Lipid delivery vehicle

Abstract

This invention relates to a lipid delivery vehicle for inducing a tolerable immune response in subjects, comprising a nuclear factor κB (NF-κB) inhibitor and a peptide antigen, wherein the lipid delivery vehicle comprises 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate glycerol (POPG).

Description

Relevant application data

[0001] This application claims priority to Australian Patent Application No. 2023904233, filed on December 22, 2023, entitled "Lipid Delivery Carrier". The entire contents of that Australian patent application are incorporated herein by reference.

[0002] sequence list This application is filed together with a sequence list, which has been submitted electronically (in XML format) and is incorporated herein by reference in its entirety. The XML copy was created on December 21, 2024, named "800861PCT sequencelisting", and is 76,991 bytes in size. Technical Field

[0003] This disclosure generally relates to a lipid delivery carrier and its uses. Background Technology

[0004] Autoantigen-specific immune tolerance induction (ASITI) is a therapeutic platform that controls cellular and humoral immune responses in autoimmune diseases by re-establishing antigen-specific tolerance without compromising the patient's normal immunity. Typically, ASITI involves co-delivering an immunomodulator and peptide antigen, encapsulated in liposomes, to antigen-presenting cells (APCs), including dendritic cells (DCs), in an attempt to induce antigen-specific tolerance in T cells.

[0005] However, the co-delivery strategy of liposome-based immunomodulators and peptide antigens faces several translational obstacles in achieving clinical benefits, including difficulties in scale-up production, long-term storage stability, stability of liposomes after exposure to plasma, and specificity of delivery to target tissues or cells.

[0006] When constructing a suitable liposome-based delivery vehicle, an important aspect to consider is the degree of protection the vehicle provides for the encapsulated active pharmaceutical ingredient (API), as this will affect the persistence and integrity of the payload in the target tissue. Unprotected peptides may degrade upon exposure to tissue or blood proteases, thus ceasing to function as effective tolerogens. Protecting the patient from the peptides before payload delivery is also crucial, and ideally, self-antigen peptides should be encapsulated within the delivery vehicle to avoid potentially triggering immune complex-mediated immune responses.

[0007] Furthermore, residual unencapsulated peptides or surface-adsorbed peptides may lead to incorrect assumptions about the concentration of liposome-encapsulated peptides that can be taken up by draining lymph node (dLN) DCs after subcutaneous administration. Prior to scaling up methods for encapsulating peptides in liposome-based delivery vehicles, free peptides were typically removed by dialysis with HEPES buffer after liposome production. However, at scale-up, peptide removal is typically and preferably performed by cross-flow filtration, which subjectes the liposomes to greater shear stress, and surface-adsorbed peptides may not be retained with the liposomes.

[0008] When considering lipid-based delivery vehicles for the growing subcutaneous API delivery market, plasma stability, storage stability, and retention of the API in liposomes before delivery of the payload are crucial. API leaching from lipid-based delivery vehicles is a known problem in this field, and placebo-controlled trials have shown that L-α-lecithin choline (EPC) and L-α-lecithin glycerol (EPG) liposomes (i.e., DEN-181) release significant amounts of encapsulated calcitriol immunomodulators into plasma. The amount of peptides released in vivo from these liposomes is unclear. The difficulties associated with developing stable liposomes for subcutaneous API administration differ significantly from those faced for APIs intended for intranasal or oral administration, which must address the unique challenges posed by the mucosa and gastrointestinal tract, respectively.

[0009] Therefore, there remains a need for lipid delivery vehicles with improved pharmacokinetic characteristics, particularly for subcutaneous delivery, especially those with improved in vivo stability, distribution, dLN DC uptake, and improved retention of lipid delivery vehicles, encapsulated peptides, and immunomodulators.

[0010] However, given the vast number of suitable components available for constructing lipid-based delivery vehicles, the difficulty in extrapolating formulation results for different routes of administration, and the particular challenges of using peptide APIs, it is nearly impossible to predict which lipid combinations can prepare lipid-based delivery vehicles that meet all these requirements. Summary of the Invention

[0011] In the course of developing this invention, the inventors unexpectedly discovered that the combination of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate glycerol (POPG) advantageously enhances the stability of lipid delivery carriers, thereby improving peptide retention within the carrier during storage and in plasma. In some forms of this disclosure, the inventors have also found that lipid delivery carriers comprising the combination of POPC and POPG result in improved targeting and uptake of liposomes by antigen-presenting cells (including dendritic cells (DCs)) in draining lymph nodes (dLNs), improved peptide retention in the dLNs, and reduced off-target effects (e.g., reduced peptide retention in the spleen and liver). The inventors have also found that the inclusion of PEGylated lipids further improves the stability of the lipid delivery carrier and peptide retention. The inventors have also found that the inclusion of POPC and POPG advantageously enhances the inhibitory effect of the lipid delivery carrier on dLN DCs. Furthermore, it has been found that constructing at room temperature improves the stability of the lipid delivery carrier and peptide retention. In some forms of this disclosure, the inventors have also discovered that lipid delivery carriers comprising a combination of POPC, POPG, PEGylated lipids, and calcitriol can lead to improved targeting and uptake of dLN, as well as reduced off-target effects (e.g., reduced hepatic uptake). Based on these findings, the inventors have developed improved lipid delivery carriers for the co-delivery of peptide antigens and immunomodulators.

[0012] Accordingly, this disclosure provides a lipid delivery carrier comprising a nuclear factor-κB (NF-κB) inhibitor and a peptide antigen, wherein the lipid delivery carrier comprises 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate glycerol (POPG).

[0013] In one instance, nuclear factor-κB (NF-κB) inhibitors and peptide antigens were encapsulated or packaged within a lipid delivery carrier.

[0014] This disclosure also provides a lipid delivery carrier comprising a nuclear factor-κB (NF-κB) inhibitor and a peptide antigen, wherein the lipid delivery carrier comprises 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC), 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate glycerol (POPG), and polyethylene glycol (PEG)-modified lipids.

[0015] This disclosure also provides a population of lipid delivery vectors disclosed herein, wherein the population of lipid delivery vectors has a polydispersity index of less than 0.20.

[0016] This disclosure also provides a pharmaceutical composition comprising the lipid delivery carrier disclosed herein and a pharmaceutically acceptable carrier.

[0017] This disclosure also provides a pharmaceutical composition for subcutaneous delivery comprising the lipid delivery carrier disclosed herein and a pharmaceutically acceptable carrier.

[0018] This disclosure also provides the lipid delivery carriers disclosed herein or the pharmaceutical compositions disclosed herein for use in therapies.

[0019] This disclosure also provides a method for inducing a tolerable immune response in a subject, including administering to the subject the lipid delivery carrier disclosed herein or the pharmaceutical composition disclosed herein.

[0020] This disclosure also provides for the use of the lipid delivery carriers disclosed herein in the preparation of medicaments for inducing a tolerable immune response in subjects.

[0021] This disclosure also provides a method for treating and / or preventing autoimmune diseases, conditions, or illnesses in a subject, including administering the lipid delivery carrier or pharmaceutical composition disclosed herein to the subject.

[0022] This disclosure also provides for the use of the lipid delivery carriers disclosed herein in the preparation of medicaments for the treatment and / or prevention of autoimmune diseases, conditions or illnesses in subjects.

[0023] Particularly preferred embodiments are described herein, including those described in the independent claims.

[0024] Brief description of the attached figures The following figures form part of this specification and are included to further illustrate certain embodiments of this disclosure. This disclosure can be better understood by referring to one or more of these figures in conjunction with the detailed description of the specific embodiments presented herein. Those skilled in the art will understand that many variations and / or modifications can be made to the above embodiments without departing from the broad general scope of this disclosure. Therefore, this disclosure should be considered illustrative rather than restrictive in all respects.

[0025] Figure 1 An experimental design (DoE) model for initial microfluidic experiments is described, which aims to identify liposome compositions that meet size, polydispersity index (PdI), and charge criteria and have optimal peptide encapsulation efficiency.

[0026] Figure 2Size exclusion columns used for size exclusion chromatography (SEC) are described. (A) After incubation of liposomes in plasma or phosphate-buffered saline (PBS), the mixture was separated into multiple fractions by SEC. (B) The particle concentrations of these fractions were then assessed using nanoparticle tracking analysis (NTA). (C) Liposome samples A and B were recovered in fractions 6–11, while plasma in PBS (sample C) contained very few nanoparticles. (D) Peptide concentrations measured using liquid chromatography-tandem mass spectrometry (LC-MS / MS) identified peaks in fractions 6–11 and 19–22. (E) Upon quantification, most peptides were recovered in fractions 6–11, with minor recoveries in fractions 12–22.

[0027] Figure 3 This describes (A) the NTA used for SEC determination and (B) the particle size distribution.

[0028] Figure 4 The biodistribution of liposomes containing POPC / POPG / 0.5% PEG / calcitriol / peptide (ASITI-201), EPC / EPG / calcitriol / peptide (DEN181), or POPC / POPG / 0.5% PEG (empty) in untreated mice was described at (A) the skin injection site, (B) the groin dLN, (C) the axillary dLN, and (D) the liver.

[0029] Figure 5 This indicates that in free peptides or those containing POPC / POPG / PEG / PI 33-63 Biodistribution of peptides after administration of calcitriol liposomes (ASITI-201). (A) 89 ZrPI 33-63 (B) Radiochemical purity in this experiment. 89 ZrPI 33-63 Quantification of the biodistribution of peptides over time. (C) Two days after subcutaneous administration of liposomes or free peptides to the left flank, it showed... 89 Whole mouse positron emission tomography-computed tomography (PET-CT) of Zr signal.

[0030] Figure 6 This study illustrates the uptake of DiI-labeled liposomes by antigen-presenting cells in (A) skin, (B) liver, (C) dLN, and (D) spleen of untreated mice. Two days after subcutaneous injection of 100 µL of DiI-labeled DEN-181 or ASITI-201, the proportions of DiI+ dendritic cells, B cells, and monocytes in the skin injection site, dLN (groin and axilla combined), liver, and spleen are shown. PBS controls represent background fluorescence in the DiI channels.

[0031] Figure 7This indicates that after subcutaneous injection at the base of the tail, free... 89 ZrPI 33-63 Peptides or those containing POPC / POPG / PEG 89 ZrPI 33-63 Calcitriol or EPC / EPG / 89 ZrPI 33-63 / Liposomes of calcitriol were biodistributed in vivo at (A) left inguinal dLN, (B) right inguinal dLN, (C) left axillary dLN, (D) right axillary dLN, (E) injection site, (F) on day 3 and (G) on day 11.

[0032] Figure 8 illustrates the experimental procedure for (A) comparing the uptake of liposomes by antigen-presenting cells (APCs) and their effects on antigen-responsive CD4+ T cells after multiple administrations in antigen-induced mice, (B) APC uptake in dLN, and (C) APC uptake in the spleen.

[0033] Figure 9 This demonstrates the effect of flow cytometry analysis on liposome uptake on (A) CD11c+ myeloid DCs in dLN, (B) plasmacytoid DCs in dLN, (C) memory B cells in dLN, (D) germinal center B cells in dLN, and (E) CD4+ stimulated by proteoglycans in the spleen. + T cell CD154 expression, (F) CD154+ T cell IFN-γ in spleen + CD154 in the spleen (G) + T cells IFN-γ + TNF + The impact.

[0034] Figure 10 This indicates that POPC / POPG / PEG / PI were present 1 hour and 72 hours after subcutaneous injection at the base of the tail. 33-63 / [3H]Calcitriol or EPC / EPG / PI 33-63 / [3H]calcitriol liposomes in vivo biodistribution at (A) injection site, (B) both inguinal dLNs, (C) both axillary dLNs, (D) blood, (E) liver, (F) spleen, (G) kidney and (H) intestine / feces. * indicates between-group differences, and * in square brackets indicates differences over time.

[0035] Key information in the sequence list Detailed Implementation

[0036] General techniques and definitions Unless otherwise specifically defined, all technical and scientific terms used herein shall have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in the fields of genomics, immunology, molecular biology, immunohistochemistry, biochemistry, oncology, and pharmacology).

[0037] The implementation of this disclosure uses conventional techniques of molecular biology, microbiology, recombinant DNA technology, and immunology (unless otherwise stated). These protocols are described, for example, in: Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratories, New York, Fourth Edition (2012), Vols. I, II, and III; DNA Cloning: A Practical Approach, Vols. Gait, ed, 1984) IRL Press, Oxford; Nucleic Acid Hybridization: A Practical Approach (BDHames&S. J. Higgins, eds., 1985) IRL Press, Oxford; Immobilized Cells and Enzymes: A Practical Approach (1986) IRL Press, Oxford; Perbal, B., APractical Guide to Molecular Cloning (1984); Current Protocols in Immunology, Eds. (John Wiley & Sons Inc, 1999–2023); and the entire series of Methods in Enzymology (S. Colowick and N. Kaplan, eds., Academic Press, Inc.).

[0038] Those skilled in the art will understand that this disclosure is susceptible to non-specific variations and modifications. It should be understood that this disclosure includes all such variations and modifications. This disclosure also includes all steps, features, compositions, and compounds mentioned or indicated in this specification, individually or collectively, and any and all combinations of any two or more of the stated steps or features. Therefore, each feature of any particular example or embodiment of this disclosure may be applied to any other example or embodiment of this disclosure with necessary modifications.

[0039] The scope of this disclosure should not be limited to the specific embodiments described herein, which are for illustrative purposes only. Functionally equivalent products, compositions, and methods are obviously within the scope of this disclosure as described herein.

[0040] Throughout this specification, unless otherwise expressly stated or required by context, references to a single step, a substance composition, a group of steps, or a group of substance compositions shall be construed as including one or more (i.e., one or more) of such steps, substance compositions, groups of steps, or groups of substance compositions.

[0041] The terms “from,” “to,” and “between” when referring to a range should be understood to include the range of the lower and upper limits. For example, “x is an integer from 0 to 6” should be understood to include the absence of x (x is 0), x is 6, and every integer value in between, i.e., x is 1, 2, 3, 4, or 5.

[0042] The singular forms “a,” “an,” and “the” used in this article include plural references unless the context clearly specifies otherwise.

[0043] The term “and / or”, such as “X and / or Y”, should be understood as either “X and Y” or “X or Y”, and should be regarded as providing explicit support for both meanings or either meaning.

[0044] Throughout this specification, the word “comprising” or variations thereof, such as “including” or “containing”, shall be understood to imply inclusion of the said element, integer, or step, or group of elements, integers, or steps, but not to exclude any other element, integer, or step, or group of elements, integers, or steps.

[0045] The term "about" regarding the numerical value x is optional and means, for example, any number within the range of 1%, 5%, or 10% of the referenced number. The term "about" also includes the exact number referenced.

[0046] "Basically composed of": In the context of (a) an amino acid sequence, it means the cited amino acid sequence together with one, two or three additional amino acids at its N- and / or C-terminus; or in the context of (b) a nucleic acid sequence, it means the cited nucleic acid sequence together with one, two or three additional nucleic acids at its 5' and / or 3' terminus.

[0047] All computer programs, algorithms, patents, and scientific literature cited in this article are incorporated herein by reference.

[0048] Any discussion of documents, actions, materials, devices, articles, or similar matters contained in this specification shall not be construed as an admission that any or all of these matters constitute part of the prior art or that such matters were common knowledge in the field relating to this disclosure prior to the priority date of each appended claim.

[0049] Lipid delivery carrier In the work leading to this invention, the inventors sought to produce lipid delivery vehicles with improved pharmacokinetic characteristics (e.g., storage stability, plasma stability), enhanced dLN targeting and uptake, and / or reduced off-target effects. Therefore, the inventors developed a lipid delivery vehicle comprising 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate glycerol (POPG), which exhibits improved pharmacokinetic characteristics (e.g., stability) in the plasma and lymphatic systems. In some forms of this disclosure, the inventors have also found that this lipid delivery vehicle improves dLN targeting and / or reduces off-target effects (e.g., reduced hepatic uptake).

[0050] Accordingly, this disclosure provides a lipid delivery carrier comprising a nuclear factor-κB (NF-κB) inhibitor and a peptide antigen, wherein the lipid delivery carrier comprises 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate glycerol (POPG).

[0051] As used herein, the term "lipid delivery carrier" should be understood to refer to lipid-based particles having at least one dimension on the nanometer scale (e.g., 1–1,000 nm). Lipid delivery carriers are formulated into compositions for delivering molecules (e.g., APIs, such as peptide antigens and / or nuclear factor-κB inhibitors) to desired targets, such as cells, tissues, organs, tumors, etc. Lipid delivery carriers may be selected from, but are not limited to: liposomes or lipid vesicles, wherein an aqueous volume is encapsulated by one or more amphiphilic lipid bilayers (e.g., single; monolayer or multiple; multilayer); micellar-like lipid nanoparticles having a non-aqueous core; and solid lipid nanoparticles, wherein the solid lipid nanoparticles lack a lipid bilayer.

[0052] When the lipid delivery carrier is a liposome or lipid vesicle for delivering one or more APIs, the API may be encapsulated in one or more amphiphilic lipid bilayers (i.e., embedded within the bilayer), or encapsulated within the carrier (i.e., encapsulated in an aqueous volume within the carrier), or a combination of both. For the purposes of this disclosure, “encapsulated within” the lipid delivery carrier is intended to cover any and all peptide antigens and / or NκB inhibitors associated with the lipid delivery carrier, except for: (a) any peptide antigens and / or NκB inhibitors that are bound only to or associated only with the surface of the lipid delivery carrier, and (b) any peptide antigens and / or NκB inhibitors that are present only in the composition containing the loaded lipid delivery carrier (i.e., the lipid delivery carrier encapsulating the API) but are not bound to or associated with the lipid delivery carrier.

[0053] In the implementation scheme, the term "lipid delivery carrier" may refer to lipid particles formed from POPC and POPG, and optionally PEGylated lipids as the only lipid-containing component.

[0054] In this embodiment, the lipid delivery carrier does not contain any phospholipids other than POPC and POPG. In this embodiment, the lipid delivery carrier does not contain any non-PEGylated lipids other than POPC and POPG.

[0055] In the implementation scheme, the lipid delivery carrier consists of or is substantially composed of POPC, POPG, and optionally PEGylated lipids as the only lipid components of the lipid delivery carrier.

[0056] The lipid delivery carriers described herein may have an average diameter of about 75 nm to about 250 nm, or about 100 nm to about 200 nm, or about 110 nm to about 200 nm, or about 120 nm to about 200 nm, or about 100 nm to about 190 nm, or about 100 nm to about 160 nm, or about 100 nm to about 170 nm, or about 100 nm to about 160 nm, or about 100 nm to about 150 nm, or about 100 nm to about 140 nm, or about 100 nm to about 130 nm, or about 110 nm to about 190 nm, or about 110 nm to about 180 nm, or about 110 nm to about 170 nm, or about 110 nm to about 160 nm, or about 110 nm to about 150 nm, or about 110 nm to about 140 nm, or about 110 nm to about 130 nm. The lipid delivery carriers described herein may have an average diameter of about 90 nm to about 160 nm. For example, the lipid delivery carriers described herein may have an average diameter of about 100 nm, or about 110 nm, or about 120 nm, or about 130 nm, or about 140 nm, or about 150 nm, or about 160 nm, or about 170 nm, or about 180 nm, or about 190 nm, or about 200 nm. The lipid delivery carriers described herein may have an average diameter of about 60 nm to about 180 nm. The lipid delivery carriers described herein may have an average diameter of about 80 nm to about 150 nm. The lipid delivery carriers described herein may have an average diameter of about 110 nm to about 150 nm. The lipid delivery carriers described herein may have an average diameter of about 80 nm to about 100 nm. For example, the lipid delivery carrier described herein may have an average diameter of about 60 nm, or about 70 nm, or about 80 nm, or about 90 nm, or about 100 nm, or about 110 nm, or about 120 nm, or about 130 nm, or about 140 nm, or about 150 nm, or about 160 nm, or about 170 nm, or about 180 nm.

[0057] The diameter of the lipid delivery carrier can be measured by dynamic light scattering (DLS), transmission electron microscopy (TEM), scanning electron microscopy (SEM) or other methods known in the art.

[0058] Electrophoretic light scattering can be used to characterize the surface charge of lipid delivery carriers at a specified pH (e.g., physiological pH). Surface charge, or zeta potential, is a measure of the strength of electrostatic repulsion / attraction between particles in a lipid delivery carrier suspension. The surface charge of a lipid delivery carrier at physiological pH (i.e., between pH 7.0 and 7.4) may be negative. For example, the surface charge may be less than 0 mV, or less than -5 mV, or less than -10 mV, or less than -15 mV, or less than -20 mV, or less than -25 mV, or less than -30 mV, or less than -40 mV, or less than -50 mV. The surface charge of a lipid delivery carrier at physiological pH (i.e., between pH 7.0 and 7.4) may be less than -20 mV. Alternatively, the surface charge of a lipid delivery carrier at physiological pH (i.e., between pH 7.0 and 7.4) may be less than -30 mV. Alternatively, the surface charge of a lipid delivery carrier at physiological pH (i.e., between pH 7.0 and 7.4) may be less than -40 mV. Alternatively, the surface charge of the lipid delivery carrier at physiological pH (i.e., between pH 7.0 and 7.4) may be less than -50 mV. Alternatively, the surface charge of the lipid delivery carrier at physiological pH (i.e., between pH 7.0 and 7.4) may be between -10 mV and -50 mV. Alternatively, the surface charge of the lipid delivery carrier at physiological pH (i.e., between pH 7.0 and 7.4) may be between -30 mV and -50 mV. Alternatively, the surface charge of the lipid delivery carrier at physiological pH (i.e., between pH 7.0 and 7.4) may be between -40 mV and -50 mV.

[0059] Dynamic light scattering (“DLS”) can be used to characterize the polydispersity index (“PdI”) and size of the lipid delivery carrier populations described herein. DLS measures the light scattering produced when a sample is exposed to a light source. The PdI determined by DLS measurements represents the distribution of particle size (around the average particle size) within the population; a perfectly homogeneous population has a PdI of zero.

[0060] The lipid delivery vehicle populations described herein can be relatively homogeneous. A polydispersity index can be used to indicate the homogeneity of the lipid delivery vehicle population. Small polydispersity indices (e.g., less than 0.2) generally indicate that the population has a narrow particle size distribution. The lipid delivery vehicle populations described herein can have a polydispersity index of about 0 to about 0.25, such as 0.01, or 0.02, or 0.03, or 0.04, or 0.05, or 0.06, or 0.07, or 0.08, or 0.09, or 0.10, or 0.11, or 0.12, or 0.13, or 0.14, or 0.15, or 0.16, or 0.17, or 0.18, or 0.19, or 0.20, or 0.21, or 0.22, or 0.23, or 0.24, or 0.25. The polydispersity index of the lipid delivery carrier population can be from about 0 to about 0.20, or from about 0.05 to 0.20. The polydispersity index of the lipid delivery carrier population can be from about 0 to about 0.15.

[0061] Cryo-electron microscopy (“cryo-EM”) can be used to determine the particle size, morphology, and structural characteristics of lipid delivery carriers.

[0062] Lipid composition analysis of lipid delivery carriers can be performed using liquid chromatography followed by electrosol detection (LC-CAD). This analysis provides a comparison between actual and theoretical lipid content.

[0063] The lipid delivery carriers in this disclosure can be selected from the group consisting of liposomes, lipid nanoparticles, lipid vesicles, and lipid-based particles. For example, a lipid delivery carrier can be a liposome. Alternatively, a lipid delivery carrier can be a lipid nanoparticle. A lipid delivery carrier can be a lipid vesicle. A lipid delivery carrier can also be a lipid-based particle.

[0064] lipids The lipid delivery carriers described herein may contain about 85 wt% to about 95 wt% of POPC. For example, the lipid delivery carriers may contain about 85 wt%, or about 86 wt%, or about 87 wt%, or about 88 wt%, or about 89 wt%, or about 90 wt%, or about 91 wt%, or about 92 wt%, or about 93 wt%, or about 94 wt%, or about 95 wt% of POPC.

[0065] Alternatively, the lipid delivery carrier described herein may contain about 85 mol% to about 95 mol% of POPC. For example, the lipid delivery carrier may contain about 85 mol%, or about 86 mol%, or about 87 mol%, or about 88 mol%, or about 89 mol%, or about 90 mol%, or about 91 mol%, or about 92 mol%, or about 93 mol%, or about 94 mol%, or about 95 mol% of POPC.

[0066] Alternatively, the lipid delivery carrier described herein may contain POPC at a concentration of about 19 mg / mL to about 37 mg / mL. For example, the lipid delivery carrier may contain POPC at a concentration of about 19 mg / mL, or about 20 mg / mL, or about 21 mg / mL, or about 22 mg / mL, or about 23 mg / mL, or about 24 mg / mL, or about 25 mg / mL, or about 26 mg / mL, or about 27 mg / mL, or about 28 mg / mL, or about 29 mg / mL, or about 30 mg / mL, or about 31 mg / mL, or about 32 mg / mL, or about 33 mg / mL, or about 34 mg / mL, or about 35 mg / mL, or about 36 mg / mL, or about 37 mg / mL.

[0067] The lipid delivery carriers described herein may contain about 5 wt% to about 15 wt% of POPG. For example, the lipid delivery carriers may contain about 5 wt%, or about 6 wt%, or about 7 wt%, or about 8 wt%, or about 9 wt%, or about 10 wt%, or about 11 wt%, or about 12 wt%, or about 13 wt%, or about 14 wt%, or about 15 wt% of POPG.

[0068] Alternatively, the lipid delivery carrier described herein may contain about 5 mol% to about 15 mol% of POPG. For example, the lipid delivery carrier may contain about 5 mol%, or about 6 mol%, or about 7 mol%, or about 8 mol%, or about 9 mol%, or about 10 mol%, or about 11 mol%, or about 12 mol%, or about 13 mol%, or about 14 mol%, or about 15 mol% of POPG.

[0069] Alternatively, the lipid delivery carrier described herein may contain POPG at a concentration of about 2.4 mg / mL to about 3.7 mg / mL. For example, the lipid delivery carrier may contain POPG at a concentration of about 2.4 mg / mL, or about 2.5 mg / mL, or about 2.6 mg / mL, or about 2.7 mg / mL, or about 2.8 mg / mL, or about 2.9 mg / mL, or about 3.0 mg / mL, or about 3.1 mg / mL, or about 3.2 mg / mL, or about 3.3 mg / mL, or about 3.4 mg / mL, or about 3.5 mg / mL, or about 3.6 mg / mL, or about 3.7 mg / mL.

[0070] In some forms of this disclosure, the inventors have also found that including PEGylated lipids can improve the targeting of dLNs and / or reduce off-target effects (e.g., reduce hepatic uptake). Therefore, the lipid delivery carrier may further include PEGylated lipids.

[0071] Those skilled in the art will understand that references to PEGylated lipids refer to lipids modified with polyethylene glycol (PEG). Exemplary PEGylated lipids include, but are not limited to: PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, and PEG-modified dialkylglycerol. Suitable PEGylated lipids may include PEG-c-DOMG, PEG-DMG (e.g., 1,2-dimyristoyl-sn-glycerol-3-methoxy polyethylene glycol (DMG-PEG2000K)), PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE lipids, and any combination thereof.

[0072] The average molecular weight of PEG in PEGylated lipids can be 5000 Da or less, or 4000 Da or less, or 3000 Da or less, or 2000 Da or less, or 1000 Da or less. For example, the average molecular weight of PEG can be between about 500 Da and about 5000 Da, or between about 1000 Da and about 4000 Da. The average molecular weight of PEG can be about 2000 Da.

[0073] The lipid delivery carriers described herein may contain about 0.1 wt% to about 5 wt% of PEGylated lipids. For example, a lipid delivery carrier may contain about 0.1 wt% to about 2 wt% of PEGylated lipids. In one example, a lipid delivery carrier may contain about 0.5 wt% to about 5 wt% of PEGylated lipids. In another example, a lipid delivery carrier may contain about 0.5 wt% to about 3 wt% of PEGylated lipids. In one example, the lipid delivery carrier may comprise about 0.1 wt%, or about 0.2 wt%, or about 0.3 wt%, or about 0.4 wt%, or about 0.5 wt%, or about 0.6 wt%, or about 0.7 wt%, or about 0.8 wt%, or about 0.9 wt%, or about 1 wt%, or about 1.1 wt%, or about 1.2 wt%, or about 1.3 wt%, or about 1.4 wt%, or about 1.5 wt%, or about 1.6 wt%, or about 1.7 wt%, or about 1.8 wt%, or about 1.9 wt%, or about 2 wt%, or about 2.1 wt%, or about 2.2 wt%, or about 2.3 wt%, or about 2.4 wt%, or about 2.5 wt%, or about 2.6 wt%, or about 2.7 wt%, or about 2.8 wt%, or about 2.9 wt%, or about 3 wt%, or about 3.1 wt%. PEGylated lipids in wt%, or about 3.2 wt%, or about 3.3 wt%, or about 3.4 wt%, or about 3.5 wt%, or about 3.6 wt%, or about 3.7 wt%, or about 3.8 wt%, or about 3.9 wt%, or about 4 wt%, or about 4.1 wt%, or about 4.2 wt%, or about 4.3 wt%, or about 4.4 wt%, or about 4.5 wt%, or about 4.6 wt%, or about 4.7 wt%, or about 4.8 wt%, or about 4.9 wt%, or about 5 wt%.

[0074] Alternatively, the lipid delivery carrier described herein may contain about 0.1 mol% to about 5 mol% of PEGylated lipids. For example, the lipid delivery carrier may contain about 0.1 mol%, or about 0.2 mol%, or about 0.3 mol%, or about 0.4 mol%, or about 0.5 mol%, or about 0.6 mol%, or about 0.7 mol%, or about 0.8 mol%, or about 0.9 mol%, or about 1 mol%, or about 2 mol%, or about 3 mol%, or about 4 mol%, or about 5 mol% of PEGylated lipids.

[0075] Alternatively, the lipid delivery carrier described herein may comprise about 0.1 mg / mL to about 1 mg / mL of PEGylated lipids. For example, the lipid delivery carrier may comprise about 0.1 mg / mL, or about 0.2 mg / mL, or about 0.3 mg / mL, or about 0.4 mg / mL, or about 0.5 mg / mL, or about 0.6 mg / mL, or about 0.7 mg / mL, or about 0.8 mg / mL, or about 0.9 mg / mL, or about 1 mg / mL of PEGylated lipids.

[0076] Alternatively, the lipid delivery carrier described herein may comprise PEGylated lipids at a concentration of about 0.3 mg / mL to about 2 mg / mL. For example, the lipid delivery carrier may comprise PEGylated lipids at a concentration of about 0.3 mg / mL, or about 0.4 mg / mL, or about 0.5 mg / mL, or about 0.6 mg / mL, or about 0.7 mg / mL, or about 0.8 mg / mL, or about 0.9 mg / mL, or about 1 mg / mL, or about 1.1 mg / mL, or about 1.2 mg / mL, or about 1.3 mg / mL, or about 1.4 mg / mL, or about 0.5 mg / mL, or about 0.6 mg / mL, or about 1.7 mg / mL, or about 1.8 mg / mL, or about 1.9 mg / mL, or about 2 mg / mL.

[0077] Lipid delivery carriers can be cholesterol-free.

[0078] surfactants Surfactants can be incorporated into or used in the manufacture of lipid delivery carriers. Suitablely, the surfactant can be a glycerol phosphate. Exemplary glycerol phosphates may include phosphatidylcholine, such as the naturally occurring surfactant dipalmitoyl L-α-phosphatidylcholine (DPPC). Surfactants can advantageously improve the surface properties of the lipid delivery carrier, for example by reducing particle-particle interactions, and can reduce the surface adhesion of the carrier. Using pulmonary endogenous surfactants avoids the need for non-physiological surfactants.

[0079] Providing surfactants on the surface of lipid delivery carriers can reduce the tendency for particle aggregation due to interactions such as electrostatic interactions, van der Waals forces, and capillary action. The presence of surfactants on the surface of lipid delivery carriers can provide increased surface roughness, thereby improving atomization by reducing the surface area available for close particle-particle interactions.

[0080] Surfactants known in the art, including any naturally occurring surfactants, can be used. Other exemplary surfactants include phospholipids such as diphosphatidylglycerol (DPPG) or phosphatidylethanolamine; fatty alcohols or fatty acids such as palmitic acid or oleic acid polyoxyethylene-9-lauryl ether; sorbitan esters such as sorbitan trioleate (Span 85); bile salts; and amphiphilic polymers such as poloxamer or proteins. Mixtures of surfactants may also be used.

[0081] peptide antigen The term "peptide antigen" should be understood to refer to all or part of a protein or peptide that can elicit an immune response in vertebrates, especially mammals. Such antigens can also react with antibodies from animals immunized with that protein or peptide.

[0082] The term “autoantigen” should be understood as a specific subset of antigens derived from proteins or peptides produced by mammals and recognized by the immune system of said mammals to elicit an undesirable immune response.

[0083] As used herein, the term “derived from” should be understood to mean an antigen that consists of or contains a fragment or portion of the peptide from which it is derived, has a sufficiently high level of sequence or structural homology with the peptide, and / or has a sufficiently high level of sequence or structural homology with a fragment or portion of the peptide, such that the antigen can elicit an immune response associated with the peptide.

[0084] "Immune response" refers to the induction of a humoral or cellular response in a subject. Humoral or cellular responses may be specific to peptide antigens. This response can be detected and / or quantified by determining the induction of antibodies and / or cellular responses in the subject. Quantitative antibody measurements can be determined. Alternatively, or additionally, qualitative antibody measurements can be determined. For example, but not limited to, measurements of one or more functional characteristics of antibodies induced in a subject administered a peptide antigen can be determined.

[0085] The term "protein" should include a single polypeptide chain, that is, a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains (i.e., polypeptide complexes) linked covalently or nonvalently to each other. For example, a series of polypeptide chains can be covalently linked using suitable chemical bonds or disulfide bonds. Examples of nonvalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions.

[0086] As used herein, the term "peptide" is intended to include compounds consisting of amino acid residues linked by amide bonds. Peptides can be natural or non-natural, ribosome-encoded or synthetically derived. Typically, peptides will consist of 2 to 200 amino acids. For example, peptide lengths can range from 10 to 20 amino acids, 10 to 30 amino acids, 10 to 40 amino acids, 10 to 50 amino acids, 10 to 60 amino acids, 10 to 70 amino acids, 10 to 80 amino acids, 10 to 90 amino acids, or 10 to 100 amino acids, including any length within said range. The terms "protein" and "peptide" are used interchangeably herein.

[0087] There are various peptide antigens associated with unwanted or harmful immune responses. For example, peptide antigens can be autoantigens, allogeneic antigens, or allergens.

[0088] Examples of autoantigens may include, but are not limited to, those derived from Ro (e.g., Ro). 401-425 Type II collagen (CII) (e.g., CII) 259-27 ), proinsulin (PI) (e.g., PI) 33-63 Insulin, chromogranin, and agglutinins (e.g., agglutinins) 200-244 ), Islet antigen 2 (IA2), Glutamate decarboxylase 65 kDalton isotype (GAD65), Heterozymous insulin peptide (HIP), Glycoprotein (gp70), Nuclear antigen, Lupus autoantigen, Smith, La, U1-RNP, Fibrillar protein, Histone, Ribosomal protein, Pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2), Hair follicle antigen, Human tropomyosin isotype 5 (hTM5), Human cartilage gp39 (HCgp39) and gp130-RAPS, DNAJp1, Citrulline Peptide antigens of proteins, citrullinated peptides, citrullinated type II collagen, citrullinated vimentin, citrullinated fibrinogen, myelin basic protein, protein lipoprotein (PLP) and myelin oligodendrocyte glycoprotein (MOG), thyroid-stimulating hormone receptor (TSH-R), acetylcholine receptor (AchR), gliadin, histones, PLP, glucose-6-phosphate isomerase, thyroglobulin, various tRNA synthetases, protease-3, human desmosome core glycoprotein 2 (DSG2), and myeloperoxidase (MPO).

[0089] As used herein, the term "citrullinated" refers to a peptide antigen that has undergone post-translational modification at arginine residues through deamidation or citrullination, such as amino acid position 64 of the full-length amino acid sequence of vimentin or amino acid position 24 of SEQ ID NO:7, amino acid position 64 of the full-length amino acid sequence of vimentin or amino acid position 27 of SEQ ID NO:8, amino acid positions 210, 230, and 235 of the full-length amino acid sequence of agglutinin or amino acid positions 11, 32, and 37 of SEQ ID NO:11, amino acid positions 876 and 877 of the full-length amino acid sequence of tendinin or amino acid positions 6 and 7 of SEQ ID NO:56, amino acid positions 1014 and 1016 of the full-length amino acid sequence of tendinin or amino acid positions 3 and 5 of SEQ ID NO:57, amino acid position 1637 of the full-length amino acid sequence of tendinin or amino acid position 5 of SEQ ID NO:58, amino acid positions 2073 and 2077 of the full-length amino acid sequence of tendinin or amino acid positions 7 and 11 of SEQ ID NO:59. For example, the citrullination used in this paper is represented by X in the amino acid sequence.

[0090] Autoantigens can be derived from CII. For example, CII can be CII. 259-273 In some instances, CII 259-273 It comprises, consists of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:1, or fragments, variants, or derivatives thereof. In some instances, CII 259-273 Variants may contain amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:1.

[0091] Autoantigens can be derived from PIs. For example, a PI can be a PI... 33-63 In some instances, PI 33-63 It comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:2 or fragments, variants, or derivatives thereof. In some instances, the PI 33-63 Variants may contain amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:2.

[0092] Autoantigens can be derived from OVAs. For example, an OVA can be an OVA. 323-339In some instances, OVA 323-339 It comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:3 or fragments, variants, or derivatives thereof. In some instances, the OVA 323-339 Variants may contain amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:3.

[0093] Autoantigens can be derived from proteoglycans. For example, proteoglycan antigens may comprise, consist of, or consist substantially of the amino acid sequence shown in any one of SEQ ID NO:4 and 11-24 and 69, or fragments, variants, or derivatives thereof. In some instances, variants of the proteoglycan may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO:4, 11-24, and 69.

[0094] In one instance, the aggregate proteoglycan can be an aggregate proteoglycan. 84-103 Cit93. In some instances, aggregated proteoglycans 84-103 Cit93 comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:4 or fragments, variants, or derivatives thereof. In some instances, the aggregated proteoglycan... 84-103 Variants of Cit93 may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:4.

[0095] In one instance, the aggregate proteoglycan can be an aggregate proteoglycan. 89-103 In some instances, agglutinins... 89-103 It comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:69 or fragments, variants, or derivatives thereof. In some instances, the aggregated proteoglycan... 89-103Variants may contain amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:69.

[0096] Autoantigens can be derived from Ro. An autoantigen can be Ro60. For example, Ro can be Ro60. 401-425 In some instances, Ro60 401-425 It comprises, consists of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:5, or fragments, variants, or derivatives thereof. In some instances, the Ro60 401-425 Variants may contain amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:5.

[0097] Autoantigens can be derived from vimentins. For example, the vimentin may comprise, consist of, or consist substantially of the amino acid sequences shown in any one of SEQ ID NOs:6-8, or fragments, variants, or derivatives thereof. In some instances, variants of the vimentin may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequences shown in any one of SEQ ID NOs:6-8, 66-68, and 70, or fragments, variants, or derivatives thereof. In some instances, variants of the vimentin may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NO: 68. For example, the vimentin may be derived from the amino acid sequence shown in SEQ ID NO: 68.

[0098] In one instance, a vimentin-derived self-antigen could be vimentin itself. 41-85 In some instances, vimentin 41-85It comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:6, or fragments, variants, or derivatives thereof. In some instances, the vimentin... 41-85 Variants may contain amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:6.

[0099] In another instance, a vimentin-derived self-antigen could be vimentin itself. 41-85 Cit64. In some instances, vimentin. 41-85 Cit64 comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:7, or fragments, variants, or derivatives thereof. In some instances, the vimentin... 41-85 Variants of Cit64 may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:7.

[0100] In another instance, a vimentin-derived self-antigen could be vimentin itself. 38-88 Cit64. In some instances, vimentin. 38-88 Cit64 comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:8, or fragments, variants, or derivatives thereof. In some instances, the vimentin... 38-88 Variants of Cit64 may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:8.

[0101] In another instance, a vimentin-derived self-antigen could be vimentin itself. 53-85 Cit64. In some instances, vimentin. 53-85 Cit64 comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:66, or fragments, variants, or derivatives thereof. In some instances, the vimentin... 53-85Variants of Cit64 may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:66.

[0102] In another instance, a vimentin-derived self-antigen could be vimentin itself. 60-85 Cit64. In some instances, vimentin. 60-85 Cit64 comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:67, or fragments, variants, or derivatives thereof. In some instances, the vimentin... 60-85 Variants of Cit64 may contain an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:67.

[0103] In another instance, a vimentin-derived self-antigen could be vimentin itself. 59-71 In some instances, vimentin 59-71 It comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:70, or fragments, variants, or derivatives thereof. In some instances, the vimentin... 59-71 Variants may contain amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:70.

[0104] The autoantigen can be derived from myeloperoxidase (MPO). For example, the MPO may comprise, consist of, or be substantially composed of the amino acid sequence shown in any one of SEQ ID NOs:9 and 10, or fragments, variants, or derivatives thereof. In some instances, variants of the MPO may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs:9 and 10.

[0105] Autoantigens can be derived from the thyroid-stimulating hormone receptor (TSH). For example, the TSH may comprise, consist of, or be substantially composed of the amino acid sequence shown in any one of SEQ ID NOs:25-55, or fragments, variants, or derivatives thereof. In some instances, variants of the TSH may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs:25-55.

[0106] The autoantigen can be derived from Tenascin C. For example, Tenascin C may comprise, consist of, or consist substantially of the amino acid sequence shown in any one of SEQ ID NOs:56-59, or fragments, variants, or derivatives thereof. In some instances, variants of Tenascin C may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs:56-59.

[0107] Autoantigens can be derived from desmoglein-2. For example, desmoglein-2 can comprise, consist of, or be substantially composed of the amino acid sequence shown in SEQ ID NO:71 or fragments, variants, or derivatives thereof. In some instances, variants of desmoglein-2 may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:71.

[0108] The peptide antigens of this disclosure may be water-soluble. As those skilled in the art will understand, "water solubility" is generally affected by the amino acid composition of the peptide, including the number of charged, uncharged, polar, or nonpolar amino acids. The water solubility of a peptide antigen can be determined by methods known in the art, including but not limited to gravimetric analysis, UV-Vis spectroscopy, HPLC, dynamic light scattering (DLS), and solubility tests. The peptide antigens of this disclosure may be water-soluble as determined by gravimetric analysis. The peptide antigens of this disclosure may be water-soluble as determined by UV-Vis spectroscopy. The peptide antigens of this disclosure may be water-soluble as determined by HPLC. The peptide antigens of this disclosure may be water-soluble as determined by dynamic light scattering (DLS). The peptide antigens of this disclosure may be water-soluble as determined by solubility tests.

[0109] The peptide antigens disclosed herein may carry a negative charge or a neutral charge. The peptide antigens disclosed herein may carry a negative charge. The peptide antigens disclosed herein may carry a neutral charge. The charge of the peptide antigen can be determined by methods known in the art, including but not limited to calculating the net charge of the peptide antigen. The charge of the peptide antigen can be changed by adjusting the pH of the solution containing the peptide antigen. For example, to prepare a peptide antigen with a neutral charge, the pH of the solution containing the peptide antigen is adjusted to the isoelectric point (pI) of the peptide antigen. Alternatively, to prepare a peptide antigen with a negative charge, the pH of the solution containing the peptide antigen is adjusted to a level higher than the pI of the peptide antigen.

[0110] A peptide antigen disclosed herein may contain multiple epitopes. For example, a multi-epitope peptide antigen may contain a total of 30 or fewer epitopes (e.g., 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, etc. or any range thereof). Alternatively, a peptide antigen may contain a total of 20 or fewer epitopes (e.g., 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, etc. or any range thereof).

[0111] As used herein, the term "epitope" refers to any protein determinant capable of specifically binding to an immunoglobulin or a fragment thereof. In some instances, the peptide antigens described herein may contain multiple epitopes. For example, the peptide antigens described herein may be contained within or housed within a multi-epitope protein or a polytope protein. A multi-epitope protein may contain one or more human leukocyte antigen (HLA) epitopes. More specifically, the multi-epitope proteins of this disclosure may include two or more different peptide antigens described herein. Those skilled in the art will understand that a particular number and / or type of constituent epitopes of the peptide antigen and / or multi-epitope protein can be readily modified while maintaining broad HLA immunogenicity. In addition to epitopes, the peptide antigen or multi-epitope protein may further contain additional or intercalated amino acids or amino acid sequences (e.g., linker sequences). Intercalated amino acids or amino acid sequences may be present between at least two epitope amino acid sequences, or between each adjacent epitope amino acid sequence. It is also contemplated that additional amino acids or amino acid sequences may be present at one or both of the N-terminus and C-terminus of the peptide antigen or multi-epitope protein.

[0112] Those skilled in the art will understand that the selection of epitopes included in peptide antigens and / or multi-epitope proteins can be tailored to suit any population, ethnicity, or other individual group.

[0113] Other criteria for including specific epitopes within peptide antigens include those epitopes that (i) have minimal or no sequence variation; and (ii) are selected from HLA epitopes with the fewest subtypes.

[0114] Isolated proteins, including the multi-epitope proteins described herein, may contain multiple epitopes derived from a variety of different protein antigens.

[0115] The peptide antigens or multi-epitope proteins described herein may have epitopes derived from one or more proteins (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more), two or more proteins, three or more proteins, four or more proteins, or five or more proteins, wherein the proteins are selected from those containing Ro (e.g., Ro). 401-425 Type II collagen (CII) (e.g., CII) 259-27 ), proinsulin (PI) (e.g., PI) 33-63 Insulin, chromogranin, and agglutinins (e.g., agglutinins) 200-244), Islet antigen 2 (IA2), glutamate decarboxylase 65 kDalton isotype (GAD65), glycoprotein (gp70), nuclear antigen, lupus autoantigen, Smith, La, U1-RNP, fibrin, histone, ribosomal protein, pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, human tropomyosin isotype 5 (hTM5), human cartilage gp39 (HCgp39) and gp130-RAPS, DNAJp1, citrullinated protein, citrullinated peptide, citrullinated type II collagen, citrullinated vimentin (e.g., vimentin) 41-85 Cit64, vimentin 53-85 Cit64 or vimentin 60-85 Cit64), citrullinated fibrinogen, citrullinated aggregates (e.g., aggregates) 200-244 Cit210, Cit230, Cit235), citrullinated tendinin C (e.g., tendinin C) 1012-1026 Cit1014, Cit1016), myelin basic protein, protein lipoprotein (PLP), and myelin oligodendrocyte glycoprotein (MOG), thyroid-stimulating hormone receptor (TSH-R, e.g., TSHR) 57-75 TSHR 70-88 TSHR 227-242 TSHR 315-334 Acetylcholine receptor (AChR), gliadin, histones, PLP, glucose-6-phosphate isomerase, thyroglobulin, various tRNA synthetases, protease 3, and myeloperoxidase (MPO, e.g., MPO) 435-465 MPO 447-459 ) and its combinations.

[0116] Multiepitope proteins may comprise two or more different epitopes derived from two or more different peptide antigens or proteins described herein. For example, a multiepitope protein may comprise an epitope derived from PI and an epitope derived from vimentin. For example, a multiepitope protein may comprise an epitope derived from type II collagen and an epitope derived from vimentin. For example, a multiepitope protein may comprise a native epitope derived from PI and a heterozygous insulin peptide (HIP) epitope derived from PI. More specifically, a multiepitope protein may comprise an epitope derived from the amino acid sequence shown in SEQ ID NO:2 (i.e., PI). 33-63 The protein comprises a first amino acid sequence and a second amino acid sequence derived from any one of the amino acid sequences shown in SEQ ID NOs:60-65. More specifically, the multi-epitope protein may comprise a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises, is composed of, or is substantially composed of SEQ ID NO:2 (i.e., PI). 33-63The second amino acid sequence consists of, is composed of, or is substantially composed of SEQ ID NO:7 (i.e., vimentin). 41-85 The protein consists of the amino acid sequence shown in SEQ ID NO:7. In another example, the multiepitope protein may comprise a first amino acid sequence derived from the amino acid sequence shown in SEQ ID NO:7 and a second amino acid sequence derived from a second or third citrullinated or non-citrullinated RA autoantigen (e.g., any one of SEQ ID NOs:1, 4, 11-24, and 56-59). In one example, the multiepitope protein may comprise a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:66, and the second amino acid sequence comprises, is composed of, or is substantially composed of the amino acid sequences shown in any one of SEQ ID NOs:1, 4-6, 11-24, and 56-59.

[0117] As used in this article, “hybrid insulin peptide” or “HIP” should be understood as a novel epitope formed by the fusion of two unrelated peptide segments. HIPs are generated by covalently cross-linking proinsulin peptide with other peptides present in β-cell secretory granules and are recognized by pathogenic CD4 T cells. Delong, T., et al., Science, 2016. 351(6274): p. 711-714 and Tran, M., et al., Journal of Biological Chemistry, Volume 300, Issue 9, 107612 (incorporated into this article by reference) both discuss how pathogenic CD4 T cells recognize HIPs in type 1 diabetes.

[0118] In one instance, the HIP comprises, consists of, or is substantially composed of the amino acid sequence shown in any one of SEQ ID NOs:60-65, or fragments, variants, or derivatives thereof. In some instances, its variants may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in any one of SEQ ID NOs:60-65.

[0119] In one instance, the HIP comprises, consists of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:60 or fragments, variants, or derivatives thereof. In some instances, its variants may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:60.

[0120] In one instance, the HIP comprises, consists of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:61 or fragments, variants, or derivatives thereof. In some instances, its variants may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:61.

[0121] In one instance, the HIP comprises, consists of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:62 or fragments, variants, or derivatives thereof. In some instances, its variants may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:62.

[0122] In one instance, the HIP comprises, consists of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:63 or fragments, variants, or derivatives thereof. In some instances, its variants may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:63.

[0123] In one instance, the HIP comprises, consists of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:64 or fragments, variants, or derivatives thereof. In some instances, its variants may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:64.

[0124] In one instance, the HIP comprises, consists of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:65 or fragments, variants, or derivatives thereof. In some instances, its variants may comprise an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acid sequence shown in SEQ ID NO:65.

[0125] Alternatively, a multiepitope protein may comprise two or more copies of a single epitope derived from the single peptide antigen described herein. In other instances, a multiepitope protein may comprise two or more different epitopes derived from the single peptide antigen described herein.

[0126] This article considers “variants” of peptide antigens. As used herein, a nucleic acid “variant” shares a definable amino acid sequence relationship with a reference amino acid sequence (e.g., any one of SEQ ID NOs: 1-71). The “variant” amino acid may have one or more amino acids of the reference amino acid sequence that are deleted, modified, conjugated, and / or substituted with different amino acids. The “variant” amino acid sequence may be truncated or extended while still retaining a definable amino acid sequence relationship with the reference amino acid sequence on a continuous segment of amino acids. It is known in the art that conserved amino acids of a protein can be substituted or deleted without altering (or only minimally altering) the protein’s activity. Suitable, the amino acid variants share at least 60% or 65%, 66%, 67%, 68%, 69%, preferably at least 70%, 71%, 72%, 73%, 74%, or 75%, more particularly at least 80%, 81%, 82%, 83%, 84%, or 85%, and even more particularly at least 90%, 91%, 92%, 93%, 94%, or 95% amino acid sequence identity with the amino acids of this disclosure (e.g., SEQ ID NOs: 1-71). The percentage sequence identity can be determined by any method known in the art, such as those described herein.

[0127] The terms commonly used in this article to describe sequence relationships between individual amino acids and nucleic acids include “comparison window,” “sequence identity,” “percentage of sequence identity,” and “substantial identity.” Since each nucleic acid / protein may contain (1) one or more portions of a complete nucleic acid / amino acid sequence shared only between nucleic acids / amino acids, and (2) one or more portions that differ between nucleic acids / amino acids, sequence comparison is typically performed by identifying and comparing local regions of sequence similarity across a “comparison window.” A “comparison window” refers to a conceptual segment, typically 6, 9, or 12 consecutive residues, used for comparison with a reference sequence. For optimal alignment of individual sequences, the comparison window may contain approximately 20% or less of additions or deletions (i.e., vacancies) compared to the reference sequence. The optimal alignment of sequences for the comparison window can be performed by the computerized implementation of the algorithm (Intelligenetics’ Geneworks program; GAP, BESTFIT, FASTA, and TFASTA in Wisconsin Genetics Package version 7.0, Genetics Computer Group, 575 Science Drive, Madison, WI, USA, incorporated herein by reference) or by examining and generating the best alignment (i.e., producing the highest percentage of homology on the comparison window) by any of the various selected methods. See also the BLAST family of programs, such as those disclosed in Altschul et al., 1997, Nucl. Acids Res. 25 3389, which is incorporated herein by reference. A detailed discussion of sequence analysis can be found in Unit 19.3 of Current Protocols in Molecular Biology, edited by Ausubel et al. (John Wiley & Sons Inc NY, 1995–1999).

[0128] The term “sequence identity” is used in its broadest sense herein, encompassing the degree to which sequences are identical on a comparison window after proper alignment using standard algorithms, taking into account the number of precise nucleotide or amino acid matches. Therefore, the “sequence identity percentage” is calculated by comparing two optimally aligned sequences on a comparison window, determining the number of positions in both sequences where the same nucleic acid base (e.g., A, T, C, G, U) or amino acid residue appears to produce a number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence identity percentage. For example, “sequence identity” can be understood as the “percentage of matches” calculated by the DNASIS computer program (Windows version 2.5; available from Hitachi Software Engineering Co., Ltd., South San Francisco, California, USA).

[0129] This article also considers amino acid fragments, such as peptide antigen fragments. A “fragment” is a segment, domain, part, or region of amino acids that constitutes less than 100% of the amino acid sequence. In specific instances, the amino acid fragment may comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 (inclusive) consecutive amino acids of, for example, the amino acid sequence described above (e.g., SEQ ID NOs: 1-71).

[0130] This article also considers nucleic acid derivatives, including peptide antigen derivatives. Such peptide antigen derivatives may include one or more modifications and / or conjugates, as described herein or known in the art.

[0131] "Allogeneic antigens" should be understood as antigens found only in a subset of members of a species, such as blood group antigens. Examples of allogeneic antigens may include, but are not limited to, glycoproteins (e.g., MHC, MNS antigens), sialic acid glycoproteins (e.g., CD43), oligosaccharides (e.g., ABO(H), secretory, Lewis, Li, and P), and sialic acid oligosaccharides (e.g., sialyl-Lewis). x Or sialoyl-Lewis a ) and proteins (e.g., rhesus monkeys).

[0132] Examples of allergens may include, but are not limited to, Fel d 1 (the feline skin and salivary gland allergen of the domestic cat Felis domesticus, whose amino acid sequence is described in WO1991006571A1 and is incorporated herein by reference), Der p I, Der p II, Der f I or Der f II (the major protein allergen of the house dust mite Dermatophagoides, whose amino acid sequence is described in WO1994024281A1 and is incorporated herein by reference).

[0133] Allergens can also be derived from, for example, any of the following: pollen from grasses, trees, and weeds (including ragweed); fungi and molds; foods such as fish, shellfish, crabs, lobsters, peanuts, nuts, wheat gluten, eggs, and milk; piercing insects such as bees, wasps, and hornets, as well as midges (non-biting midges); other insects such as houseflies, fruit flies, blowflies, spiral fly, cereal weevils, silkworms, bees, non-biting midge larvae, wasp moth larvae, mealworms, cockroaches, and Tenebrio molitor beetle larvae; spiders and mites, including house dust mites; allergens found in the dander, urine, saliva, blood, or other bodily fluids of mammals such as cats, dogs, cattle, pigs, sheep, horses, rabbits, rats, guinea pigs, mice, and gerbils; particulate matter in the general air; latex; and protein detergent additives.

[0134] Peptide antigens can be isolated from natural sources or prepared using recombinant techniques known in the art. For example, peptide antigens can be eluted from the major histocompatibility complex (MHC) and other antigen-presenting molecules of cell populations or tissues from which a modified immune response is desired (e.g., allogeneic tissues or cell populations in transplant medicine). The eluted peptides can be purified using standard protein purification techniques known in the art (Rawson et al., 2000, Cancer Res60(16), 4493-4498). If desired, the purified peptides can be sequenced, and synthetic versions of the peptides can be produced using standard protein synthesis techniques, such as those described below.

[0135] Alternatively, a crude antigen preparation can be prepared by isolating a sample of cell populations or tissues from which a modified immune response is desired, and lysing the sample or subjecting it to conditions that induce apoptosis (e.g., irradiation with ultraviolet or gamma rays, viral infection, cytokines, or by depriving the cell culture medium of nutrients, incubation with hydrogen peroxide, or incubation with drugs such as dexamethasone, ceramide chemotherapeutic agents, and anti-hormonal agents such as leuprorelin or tamoxifen). The lysate or apoptotic cells can then be used as a source of crude antigen for contact with antigen-presenting cells.

[0136] Peptide antigens can be conveniently prepared in recombinant form using standard protocols, such as those described in Sambrook et al., *Molecular Cloning: A Laboratory Manual* (Cold Spring Harbor Press, 1989), particularly Sections 16 and 17; Ausubel et al., *Current Protocols in Molecular Biology* (John Wiley & Sons, Inc., 1994–1998), particularly Chapters 10 and 16; and Coligan et al., *Current Protocols in Protein Science* (John Wiley & Sons, Inc., 1995–1997), particularly Chapters 1, 5, and 6.

[0137] Peptide antigens can be prepared by a method comprising the following steps: (a) providing an expression vector from which the peptide antigen or an analogue or mimic thereof can be expressed; (b) introducing the vector into a suitable host cell; (c) culturing the host cell to express the recombinant polypeptide from the vector; and (d) isolating the recombinant polypeptide.

[0138] Alternatively, peptide antigens can be synthesized using liquid-phase or solid-phase synthesis, as described by Atherton and Sheppard (Solid Phase Peptide Synthesis: A Practical Approach, IRL Press at Oxford University Press, Oxford, England, 1989) or Roberge et al. (1995, Science 269: 202).

[0139] The peptide antigen may be present in the lipid delivery carrier described herein at an amount of about 1 µg / mL to about 50 µg / mL. For example, the peptide antigen may be present in the lipid delivery carrier described herein at an amount of about 10 µg / mL to about 30 µg / mL. For example, peptide antigens can be expressed in concentrations of approximately 1 µg / mL, or approximately 2 µg / mL, or approximately 3 µg / mL, or approximately 4 µg / mL, or approximately 5 µg / mL, or approximately 6 µg / mL, or approximately 7 µg / mL, or approximately 8 µg / mL, or approximately 9 µg / mL, or approximately 10 µg / mL, or approximately 11 µg / mL, or approximately 12 µg / mL, or approximately 13 µg / mL, or approximately 14 µg / mL, or approximately 15 µg / mL, or approximately 16 µg / mL, or approximately 17 µg / mL, or approximately 18 µg / mL, or approximately 19 µg / mL, or approximately 20 µg / mL, or approximately 21 µg / mL, or approximately 22 µg / mL, or approximately 23 µg / mL, or approximately 24 µg / mL, or approximately 25 µg / mL, or approximately 6 µg / mL, or approximately 27 µg / mL, or approximately 28 µg / mL. It is present in amounts of µg / mL, or about 29 µg / mL, or about 30 µg / mL, or about 31 µg / mL, or about 32 µg / mL, or about 33 µg / mL, or about 34 µg / mL, or about 35 µg / mL, or about 36 µg / mL, or about 37 µg / mL, or about 38 µg / mL, or about 39 µg / mL, or about 40 µg / mL, or about 41 µg / mL, or about 42 µg / mL, or about 43 µg / mL, or about 44 µg / mL, or about 45 µg / mL, or about 46 µg / mL, or about 47 µg / mL, or about 48 µg / mL, or about 49 µg / mL, or about 50 µg / mL.

[0140] For example, an autoantigen derived from proinsulin (e.g., SEQ ID NO: 2) may be present in the lipid delivery carrier described herein in an amount from about 1 µg / mL to about 50 µg / mL. For example, an autoantigen derived from proinsulin (e.g., SEQ ID NO: 2) may be present in the lipid delivery carrier described herein in an amount from about 10 µg / mL to about 30 µg / mL. For example, an autoantigen derived from proinsulin (e.g., SEQ ID NO: 2) may be present in concentrations of about 1 µg / mL, or about 2 µg / mL, or about 3 µg / mL, or about 4 µg / mL, or about 5 µg / mL, or about 6 µg / mL, or about 7 µg / mL, or about 8 µg / mL, or about 9 µg / mL, or about 10 µg / mL, or about 11 µg / mL, or about 12 µg / mL, or about 13 µg / mL, or about 14 µg / mL, or about 15 µg / mL, or about 16 µg / mL, or about 17 µg / mL, or about 18 µg / mL, or about 19 µg / mL, or about 20 µg / mL, or about 21 µg / mL, or about 22 µg / mL, or about 23 µg / mL, or about 24 µg / mL, or about 25 µg / mL, or about 6 µg / mL. It is present in amounts of µg / mL, or about 27 µg / mL, or about 28 µg / mL, or about 29 µg / mL, or about 30 µg / mL, or about 31 µg / mL, or about 32 µg / mL, or about 33 µg / mL, or about 34 µg / mL, or about 35 µg / mL, or about 36 µg / mL, or about 37 µg / mL, or about 38 µg / mL, or about 39 µg / mL, or about 40 µg / mL, or about 41 µg / mL, or about 42 µg / mL, or about 43 µg / mL, or about 44 µg / mL, or about 45 µg / mL, or about 46 µg / mL, or about 47 µg / mL, or about 48 µg / mL, or about 49 µg / mL, or about 50 µg / mL.

[0141] A surprising feature of the disclosed lipid delivery carrier containing 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate glycerol (POPG) is its ability to retain encapsulated peptides and / or nuclear factor-κB (NF-κB) inhibitors within the lipid delivery carrier. Given the notorious reputation of lipid delivery carriers for “leakage” (i.e., APIs are typically not well retained, and this leakage worsens over time, causing storage stability issues, which are further exacerbated by incubation in biological fluids, potentially reducing the effectiveness of targeted subcutaneous delivery), the retention of encapsulated peptides and / or NF-κB inhibitors within the lipid delivery carrier is remarkable. Therefore, the lipid delivery carriers of this disclosure are advantageous for storing aqueous formulations / compositions containing loaded lipid delivery carriers, and are particularly advantageous for aqueous formulations / compositions containing loaded lipid delivery carriers intended for subcutaneous delivery, since these carriers must retain the encapsulated peptides and / or nuclear factor-κB (NF-κB) inhibitors within the lipid delivery carrier during recipient plasma circulation.

[0142] As used herein, “retention” of peptide antigens and / or NF-κB inhibitors refers to the encapsulation stability or efficiency of APIs (e.g., peptide antigens and / or NF-κB inhibitors) in vitro or in vivo, and can be measured using any method known in the art, including methods described or developed herein (e.g., concentration after in vitro storage at 4°C or -20°C, or in vivo stability in plasma estimated by SEC after incubation with PBS or plasma at 37°C, and / or peptidase assay).

[0143] The liposome concentrations of peptide antigens and / or NF-κB inhibitors can be measured using any method known in the art, including those described herein (e.g., mass spectrometry, enzyme-linked immunosorbent assay (ELISA)).

[0144] The retention of peptide antigens can be measured using mass spectrometry (e.g., as described in the examples) before and / or after incubation with plasma. The retention of peptide antigens can be calculated by measuring the retention before incubation with plasma using mass spectrometry and measuring the retention after incubation with plasma using mass spectrometry, and determining the difference in the amount of peptide antigens.

[0145] The retention of NF-κB inhibitors can be measured using an ELISA (e.g., as described in the examples) before and / or after incubation with plasma. The retention of NF-κB inhibitors can be calculated by measuring the retention of NF-κB inhibitors before and after incubation with plasma using an ELISA, and by determining the difference in the amount of NF-κB inhibitors.

[0146] When expressed as a percentage, the retention percentage of peptide antigens and / or NF-κB inhibitors refers to the liposome concentration of peptide antigens and / or NF-κB inhibitors after incubation for a specific period of time under specific conditions (i.e., 15 minutes in plasma at 37°C; or 1 hour in PBS at 20°C), relative to the pre-incubation liposome concentration of peptide antigens and / or NF-κB inhibitors. This can also be expressed as the percentage of peptide antigens and / or NF-κB inhibitors retained within the lipid delivery carrier.

[0147] The retention of peptide antigens within the lipid delivery carrier can be between 25% and 100%. The retention of peptide antigens within the lipid delivery carrier can be between 50% and 100%. The retention of peptide antigens within the lipid delivery carrier can be between 70% and 100%. The retention of peptide antigens within the lipid delivery carrier can be between 70% and 90%. The retention of peptide antigens within the lipid delivery carrier can be at least 25%, for example, about 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100%. In some instances, the retention can be at least 40%. In some instances, the retention can be at least 50%.

[0148] NF-κB inhibitors As used herein, the terms “nuclear factor-κB” or “NF-κB” should be understood as referring to a protein complex (e.g., an immunomodulator) that controls DNA transcription. NF-κB is present in most cell types and participates in cellular responses to stimuli such as stress, cytokines, free radicals, ultraviolet radiation, oxidized low-density lipoprotein, and bacterial or viral antigens. NF-κB plays a crucial role in regulating the immune response to infection, and dysregulation of NF-κB is associated with a variety of diseases. The NF-κB signaling pathway and its components are known in the art.

[0149] The term "nuclear factor-κB inhibitor" or "NF-κB inhibitor" should be understood to refer to any molecule or compound that increases or decreases the level or functional activity of NF-κB signaling or NF-κB signaling pathway proteins. For example, an NF-κB inhibitor can be an inhibitor that has been identified as inhibiting the activity of NF-κB proteins. Alternatively, an NF-κB inhibitor can be a regulator of pathway proteins that inhibit active NF-κB signaling.

[0150] As used herein, the term "reduction" should be understood to mean a decrease in the level and / or functional activity of NF-κB signaling pathway members or proteins compared to their native levels and / or functional activity. For example, a "reduction" in the level and / or functional activity of NF-κB signaling pathway members or proteins is a reduction compared to their native levels and / or functional activity prior to administration of an NF-κB inhibitor.

[0151] As used herein, the term “increase” should be understood to mean an increase in the level and / or functional activity of NF-κB signaling pathway members or proteins compared to their native levels and / or functional activity. For example, an “increase” in the level and / or functional activity of NF-κB signaling pathway members or proteins is an elevation compared to their native levels and / or functional activity prior to administration of an NF-κB inhibitor.

[0152] NF-κB inhibitors can reduce the levels and / or functional activity of NF-κB signaling pathway members selected from the following group: BTK, LYN, BCR Igα, BCR Igβ, Syk, Bink, PLCγ2, PKCβ, DAG, CARMA1, BCL1O, MALT1, PI3K, PIP3, AKT, p38 MAPK, ERK, COT, IKKα, IKKβ, IKKγ, NIK, RelA / p65, P105 / p50, cRel, RelB, p52, NIK, Leu13, CD81, CD19, CD21 and their ligands in the complement and coagulation cascade, TRAF6, ubiquitin ligase, Tab2, TAK1, NEMO, NOD2, RIP2, Lek, fyn, Zap70, LAT, GRB2, SOS, CD3 Zeta, Slp-76, GADS, ITK, PLCγ1, PKCθ, ICOS, CD28, SHP2, SAP, SLAM and 2B4 and their combinations.

[0153] NF-κB inhibitors can reduce the level and / or functional activity of NF-κB family proteins selected from the following group: Rel-A (p65), Rel-B, Rel (c-Rel), NF-κB1 (p50 / p105) and NF-κB2 (p52 / p100) and combinations thereof.

[0154] Appropriately, NF-κB inhibitors block, inhibit, or otherwise antagonize at least one function or activity of a member of the NF-κB signaling pathway.

[0155] NF-κB inhibitors can increase the level and / or functional activity of NF-κB signaling pathway members selected from the following group: SHP1, SHIP, PIR-B, CD22, CD72, FcgRIIB, IκB, P100, CTLA4, PD-1, Chi, KIR3DL1, KIR3DL2, KIR2DL and Csk and combinations thereof.

[0156] Appropriately, NF-κB inhibitors increase, stimulate, or otherwise activate at least one function or activity of members of the NF-κB signaling pathway.

[0157] NF-κB inhibitors can be vitamin D, active metabolites of vitamin D, provitamin D, or synthetic vitamin D analogs. Vitamin D and related molecules, whether naturally occurring or synthetic, are known to be NF-κB inhibitors and are used in a range of other therapeutic applications. However, naturally occurring vitamin D is not always suitable for the numerous indications for which vitamin D has been investigated and / or involved, leading to the discovery and development of a large number of vitamin D alternatives—molecules that serve as prodrug or prodrug versions of vitamin D—as well as vitamin analogs with superior drugability profiles. As this group of “alternatives” will only increase over time, it should be understood that this disclosure is intended to include any currently and future discovered or synthesized vitamin D, active metabolites of vitamin D, provitamin D, or synthetic vitamin D analogs that have properties similar to or even superior to calcitriol, the vitamin D analog exemplified herein.

[0158] NF-κB inhibitors may be selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, idecalciferol, inenacalciferol, siocalciferol, calcipotriene, mascaracalciferol, calcidiol, calcipotriol; their synthetic analogs and / or side-chain analogs; and combinations thereof.

[0159] NF-κB inhibitors may be selected from the following group: calcitriol, rapamycin, leflunomide, teriflunomide, bevacizumab, thiochlorophen, bortezomib, doxorubicin hydrochloride, cantharidin, carfilzomib, chromomycin, daunorubicin, digoxin, sucrose, emetine, erlotinib hydrochloride, gemcitabine, irinotecan, quinacrine dihydrochloride, fluxalen, manidipine hydrochloride, methylsalicylic acid, lentatinib, ouabain, pemetrexed disodium, sorafenib tosylate, sunitinib malate, tiaconazole, topotecan, tribromosalinomycin, triclobenzazole, zafirlukast, solanine A, quercetin, curcumin, Bay 11-7085, Bay 11-7082, dexamethasone, and combinations thereof. For example, an NF-κB inhibitor may be calcitriol. Alternatively, an NF-κB inhibitor may be rapamycin. NF-κB inhibitors can include leflunomide, teriflunomide, bevacizumab, thiochlorophen, bortezomib, doxorubicin hydrochloride, cantharidin, carfilzomib, chromomycin / daunorubicin, digoxin, sucralose, emetine, erlotinib hydrochloride, gemcitabine, irinotecan, quinacrine dihydrochloride, fluxalam, manidipine hydrochloride, methylsalicylate, and lenatatinib. NF-κB inhibitors can include: ouabain, pemetrexed disodium, sorafenib tosylate, sunitinib malate, tiaconazole, topotecan, tribromosalam, triclobenzazole, zafirlukast, solanine A, quercetin, curcumin, Bay 11-7085, Bay 11-7082, and dexamethasone.

[0160] NF-κB inhibitors can be non-toxic to subjects. For example, NF-κB inhibitors can produce minimal or negligible side effects in subjects. NF-κB inhibitors can block the alternative NF-κB pathway.

[0161] The NF-κB inhibitor may be present in the lipid delivery vehicle described herein at an amount of about 10 ng / mL to about 600 ng / mL. Alternatively, the NF-κB inhibitor may be present in the lipid delivery vehicle described herein at an amount of about 10 ng / mL to about 100 ng / mL. In another alternative, the NF-κB inhibitor may be present in the lipid delivery vehicle described herein at an amount of about 300 ng / mL to about 600 ng / mL. Alternatively, the NF-κB inhibitor may be present in the lipid delivery vehicle described herein at an amount of about 100 ng / mL to about 300 ng / mL. For example, NF-κB inhibitors can be administered at doses of approximately 10 ng / mL, or approximately 20 ng / mL, or approximately 30 ng / mL, or approximately 40 ng / mL, or approximately 50 ng / mL, or approximately 60 ng / mL, or approximately 70 ng / mL, or approximately 80 ng / mL, or approximately 90 ng / mL, or approximately 100 ng / mL, or approximately 110 ng / mL, or approximately 120 ng / mL, or approximately 130 ng / mL, or approximately 140 ng / mL, or approximately 150 ng / mL, or approximately 160 ng / mL, or approximately 170 ng / mL, or approximately 180 ng / mL, or approximately 190 ng / mL, or approximately 200 ng / mL, or approximately 210 ng / mL, or approximately 220 ng / mL, or approximately 230 ng / mL, or approximately 240 ng / mL, or approximately 250 ng / mL. ng / mL, or about 260 ng / mL, or about 270 ng / mL, or about 280 ng / mL, or about 290 ng / mL, or about 300 ng / mL, or about 310 ng / mL, or about 320 ng / mL, or about 330 ng / mL, or about 340 ng / mL, or about 350 ng / mL, or about 360 ng / mL, or about 370 ng / mL, or about 380 ng / mL, or about 390 ng / mL, or about 400 ng / mL, or about 410 ng / mL, or about 420 ng / mL, or about 430 ng / mL, or about 440 ng / mL, or about 450 ng / mL, or about 460 ng / mL, or about 470 ng / mL, or about 480 ng / mL, or about 490 ng / mL It is present in amounts of ng / mL, or about 500 ng / mL, or about 510 ng / mL, or about 520 ng / mL, or about 530 ng / mL, or about 540 ng / mL, or about 550 ng / mL, or about 560 ng / mL, or about 570 ng / mL, or about 580 ng / mL, or about 590 ng / mL, or about 600 ng / mL.

[0162] Calcitriol may be present in amounts from about 100 ng / mL to about 600 ng / mL. Alternatively, calcitriol may be present in amounts from about 300 ng / mL to about 600 ng / mL. For example, calcitriol can be administered at concentrations of approximately 100 ng / mL, or approximately 110 ng / mL, or approximately 120 ng / mL, or approximately 130 ng / mL, or approximately 140 ng / mL, or approximately 150 ng / mL, or approximately 160 ng / mL, or approximately 170 ng / mL, or approximately 180 ng / mL, or approximately 190 ng / mL, or approximately 200 ng / mL, or approximately 210 ng / mL, or approximately 220 ng / mL, or approximately 230 ng / mL, or approximately 240 ng / mL, or approximately 250 ng / mL, or approximately 260 ng / mL, or approximately 270 ng / mL, or approximately 280 ng / mL, or approximately 290 ng / mL, or approximately 300 ng / mL, or approximately 310 ng / mL, or approximately 320 ng / mL, or approximately 330 ng / mL. ng / mL, or approximately 340 ng / mL, or approximately 350 ng / mL, or approximately 360 ng / mL, or approximately 370 ng / mL, or approximately 380 ng / mL, or approximately 390 ng / mL, or approximately 400 ng / mL, or approximately 410 ng / mL, or approximately 420 ng / mL, or approximately 430 ng / mL, or approximately 440 ng / mL, or approximately 450 ng / mL, or approximately 460 ng / mL, or approximately 470 ng / mL, or approximately 480 ng / mL, or approximately 490 ng / mL, or approximately 500 ng / mL, or approximately 510 ng / mL, or approximately 520 ng / mL, or approximately 530 ng / mL, or approximately 540 ng / mL, or approximately 550 ng / mL, or approximately 560 ng / mL, or approximately 570 ng / mL. It is present in amounts of ng / mL, or about 580 ng / mL, or about 590 ng / mL, or about 600 ng / mL.

[0163] The retention of NF-κB inhibitors within the lipid delivery vehicle can be between 25% and 100%. The retention of NF-κB inhibitors within the lipid delivery vehicle can be between 50% and 100%. The retention of NF-κB inhibitors within the lipid delivery vehicle can be between 70% and 100%. The retention of NF-κB inhibitors within the lipid delivery vehicle can be between 70% and 90%. The retention of NF-κB inhibitors within the lipid delivery vehicle can be at least 25%, for example, about 20%, or 25%, or 30%, or 35%, or 40%, or 45%, or 50%, or 60%, or 70%, or 80%, or 90%, or 95%, or 96%, or 97%, or 98%, or 99%, or 100%. In some instances, the retention can be at least 95%. In some instances, the retention can be at least 99%.

[0164] The lipid delivery carrier may comprise an NF-κB inhibitor selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inanecalciferol, ciorcalciferol, calcipotriene, mascalcalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof, as well as a peptide antigen, wherein the lipid delivery carrier comprises POPC, POPG, and PEGylated lipids.

[0165] The lipid delivery carrier may contain NF-κB inhibitors selected from the group consisting of: calcitriol, rapamycin, leflunomide, teriflunomide, bevacizumab, thiochlorophen, bortezomib, doxorubicin hydrochloride, cantharidin, carfilzomib, chromomycin, daunorubicin, digoxin, sucrose, emetine, erlotinib hydrochloride, gemcitabine, irinotecan, quinacrine dihydrochloride, fluxalen, manidipine hydrochloride, methylsalicylic acid, lentatinib, ouabain, pemetrexed disodium, sorafenib tosylate, sunitinib malate, tiaconazole, topotecan, tribromosalamin, triclobenzazole, zafirlukast, solanine A, quercetin, curcumin, Bay 11-7085, Bay 11-7082, dexamethasone and combinations thereof, and peptide antigens, wherein the lipid delivery carriers include POPC and POPG. The lipid delivery carrier may further contain PEGylated lipids.

[0166] The lipid delivery carrier may comprise an NF-κB inhibitor selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inenaciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; combinations thereof; and an autoantigen derived from CII, wherein the lipid delivery carrier comprises POPC, POPG, or PEGylated lipids. For example, the autoantigen derived from CII may be CII. 259-273 Antigen (e.g., SEQ ID NO: 1).

[0167] The lipid delivery carrier may comprise an NF-κB inhibitor selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inenaciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; combinations thereof; and an autoantigen derived from PI, wherein the lipid delivery carrier comprises POPC, POPG, or PEGylated lipids. For example, the autoantigen derived from PI may be PI33-63 Antigen (e.g., SEQ ID NO: 2).

[0168] The lipid delivery carrier may comprise an NF-κB inhibitor selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inenaciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; combinations thereof; and an autoantigen derived from proteoglycans, wherein the lipid delivery carrier comprises POPC, POPG, or PEGylated lipids. For example, the autoantigen derived from proteoglycans may be proteoglycans. 84-103 Cit93 antigen (e.g., SEQ ID NO: 4). For example, an autoantigen derived from agglutinin can be agglutinin. 89-103 Cit93 antigen (e.g., SEQ ID NO: 69).

[0169] The lipid delivery carrier may comprise an NF-κB inhibitor selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, idecalciferol, inanecalciferol, ciocalciferol, calcipotriene, mascalcalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; combinations thereof, and an autoantigen derived from proteoglycans, wherein the lipid delivery carrier comprises POPC, POPG, or PEGylated lipids. For example, the autoantigen derived from proteoglycans may comprise, consist of, or substantially consist of the amino acid sequence shown in any one of SEQ ID NOs 11-24.

[0170] The lipid delivery carrier may comprise an NF-κB inhibitor selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inenaciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; combinations thereof; and an autoantigen derived from Ro, wherein the lipid delivery carrier comprises POPC, POPG, or PEGylated lipids. For example, the autoantigen derived from Ro may be Ro. 401-425 Antigen (e.g., SEQ ID NO: 5).

[0171] The lipid delivery vehicle may comprise an NF-κB inhibitor selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inenaciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; combinations thereof; and a self-antigen derived from vimentin, wherein the lipid delivery vehicle comprises POPC, POPG, or PEGylated lipids. For example, the self-antigen derived from vimentin may be vimentin itself. 41-85 Antigens (e.g., SEQ ID NO: 6). In another instance, an autoantigen derived from vimentin could be vimentin itself. 41-85 Cit64 antigen (e.g., SEQ ID NO: 7). In another instance, an autoantigen derived from vimentin could be vimentin itself. 38-88 Cit64 antigen (SEQ ID NO: 8). In another instance, an autoantigen derived from vimentin could be vimentin itself. 53-85 Cit64 antigen (SEQ ID NO: 66). In another instance, an autoantigen derived from vimentin could be vimentin itself. 60-85 Cit64 antigen (SEQ ID NO: 67). In another instance, an autoantigen derived from vimentin could be vimentin itself. 59-71 Antigen (SEQ ID NO: 71).

[0172] The lipid delivery carrier may comprise an NF-κB inhibitor selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, idecalciferol, inanecalciferol, ciorcalciferol, calcipotriene, mascaracalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; combinations thereof, and an MPO-derived autoantigen, wherein the lipid delivery carrier comprises POPC, POPG, or PEGylated lipids. For example, an MPO-derived autoantigen may comprise, consist of, or substantially consist of the amino acid sequence shown in any one of SEQ ID NOs 9 and 10.

[0173] The lipid delivery carrier may comprise an NF-κB inhibitor selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inanecalciferol, ciocalciferol, calcipotriene, mascalcalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; combinations thereof, and a TSH-derived autoantigen, wherein the lipid delivery carrier comprises POPC, POPG, or PEGylated lipids. For example, a TSH-derived autoantigen may comprise, consist of, or substantially consist of the amino acid sequence shown in any one of SEQ ID NOs:25-55.

[0174] The lipid delivery vehicle may comprise an NF-κB inhibitor selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inanecalciferol, ciocalciferol, calcipotriene, mascalcalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; combinations thereof, and an autoantigen derived from tendinin C, wherein the lipid delivery vehicle comprises POPC, POPG, or PEGylated lipids. For example, the autoantigen derived from tendinin C may comprise, consist of, or consist substantially of the amino acid sequence shown in any one of SEQ ID NOs: 56-59.

[0175] The lipid delivery carrier may comprise an NF-κB inhibitor selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inanecalciferol, ciocalciferol, calcipotriene, mascalcalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; combinations thereof, and a self-antigen derived from human desmosome core protein 2, wherein the lipid delivery carrier comprises POPC, POPG, or PEGylated lipids. For example, the self-antigen derived from human desmosome core protein 2 may comprise, consist of, or substantially consist of the amino acid sequence shown in SEQ ID NO: 70.

[0176] The lipid delivery carrier may comprise an NF-κB inhibitor selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, idecalciferol, inelciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof, as well as a heterozygous insulin peptide (HIP), wherein the lipid delivery carrier comprises POPC, POPG, or PEGylated lipids. For example, the heterozygous insulin peptide (HIP) may comprise, consist of, or consist substantially of the amino acid sequence shown in any one of SEQ ID NOs: 60-65.

[0177] The lipid delivery carrier may contain calcitriol and peptide antigens, wherein the lipid delivery carrier contains POPC and POPG. The lipid delivery carrier may further contain PEGylated lipids.

[0178] Lipid delivery carriers may contain calcitriol and peptide antigens, wherein the lipid delivery carriers contain POPC, POPG and PEGylated lipids.

[0179] The lipid delivery carrier may contain calcitriol and a CII-derived autoantigen, wherein the lipid delivery carrier comprises POPC and POPG. For example, the CII-derived autoantigen may be CII. 259-273 Antigen (e.g., SEQ ID NO: 1). The lipid delivery carrier may further contain PEGylated lipids.

[0180] The lipid delivery vehicle may contain calcitriol and a PI-derived autoantigen, wherein the lipid delivery vehicle comprises POPC and POPG. For example, the PI-derived autoantigen may be PI... 33-63 Antigen (e.g., SEQ ID NO: 2). The lipid delivery carrier may further contain PEGylated lipids.

[0181] The lipid delivery carrier may contain calcitriol and an autoantigen derived from aggregated proteoglycans, wherein the lipid delivery carrier comprises POPC and POPG. For example, the autoantigen derived from aggregated proteoglycans may be aggregated proteoglycans themselves. 84-103 Cit93 antigen (e.g., SEQ ID NO: 4). For example, an autoantigen derived from agglutinin can be agglutinin. 89-103 Cit93 antigen (e.g., SEQ ID NO: 69). The lipid delivery carrier may further contain PEGylated lipids.

[0182] The lipid delivery carrier may comprise calcitriol and an autoantigen derived from aggregated proteoglycans, wherein the lipid delivery carrier comprises POPC and POPG. For example, the autoantigen derived from aggregated proteoglycans may comprise, consist of, or consist substantially of the amino acid sequences shown in any one of SEQ ID NOs: 11-24. The lipid delivery carrier may further comprise PEGylated lipids.

[0183] The lipid delivery vehicle may contain calcitriol and a Ro-derived autoantigen, wherein the lipid delivery vehicle comprises POPC and POPG. For example, the Ro-derived autoantigen may be Ro. 401-425 Antigen (e.g., SEQ ID NO: 5). The lipid delivery carrier may further contain PEGylated lipids.

[0184] The lipid delivery vehicle may contain calcitriol and a vimentin-derived autoantigen, wherein the lipid delivery vehicle comprises POPC and POPG. For example, the vimentin-derived autoantigen may be vimentin itself. 41-85 Antigens (e.g., SEQ ID NO: 6). In another instance, an autoantigen derived from vimentin could be vimentin itself. 41-85 Cit64 antigen (e.g., SEQ ID NO: 7). In another instance, an autoantigen derived from vimentin could be vimentin itself. 38-88 Cit64 antigen (SEQ ID NO: 8). In another instance, an autoantigen derived from vimentin could be vimentin itself. 53-85 Cit64 antigen (SEQ ID NO: 66). In another instance, an autoantigen derived from vimentin could be vimentin itself. 60-85 Cit64 antigen (SEQ ID NO: 67). In another instance, an autoantigen derived from vimentin could be vimentin itself. 59-71 Antigen (SEQ ID NO: 71). The lipid delivery carrier may further contain PEGylated lipids.

[0185] The lipid delivery carrier may comprise calcitriol and an MPO-derived autoantigen, wherein the lipid delivery carrier comprises POPC and POPG. For example, the MPO-derived autoantigen may comprise, consist of, or consist substantially of the amino acid sequence shown in either SEQ ID NOs: 9 and 10. The lipid delivery carrier may further comprise PEGylated lipids.

[0186] The lipid delivery carrier may comprise calcitriol and a TSH-derived autoantigen, wherein the lipid delivery carrier comprises POPC and POPG. For example, the TSH-derived autoantigen may comprise, consist of, or consist substantially of the amino acid sequences shown in any one of SEQ ID NOs: 25-55. The lipid delivery carrier may further comprise PEGylated lipids.

[0187] The lipid delivery vehicle may comprise calcitriol and an autoantigen derived from tendinin C, wherein the lipid delivery vehicle comprises POPC and POPG. For example, the autoantigen derived from tendinin C may comprise, consist of, or consist substantially of the amino acid sequence shown in any one of SEQ IDNOs: 56-59. The lipid delivery vehicle may further comprise PEGylated lipids.

[0188] The lipid delivery vehicle may comprise calcitriol and an autoantigen derived from human desmosome core protein 2, wherein the lipid delivery vehicle comprises POPC and POPG. For example, the autoantigen derived from human desmosome core protein 2 may comprise, consist of, or consist substantially of the amino acid sequence shown in SEQ ID NO: 70. The lipid delivery vehicle may further comprise PEGylated lipids.

[0189] The lipid delivery carrier may comprise calcitriol and a heterozygous insulin peptide (HIP), wherein the lipid delivery carrier comprises POPC and POPG. For example, the heterozygous insulin peptide (HIP) may comprise, consist of, or consist substantially of the amino acid sequence shown in any one of SEQ ID NOs: 60-65. The lipid delivery carrier may further comprise PEGylated lipids.

[0190] Preparation method Suitable methods for producing the lipid delivery carriers of this disclosure will be apparent to those skilled in the art and / or described herein. For example, methods known in the formulation art can be used to prepare the lipid delivery carriers of this disclosure. Suitable lipid delivery carriers can be formed using mixing processes, such as microfluidics, including herringbone micromixing and T-connector mixing of two fluids, one containing a peptide antigen (typically in an aqueous solution) and the other containing various desired lipid components, including NF-κB inhibitors (typically in ethanol).

[0191] Lipid delivery carriers can be prepared by mixing phospholipids (e.g., POPC or POPG, available from various commercial sources, including Avanti Polar Lipids, Alabaster, AL) and PEGylated lipids (e.g., 1,2-dimyristoyl-sn-glycerol methoxy polyethylene glycol, also known as PEG-DMG 2000K, available from various commercial sources, including Avanti Polar Lipids, Alabaster, AL) in ethanol. For storage, the lipid delivery carriers can be suspended in a cryoprotectant buffer (e.g., 20 nM tris, 10% sucrose) and frozen, for example, at -20°C ± 5°C. Various lipids can be combined to achieve the desired weight ratios and diluted with water and ethanol to the final desired lipid concentration.

[0192] Lipid delivery carriers containing peptide antigens and NF-κB inhibitors can be prepared by combining a lipid solution containing an NF-κB inhibitor with a solution containing the peptide antigen. The lipid solution can be rapidly injected into a DNA solution at a flow rate of approximately 3 ml / min to approximately 18 ml / min using a NanoAssemblr microfluidic system to produce a suspension with a water-to-ethanol ratio of approximately 1:1 to approximately 4:1. The pH of the solution containing the peptide antigen can be adjusted to be equal to or higher than the isoelectric point (pI) of the peptide antigen. As those skilled in the art will understand, increasing the pH of the solution to the pI of the peptide antigen will produce a peptide antigen with a neutral charge, while increasing the pH of the solution to a level higher than the pI of the peptide antigen will produce a peptide antigen with a negative charge.

[0193] Lipid delivery vehicles containing peptide antigens and NF-κB inhibitors can be prepared at room temperature. For example, lipid delivery vehicles containing peptide antigens and NF-κB inhibitors can be prepared at about 20°C, or about 21°C, or about 22°C, or about 23°C, or about 24°C, or about 25°C, or about 26°C. Lipid delivery vehicles containing peptide antigens and NF-κB inhibitors can be prepared at about 22°C to about 24°C.

[0194] Lipid delivery vehicles containing peptide antigens and NF-κB inhibitors can be prepared below freezing point. For example, lipid delivery vehicles containing peptide antigens and NF-κB inhibitors can be prepared between about -20°C, or about -25°C, or about -30°C, or about -35°C, or about -20°C to about -30°C. Lipid delivery vehicles containing peptide antigens and NF-κB inhibitors can be prepared at about -28°C to about -32°C.

[0195] As used herein, the term “encapsulation” refers to the process or result of confining one or more payloads or reagents (e.g., peptide antigens and / or NF-κB inhibitors) within a lipid delivery vehicle. As used herein, the terms “encapsulation” and “loading” are used interchangeably. Encapsulation can occur within the core of a lipid delivery vehicle and / or within a lipid bilayer and / or between the individual lipid bilayers of a multilayered vesicle.

[0196] As used herein, “encapsulation efficiency” refers to the amount of peptide antigen and / or NF-κB inhibitor that becomes part of the lipid delivery carrier relative to the initial total amount of peptide antigen and / or NF-κB inhibitor used to prepare the lipid delivery carrier. For example, if the lipid delivery carrier contains 92 mg of peptide antigen and / or immunomodulator, and 100 mg of peptide antigen and / or immunomodulator was initially provided to form the composition, the encapsulation efficiency can be expressed as 92%.

[0197] The encapsulation efficiency of the peptide antigen and / or NF-κB inhibitor within the lipid delivery carrier can be at least 50%, for example, about 50%, or 55%, or 60%, or 65%, or 70%, or 75%, or 80%, or 85%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99%. In some instances, the encapsulation efficiency can be at least 80%. In some instances, the encapsulation efficiency can be at least 90%. In some instances, the encapsulation efficiency can be between 25% and 100%. In some instances, the encapsulation efficiency can be between 50% and 100%. In some instances, the encapsulation efficiency can be between 70% and 100%. In some instances, the encapsulation efficiency can be between 70% and 90%.

[0198] Pharmaceutical Composition This disclosure envisions lipid delivery carriers being used in pharmaceutical compositions.

[0199] The pharmaceutical compositions disclosed herein may contain one or more peptide antigens. For example, the pharmaceutical compositions disclosed herein may contain one or more identical peptide antigens. Alternatively, the pharmaceutical compositions disclosed herein may contain one or more different peptide antigens. For example, the pharmaceutical compositions disclosed herein may contain at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight identical peptide antigens.

[0200] The pharmaceutical compositions disclosed herein may comprise one or more lipid delivery carriers. For example, the pharmaceutical compositions disclosed herein may comprise one or more lipid delivery carriers, each comprising a different peptide antigen. For example, the pharmaceutical compositions disclosed herein may comprise at least two, or at least three, or at least four, or at least five, or at least six, or at least seven, or at least eight delivery carriers, each comprising a different peptide antigen.

[0201] Suitablely, in compositions or methods for administering the lipid delivery carrier of this disclosure to a subject, the lipid delivery carrier is combined with a pharmaceutically acceptable carrier as understood in the art. Therefore, one example of this disclosure provides a composition (e.g., a pharmaceutical composition) comprising a combination of the lipid delivery carrier of this disclosure and a pharmaceutically acceptable carrier.

[0202] "Pharmaceutically acceptable carriers, diluents, or excipients" means solid or liquid fillers, diluents, or encapsulating substances that can be safely used for systemic administration. Depending on the specific route of administration, a variety of carriers, diluents, and excipients known in the art can be used. These can be selected from the group consisting of sugars, starches, cellulose and their derivatives, malt, gelatin, talc, calcium sulfate, vegetable oils, synthetic oils, polyols, alginic acid, phosphate buffer solutions, emulsifiers, isotonic saline solutions, and salts such as inorganic acid salts including hydrochlorides, bromides, and sulfates, organic acids such as acetates, propionates, and malonates, water, and pyrogen-free water.

[0203] A useful reference describing acceptable carriers, diluents, and excipients is Remington Pharmaceutical Sciences (Mack Publishing Co. NJ USA, 1991), which is incorporated herein by reference.

[0204] The lipid delivery carriers disclosed herein can be administered via any safe route, including but not limited to oral, rectal, parenteral, sublingual, buccal, intravenous, intra-articular, intramuscular, intradermal, subcutaneous, inhalation, intranasal, intraocular, intraperitoneal, intraventricular, local, mucosal, and transdermal administration. For example, the lipid delivery carriers can be administered parenterally, such as intramuscularly, subcutaneously, or intravenously.

[0205] Lipid delivery carriers can be administered subcutaneously. Subcutaneously administered pharmaceutical compositions can be formulated with a pH between about 7.0 and about 8.0.

[0206] The formulation of the lipid delivery carrier to be administered will vary depending on the chosen route of administration and dosage form (e.g., solid, semi-solid, liquid, or gaseous form, such as tablets, capsules, powders, granules, ointments, solutions, suspensions, suppositories, injections, inhalers, gels, microspheres, and aerosols). Suitable pharmaceutical compositions containing the lipid delivery carrier to be administered can be prepared in physiologically acceptable carriers. For solutions or emulsions, suitable carriers include, for example, aqueous or alcohol / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral carriers may include sodium chloride solution, Ringer's glucose, glucose and sodium chloride, lactated Ringer's solution, or non-volatile oils. A variety of suitable aqueous carriers are known to those skilled in the art, including water, buffered water, buffered saline, polyols (e.g., glycerol, propylene glycol, liquid polyethylene glycol), glucose solutions, and glycine. Intravenous delivery carriers may include various additives, preservatives, or fluids, nutrients, or electrolyte supplements (see, generally, Remington Pharmaceutical Science, 16th edition, Mack, Ed. 1980). If desired, the composition may optionally contain pharmaceutically acceptable excipients to approximate physiological conditions, such as pH adjusters and buffers, and toxicity modifiers, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, and sodium lactate. Lipid delivery carriers may be stored in a liquid stage or may be lyophilized and reconstituted in a suitable carrier prior to use according to lyophilization and reconstitution techniques known in the art.

[0207] The optimal concentration of the active ingredient (i.e., peptide antigen and immunomodulator) in the selected medium can be determined empirically based on procedures known to those skilled in the art and will depend on the desired final pharmaceutical formulation.

[0208] After formulation, the compositions of this disclosure will be administered in a dosage form compatible with and at a therapeutically / preventively effective amount. The dosage range used for administering the lipid delivery carrier of this disclosure is sufficiently wide to produce the desired effect. For example, the pharmaceutical composition may contain an effective amount of encapsulated peptide antigen and immunomodulator. Alternatively, or additionally, the pharmaceutical composition may contain a therapeutically effective amount of peptide antigen and NF-κB inhibitor. The pharmaceutical composition may contain a preventively effective amount of peptide antigen and immunomodulator.

[0209] The dosage should not be too high to cause adverse side effects. Generally, the dosage will vary depending on the patient's age, condition, sex, and severity of the disease, and can be determined by a person skilled in the art. In the event of any complications, an individual physician may adjust the dosage.

[0210] Any suitable procedure is considered for the production of the lipid delivery carriers and / or pharmaceutical compositions described herein.

[0211] Dosage forms include tablets, dispersants, suspensions, injections, solutions, syrups, lozenges, capsules, nasal sprays, suppositories, aerosols, transdermal patches, etc. These dosage forms may also include injection or implantable controlled-release devices specifically designed for this purpose, or other forms of implants modified to function additionally in this way. Controlled release can be achieved by coating with a hydrophobic polymer, including acrylic resins, waxes, higher fatty alcohols, polylactic acid and polyglycolic acid, and certain cellulose derivatives such as hydroxypropyl methylcellulose. Furthermore, controlled release can be achieved by using other polymer matrices, liposomes, and / or microspheres.

[0212] The compositions can be presented as discrete units, such as capsules, sachets, pre-filled syringes, vials, ampoules, functional foods / feeds, or tablets, each containing a predetermined amount of one or more therapeutic agents of this disclosure, as powders or granules, or as solutions or suspensions in aqueous liquids, non-aqueous liquids, oil-in-water emulsions, or water-in-oil liquid emulsions. Such compositions can be prepared by any pharmaceutical method, but all methods include the step of combining one or more agents as described above with a carrier constituting one or more essential components. Typically, the compositions are prepared by uniformly and closely mixing the agents of this disclosure with a liquid carrier or a finely dispersed solid carrier, or both, and then, if desired, shaping the product into the desired presentation form.

[0213] The above-described compositions can be administered in a dosage form compatible manner and in an effective amount. In the context of this disclosure, the dose administered to the subject should be sufficient to influence a beneficial response (e.g., to induce a tolerant immune response) within an appropriate time period. The amount of reagent to be administered may depend on the subject to be treated, including their age, sex, weight, and general health condition, which will depend on the practitioner's judgment. In determining an effective dose that can be administered to the subject, inflammation, pro-inflammatory cytokine levels, lymphocyte proliferation, cytolytic T lymphocyte activity, and regulatory T lymphocyte function can be assessed.

[0214] The pharmaceutical compositions disclosed herein may further comprise a buffer. The buffer may be any suitable buffer known in the art. For example, the buffer may be a TRIS, acetate, glutamate, lactate, maleate, tartrate, phosphate, citrate, carbonate, glycine, histidine, glycine, succinate, and triethanolamine buffer, or a phosphate buffer. The buffer may be a phosphate buffer. Alternatively, the buffer may be a succinate buffer. The buffer may be a histidine buffer. The buffer may be a citrate buffer.

[0215] Buffers may be selected from those compatible with the United States Pharmacopeia (USP) for parenteral use, particularly when the pharmaceutical preparation is intended for parenteral administration. For example, buffers may be selected from the following group: monocarboxylic acids such as acetic acid, benzoic acid, gluconic acid, glyceric acid, and lactic acid; dicarboxylic acids such as aconitic acid, adipic acid, ascorbic acid, carbonic acid, glutamic acid, malic acid, succinic acid, and tartaric acid; polycarboxylic acids such as citric acid and phosphoric acid; and bases such as ammonia, diethanolamine, glycine, triethanolamine, and TRIS.

[0216] This document also describes a container for containing a sterile liquid formulation or lyophilized composition of the pharmaceutical composition described herein. Any suitable container known in the art can be used. For example, the container may be selected from the group consisting of: vials, syringes, ampoules, flasks, fermenters, bioreactors, bags, cans, ampoules, cartridges, and disposable pens. The container may be a syringe, vial, or ampoule.

[0217] Containers may be made of glass, metals (e.g., steel, stainless steel, aluminum, etc.) and / or polymers (e.g., thermoplastics, elastomers, thermoplastic elastomers). Containers may be at least partially siliconeized. These containers should be sterile.

[0218] The kit or composition may be packaged together (e.g., in the same box) with instructions for use containing detailed information about the drug composition (e.g., instructions for dosing, details of antigens and immunomodulators in the composition, etc.). The instructions may also include warnings, such as the availability of epinephrine solution in case of an allergic reaction following vaccination.

[0219] Treatment and prevention methods The lipid delivery carriers and pharmaceutical compositions disclosed herein are suitable for administration to human or non-human animal subjects, thereby providing methods for inducing a tolerable immune response and / or treating and / or preventing an unwanted or harmful immune response in subjects.

[0220] This disclosure also provides for the use of the compositions described herein as pharmaceuticals, and provides for the use of such compositions in the preparation of pharmaceuticals for inducing a tolerant immune response and / or preventing and / or treating an undesirable or harmful immune response in a subject.

[0221] This disclosure provides a method for inducing a tolerable immune response and / or treating and / or preventing an unwanted or harmful immune response in a subject, the method comprising administering a composition as described herein.

[0222] As described in this article, undesirable or harmful immune responses include, but are not limited to, transplant rejection, allergies, parasitic diseases and autoimmune diseases, conditions or illnesses, and combinations thereof.

[0223] Examples of transplant rejection that can be treated or prevented using the lipid delivery carriers or pharmaceutical compositions described herein include, but are not limited to, rejections and combinations thereof associated with stem cell, bone marrow, and organ (e.g., heart, liver, pancreas, kidney, lung, eye, skin, etc.) transplants.

[0224] Examples of allergies that can be treated or prevented using the lipid delivery carriers or pharmaceutical compositions described herein include, but are not limited to, seasonal respiratory allergies, allergies to airborne allergens (e.g., hay fever), allergies that can be treated by reducing serum IgE and eosinophilia, asthma, eczema, animal allergies, food allergies, latex allergies, dermatitis, or allergies that can be treated by allergy desensitization.

[0225] Examples of autoimmune diseases, conditions, or illnesses that can be treated or prevented using the lipid delivery carriers or pharmaceutical compositions described herein include, but are not limited to, psoriasis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus (SLE), myasthenia gravis, stiff-person syndrome, rheumatic heart disease, Sidnam's chorea, rheumatoid arthritis, type 1 diabetes, Crohn's disease, chronic inflammatory eye diseases including uveitis and shotgun retinopathy, chronic inflammatory lung disease and chronic inflammatory liver disease, autoimmune hemolytic anemia, idiopathic leukopenia, ulcerative colitis, dermatomyositis, scleroderma, mixed connective tissue disease, multiple sclerosis, neuromyelitis optica, vitiligo, and alopecia areata. Guillain-Barré syndrome, antiphospholipid syndrome, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, pulmonary hemorrhage-nephritis syndrome, Behçet's syndrome, Sjögren's syndrome, juvenile inflammatory arthritis, antineutrophil-associated cytoplasmic antibody-associated vasculitis, sympathetic ophthalmia, Hashimoto's disease, hypothyroidism, celiac disease, herpetic dermatitis, demyelinating diseases, primary biliary cirrhosis, autoimmune chronic active hepatitis, Graves' disease, hyperthyroidism, chronic idiopathic thrombocytopenic purpura, pemphigus vulgaris, bullous pemphigoid, antineutrophil-associated vasculitis, neuromyelitis optica, and checkpoint inhibitor-induced autoimmune diseases and combinations thereof.

[0226] This disclosure provides a method for treating and / or preventing autoimmune diseases, conditions, or illnesses in a subject, the method comprising administering to the subject a therapeutically effective amount of the lipid delivery carrier or pharmaceutical composition described herein.

[0227] This disclosure also provides a method for inducing a tolerable immune response in a subject, the method comprising administering to the subject a therapeutically effective amount of the lipid delivery carrier or pharmaceutical composition described herein.

[0228] This disclosure also provides the use of lipid delivery carriers in the preparation of medicaments for the treatment and / or prevention of autoimmune diseases, conditions or illnesses in subjects.

[0229] This disclosure also provides the lipid delivery carriers or pharmaceutical compositions described herein for the treatment and / or prevention of autoimmune diseases, conditions or illnesses in subjects.

[0230] As described herein, the terms “subject,” “patient,” and “individual” include, but are not limited to, mammals, including humans, racing animals (e.g., horses, camels, greyhounds), livestock (e.g., cattle, sheep, horses), and companion animals (e.g., cats and dogs). For example, a subject can be a person.

[0231] "Inducing a tolerance immune response" refers to the generation or stimulation of the immune system's tolerance to a target antigen. The tolerance immune response described herein can include antigen-specific induction by exposing antigen-presenting cells (APCs), such as dendritic cells, to a target antigen to induce T cell tolerance. This may result in T cell clonal loss, T cell anergy, or proliferation or differentiation of regulatory T cells (Tregs).

[0232] The term "therapeuticly effective amount" refers to an amount that, upon administration, at least partially improves, eliminates, or reduces symptoms or pathological signs of an unwanted or harmful immune response. Alternatively, a therapeutically effective amount refers to an amount that, upon administration, prevents the worsening of one or more symptoms or pathological signs of an unwanted or harmful immune response. The amount to be administered will depend on the characteristics of the subject, such as general health status, other medical conditions, age, sex, genotype, and weight. Those skilled in the art will be able to determine an appropriate dosage based on these and other factors. Therefore, this term should not be construed as limiting this disclosure to a specific quantity, such as the weight or amount of the compound.

[0233] As is commonly used herein, the terms “immune” and “immunization” refer to methods and / or compositions capable of evoking a tolerant immune response to a peptide antigen, thereby preventing or minimizing, at least partially, subsequent transplantation, allergic reactions, parasitic or autoimmune diseases, conditions or illnesses.

[0234] As used herein, “treatment,” “curing,” or “management” means a therapeutic intervention that, after the onset of development, at least partially improves, eliminates, or reduces symptoms or pathological signs of an undesirable or harmful immune response. Treatment need not be absolutely beneficial to the subject. Beneficial effects can be determined using any method or standard known to those skilled in the art. “Prophylactic” treatment is administered to subjects who do not exhibit signs of an undesirable or harmful immune response, or who exhibit only early signs, with the aim of reducing the risk of developing symptoms or pathological signs of an undesirable or harmful immune response. Therefore, the methods described herein include prophylactic treatment methods.

[0235] As used herein, “prevention,” “avoidance,” or “prevention” refers to a process of action initiated before and / or before the appearance of symptoms or pathological signs of an undesirable or harmful immune response, arising from exposure to a graft, allergic reaction, parasitic disease, or subject at risk, in order to prevent an undesirable or harmful immune response and / or alleviate symptoms or pathological signs. It should be understood that such prevention need not be absolutely beneficial to the subject.

[0236] As used in this article, “subjects at risk” can refer to subjects who are positive for autoantibodies and / or have a human leukocyte antigen (HLA) haplotype or have a family history of being susceptible to and / or stimulating unwanted or harmful immune responses.

[0237] For example, a method for treating and / or preventing Sjögren's syndrome or systemic lupus erythematosus (SLE) in a subject may include administering to the subject a lipid delivery carrier or pharmaceutical composition described herein, comprising an NF-κB inhibitor (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, idecalciferol, inenaciferol, ciorcalciferol, calcipotriene, mascaracalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and an autoantigen derived from Ro (e.g., Ro). 401-425 Antigen; SEQ ID NO: 5).

[0238] This article describes NF-κB inhibitors (e.g., selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inenaciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogues and / or side-chain analogues thereof; and combinations thereof) and autoantigens derived from Ro (e.g., Ro). 401-425 The lipid delivery carrier of the antigen (SEQ ID NO:5) can be used to prepare a drug for the treatment and / or prevention of Sjögren's syndrome or SLE in subjects.

[0239] A method for treating and / or preventing rheumatoid arthritis in a subject may include administering to the subject a lipid delivery carrier or pharmaceutical composition described herein, comprising an NF-κB inhibitor (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inenaciferol, ciorcalciferol, calcipotriene, mascaracalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and an autoantigen derived from CII (e.g., CII). 259-273 Antigen; SEQ ID NO: 1).

[0240] This article describes NF-κB inhibitors (e.g., selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inenaciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and autoantigens derived from CII (e.g., CII). 259-273 A lipid delivery carrier of an antigen (SEQ ID NO:1) that can be used to prepare a drug for the treatment and / or prevention of rheumatoid arthritis in subjects.

[0241] A method for treating and / or preventing rheumatoid arthritis in a subject may include administering to the subject a lipid delivery carrier or pharmaceutical composition described herein, comprising an NF-κB inhibitor (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inenaciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and a self-antigen derived from vimentin (e.g., vimentin). 41-85 ; SEQ ID NO: 6, or vimentin 41-85 Cit64 antigen (SEQ ID NO: 7 or SEQ ID NO: 8).

[0242] This article describes NF-κB inhibitors (e.g., selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inenaciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and autoantigens derived from vimentin (e.g., vimentin). 41-85; SEQ ID NO: 6, or vimentin 41-85 A lipid delivery carrier of Cit64 antigen (SEQ ID NO: 7 or SEQ ID NO: 8) can be used to prepare a medicament for the treatment and / or prevention of rheumatoid arthritis in subjects.

[0243] A method for treating and / or preventing type 1 diabetes in a subject may include administering to the subject a lipid delivery carrier or pharmaceutical composition described herein, comprising an NF-κB inhibitor (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, idecalciferol, inenaciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and an autoantigen derived from proinsulin (e.g., proinsulin). 33-63 Antigen; SEQ ID NO: 2).

[0244] This article describes NF-κB inhibitors (e.g., selected from the group consisting of: calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inelciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogues and / or side-chain analogues thereof; and combinations thereof) and autoantigens derived from proinsulin (e.g., proinsulin). 33-63 The antigen (SEQ ID NO: 2) is a lipid delivery carrier that can be used to prepare a drug for the treatment and / or prevention of type 1 diabetes in subjects.

[0245] A method for treating and / or preventing autoimmune diseases in a subject may include administering to the subject a lipid delivery carrier or pharmaceutical composition described herein, comprising an NF-κB inhibitor (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, edicalciferol, inenaciferol, ciorcalciferol, calcipotriene, mascalcalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and an autoantigen derived from aggregated proteoglycans (e.g., SEQ ID NOs: 4 and 11-24).

[0246] The lipid delivery carriers described herein, comprising NF-κB inhibitors (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, edicalciferol, inelciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and autoantigens derived from aggregate proteoglycans (e.g., SEQ ID NOs: 4 and 11-24), may be used to prepare medicaments for the treatment and / or prevention of autoimmune diseases in subjects.

[0247] A method for treating and / or preventing Graves' disease in a subject may include administering to the subject a lipid delivery carrier or pharmaceutical composition described herein, comprising an NF-κB inhibitor (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, edicalciferol, inelciferol, ciorciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and a TSH-derived autoantigen (e.g., SEQ ID NOs: 25-55).

[0248] The lipid delivery carriers described herein, comprising NF-κB inhibitors (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, edicalciferol, inelciferol, siocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and TSH-derived autoantigens (e.g., SEQ ID NOs: 25-55), may be used to prepare medicaments for the treatment and / or prevention of Graves' disease in subjects.

[0249] A method for treating and / or preventing antineutrophil-associated vasculitis in a subject may include administering to the subject a lipid delivery carrier or pharmaceutical composition described herein, comprising an NF-κB inhibitor (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, edicalciferol, inelciferol, cefixime, masalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and an MPO-derived autoantigen (e.g., SEQ ID NOs: 9 and 10).

[0250] The lipid delivery carriers described herein, comprising NF-κB inhibitors (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, edicalciferol, inelciferol, siocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and MPO-derived autoantigens (e.g., SEQ ID NOs: 9 and 10), may be used to prepare medicaments for the treatment and / or prevention of antineutrophil-associated vasculitis in subjects.

[0251] A method for inducing a tolerable immune response and / or treating and / or preventing an unwanted or harmful immune response in a subject may include administering to a subject a lipid delivery carrier or pharmaceutical composition described herein comprising an NF-κB inhibitor (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, edicalciferol, inelciferol, ciorciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and an autoantigen derived from tendinin C (e.g., SEQ ID NOs: 56-59).

[0252] The lipid delivery carriers described herein, comprising NF-κB inhibitors (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, edicalciferol, inelciferol, siocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and autoantigens derived from tendinin C (e.g., SEQ ID NOs: 56-59), may be used to prepare medicaments for inducing a tolerable immune response and / or treating and / or preventing unwanted or harmful immune responses in subjects.

[0253] A method for inducing a tolerable immune response and / or treating and / or preventing an unwanted or harmful immune response in a subject may include administering to a subject a lipid delivery carrier or pharmaceutical composition described herein comprising an NF-κB inhibitor (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, idecalciferol, inelciferol, ciorciferol, calcipotriene, mascaracalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and an autoantigen derived from human desmosome core protein 2 (e.g., SEQ ID NO: 70).

[0254] The lipid delivery carriers described herein, comprising NF-κB inhibitors (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorotriol, idecalciferol, inelciferol, siocalciferol, calcipotriene, mascalcalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and an autoantigen derived from human desmosome core protein 2 (e.g., SEQ ID NO: 70), may be used to prepare medicaments for inducing a tolerable immune response and / or treating and / or preventing unwanted or harmful immune responses in subjects.

[0255] A method for inducing a tolerable immune response and / or treating and / or preventing an unwanted or harmful immune response in a subject may include administering to a subject a lipid delivery carrier or pharmaceutical composition described herein comprising an NF-κB inhibitor (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, idecalciferol, inelciferol, ciocalciferol, calcipotriene, mascalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and an autoantigen derived from a heterozygous insulin peptide (HIP) (e.g., SEQ ID NOs: 60-65).

[0256] The lipid delivery carriers described herein, comprising NF-κB inhibitors (e.g., selected from the group consisting of calcitriol, calciferol, ergocalciferol, alfacalciferol, ducalciferol, calcidiol, 22-oxacalcitriol, paricalciferol, tacalciferol, fluorocalcitriol, idecalciferol, inelciferol, siocalciferol, calcipotriene, mascalcalciferol, calcidiol, calcipotriol; synthetic analogs and / or side-chain analogs thereof; and combinations thereof) and autoantigens derived from heterozygous insulin peptides (HIP) (e.g., SEQ ID NOs: 60-65), may be used to prepare medicaments for inducing a tolerable immune response and / or treating and / or preventing unwanted or harmful immune responses in subjects.

[0257] In order to fully understand and put into practice the preferred embodiments of this disclosure, reference is made to the following non-limiting embodiments.

[0258] The present invention is further disclosed in the following numbered paragraphs: 1. A lipid delivery carrier comprising a nuclear factor-κB (NF-κB) inhibitor and a peptide antigen, wherein the lipid delivery carrier comprises 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate glycerol (POPG).

[0259] 2. The lipid delivery carrier according to paragraph 1 further comprises PEGylated lipids.

[0260] 3. The lipid delivery carrier according to paragraph 2, wherein the PEGylated lipid is selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, and combinations thereof.

[0261] 4. The lipid delivery carrier according to paragraph 2 or 3, wherein the lipid delivery carrier comprises about 0.5 mol% to about 5 mol% of the PEGylated lipid.

[0262] 5. The lipid delivery carrier according to any one of paragraphs 1 to 4, wherein the lipid delivery carrier comprises about 85 mol% to 95 mol% POPC.

[0263] 6. The lipid delivery carrier according to any one of paragraphs 1 to 5, wherein the lipid delivery carrier comprises about 5 mol% to 15 mol% of POPG.

[0264] 7. The lipid delivery carrier according to any one of paragraphs 1 to 6, wherein the average diameter of the lipid delivery carrier is between about 75 nm and about 250 nm, and preferably between about 80 nm and about 150 nm.

[0265] 8. The lipid delivery carrier according to any one of paragraphs 1 to 7, wherein the lipid delivery carrier has a negative surface charge.

[0266] 9. The lipid delivery carrier according to paragraph 8, wherein the surface charge is less than -15 mV, and preferably less than -20 mV.

[0267] 10. The lipid delivery carrier according to any one of paragraphs 1 to 9, wherein the polydispersity index of the lipid delivery carrier is less than 0.20.

[0268] 11. The lipid delivery carrier according to any one of paragraphs 1 to 10, wherein the lipid delivery carrier is selected from the group consisting of liposomes, lipid nanoparticles, lipid vesicles, and lipid-based particles.

[0269] 12. The lipid delivery carrier according to paragraph 11, wherein the lipid delivery carrier is a liposome.

[0270] 13. The lipid delivery carrier according to any one of paragraphs 1 to 12, wherein the peptide antigen is selected from the group consisting of autoantigens, allogeneic antigens, and allergens.

[0271] 14. The lipid delivery carrier according to paragraph 13, wherein the autoantigen is selected from the group consisting of: Ro, type II collagen (CII), proinsulin (PI), insulin, hybrid insulin peptide, chromogranin, agglutinin glycan, islet antigen 2 (IA2), glutamate decarboxylase 65 kDalton isoform (GAD65), glycoprotein (gp70), nuclear antigen, lupus autoantigen, Smith, La, U1-RNP, fibrin, histone, ribosomal protein, pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, human tropomyosin isoform 5 (hTM5), human cartilage gp 39 (HCgp39) and gp130-RAPS, dnaJp1, citrullinated protein, citrullinated peptide, citrullinated type II collagen, citrullinated vimentin, citrullinated fibrinogen, citrullinated agglutinin glycan, citrullinated tendinin C, myelin basic protein, protein lipid protein (PLP) and myelin oligodendrocyte glycoprotein (MOG), thyroid-stimulating hormone receptor (TSH-R), acetylcholine receptor (AchR), gliadin, histone, PLP, glucose-6-phosphate isomerase, thyroglobulin, various tRNA synthetases, protease 3, human desmosome core protein 2 (DSG2) and myeloperoxidase.

[0272] 15. The lipid delivery carrier according to any one of paragraphs 1 to 14, wherein the peptide antigen comprises, is composed of, or is substantially composed of an amino acid sequence, fragment, variant or derivative thereof, or any combination thereof shown in any one of SEQ ID NOs: 1-59.

[0273] 16. The lipid delivery carrier according to any one of paragraphs 1 to 15, wherein the peptide antigen comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO: 2 or fragments, variants or derivatives thereof.

[0274] 17. A lipid delivery vehicle according to any one of paragraphs 1 to 16, wherein the NF-κB inhibitor reduces the level and / or functional activity of NF-κB signaling pathway members selected from the group consisting of: BTK, LYN, BCR Igα, BCR Igβ, Syk, Bink, PLCγ2, PKCβ, DAG, CARMA1, BCL1O, MALT1, PI3K, PIP3, AKT, p38 MAPK, ERK, COT, IKKα, IKKβ, IKKγ, NIK, RelA / p65, P105 / p50, cRel, RelB, p52, NIK, Leu13, CD81, CD19, CD21 and their ligands in the complement and coagulation cascade, TRAF6, ubiquitin ligase, Tab2, TAK1, NEMO, NOD2, RIP2, Lek, fyn, Zap70, LAT, GRB2, SOS, CD3 zeta, Slp-76, GADS, ITK, PLCγl, PKCθ, ICOS, CD28, SHP2, SAP, SLAM and 2B4.

[0275] 18. The lipid delivery vehicle according to any one of paragraphs 1 to 17, wherein the NF-κB inhibitor increases the level and / or functional activity of NF-κB signaling pathway members selected from the group consisting of: SHP1, SHIP, PIR-B, CD22, CD72, FcgRIIB, IκB, P100, CTLA4, PD-1, Chi, KIR3DL1, KIR3DL2, KIR2DL, and Csk.

[0276] 19. The lipid delivery vehicle according to any one of paragraphs 1 to 18, wherein the NF-κB inhibitor reduces the level and / or functional activity of NF-κB family proteins selected from the group consisting of: Rel-A (p65), Rel-B, Rel (c-Rel), NF-κB1 (p50 / p105) and NF-κB2 (p52 / p100).

[0277] 20. The lipid delivery vehicle according to any one of paragraphs 1 to 19, wherein the NF-κB inhibitor is selected from the group consisting of: calcitriol, rapamycin, leflunomide, teriflunomide, bevacizumab, thiochlorophen, bortezomib, doxorubicin hydrochloride, cantharidin, carfilzomib, chromomycin, daunorubicin, digoxin, sucrose, emetine, erlotinib hydrochloride, gemcitabine, irinotecan, quinacrine dihydrochloride, fluxalen, manidipine hydrochloride, methylsalicylate, lentatinib, ouabain, pemetrexed disodium, sorafenib tosylate, sunitinib malate, tiaconazole, topotecan, tribromosalamin, triclobenzazole, zafirlukast, solanine A, quercetin, curcumin, BAY 11-7085, and BAY 11-7082.

[0278] 21. The lipid delivery carrier according to any one of paragraphs 1 to 20, wherein the NF-κB inhibitor is calcitriol.

[0279] 22. The lipid delivery carrier according to any one of paragraphs 1 to 21, wherein the peptide antigen comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO: 2 or a fragment, variant, or derivative thereof, and the NF-κB inhibitor is calcitriol or a fragment, variant, or derivative thereof.

[0280] 23. The lipid delivery carrier according to paragraph 22, comprising about 1 µg / mL to about 50 µg / mL of the peptide antigen, the peptide antigen comprising, consisting of or substantially consisting of the amino acid sequence shown in SEQ ID NO: 2 or fragments, variants or derivatives thereof, and about 400 ng / mL to about 600 ng / mL of calcitriol or fragments, variants or derivatives thereof.

[0281] 24. The lipid delivery carrier according to any one of paragraphs 1 to 21, wherein the peptide antigen comprises, is composed of, or is substantially composed of an amino acid sequence shown in any one of SEQ ID NOs: 6-8 or a fragment, variant, or derivative thereof, and the NF-κB inhibitor is calcitriol or a fragment, variant, or derivative thereof.

[0282] 25. A pharmaceutical composition comprising any one of paragraphs 1 to 24 of a lipid delivery carrier and a pharmaceutically acceptable carrier.

[0283] 26. The lipid delivery carrier of any one of paragraphs 1 to 24 or the pharmaceutical composition of paragraph 25 for use in a therapy.

[0284] 27. A method for inducing a tolerable immune response in a subject, comprising administering to the subject any of the lipid delivery carriers described in paragraphs 1 to 24 or the pharmaceutical composition described in paragraph 25.

[0285] 28. Use of any of the lipid delivery carriers described in paragraphs 1 to 24 in the preparation of a medicament for inducing a tolerable immune response in a subject.

[0286] 29. A method of treating and / or preventing an autoimmune disease, condition or illness in a subject, comprising administering to the subject any of the lipid delivery carriers described in paragraphs 1 to 24 or the pharmaceutical composition described in paragraph 25.

[0287] 30. Use of any of the lipid delivery carriers described in paragraphs 1 to 24 in the preparation of a medicament for the treatment and / or prevention of an autoimmune disease, condition or illness in a subject.

[0288] 31. According to the method described in paragraph 29 or the use described in paragraph 30, the autoimmune disease mentioned is selected from the group consisting of: psoriasis, psoriatic arthritis, ankylosing spondylitis, systemic lupus erythematosus (SLE), myasthenia gravis, stiff-person syndrome, rheumatic heart disease, Sidnam's chorea, rheumatoid arthritis, type 1 diabetes, Crohn's disease, chronic inflammatory eye diseases including uveitis and shotgun retinopathy, chronic inflammatory lung disease and chronic inflammatory liver disease, autoimmune hemolytic anemia, idiopathic leukopenia, ulcerative colitis, dermatomyositis, scleroderma, mixed connective tissue disease, multiple sclerosis, neuromyelitis optica, vitiligo, alopecia areata, Guillain-Barré syndrome. - Barre syndrome, antiphospholipid syndrome, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, pulmonary hemorrhage-nephritis syndrome, Behçet's syndrome, Sjögren's syndrome, juvenile inflammatory arthritis, antineutrophil-associated cytoplasmic antibody-associated vasculitis, sympathetic ophthalmia, Hashimoto's disease, hypothyroidism, celiac disease, herpetic dermatitis, demyelinating diseases, primary biliary cirrhosis, autoimmune chronic active hepatitis, Graves' disease, hyperthyroidism, chronic idiopathic thrombocytopenic purpura, pemphigus vulgaris, bullous pemphigoid, antineutrophil-associated vasculitis, neuromyelitis optica, and checkpoint inhibitor-induced autoimmune diseases.

[0289] Example Example 1 – Method Preparation of lipid delivery carriers Lipid delivery carriers (referred to as "liposomes" in the examples below) were prepared using microfluidic technology with a benchtop NanoAssemblr Ignite instrument (Precision Nano-Systems Inc.). However, this should not be construed as requiring the lipid delivery carriers to have any particular morphology. The methanol lipid phase (containing L-α-phosphatidylcholine (EPC): L-α-phosphatidylglycerol (EPG) (35:4 w:w); 1,2-dipalmitoyl-sn-glycerol-3-phosphate choline (DPPC): 1,2-dipalmitoyl-sn-glycerol-3-phosphate DPPG (6.3:1.6 w:w); or 1-palmitoyl-2-oleoyl-glycerol-3-phosphate choline (POPC): 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate-(1'-rac-glycerol)(POPG) (35:4 w:w); 1,2-dioleoyloxy-3-trimethylammonium propane chloride (DOTAP); polyethyleneimine (PEI): POPC:POPG; or DOTAP:POPG-DSPC:cholesterol) was mixed with HEPES buffer in a microfluidic tube at a 1:1 ratio and a flow rate of 12 ml / min.

[0290] For drug-loaded liposomes, calcitriol (Dishman, Veendaal, Netherlands) was added to the lipid phase at 2 µg / mL, and peptides [collagen] were added. 259-273 II (GIAGFKGEQGPKGEB; SEQ ID NO: 1), proinsulin 33-63 (EAEDLQVGQVELGGGPGAGSLQPLALEGSLQ; SEQ ID NO: 2), ovalbumin 323-339 (ISQAVHAAHAEINEAGR; SEQ ID NO: 3), Aggregate proteoglycan 89-103 (ATEGRVRVNSAYQDK; SEQ IDNO: 4), Ro60 401-425 (MVVTRTEKDSYVVAFSDEMVPCPVT; SEQ ID NO: 5), human desmosome core protein 2 (hDSG2) (SEQ ID NO: 70), vimentin 41-85 (SEQ ID NO: 6), vimentin 41-85 Cit64 (SEQ ID NO: 7), vimentin 59-71[(SEQ ID NO: 71)] was added to the HEPES buffered aqueous stream at concentrations of 120 µg / mL, 60 µg / mL, or 30 µg / mL (Table 16). The liposomes were dialyzed against the HEPES buffer using a 10 or 100 kDa filtration membrane to remove free drug.

[0291] Assess the hydrophilicity of the peptide antigen. If necessary, dilute the peptide antigen with 2%, 1%, 0.5%, or 0.1% ethanol:HEPES buffer solution to enhance its solubility in water. Additionally, the pH of the peptide antigen can be adjusted to make it negatively charged, thereby further increasing its solubility in water.

[0292] Adding surfactants to semi-synthetic phospholipid formulations 2-Dimyristicoyl-racemic-glycerol-3-methoxypolyethylene glycol-2000 (PEG-DMG 2000) (varying from 0 mol% to 5 mol%) was added to the lipid phase.

[0293] plasma incubation Each liposome sample was incorporated into pooled healthy human plasma (CryoCheck Pooled Normal Plasma CCN-10, Precision Biologic USA) at a ratio of one part liposome stock solution to three parts plasma. The incorporated plasma was incubated at 37°C for 15 minutes and then reconstituted in PBS to a total volume of 500 µL. Size exclusion columns were internally packed into 10 mL nominal volumes of syringes by gravity flow. These columns consisted of 10 mL of Sepharose 4B resin (Merck, Australia) with 30 µm top and bottom sieves. The columns were equilibrated with at least one volume of PBS before use. The sample was then applied to the top of the SEC column to ensure complete infiltration into the column matrix, followed by stepwise application of PBS to the top of the column to elute the sample. Twenty-two 0.5 mL fractions were collected for a total elution volume of 11 mL. The collected fractions were stored at 4°C for further analysis. The column was washed with two volumes of PBS + 0.1% Tween-20 to remove any residual sample.

[0294] Nanoparticle tracking analysis (NTA) to determine peptide antigen / NF-κB inhibitor retention NTA analysis was performed on a NanoSight NS300 instrument, and data were processed using NTA Software v3.4 Build 3.4.4 (Malvern Panalytical, UK). Equal volumes of SEC fractions 6–11 (2.5–5.5 mL elution volume) and fractions 16–22 (7.5–11 mL elution volume) were combined and thoroughly mixed. The combined sample was then diluted in PBS to a total volume of 1 mL for NTA measurement. Specific NTA settings were as follows: temperature = 25°C, camera level = 15, syringe pump load = 100, detection threshold = 5, number of measurements = 3 x 60 seconds. All other settings were determined automatically by the instrument. Measurements with an average of ≤20 particles detected per frame were considered to have negligible particle content above the background.

[0295] Calcitriol concentration was measured using a 1,25-dihydroxyvitamin D ELISA (Immunodiagnostic Systems) according to the manufacturer’s instructions (incorporated herein by reference), with a sensitivity of 6 pmol / L.

[0296] Liposome biodistribution BALB / c mice were purchased from OzGene Australian Research Council (BALB / cArc) or Charles River (BALB / cAnNCrl). All mice were housed under specific pathogen-free conditions at the Biological Research Facilities of Translational Research Institute.

[0297] 89 Zr peptide labeling Deferroamine (DFO)-NH2 was linked to PI using HATU activation. 33-63 The DFO was then dialyzed onto COOH to remove free DFO and avoid metal exposure. DFO binding was confirmed by MALDI-TOF. After sonication, the resulting peptide was dissolved in ethanol. The DFO-PI was then... 33-63 Added to HEPES buffer 89 In Zr, the final ethanol concentration was 45%, and then it was incubated at 37°C for 1 hour. Incubation with an excess of diethylenetriaminepentaacetic acid (DTPA) was then performed to bind free [unspecified substance]. 89 After Zr, the sample was run on high-performance liquid chromatography (HPLC) for identification. 89 Zr-DTPA and 89 Zr-DFO-NH2 and Zr-PI 33-63-DFO. The elution time for peptides is comparable to that of cold peptides. 89 Zr-DTPA and DFO-NH2 eluted earlier than cold peptides.

[0298] Biodistribution of a single liposome dose in initial mice Liposome uptake was assessed by flow cytometry using female BALB / c-cArc mice (8 weeks old, n=6) and BALB / cAnNCrl mice (18 months old, n=7). Liposomes were labeled with the lipophilic membrane dye DiI (Molecular Probes) at a final concentration of 25 µg / ml for at least 15 minutes at room temperature. Five days prior to liposome injection, mice received 40 µg of QuilA adjuvant (subcutaneously; sc) at the base of the tail. Mice were then given 100 µl of DiI-labeled liposomes (DiI-DEN-181 or DiI-ASITI-201) or PBS (50 µl per side, sc) at the flank.

[0299] Biodistribution of proteoglycan-sensitized mice after multiple liposome administration Liposome uptake was assessed by flow cytometry in male BALB / cAnNCrl mice (n=10). Liposomes were labeled with the lipophilic membrane dye DiD (Molecular Probes) at a final concentration of 25 µg / ml for at least 15 minutes at room temperature. Mice received 80 μg of recombinant human agglutinin G1 proteoglycan (rhG1-PG)-Xa-mFc2a fusion protein intraperitoneally (ip), emulsified with 2 mg dimethyl dioctadecyl ammonium bromide (DDA; Sigma-Aldrich). At days 7, 11, and 14, 100 µl of liposomes formulated with POPC / POPG / PEG (empty or containing 20 μg / ml agglutinin) were subcutaneously injected. 89-103 (and 400 ng / ml calcitriol) or EPC / EPG (containing 20 μg / ml proteoglycan) 89-103 400 ng / ml calcitriol was injected into the lateral abdomen. The liposome dose injected on day 14 was labeled with DiD (50 µl per side, subcutaneously).

[0300] Flow cytometry For flow cytometry analysis, mice were euthanized with CO2, and spleens, livers, lymph nodes (groin and axilla) and skin (approximately 1 x 1 cm area) at the injection site were collected (initially induced mice) or lymph nodes (groin and axilla) and spleen (in rhG1-PG sensitized mice). These were placed in RPMI containing sodium pyruvate and penicillin-streptomycin-glutamine. Spleens and livers were transferred to 6-well plates containing 1 ml of digestion buffer (0.01 mg / ml DNase I and 1 mg / ml collagenase D in saline), followed by another 1 ml of digestion buffer. The plates were then incubated at 37°C for 30 minutes, followed by lysis through a 70 µm filter and washing with saline + 10% fetal bovine serum (FBS). To remove hepatocytes, the livers were first centrifuged at 50 × g for 3 minutes, and the supernatant containing immune cells was collected; the hepatocyte pellet was discarded. The spleen and liver were then centrifuged (1500 rpm, 6 minutes, 4°C), incubated in 1 ml ACK lysis buffer to lyse red blood cells (3 minutes at room temperature), and washed with saline for staining.

[0301] Skin samples from both sides of each mouse were combined into 2 ml of saline containing 2 mg / ml Dispase II and minced with scissors. The samples were then incubated on a shaker at 37°C and 200 rpm for 1.5 h, followed by the addition of DNase I (final concentration 0.01 mg / ml) and collagenase D (final concentration 1 mg / ml), and shaken for another 45 min (37°C). The skin and lymph nodes were then ground through a 70 µm filter, washed with saline + 10% FBS, and resuspended in saline for staining.

[0302] Cells were then transferred to FACS tubes, washed with saline (1500 rpm, 5 min, 4°C), and then stained with FVS700 for live / dead cells (1 / 40,000, 20 min, 4°C). Next, the samples were washed with FACS buffer (1x PBS containing 0.4% EDTA and 20 mM BSA), incubated with anti-CD16 / 32 to block Fc receptors (1 / 200, 10 min, 4°C), and then stained with primary antibody at 4°C for 20 min. Cells were then washed with FACS buffer and fixed with 1% PFA (4°C, 40 min). Finally, cells were washed twice with FACS buffer, resuspended in 300 µl of FACS buffer, and harvested the next day on a BD Fortessa.

[0303] Fluorescence in vivo imaging In vivo imaging experiments were performed using female BALB / cArc mice (12–14 weeks old) (n=27). Liposomes were labeled with a lipophilic membrane dye (DiD, Molecular Probes, 25 µg / ml final concentration) as described above. Four days prior to liposome injection, mice were sensitized with 40 µg QuilA (sc, tail base). Mice were then administered 100 µl of DiD-DEN-181, DiD-ASITI-201, DiD-empty liposomes (POPC:POPG:PEG), or PBS only in the left ventral region (sc). The distribution of DiD-labeled liposomes in whole-body images and subsequently in vivo in isolated organs (spleen and liver) and lymph nodes (groin, axilla, and mesentery) at 1, 2, 7, or 14 days post-liposome injection was assessed using the IVIS Spectrum in vivo imaging system (Perkin Elmer). Total radiation efficiency ([p / s] / [µW / cm²)) was calculated for each organ. 2 ]).

[0304] Statistical analysis All experiments were analyzed using either univariate (multiple groups) or two-way ANOVA (multiple groups and conditions). * p < 0.05, ** p < 0.01, *** p < 0.005, **** p < 0.001.

[0305] Example 2 – Results Liposome composition The initial experiment used a Design of Experiments (DoE) model ( Figure 1 The liposomes were designed and optimized using microfluidic technology to possess the following characteristics (target characteristics) that enhance passive targeting of phagocytic DCs in dLN, improve storage stability without particle aggregation (Moghimi 2006, Biomaterials;27(1):136-44; Hoshyar et al. 2016, Nanomedicine;11(6):673-92): size 90-160 nm, PdI < 0.15, zeta potential < -50; and improve the stability (retention) of peptide and calcitriol encapsulation in vitro and in vivo.

[0306] Several possible semi-synthetic compositions were evaluated. Encapsulated CII was generated. 259-273 Liposomes of calcitriol with different concentrations of DPPC:DPPG:cholesterol, POPC:POPG, or POPC:POPG:MBP-PE were then tested for peptide retention in vitro for 2 weeks.

[0307] For an initial composition containing DPPC:DPPG:cholesterol, the optimal CII is achieved. 259-273The retention rates were 30% after 2 weeks and 15% after 4 weeks. Compared with DPPC:DPPG:cholesterol, the peptide retention in compositions containing EPC:EPG, POPC:POPG, and POPC:POPG:MBP was improved (Table 1).

[0308] Table 1. Collagen II 259-273 Comparison of stability in liposomes at -30°C for 2 weeks and 4 weeks.

[0309] Comparison of PI 33-63 The stability of calcitriol in compositions containing EPC:EPC, POPC:POPG, DPPC:DPPG-Chol, or DOTAP or PEI was assessed (Table 2). In compositions containing PEI, DOTAP, ionizable lipids, or DPPC:DPPG-Chol, the retention of size, charge, and peptide encapsulation (stability over time) did not meet the target characteristics. For EPC:EPG and POPC:POPG, size, charge, and peptide encapsulation were adequate, but the peptide retention in the EPC:EPG composition was relatively low at 2 weeks, and calcitriol also showed low retention at 2 weeks.

[0310] Table 2. PI in liposomes with different compositions 33-63 Comparison of size, charge, and encapsulation with calcitriol at -30°C

[0311] ND: Not detected. NL: Not loaded.

[0312] PEG-DMG 2000 surfactant The stability of liposome compositions containing POPC:POPG and PEG content between 0% and 5% was compared. In the absence of PEG-DMG 2000, collagen II... 259-273 (SEQ ID NO.1) showed poor retention at 2 weeks. Surprisingly, the highest retention of peptide antigen and calcitriol occurred at a PEG concentration of only 0.5% (Tables 3 and 4). In PI 33-63 In liposomes, calcitriol retention decreased with increasing PEG concentration above 0.5%, but remained high in collagen II. 259-273 This is not the case in liposomes.

[0313] Table 3. Retention of peptide antigens and calcitriol in POPC:POPG liposomes with different % PEG at -30°C

[0314] Table 4. PIs with different concentrations of PEG constructed at 60℃ or room temperature (RT) 33-63 Comparison of calcitriol POPC and POPG liposomes

[0315] *: After peptidase assay. LOQ: Limit of quantitation.

[0316] Table 5. Ro60 in POPC:POPG liposomes with different % PEG 401-425 and the retention of calcitriol at -30°C

[0317] Table 6. Vimentin in POPC:POPG liposomes with different % PEG 38-88 Retention of Cit64 and calcitriol at -30°C

[0318] Table 7. Vimentin 41-85 Retention of CIT64 and calcitriol in POPC:POPG 0.5% PEG liposomes at -30°C

[0319] Table 8. PI 33-63 Retention of calcitriol and POPC:POPG liposomes containing 0.5% PEG at -30°C

[0320] Table 9. PI 33-63 Retention of calcitriol in EPC:EPG liposomes at -30°C

[0321] Table 10. PI 33-63 Retention of teriflunomide in POPC:POPG 0.5% PEG liposomes at -30°C

[0322] Table 11. Vimentin 41-85 Retention of CIT64 and calcitriol in POPC:POPG 3% PEG liposomes at -30°C

[0323] Distinguishing between free or surface-adsorbed peptides and liposome-encapsulated peptides. An important aspect to consider when constructing nanocarriers is the level of protection provided by the nanoparticles for the encapsulated APIs (e.g., peptide antigens and NF-κB inhibitors), as this will affect the integrity of the payload in the target tissue. Ideally, the antigenic peptide should be protected by liposome encapsulation to avoid potentially triggering immune complex-mediated immune responses. Unprotected peptides may degrade upon exposure to tissue or blood proteases, thus ceasing to be effective tolerogens. Furthermore, residual unencapsulated peptides or surface-adsorbed peptides may lead to incorrect assumptions about the concentration of peptides encapsulated by the liposomes. Prior to scale-up, free peptides are typically removed post-liposome production by dialysis with HEPES buffer. At scale-up, peptides are removed by cross-flow filtration, which subjectes the liposomes to greater shear stress, and surface-adsorbed peptides may not remain attached to the liposomes.

[0324] The inventors compared PI 33-63 / Calcitriol POPC:POPG 1.4% PEG liposomes retain peptides before and after dialysis through 10 kDa or 100 kDa membranes. Due to PI 33-63 At approximately 3 kDa, a larger pore size is required to remove peptide aggregates. Data showed that a 10 kDa membrane removed 17% of the peptides compared to overnight storage in PBS. In contrast, >80% of the peptides were removed by a 100 kDa membrane, indicating that aggregated peptides had been adsorbed onto the liposome surface. Lowering the temperature at which the lipids were incorporated into the methanol phase and mixing with the peptides at room temperature instead of 60°C enhanced peptide retention in the liposomes, even after dialysis with 100 kDa (Table 4). The inventors found that, although the optimal PEG concentration varies, the presence of PEG itself improves the stability of the lipid delivery carrier (Tables 3 to 11).

[0325] To verify the concentration of liposome-encapsulated peptides, the inventors developed an in vitro protease digestion assay based on the concept that unincorporated or surface-adsorbed peptides are digested by specific in vitro proteases, while peptides in the aqueous core are protected from protease digestion. After protease incubation, the liposomal peptides are quantified by liquid chromatography-tandem mass spectrometry (LC-MS / MS). PI 33-63 Digested with chymotrypsin.

[0326] With PI 33-63 Consistent with the in vitro stability data of calcitriol liposomes (Table 3), the percentage of peptides encapsulated in POPC:POPG 0.5% PEG-DMG 2000 liposomes (74%) was higher than that in POPC:POPG alone (44%) or POPC:POPG 1.4% PEG (64%) (Table 12). Positive control: naked PI 33-63 Peptides are completely degraded by the protease chymotrypsin during digestion.

[0327] Table 12. Chymotrypsin digestion to determine the encapsulated liposome PI 33-63 Estimating in vivo stability in plasma To assess the effect of plasma on stability, the inventors developed an assay to evaluate the retention of peptides and calcitriol in liposomes after brief incubation with plasma or PBS. After dialysis with HEPES buffer at 100 kDa, particles were incubated with plasma for 15 min and then fractionated according to size exclusion chromatography (SEC) based on a method used for purifying extracellular vesicles (Lane et al. Methods Mol Biol. 2017;1660:111-30). Liposome particles within the expected size range (approximately 100 nm measured using NanoSight) were recovered in fractions 6–11, while free peptides but almost no liposomes were collected in fractions 16–22. Figure 2 After collecting the fractions, the concentrations of peptides and calcitriol were quantified by mass spectrometry and sensitive ELISA, respectively, to calculate the retention rate of each fraction (fractions 6-11, 16-22) relative to the total peptides and calcitriol recovered from all fractions (fractions 6-22).

[0329] To test the effect of plasma incubation, PI was used. 33-63 / calcitriol / 0.5% PEG liposomes were incubated with plasma or PBS for 15 or 60 minutes, then separated by SEC fractionation, and peptides were quantified and particles evaluated by NTA. With prolonged incubation in plasma, the number of recovered particles decreased, and the % peptide retention rate decreased (Table 13). Figure 3 ).

[0330] Table 13. POPC:POPG:PI after SEC measurement 33-63 Peptides retained in calcitriol liposomes

[0331] To determine the retention of post-SEC peptides and calcitriol, PI produced during scale-up production at a TFF flow rate of 12 mL / min will be used. 33-63 / Calcitriol / 0.5% PEG liposomes were incubated with plasma for 15 minutes, and then all fractions were collected by SEC. The percentage of encapsulated peptides and calcitriol in fractions 6-11 was calculated relative to the total recovery. After plasma incubation and separation by SEC, 50.5% of the peptides and 33% of the calcitriol were retained in fractions 6-11 (Table 14).

[0332] In protease assays performed on the same liposomes, peptide retention was 48%, and calcitriol retention was 100% (Table 14). These data collectively indicate that peptide retention estimated using SEC and peptidase assays is very similar. Therefore, both assays are likely capable of separating liposome-encapsulated peptides from free or liposome-adhered peptides. Calcitriol retention was unaffected by protease incubation but gradually decreased with plasma incubation. This is expected to be due to the presence of vitamin D-binding protein (DBP) in plasma (Chun et al. Front Endocrinol (Lausanne). 2019;10:718), which binds to vitamin D with high affinity. DBP will bind and remove calcitriol exposed within the liposome bilayer. Calcitriol bound to DBP is inactive in most cells. Although subcutaneous DBP concentrations may be lower than in plasma, some liposomal calcitriol activity may be lost en route to the dLN after subcutaneous administration, which should be considered in biodistribution and toxicokinetic studies (Sonigra et al. 2022, JCI Insight 2022;7:e160964). It should be noted that commercially available calcitriol assays measure total calcitriol, including both DBP-bound and free calcitriol. DBP-calcitriol is taken up and metabolized by the epithelial cells of the proximal tubule (Chun et al. Front Endocrinol (Lausanne). 2019;10:718).

[0333] Table 14. POPC:POPG:PI 33-63 / Peptides and calcitriol retained in calcitriol liposomes: SEC estimates in vivo stability in plasma

[0334] Table 15. POPC:POPG:PI 33-63 Peptides and calcitriol retained in calcitriol liposomes: after peptidase incubation

[0335] Table 16. POPC:POPG liposomes containing various peptide antigens

[0336] Biological distribution To assess in vivo performance, the biodistribution of EPC / EPG / peptide / calcitriol (DEN-181, e.g., as described by Sonigra et al. JCI Insight 2022;7:e160964) liposomes was compared with that of POPC / POPG / 0.5%PEG / peptide / calcitriol (ASITI-201) and POPC / POPG / 0.5%PEG (empty) liposomes. Liposomes were fluorescently labeled with the lipophilic dye DiD and administered subcutaneously to the tail root of naïve mice 4 days after local injection with QuilA adjuvant to expand dLN for flow cytometry. Radiation intensity in whole-body and individual lymphoid organs was quantified using in vivo imaging (using IVIS Spectrum). PBS injection served as a control. Whole-body imaging at the skin injection site in all mice administered liposomes but not PBS detected signals at days 1 and 2 post-injection. No signals were observed at days 7 and 14. Signals were also observed in individual organs up to day 2, but not at days 7 or 14. Figure 4 The results show the quantification of radiation intensity in 2–3 mice per group that received liposomes, after subtracting the PBS background. No radiation intensity was observed in the skin or dLN on the uninjected side (not shown). In all groups, the DiD intensity at the skin site was highest on day 1 and decreased on day 2.

[0337] Surprisingly, the DiD intensity of ASITI-201 (POPC / POPG / 0.5% PEG / peptide / calcitriol) was higher in dLN on day 1 than that of DEN-181 (EPC / EPG / peptide / calcitriol) or empty liposomes (POPC / POPG / 0.5% PEG). Figure 4 This indicates that both the ASITI-201 liposomal formulation and its payload enhanced uptake in dLN. ASITI-201 uptake in the liver was also lower than that of DEN-181, suggesting that the liposomal composition has a beneficial effect on dLN targeting relative to systemic lymphoid organs. Equally surprising is that the DiD intensity of empty liposomes continued to increase in both dLN and liver until day 2, while the DiD intensity of DEN-181 and ASITI-201 decreased. Figure 4 Given that topical application of vitamin D has been shown to reduce the number of dendritic cells (DCs) in the skin and enhance DC migration to dLN (Gorman et al. J Steroid Biochem Mol Biol. 2010;121(1-2):247-9), the inventors hypothesize that subcutaneous application of calcitriol-containing liposomes would synergistically increase the infiltration of migratory DCs into dLN and liposome uptake compared to the relatively delayed uptake of dLN DCs by empty liposomes that migrate steadily.

[0338] To assess the biodistribution of peptides, PI was used.33-63 Conjugated with DFO-NH2, and then used as described in this article. 89 Zr labeling. Then it is formulated as POPC / POPG / 0.5% PEG / PI. 33-36 / calcitriol liposomes and dialysis. Two weeks after subcutaneous administration of liposomes or free radiolabeled peptides to the left ventral region of BALB / c mice, in vivo mouse imaging was performed using PET-CT. Figure 5 C). Radioactive-HPLC showed 87.6% 89 Zr and DFO-PI 33-36 Combination. Liposome characteristics and the use of cold PI 33-36 The generated liposomes were comparable. High-intensity radiation was observed at the skin injection site, left axilla, and groin dLN after liposome injection. The signal was first detected at 2 hours and persisted at these sites for 14 days. 89 The detectable maximum half-life of Zr. Given that the fluorescent lipid signal disappears after 2 days, the retention of the peptide in the skin and dLN after liposome delivery is quite surprising. Figure 5 C). Liposome peptides are much more efficient at retention in dLN than individual peptides because subcutaneously delivered radiolabeled peptides are observed in the skin but not in dLN ( Figure 5 B). In mice injected with liposomes or naked peptides, radiation signals were also detected in the kidneys (the expected site of clearance for radiolabeled chemicals).

[0339] Cellular uptake To identify cells that took up liposomes, ASITI-201 or DEN-181 was labeled with the fluorescent dye DiI and administered subcutaneously to naïve mice. The proportions of DiI+ DCs, monocytes, and B cells at the skin injection site, dLN, liver, and spleen were then quantified by flow cytometry. No DiI staining above background was observed in the spleen. In the skin, 20% of all viable cells took up the liposomes, including 70% of CD8+ cells. + DC, 40-60% CD301b - DC and 90% of CD301b + Genuine leather DC.

[0340] It has been proven that the antigen presents CD301b + dDC induces antigen-specific Tregs via retinoic acid-induced ALDH and is essential for controlling Tfh during the immune process (Weckel et al., Immunity. 2023;56(6):1239-54e7;Kumamoto et al. Elife. 2016;5). In dLN, liposome uptake varies among mice, but on average is 5% pDC and 10% CD8.+ cDC1, 5% CD301b, cDC2, and 2% CD301b + cDC2. In the liver, 9-14% of CD301b... + DC is DiI+, while a small portion of pDC and CD8 + DC and CD301b - DC is DiI + These data indicate that ASITI-201 and DEN-181 liposomes are taken up by dendritic cells (DCs) in the skin, dLN, and liver, with a preference for the tolerant CD301 in the skin and liver. + Genuine leather DC ( Figure 6 ).

[0341] in conclusion Using multiple assays, POPC / POPG / 0.5%PEG / peptide / calcitriol demonstrated significantly superior peptide encapsulation and stability in vitro compared to any other liposome composition. These liposomes are well-suited for scale-up and production. In vivo, POPC / POPG / 0.5%PEG / peptide / calcitriol liposomes exhibited better and faster dLN targeting than EPC / EPG / peptide / calcitriol liposomes or empty POPC / POPG / 0.5%PEG liposomes, indicating that this lipid composition synergizes with calcitriol to enhance dLN targeting, thereby minimizing off-target effects. Following subcutaneous application of POPC / POPG / 0.5%PEG / peptide / calcitriol liposomes, the peptides were retained in the skin and dLN by DC presentation for at least 14 days. Figure 5 B and 5C). Expected target DCs, including CD301b. + DC, liposomes are taken up in the skin, dLN and liver ( Figure 6 ).

[0342] Example 3 – In vivo peptide biodistribution To compare the peptide biodistribution after administration of liposomes containing different compositions, PI 33-63 (SEQ ID NO. 2) is conjugated with DFO-NH2, and then used as described herein. 89 Zr mark.

[0343] method PI 33 -DFO Synthesis PI was prepared in 1.5 mL Eppendorf tubes. 33-63A solution of p-SCN-deferoxamine (1.0 mg, 0.33 µmol) in 0.1 M NaHCO3 (500 µL) was prepared. A solution of p-SCN-deferoxamine (1.24 mg, 1.65 mmol) in dimethyl sulfoxide (DMSO) (300 µL) was gently heated and then transferred to a reaction vessel. The tube was placed in an Eppendorf ThermoMixer® and shaken at 37 °C for 16 h. The crude mixture was purified by semi-preparative HPLC using standard methods. The fractions were collected and lyophilized to give a white solid PI. 33 -DFO.

[0344] Radiolabeling of peptides To PI 33 Add 1M HEPES (pH 7.4, 188 µL) to DFO (192.4 µL, 96 µg), then add buffer. 89 Zr (120 µL, 42 MBq). The reaction mixture was briefly centrifuged to ensure homogeneity, and then incubated at 37 °C and 500 rpm for 60 min. Aliquots (5 µL) of the solution were added to 5 mM EDTA (5 µL) and analyzed by RP-HPLC 5-100% MeCN (0.1% TFA) over 15 min. Then conjugated and labeled PI were... 33-36 Formulated as POPC / POPG / 0.5% PEG / PI 33-63 Calcitriol or EPC / EPG / PI 33-63 / Calcitriol liposomes and dialysis.

[0345] Mice were subcutaneously injected with approximately 150 kBq of radioactive material at the base of the tail. This procedure ensured that the amount of active material injected remained consistent across different compounds, taking into account variations in their specific activity. Following injection at the base of the tail, liposome uptake was expected in the left and right inguinal lymph nodes. Depending on the location of the subcutaneous liposome reservoir, a higher proportion of lymph node uptake may occur on one side.

[0346] Longitudinal PET-CT scans were performed on live mice on days 3, 5, 7, and 11 post-injection, and in vitro biodistribution analysis was performed on days 3 and 11.

[0347] result Radioactive-HPLC showed 97-99% purity. 89 Zr binds to DFO-proinsulin peptide. Liposome characteristics and use with cold PI 33-36 The generated liposomes are comparable. Application of POPC / POPG / 0.5% PEG / PI 33-63Following calcitriol liposome administration, compared to peptide administration alone, radiation in the left and right groins and surprisingly also in the left and right axillae was significantly higher on days 3, 5, and 11. Figure 7 AD).

[0348] Application of EPC / EPG / PI 33-63 Following calcitriol liposome administration, radiation in the axillary dLN was low at all time points and not significantly higher than with peptide alone at any time point. Figure 7 C and 7D). Application of EPC / EPG / PI 33-63 Following calcitriol liposome administration, radiation was observed only in the right inguinal dLN compared to peptides alone. Figure 7 B). Application of POPC / POPG / 0.5% PEG / PI 33-63 Calcitriol or EPC / EPG / PI 33-63 Following calcitriol liposome administration, radiation levels at the injection site were higher at 4 hours and 3 days than after administration of the peptide alone. By day 3, POPC / POPG / 0.5% PEG / PI 33-63 Radiation at the injection site after calcitriol is significantly lower than that at EPC / EPG / PI. 33-63 Calcitriol liposomes reflected a greater proportion of peptides present in the dLN. Biodistribution data on day 3 confirmed that POPC liposomes resulted in a greater distribution of peptides in the axillary dLN compared to EPC liposomes. The biodistribution was even more significant on day 11, with POPC / POPG / 0.5% PEG / PI... 33-63 Calcitriol post-peptide is distributed at higher levels across all dLNs than EPC / EPG / PI. 33-63 / Calcitriol liposomes. In POPC / POPG / 0.5% PEG / PI 33-63 Following calcitriol liposome administration, the peptide distribution levels in the liver, spleen, and kidneys were very low on day 3. On day 3, POPC / POPG / 0.5% PEG / PI 33-63 Following calcitriol administration, the distribution of peptides intended for excretion to the kidneys was significantly lower than that of EPC / EPG / PI. 33-63 / Calcitriol liposomes or individual peptides.

[0349] in conclusion When peptides are encapsulated in liposomes, their persistence in the skin and dLN is significantly longer than when delivered in free form. Surprisingly, compared to peptides in EPC / EPG liposomes, POPC / POPG / PEG liposome peptides migrated significantly to the more distal (axillary) dLN within 3 days and persisted at significantly higher levels in the dLN for a longer period of time, while at lower levels in the kidneys. Figure 7These data show that EPC / EPG / peptide / calcitriol liposomes have better in vitro stability and peptide retention compared to POPC / POPG / PEG / peptide / calcitriol liposomes.

[0350] Example 4 – Liposomes are taken up by antigen-presenting cells in vivo To compare the effects of liposomes on antigen-presenting cells (APCs) after multiple subcutaneous administrations of liposomes with different compositions in the early stages of a proteoglycan-induced mouse arthritis model (PGIA) (e.g., as described in Galea R et al. JCI Insight 2019, 4(18):e126025), liposomes containing POPC / POPG / 0.5% PEG / aggregated proteoglycan were prepared. 89-103 Calcitriol, EPC / EPG / aggregated proteoglycans 89-103 Liposomes of calcitriol and POPC / POPG / 0.5% PEG (empty) were compared. Liposomes were administered subcutaneously to the flank on days 7, 11, and 14 after sensitization with recombinant human phosphatidylglycerol (rh-PG). On day 14, the liposomes were fluorescently labeled with the lipophilic dye DiD. Mice were sacrificed on day 15, and the dLN in the axilla and groin, as well as the spleen, were analyzed by flow cytometry (Fig. 8A). Aggregates and proteoglycans were also analyzed. 89-103 This corresponds to SEQ ID NO.69.

[0351] result Surprisingly, in the dLN, the uptake of POPC / POPG / aggregatin / calcitriol liposomes, and usually also empty POPC / POPG liposomes, was significantly higher than that of EPC / EPG / aggregatin / calcitriol liposomes, in total CD19+, memory and germinal center B cells, and all DC subsets and other myeloid cells (Fig. 8B). In the spleen, the uptake of POPC / POPG / aggregatin / calcitriol liposomes and empty POPC / POPG liposomes was significantly higher than that of EPC / EPG / aggregatin / calcitriol liposomes, but only in cDC1, cDC2 dendritic cells and other myeloid cells (Fig. 8C).

[0352] in conclusion Following multiple subcutaneous administrations of liposomes in the early stages of an arthritis animal model, liposome uptake by antigen-presenting B cells and dendritic cells (DCs) in the dLN was higher than that in EPC / EPG liposomes when the liposome composition contained POPC / POPG / PEG. In the spleen, this was only observed in DCs. Consistent with studies of the biodistribution of radiolabeled peptides, these uptake data in the dLN confirm that the POPC / POPG / PEG liposome composition is more effective at targeting the dLN than the EPC / EPG liposome composition. The identification of DiD markers in splenic DCs in cases where the peptides did not biodistribute to the spleen indicates that, following liposome uptake, dLN DCs process their peptide payload for antigen presentation and migrate from the dLN to the spleen, thus promoting a broader mechanism of action compared to EPC / EPG liposomes.

[0353] Example 5 – Flow Cytometry Analysis of Antigen-Presenting Cell Function and T Cell Response method Drainage LN samples from Example 4 were stained with a set of markers to identify CD19+ B cells, CD11c+ conventional DCs, and CD11c- plasmacytoid (p) DCs, as well as the levels of MHC class II, CD80, CD86, and PD-L1 on DiD-expressing cells (e.g., as published in Galea R et al. JCI Insight 2019, 4(18):e126025), and then analyzed by flow cytometry. Aggregate proteoglycans were also used. 89-103 The peptide restimulated spleen cells. The next day, CD154-expressing antigen-activated CD4+ T cells were identified, and the expression of IFN-γ and TNF in each group of mice was compared. Figure 9 (Small Figure A).

[0354] result In dLN, after uptake of POPC / POPG / calcitriol / aggregatin liposomes, the MHC class II expression levels of myeloid and plasmacytoid DiD+ DCs were lower than those after uptake of EPC / EPG / calcitriol / aggregatin liposomes. Figure 9 A and 9B), while memory and germinal center B cells expressed higher levels of MHC class II (A and 9B). Figure 9 C and 9D). PD-L1 expression was also different between myeloid and plasmacytoid DCs after POPC / POPG / calcitriol / aggregatin liposome uptake compared to EPC / EPG / calcitriol / aggregatin liposome uptake. Figure 9 A and 9B).

[0355] In splenic T cells, the frequency of cells responding to in vitro restimulation of aggregate-glycan peptide via CD154 expression was comparable in mice administered POPC / POPG / calcitriol / aglycan or EPC / EPG / calcitriol / aglycan liposomes. Figure 9 E), but in mice treated with POPC / POPG / calcitriol / aggregatin glycan liposomes, the levels of IFN-γ or IFN-γ and TNF produced by CD154+ antigen-reactive T cells were significantly lower than in mice treated with EPC / EPG / calcitriol / aggregatin glycan liposomes ( Figure 9 F and 9G).

[0356] in conclusion In antigen-sensitized mice in the early stages of inflammatory arthritis, compared with mice administered multiple doses of EPC / EPG / calcitriol / aggregatin liposomes, multiple doses of POPC / POPG / calcitriol / aggregatin liposomes exhibited significantly stronger inhibitory effects on dendritic cell MHC class II and lower levels of pro-inflammatory cytokines secreted by antigen-responsive T cells. This is consistent with the better uptake of POPC / POPG / calcitriol / peptide liposomes and their payload due to the higher stability of the liposomes.

[0357] Example 6 – Biodistribution of calcitriol in vitro method Tritium-modified calcitriol ([3H]calcitriol) was purchased from American Radiolabeled Chemicals (St. Louis, MO) and had a specific activity of 20 Ci / mmol. 8 µCi of [3H]calcitriol was incorporated into an excess (>10 times by weight) of unlabeled calcitriol and then incorporated into POPC / POPG / 0.5% PEG / PI. 33-63 Calcitriol or EPC / EPG / PI 33-63 The liposome formulation was then dialyzed overnight against HEPES buffer, recovered, and its specific activity was determined by scintillation counting of 10 µL aliquots.

[0358] Male BALB / c mice (8 weeks old, n=3 per compound at each time point) were subcutaneously administered 100 µL of [3H]calcitriol-labeled liposomal formulation (approximately 0.25 µCi) per mouse near the tail root. Mice were then sacrificed at 1 hour and 72 hours post-administration to assess biodistribution. 50 µL of blood was collected via cardiac puncture, and inguinal and axillary lymph nodes, along with all major tissues, were collected and weighed. 2 mL of milliQ water was added to each liver and intestinal sample, followed by homogenization, and 500 µL of the sample was taken for analysis. All tissues were then dissolved by incubating 1 mL (lymph nodes) or 2 mL (all other tissues) of SOLVABLE (Perkin Elmer) overnight in a 60°C oven. Samples were then bleached with 30% hydrogen peroxide and mixed with Ultima Gold (Perkin Elmer) scintillation solution. Samples were incubated at 4°C for 2 days and then analyzed on a Tri-Carb 4910TR 110V liquid scintillation counter. All collected values ​​were background corrected by subtracting values ​​obtained from injected saline animal tissues and then normalized to the injection dose of triplicate samples run on the same day. These values ​​were then normalized per gram of tissue to obtain the percentage of injection dose per gram of tissue (%ID / g).

[0359] result When applying POPC / POPG / 0.5% PEG / PI 33-63 Calcitriol or EPC / EPG / PI 33-63 One hour after administration of calcitriol liposomes, the highest radiation levels were observed at the skin injection site, affecting any organ. At 72 hours, radiation levels in all tissues were at background levels, consistent with the complete metabolism of the administered [3H]calcitriol at this time. In mice receiving EPC liposomes, some residual radiation in the intestines at 72 hours reflected slower fecal excretion in this group.

[0360] Radiation levels at 1 hour were significantly higher than at 72 hours at the injection site, liver, kidneys, axillary lymph nodes, and intestines, indicating that calcitriol was distributed to these organs. The two formulations, POPC / POPG / 0.5% PEG / PI, were compared within the first hour. 33-63 Following calcitriol liposome administration, radiation levels at the injection site were significantly higher, while radiation levels in the liver, spleen, and kidneys were significantly lower than those in EPC / EPG / PI. 33-63 Calcitriol liposomes showed that, compared to POPC / POPG / PEG liposomes, EPC / EPG liposomes resulted in greater systemic distribution of calcitriol and less retention at the injection site. Radiation in the groin or axillary draining lymph nodes was comparable one hour after injection of either POPC / POPG / PEG or EPC / EPG liposome formulations.

[0361] in conclusion These calcitriol biodistribution data support the conclusion that the calcitriol payload of EPC / EPG liposomes is not as stably retained as that of POPC / POPG / PEG liposomes, resulting in greater systemic diffusion within the first hour after subcutaneous injection.

[0362] The sequence of this disclosure

Claims

1. A lipid delivery carrier comprising a nuclear factor-κB (NF-κB) inhibitor and a peptide antigen, wherein the lipid delivery carrier comprises 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate choline (POPC) and 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphate glycerol (POPG).

2. The lipid delivery carrier according to claim 1, further comprising PEGylated lipids, wherein the PEGylated lipids are selected from the group consisting of PEG-modified phosphatidylethanolamine, PEG-modified phosphatidic acid, PEG-modified ceramide, PEG-modified dialkylamine, PEG-modified diacylglycerol, PEG-modified dialkylglycerol, PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, PEG-DSPE, and combinations thereof.

3. The lipid delivery carrier according to claim 2, wherein the lipid delivery carrier comprises about 85 mol% to 95 mol% of POPC, about 5 mol% to 15 mol% of POPG and about 0.1 mol% to about 5 mol% of the PEGylated lipid.

4. The lipid delivery carrier according to claim 3, wherein the lipid delivery carrier has an average diameter of about 60 nm to about 180 nm.

5. The lipid delivery carrier according to claim 4, wherein the lipid delivery carrier has a negative surface charge of less than about -30 mV.

6. The lipid delivery carrier according to any one of claims 1 to 5, wherein the peptide antigen is an autoantigen derived from a protein selected from the group consisting of: Ro, type II collagen (CII), proinsulin (PI), insulin, ovalbumin, tendinin C, chromogranin, agglutinin glycan, islet antigen 2 (IA2), glutamate decarboxylase 65 kDalton isoform (GAD65), glycoprotein (gp70), nuclear antigen, lupus autoantigen, Smith, La, U1-RNP, fibrin, histone, ribosomal protein, pyruvate dehydrogenase dihydrolipoamide acetyltransferase (PCD-E2), hair follicle antigen, human tropomyosin isoform 5 (hTM5), human... Cartilage gp39 (HCgp39) and gp130-RAPS, DNAJp1, citrullinated protein, citrullinated peptide, citrullinated type II collagen, citrullinated vimentin, citrullinated fibrinogen, citrullinated agglutinin glycan, citrullinated tendinin C, myelin basic protein, protein lipoprotein (PLP) and myelin oligodendrocyte glycoprotein (MOG), thyroid-stimulating hormone receptor (TSH-R), acetylcholine receptor (AchR), gliadin, histone, PLP, glucose-6-phosphate isomerase, thyroglobulin, various tRNA synthetases, protease-3, human desmosome core glycoprotein 2 (hDSG2) and myeloperoxidase (MPO).

7. The lipid delivery carrier according to claim 6, wherein the peptide antigen is an autoantigen derived from a protein selected from the group consisting of: collagen, proinsulin, ovalbumin, insulin, agglutinin, Ro, vimentin, thyroid-stimulating hormone receptor (TSH-R), myeloperoxidase (MPO), tendinin C, human desmosome core glycoprotein 2 (hDSG2), citrullinated vimentin, citrullinated agglutinin, and citrullinated tendinin C.

8. The lipid delivery carrier according to claim 7, wherein the peptide antigen is an autoantigen derived from a protein selected from the group consisting of: collagen, proinsulin, ovalbumin, insulin, agglutinin, Ro, vimentin, human desmosome core glycoprotein 2 (hDSG2), citrullinated vimentin, and citrullinated agglutinin.

9. The lipid delivery carrier according to claim 8, wherein the peptide antigen is derived from proinsulin or an autoantigen of insulin.

10. The lipid delivery carrier according to claim 8, wherein the peptide antigen is an autoantigen derived from proteoglycans or citrullinated proteoglycans.

11. The lipid delivery carrier of claim 8, wherein the peptide antigen is an autoantigen derived from vimentin or citrullinated vimentin.

12. The lipid delivery carrier of claim 8, wherein the peptide antigen comprises, is composed of, or is substantially composed of an amino acid sequence or a variant thereof shown in any one of SEQ ID NO: 1-71, wherein the variant has at least 80% sequence identity with the amino acid sequence.

13. The lipid delivery carrier of claim 12, wherein the peptide antigen comprises, is composed of, or is substantially composed of an amino acid sequence or a variant thereof shown in any one of SEQ ID NO: 4, 11-24 or 69, wherein the variant has at least 80% sequence identity with the amino acid sequence.

14. The lipid delivery carrier of claim 12, wherein the peptide antigen comprises, is composed of, or is substantially composed of the amino acid sequence shown in any one of SEQ ID NO: 6-8, 66-68 or 71 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence shown in SEQ ID.

15. The lipid delivery carrier of claim 12, wherein the peptide antigen comprises, is composed of, or is substantially composed of the amino acid sequence shown in any one of SEQ ID NO:2 or 60-65 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence shown in SEQ ID.

16. The lipid delivery carrier of claim 12, wherein the peptide antigen comprises, is composed of, or is substantially composed of the amino acid sequence shown in SEQ ID NO:2 or a variant thereof, wherein the variant has at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

2.

17. The lipid delivery carrier according to claim 6, wherein the NF-κB inhibitor is selected from the group consisting of: calcitriol, rapamycin, leflunomide, teriflunomide, bevacizumab, thiochlorophen, bortezomib, doxorubicin hydrochloride, cantharidin, carfilzomib, chromomycin donomycin, digoxin, sucrose, emetine, erlotinib hydrochloride, gemcitabine, irinotecan, quinacrine dihydrochloride, fluxalen, manidipine hydrochloride, methylsalicylic acid, lentatinib, ouabain, pemetrexed disodium, sorafenib tosylate, sunitinib malate, tiaconazole, topotecan, tribromosalamin, triclobenzazole, zafirlukast, solanine A, quercetin, curcumin, BAY 11-7085 and BAY 11-7082, and / or their active metabolites and synthetic analogues, and combinations thereof.

18. The lipid delivery carrier of claim 17, wherein the NF-κB inhibitor is teriflunomide, or leflunomide and / or its active metabolite or synthetic analogue.

19. The lipid delivery carrier according to claim 6, wherein the NF-κB inhibitor is vitamin D, an active metabolite of vitamin D, previtamin D, or a synthetic vitamin D analog.

20. The lipid delivery carrier of claim 19, wherein the NF-κB inhibitor is selected from the group consisting of: calcitriol, calcitriol, ergocalcitriol, alfacalcitriol, ducalcitriol, calcidiol, 22-oxacalcitriol, paricalcitriol, tacalcitriol, fluorocalcitriol, idecalcitriol, inanecalcitriol, cefixime, masalcalcitriol, calcidiol, calcitriol; synthetic analogs thereof and / or side-chain analogs thereof; and combinations thereof.

21. The lipid delivery carrier of claim 20, wherein the NF-κB inhibitor is calcitriol, its side-chain analogue, or a synthetic analogue thereof.

22. The lipid delivery carrier according to any one of claims 1 to 3, wherein the peptide antigen is an autoantigen derived from proinsulin or insulin, and the NF-κB inhibitor is vitamin D, an active metabolite of vitamin D, previtamin D, or a synthetic vitamin D analog, and wherein the lipid delivery carrier comprises about 1 µg / mL to about 50 µg / mL of the peptide antigen and about 400 ng / mL to about 600 ng / mL of the NF-κB inhibitor.

23. The lipid delivery carrier of claim 22, wherein at least 50% of the NF-κB inhibitor and at least 50% of the peptide antigen are encapsulated within the lipid delivery carrier.

24. The lipid delivery carrier of claim 23, wherein when incubated in plasma at 37°C for 15 minutes, at least 50% of the encapsulated NF-κB inhibitor and at least 50% of the encapsulated peptide antigen are retained inside the lipid delivery carrier.

25. A population of lipid delivery carriers according to any one of claims 1 to 24, wherein the population has a polydispersity index of less than 0.

20.

26. A pharmaceutical composition for subcutaneous delivery comprising a lipid delivery carrier and a pharmaceutically acceptable carrier according to any one of claims 22 to 24.

27. A method for inducing a tolerable immune response in a subject, comprising administering to the subject a lipid delivery vehicle according to any one of claims 1 to 24.

28. Use of the lipid delivery carrier according to any one of claims 1 to 24 in the preparation of a medicament for inducing a tolerable immune response in a subject.

29. A method for treating and / or preventing an autoimmune disease, condition, or illness in a subject, comprising administering to the subject a lipid delivery carrier according to any one of claims 1 to 24.

30. Use of the lipid delivery carrier according to any one of claims 1 to 24 in the preparation of a medicament for treating and / or preventing autoimmune diseases, conditions or illnesses in a subject.

31. The method of claim 27 or 29, wherein the lipid delivery carrier is administered subcutaneously.

32. The use according to claim 28 or 30, wherein the drug is formulated for subcutaneous administration.

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