., International Journal of Pharmaceutics 95 (1993) 51-56).

[0100] In one embodiment, liposomes carrying the antigen can be prepared by the following procedure, which includes: (a) preparing a lipid mixture in a suitable solvent (such as chloroform), (b) removing the solvent, for example, by vacuum evaporation, and (c) hydrating the lipid blend with a suitable buffer containing the antigen (such as phosphate buffer) to obtain antigen-containing liposomes.

[0101] In another embodiment, antigen-carrying liposomes can be prepared by the following procedure, which includes: (a) preparing a lipid mixture in a suitable solvent (such as ethanol), and (b) injecting the liposome blend into a solution containing at least one antigen and a suitable buffer (such as phosphate buffer) to obtain antigen-containing liposomes.

[0102] When the liposomes carry more than one antigen, the hydration step (c) or the injection step (b) is carried out in the presence of a buffer of a mixture containing the antigens in the desired proportions. This proportion takes into account the specific encapsulation efficiency of each antigen.

[0103] In some embodiments, the weight ratio between the total amount of lipids forming the liposome membrane and the total amount of antigen is from 1000:1 to 1:1, or from 500:1 to 2:1, or from 350:1 to 10:1, or from 250:1 to 15:1, or from 210:1 to 20:1, or from 190:1 to 30:1. In another particular embodiment, the weight ratio between the total amount of lipids forming the liposome membrane and the total amount of antigen is from 350:1 to 10:1. In an even more particular embodiment, the weight ratio between the total amount of lipids forming the liposome membrane and the total amount of antigen is from 250:1 to 20:1. In an even more particular embodiment, the weight ratio between the total amount of lipids forming the liposome membrane and the total amount of antigen is from 210:1 to 25:1. In an even more particular embodiment, the weight ratio between the total amount of lipids forming the liposome membrane and the total amount of antigen is from 90:1 to 30:1.

[0104] Other methods known in the art can also be used to obtain antigen-carrying liposomes. For example, some embodiments contemplate first obtaining liposomes and then incorporating the antigen. There are well-known methods in the prior art for incorporating compounds into liposomes (see Maurer N. et al., Expert Opin Biol Ther, 2001, Vol. 1(6), pp. 923-47; Waterhouse D.N. et al., Methods Enzymol., 2005; Vol. 391, pp. 40-57; Urbán P. et al., Nanosc.Res.Lett., 2011, Vol. 6, p. 620).

[0105] The resulting antigen-containing liposomes obtained as described above or by any other method known to the person skilled in the art can be subjected to additional 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, the purification step for removing unencapsulated antigen can be carried out by filtration through a 100 Kda filter. The antigen-containing liposomes can be further homogenized or separated according to size. Extrusion can be used to homogenize the liposome size, i.e., to produce liposomes with a predetermined average size by pushing the liposomes through a filter with a specific pore size under pressure. Filtration such as tangential flow filtration can also be used to purify and separate the liposomes according to their size, i.e., to produce a liposome population with fewer impurities and a desired size distribution. Other methods for separating liposome populations according to their size are centrifugation (e.g., ultracentrifugation), size exclusion chromatography, gel permeation chromatography, and combinations thereof.

[0106] In one embodiment, the liposome composition of the first aspect is prepared by a process comprising: obtaining antigen-carrying liposomes by any of the above methods and optionally additional purification, separation and / or enrichment steps. In a particular embodiment, the process further comprises a purification step for removing unencapsulated peptides. In another embodiment, the process further comprises a homogenization step. In another embodiment, the process further comprises a separation step. In some embodiments, the process further comprises an enrichment step. For example, a portion of the composition obtained by any of the above methods can be separated to isolate a liposome population, e.g., a liposome population with a size less than 200 nm. Then, the isolated liposome population can be added to a composition containing two populations, thereby enriching the selected liposome population, e.g., a liposome population with a size below 200 nm. In other embodiments, extrusion, physical separation or sonication is used to homogenize the liposome size or to obtain a desired liposome population. Then, the homogenized liposomes or the separated liposome population can be added to a liposome composition containing the population of interest defined in the first aspect, whereby the composition is enriched in a specific liposome population.

[0107] The composition of the first aspect of the present disclosure can be obtained by separately preparing each of the two liposome populations defined in the first aspect and then mixing them in a suitable ratio. The composition can also be obtained by a method that produces a composition already containing the two populations defined in the first aspect.

[0108] In one embodiment, the liposome composition of the first aspect can be prepared by the following steps, which include: (a) obtaining antigen-carrying liposomes, for example, by any of the above methods; (b) separating liposomes with a size of 2 - 200 nm; (c) separating liposomes with a size of 500 - 2000 nm; (d) mixing the liposomes with a size of 2 - 200 nm and the liposomes with a size of 500 - 2000 nm.

[0109] In another embodiment, the liposome composition of the first aspect is prepared by the following steps, which include: obtaining antigen-carrying liposomes, for example, by any of the above methods; (b) performing a separation step on a part of the liposome composition obtained in (a) to separate liposomes with a size of 2 - 200 nm; (c) adding the separated liposomes with a size of 2 - 200 nm to the liposome composition obtained in (a) to obtain a liposome composition rich in liposomes with a size of 2 - 200 nm.

[0110] The present disclosure contemplates liposome compositions obtainable by any of the above methods.

[0111] In a particular embodiment, a liposome composition having a wide size polydispersity and carrying an antigen can be prepared by the solvent injection method, which includes the following steps: preparing a lipid blend in a suitable solvent (such as ethanol), and injecting the lipid blend into a solution containing at least one antigen and a suitable buffer (such as phosphate buffer) to obtain antigen-containing liposomes. A low injection rate, a specific number of injections, and gentle shaking instead of vortexing or high-speed stirring promote the formation of larger-sized liposomes. By not applying any subsequent homogenization step (such as pressure extrusion) and not applying any additional purification step for removing smaller liposomes (such as tangential flow filtration) thereafter, the liposome composition will exhibit a wide size distribution. To reduce the average diameter of the liposome composition, some consecutive extrusion cycles can be applied to the heterogeneous and polydisperse composition. To achieve this, the liposomes are sequentially filtered through a series of polycarbonate membranes with pores of decreasing diameter, thereby conveniently and reproducibly obtaining liposomes with an average size close to the pore size of the membrane. In addition, directly applying simple sonication to the preparation with a needle at room temperature or indirectly in a water bath sonicator also reduces the average diameter of the liposomes. Other industrial homogenization methods for breaking large liposomes are microfluidization, high-pressure homogenization, and shear force-induced homogenization techniques. In one embodiment, the present invention also contemplates liposome compositions obtainable by this particular method.

[0112] Preparation

[0113] In one embodiment, the liposomal composition of the first aspect is a pharmaceutical composition. The present invention thus 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 an 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 invention provides a pharmaceutical or veterinary composition consisting of an effective amount of the liposomal composition defined in the first aspect and a suitable pharmaceutically or veterinarily acceptable excipient or carrier.

[0114] As used herein, the expression "therapeutically effective amount" refers to the amount of liposomes that, when administered, is sufficient to prevent or to some extent alleviate the development of the symptoms of one or more of the disorders addressed, particularly the amounts of the first liposomal population and the second liposomal population of the composition of the first aspect. The specific dose of the compound administered according to the present disclosure will obviously depend on the particular circumstances, including the antigen administered, the route of administration, the particular disorder being treated, and similar considerations. In one embodiment, the therapeutically effective amount in the sense of the present invention relates to reducing the level of an unwanted immune response. In other embodiments, the therapeutically effective amount relates to completely preventing an unwanted immune response. In other embodiments, the therapeutically effective amount relates to delaying the onset of an unwanted immune response. In the sense of the present disclosure, the therapeutically effective amount results in a tolerant immune response of the subject to the antigen. The achievement of any of the above can be monitored by conventional methods.

[0115] The present disclosure also contemplates compositions in which the liposomes carry more than one antigen 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 immune disorder.

[0116] In the present invention, the term "pharmaceutically acceptable excipient or carrier" refers to a pharmaceutically acceptable material, composition, or carrier. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It must also be suitable for contact with the tissues or organs of humans and animals without producing excessive toxicity, irritation, allergic responses, antigenicity, or other problems or complications commensurate with a reasonable benefit / risk ratio. Similarly, the term "veterinarily acceptable" means suitable for contact with non-human animals.

[0117] The antigen-containing liposomes of the present composition are themselves considered to be active pharmaceutical agents, and thus antigen-containing liposomes within a specific size range play a role in promoting tolerance to the specific antigen contained therein. Therefore, it is noteworthy that no other active pharmaceutical agents for inducing tolerance are required. Importantly, the composition of the present invention does not need to be used in combination with other immunomodulators. In particular, the composition does not need to contain an immunosuppressant or be used in combination therewith. In a particular embodiment, the composition of the first aspect does not contain an immunosuppressant. In other words, the composition of the first aspect is completely free of immunosuppressants.

[0118] Nevertheless, although not required to achieve the technical effect, the present disclosure also contemplates using the liposome compositions described herein in combination with other active pharmaceutical agents. Thus, the pharmaceutical compositions contemplated herein may contain other active pharmaceutical agents, such as other immunomodulators, such as immunosuppressants. Examples of immunosuppressants include glucocorticoids (prednisolone, methylprednisolone, betamethasone), cytostatic agents (methotrexate...), antibodies (rituximab...), drugs acting on immunophilins (cyclosporine, tacrolimus, sirolimus, everolimus...), interferons, TNF-binding proteins, mycophenolate mofetil, and small biologics (fingolimod, myriocin).

[0119] The formulation of the composition of the present disclosure depends to a large extent on the route of administration. In one embodiment, the pharmaceutical composition is administered orally to a patient. Oral compositions include tablets, powders, capsules, cachets, and liquid syrups, suspensions, and elixirs, all of which can be formulated by methods well known in the art. The composition of the present invention can also be administered to a patient by intravenous, intra-arterial, intraperitoneal (i.p.), subcutaneous, intramuscular, or intradermal routes. Compositions suitable for these routes of administration are also well known in the art and include injection solutions, perfusion solutions, powders for reconstitution of liquid injections, and prefilled syringes. In the sense of the present disclosure, the compositions disclosed herein can also be formulated in the form of, for example, creams, gels, ointments, or skin patches for intranasal or inhaled administration, rectal administration, or for topical application. The methods for preparing these formulations are known in the art. In addition, the compositions of the present disclosure can be formulated into controlled-release dosage forms. Controlled-release dosage forms are known in the art and are particularly advantageous for treating chronic diseases or for administering active pharmaceutical agents that may be toxic at high doses or exhibit a low half-life pattern upon administration to a patient.

[0120] Also disclosed herein is a kit comprising:

[0121] (a) a liposome composition as defined above, optionally together with a pharmaceutically acceptable excipient or carrier

[0122] (b) an optional additional active pharmaceutical agent; and

[0123] (c) Optional instructions for use.

[0124] The present invention also discloses a container or an injection device, which comprises the liposome composition as defined above, preferably together with a pharmaceutically acceptable excipient or carrier.

[0125] Therapeutic use

[0126] As described above, the liposome compositions of the present invention can be used for treatment because they promote tolerance to the antigen contained in the composition. In one embodiment, the induction of tolerance includes B cell-mediated tolerance. In another embodiment, the induction of tolerance includes T cell-mediated tolerance, particularly tolerance mediated by the tolerogenic presentation of the antigen by dendritic cells. When referring to the medical use of the liposome compositions of the present invention, it is contemplated that the compositions can be pharmaceutical compositions which comprise a therapeutically effective amount of the liposome composition as defined in the first aspect, optionally together with other suitable pharmaceutically or veterinarily acceptable excipients or carriers.

[0127] The liposome compositions of the present invention, as broadly described above, contain two populations of liposomes of different sizes, both containing antigen and having specific membrane components. These liposome populations, without the addition of additional active ingredients (such as immunosuppressants), themselves constitute active ingredients that induce tolerance and have the effect of treating disorders associated with immune response dysregulation (such as exacerbation). Each liposome population targets a different tolerogenic mechanism. While the liposome population with a size above 500 nm induces antigen-specific tolerance through a mechanism similar to efferocytosis and involving tolerogenic presentation by dendritic cells, the liposome population with a size of 2 - 200 nm is capable of inducing tolerance through B cells. The dual effect has not been previously disclosed and provides an improved tolerogenic effect. In addition, the liposome compositions disclosed herein effectively treat immune disorders that cannot be treated by previous tolerance inducers due to this dual effect.

[0128] The liposome composition as defined above is used for immune regulation, and more particularly, for suppressing an excessive immune response to a specific antigen. In some embodiments, the liposome composition as defined above is used for treating disorders associated with abnormal (usually excessive) immune responses. Importantly, the tolerogenic / immune regulatory effect of the liposome composition is antigen-specific. Thus, in certain embodiments, the liposome composition as defined above is used for treating disorders associated with abnormal (usually excessive) immune responses, wherein the liposome composition restores tolerance to the antigen contained in the liposome composition.

[0129] In one embodiment, the disorders associated with an abnormal (usually excessive) immune response are selected from autoimmune diseases, allergies, drug hypersensitivity reactions, and transplant rejection.

[0130] For the purposes of the present invention, the term "treatment" includes prophylactic 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 a particular embodiment, the treatment is prophylactic treatment. In one embodiment, prophylactic treatment includes partial or complete prevention of an immune disorder. In one embodiment, an abnormal immune response to a self-antigen is prevented, thereby not triggering the pathogenic events underlying the abnormal immune response. In another particular embodiment, the treatment includes improving, slowing down, arresting, or reversing the pathological mechanisms underlying the immune disorder. In a particular embodiment, the treatment includes improving, slowing down, arresting, delaying, or reversing the clinical symptoms of the immune disorder.

[0131] The present disclosure contemplates treating immune disorders in patients who, despite having an abnormal immune response and some tissue damage, do not exhibit clinical symptoms or exhibit only very few clinical symptoms of the disease. This stage is commonly referred to as the "preclinical" stage and is typical for transplant rejection and many autoimmune diseases, such as prediabetes in T1D. In prediabetes, pancreatic beta cells are damaged to a certain extent but only meet some of the diagnostic criteria for diabetes. Such diseases in the preclinical stage can be effectively treated with the liposomal compositions of the present invention. Thus, one embodiment is directed to treating autoimmune diseases in the preclinical stage. In addition, for the advanced stages of immune diseases with severe tissue damage and obvious clinical symptoms, they can also be effectively treated by administering an effective amount of the liposomal compositions of the present disclosure.

[0132] In particular embodiments, the disorder is an autoimmune disease selected from the group consisting of: 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, Crohn's disease, aphthous ulcers, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell aplasia, idiopathic thrombocytopenia, immune-mediated thrombotic thrombocytopenic purpura, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic steatorrhea, lichen planus, sarcoidosis, primary biliary cirrhosis, posterior uveitis, and pulmonary interstitial fibrosis.

[0133] In particular embodiments, the present disclosure provides the liposome composition or a pharmaceutical composition thereof as defined in the first aspect for treating MS. In another embodiment, the autoimmune disease to be treated is T1D. In another embodiment, the present invention provides the liposome composition or a pharmaceutical composition thereof as defined in the first aspect for treating T1D in a pre-diabetic subject. In another particular embodiment, the autoimmune disease to be treated is myasthenia gravis. In another particular embodiment, the autoimmune disease to be treated is selected from the group consisting of: rheumatoid arthritis, neuromyelitis optica, myositis or anti-synthetase syndrome, thrombotic thrombocytopenic purpura, and celiac disease.

[0134] In another particular embodiment, the disorder is an allergy or allergic disorder. "Allergy" or "allergic disorder" includes, but is not limited to, allergic asthma, hay fever, urticaria, eczema, plant allergy, bee sting allergy, pet allergy, latex allergy, mold allergy, cosmetic allergy, food allergy, allergic rhinitis (rhinitis or coryza), local allergic reaction, anaphylaxis, atopic dermatitis, hypersensitivity reaction, and other allergic symptoms. In some embodiments, the allergy is a food allergy. Food allergies include, but are not limited to, milk allergy, egg allergy, nut allergy, fish allergy, shellfish allergy, soy allergy, or wheat allergy.

[0135] The response to therapeutic drugs can also produce adverse immunogenicity. Thus, in another specific embodiment, the disorder is drug hypersensitivity. "Drug hypersensitivity" refers to an immune-mediated reaction to a drug. The symptoms range from mild to severe and include rashes, allergic reactions, and serum sickness. In the context of the present invention, there are no particular limitations on the drugs that cause drug allergies.

[0136] Ongoing efforts are being made to reduce the immunogenicity of life-saving gene therapy vectors. Adverse immune responses to gene therapy vectors often cause severe toxic reactions and form neutralizing antibodies, preventing any necessary readministration. Thus, in one embodiment, the use of liposome compositions for preventing or treating adverse reactions caused by gene therapy. In another specific embodiment, the use of liposome compositions for preventing or treating adverse reactions caused by viral vectors, particularly viral transfer vectors. The present invention also contemplates the use of the liposome compositions disclosed herein for preventing or treating adverse reactions caused by any drug, such as therapeutic proteins.

[0137] In another specific embodiment, the disorder is transplant rejection. "Transplant rejection" occurs when the recipient's immune system rejects the transplanted tissue, which can damage the transplanted tissue. "Transplantation" and its variants refer to the implantation of a graft (also known as a transplant) into a recipient, whether the transplant is syngeneic (the donor and recipient have the same genes), allogeneic (the donor and recipient have different genetic origins but belong to the same species), or xenogeneic (the donor and recipient are from different species). The term "allograft" or "allogeneic graft" or "allogenic graft" refers to a transplanted tissue such as an organ from a donor who belongs to the same species as the recipient but has a different genetic origin. For example, an allograft can be a solid organ, particularly a kidney, or it can 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. It will be apparent to those skilled in the art that the allografts covered can also be organ tissues, such as kidney tissue, lung tissue, heart tissue, liver tissue, etc. As used herein, the terms "graft rejection" or "transplant rejection" cover acute and chronic transplant rejection reactions and refer to the rejection of transplanted tissue by the recipient's immune system. The present invention contemplates any type of transplant rejection reaction for any type of graft. In a specific embodiment, the immune disorder is graft-versus-host disease

[0138] The specific dose of the liposome composition administered in the present invention can be determined according to specific circumstances, including the antigen administered, the route of administration, the specific disorder being treated, and similar considerations. In addition, it may also be necessary to take into account the clinical stage of the immune disorder to be treated to determine the appropriate dose of the liposome composition to be administered.

[0139] In summary, the dose of the liposomal composition to be administered is determined according to various circumstances. In some embodiments, the dose is calculated based on the amount of liposomes per Kg body weight (mg liposomes / Kg body weight). In one embodiment, the dose is from 0.025 to 50 mg of liposomes / Kg body weight, particularly from 0.25 to 10 mg of liposomes / Kg body weight.

[0140] In addition, medical practitioners will determine how much of the drug to administer to a subject in need to treat an immune disorder. In this regard, it is noteworthy that the liposomal composition therapy developed by the present inventors does not require long-term use. Instead, by administering the liposomal compositions of the present disclosure, a lasting restoration of tolerance can be achieved, thereby enabling effective treatment of immune diseases. This is in stark contrast to known immunomodulatory or anti-inflammatory treatments for immune disorders, which are typically life-long treatments. The liposomal compositions of the present disclosure can achieve a lasting tolerogenic effect after a single administration or alternatively after 2 - 4 administrations. However, medical practitioners may decide that more doses are needed to treat advanced stages of the disease or for any other reason. In one embodiment, the treatment comprises administering 1 to 10 doses of the liposomal composition to the subject, such as 2, 3, 4, 5, 6, 7, or 8 doses.

[0141] In one embodiment, the subject requires 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 received, is receiving, or will receive a therapeutic protein for an unwanted immune response that they have experienced, are experiencing, or expect to experience.

[0142] As described above, the present invention does not exclude the use of the liposomal compositions described herein in combination with other active agents. Thus, in one embodiment, the liposomal composition for any of the uses described above is for use in combination with another active agent. The liposomal composition and the additional active agent can be administered in the same composition or in different compositions, and in the latter case, they can be administered sequentially, simultaneously, or within a treatment interval. In a particular embodiment, the additional active agent is another immunomodulator, such as an immunosuppressant.

[0143] For completeness, the present specification also discloses the following numbered embodiments:

[0144] 1. A composition comprising two populations of liposomes, wherein:

[0145] - The first population of liposomes has a size that is equal to or less than 200 nm,

[0146] - The second population of liposomes has a size that is equal to or greater than 500 nm,

[0147] - The first liposome population and the second liposome population carry one or more antigens, and

[0148] - The liposomal membrane of each liposome in the first liposome population and the second liposome population contains phosphatidylserine in an amount of 20 wt% to 60 wt% relative to the total composition of the membrane of the liposome.

[0149] 2. A composition consisting essentially of two liposome populations, wherein:

[0150] - The size of the first liposome population is equal to or less than 200 nm,

[0151] - The size of the second liposome population is equal to or greater than 500 nm,

[0152] - The first liposome population and the second liposome population carry one or more antigens, and

[0153] - The liposomal membrane of each liposome in the first liposome population and the second liposome population contains phosphatidylserine in an amount of 20 wt% to 60 wt% relative to the total composition of the membrane of the liposome.

[0154] 3. The composition according to any one of the preceding embodiments, wherein

[0155] - The size of the first liposome population is 2 to 200 nm, and

[0156] - The size of the second liposome population is 500 to 2000 nm.

[0157] 4. The composition according to any one of the preceding embodiments, wherein 15 - 75% of the liposomes in the composition correspond to the first population.

[0158] 5. The composition according to the preceding embodiment, wherein 15 - 55% of the liposomes in the composition correspond to the first population.

[0159] 6. The composition according to the preceding embodiment, wherein 20 - 50% of the liposomes in the composition correspond to the first population.

[0160] 7. The composition according to any one of the preceding embodiments, wherein 2 - 40% of the liposomes in the composition correspond to the second population.

[0161] 8. The composition according to the preceding embodiment, wherein 2 - 25% of the liposomes in the composition correspond to the second population.

[0162] 9. The composition according to the preceding embodiment, wherein 2 - 15% of the liposomes in the composition correspond to the second population.

[0163] 10. The composition according to any one of the foregoing embodiments, wherein the amount of phosphatidylserine in the membrane of the liposome is 35 to 45% by weight based on the total components of the membrane of the liposome.

[0164] 11. The composition according to the foregoing embodiment, wherein the amount of phosphatidylserine in the membrane of the liposome is 35 to 45% by weight based on the total components of the membrane of the liposome. For example, it is 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, or 44% by weight based on the total components of the membrane of the liposome.

[0165] 12. The composition according to any one of the foregoing 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, 1-oleoyl-2-palmitoyl-phosphatidylserine, 1-oleoyl-2-stearoyl-phosphatidylserine, 1-palmitoyl-2-oleoyl-phosphatidylserine, 1-stearoyl-2-oleoyl-phosphatidylserine, and combinations thereof.

[0166] 13. The composition according to any one of the foregoing embodiments, wherein the liposome membrane further comprises phosphatidylcholine (PC).

[0167] 14. The composition according to the foregoing embodiment, wherein the amount of PC in the membrane of the liposome is 20 to 50% by weight based on the total components of the membrane of the liposome.

[0168] 15. The composition according to the foregoing embodiment, wherein the amount of PC in the membrane of the liposome is 32 to 42% by weight based on the total components of the membrane of the liposome. For example, it is 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, or 41% by weight based on the total components of the membrane of the liposome.

[0169] 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), dipalmitoyl phosphatidylcholine (DPPC), dioleoyl phosphatidylcholine, 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.

[0170] 17. The composition according to any one of the foregoing embodiments, wherein the liposomal membrane further comprises a sterol lipid or a steroid, particularly cholesterol (CHOL).

[0171] 18. The composition according to the foregoing embodiment, wherein the amount of CHOL in the liposomal membrane is 10 to 40% by weight relative to the total composition of the liposomal membrane.

[0172] 19. The composition according to the foregoing embodiment, wherein the amount of CHOL in the liposomal membrane is 20 to 35% by weight relative to the total composition of the liposomal membrane, for example, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28% or 29% by weight relative to the total composition of the liposomal membrane.

[0173] 20. The composition according to any one of the foregoing embodiments, wherein the liposomal membrane comprises PS, PC and CHOL.

[0174] 21. The composition according to the foregoing embodiment, wherein the liposomal membrane consists essentially of PS, PC and CHOL.

[0175] 22. The composition according to any one of Embodiments 1-21, wherein the molar ratio of PS:PC:CHOL is 1:(0.2-4):(0.2-5).

[0176] 23. The composition according to the foregoing embodiment, wherein the molar ratio of PS:PC:CHOL is 1:(0.6-1.8):(0.7-2.5), particularly 1:(0.7-1.5):(0.9-2).

[0177] 24. The composition according to the foregoing embodiment, wherein the molar ratio of PS:PC:CHOL is 1:(0.8-1.4):(1.1-1.9), particularly 1:(0.9-1.3):(1.2-1.7).

[0178] 25. The composition according to any one of the foregoing embodiments, wherein the first population of liposomes provides 5 to 30% by weight of the total liposomal PS in the composition, particularly 5 to 20% by weight of the total liposomal PS in the composition.

[0179] 26. The composition according to any one of the foregoing embodiments, wherein the second population of liposomes provides 5 to 75% of the total liposomal PS in the composition, particularly 5 to 55% by weight of the total liposomal PS in the composition.

[0180] 27. The composition according to any one of the foregoing embodiments, wherein the liposomes contain an antigen.

[0181] 28. The composition according to any one of the foregoing embodiments, wherein the liposome contains more than one antigen.

[0182] 29. The composition according to any one of the foregoing embodiments, wherein the antigen is a peptide.

[0183] 30. The composition according to the foregoing embodiment, wherein the antigen peptide has a size of 5 to 1000 amino acids.

[0184] 31. The composition according to the foregoing embodiment, wherein the antigen peptide has a size of 5 to 200 amino acids, such as 15 to 100 amino acids.

[0185] 32. The composition according to any one of the foregoing embodiments, wherein the antigen is an autoantigen.

[0186] 33. The composition according to the foregoing embodiment, wherein the autoantigen is associated with an autoimmune disease 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 ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell aplasia, idiopathic thrombocytopenia, immune-mediated thrombotic thrombocytopenic purpura, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic steatorrhea, lichen planus, sarcoidosis, primary biliary cirrhosis, posterior uveitis, and interstitial pulmonary fibrosis.

[0187] 34. The composition according to any one of embodiments 32 - 33, wherein the autoantigen is selected from the group consisting of: insulin, proinsulin, protein tyrosine phosphatase (IA2), glutamate 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), human cartilage glycoprotein 39, chromogranin A, gp130 - RAPS, vimentin, citrullinated vimentin, ADAMST13, aquaporin 4, proteolipid protein, fibrillarin, nucleoprotein, nucleolar protein (e.g., small nucleolar protein), histidyl - tRNA synthetase (HisRS), histidine - tRNA synthetase (HARS1), jo - 1, thyroid - stimulating factor receptor, histone, glycoprotein gp70, ribosomal protein, pyruvate dehydrogenase dihydrolipoamide acetyltransferase, hair follicle antigen, human tropomyosin isoform 5, mitochondrial protein, islet β - cell protein, 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, antigen fragments disclosed in Table 3.

[0188] 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 antigenic fragments or derivatives of the antigens in Table 1 or Table 2.

[0189] 36. The composition according to any one of embodiments 1 - 31, wherein the antigen is a drug, including a therapeutic protein.

[0190] 37. The composition according to any one of embodiments 1 - 31, wherein the antigen is an allergen.

[0191] 38. The composition according to any one of embodiments 1 - 31, wherein the antigen is an alloantigen.

[0192] 39. The composition according to any one of Embodiments 1-31, wherein the antigen is a viral antigen, particularly a viral vector, and more particularly, the antigen is a viral envelope protein or a viral capsid protein, such as selected from VP1, VP2, and VP3.

[0193] 40. The composition according to any one of Embodiments 36-39, wherein the antigen is a protein fragment, preferably an immunogenic fragment.

[0194] 41. The composition according to any one of the foregoing embodiments, wherein the antigen is a polynucleotide encoding an antigenic protein or a fragment thereof.

[0195] 42. The composition according to any one of the foregoing embodiments, wherein the composition does not contain an immunosuppressant.

[0196] 43. A method for preparing the liposome composition as defined in any one of the foregoing embodiments, the method comprising the following steps:

[0197] (a) preparing a lipid blend in a suitable solvent, and

[0198] (b) injecting the lipid blend into a solution containing at least one antigen and a suitable buffer.

[0199] 44. The method according to Embodiment 43, wherein the liposome mixture comprises PS, PC, and CHOL.

[0200] 45. The method according to any one of Embodiments 43-44, wherein the molar ratio of PS:PC:CHOL is 1:(0.8-1.4):(1.1-1.9), particularly 1:(0.9-1.3):(1.2-1.7).

[0201] 46. The method according to any one of Embodiments 43-45, wherein the solvent in step (a) is ethanol.

[0202] 47. The method according to any one of Embodiments 43-46, wherein the buffer in step (b) is phosphate buffered saline.

[0203] 48. The method according to any one of Embodiments 43-47, wherein step (b) comprises slow injection and gentle oscillation.

[0204] 49. The method according to any one of Embodiments 43-48, further comprising a separation step selected from filtration (such as tangential flow filtration), centrifugation (such as ultracentrifugation), size exclusion chromatography, gel permeation chromatography, and combinations thereof.

[0205] 50. A liposome composition obtainable by the method as defined in any one of Embodiments 43-49.

[0206] 51. A composition according to any one of embodiments 1 - 42 or 50, which is a pharmaceutical composition and comprises a pharmaceutically acceptable excipient and carrier.

[0207] 52. A composition according to the foregoing embodiments, which is for intravenous administration, intra - arterial administration, intra - peritoneal administration (i.p.), subcutaneous administration, intramuscular administration or intradermal administration.

[0208] 53. A composition according to any one of embodiments 51 - 52 further comprises an additional active ingredient.

[0209] 54. A kit comprising:

[0210] (a) A composition according to any one of embodiments 1 - 42 or 50 - 53

[0211] (b) Optionally, an additional active agent; and

[0212] (c) Optionally, instructions for use.

[0213] 55. A composition as defined in any one of embodiments 1 - 42 or 50 - 53 is used as a medicine.

[0214] 56. A composition as defined in any one of embodiments 1 - 42 or 50 - 53 is used for inducing tolerance to an antigen.

[0215] 57. A composition for use in the application of embodiment 56, wherein the induction of tolerance includes B - cell - mediated and T - cell - mediated tolerance to the antigen.

[0216] 58. A composition as defined in any one of embodiments 1 - 42 or 50 - 53 is used for immunomodulation.

[0217] 59. A composition as defined in any one of embodiments 1 - 42 or 50 - 53 is used for suppressing an excessive immune response.

[0218] 60. A composition as defined in any one of embodiments 1 - 42 or 50 - 53 is used for treating disorders associated with abnormal immune responses, wherein the liposomal composition restores tolerance to the antigen contained in the liposomal composition.

[0219] 61. A composition as defined in any one of embodiments 1 - 42 or 50 - 53 is used for treating autoimmune diseases.

[0220] 62. The composition for use as described in any of the foregoing embodiments, wherein the autoimmune disease is selected from the group consisting of: T1D, lupus erythematosus, arthritis (including 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 ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell aplasia, idiopathic thrombocytopenia, immune-mediated thrombotic thrombocytopenic purpura, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic steatorrhea, lichen planus, sarcoidosis, primary biliary cirrhosis, posterior uveitis, and pulmonary interstitial fibrosis.

[0221] 63. The composition for use as described in any of the foregoing embodiments, wherein the autoimmune disease is selected from the group consisting of: T1D, multiple sclerosis, rheumatoid arthritis, neuromyelitis optica, myasthenia gravis, myositis or anti-synthetase syndrome, thrombotic thrombocytopenic purpura, and celiac disease.

[0222] 64. The composition as defined in any of embodiments 1 - 42 or 50 - 53 is used for treating allergy.

[0223] 65. The composition as defined in any of embodiments 1 - 42 or 50 - 53 is used for treating hypersensitivity reactions (such as drug hypersensitivity reactions).

[0224] 66. The composition as defined in any of embodiments 1 - 42 or 50 - 53 is used for treating transplant rejection.

[0225] 67. The composition as defined in the foregoing embodiments, wherein the transplant rejection is graft-versus-host disease.

[0226] 68. The composition as defined in any of embodiments 1 - 42 or 50 - 53 is used for treating adverse immune effects triggered by gene therapy.

[0227] 69. The composition as described in any of embodiments 1 - 42 or 50 - 53 is used in combination with an additional active ingredient.

[0228] 70. A composition for the combined use of applications as described in the foregoing embodiments, wherein the composition defined in any one of Embodiments 1-42 or 50-53 and an additional active ingredient are administered sequentially, simultaneously, or within a treatment interval.

[0229] 71. A composition for the combined use of applications as described in any one of Embodiments 69-70, wherein the additional active ingredient is not an immunosuppressant.

[0230] 72. A composition for the application as described in any one of Embodiments 55-71, wherein the dose of the liposomal composition is 0.25 to 50 mg of liposomes / Kg body weight

[0231] 73. A composition for the application as described in any one of Embodiments 60-72, wherein the treatment is a prophylactic treatment.

[0232] Throughout the specification and claims, the word "comprising" and its variants are not intended to exclude other technical features, additives, ingredients, or steps. Additionally, the word "comprising" also encompasses the cases of "consisting of" and "consisting essentially of". Other objects, advantages, and features of the present invention will become apparent to those skilled in the art upon studying the specification of the present invention, or those skilled in the art can understand them by implementing the present invention. The following examples and drawings are provided for illustration only, and they are not intended to limit the present invention. Furthermore, the present invention encompasses all possible combinations of the specific embodiments and preferred embodiments described herein.

[0233] Examples

[0234] 1. Materials and methods

[0235] 1.1 Manufacture of Liposomes

[0236] Antigenic peptides (antigens) were selected based on their reported correlation with the development of autoimmune diseases. Peptides - human mutant citrullinated vimentin (MCV) (SEQ ID NO: 44), MOG 35-55 (SEQ ID NO: 4) and human AChR 146-162 (SEQ ID NO: 57) were purchased from the Peptide Synthesis Facility (Department of Experimental and Health Sciences, Pompeu Fabra University, Spain), with a purity of >95%; human insulin (CAS number: 11061-68-0; Ins(h)) was purchased from Sigma Aldrich (USA). It was resuspended in phosphate-buffered saline (DPBS, Fisher Scientific, Spain) at 0.5 mg / ml. In the clean room of Ahead Therapeutic (Spain, ) Liposomes were manufactured under GMP-like conditions. The 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).

[0237] 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 desired antigen, with a final lipid concentration of 30 mM. The liposome emulsion was extruded twice through a polycarbonate membrane with a 1-μm pore size using a Lipex Thermobarrel Extruder (Evonik, Canada) and sonicated for 10 minutes in a water bath sonicator (Bandelin, Sonorex RK100) to obtain the liposome composition of the present invention (abbreviated as PS-PC-Chol liposomes 200&500).

[0238] PS-PC-Chol liposomes smaller than 200 nm (abbreviated as PS-PC-Chol liposomes <200) were obtained by multi-step extrusion through a 200-nm or 100-nm pore size membrane.

[0239] PS-PC-Chol liposomes with a diameter greater than 500 nm (abbreviated as PS-PC-Chol liposomes >500) were prepared as described in WO2015107140A1.

[0240] Fluorescent liposomes were prepared with 3-hexanoyl-nitrophenyl oxadiazole cholesterol (NBD, Cayman Chemical, USA) or Alexa Fluor 750 dye (ThermoFisher Scientific, USA). The DOPE-AF750 dye was obtained by incubating 0.11 μL of TEA and 4 mg of 1,2-dioleoyl-sn-glycero-phosphoethanolamine (DOPE) in a 0.5 mg / mL Alexa Fluor 750 ethanol solution. The solution was stirred for 5 hours. Before injection into the DPBS solution, 0.15 mol% of 3-hexanoyl-NBD cholesterol or DOPE-AF750 dye was added to the ethanol solution of DOPS, DMPC, and cholesterol.

[0241] The particle size was determined by nanoparticle tracking analysis (NTA) using a Malvern Panalytical Nanosight NS300, and the ζ-potential was measured by dynamic light scattering using a Zetasizer Nano ZS (Malvern, Instruments Ltd, UK). The peptide encapsulation efficiency (EE) was indirectly calculated by the following equation: EE (%) = [(total peptide – free peptide) / total peptide] × 100, where the total peptide is the total concentration of the peptide in the formulation and the free peptide is the concentration of the unencapsulated peptide. To measure the free peptide, the liposome emulsion was centrifuged at 3700 g for 20 minutes at room temperature, and the concentration of the unencapsulated peptide in the supernatant was evaluated using a PIERCE BCA protein assay kit (Thermo Fisher Scientific Inc., USA).

[0242] Unencapsulated peptide was removed from the liposome suspension using a 100KDa Spectra / Por 7 dialysis tube (SPECTRUM; 131420). The suspension was dialyzed against 50 - 60 volumes of DPBS Ca / Mg with gentle stirring at room temperature for 24 hours, and the dialysis buffer was changed once.

[0243] 1.2 Mice

[0244] Wild-type non-obese diabetic (NOD) mice were purchased from the Jackson Laboratory (USA) and bred under specific pathogen-free (SPF) conditions at the Center for Comparative Medicine and Bioimaging (Badalona, Spain). These mouse strains spontaneously develop autoimmune diabetes after 12 weeks of age. Female C57BL / 6J mice, 8 weeks old, purchased from Envigo Laboratories (Milan, Italy), were used for experimental autoimmune encephalomyelitis (EAE) experiments.

[0245] All mice were housed in a temperature- and humidity-controlled facility, had free access to food and water, and were subjected to a 12-hour light / dark cycle. All experiments using animal models were conducted in accordance with the regulations of the animal facility, fully compliant with the recommendations set forth in the Declaration of Helsinki for Animal Experimentation Research and the Principles of Laboratory Animal Care of the National Institutes of Health of the United States. The research protocol has been approved by the government animal ethics committee of the institutions involved in the study.

[0246] 1.3 Interaction of Liposomes with Mouse B Cells (Spleen Cells)

[0247] Spleens were collected from non-obese diabetic (NOD) mice (Jackson Laboratory, Bar Harbor, ME, USA) at 12 weeks and mechanically disrupted. The cell suspension was homogenized and washed twice before cell counting by flow cytometry using 7-AAD (BD Biosciences, San Jose, CA, USA). Then, 10 6 cells / ml of splenocytes were cultured in RPMI-1640 medium (Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (ThermoFisher Scientific, Waltham, MA, USA), 100 IU / ml penicillin (NormonSA, Madrid, Spain), and 100 μg / ml streptomycin (Laboratorio Reig Jofré, Sant Joan Despí, Spain), and incubated with 1 mM of PS-PC-Chol-Insh liposomes <200 (non-fluorescent) or 1 mM PS-PC-Chol-NBD-Insh liposomes <200 (fluorescent) for 4 hours at 37 °C and 5% CO2 without stimulation. After incubation, cells were collected, washed, and stained with 7-AAD, B220 BV510, CD19 APC-R700 (BD Biosciences), and CD1d PECy7 (BioLegend, San Diego, CA) for 20 minutes at 4 °C. After washing, cells and NBD positivity were analyzed by flow cytometry using a FACS Fortessa (BD Biosciences), and data were analyzed with FlowJo software (Tree Star, Ashland, OR, USA). The experiment was repeated three times.

[0248] 1.4 EAE induction and clinical follow-up

[0249] Anesthetized mice were injected subcutaneously with 100 μl of 100 μg of mouse MOG peptide 35-55 (MOG 35–55() Immunized with phosphate-buffered saline (PBS) from the Proteomics Section, Universitat Pompeu Fabra, Barcelona, Spain. On days 0 and 2 post-immunization (p.i.), mice were injected intravenously with 250 ng of pertussis toxin (Sigma Chemicals). Mice were weighed daily and neurobiological signs were examined using the following criteria: 0 = no clinical symptoms; 0.5 = partial loss of tail rigidity for 2 consecutive days; 1 = complete tail paralysis; 2 = mild paralysis of one or both hind limbs; 2.5 = severe paralysis or paraplegia; 3 = mild quadriplegia; 4 = quadriplegia (severe in hind limbs); 4.5 = severe quadriplegia; 5 = tetraplegia; 6 = death (Gutierrez et al., Mol Neurobiol, 2017). All data presented were according to the recommendations of the EAE publication guidelines (Baker D et al., J. Neuroimmunol, 2012). Weight loss was calculated as the percentage of daily weight change compared to the initial weight on the day of immunization. A score of 5 and weight loss > 30% were defined as the end-point criteria to minimize suffering and ensure animal welfare. Clinical scores were monitored for 28 days.

[0250] 1.5 Liposomal biodistribution in NOD mice

[0251] 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). Pre-diabetic (< 12-week-old) NOD mice were treated with a single dose of 100 μl of empty fluorescent DOPE-AF750-PS liposomes via the intravenous (i.v.) route. In vivo imaging was performed at 1 hour, 6 hours, and 24 hours post-injection. Axillary lymph nodes (LN), bladder, brain, heart, hind limb bones, kidneys, liver, lungs, mediastinal lymph nodes (MDLN), pancreas, perigonadal adipose tissue (PAT), pancreatic lymph nodes (PLN), salivary glands, spleen, stomach, and thymus were harvested, washed with DPBS, and finally imaged ex vivo using the Pearl Impulse system (LI-COR). Background fluorescence was subtracted to normalize the fluorescence values. Fluorescence signals were expressed as relative fluorescence units (RFU) per gram of tissue for each organ.

[0252] 1.6 In vivo capture of fluorescent liposomes in NOD mice

[0253] To characterize the in vivo interaction of liposomes with B cells, dendritic cells, and other APCs (such as macrophages and LSECs), pre-diabetic NOD mice were treated with fluorescently labeled empty NBD-PS liposomes or NBD-PSIns(h) liposomes, administered as a single dose of 100 μl by intravenous injection. Spleens were collected 1 hour and 6 hours after injection. Splenocytes were obtained after mechanical disruption and red blood cell lysis, and then labeled with monoclonal antibodies for 20 minutes at 4 °C. The antibody combinations used in this experiment included CD11c BV786, B220 / CD45R BV510, CD205 / DEC-205BV421, CD8a PECy7, MHC-II / MHC-I-A[d]PE, CD19 APCCy7. Fixable viability stain 575V (BD Biosciences, USA) was used to stain to exclude dead cells. Cells were acquired using a flow cytometer (FACS LSR Fortessa, BD Biosciences), and corresponding fluorescence minus one (FMO) staining was used as a control. Data analysis was performed using FlowJo software (Tree Star, OR, USA).

[0254] Table 4. Immune cell subsets determined by flow cytometry analysis

[0255] Subpopulation name Phenotype B cells <![CDATA[CD19 + > <![CDATA[CD1d high CD5 + B cell]]> <![CDATA[CD19 + CD1d high CD5 + > B1a B cells <![CDATA[CD19 + CD5 + CD43 + > B1b B cells <![CDATA[CD19 + CD5 - CD43 + > Marginal zone (MZ) B cells <![CDATA[CD19 + CD21 high CD23 - > <![CDATA[Foxp3 + B cell]]> <![CDATA[CD19 + Foxp3 + > <![CDATA[CD1d + LAG3 + B cell]]> <![CDATA[CD19 + CD1d + LAG3 + > CDC (cDC) <![CDATA[CD11c + MHC-II + CD205 + CD8a + > pDCs CD11clow MHCII+ B220+ T cells CD45+ CD3+ Macrophages F4 / 80+ Kupffer cells F4 / 80+ CD68+ LSECs CD206+ F4 / 80-

[0256] DC, dendritic cell.

[0257] 1.7 In vitro liposome capture of splenocytes from NOD was evaluated by flow cytometry

[0258] After mechanical disruption and red blood cell lysis, splenocytes from female NOD mice were obtained. Then, in a 96-well round-bottom plate, 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 potassium penicillin salt (Lonza), and 100 μg streptomycin sulfate (Lonza), cells were cultured at 3×10 5 cells / well. They were cultured with 1 mM NBD-PS liposomes or NBD-PSIns(h) liposomes at 37 °C and 5% CO2 for 2 hours and kept overnight. IL-10 was stained using an APC mouse IL-10 secretion detection kit (Miltenyi Biotech, Germany) according to the manufacturer's instructions +The cells were then re-incubated with 1 mM NBD-PS liposomes or NBD-PSIns(h) liposomes for 1 hour to enhance NBD labeling. B cell subsets were identified by monoclonal antibody immunophenotyping, with the combination design as follows: (1) CD19 BV510, CD5 BV421, CD1d PECy7, CD43 PerCP-Cy5.5, LAG3 APC; (2) CD19 BV510, CD21 APC Cy7, CD23 BV421. Cells were acquired using a flow cytometer (FACS LSR Fortessa, BD Biosciences), and corresponding FMO staining was used as a control. Data analysis was performed using FlowJo software (Tree Star).

[0259] 1.8 Analysis of IL-10 and TGF-β in PMBC

[0260] Twenty microliters of peripheral blood was obtained from adult healthy blood donors in heparin tubes (BD Biosciences) by venipuncture. 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 PBMC. The 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 Jofré). After counting and assessing viability by flow cytometry using 7aad (BD Biosciences), the cells were cultured with different liposome compositions at 37 °C and 5% CO2 for 24 hours. After incubation, the cells were collected, washed, and stained with CD19 BV785, CD14 BV711, CD3 BV650, CD4 BV570, CD8 APC Cy7, CD68 BV421 (BioLegend), TGF-β / LAP PE (Miltenyi Biotec), CD11c PECy7, and 7aad (BD Biosciences). IL-10 secretion was analyzed using a human IL-10 secretion detection kit (Miltenyi Biotec) according to the manufacturer's instructions. Subsets, NBD positivity rate, IL-10 secretion, and TGF-β expression in PBMC were analyzed using a FACS Fortessa (BD Biosciences), and data were analyzed using FlowJo software (Tree Star). The experiment was repeated three times.

[0261] 1.9 Statistical analysis

[0262] Statistical analysis was performed using Prism 9.0 software (GraphPad Software Inc., San Diego, CA). For comparisons between unpaired data, parametric two-tailed Student's t-test (Gaussian distribution) or non-parametric Mann-Whitney test was used. For comparisons among multiple groups, one-way analysis of variance (Gaussian distribution) or two-way analysis of variance with corresponding multiple comparison tests was performed. A P value < 0.05 was considered significant.

[0263] 2. Results

[0264] 2.1 PS-PC-Chol liposomes smaller than 200 nm interact with B cells and induce IL-10 secretion

[0265] PS-PC-Chol-NBD-Ins(h) liposomes <200 were manufactured by ethanol injection and extrusion 10 times through a 200 nm pore membrane, yielding a composition with 98% of the liposomes having a diameter less than 200 nm and a ζ potential of -20.0 mV as determined by NTA. These liposomes were incubated with splenocytes for 4 hours and the percentage of interacting B cells (CD19+) was evaluated. Surprisingly, 6.2% of CD19+ cells were found to interact with PS-PC-Chol-NBD-Ins(h) liposomes <200, and even more significantly, when the interaction in the putative Breg compartment CD19+CD1dhi was evaluated, this percentage increased to 28% ( Figure 1 ).

[0266] To determine that this interaction has biological significance in inducing tolerance, the expression of the tolerogenic cytokine IL-10 in B cells from PBMC samples was analyzed after incubation with PS-PC-Chol-Ins(h) liposomes <200 (97.5% 10 - 200 nm; 0% >500 nm; ζ potential -20.0 mV) for 24 hours. As shown, PS-PC-Chol-insulin-liposomes <200 induced ex vivo expression of IL-10 in human B cells, as an increase in the expression of this cytokine in B cells was observed by 30% after incubation with these liposomes.

[0267] 2.2 The PS-PC-Chol liposome composition of the present invention has higher efficacy than PS-PC-Chol liposomes >500 nm

[0268] EAE was induced by MOG immunization as described above and on the 5th day after immunization, 50 μL of PS-PC-Chol-MOG was administered via the intravenous route using a 1 mL syringe and a 30G needle36-55 Liposomes - 200&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 Liposomes >500 (10 - 200 nm 0%; >500 nm 95.2%; ζ potential - 24.7 mV), PS - PC - Chol empty - liposomes 200&500 (10 - 200 nm 26%; >500 nm 8.2%; ζ potential - 22.7 mV), or PS - PC - Chol empty - liposomes >500 (10 - 200 nm 0%; >500 nm 93.1%; ζ potential - 23.2 mV), and the clinical scores were monitored for 28 days.

[0269] The differences in the area under the curve (AUC) of the clinical scores were calculated to compare the beneficial effects of the two liposome compositions. The AUC score of each MOG - bearing liposome - treated mouse was subtracted from the AUC score of each control mouse (blank group). A positive AUC difference indicates that the AUC score of the empty - liposome - treated mouse is higher than that of the MOG - bearing liposome - treated mouse; that is, the clinical course of EAE in the MOG - bearing liposome - treated mouse is less severe. As Figure 2 shown, PS - PC - Chol - MOG 36-55 Liposomes - 200&500 showed higher efficacy in controlling disease progression and clinical symptoms compared to PS - PC - Chol - MOG 36-55 Liposomes >500.

[0270] 2.3. Fluorescently labeled PS - PC - Chol liposomes - 200&500 are distributed in target organs and captured by phagocytes in NOD mice

[0271] The biodistribution of PS - PC - Chol liposomes - 200&500 was studied using PS - PC - Chol empty - liposomes - 200&500 labeled with AF750. Thus, PS - PC - Chol - AF750 empty - liposomes - 200&500 (10 - 200 nm 45.1%; >500 nm 4.7%; ζ potential - 24.7 mV) were administered via the intravenous route, and the fluorescence signals in various organs were evaluated at 1, 6, and 24 hours after administration ( Figure 3 ). The intravenous route showed a peak at 1 hour after injection, and the signal decreased over time and was negligible at the 24 - hour checkpoint. As shown, when PS - PC - Chol - AF750 empty - liposomes - 200&500 were administered intravenously, the fluorescence specifically accumulated in the liver, lung, MDLN, spleen, and stomach.

[0272] To confirm which immune cells interact with the composition of the present invention in vivo, NOD mice were administered a single dose of PS-PC-Chol-NBD-insulin-liposomes-200&500 (10 - 200 nm 37.1%; >500 nm 16.3%; ζ potential 24.3 mV) via the intravenous route. Spleen and liver were collected 1 hour and 6 hours after administration. The spleen cell viability was 98.8 ± 0.23 (mean ± SEM, data not shown). In the spleen, conventional DC (cDC) (8.6 ± 2.1%) showed NBD signal, and 1.4 ± 0.5% of B cells also showed positive NBD staining, indicating that the formulation of the present invention directly interacts with these two types of immune cells. In the liver, in addition to confirming the interaction with DC and B cells, among the immune cells studied, macrophages showed the highest percentage in terms of the parental population interacting with the liposomes. Notably, LSECs were also found to interact with the liposomes ( Figure 4 ).

[0273] 2.4 PS liposomes interact with Breg cell subsets and promote IL-10 secretion

[0274] Since B cells play an important role as antigen-presenting cells (APCs), we confirmed the tolerogenic effect of PS-PC-Chol-Ag-liposomes-200&500 in B cells. Therefore, spleen cells from NOD mice were co-cultured with PS-PC-Chol-NBD-insulin-liposomes-200&500 (10 - 200 nm 36.4%; >500 nm 18%; ζ potential -25 mV). Among all B lymphocytes, 34.8 ± 2.0% showed NBD fluorescence. When dissecting the NBD fluorescence in different B cell subsets with the above regulatory potential, CD5 + CD1d high cells showed 48.1 ± 2.3% NBD signal (relative to the entire CD5 + CD1d high subset); NBD + B1a B lymphocytes, 58.7 ± 1.4% of all B1a B lymphocytes; NBD + B1b B lymphocytes, 24.8 ± 1.5% of all B1b B lymphocytes, and NBD + marginal zone (MZ) B cells, 67.5 ± 0.4% of all MZ B cells ( Figure 5 ). To demonstrate their regulatory function after treatment, positive IL-10-secreting cells were found in each of the following subsets: 13.1 ± 1.4% of 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. In each subset, when compared to non-fluorescent B cells, IL-10 secretion in NBD-fluorescent B cells showed a higher biological tendency.

[0275] The liposomes of the composition of the present invention induce B cells and dendritic cells from PBMC to secrete IL-10 and TGF-β.

[0276] To confirm the tolerance-inducing ability of the composition of the present invention in human cells, PBMC from healthy donors were incubated in the presence or absence of 100 ng / ml lipopolysaccharide in 1 mM PS-PC-Chol-NBD-Ins(h)-liposomes-200&500 (10 - 200 nm 37.0%; >500 nm 2.8%; ζ potential -22.3 mV), 1 mM PS-PC-Chol-NBD-MCV-liposomes-200&500 (10 - 200 nm 39.3%; >500 nm 3.6%; ζ potential -24.7 mV) or 1 mM PS-PC-Chol-NBD-AChR-liposomes-200&500 (10 - 200 nm 18.1%; >500 nm 13.6%; ζ potential 24.9 mV) or PS-PC-Chol-MCV-200&500 (10 - 200 nm 28.6%; >500 nm 9.4%; ζ potential 23.7 mV) labeled with amine and pHrodo TM green-stained according to the manufacturer's instructions for 24 hours, and the expression of tolerance-related cytokines IL-10 and TGF-β in NBD-positive and -negative subsets of B cells and DCs was analyzed. The expression of these two cytokines was also evaluated in pHrodo-positive and -negative subsets of B cells.

[0277] As shown, interaction with liposomes of different loads induced a significant increase in the expression of IL-10 and TGF-β in the CD19+ region, and DCs that captured liposomes did express a higher level of IL-10 than DCs that did not phagocytose liposomes, but no difference was observed in the case of TGF-β expression. In addition, by pHrodo staining that is sensitive to pH and indicates direct phagocytosis of cells, if positive, we confirmed that the regulation of B cells by liposome treatment was due to phagocytosis of liposomes rather than indirect interaction with the membrane. These data suggest that the mechanisms of action for inducing tolerance through B cells and DCs are independent of the antigen loaded in the PS liposomes.

[0278] List of references

[0279] Patent documents:

[0280] -WO2015107140

[0281] Non-patent documents:

[0282] -Chen et al., J Immunol 2020, 204(2): 335–347. https: / / doi.org / 10.4049 / jimmunol.1801677

[0283] -Roep BO, Peakman M. Cold Spring Harb Perspect Med, 2012, Vol. 2(4): a007781. doi: 10.1101 / cshperspect.a007781

[0284] -Lernmark A. J. Clin Invest, 2001, Vol. 108, pp. 1091-1096

[0285] -Bangham et al., J. Mol. Biol., 13, 238(1965)

[0286] -Pons et al International Journal of Pharmaceutics 95(1993)51-56)

[0287] -Maurer N. et al., Expert Opin Biol Ther, 2001, Vol. 1(6), pp. 923-47

[0288] -Waterhouse D.N. et al., Methods Enzymol., 2005; Vol. 391, pp. 40-57

[0289] -Urbán P. et al., Nanosc.Res.Lett., 2011, Vol. 6, p. 620

[0290] -Gutierrez et al., Mol Neurobiol. 2017 Aug; 54(6): 4820-4831. doi: 10.1007 / s12035-016-0154-2.

[0291] -Baker D. et al., J.Neuroimmunol. 2012 Jan 18; 242(1-2): 78-83. doi: 10.1016 / j.jneuroim.2011.11.003

[0292] -Whitepaper on Nanoscale Material Characterization:a Review of the use of Nanoparticle Tracking Analysis (NTA), 2015 Malvern Instruments Limited

Claims

1. A composition comprising two liposome populations, wherein: - The first liposome population has a size of 2 nm to 200 nm, - The second liposome population has a size of 500 nm to 2000 nm, - The first liposome population and the second liposome population carry one or more antigens, and - The liposome membrane of each liposome in the first liposome population and the second liposome population contains phosphatidylserine, and the amount of phosphatidylserine is 20% to 60% by weight relative to the total composition of the liposome membrane.

2. The composition according to claim 1, wherein 15 - 75% of the liposomes in the composition correspond to the first population, and 2% to 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 relative to the total composition of the liposome membrane.

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

5. The composition according to claim 4, wherein The liposome membrane contains PS, PC, and CHOL, and the PS:PC:CHOL molar ratio is 1:(0.6 - 1.8):(0.7 - 2.5).

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

7. The composition according to any one of claims 1 to 6, wherein The antigen is selected from the group consisting of: autoantigens, drugs including therapeutic proteins, viral vectors including viral capsid proteins, allergens, and alloantigens.

8. The composition according to claim 7, wherein, The antigen is an autoantigen associated with an autoimmune disease. 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 - L - fucose synthase, acetylcholine receptor (AChR), muscle - specific tyrosine kinase (MuSK), agrin, lipoprotein - related protein 4 (LRP4), cortactin, transglutaminase, deamidated gliadin, thyroglobulin, collagen (such as type 11 collagen), human cartilage glycoprotein 39, chromogranin A, gp130 - RAPS, vimentin, citrullinated vimentin, ADAMST13, aquaporin 4, proteolipid protein, fibrillarin, nucleoprotein, nucleolar protein (such as small nucleolar protein), histidyl - tRNA synthetase (HisRS), histidine - tRNA synthetase (HARS1), jo - 1, thyroid - stimulating factor receptor, histone, glycoprotein gp70, ribosomal protein, pyruvate dehydrogenase dihydrolipoamide acetyltransferase, hair follicle antigen, human tropomyosin isoform 5, mitochondrial protein, islet β - cell protein, gluten, and antigenic fragments or derivatives of any of the above, and immunogenic fragments or derivatives of any of the above.

9. The composition according to claim 7, wherein, The antigen is a viral capsid protein, particularly 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 according to any one of claims 1 to 10, which is a pharmaceutical composition and contains a pharmaceutically acceptable excipient and carrier.

12. The composition according to any one of claims 1 to 11, which is used as a drug.

13. The composition according to any one of claims 1 to 11, for use in inducing tolerance to an antigen, in particular, wherein, The induction of the tolerance includes B cell-mediated tolerance to the antigen and T cell-mediated tolerance to the antigen.

14. The composition according to any one of claims 1 to 11, which is used for treating a disorder selected from the group consisting of: autoimmune diseases, allergies, drug hypersensitivity, transplant rejection, adverse immune effects triggered by gene therapy.

15. The composition for use as claimed in claim 14, wherein, The disorder is an autoimmune disease, and the autoimmune disease is selected from the group consisting of: T1D, lupus erythematosus, arthritis (including 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 ulcer, iritis, conjunctivitis, keratoconjunctivitis, ulcerative colitis, autoimmune uveitis, allergic encephalomyelitis, acute necrotizing hemorrhagic encephalopathy, idiopathic bilateral progressive sensorineural hearing loss, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, immune-mediated thrombotic thrombocytopenic purpura, polychondritis, Wegener's granulomatosis, chronic active hepatitis, Stevens-Johnson syndrome, idiopathic steatorrhea, 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 in the application according to any one of claims 12 to 15, wherein, The size of the liposome is determined by nanoparticle tracking analysis (NTA).

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