Immunomodulatory combinations of antigen and drug-lipid conjugates

Abstract

(i) a lipid conjugate comprising an immunomodulatory agent covalently linked to a lipophilic moiety by a cleavable linkage or via a cleavable linker; and (ii) an immunomodulatory combination comprising an antigen and / or one or more nucleic acids(s) encoding the antigen. The antigen is a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, or polysaccharide. The lipid conjugate and the antigen and / or one or more nucleic acids(s) encoding the antigen are formulated in separate delivery vehicles or are co-formulated in the same delivery vehicle.

Description

[Technical field]

[0001] The present disclosure relates to combinations and / or combinations of antigens and immunomodulator-lipid conjugates for immunomodulation, for example, to treat, prevent and / or ameliorate antigen-induced disorders. [Background technology]

[0002] Antigen-presenting cells (APCs) direct the adaptive immune system. APCs take up exogenous (and self) antigens, process them, and load them onto MHC (or HLA molecules) on the cell surface, which can then be recognized by various classes of T lymphocytes to induce an appropriate immune response. While engaging antigen-specific T cells via their T cell receptors that recognize the antigen:MHC complex, APCs deliver additional signals to these T cells depending on whether the antigen was taken up in the presence of proinflammatory or homeostatic / regulatory signals. In the event that the antigen was taken up in the presence of proinflammatory signals (e.g., from infectious pathogens), APCs are matured / activated and are often characterized by high surface levels of costimulatory molecules. These mature APCs deliver costimulatory signals to antigen-specific T cells to induce downstream humoral and / or cellular immune responses against the antigen. In contrast, when antigens are internalized in the absence of inflammatory cues, APCs remain immature or express co-inhibitory molecules that dampen antigen-specific T cell responses and induce regulatory T cell responses against the antigen. Thus, APCs have a central role in directing antigen-specific immune responses.

[0003] Many pathological conditions are associated with undesirable or inappropriate antigen-specific immune responses. These immune responses are diverse and range from allergic diseases to autoimmune diseases to transplant rejection. Existing treatments cause a generalized, non-discriminatory global suppression of the immune system, with increased risk of infection and / or cancer, as well as the additional adverse effects of immunosuppressive drugs. Moreover, none of these treatments are curative, requiring chronic use of treatments that further exacerbate the adverse reactions and, importantly, fail to completely control the disease.

[0004] Tolerizing APC to induce tolerance to specific antigen is another approach to treat antigen-induced disorders.Such an approach can avoid the drawback of non-specific suppression of immune system associated with immunosuppressants.However, one of the main limitations of tolerizing APC for therapeutic treatment is the need to remove APC from subject's body and tolerize ex vivo, and the limited transport of these APC to relevant disease tissue / site when re-implanted.

[0005] Thus, there is a need in the art for immunomodulatory treatments that address the shortcomings of known approaches to modify a subject's immune response to a particular antigen, for example, to treat, prevent and / or ameliorate antigen-induced disorders. Summary of the Invention

[0006] The present disclosure seeks to address one or more limitations of the known prior art or to provide a useful alternative.

[0007] Various embodiments disclosed herein relate to immunomodulatory combinations and / or combinations comprising a lipid conjugate comprising an immunomodulatory agent covalently linked to a lipophilic moiety by a cleavable linkage or via a cleavable linker; and an antigen and / or one or more nucleic acids encoding the antigen, where the antigen is a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, or polysaccharide, and the lipid conjugate and the antigen and / or one or more nucleic acids encoding the antigen are formulated in separate delivery vehicles or are formulated together in the same delivery vehicle. In some embodiments, the delivery vehicle is a lipid nanoparticle and / or liposome. In some of these embodiments, the delivery vehicle is a lipid nanoparticle that delivers to an antigen-presenting cell (APC). In some embodiments, the antigen or one or more nucleic acids encoding the antigen are entrapped within the lipid nanoparticle or liposome and have a net charge opposite to the net charge of the lipid in the lipid nanoparticle, or the antigen is lipophilic and is incorporated into the lipid compartment of the lipid nanoparticle or liposome. In other embodiments, the antigen is hydrophilic and is entrapped in liposomes that comprise an aqueous core.

[0008] In some embodiments, the lipid conjugate is co-formulated in the same delivery vehicle as the antigen or one or more nucleic acids encoding the antigen. The immunomodulatory combination may include two or more (e.g., two, three, or more) lipid conjugates. In some embodiments, the lipid conjugate is a first lipid conjugate, and the immunomodulatory combination further includes a second lipid conjugate, the second lipid conjugate includes an immunomodulatory agent covalently linked to a lipophilic moiety by a cleavable linkage or via a cleavable linker, the immunomodulatory agent of the second lipid conjugate is different from the immunomodulatory agent of the first lipid conjugate, and the first lipid conjugate and the second lipid conjugate are formulated in separate delivery vehicles or are co-formulated in the same delivery vehicle. In some such embodiments, the immunomodulatory agent of the second lipid conjugate targets a different immune pathway than the immunomodulatory agent of the first lipid conjugate. In some embodiments, the first lipid conjugate and the second lipid conjugate are co-formulated in the same delivery vehicle.

[0009] In various embodiments, the immunomodulatory combinations disclosed herein may be used to treat a subject having an antigen-induced disorder or an unwanted antigen-driven immune response, or may be used in the manufacture of a medicament for treating a subject. Thus, the present disclosure provides a method of treating a subject having an antigen-induced disorder or an unwanted antigen-specific immune response, comprising administering to the subject an immunomodulatory combination as defined herein, wherein the antigen or one or more nucleic acids encoding the antigen, and the lipid conjugate are administered together or sequentially.

[0010] According to another aspect of the present disclosure, there is provided a vaccine formulation comprising at least one prodrug comprising an immunomodulatory agent conjugated to a lipid moiety; and at least one antigen and / or a nucleic acid encoding an antigen, wherein the at least one antigen is a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, or polysaccharide, and wherein the at least one prodrug and the antigen and / or the nucleic acid encoding the antigen are formulated in separate delivery vehicles or co-formulated in the same delivery vehicle in the formulation.

[0011] According to another aspect of the present disclosure, there is provided a method of treating a subject having an antigen-induced disorder or an unwanted antigen-driven immune response comprising administering at least one prodrug comprising an immunomodulatory agent conjugated to a lipophilic moiety; and administering at least one antigen and / or nucleic acid encoding an antigen, wherein the at least one antigen is a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, or polysaccharide, and the at least one prodrug and the antigen and / or nucleic acid encoding the antigen are formulated separately in a delivery vehicle or co-formulated in the same delivery vehicle, and the at least one prodrug and the at least one antigen and / or nucleic acid encoding the antigen are administered together or sequentially.

[0012] According to another aspect of the present disclosure, there is provided a use of a prodrug comprising an immunomodulatory agent conjugated to a lipophilic moiety for treating a subject having an antigen-induced disorder or an unwanted antigen-driven immune response in combination with at least one antigen and / or a nucleic acid encoding at least one antigen, wherein the at least one antigen is a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, or polysaccharide, and the at least one prodrug and the antigen and / or the nucleic acid encoding the antigen are (i) co-formulated in the same delivery vehicle in at least one formulation, or (ii) formulated in separate delivery vehicles for sequential or co-administration to the subject.

[0013] According to another aspect of the present disclosure, there is provided a combination of a prodrug comprising an immunomodulatory agent conjugated to a lipophilic moiety and at least one antigen and / or a nucleic acid encoding at least one antigen for treating a subject having an antigen-induced disorder or an unwanted antigen-driven immune response, wherein the at least one antigen is a protein, a polypeptide, a peptide, a lipoprotein, a glycolipid, a polynucleotide, or a polysaccharide, and the at least one prodrug and the antigen and / or the nucleic acid encoding the antigen are (i) co-formulated in a delivery vehicle or (ii) formulated in separate delivery vehicles for sequential or co-administration to a subject.

[0014] According to a further aspect of the present disclosure, a formulation is provided comprising at least two prodrugs comprising an immunomodulatory agent conjugated to a lipophilic moiety, the at least two prodrugs being formulated in separate delivery vehicles or co-formulated in the same delivery vehicle in a formulation, the at least two prodrugs targeting different immune pathways. Optionally, the formulation comprises at least one antigen and / or a nucleic acid encoding the antigen, the at least one antigen being a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, or polysaccharide. The antigen or the nucleic acid encoding the antigen may be formulated in a delivery vehicle, including being co-formulated with the two prodrugs in one delivery vehicle or being formulated in its separate delivery vehicle.

[0015] According to any one of the aforementioned aspects of the present disclosure, the delivery vehicle can be a lipid nanoparticle.

[0016] According to any one of the foregoing aspects or embodiments of the present disclosure, the at least one prodrug may be co-formulated in the same delivery vehicle as the antigen or a nucleic acid encoding the antigen.

[0017] According to any one of the foregoing aspects or embodiments of the present disclosure, two prodrugs may be present in the formulation, and may be formulated separately or may be co-formulated in the same delivery vehicle.

[0018] According to any one of the foregoing aspects or embodiments of the present disclosure, the immunomodulatory agent may be a tolerogenic agent or an anti-inflammatory agent.

[0019] According to any one of the foregoing aspects or embodiments of the present disclosure, the immunomodulatory agent may be an immunostimulant or an immunosuppressant.

[0020] Further, according to any one of the foregoing aspects or embodiments of the disclosure, the at least one prodrug immunomodulatory agent may be selected from the group consisting of prednisone, budesonide, prednisolone, methylprednisolone, hydrocortisone, cortisone, betamethasone, budesonide, triamcinolone, flunisolide, beclomethasone, fluticasone, mometasone, fludrocortisone, flumethasone, triamcinolone acetonide, isoflupredone, corticosterone, desoxycortone acetate, desoxycortone enanthate, 11-deoxycorticosterone ... 1-Deoxycortisol, Aldosterone, Dexamethasone, Calcitriol, Acetylsalicylic Acid, Salicylate, Mycophenolic Acid, Sirolimus, Tacrolimus, Cholecalciferol, Calcifediol, Alfacalcidol, Calcipotriol, Falecalcitriol, Maxacalcitol, Paricalcitol, Doxercalciferol, 22-Oxacalcitriol, Tacalcitol, Eldecalcitol, Elocalcitol, Inecalcitol, Becocalcidiol, Seocalcitol, Ergocalciferol, Leki Sacalcitol, retinoic acid, cyclophosphamide (nitrogen mustard), filgotinib, baricitinib, tofacitinib, ruxolitinib, upadacitinib, oclacitinib, peficitinib, fedratinib, delgocitinib, deuclavacitinib, abrocitinib, auranofin, apremilast, azathioprine, chloroquine, hydroxychloroquine, cyclosporine, leflunomide, methotrexate, minocycline, sulfasalazine, salicylic acid, diflunisal, salsalate, naproxen, ibuprofen, oncolytic acid, cyclosporine ... Xaprozin, loxoprofen, zaltoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, bromfenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, phenylbutazone, celecoxib, firocoxib, parecoxib, etoricoxib, clonixin, licofelone, MCC950, glyburide, CY-09, tranilast, oridonin, BOT-4-one (2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one), INF39 (ethyl 2-(2-chlorobenzyl)acrylate, MNS (3,4-methylenedioxy-β-nitrostyrene), fenamic acid, beta-hydroxybutyric acid, quercetin, JC-171, ibrutinib, OLT1177, FC11A-2, INF58, JC124, ethyl 2-((2-chlorophenyl)(hydroxy)methyl)acrylate, or a combination thereof.

[0021] According to any one of the aforementioned aspects or embodiments of the present disclosure, two or more antigens and / or nucleic acids encoding antigens may be present in the formulation and may be formulated separately or may be co-formulated in the same delivery vehicle.

[0022] According to any one of the aforementioned aspects or embodiments of the present disclosure, the antigen may be a peptide, a polypeptide or a protein.

[0023] Other objects, features and advantages of the present disclosure will become apparent to those skilled in the art from the following detailed description and figures. [Brief description of the drawings]

[0024] [Figure 1A] Figure 1A shows gate plots (gated on live cells) from pancreatic lymph nodes of mice assessed for APC markers, CD11b and CD11c, by flow cytometry 48 hours after administration of DiO (3,3'-dioctadecyloxacarbocyanine perchlorate)-labeled lipid nanoparticles (54 / 45 / 1 mol:mol LNPs, DSPC / cholesterol / DSPE-PEG). Mice received two injections, 24 hours apart, of LNPs injected ip at a dose of 600 mg / kg.

[0025] [Figure 1B] FIG. 1B shows the enumeration of DiO-positive APCs (gated on CD11b+CD11c+ cells) in the pancreatic lymph nodes of mice injected with DiO-labeled LNPs.

[0026] [Figure 1C] FIG. 1C shows a gate plot (gated on live cells) from mouse islets assessed for APC markers, CD11b and CD11c, by flow cytometry 48 hours after administration of DiO-labeled lipid nanoparticles (54 / 45 / 1 mol:mol DSPC / cholesterol / DSPE-PEG).

[0027] [Figure 1D] FIG. 1D shows the counting of DiO-positive APCs (gated on CD11b+CD11c+ cells) in the pancreatic islets of mice injected with DiO-labeled LNPs.

[0028] [Figure 2A] Figure 2A shows the counting of DiO positive cells in islet macrophages (CD11b+CD11c+) from mice injected with PBS control, DSPC / cholesterol / DSPE-PEG LNPs (DSPC / cholesterol / DSPE-PEG at 54 / 45 / 1 mol:mol) or ionizable LNPs (A002 / DSPC / cholesterol / PEG-DMG at 50 / 10 / 38.5 / 1.5 mol:mol). Mice were injected with 150 mg / kg DiO-loaded LNP formulations and 24 hours later, islets, pancreatic lymph nodes and splenocytes were isolated.

[0029] [Figure 2B] FIG. 2B is a bar graph showing the percentage of DiO-positive APCs (CD11b+CD11c+ cells) in pancreatic islets, pancreatic lymph nodes, and spleens from mice injected with DSPC / cholesterol LNPs (horizontal hatching) and ionizable LNPs (diagonal hatching).

[0030] [Diagram 3]FIG. 3 is a bar graph showing the percentage of lipid conjugate (prodrug) remaining in LNPs (49 / 40 / 10 / 1 mol:mol DSPC / Cholesterol / Prodrug / DSPE-PEG) co-formulated with dexamethasone (D045) and calcitriol (D053, D068, D083) lipid conjugates (prodrugs) after 2 hours of incubation in human plasma (hPlasma).

[0031] [Figure 4] 4 is a bar graph showing the percentage of myeloid dendritic cells (BMDCs) with costimulatory molecules on their surface after aLNP lipid conjugate (prodrug) treatment. LNPs were formulated with D034 or D045 dexamethasone lipid conjugates (prodrugs) and / or D053 and D083 calcitriol lipid conjugates (prodrugs) as indicated. BMDCs were treated for 48 hours, with LNPs containing various calcitriol and dexamethasone lipid conjugates (alone or in combination). BMDCs were then challenged with lipopolysaccharide (LPS) stimulation for 24 hours to determine whether the lipid conjugate (prodrug) formulations could prevent LPS-mediated activation (i.e., tolerate BMDCs). Costimulatory markers on BMDCs were characterized as CD80-CD86- (vertical lines), CD80+CD86+dim (horizontal lines) and CD80+CD86+ (diagonal lines). Treatments are shown in comparison to BMDCs treated with LPS only (control) and without LPS (untreated).

[0032] [Diagram 5]Figure 5 is a bar graph showing the percentage of proliferation of CD4+ T cells for various LNP formulations of prodrugs of dexamethasone (INT-D034 and INT-D045) and calcitriol (INT-D053 and INT-D083) at molar % ranging from 10 to 99%, as shown in a mixed leukocyte (MLR) response assay. Bone marrow-derived dendritic cells (BMDCs) from C57Bl / 6 mice were first treated with LNPs containing various molar % of dexamethasone or calcitriol conjugates for 48 h and then activated by incubation with LPS for 24 h. They were then harvested and mixed with CD4+ T cells isolated from Balb / cJ mice (Jackson Laboratories) at a T:BMDC ratio of 5:1 or 10:1. Treatments are shown in comparison to BMDCs treated with LPS and empty LNP (control), with LPS and without LNP (+LPS-LNP), and without LPS and without LNP (untreated).

[0033] [Figure 6] Figure 6 is a bar graph showing the T cell proliferation percentage for various lipid conjugate (prodrug) LNPs formulated with dexamethasone (D034, D045) and calcitriol (D083) lipid conjugates (prodrugs) and combinations of these prodrugs. C57Bl / 6 BMDCs were treated with LNPs containing single lipid conjugates (prodrugs) or combinations of lipid conjugates (prodrugs) (DSPC or DMPC, cholesterol, prodrug, and PEG-DSPE (49 / 40 / 10 / 1 molar ratio) for 48 hours, then washed and co-cultured with Balb / c CD4+ T cells. The lipid concentration was 30 μM for all treatments. Data are presented as the mean + SD of the % proliferation (by CFSE dilution) in CD4+ cells in triplicates.

[0034] [Figure 7]FIG. 7 is a bar graph showing T cell proliferation as a function of different ratios of dexamethasone (D045) and calcitriol (D083) lipid conjugates (prodrugs) co-formulated into LNPs. Different ratios of dexamethasone (D045) and calcitriol (D083) lipid conjugates (prodrugs) in LNPs affect T cell proliferation. C57BL / 6 BMDCs were treated with LNPs containing various molar ratios of D045 and D083 for 48 hours, then washed and co-cultured with Balb / c CD4+ T cells. Lipid concentration was 30 uM for all treatments. Data are presented as mean + SD of % proliferation (by CFSE dilution) in CD4+ cells in triplicates.

[0035] [Figure 8] Figure 8 is a bar graph showing T cell proliferation of CD4+ T cells from OT-II mice after mixing with BMDCs pretreated with LNPs formulated with lipid conjugates containing calcitriol (D053) at the concentrations indicated in the legend, untreated or control LNPs (Ctr LNPs). After LNP pretreatment, BMDCs were pulsed with various concentrations of free ovalbumin 323-339 peptide (OVA) and co-cultured with CD4+ T cells. Data are presented as mean + SD of % proliferation (by CFSE dilution) in CD4+ cells in triplicates.

[0036] [Figure 9] 9 is a bar graph showing T cell proliferation of CD4+ T cells harvested from OT-II mice after mixing with C57BL / 6 BMDCs treated with or without OVA, control LNPs, LNPs encapsulated with OVA, or LNPs co-formulated with calcitriol and OVA (D053-LNP-OVA). Data are presented as mean + SD of % proliferation (by CFSE dilution) in CD4+ cells in triplicates.

[0037] [Figure 10A] FIG. 10A shows the structure of an exemplary immunomodulatory agent-lipid conjugate. [Figure 10B]FIG. 10B shows the structure of an exemplary immunomodulatory agent-lipid conjugate. [Figure 10C] FIG. 10C shows the structure of an exemplary immunomodulatory agent-lipid conjugate. [Figure 10D] FIG. 10D shows the structure of an exemplary immunomodulatory agent-lipid conjugate. [Figure 10E] FIG. 10E shows the structure of an exemplary immunomodulatory agent-lipid conjugate. [Figure 10F] FIG. 10F shows the structure of an exemplary immunomodulatory agent-lipid conjugate. [Figure 10G] FIG. 10G shows the structure of an exemplary immunomodulatory agent-lipid conjugate. [Figure 10H] FIG. 10H shows the structure of an exemplary immunomodulatory agent-lipid conjugate. [Figure 10I] FIG. 10I shows the structures of exemplary immunomodulatory agent-lipid conjugates. [Figure 10J] FIG. 10J shows the structure of an exemplary immunomodulatory agent-lipid conjugate. [Figure 10K] FIG. 10K shows the structure of an exemplary immunomodulatory agent-lipid conjugate. [Figure 10L] FIG. 10L shows the structure of an exemplary immunomodulatory agent-lipid conjugate. [Figure 10M] FIG. 10M shows the structure of an exemplary immunomodulatory agent-lipid conjugate.

[0038] [Figure 11] 11 is a bar graph of antigen-specific T cell proliferation in co-culture of Ova-specific CD4+ T cells (from OTII mice) with C57BL / 6 BMDCs pretreated with LNPs separately loaded with Ova mRNA and lipid conjugates. Error bars represent mean ± SD.

[0039] [Figure 12]12 is a series of bar graphs showing the proliferation of CFSE-labeled Ova-specific CD4+ T cells (from OTII mice) after co-culture with C57BL / 6 BMDCs pretreated with various LNPs co-loaded with mRNA encoding the Ova antigen and lipid conjugates (prodrugs) of the invention. Data are presented as mean ± SD of marker positive cells as a percentage of CD4+ cells (Y-axis).

[0040] [Figure 13A] Figure 13A is a set of bar graphs showing cytokines measured in supernatants of CFSE-labeled OTII CD4+ T cells co-cultured with C57BL / 6 BMDCs pre-treated with LNPs co-loaded with mRNA encoding the Ova antigen and a lipid conjugate (prodrug) of the invention. Data are presented as mean ± SD of marker positive cells as a percentage of CD4+ cells (Y-axis). [Figure 13B] Figure 13B is a set of bar graphs showing cytokines measured in supernatants of CFSE-labeled OTII CD4+ T cells co-cultured with C57BL / 6 BMDCs pre-treated with LNPs co-loaded with mRNA encoding the Ova antigen and a lipid conjugate (prodrug) of the invention. Data are presented as mean ± SD of marker positive cells as a percentage of CD4+ cells (Y-axis).

[0041] [Figure 14] 14 is a bar graph showing antibody production in mice in response to injection of LNPs further loaded with lipid conjugate D034 versus LNPs carrying Ova mRNA. Error bars represent ±SD, statistical analysis was performed by one-way ANOVA with Tukey's multiple comparison test, *P<0.5, ****P<0.0001, n>7 mice per group from two independent experiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] Provided herein are immunomodulatory combinations (alternatively referred to as vaccine formulations), their uses and methods. The immunomodulatory combinations include (i) a lipid conjugate comprising an immunomodulatory agent linked to a lipophilic moiety and formulated into a delivery vehicle (e.g., lipid nanoparticles (LNPs), liposomes, etc.), and (ii) an antigen or one or more nucleic acids encoding the antigen, formulated into the delivery vehicle. The lipid conjugate and the antigen and / or the nucleic acid encoding it can be formulated into separate delivery vehicles or can be co-formulated into the same delivery vehicle.

[0043] In some embodiments, the immunomodulatory agent is taken up by the APCs as demonstrated by either in vitro or in vivo assays. By way of illustration and not by way of limitation, the present disclosure shows that the immunomodulatory combinations disclosed herein can induce a tolerizing phenotype in myeloid cells (e.g., myeloid dendritic cells (BMDCs)). Furthermore, these tolerizing APCs can suppress proliferation of antigen-specific T cells, induce antigen-specific regulatory T cells, reduce Th1 and Th2 cytokine secretion, and reduce antigen-specific antibody production.

[0044] definition

[0045] "Vaccine formulation" refers to any pharmaceutical formulation comprising one or more of the same or different delivery vehicles as described herein for treating, preventing and / or ameliorating an antigen-induced disorder in a subject. The term includes formulations prepared with any suitable pharma- ceutically acceptable salts and / or excipients.

[0046] "Immunomodulator" refers to an agent capable of altering the immune response in a subject. In one non-limiting embodiment, an immunomodulator is an immunostimulant that enhances the immune response in a subject. In another non-limiting embodiment, an immunomodulator is an immunosuppressant that prevents or reduces the immune response in a subject. An immunomodulator can modulate myeloid cells (monocytes, macrophages, dendritic cells, megakaryocytes, and granulocytes) or lymphoid cells (T cells, B cells, and natural killer (NK) cells) and any further differentiated cells thereof.

[0047] "Tolerogenic agent" refers to an agent that suppresses an immune response or induces tolerance to an antigen. In some embodiments, a tolerogenic agent enhances the suppression of an immune response to an antigen and / or enhances the induction of tolerance to an antigen. For example, a tolerogenic agent can promote tolerogenic presentation of an antigen by APCs.

[0048] "Antigen-induced disorder or unwanted antigen-driven immune response" refers to a condition in a subject associated with antigen-specific immune stimulation, e.g., stimulation of the immune system against an antigen by antigen-presenting cells. Such disorders include any unwanted stimulation of the immune system, including, but not limited to, allergies, autoimmune diseases, transplant rejection, anti-drug antibody responses, and the like.

[0049] "Antigen" has its usual meaning in the art and refers to a molecule or molecular complex that can bind to a specific antibody or T-cell receptor and modify the immune system. In some embodiments, the antigen is a foreign or non-foreign protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, or polysaccharide. In some embodiments, the antigen induces an antigen-induced injury.

[0050] "Delivery vehicle" refers to any suitable particle in which an immunomodulatory agent-lipid conjugate (e.g., a prodrug) can be formulated. Non-limiting examples include lipid nanoparticles, liposomes, and the like.

[0051] "Lipophilic moieties," in reference to moieties linked to immunomodulatory agents as part of a lipid conjugate (e.g., a prodrug) or lipophilic moieties of ionizable or permanently charged lipids, include, but are not limited to, lipids or other lipophilic groups that confer sufficient hydrophobicity to the immunomodulatory agent, prodrug, or lipid to facilitate its formulation into a suitable delivery vehicle.

[0052] "Scaffold moiety" refers to the hydrocarbon chain of a lipid conjugate, a prodrug, or a lipophilic portion of an ionizable or permanently charged lipid, which is linked via its one or more hydrocarbon chains, or one or more respective biodegradable groups.

[0053] As used herein, "prodrug", "lipid prodrug", "immunomodulatory agent-lipid conjugate", "lipid conjugate", "drug-lipid conjugate" or "prodrug conjugate" refers to an immunomodulatory agent linked to a lipophilic moiety via any suitable linkage or linker, including covalent and non-covalent bonds. In some embodiments, the linkage or linker is covalently attached. The immunomodulatory agent may be activated upon release from the lipophilic moiety.

[0054] Lipid conjugates

[0055] The lipid conjugate comprises an immunomodulatory agent linked to a lipophilic moiety.

[0056] In some embodiments, the immunomodulatory agent is a tolerogenic agent that suppresses the immune response or induces tolerance to an antigen. In some embodiments, the immunomodulatory agent is an immunosuppressant. In some embodiments, the immunomodulatory agent is an immunostimulatory agent.

[0057] Immunomodulators exert their immunomodulatory effects in subjects by targeting various molecules in the upstream and downstream immune pathways. Upstream are targets that are directly modified by the drug, and downstream are important pathways by which the immunomodulator inhibits inflammation. Many of these drugs converge / overlap with the same downstream pathways. Upstream targets include, but are not limited to, glucocorticoid receptor, mammalian target of rapamycin (mTOR), COX1 / COX2 (by direct acetylation, such as by ASA, or by inhibiting expression, such as by salicylate), vitamin D receptor, JAK1, JAK2, JAK3, TYK2, and calcineurin. Downstream targets include, but are not limited to, inhibition of expression / activity of NF-kappa B complex, inhibition of expression / activity of AP-1, inhibition of p38 MAP kinase pathway, and inhibition of NFAT family.

[0058] Examples of immunomodulatory agents for inclusion in the lipid conjugate include nonsteroidal anti-inflammatory drugs (NSAIDs), inflammasome inhibitors, Janus kinase (JAK) inhibitors, corticosteroids, mTOR inhibitors, DMARDs (disease-modifying antirheumatic drugs), calcineurin inhibitors, and / or vitamin D receptor agonists. In some embodiments, each immunomodulatory agent is independently selected from an NSAID, an inflammasome inhibitor, a JAK inhibitor, a corticosteroid, an mTOR inhibitor, a DMARD, a calcineurin inhibitor, or a vitamin D receptor agonist.In some embodiments, each immunomodulatory agent is selected from the group consisting of dexamethasone, calcitriol, acetylsalicylic acid, salicylate, mycophenolic acid, sirolimus, tacrolimus, cholecalciferol, calcifediol, alfacalcidol, calcipotriol, falecalcitriol, maxacalcitol, paricalcitol, doxercalciferol, 22-oxacalcitriol, tacalcitol, eldecalcitol, elocalcitol, inecalcitol, becocalcidiol, seocalcitol, ergocalcitrate ... Luciferol, lexacalcitol, retinoic acid, cyclophosphamide (nitrogen mustard), filgotinib, baricitinib, tofacitinib, ruxolitinib, upadacitinib, oclacitinib, peficitinib, fedratinib, delgocitinib, deuclavacitinib, abrocitinib, auranofin, apremilast, azathioprine, chloroquine, hydroxychloroquine, cyclosporine, leflunomide, methotrexate, minocycline, sulfasalazine, salicylic acid, diflunisal, monkey Salate, naproxen, ibuprofen, oxaprozin, loxoprofen, zaltoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, bromfenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, phenylbutazone, celecoxib, firocoxib, parecoxib, etoricoxib, clonixin, licofelone, MCC950, glyburide, CY-09, tranilast, oridoni , BOT-4-one (2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one), INF39 (ethyl 2-(2-chlorobenzyl)acrylate), MNS (3,4-methylenedioxy-β-nitrostyrene), fenamic acid, beta-hydroxybutyric acid, quercetin, JC-171, ibrutinib, OLT1177, FC11A-2, INF58, JC124, or ethyl 2-((2-chlorophenyl)(hydroxy)methyl)acrylate.

[0059] In one embodiment, the immunomodulatory agent is selected from dexamethasone, calcitriol, acetylsalicylic acid, mycophenolic acid, sirolimus, and / or tacrolimus.

[0060] In another embodiment, the immunomodulatory agent is a JAK inhibitor, a DMARD (disease-modifying antirheumatic drug), an NSAID, an inflammasome inhibitor, and / or a vitamin D receptor agonist.

[0061] For example, the JAK inhibitor may be selected from filgotinib, baricitinib, tofacitinib, ruxolitinib, upadacitinib, oclacitinib, peficitinib, fedratinib, delgocitinib, deuclavacitinib and / or abrocitinib.

[0062] The DMARD may be selected from auranofin, apremilast, azathioprine, chloroquine, hydroxychloroquine, cyclosporine, leflunomide, methotrexate, minocycline and / or sulfasalazine.

[0063] Examples of NSAIDs that can be incorporated into the lipid conjugate include salicylic acid, diflunisal, salsalate, naproxen, ibuprofen, oxaprozin, loxoprofen, zaltoprofen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, bromfenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, phenylbutazone, celecoxib, firocoxib, parecoxib, etoricoxib, clonixin, and / or licofelone.

[0064] Inflammasome inhibitors for incorporation into lipid conjugates include MCC950, glyburide, CY-09, tranilast, oridonin, BOT-4-one (2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one), INF39 (ethyl 2-(2-chlorobenzyl)acrylate), MNS (3,4-methylenedioxy-β-nitrostyrene), fenamic acid, beta-hydroxybutyric acid, quercetin, JC-171, ibrutinib, OLT1177, FC11A-2, INF58, JC124, and / or ethyl 2-((2-chlorophenyl)(hydroxy)methyl)acrylate.

[0065] Examples of vitamin D receptor agonists include calcitriol, cholecalciferol, calcifediol, alphacalcidol, calcipotriol, falecalcitriol, maxacalcitol, paricalcitol, doxercalciferol, 22-oxacalcitriol, tacalcitol, eldecalcitol, erocalcitol, inecalcitol, becocalcidiol, seocalcitol, ergocalciferol, and / or lexacalcitol.

[0066] The lipophilic portion of the lipid conjugate confers sufficient hydrophobicity to the immunomodulatory agent to facilitate its formulation in a suitable delivery vehicle. Examples of suitable lipid moieties include those described in co-owned and co-pending WO2020 / 191477 (PCT / CA2020 / 000039; incorporated herein by reference).

[0067] Two or more lipid conjugates comprising an immunomodulatory agent may be formulated in the same or separate delivery vehicles. In such embodiments, two or more immunomodulatory agents may be formulated such that the two (or more) agents are stably retained in the same delivery vehicle in a molar ratio that is additive or synergistic. For example, two, three, four, or more lipid conjugates (e.g., two, three, four, or more prodrugs) may be formulated together or in separate delivery vehicles. In some embodiments, the immunomodulatory combination comprises a plurality of lipid conjugates, the plurality of lipid conjugates comprising three, four, five, six, seven, eight, nine, ten, or more than ten immunomodulatory agents, each immunomodulatory agent of the plurality of lipid conjugates being different, and each lipid conjugate of the plurality of lipid conjugates being independently formulated in a separate delivery vehicle from the other components of the immunomodulatory combination or being co-formulated with one or more of the other components of the immunomodulatory combination. The additive or synergistic effect between two or more immunomodulatory agents can be determined by any suitable technique, including the Chou Talalay method known to those skilled in the art. In some embodiments, two lipid conjugates (e.g., two prodrugs) are co-formulated into the same delivery vehicle. The lipid conjugates herein are particularly suitable for formulation into delivery vehicles with high encapsulation efficiency, for example, up to 90% or more encapsulation efficiency.

[0068] In some embodiments, the lipid conjugate is a first lipid conjugate, and the immunomodulatory combination further comprises a second lipid conjugate, the second lipid conjugate comprising an immunomodulatory agent covalently linked to a lipophilic moiety by a cleavable linkage or via a cleavable linker, the immunomodulatory agent of the second lipid conjugate being different from the immunomodulatory agent of the first lipid conjugate, and the first lipid conjugate and the second lipid conjugate being formulated in separate delivery vehicles or co-formulated in the same delivery vehicle. In some embodiments, the immunomodulatory agent of the second lipid conjugate targets a different immune pathway than the immunomodulatory agent of the first lipid conjugate. In some embodiments, the first lipid conjugate and the second lipid conjugate are co-formulated in the same delivery vehicle.

[0069] In still further embodiments, the immunomodulatory combination further comprises a third lipid conjugate. In some embodiments, the immunomodulatory agent of the third lipid conjugate targets a different immune pathway than the immunomodulatory agent of the first and / or second lipid conjugate. In still further embodiments, the immunomodulatory combination further comprises more than four lipid conjugates.

[0070] Immunomodulatory agents may be covalently or non-covalently linked to a lipid moiety, such as a fatty acid, glyceride, phospholipid, or other hydrophobic moiety, including those produced by organic synthesis. Linking a lipophilic moiety to the immunomodulatory agent typically increases the hydrophobicity of the immunomodulatory agent.

[0071] The LogP of the lipid conjugate may be sufficient to impart a desired degree of hydrophobicity to the immunomodulatory agent, hi one embodiment, the predicted LogP of the lipid conjugate is between 5-30, between 6-28, or between 7-25.

[0072] In some embodiments, the immunomodulatory agent is covalently linked to the lipophilic moiety, hi some embodiments, the immunomodulatory agent is covalently linked to the lipophilic moiety by a cleavable linkage or via a cleavable linker.

[0073] The immunomodulatory agent may be bioactive when linked to the lipophilic moiety or may be bioactive upon cleavage from the lipophilic moiety following administration to a subject. In this regard, the lipid conjugate may include one or more biodegradable groups that are cleavable upon administration of the lipid conjugate to a subject.

[0074] The biodegradable groups may be independently selected from linkages that include one or more functional groups selected from esters, amides, amidines, hydrazones, disulfides, ethers, carbonates, carbamates, thionocarbamates, guanidines, guanines, oximes, isoureas, acylsulfonamides, phosphoramides, phosphonamides, phosphoramidates, phosphates, phosphonates, phosphodiesters, phosphate phosphonooxymethyl ethers, N-Mannich adducts, N-acyloxyalkylamines, sulfonamides, imines, azos; carbon-based functional groups including alkanes, alkenes, or alkynes; methylene (CH2), or ureas.

[0075] In one embodiment, the lipophilic moiety may be derived from a precursor fatty acid or other lipophilic molecule having, for example, from 5 to 30 carbon atoms, from 14 to 20 carbon atoms, or from 16 to 18 carbon atoms.

[0076] In another embodiment, the lipophilic moiety is a linear or branched lipophilic chain having up to 3, 4, 5, or 6 biodegradable groups. In one embodiment, at least one of the biodegradable groups is selected from at least one of esters, amides, amidines, hydrazones, disulfides, ethers, carbonates, carbamates, thionocarbamates, and combinations thereof. In one embodiment, the biodegradable group is an ester that is cleavable by esterases in vivo.

[0077] In certain embodiments, the lipid conjugate is formulated into a delivery vehicle that comprises a linear or branched lipophilic moiety conjugated to an immunomodulatory agent, the lipophilic moiety having the structure of Formula I: [ka] (In the formula, L is represented by L1+L2+L3+L4+L5+L6, and L contains 2 to 100, 2 to 75, 2 to 80, 3 to 60, 4 to 50, 5 to 45, or 5 to 40 carbon atoms and 0 to 6 cis or trans C═C double bonds; L1 is a carbon chain having 0-40, 1-40, 1-35, or 3-30 carbon atoms, optionally L1 having one or more cis or trans C=C double bonds or 0-2 cis or trans C=C double bonds; L2 and L4 are carbon atoms; L3 is 0 to 20 carbon atoms and contains 0 to 2 cis or trans C=C double bonds; L5 is 0-20 carbon atoms and contains 0-2 cis or trans C=C double bonds; L6 is -CH3, ═CH2 or H; each R is independently a straight or branched hydrocarbon chain having 0-30 carbon atoms and 0-3 cis or trans C=C double bonds, optionally 0-2 cis or trans C=C double bonds, where if one or more of the R's are branched, each branch point optionally contains an X2 functional group or is a carbon atom; n is 0 to 8, p is 0 to 8, and n+p is 0 or ≧1 or 1 to 8, 2 to 6, or 2 to 4; When present, each X2 is independently an ester, amide, amidine, hydrazone, ether, carbonate, carbamate, thionocarbamate, guanidine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, phosphoramidate, phosphate, phosphonate, phosphodiester, phosphate phosphonooxymethyl ether, N-Mannich adduct, N-acyloxyalkylamine, sulfonamide, imine, azo; a carbon-based functional group including an alkane, alkene, or alkyne; methylene (CH2) or urea; Alternatively, X2 is a linkage that includes at least one hydrogen bond.

[0078] In one embodiment, the lipid conjugate comprises a scaffold moiety, which in one embodiment is represented by L of formula I above, where at least one R is present as a hydrocarbon side chain and n+p is 1 or 1-8 or 1-7 or 1-6 or 1-5 or 1-4 or 1-3.

[0079] In some embodiments, the lipid conjugate optionally comprises one lipophilic moiety having the structure of formula I. In some embodiments, the lipid conjugate comprises two lipophilic moieties, each of which independently has the structure of formula I, which may be the same or different, and each lipophilic moiety is linked to the immunomodulatory agent via a separate linkage or linker or to the same linkage or linker. In certain embodiments, each linkage is an ester. In some embodiments, the lipid conjugate comprises more than two lipophilic moieties.

[0080] In some embodiments, each lipophilic moiety of formula I is independently defined by: L1 is a carbon chain having 3-30 carbon atoms and 0-3 cis or trans C=C double bonds; n is 0, L3 does not exist, p is 1, X2 is a carbonate or an ester (optionally [ka] ), R is a linear or branched carbon chain having 1 to 20 carbon atoms and 0 to 3 cis or trans C=C double bonds; L5 is a carbon chain having 1 to 10 carbon atoms and containing 0 to 1 cis or trans C=C double bonds; L6 is -CH3; Or L is a carbon chain having 5 to 20 carbon atoms and 0 C=C double bonds.

[0081] In some embodiments, each lipophilic moiety of formula I is independently defined by: L1 is a carbon chain having 5 to 20 carbon atoms (optionally 8 to 15 carbon atoms) and 1 or 2 cis or trans C=C double bonds (optionally 1 cis or trans C=C double bond); optionally, L1 is -C 6-9 -C=CC-, n is 0, L3 does not exist, p is 1, X2 is a carbonate or an ester (optionally [ka] ), R is a straight or branched carbon chain having 1 to 20 carbon atoms and 0 to 2 cis or trans C=C double bonds (optionally R is -C 1-6 Or C 5-8 -C=CCC=CC 4-6 ) and L5 is a carbon chain having 1 to 10 carbon atoms and containing 0 to 1 cis or trans C=C double bonds (optionally 0 C=C double bonds); L6 is -CH3; Or L is a carbon chain having 5 to 15 carbon atoms and 0 C=C double bonds.

[0082] L1 can be linked to the immunomodulatory agent via an X1 linkage by a covalent linkage or via hydrogen bonding.

[0083] In some embodiments, the X1 linkage is biodegradable, which means that it can be cleaved after administration to a subject.Without being limited thereto, the ester bond can be hydrolyzed by esterase after administration to a patient, thereby releasing the immunomodulator from the lipophilic moiety.However, other X1 linkages can be utilized for tailored drug release based on their release properties when exposed to the environment at the disease site.

[0084] In some embodiments, X1 may be cleavable by esterases, alkaline phosphatases, amidases, peptidases, or upon exposure to a reducing environment and / or high or low pH.

[0085] The chemical linkage of X1 in certain embodiments is most advantageously a linker. A wide variety of chemical linkers are known to those skilled in the art and may be used in certain embodiments described herein. The linker may have 0-12 carbon atoms and at least one cleavable functional group. In one embodiment, the linker has at least two functional groups, a first functional group for conjugating one end of the linker to an immunomodulatory agent and a second functional group for conjugating the other end of the linker to a carbon atom on L of formula I. The two functional groups may each be independently selected from ester, amide, amidine, hydrazone, ether, carbonate, carbamate, thionocarbamate, guanidine, guanine, oxime, isourea, acylsulfonamide, phosphoramide, phosphonamide, phosphoramidate, phosphate, phosphonate, phosphodiester, phosphate phosphonooxymethyl ether, N-Mannich adduct, N-acyloxyalkylamine, sulfonamide, imine, azo; a carbon-based functional group such as an alkane, alkene, or alkyne; methylene (CH2) or urea.

[0086] The linker may provide enhanced release of the immunomodulator by the introduction of a biodegradable group. A linker having one or more ester bonds may be hydrolyzed by esterases after administration to a patient, thereby releasing the immunomodulator from the lipid conjugate. Similar to the linkage resulting from a direct reaction between the immunomodulator and L, a linker that introduces a hydrazone bond between the immunomodulator and a lipophilic moiety may confer pH-sensitive release of the immunomodulator from the lipid conjugate.

[0087] However, it will be understood that the foregoing is merely illustrative. Additional examples of linkers are provided in U.S. Patent No. 5,149,794, which is incorporated herein by reference. Non-limiting examples of linkers described in U.S. Patent No. 5,149,794 include aminohexanoic acid, polyglycine, polyamide, polyethylene, and short functionalized polymers having a carbon backbone that is 1 to 12 carbon atoms in length.

[0088] Still further examples of linkers suitable for use in the lipid conjugates described herein are provided in the following references: JPEG2024533322000005.jpg112161

[0089] Each of the above references is incorporated herein by reference in its entirety. In further embodiments, the chemical linkage of X1 includes both a functional group and a separate linker. Various combinations of linkers and functional groups can be incorporated into the lipid conjugate.

[0090] In one embodiment, at least a second functional group conjugating one end of the linker to L1 is an ester or amide linkage. In another embodiment, the functional group of the linker can be hydrolyzed by an enzyme, such as an esterase. In a further embodiment, both functional groups of the linker are ester linkages.

[0091] Some exemplary lipid conjugates are shown in Figures 10A-10M. The preparation of these and other lipid conjugates for use in the immunomodulatory combinations disclosed herein is known in the art and has been previously described. See, for example, WO / 2020 / 191477, which is incorporated herein by reference in its entirety. The synthesis procedures for some lipid conjugates are described in the examples.

[0092] Briefly, an immunomodulatory agent can be attached to a lipid moiety by conjugation to a reactive group or linker group of the scaffold L to form a chemical linkage X1. In one embodiment, an immunomodulatory agent loses a hydroxyl group or a hydrogen atom upon conjugation to a lipophilic moiety (e.g., Formula I) or linker to form a lipid conjugate. An immunomodulatory agent can be derived from a chemical structure that includes one or more reactive functional groups, such as -(C=O)O, -OH, -NH2, -NHR, -PO3H2, among others known to those of skill in the art, and is not limited by the orientation of the atoms.

[0093] For example, a lipid conjugate can be formed (directly or via one or more intermediates) by conjugation between a (C=O)OH group of an immunomodulatory agent and a hydroxyl group of a precursor scaffold P. The general reaction is shown below for a molecule of interest (e.g., an immunomodulatory agent): [ka]

[0094] In the above exemplary embodiment, the chemical linkage of X is an ester and has the following structure: [ka] .

[0095] In another illustrative example, the immunomodulatory agent may have a hydroxyl group (-OH) that reacts with a carboxyl group ((C=O)OH) of the linker. A second carboxyl group ((C=O)OH) of the linker may react with a hydroxyl group on a carbon atom of the precursor scaffold P via a condensation reaction. The following reaction shows the use of succinic acid as a linker. The use of such a linker results in a lipid conjugate with two ester groups by the following reaction: [ka]

[0096] In the above non-limiting examples, the chemical linkage of X1 has the following structure: [ka] .

[0097] It should be appreciated that the above reaction may proceed in two steps, i.e., the immunomodulatory agent may first be conjugated to the linker, and the resulting drug-linker conjugate may then be reacted with the precursor scaffold P to generate the lipid-conjugate (prodrug) reaction product.

[0098] The foregoing are provided for illustrative purposes only as a variety of different linkers, other than that succinic acid can be used to generate lipid conjugates.

[0099] In another example, the immunomodulatory agent or linker may have a carboxyl group ((C=O)O) for conjugation with an amine group of L to form an amide or amide-containing linkage X1 between the immunomodulatory agent and L. As discussed below, other reactions between functional groups of the drug or linker and scaffold L to generate the chemical linkage of X1 can be envisioned by one of skill in the art.

[0100] Certain immunomodulatory agents may contain more than one reactive functional group for linkage to the precursor scaffold P. In such embodiments, as can be appreciated by one of skill in the art, protecting groups can be used during synthesis of the drug-lipid conjugate to selectively conjugate certain groups of the drug to the scaffold L while leaving other groups unconjugated.

[0101] Antigen or nucleic acid(s) encoding the antigen

[0102] In some embodiments, the antigen is a protein, a polypeptide, a peptide, a lipoprotein, a glycolipid, a polynucleotide, or a polysaccharide. In certain embodiments, the antigen is a protein, a polypeptide, or a peptide. Antigens may be formulated in the delivery vehicles herein in the same forms as those known to elicit an unwanted immune response, including, but not limited to, fragments or derivatives thereof. Antigens may originate from within the subject's body, referred to as auto or "self," or from the external environment, referred to as foreign or "non-self."

[0103] Antigens include, but are not limited to, allergens, superantigens, tolerogens, T-dependent antigens, T-independent antigens, or immunodominant antigens. In another embodiment, antigens are characterized by their source and include exogenous antigens, endogenous antigens, autoantigens, or neoantigens.

[0104] An antigen can have a single epitope or can include more than one epitope (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 100, or more than 100 epitopes).

[0105] In some embodiments, antigens are presented on antigen presenting cells (APCs) and can activate T cells of the immune system using the delivery vehicles described herein.

[0106] In some embodiments, the antigen is associated with allergic reactions, autoimmune diseases, organ or tissue rejection, graft-versus-host disease, anti-drug antibodies, or gene / protein replacement therapy.One or more of these disorders may also be referred to as inflammatory disorders.When the antigen is associated with inflammatory reactions, the antigen may include, but is not limited to, non-self antigens that are allergens as described, or self or non-self antigens that induce unwanted immune responses.

[0107] Where an antigen is associated with an allergic reaction, the antigen may include, but is not limited to, non-self antigens (also referred to as allergens) derived from animal sources including animal substances or foods from terrestrial or aquatic animals, plant sources such as plant pollen or gluten, drugs, foods, insect stings, fungal sources such as mold spores, metals, latex, etc.

[0108] Non-limiting examples of allergies include allergic asthma, hay fever, urticaria, eczema, plant allergies, insect bite allergies, pet allergies, latex allergies, mold allergies, cosmetic allergies, food allergies, allergic rhinitis or coryza, topical allergic reactions, anaphylaxis, atopic dermatitis, hypersensitivity reactions and other allergic conditions. Non-limiting examples of food allergies include milk allergies, egg allergies, peanut / regum allergies, tree nut allergies, fish allergies, shellfish allergies, soy allergies and gluten allergies.

[0109] Examples of inflammatory diseases include Alzheimer's disease, arthritis, asthma, atherosclerosis, Crohn's disease, colitis, cystic fibrosis, dermatitis, diverticulitis, hepatitis, irritable bowel syndrome (IBS), lupus erythematosus, muscular dystrophy, nephritis, e.g. glomerulonephritis Parkinson's disease, shingles and ulcerative colitis, cardiovascular disease, idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease (COPD), bronchiectasis, chronic cholecystitis, tuberculosis, sepsis, sarcoidosis, silicosis and other pneumoconiosis diseases, uveitis, orchitis, oophoritis, pancreatitis, gastritis, rheumatic fever.

[0110] Autoimmune diseases include, but are not limited to, rheumatoid arthritis, multiple sclerosis, myelin oligodendrocyte glycoprotein antibody disorder, primary biliary cholangitis, immune-mediated or type I diabetes mellitus, systemic lupus erythematosus, psoriasis, scleroderma, autoimmune thyroid diseases such as Hashimoto's thyroiditis and primary myxedema, alopecia areata, Graves' disease, Guillain-Barre syndrome, celiac disease, Sjogren's syndrome, autoimmune atrophic gastritis, autoimmune hepatitis, autoimmune pancreatitis, phacogenic uveitis, neuromyelitis optica, myasthenia gravis, pernicious anemia, autoimmune hemolytic anemia, Addison's disease, scleroderma, Goodpasture's syndrome, anti-glomerular basement membrane disease, psoriasis, pemphigus vulgaris, pemphigoid, sympathetic ophthalmia, thrombocytopenic purpura, autoimmune neutropenia, vitiligo, autoimmune vasculitis, and dermatomyositis.

[0111] Antigens may also be used to treat, prevent and / or ameliorate symptoms associated with organ or tissue rejection. Such antigens include those derived from allogeneic cells, e.g., antigens derived from extracts of allogeneic cells, and antigens derived from other cells, such as endothelial cell antigens.

[0112] In further embodiments, antigens can be used to treat, prevent and / or ameliorate undesirable symptoms associated with transplantable grafts. Such antigens are associated with undesirable immune responses in recipients of transplantable grafts. In some embodiments, transplantation antigens include those associated with organ or tissue rejection or graft-versus-host disease. Such antigens can also be obtained or derived from cells of biological material or from information about the transplantable graft. Transplantation antigens generally include those contained or expressed in cells. Information about the transplantable graft can include, but is not limited to, sequence information, antigen type or class and / or their MHC class I, MHC class II, or B cell presentation constraints. In other embodiments, information used to generate antigens includes the type of transplantable graft (e.g., autograft, allograft, xenograft), the molecular and cellular composition of the graft, the body location from which the graft originates or to which the graft is implanted (e.g., whole or partial organs, skin, bone, nerves, tendons, neurons, blood vessels, fat, cornea, etc.).

[0113] According to certain embodiments, the antigen is encoded by a nucleic acid capable of directly or indirectly expressing an antigenic protein, peptide, polypeptide or fragment thereof. The antigen may be encoded by a nucleic acid sequence, including but not limited to DNA or RNA and hybrids thereof. DNA includes any vector capable of expressing the antigen. RNA may in turn encode an mRNA or a self-amplifying RNA that encodes the antigenic protein, peptide, polypeptide or fragment thereof.

[0114] The DNA encoding the antigen is typically circular (e.g., a plasmid, minicircle, or nanoplasmid), although linear DNA is also contemplated in certain embodiments herein. In some embodiments, the DNA encoding the antigen is autonomously replicating. Autonomously replicating DNA has an origin of replication or an autonomously replicating sequence (ARS) that is functional in the host cell. In another embodiment, the DNA encoding the antigen may be replicated by being inserted into the genome of the host cell of interest using known techniques.

[0115] The DNA encoding the antigen may encode regulatory regions such as promoter sequences and termination regions. The DNA encoding the antigen may be cloned into a suitable microorganism (e.g., E. coli) and then formulated into a delivery vehicle disclosed herein for in vitro or in vivo expression. The DNA sequence may include a reporter gene sequence, although including the reporter gene sequence in the formulation for administration is optional. Such sequences may be incorporated into the DNA for studies in animal models.

[0116] The DNA encoding the antigen may be single-stranded, double-stranded, or in some embodiments, is a DNA-RNA hybrid. Single-stranded nucleic acids include antisense oligonucleotides (complementary to DNA and RNA), ribozymes, and triplex-forming oligonucleotides. To have long-term activity, the single-stranded nucleic acid in some embodiments may have some or all of the nucleotide linkages replaced with stable non-phosphodiester linkages.

[0117] The DNA encoding the antigen may include nucleic acids with modifications at one or more sugar moieties and / or at one or more of the pyrimidine or purine bases. In further embodiments, the DNA vector may be modified with a peptide, protein, steroid or sugar moiety. Such modifications may facilitate delivery to the desired target site.

[0118] The nucleic acids used in the immunomodulatory combinations can be isolated from natural sources, obtained from sources such as ATCC or GenBank libraries, or prepared by synthetic methods. Synthetic nucleic acids can be prepared by a variety of solution or solid-phase methods. In general, solid-phase synthesis is preferred. Detailed descriptions of procedures for solid-phase synthesis of nucleic acids by phosphite-triester, phosphotriester, and H-phosphonate chemistry are widely available.

[0119] In one embodiment, the DNA vector is double stranded DNA and comprises more than 700 base pairs, more than 800 base pairs, or more than 900 base pairs, or more than 1000 base pairs.

[0120] As discussed above, the nucleic acid may be an RNA that encodes an antigen, such as an mRNA or a self-amplifying RNA. The RNA may be purified from a natural source, produced using a recombinant expression system, optionally purified, or chemically synthesized. In certain embodiments, the RNA that encodes an antigen includes both modified and unmodified RNA.

[0121] In those embodiments in which the RNA is chemically synthesized, the RNA may include nucleoside analogs, such as analogs with chemically modified bases or sugars, and / or backbone modifications. In some embodiments, the RNA may include natural nucleosides (e.g., adenosine, guanosine, cytidine, uridine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyladenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, the bases being or containing: 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanine, 2-thiocytidine, pseudouridine, 5-methoxyuridine, and 5-methylcytidine; chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0122] The RNA encoding the antigen can be synthesized according to any of a variety of known methods. For example, in certain embodiments, the RNA can be synthesized via in vitro transcription (IVT). Briefly, IVT is typically performed using a linear or circular DNA template that includes a promoter, a pool of ribonucleotide triphosphates, a buffer system that may include DTT and magnesium ions, and an appropriate RNA polymerase (e.g., T3, T7 or SP6 RNA polymerase), DNAse I, pyrophosphatase, and / or RNAse inhibitor.

[0123] In some embodiments, the in vitro synthesized RNA encoding an antigen can be purified prior to formulation and encapsulation to remove unwanted impurities, including various enzymes and other reagents used during RNA synthesis.

[0124] In one embodiment, the RNA comprises one or more coding and non-coding regions.

[0125] RNA can be of various lengths. In some embodiments, the present disclosure can be used to formulate in vitro synthesized RNA ranging in length from about 0.1-20 kb, 1-20 kb, about 1-15 kb, about 1-10 kb, about 5-20 kb, about 5-15 kb, about 5-12 kb, about 5-10 kb, about 8-20 kb, or about 8-15 kb. Some antigens can be as small as 8-12 amino acids in length.

[0126] Typically, RNA synthesis involves adding a "cap" to the 5' end and a "tail" to the 3' end. The presence of the cap is important in providing resistance to nucleases found in most eukaryotic cells. The presence of the "tail" helps protect the RNA from exonuclease degradation.

[0127] In some embodiments, the RNA encoding the antigen comprises a 5' and / or a 3' untranslated region. In some embodiments, the 5' untranslated region comprises one or more elements that affect the stability or translation of the RNA, such as an iron-responsive element. In some embodiments, the 5' untranslated region can be between about 50-500 nucleotides in length.

[0128] In some embodiments, the 3' untranslated region comprises one or more of a polyadenylation signal, a binding site for a protein that affects the stability of the RNA at a location in the cell, or one or more binding sites for the RNA, hi some embodiments, the 3' untranslated region can be between about 50 and 500 or more nucleotides in length.

[0129] In certain embodiments, RNA provided from an in vitro transcription reaction may be desirable, although other sources of RNA are contemplated, such as RNA produced from bacteria, fungi, plants, and / or animals.

[0130] The RNA sequence may include a reporter gene sequence, although including a reporter gene sequence in the pharmaceutical formulation for administration is optional. Such sequences can be incorporated into RNA for in vivo studies in animal models to assess biodistribution.

[0131] In some embodiments, the immunomodulatory combination comprises a single antigen or one or more nucleic acids encoding a single antigen. In other embodiments, the immunomodulatory combination comprises a single antigen or one or more nucleic acids encoding more than one antigen. The immunomodulatory combination may comprise a combination of antigen(s) and nucleic acid(s) encoding the antigen(s). In certain embodiments, the more than one antigen comprises two antigens. In certain embodiments, the more than one antigen comprises more than two antigens.

[0132] In certain embodiments, the immunomodulatory combination comprises an antigen or one or more nucleic acids encoding the antigen. In some of these embodiments, the antigen is a first antigen, and the immunomodulatory combination further comprises a second antigen or one or more nucleic acids encoding the second antigen, where the first antigen is different from the second antigen, and the second antigen or one or more nucleic acids encoding the second antigen are formulated in a delivery vehicle separate from the other components of the immunomodulatory combination or are co-formulated with one or more of the other components of the immunomodulatory combination. In certain embodiments, the one or more nucleic acids encoding the first antigen and the one or more nucleic acids encoding the second antigen are included in a single nucleic acid, and the first antigen and the second antigen are co-formulated in the same delivery vehicle.

[0133] In certain embodiments, the immunomodulatory combination comprises multiple antigens, or one or more nucleic acids encoding multiple antigens, or a combination of antigens and nucleic acid(s) encoding the antigens to provide multiple antigens. Each antigen or nucleic acid encoding an antigen is formulated independently in a delivery vehicle separate from the other components of the immunomodulatory combination, or is co-formulated with one or more of the other components of the immunomodulatory combination. Each antigen may comprise one or more epitopes. In alternative embodiments, the multiple antigens comprise 3, 4, 5, 6, 7, 8, 9, 10, or more than 10 antigens.

[0134] Delivery Vehicle

[0135] A variety of delivery vehicles can be used to prepare the immunomodulatory combinations (alternatively referred to as "vaccine formulations"), including, but not limited to, nanoparticles, including lipid nanoparticles (LNPs), liposomes, lipid-containing polymeric nanoparticles, polymer-based nanoparticles, emulsions, and micelles.

[0136] The lipid conjugates of the present disclosure are particularly suitable for incorporation into nanoparticles that contain lipids or other hydrophobic components (e.g., lipid nanoparticles, liposomes, or polymer-based systems). The lipid-like properties of the lipid conjugates in certain embodiments may facilitate their loading into these or other delivery vehicles. For example, in some embodiments, the loading efficiency into a given nanoparticle is 75%-100%, 80%-100%, or most advantageously 90%-100%. In some embodiments, the delivery vehicle(s) is a liposome and / or a lipid nanoparticle.

[0137] In one embodiment, the lipid conjugate and the antigen or the nucleic acid encoding same are loaded into lipid nanoparticles or liposomes by mixing with lipid formulation components including vesicle-forming lipids and optionally sterols. As a result, lipid nanoparticles and / or liposomes incorporating cargo can be prepared using a wide variety of well-described formulation methodologies known to those skilled in the art, including but not limited to extrusion, ethanol injection, and in-line mixing. Such methods are described in Maclachlan, I. and P. Cullis, "Diffusible-PEG-lipid Stabilized Plasmid Lipid Particles", Adv. Genet., 2005.53PA:157-188; Jeffs, LB et al., "A Scalable, Extrusion-free Method for Efficient Liposomal Encapsulation of Plasmid DNA", Pharm Res, 2005.22(3):362-72; and Leung, AK et al., "Lipid Nanoparticles Containing siRNA Synthesized by Microfluidic Mixing Exhibit an Electron-Dense Nanostructured Core", The Journal of Physical Chemistry.C, Nanomaterials and Interfaces, 2012, 116(34):18440-18450, each of which is incorporated herein by reference in its entirety.

[0138] Liposomes contain an internal aqueous solution surrounded by a phospholipid bilayer, whereas lipid nanoparticles may alternatively contain a lipophilic core. Such lipophilic cores can serve as reservoirs for lipid conjugates (e.g., prodrugs) and / or antigens or nucleic acids encoding same. Solid and liquid lipid nanoparticles can be used for delivery of lipid conjugate(s) and / or antigen(s) or nucleic acid(s) encoding same as described herein.

[0139] In one embodiment, a liposome or lipid nanoparticle is provided that comprises a phospholipid bilayer, and the lipid conjugate forms a hydrophobic oil phase within the bilayer. Such a delivery vehicle is described in WO2020 / 191477 (PCT / CA2020 / 000039), which is incorporated herein by reference. In another embodiment, the delivery vehicle is a liposome.

[0140] In some embodiments, at least one delivery vehicle is a lipid nanoparticle, and optionally at least one lipid conjugate is incorporated into the lipid compartment of the lipid nanoparticle. In some embodiments, at least one delivery vehicle is a liposome, and optionally at least one lipid conjugate is incorporated into the oil phase of the lipid bilayer of the liposome. In certain embodiments, at least one antigen or one or more nucleic acids encoding at least one antigen are entrapped in the lipid nanoparticle or liposome and have a net charge opposite to the net charge of the lipid in the lipid nanoparticle. In certain embodiments, at least one antigen is lipophilic and is entrapped in the lipid compartment of the lipid nanoparticle or liposome. In certain embodiments, at least one antigen is hydrophilic and is entrapped in a liposome that comprises an aqueous core.

[0141] The delivery vehicle can also be a nanoparticle (e.g., liposome or LNP) that contains a lipid core stabilized by a surfactant. Vesicle-forming lipids can be utilized as stabilizers. In another embodiment, the delivery vehicle is a polymer-lipid hybrid system that contains a polymer nanoparticle core surrounded by stabilizing lipids.

[0142] Alternatively, nanoparticles may be prepared from lipid-free polymers. Such nanoparticles may contain a concentrated core of drug surrounded by a polymer shell, or may have a solid or liquid dispersed throughout the polymer matrix.

[0143] The lipid conjugates and / or antigens or nucleic acids encoding the same described herein can also be incorporated into emulsions, which are drug delivery vehicles that contain oil droplets or oil cores.The emulsions can be lipid-stabilized.For example, the emulsions can contain an oil-filled core stabilized by emulsifying components such as lipid monolayers or bilayers.

[0144] Micelles are self-assembling particles composed of amphiphilic lipid or polymer components that are utilized for the delivery of drugs present in the hydrophobic core. Drug loading into micelles can be improved by conjugating the drug to a scaffold molecule L as described herein and to a hydrophobic group R.

[0145] A further class of drug delivery vehicles known to those of skill in the art that can be used to encapsulate the lipid conjugates herein are carbon nanotubes.

[0146] A variety of methods for preparing the aforementioned delivery vehicles and incorporating lipid-conjugated immunomodulatory agents therein are available and may be readily performed by one of skill in the art.

[0147] Certain lipid conjugates encompassed by the present disclosure may form part of a carrier-free system. In such embodiments, the lipid conjugates may self-assemble into particles. Without being limited thereto, when the immunomodulatory agent is hydrophilic, the amphiphilic prodrug may be assembled into nanoparticles with or without the use of a stabilizer.

[0148] LNPs can be made using a variety of well-described formulation methodologies, including high-pressure extrusion, ethanol injection, microfluidic mixing, and in-line mixing.

[0149] In some embodiments, the polydispersity index (PdI) of the drug delivery vehicle comprising the lipid conjugate (prodrug) and / or the antigen or a nucleic acid encoding same is less than 0.40, 0.35, 0.30, 0.25, 0.20, or 0.15.

[0150] The lipid conjugates described herein are particularly suitable for high encapsulation efficiency in drug delivery vehicles. In one embodiment, the encapsulation efficiency of the lipid conjugate (prodrug) is 10-99%, 15-99%, 20-99%, or 25-99%.

[0151] A delivery vehicle containing the lipid conjugate(s) may be co-formulated with the antigen(s) or nucleic acid(s) encoding same, or the lipid conjugate(s) and the antigen(s) or nucleic acid(s) may be formulated in separate delivery vehicles. In some embodiments, the lipid conjugate(s) is co-formulated in the same delivery vehicle as the antigen(s) or one or more nucleic acids encoding the antigen(s).

[0152] The antigen or the nucleic acid encoding it can be formulated into a delivery vehicle such as lipid nanoparticles that contain ionizable or permanently charged lipids. When an antigen that is a peptide, polypeptide or protein is encapsulated in lipid nanoparticles, the ionizable or permanently charged lipid can be cationic or anionic, depending on the charge of the antigen at physiological pH and temperature. The charged lipid can include a lipophilic moiety that includes a scaffold and one or more hydrocarbon side chains linked thereto by a biodegradable group, as described, for example, in co-owned WO2021 / 026647; Application No. PCT / CA2020 / 051098, which are incorporated herein by reference.

[0153] Charged antigens (e.g., proteins, peptides and / or nucleic acids) can be trapped in liposomes or LNPs using ionic interactions. If the antigen is anionic, cationic lipids can be used, and vice versa. If a nucleic acid is formulated into the delivery vehicle, the ionizable or permanently charged lipids are typically positively charged at physiological pH. Similarly, if the antigen is charged, the ionizable or permanently charged lipids typically have an opposite charge to that of the antigen to facilitate its formulation in lipid nanoparticles. For example, if the antigen is a charged peptide, polypeptide or protein, the ionizable or permanently charged lipids can typically have an opposite net charge at physiological pH.

[0154] Lipophilic or hydrophobic antigens can be directly loaded into liposomes or lipid nanoparticles by adding them to the lipid mixture prior to the mixing process (e.g., extrusion, ethanol injection, in-line mixing, microfluidics). The hydrophobicity of the cargo allows for spontaneous entrapment within the lipid compartment of the nanoparticle as it is formed. This method can be applied to cargoes that are lipophilic or hydrophobic in nature.

[0155] Water-soluble or hydrophilic antigens can be passively loaded into liposomes containing an aqueous core. The antigen is added directly to the buffered aqueous solution used to form the vesicles. The liposomes are made using established methods (e.g., extrusion, ethanol injection, in-line mixing, microfluidics, etc.). This method generally results in low entrapment, but the amount of cargo can be controlled by the concentration used. Small amounts of antigen are used to induce tolerance.

[0156] Examples of formulations for liposomal vaccine systems are provided in: Schewendener 2014 Ther Adv Vaccines 2:159-182; Schmidt et al., 2016 Pharmaceutics 8:7; and Kersten 1995 Biochim Biophys Acta 1241:117-138.

[0157] In certain embodiments, the permanently charged or ionizable lipid of an LNP or liposome delivery vehicle comprises a head group and a linear or branched lipophilic moiety, for example, having the structure of Formula I above.

[0158] In one embodiment, the permanently charged or ionizable lipid comprises a scaffold moiety, which in one embodiment is represented by L(L1+L2+L3+L4+L5) of formula I above, where at least one R is present as a hydrocarbon side chain and n+p is 1 or 1-8 or 1-7 or 1-6 or 1-5 or 1-4 or 1-3.

[0159] L1 in formula I may be linked to the head group directly or via a linker. The linker may be a linear, branched or cyclic structure. Examples of suitable linker groups for ionizable or permanently charged lipids are well known in the art, as provided in WO2021 / 026647 (PCT / CA2020 / 051098), which is incorporated herein by reference.

[0160] Examples of head groups that may be linked directly or indirectly to L1 via a linker region include: (i) an ionizable cationic moiety selected from the group consisting of: [ka] (ii) a permanently charged moiety selected from the group consisting of: [ka] (iii) an ionizable anionic moiety selected from the group consisting of: [ka] or (iv) a zwitterionic moiety selected from the group consisting of: [ka] .

[0161] Selective delivery to antigen-presenting cells (APCs)

[0162] The compositions described herein can be used for delivery to APCs. By way of example, and not by way of limitation, in vivo APC uptake of the delivery vehicle can be demonstrated in pancreatic islets (e.g., where APCs interact with beta cells to take up beta cell antigens in the case of type 1 diabetes) or pancreatic lymph nodes (e.g., where APCs interact with T cells to present antigens and direct the type of T cell response that can be initiated against the antigen). In one embodiment, uptake of the delivery vehicle is selective for APCs and limited uptake of non-APC immune cells. Determining whether uptake is selective for APCs can be performed using the method of Example 1 herein. For example, delivery of the delivery vehicle can be limited to endocrine cells of pancreatic islets and non-APC immune cells (e.g., T cells) of lymph nodes.

[0163] For example, the total population of delivery vehicles, such as lipid nanoparticles in the formulations described herein, that are delivered to non-APCs may be less than 20%, less than 15%, or less than 10%, as measured using the techniques of Example 1.

[0164] In some embodiments, the delivery vehicle is a lipid nanoparticle that preferentially delivers to antigen presenting cells (APCs) in pancreatic islets and / or lymph nodes.

[0165] Method of administration

[0166] In some embodiments, the delivery vehicle comprising the lipid conjugate is part of a pharmaceutical formulation that is administered to treat, prevent, and / or ameliorate an antigen-induced disorder or an unwanted antigen-driven immune response in a subject. The pharmaceutical formulation may be administered in any suitable dosage and may include pharma- ceutical acceptable excipients.

[0167] In the embodiment where the lipid conjugate(s) is formulated in a first delivery vehicle and the antigen or nucleic acid encoding the antigen is formulated in a second delivery vehicle, the first and second delivery vehicles can be administered separately or together. When administered separately, the first and second delivery vehicles can be administered sequentially to the subject. That is, the first delivery vehicle can be administered before the second, and vice versa. The time frame between the administration of the first and second delivery vehicles can be selected based on the patient's requirements. The first and second delivery vehicles are typically each part of a pharmaceutical formulation that includes suitable excipients and pharma-ceutically acceptable salts.

[0168] In one embodiment, the one or more pharmaceutical preparations are administered parenterally, i.e., intraarterially, intravenously, subcutaneously or intramuscularly. In yet a further embodiment, the pharmaceutical composition is for intratumoral or intrauterine administration. In another embodiment, the pharmaceutical composition is administered intranasally, intravitreally, subretinal, intrathecal, or via other local routes.

[0169] The compositions described herein can be administered to a subject, including a patient, including a human or non-human subject. In some embodiments, the subject is a human.

[0170] The subject selected for treatment may have a pathological condition associated with antigen-specific immune stimulation or an undesired antigen-driven immune response. These immune disorders and undesired antigen-driven immune responses are diverse and range from allergic reactions to autoimmune diseases to transplant rejection. In some embodiments, the antigen-induced disorder is selected from autoimmune diseases (T cell and / or antibody responses against self-antigens), allergic diseases (T cell and IgE responses against environmental or food antigens), transplantation (T cell responses against major and minor histocompatibility antigens in donor tissues / organs / cells), anti-drug antibody responses (antibody responses that reduce the effectiveness of therapeutic drugs), or gene / protein replacement therapy (T cell / antibody responses against therapeutically replaced proteins in genetic protein deficiencies). In some embodiments, the antigen-induced disorder is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, myelin oligodendrocyte glycoprotein antibody disorder, vitiligo, type 1 diabetes, primary biliary cholangitis, anti-GBM nephritis / Goodpasture's disease, celiac disease, psoriasis, myasthenia gravis, immune thrombocytopenic purpura, Graves' disease, neuromyelitis optica, pemphigus vulgaris, bullous pemphigoid, cicatricial pemphigoid, systemic lupus erythematosus (SLE), lupus including SLE, autoimmune liver disease, myositis, Evans syndrome, transverse myelitis, Guillain-Barre syndrome, warm autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, chronic myelopathy ... The allergy is selected from allergies caused by mesenteric disorders, autoimmune autonomic neuropathy, autoimmune angioedema, Hashimoto's thyroiditis, Lambert-Eaton syndrome, peanut / regum allergy, tree nut allergy (antigens derived from any of cashews, pistachios, hazelnuts, walnuts, and almonds), egg allergy, milk allergy, soy allergy, fish allergy, shellfish allergy, sesame allergy, wheat allergy, allergic airway disease, or environmental allergens (antigens derived from pollen, dust, pet dander, mold, and cockroaches).

[0171] Exemplary embodiments

[0172] Various non-limiting embodiments disclosed herein are defined below: Embodiment 1. An immunomodulatory combination comprising a lipid conjugate comprising an immunomodulatory agent covalently linked to a lipophilic moiety by a cleavable linkage or via a cleavable linker; and an antigen and / or one or more nucleic acids encoding the antigen, wherein the antigen is a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, or polysaccharide, and wherein the lipid conjugate and the antigen and / or one or more nucleic acids encoding the antigen are formulated in separate delivery vehicles or are co-formulated in the same delivery vehicle. Embodiment 2. The immunomodulatory combination of embodiment 1, wherein the delivery vehicle is a lipid nanoparticle and / or a liposome, optionally wherein the delivery vehicle is a lipid nanoparticle that effectively delivers to antigen presenting cells (APCs) in pancreatic islets and / or lymph nodes. Embodiment 3. An immunomodulatory combination according to embodiment 2, wherein the antigen or one or more nucleic acids encoding the antigen are entrapped within a lipid nanoparticle or liposome and have a net charge opposite to the net charge of the lipid in the lipid nanoparticle, or the antigen is lipophilic and is incorporated into the lipid compartment of the lipid nanoparticle or liposome, or the antigen is hydrophilic and is entrapped in a liposome that comprises an aqueous core. Embodiment 4. An immunomodulatory combination according to any one of embodiments 1 to 3, wherein the lipid conjugate is co-formulated in the same delivery vehicle as the antigen or one or more nucleic acids encoding the antigen. Embodiment 5. The immunomodulatory combination according to any one of embodiments 1 to 4, wherein the lipid conjugate is a first lipid conjugate, and the immunomodulatory combination further comprises a second lipid conjugate, wherein the second lipid conjugate comprises an immunomodulatory agent covalently linked to a lipophilic moiety by a cleavable linkage or via a cleavable linker, wherein the immunomodulatory agent of the second lipid conjugate is different from the immunomodulatory agent of the first lipid conjugate, and wherein the first lipid conjugate and the second lipid conjugate are formulated in separate delivery vehicles. immunomodulatory combinations, optionally comprising a lipid conjugate comprising 3, 4, 5, 6, 7, 8, 9, or 10 immunomodulatory agents, wherein each immunomodulatory agent of the plurality of lipid conjugates is different, and each lipid conjugate of the plurality of lipid conjugates is formulated independently in a delivery vehicle separate from the other components of the immunomodulatory combination or is co-formulated in the same delivery vehicle with one or more of the other components of the immunomodulatory combination. Embodiment 6. The immunomodulatory combination of embodiment 5, wherein the immunomodulatory agent of the second lipid conjugate targets a different immune pathway than the immunomodulatory agent of the first lipid conjugate. Embodiment 7. The immunomodulatory combination of embodiment 5 or 6, wherein the first lipid conjugate and the second lipid conjugate are co-formulated in the same delivery vehicle. Embodiment 8. An immunomodulatory combination according to any one of embodiments 1 to 7, wherein each immunomodulatory agent is a tolerogenic agent or an anti-inflammatory agent. Embodiment 9. An immunomodulatory combination according to any one of embodiments 1 to 7, wherein each immunomodulatory agent is an immunostimulant or an immunosuppressant. Embodiment 10. The immunomodulatory combination of any one of embodiments 1-9, wherein each immunomodulatory agent is independently a nonsteroidal anti-inflammatory drug (NSAID), an inflammasome inhibitor, a Janus kinase (JAK) inhibitor, a corticosteroid, an mTOR inhibitor, a DMARD (disease-modifying antirheumatic drug), a calcineurin inhibitor, or a vitamin D receptor agonist. Embodiment 11. Each immunomodulatory agent is independently selected from the group consisting of prednisone, budesonide, prednisolone, methylprednisolone, hydrocortisone, cortisone, betamethasone, budesonide, triamcinolone, flunisolide, beclomethasone, fluticasone, mometasone, fludrocortisone, flumethasone, triamcinolone acetonide, isoflupredone, corticosterone, desoxycortone acetate, desoxycortone enanthate, 11-deoxycorticosterone, 11-deoxycortisol, aldosterone, dexamethasone, calcitonin, acetaminophen ... citriol, acetylsalicylic acid, salicylate, mycophenolic acid, sirolimus, tacrolimus, cholecalciferol, calcifediol, alfacalcidol, calcipotriol, falecalcitriol, maxacalcitol, paricalcitol, doxercalciferol, 22-oxacalcitriol, tacalcitol, eldecalcitol, erocalcitol, inecalcitol, becocalcidiol, seocalcitol, ergocalciferol, lexacalcitol, retinoic acid, cyclophosphamide (na Itrogen mustard), filgotinib, baricitinib, tofacitinib, ruxolitinib, upadacitinib, oclacitinib, peficitinib, fedratinib, delgocitinib, deuclavacitinib, abrocitinib, auranofin, apremilast, azathioprine, chloroquine, hydroxychloroquine, cyclosporine, leflunomide, methotrexate, minocycline, sulfasalazine, salicylic acid, diflunisal, salsalate, naproxen, ibuprofen, oxaprozin, loxoprofen, zaltoprolol Fen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, bromfenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, phenylbutazone, celecoxib, firocoxib, parecoxib, etoricoxib, clonixin, licofelone, MCC950, glyburide, CY-09, tranilast, oridonin, BOT-4-one (2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo[1,3] oxathiol-4-one), INF39 (ethyl 2-(2-chlorobenzyl)acrylate), MNS (3,4-methylenedioxy-β-nitrostyrene), fenamic acid, beta-hydroxybutyric acid, quercetin, JC-171, ibrutinib, OLT1177, FC11A-2, INF58, JC124, or ethyl 2-((2-chlorophenyl)(hydroxy)methyl)acrylate. Embodiment 12. The immunomodulatory combination of any one of embodiments 1-11, wherein the antigen is a first antigen, and the immunomodulatory combination further comprises a second antigen or one or more nucleic acids encoding the second antigen, wherein the first antigen is different from the second antigen, and wherein the second antigen or one or more nucleic acids encoding the second antigen are formulated in a delivery vehicle separate from the other components of the immunomodulatory combination or are co-formulated with one or more of the other components of the immunomodulatory combination, optionally wherein the immunomodulatory combination comprises multiple antigens, or comprises one or more nucleic acids encoding multiple antigens, or comprises a combination of an antigen and a nucleic acid(s) encoding an antigen to provide the multiple antigens, wherein the multiple antigens comprises 3, 4, 5, 6, 7, 8, 9, or 10 antigens, and each antigen or nucleic acid encoding an antigen is formulated independently in a delivery vehicle separate from the other components of the immunomodulatory combination or is co-formulated with one or more of the other components of the immunomodulatory combination. Embodiment 13. The immunomodulatory combination of embodiment 12, wherein the one or more nucleic acids encoding the first antigen and the one or more nucleic acids encoding the second antigen are contained within a single nucleic acid, and the first antigen and the second antigen are co-formulated in the same delivery vehicle. Embodiment 14. An immunomodulatory combination according to any one of embodiments 1 to 13 for use in treating a subject with an antigen-induced disorder or an unwanted antigen-driven immune response or for use in the manufacture of a medicament for treating a subject, optionally wherein the antigen-induced disorder or the unwanted antigen-driven immune response is selected from autoimmune diseases (T cell and / or antibody responses against self-antigens), allergic diseases (T cell and IgE responses against environmental or food antigens), transplantation (T cell responses against major and minor histocompatibility antigens in donor tissues / organs / cells), anti-drug antibody responses (antibody responses that reduce the efficacy of therapeutic drugs), gene / protein replacement therapy (T cell / antibody responses against therapeutically replaced proteins in genetic protein deficiencies), and optionally wherein the antigen-induced disorder or the unwanted antigen-driven immune response is selected from multiple sclerosis, rheumatoid arthritis, myelin oligodendrocyte glycoprotein antibody disorder, vitiligo, type 1 diabetes, primary biliary cholangitis, anti-inflammatory and anti-inflammatory diseases. GBM nephritis / Goodpasture's disease, Celiac disease, Psoriasis, Myasthenia gravis, Immune thrombocytopenic purpura, Graves' disease, Neuromyelitis optica, Pemphigus vulgaris, Bullous pemphigoid, Cicatricial pemphigoid, Lupus including systemic lupus erythematosus (SLE), Autoimmune liver disease, Myositis, Evans syndrome, Transverse myelitis, Guillain-Barre syndrome, Warm autoimmune hemolytic anemia, Chronic inflammatory demyelinating polyneuropathy, Autoimmune autonomic neuropathy, Autoimmune angioedema, Hashimoto's thyroiditis, Lambert-Eaton Immunomodulatory combinations selected from allergies caused by allergens such as maltose, peanut / regum allergy, tree nut allergy (antigens derived from any of the following: cashew, pistachio, hazelnut, walnut, almond), egg allergy, milk allergy, soy allergy, fish allergy, shellfish allergy, sesame allergy, wheat allergy, allergic airway disease, and environmental allergens (antigens derived from pollen, dust, pet dander, mold, and cockroaches). Embodiment 15. A method of treating a subject having an antigen-induced disorder or an undesired antigen-driven immune response comprising administering to the subject an immunomodulatory combination according to any one of embodiments 1 to 13, wherein an antigen, or one or more nucleic acids encoding an antigen, and a lipid conjugate are administered together or sequentially, and optionally the antigen-induced disorder or the undesired antigen-driven immune response is selected from autoimmune diseases (T cell and / or antibody responses against self-antigens), allergic diseases (T cell and IgE responses against environmental or food antigens), transplantation (T cell responses against major and minor histocompatibility antigens in donor tissues / organs / cells), anti-drug antibody responses (antibody responses that reduce the efficacy of therapeutic drugs), gene / protein replacement therapy (T cell / antibody responses against therapeutically replaced proteins in genetic protein deficiencies), and optionally the antigen-induced disorder or the undesired antigen-driven immune response is selected from multiple sclerosis, rheumatoid arthritis, myelin oligodendrocyte glycoprotein antibody disorder, vitiligo, Type 1 diabetes, primary biliary cholangitis, anti-GBM nephritis / Goodpasture's disease, celiac disease, psoriasis, myasthenia gravis, immune thrombocytopenic purpura, Graves' disease, neuromyelitis optica, pemphigus vulgaris, bullous pemphigoid, cicatricial pemphigoid, lupus including systemic lupus erythematosus (SLE), autoimmune liver disease, myositis, Evans syndrome, transverse myelitis, Guillain-Barré syndrome, warm autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, autoimmune autonomic neuropathy, autoimmune angioedema, Hashimoto's thyroiditis , Lambert-Eaton syndrome, peanut / regum allergy, tree nut allergy (antigens derived from any of cashews, pistachios, hazelnuts, walnuts, and almonds), egg allergy, milk allergy, soy allergy, fish allergy, shellfish allergy, sesame allergy, wheat allergy, allergic airway disease, and allergies caused by environmental allergens (antigens derived from pollen, dust, pet dander, mold, and cockroaches). Embodiment 16. A vaccine formulation comprising at least one prodrug comprising an immunomodulatory agent conjugated to a lipid moiety; and at least one antigen and / or a nucleic acid encoding an antigen, wherein the at least one antigen is a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, or polysaccharide, and wherein the at least one prodrug and the antigen and / or the nucleic acid encoding the antigen are formulated in separate delivery vehicles or are co-formulated in the same delivery vehicle in the formulation. Embodiment 17 The vaccine formulation of embodiment 16, wherein the delivery vehicle is a lipid nanoparticle. Embodiment 18. The vaccine formulation of embodiment 16 or 17, wherein at least one prodrug is co-formulated in the same delivery vehicle as the antigen or a nucleic acid encoding the antigen. Embodiment 19. The vaccine formulation of embodiment 16, 17, or 18, wherein two prodrugs are present in the formulation, either formulated separately or co-formulated in the same delivery vehicle. Embodiment 20. A vaccine formulation according to any one of embodiments 16 to 19, wherein the immunomodulatory agent is a tolerogenic agent. Embodiment 21. A vaccine formulation according to any one of embodiments 16 to 19, wherein the immunomodulatory agent is an immunostimulant or an immunosuppressant. Embodiment 22. The at least one prodrug immunomodulator is selected from the group consisting of dexamethasone, calcitriol, acetylsalicylic acid, mycophenolic acid, sirolimus, tacrolimus, cholecalciferol, calcifediol, retinoic acid, cyclophosphamide (nitrogen mustard), filgotinib, baricitinib, tofacitinib, ruxolitinib, upadacitinib, oclacitinib, peficitinib, fedratinib, delgocitinib, duke's. Rabasitinib, Abrocitinib, Auranofin, Apremilast, Azathioprine, Chloroquine, Hydroxychloroquine, Cyclosporine, Leflunomide, Methotrexate, Minocycline, Sulfasalazine, Salicylic Acid, Diflunisal, Salsalate, Naproxen, Ibuprofen, Oxaprozin, Loxoprofen, Zaltoprofen, Indomethacin, Tolmetin, Sulindac, Etodolac, Ketorolac, Diclofenac, Aceclofe Nac, bromfenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, phenylbutazone, celecoxib, firocoxib, parecoxib, etoricoxib, clonixin, licofelone, MCC950, glyburide, CY-09, tranilast, oridonin, BOT-4-one (2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo[1,3]oxathiol-4-one 22. The vaccine formulation of any one of embodiments 16 to 21, wherein the hydroxybutyric acid is selected from the group consisting of benzodiazepine, benzoyl ketone ... Embodiment 23. A vaccine formulation according to any one of embodiments 16 to 22, wherein two antigens and / or nucleic acids encoding the antigens are present in the formulation, either formulated separately or co-formulated in the same delivery vehicle. Embodiment 24. A method of treating a subject having an antigen-induced disorder comprising administering at least one prodrug comprising an immunomodulatory agent conjugated to a lipophilic moiety; and administering at least one antigen and / or nucleic acid encoding an antigen, wherein the at least one antigen is a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, or polysaccharide, and the at least one prodrug and the antigen and / or nucleic acid encoding an antigen are formulated separately in a delivery vehicle or co-formulated in the same delivery vehicle, and the at least one prodrug and the at least one antigen and / or nucleic acid encoding an antigen are administered together or sequentially. Embodiment 25 The method of embodiment 24, wherein the delivery vehicle is a lipid nanoparticle. Embodiment 26 The method of embodiment 24 or 25, wherein at least one prodrug is co-formulated with the antigen and / or a nucleic acid encoding the antigen in the delivery vehicle. Embodiment 27. The method of embodiment 24, 25, or 26, wherein two prodrugs are present in the formulation, either formulated separately or co-formulated in the same delivery vehicle. Embodiment 28. The method of any one of embodiments 24 to 27, wherein the immunomodulatory agent is a tolerogenic agent. Embodiment 29. The method of any one of embodiments 24 to 28, wherein the immunomodulatory agent is an immunostimulant or an immunosuppressant. Embodiment 30. The at least one prodrug immunomodulator is selected from the group consisting of dexamethasone, calcitriol, acetylsalicylic acid, mycophenolic acid, sirolimus, tacrolimus, cholecalciferol, calcifediol, retinoic acid, cyclophosphamide (nitrogen mustard), filgotinib, baricitinib, tofacitinib, ruxolitinib, upadacitinib, oclacitinib, peficitinib, fedratinib, delgocitinib, dew. Clavacitinib, Abrocitinib, Auranofin, Apremilast, Azathioprine, Chloroquine, Hydroxychloroquine, Cyclosporine, Leflunomide, Methotrexate, Minocycline, Sulfasalazine, Salicylic Acid, Diflunisal, Salsalate, Naproxen, Ibuprofen, Oxaprozin, Loxoprofen, Zaltoprofen, Indomethacin, Tolmetin, Sulindac, Etodolac, Ketorolac, Diclofenac, Aceclo Fenac, bromfenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, phenylbutazone, celecoxib, firocoxib, parecoxib, etoricoxib, clonixin, licofelone, MCC950, glyburide, CY-09, tranilast, oridonin, BOT-4-one (2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo[1,3]oxathiol-4 30. The method of any one of embodiments 24-29, wherein the active ingredient is selected from the group consisting of ethyl 2-((2-chlorophenyl)(hydroxy)methyl)acrylate, ... Embodiment 31. The method of any one of embodiments 24 to 30, wherein two antigens and / or nucleic acids encoding antigens are present in the formulation, either formulated separately or co-formulated in the same delivery vehicle. Embodiment 32. Use of a prodrug comprising an immunomodulatory agent conjugated to a lipophilic moiety in combination with at least one antigen and / or a nucleic acid encoding at least one antigen to treat a subject having an antigen-induced disorder, wherein the at least one antigen is a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, or polysaccharide, and the at least one prodrug and the antigen and / or the nucleic acid encoding the antigen are (i) co-formulated in the same delivery vehicle in at least one vaccine formulation, or (ii) formulated in separate delivery vehicles for sequential or co-administration to the subject. Embodiment 33 The use according to embodiment 32, wherein the delivery vehicle is a lipid nanoparticle. Embodiment 34. The use according to embodiment 32 or 33, wherein at least one prodrug is co-formulated in the same delivery vehicle as the antigen or a nucleic acid encoding such antigen. Embodiment 35. The use according to embodiment 32, 33 or 34, wherein two prodrugs are present in the formulation, either formulated separately or co-formulated in the same delivery vehicle. Embodiment 36. The use according to any one of embodiments 32 to 35, wherein the immunomodulatory agent is a tolerogenic agent. Embodiment 37. The use according to any one of embodiments 32 to 35, wherein the immunomodulatory agent is an immunostimulant or an immunosuppressant. Embodiment 38. The at least one prodrug immunomodulator is selected from the group consisting of dexamethasone, calcitriol, acetylsalicylic acid, mycophenolic acid, sirolimus, tacrolimus, cholecalciferol, calcifediol, retinoic acid, cyclophosphamide (nitrogen mustard), filgotinib, baricitinib, tofacitinib, ruxolitinib, upadacitinib, oclacitinib, peficitinib, fedratinib, delgocitinib, dew. Clavacitinib, Abrocitinib, Auranofin, Apremilast, Azathioprine, Chloroquine, Hydroxychloroquine, Cyclosporine, Leflunomide, Methotrexate, Minocycline, Sulfasalazine, Salicylic Acid, Diflunisal, Salsalate, Naproxen, Ibuprofen, Oxaprozin, Loxoprofen, Zaltoprofen, Indomethacin, Tolmetin, Sulindac, Etodolac, Ketorolac, Diclofenac, Aceclo Fenac, bromfenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, phenylbutazone, celecoxib, firocoxib, parecoxib, etoricoxib, clonixin, licofelone, MCC950, glyburide, CY-09, tranilast, oridonin, BOT-4-one (2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo[1,3]oxathiol-4 36. The use according to any one of embodiments 32 to 35, wherein the active ingredient is selected from: 1,2-dichlorophenyl)-2-((2-chlorophenyl)(hydroxy)methyl)acrylate, 1,2-dichlorophenyl)-2-((2-chlorophenyl)-2-propanediol), 1,2-dichlorophenyl)-2-propanediol, ... Embodiment 39. The use according to any one of embodiments 32 to 38, wherein two antigens and / or nucleic acids encoding antigens are present in the formulation, either formulated separately or co-formulated in the same delivery vehicle. Embodiment 40. A combination of a prodrug comprising an immunomodulatory agent conjugated to a lipophilic moiety and at least one antigen and / or a nucleic acid encoding at least one antigen for treating a subject having an antigen-induced disorder, wherein the at least one antigen is a protein, polypeptide, peptide, lipoprotein, glycolipid, polynucleotide, or polysaccharide, and the at least one prodrug and the antigen and / or the nucleic acid encoding the antigen are (i) co-formulated in the same delivery vehicle or (ii) formulated in separate delivery vehicles for sequential or co-administration to the subject. EXAMPLES

[0173] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention.

[0174] Synthesis of lipid conjugates

[0175] Various lipid conjugates were prepared using synthetic procedures A to E shown below.

[0176] All reagents and solvents, except for THF (freshly distilled from Na / benzophenone under nitrogen) and EtN, DMF, and CHCl (freshly distilled from CaH under nitrogen), were purchased from commercial suppliers and used without further purification unless otherwise noted. USP grade castor oil was purchased from a local pharmacy (Life™ brand) and used as received. For NMR, chemical shifts are reported in parts per million (ppm) on the δ scale, and coupling constants J are in Hertz (Hz). Multiplicities are reported as "s" (singlet), "d" (doublet), "dd" (doublet of doublets), "dt" (doublet of triplets), "ddd" (doublet of doublets of doublets), "t" (triplet), "td" (triplet of doublets), "q" (quartet), "quin" (quintet), "sex" (sexlet), "m" (multiplet), and further qualified as "app" (apparent) and "br" (broad).

[0177] The general synthetic steps for lipid conjugates based on hydroxy and carboxy derivatives of castor oil (ricinolein) are provided below in Scheme 1. This is followed by Scheme 1, designated General Procedures A-E, which describes the steps for producing lipid conjugates of Examples E, S, T and W below.

[0178] Scheme 1: General synthesis of lipid conjugates based on hydroxy and carboxy derivatives of castor oil (ricinolein). [ka]

[0179] According to the synthetic reactions described above in Scheme I, castor oil, also known as ricinolein (the glyceride of ricinoleic acid), is the starting material for the synthesis of the prodrug shown in FIG.

[0180] In step 1) above, sodium methoxide (2.0 mL of a 3.0 M solution in MeOH, 6.00 mmol, 0.20 equiv) was added to a stirring room temperature 1:1 THF / MeOH (30 mL) solution of castor oil (28.0 g, 30.0 mmol, 1.00 equiv) in a round bottom flask under argon. After 14 h, the reaction mixture was quenched with saturated aqueous NH4Cl and extracted with Et2O (3 x 150 mL). The combined organic layers were washed with water (1 x 150 mL), brine (1 x 150 mL), dried over Na2SO4, and concentrated to give methyl (12R)-hydroxyoleate 1 (28.0 g, quantitative yield) as a clear, colorless oil, which was used without further purification. The structure of methyl (12R)-hydroxyoleate and its physical properties are shown below: Methyl (12R)-hydroxyoleate (1): [ka] R f = 0.50 (SiO2, 70:30 hexane / EtOAc);

number

[0181] According to 2) of the reaction scheme above, a room temperature solution of methyl (12R)-hydroxyoleate (9.37 g, 30.0 mmol) in THF (15 mL) was added via addition funnel over 20-30 min to a stirred ice-cold THF (90 mL) suspension of LiAlH4 (1.25 g, 33.0 mmol, 1.10 equiv) in a round-bottom flask under argon. After the addition was complete, the cold bath was removed. After 14 h, the reaction mixture was cooled in an ice bath, diluted with Et2O (150 mL), quenched with quench solution (1.25 mL of HO, 1.25 mL of 1 M aqueous NaOH, 3.75 mL of HO), stirred at room temperature for 1 h, and filtered through Celite, washing extensively with Et2O. The filtrate was concentrated on a rotary evaporator to give the crude diol as a pale yellow oil (quantitative yield), which was used without further purification.

[0182] According to 3) of the reaction scheme above, a room temperature solution of tert-butyldimethylsilyl chloride (3.96 g, 26.2 mmol, 1.00 equiv) in DMF (20 mL) was added via addition funnel over 30 min to a 10-15 °C DMF (25 mL) solution of the above diol (8.21 g, 28.9 mmol, 1.10 equiv) and i-Pr2Net (5.73 mL, 32.8 mmol, 1.25 equiv) in a round bottom flask under argon. The reaction mixture was warmed for 14 h, then quenched with saturated aqueous NH4Cl and extracted with 1:1 Et2O / hexanes (3 x 100 mL). The combined organic layers were washed with H2O (3 x 100 mL), brine (1 x 100 mL), dried over Na2SO4, and concentrated on a rotary evaporator to give the crude first silyl ether as a pale yellow oil. The crude product was purified by filtration through a plug of silica gel (220 mL of SiO, 99:1→95:5 hexanes / EtOAc) to give a clear, colorless oil consisting of silyl ether 2 (8.38 g, 80% yield). The structure of silyl ether 2, as well as its physical properties, are shown below:

[0183] I-1-(tert-butyldimethylsilyl)-12-hydroxyoleyl alcohol (2): [ka] R f = 0.16 (SiO2, 95:5 hexane / EtOAc);

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[0184] According to 4) of the reaction scheme above, N,N'-dicyclohexylcarbodiimide (DCC) (495 mg, 2.40 mmol, 1.20 equiv.) was added to an ice-cold solution of RCOH (279 mg, 2.40 mmol, 1.20 equiv.) in CHCl (6 mL) in a round-bottom flask under argon, after which the ice bath was removed and the resulting mixture was stirred for 15 min. In this example, RCOH was hexanoic acid, but other acyl groups can be utilized to generate the desired hydrocarbon side chain S. The reaction mixture was again cooled in an ice bath and a solution of the silyl ether, I-1-(tert-butyldimethylsilyl)-12-hydroxyoleyl alcohol 2 (797 mg, 2.00 mmol) in CHCl (2 mL) was added, followed by DMAP (366 mg, 3.00 mmol, 1.50 equiv.) and the reaction mixture was allowed to warm to room temperature over 14 h. The reaction mixture was diluted with EtO, stirred for 10 min, and then filtered through Celite. The filtrate was concentrated on a rotary evaporator to give the crude ester as a white semi-solid. The crude product was purified by filtration through a plug of silica gel (20 mL SiO, 95:5 hexanes / EtOAc) to give R f = 0.53 (SiO2, 90:10 hexanes / EtOAc) as the intermediate ester (quantitative yield).

[0185] According to 5) of the reaction scheme above, a neat HF·pyridine solution (0.74 mL of 70% HF in pyridine, 6.00 mmol, 3.00 equiv.) was added to a stirred ice-cold solution of pyridine (0.48 mL, 6.00 mmol, 3.00 equiv.) and the above silyl ether (2.00 mmol) in THF (6 mL) in a round-bottom flask under argon. After 2 h, the reaction mixture was quenched with saturated aqueous NaHCO3. The mixture was extracted with Et2O (2 × 10 mL), and the combined organic extracts were then washed with H2O (1 × 10 mL), brine, dried over Na2SO4, and concentrated on a rotary evaporator to give the crude primary alcohol. The crude product was purified by filtration through a plug of silica gel (20 mL, 90:10 hexanes / EtOAc) to give the primary alcohol 3 (quantitative yield) as a clear, colorless oil with the following structure and physical properties: (12R)-Hexanoyloxyoleyl alcohol (3): [ka]

[0186] According to 6) of the reaction scheme above, solid succinic anhydride (400 mg, 4.00 mmol, 2.00 equiv) and DMAP (611 mg, 5.00 mmol, 2.50 equiv) were added to a stirring room temperature solution of (12R)-hexanoyloxyoleyl alcohol (3) (765 mg, 2.00 mmol, 1.00 equiv) in CHCl (6 mL) in a round bottom flask under argon. After 14 h, the reaction was quenched with 1M aqueous HCl and extracted with CHCl (2×15 mL). The combined organic extracts were then washed with 1M aqueous HCl (1×15 mL), H0 (2×15 mL), dried over NaSO, and concentrated on a rotary evaporator to give the intermediate hemisuccinate (quantitative yield) as a pale yellow oil, which was used without further purification. The intermediate was R f = 0.32 (SiO2, 50:50 hexanes / EtOAc).

[0187] According to 7) of the reaction scheme, solid DCC (99 mg, 0.48 mmol, 1.20 equiv) was added to a stirred ice-cold solution of the above hemisuccinate (232 mg, 0.48 mmol, 1.20 equiv) in CHCl (2 mL) in a round-bottom flask under argon, then the ice bath was removed and the resulting mixture was stirred for 15 min. The reaction mixture was cooled again in an ice bath and solid dexamethasone (157 mg, 0.40 mmol) and DMAP (73 mg, 0.60 mmol, 1.50 equiv) were added. The reaction mixture was warmed for 14 h, diluted with EtO, stirred for 10 min, and then filtered through Celite. The filtrate was concentrated to give the crude product as a pale yellow oil. The crude product was purified by flash column chromatography (50 mL SiO, 80:20→50:50 hexanes / EtOAc) to give a clear, colorless oil as the desired prodrug 4 (328 mg, 95% yield) with the following structure and properties: 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]ctadic-9-en-17-yl)-2-oxoethyl((R,Z)-12-(hexanoyloxy)ctadic-9-en-1-yl)succinate (4): [ka] R f = 0.38 (SiO2, 50:50 hexane / EtOAc);

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[0188] The lipid conjugate is based on the ricinoleyl scaffold L with a hexanoyl (C6:0) side chain conjugated to dexamethasone by a succinate linker (INT-D034). In the above example, the RCO2H added in step 4) of the above reaction to generate the hexanoyl side chain (C6:0) was hexanoic acid, however, other fatty acids can be utilized to generate the desired hydrocarbon side chain R on the ricinoleyl scaffold.

[0189] General Procedure A - Acylation of (R)-1-(tert-butyldimethylsilyl)-12-hydroxyoleyl alcohol 3 (4a-h):

[0190] DCC (1.20 equiv.) was added to a stirring ice-cold solution of the desired carboxylic acid (1.20 equiv.) in a round-bottom flask under argon, then the ice bath was removed and the resultant stirred for 15 min. The reaction mixture was cooled again in an ice bath and a solution of alcohol 3 (1.00 equiv., 0.25 M in CH2Cl2) in CH2Cl2 was added, followed by DMAP (1.50 equiv.) and the reaction mixture was allowed to warm to room temperature over 14 h. The reaction mixture was diluted with Et2O, stirred for 10 min, then filtered through Celite®. The filtrate was concentrated on a rotary evaporator to give the crude ester as a white semi-solid. The crude product was purified by filtration through a plug of silica gel (95:5 hexanes / EtOAc) to give the pure ester.

[0191] General Procedure B - Desilylation-succinylation of (12R)-acyloxyoleyl alcohols 4a-h (5a-h):

[0192] A solution of HF·pyridine (3.00 equiv. of 70% HF in pyridine) was added to a stirring ice-cold THF (0.30 M with respect to starting silyl ether) solution of pyridine (3.00 equiv.) and 12-acylcinoyl alcohol silyl ether (1.00 equiv.) in a round-bottom flask under argon. When TLC indicated consumption of starting material (2–8 h), the reaction mixture was quenched with saturated aqueous NaHCO3. The mixture was extracted with Et2O (2×10 mL), and the combined organic extracts were then washed with H2O (1×10 mL), brine, dried over Na2SO4, and concentrated on a rotary evaporator to give the crude primary alcohol. The crude product was purified by filtration through a plug of silica gel (90:10 hexanes / EtOAc), concentrated on a rotary evaporator, and dried under high vacuum to give the primary alcohol as a clear, colorless oil that was used in subsequent succinylations without further purification.

[0193] Solid succinic anhydride (2.00 equiv.) and DMAP (2.50 equiv.) were added to a stirring room temperature solution of 12-acylcinoyl alcohol (1.00 equiv.) in CHCl (0.30 M relative to starting primary alcohol) in a round bottom flask under argon. After 14 h, the reaction was quenched with 1 M aqueous HCl and extracted with CHCl (2×15 mL). The combined organic extracts were then washed with 1 M aqueous HCl (1×15 mL), H0 (2×15 mL), dried over NaSO, and concentrated on a rotary evaporator. The residue was redissolved in hexane, treated with activated charcoal, filtered through Celite®, and the filtrate was concentrated to give the intermediate hemisuccinate as a colorless to pale yellow oil, which was used without further purification.

[0194] General procedure for acylation of C-methyl (12R)-ricinoleate 2 (6a-c):

[0195] DCC (1.20 equiv.) was added to a stirring ice-cold CHCl solution of the desired carboxylic acid (1.20 equiv.) in a round-bottom flask under argon, then the ice bath was removed and the resultant stirred for 15 min. The reaction mixture was cooled again in an ice bath and a CHCl solution of methyl (12R)-ricinoleate (1.00 equiv., 0.30 M in CHCl) was added, followed by DMAP (1.50 equiv.), and the reaction mixture was allowed to warm to room temperature over 14 h. The reaction mixture was diluted with hexanes, stirred for 10 min, and then filtered through Celite®. The filtrate was concentrated on a rotary evaporator to give the crude diester as a white semi-solid, which was purified by filtration through a plug of silica gel (95:5 hexanes / EtOAc) to give the pure ester.

[0196] General Procedure 5a-h Conjugation of Dexamethasone to D-Hemisuccinate:

[0197] DCC (1.20 equiv.) was added to a stirring ice-cold solution of 12-acylricinoleyl hemisuccinate (1.20 equiv.) in CHCl (0.2 M in dexamethasone) in a round-bottom flask under argon, then the ice bath was removed and the resultant was stirred for 15 min. The reaction mixture was cooled again in an ice bath and solid dexamethasone (1.00 equiv.) and DMAP (1.50 equiv.) were added. The reaction mixture was warmed for 14 h, diluted with EtO, stirred for 10 min, and then filtered through Celite®. The filtrate was concentrated to give the crude product as a pale yellow oil, which was then purified by flash column chromatography (SiO, 80:20→50:50 hexane / EtOAc) to give a clear, colorless oil as the desired dexamethasone conjugate.

[0198] General Procedure E - Conjugation of Dexamethasone to Ricinoleic Acid 12a-b, 13:

[0199] DCC (1.10 equiv.) was added to a stirring ice-cold solution of acyloxystearic acid (1.10 equiv.) in CHCl (0.1 M in dexamethasone) in a round-bottom flask under argon, then the ice bath was removed and the resultant was stirred for 15 min. The reaction mixture was cooled again in an ice bath and solid dexamethasone (1.00 equiv.) and DMAP (1.50 equiv.) were added. The reaction mixture was warmed for 14 h, diluted with EtO, stirred for 10 min, and then filtered through Celite®. The filtrate was concentrated to give the crude product as a pale yellow oil, which was then purified by flash column chromatography to give a clear, colorless oil as the desired conjugate.

[0200] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl acetate (4a):

[0201] Acetyl chloride (0.43 mL, 6.00 mmol, 1.20 equiv) was added dropwise to a stirred ice-cold solution of silyl ether 3 (2.00 g, 5.00 mmol, 1.00 equiv), acetyl chloride (0.43 mL, 6.00 mmol, 1.20 equiv), triethylamine (0.83 mL, 6.00 mmol, 1.2 equiv) and DMAP (733 mg, 6.00 mmol, 1.20 equiv) in CHCl (10 mL) under argon in a round-bottom flask, which was allowed to warm to room temperature. After 14 h, the reaction mixture was diluted with CHCl, washed with saturated aqueous NHCl (1×15 mL), water (2×15 mL), dried over NaSO, and concentrated on a rotary evaporator. The residue was redissolved in eluent and passed through a plug of silica gel (30 mL of SiO.sub.2, 97:3 hexanes / EtOAc) to give the ester 4a (1.83 g, 83%) as a pale yellow oil. R f = 0.45 (SiO2, 95:5 hexane / EtOAc);

number

[0202] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl hexanoate (4b):

[0203] Following general procedure A, silyl ether 3 (2.00 g, 5.00 mmol), hexanoic acid (697 mg, 6.00 mmol), DCC (1.24 g, 6.00 mmol) and DMAP (916 mg, 7.50 mmol) in CH2Cl2 (15 mL) gave 2.37 g of ester 4b (2.39 g, quantitative yield) as a clear, colorless oil. R f = 0.43 (SiO2, 95:5 hexane / EtOAc);

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[0204] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl laurate (4c):

[0205] Following general procedure A, silyl ether 3 (997 mg, 2.50 mmol), lauric acid (601 mg, 3.00 mmol), DCC (619 mg, 3.00 mmol) and DMAP (458 mg, 3.75 mmol) in CH2Cl2 (8 mL) gave ester 4c (1.38 g, quantitative yield) as a clear, colorless oil. R f = 0.56 (SiO2, 95:5 hexane / EtOAc);

number

[0206] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl stearate (4d):

[0207] Following general procedure A, silyl ether 3 (997 mg, 2.50 mmol), stearic acid (853 mg, 3.00 mmol), DCC (619 mg, 3.00 mmol) and DMAP (458 mg, 3.75 mmol) in 2:1 THF / CH2Cl2 (6 mL) gave ester 4d (1.56 g, 94%) as a clear, colorless oil. R f = 0.48 (SiO2, 90:10 hexane / EtOAc);

number

[0208] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl oleate (4e):

[0209] Following general procedure A, silyl ether 3 (997 mg, 2.50 mmol), oleic acid (847 mg, 3.00 mmol), DCC (619 mg, 3.00 mmol) and DMAP (458 mg, 3.75 mmol) in CH2Cl2 (10 mL) gave ester 4e (1.64 g, quantitative) as a clear, colorless oil. R f = 0.41 (SiO2, 95:5 hexane / EtOAc);

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[0210] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl linoleate (4f):

[0211] Following general procedure A, silyl ether 3 (847 mg, 2.12 mmol), linoleic acid (715 mg, 2.55 mmol), DCC (526 mg, 2.55 mmol) and DMAP (389 mg, 3.19 mmol) in CH2Cl2 (7 mL) gave ester 4f (1.06 g, 76%) as a clear, colorless oil. R f = 0.46 (SiO2, 95:5 hexane / EtOAc);

number

[0212] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl linolenate (4g):

[0213] Following general procedure A, the silyl ether 3 (997 mg, 2.50 mmol), linolenic acid (835 mg, 3.00 mmol), DCC (619 mg, 3.00 mmol) and DMAP (458 mg, 3.75 mmol) in CH2Cl2 (8 mL) gave the ester 4g (1.52 g, 92%) as a clear, colorless oil. R f = 0.34 (SiO2, 95:5 hexane / EtOAc);

number

[0214] (R,Z)-18-((tert-butyldimethylsilyl)oxy)octadec-9-en-7-yl arachidonate (4h):

[0215] Following general procedure A, silyl ether 3 (797 mg, 2.00 mmol), arachidonic acid (670 mg, 2.20 mmol), DCC (227 mg, 2.20 mmol) and DMAP (366 mg, 3.00 mmol) in CH2Cl2 (7 mL) gave ester 4h (730 mg, 53%) as a clear, colorless oil after flash column chromatography (99:1→95:5 hexanes / EtOAc). R f = 0.57 (SiO2, 95:5 hexane / EtOAc);

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[0216] (R,Z)-4-((12-acetoxyoctadec-9-en-1-yl)oxy)-4-oxobutanoic acid (5a):

[0217] Following general procedure B, silyl ether 4a (1.79 g, 4.07 mmol) was desilylated using HF·pyridine solution (1.52 mL, 12.2 mmol), pyridine (0.98 mL, 12.2 mmol) and THF (10 mL) to give the intermediate primary alcohol (1.34 g), which was subjected to acylation with succinic anhydride (814 mg, 8.14 mmol), DMAP (1.24 g, 10.2 mmol) and CHCl (10 mL) to give carboxylic acid 5a (1.72 g, quantitative yield). R f = 0.23 (SiO2, 50:50 hexane / EtOAc);

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[0218] (R,Z)-4-((12-hexanoyloxy)octadec-9-en-1-yl)oxy)-4-oxobutanoic acid (5b):

[0219] Following general procedure B, silyl ether 4b (2.35 g, 5.00 mmol) was desilylated using HF·pyridine solution (1.86 mL, 15.0 mmol), pyridine (1.21 mL, 15.0 mmol), and THF (13 mL) to give the intermediate primary alcohol (2.01 g), which was subjected to acylation with succinic anhydride (1.00 g, 10.0 mmol), DMAP (1.53 g, 12.5 mmol), and CHCl (13 mL) to give carboxylic acid 5b (2.20 g, 92% yield). R f = 0.32 (SiO2, 50:50 hexane / EtOAc);

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[0220] (R,Z)-4-((12-lauroyloxy)octadec-9-en-1-yl)oxy)-4-oxobutanoic acid (5c):

[0221] Following general procedure B, silyl ether 4c (1.38 g, 2.50 mmol) was desilylated using HF·pyridine solution (0.93 mL, 7.50 mmol), pyridine (0.60 mL, 7.50 mmol) and THF (8 mL) to give the intermediate primary alcohol (1.21 g), which was subjected to acylation with succinic anhydride (500 mg, 5.00 mmol), DMAP (764 mg, 6.25 mmol) and CHCl (8 mL) to give the carboxylic acid 5c (1.33 g, 94%). R f = 0.44 (SiO2, 50:50 hexane / EtOAc);

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[0222] (R,Z)-4-oxo-4-((12-(stearoyloxy)octadec-9-en-1-yl)oxy)butanoic acid (5d):

[0223] Following general procedure B, silyl ether 4d (1.66 g, 2.50 mmol) was desilylated using HF·pyridine solution (0.93 mL, 7.50 mmol), pyridine (0.60 mL, 7.50 mmol), and THF (8 mL) to give the intermediate primary alcohol (1.30 g), which was subjected to acylation with succinic anhydride (500 mg, 5.00 mmol), DMAP (764 mg, 6.25 mmol), and CHCl (8 mL) to give the carboxylic acid 5d (1.29 g, 79% yield). R f = 0.35 (SiO2, 50:50 hexane / EtOAc);

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[0224] (R,Z)-4-oxo-4-((12-(oleoyloxy)octadec-9-en-1-yl)oxy)butanoic acid (5e):

[0225] Following general procedure B, the silyl ether 4e (663 mg, 1.00 mmol) was desilylated using HF·pyridine solution (0.37 mL, 3.00 mmol), pyridine (0.24 mL, 3.00 mmol) and THF (5 mL) to give the intermediate primary alcohol (546 mg), which was subjected to acylation with succinic anhydride (200 mg, 2.00 mmol), DMAP (305 mg, 2.50 mmol) and CHCl (5 mL) to give the carboxylic acid 5e (630 mg, 97% yield). R f= 0.42 (SiO2, 50:50 hexane / EtOAc);

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[0226] 4-(((R,Z)-12-(linoleoyloxy)octadec-9-en-1-yl)oxy)-4-oxobutanoic acid (5f):

[0227] Following general procedure B, silyl ether 4f (1.06 g, 1.60 mmol) was desilylated using HF·pyridine solution (0.60 mL, 4.80 mmol), pyridine (0.39 mL, 4.80 mmol) and THF (8 mL) to give the intermediate primary alcohol (890 mg), which was subjected to acylation with succinic anhydride (320 mg, 3.20 mmol), DMAP (489 mg, 4.00 mmol) and CHCl (8 mL) to give the carboxylic acid 5f (1.04 g, quantitative yield). R f = 0.35 (SiO2, 50:50 hexane / EtOAc);

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[0228] 4-(((R,Z)-12-(linolenoyloxy)octadec-9-en-1-yl)oxy)-4-oxobutanoic acid (5g):

[0229] Following general procedure B, the silyl ether 4g (1.54 g, 2.34 mmol) was desilylated using HF·pyridine solution (0.87 mL, 7.01 mmol), pyridine (0.57 mL, 7.01 mmol) and THF (6 mL) to give the intermediate primary alcohol (1.31 g), which was subjected to acylation with succinic anhydride (468 mg, 4.68 mmol), DMAP (714 mg, 5.84 mmol) and CHCl (6 mL) to give the carboxylic acid 5g (1.47 g, quantitative yield). R f = 0.35 (SiO2, 50:50 hexane / EtOAc);

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[0230] 4-(((R,Z)-12-(arachidonoyloxy)octadec-9-en-1-yl)oxy)-4-oxobutanoic acid (5h):

[0231] Following general procedure B, silyl ether 4h (711 mg, 1.04 mmol) was desilylated using HF·pyridine solution (0.39 mL, 3.11 mmol), pyridine (0.25 mL, 3.11 mmol) and THF (5 mL) to give the intermediate primary alcohol (593 mg), which was subjected to acylation with succinic anhydride (201 mg, 2.01 mmol), DMAP (306 mg, 2.51 mmol) and CHCl (5 mL) to give the carboxylic acid 5h (582 mg, 87% yield). R f = 0.31 (SiO2, 50:50 hexane / EtOAc);

number

[0232] Methyl (12R)-hexanoyloxyoleate (6a):

[0233] Following general procedure C, methyl ricinoleate (2.00 g, 6.40 mmol), hexanoic acid (898 mg, 7.68 mmol), DCC (1.58 g, 7.68 mmol) and DMAP (1.17 g, 9.60 mmol) in CH2Cl2 (10 mL) afforded ricinoleate 6a (2.52 g, 96% yield) as a clear, colorless oil after filtration through silica gel (95:5 hexane / EtOAc). R f = 0.62 (SiO2, 70:30 hexane:EtOAc);

number

[0234] Methyl (12R)-linoleoyloxyoleate (6b):

[0235] Following general procedure C, methyl ricinoleate (500 mg, 1.60 mmol), linoleic acid (538 mg, 1.92 mmol), DCC (396 mg, 1.92 mmol) and DMAP (293 mg, 2.40 mmol) in CH2Cl2 (5 mL) afforded ricinoleate 6c (875 g, 93% yield) as a pale yellow oil after filtration through silica gel (95:5 hexane / EtOAc). R f = 0.67 (SiO2, 80:20 hexane:EtOAc);

number

[0236] (12R)-Hexanoyloxyoleic acid (7a):

[0237] An argon-flushed round-bottom flask was charged with the methyl ester 6a (1.97 g, 4.79 mmol, 1.00 equiv) and t-BuOH (12 mL), followed by 2.0 M aqueous NaOH (1.80 mL, 3.60 mmol, 0.75 equiv). After 17 h, the pH of the reaction solution was adjusted to 2 using 1 M aqueous HCl and extracted with Et2O (3×30 mL). The combined organics were washed with water (1×30 mL), brine (1×30 mL), dried over Na2SO4, and concentrated on a rotary evaporator under reduced pressure. The residue was filtered through a plug of silica (98:2:0→50:45:5 hexanes:EtOAc:MeOH) to give the carboxylic acid 7a (1.30 g, 92% yield) as a pale yellow oil. R f = 0.24 (SiO2, 75:20:5 hexane / EtOAc / MeOH);

number

[0238] (12R)-Linoleoyloxyoleic acid (7b):

[0239] An argon-flushed round-bottom flask was charged with the methyl ester 6b (5.97 g, 10.4 mmol, 1.00 equiv) and t-BuOH (26 mL), followed by 2.0 M aqueous NaOH (4.70 mL, 9.30 mmol, 0.90 equiv). After 17 h, the pH of the reaction solution was adjusted to 2 using 1 M aqueous HCl and extracted with Et2O (3×30 mL). The combined organics were washed with water (1×30 mL), brine (1×30 mL), dried over Na2SO4, and concentrated on a rotary evaporator under reduced pressure. The residue was purified by flash column chromatography (SiO2, 95:5:0→80:15:5 hexanes:EtOAc:MeOH) to give the carboxylic acid 7b (4.48 g, 85% yield) as a pale yellow oil. R f= 0.35 (SiO2, 75:20:5 hexane / EtOAc / MeOH);

number

[0240] Methyl 9,10-dihydroxystearate (8):

[0241] KOH (7.01 g, 125 mmol, 5.00 equiv) was added to a rapidly stirred room temperature mixture of oleic acid (7.06 g, 25.0 mmol) and water (175 mL) in a 500 mL Erlenmeyer flask and then cooled to about 10° C. A solution of KMnO4 (7.11 g, 45.0 mmol, 1.80 equiv) in water (75 mL) was added dropwise over 10 min. After stirring for an additional 10-15 min, the reaction was quenched by the addition of saturated aqueous NaHSO3 and then adjusted to pH≦2 by the addition of concentrated HCl with the aid of a cooling bath. The white fluffy mixture was stirred at room temperature for 1 h, then the solid was collected by suction filtration and air dried overnight. The resulting white solid was hot gravity filtered and recrystallized from EtOH to give (±)-syn-9,10-dihydroxystearic acid as white crystals (5.86 g, 74% yield).

[0242] Concentrated H2SO4 (0.06 mL, 1.00 mmol, 0.05 equiv) was added to a suspension of the above dihydroxy acid (6.33 g, 20.0 mmol) in MeOH (50 mL) and the resulting mixture was heated to reflux. After 14 h, the mixture was cooled to room temperature, concentrated on a rotary evaporator under reduced pressure, and the resulting residue was partitioned between EtOAc and saturated aqueous NaHCO3. The organic layer was washed with water (1 x 75 mL), brine, dried over Na2SO4, and concentrated on a rotary evaporator under reduced pressure to give the methyl ester 8 (6.44 g, 97% yield) as a white solid. R f= 0.45 (SiO2, 50:50 hexane / EtOAc);

number

[0243] Methyl 9,10,12R-trihydroxystearate (9):

[0244] KOH (5.61 g, 100 mmol, 2.00 equiv.) was added to a rapidly stirred room temperature mixture of ricinoleic acid (14.9 g, 50.0 mmol) and water (500 mL) in a 1 L Erlenmeyer flask and then cooled to approximately 10°C. A solution of KMnO4 (13.4 g, 85.0 mmol, 1.70 equiv.) in water (250 mL) was added dropwise over 15 min. After stirring for an additional 10-15 min, the reaction was quenched by the addition of saturated aqueous Na2SO3 and then adjusted to pH ≤ 2 by the addition of concentrated HCl with the aid of a cooling bath. The white fluffy mixture was stirred at room temperature for 4 h, then the solid was collected by suction filtration and air dried overnight. The resulting white solid was hot gravity filtered with EtOH to give crude 9,10,12-trihydroxystearic acid, which was used without further purification.

[0245] Concentrated H2SO4 (0.13 mL, 2.50 mmol, 0.05 equiv) was added to a suspension of the above dihydroxy acid (6.33 g, 20.0 mmol) in MeOH (120 mL) and the resulting mixture was heated to reflux. After 14 h, the mixture was cooled to room temperature, concentrated on a rotary evaporator under reduced pressure, and the resulting residue was partitioned between hot EtOAc and saturated aqueous NaHCO3. The organic layer was washed with water (1 x 75 mL), brine, dried over Na2SO4, and concentrated on a rotary evaporator under reduced pressure. The resulting pale yellow solid was triturated four times with hot Et2O to give the methyl ester 9 (9.52 g, 55% yield) as a white solid. R f= 0.33 (SiO2, 50:50 hexane / EtOAc);

number

[0246] Methyl 9,10-dihexanoyloxystearate (10a):

[0247] DCC (2.27 g, 11.0 mmol, 2.20 equiv.) was added to a stirring ice-cold solution of hexanoic acid (1.28 g, 11.0 mmol, 2.20 equiv.) in CHCl (13 mL) in a round-bottom flask under argon, then the ice bath was removed and the resultant was stirred for 15 min. The reaction mixture was cooled again in an ice bath and diol 8 (1.65 g, 5.00 mmol) was added followed by DMAP (1.53 g, 12.5 mmol, 2.50 equiv.) and the reaction mixture was allowed to warm to room temperature over 14 h. The reaction mixture was diluted with EtO, stirred for 10 min, then filtered through Celite®. The filtrate was washed with 1M aqueous HCl (2 × 30 mL), 1M aqueous NaOH (2 × 30 mL), HO (1 × 30 mL), brine, dried over NaSO, and concentrated under reduced pressure on a rotary evaporator to give triester 10a (2.61 g, quantitative yield) as a clear, colorless oil. R f = 0.66 (SiO2, 70:30 hexane / EtOAc);

number

[0248] Methyl 9,10-dilinoleoyloxystearate (10b):

[0249] DCC (4.33 g, 21.0 mmol, 2.10 equiv.) was added to a stirred ice-cold solution of linoleic acid (5.89 g, 21.0 mmol, 2.20 equiv.) in CHCl (25 mL) in a round-bottom flask under argon, then the ice bath was removed and the resultant was stirred for 15 min. The reaction mixture was cooled again in an ice bath and diol 8 (3.30 g, 10.0 mmol) was added followed by DMAP (3.05 g, 25.0 mmol, 2.50 equiv.) and the reaction mixture was allowed to warm to room temperature over 14 h. The reaction mixture was diluted with hexanes, stirred for 10 min, then filtered through Celite®. The filtrate was concentrated on a rotary evaporator to give the crude product as a white semi-solid, which was purified by filtration through a plug of silica gel (95:5 hexanes / EtOAc) to give triester 10b (7.24 g, 85% yield) as a clear, colorless oil. R f = 0.57 (SiO2, 70:30 hexane / EtOAc);

number

[0250] Methyl 9,10,12R-trihexanoyloxystearate (11):

[0251] DCC (2.64 g, 12.8 mmol, 3.20 equiv.) was added to a stirring ice-cold solution of hexanoic acid (1.49 g, 12.8 mmol, 3.20 equiv.) in CHCl (13 mL) in a round-bottom flask under argon, then the ice bath was removed and the resultant stirred for 15 min. The reaction mixture was cooled again in an ice bath and triol 9 (1.39 g, 4.00 mmol) was added followed by DMAP (1.71 g, 14.0 mmol, 3.50 equiv.) and the reaction mixture was allowed to warm to room temperature over 14 h. The reaction mixture was diluted with hexanes, stirred for 10 min, then filtered through Celite®. The filtrate was washed with 1M aqueous HCl (2 × 30 mL), 1M aqueous NaOH (2 × 30 mL), HO (1 × 30 mL), brine, dried over NaSO, and concentrated under reduced pressure on a rotary evaporator to give the triester 11 (1.99 g, 78% yield) as a clear, colorless oil. R f = 0.77 (SiO2, 70:30 hexane / EtOAc);

number

[0252] 9,10-Dihexanoyloxystearic acid (12a):

[0253] A 2.0 M aqueous solution of KOH (0.91 mL, 1.82 mmol, 1.00 equiv) was added to a room temperature solution of triester 10a (1.05 g, 2.00 mmol, 1.10 equiv) in t-BuOH (7 mL) in a round-bottom flask under argon. After stirring for 20 h, the reaction mixture was acidified to pH ≤ 2 by addition of 3 M aqueous HCl and extracted with Et2O (3 x 20 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated on a rotary evaporator under reduced pressure. The crude residue was purified by flash column chromatography (90:5:5 to 85:10:5 hexanes / EtOAc / MeOH) to give the carboxylic acid 12a (802 mg, 86% yield) as a clear, colorless oil. R f = 0.22 (SiO2, 85:10:5 hexane / EtOAc / MeOH);

number

[0254] 9,10-Dilinoleoyloxystearic acid (12b):

[0255] A 2.0 M aqueous solution of KOH (3.00 mL, 6.00 mmol, 1.00 equiv.) was added to a room temperature solution of triester 10b (5.64 g, 6.60 mmol, 1.10 equiv.) in t-BuOH (7 mL) in a round-bottom flask under argon. After stirring for 20 h, the reaction mixture was acidified to pH ≤ 2 by addition of 3 M aqueous HCl and extracted with hexanes (3 x 75 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated on a rotary evaporator under reduced pressure. The crude residue was purified by flash column chromatography (90:10:0 to 85:10:5 hexanes / EtOAc / MeOH) to give the carboxylic acid 12b (2.39 g, 68% yield) as a clear, colorless oil. R f = 0.33 (SiO2, 85:10:5 hexane / EtOAc / MeOH);

number

[0256] 9,10,12R-trihexanoyloxystearic acid (13):

[0257] Aqueous 2.0 M KOH (1.47 mL, 2.94 mmol, 1.00 equiv) was added to a room temperature solution of tetraester 11 (1.98 g, 3.10 mmol, 1.10 equiv) in t-BuOH (10 mL) in a round-bottom flask under argon. After stirring for 20 h, the reaction mixture was acidified to pH ≤ 2 by addition of aqueous 3 M HCl and extracted with hexanes (3 × 30 mL). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated on a rotary evaporator under reduced pressure. The crude residue was purified by flash column chromatography (90:10:0 → 85:10:5 → 75:20:5 hexanes / EtOAc / MeOH) to give the carboxylic acid 13 (1.40 g, 78% yield) as a clear, colorless oil. R f = 0.32 (SiO2, 80:15:5 hexane / EtOAc / MeOH);

number

[0258] Example E: Synthesis of INT-D045 [ka] 2-((8S,9R,10S,11S,13S,14S,16R,17R)-9-fluoro-11,17-dihydroxy-10,13,16-trimethyl-3-oxo-6,7,8,9,10,11,12,13,14,15,16,17-dodecahydro-3H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl((R,Z)-12-(linoleoyloxy)octadec-9-en-1-yl)succinate (INT-D045):

[0259] Following general procedure D, dexamethasone (157 mg, 0.40 mmol), hemisuccinate 5f (310 mg, 0.48 mmol), DCC (99 mg, 0.48 mmol), DMAP (73 mg, 0.60 mmol) and CH2Cl2 (2 mL) afforded INT-D045 (278 mg, 68% yield) as a clear, colorless oil after flash column chromatography (SiO2, 80:20→50:50 hexanes / EtOAc). R f = 0.50 (SiO2, 50:50 hexane / EtOAc);

number

[0260] Example S: Synthesis of INT-D053 [ka] (1R,3S,Z)-3-Hydroxy-5-(2-((1R,3aS,7aR,E)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methyloctahydro-4H-inden-4-ylidene)ethylidene)-4-methylenecyclohexyl (R,Z)-12-acetoxyoctadec-9-enoate and (1S,5R,Z)-5 -Hydroxy-3-(2-((1R,3aS,7aR,E)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methyloctahydro-4H-inden-4-ylidene)ethylidene)-2-methylenecyclohexyl (R,Z)-12-acetoxyoctadec-9-enoate (INT-D053): JZ-25-057, 029

[0261] DCC (50 mg, 0.24 mmol, 1.20 equiv) was added to a stirred ice-cold 1:1 CHCl / THF (4 mL) solution of (12R)-acetoxyoleic acid (82 mg, 0.24 mmol, 1.20 equiv) in a round-bottom flask under argon, then the ice bath was removed and the resultant stirred for 15 min. The reaction mixture was cooled again in an ice bath and solid calcitriol (83 mg, 0.20 mmol) and DMAP (29 mg, 0.24 mmol, 1.20 equiv) were added. The reaction mixture was warmed for 14 h, diluted with EtOAc, stirred for 10 min, then filtered through Celite®. The filtrate was concentrated to give the crude product as a pale yellow oil, which was then purified by flash column chromatography (SiO, 80:20→65:35 hexanes / EtOAc) to give a ca. 1:1 mixture of 1- and 3-acetylated conjugates (61 mg, 41% yield) as a clear, colorless oil. R f = 0.33 (SiO2, 60:40 hexane / EtOAc);

number

[0262] Example T: Synthesis of INT-D068 [ka] (R,Z)-18-(((1R,3S,Z)-3-hydroxy-5-(2-((1R,3aS,7aR,E)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methyloctahydro-4H-inden-4-ylidene)ethylidene)-4-methylenecyclohexyl)oxy)-18-oxooctadec-9-en-7-yl linoleate and (R,Z )-18-(((1S,5R,Z)-5-hydroxy-3-(2-((1R,3aS,7aR,E)-1-((R)-6-hydroxy-6-methylheptan-2-yl)-7a-methyloctahydro-4H-inden-4-ylidene)ethylidene)-2-methylenecyclohexyl)oxy)-18-oxooctadec-9-en-7-yl linoleate (INT-D068):

[0263] DCC (50 mg, 0.24 mmol, 1.20 equiv) was added to a stirred ice-cold 1:1 CHCl / THF (4 mL) solution of (12R)-linoleoyloxyoleic acid (135 mg, 0.24 mmol, 1.20 equiv) in a round-bottom flask under argon, then the ice bath was removed and the resultant stirred for 15 min. The reaction mixture was cooled again in an ice bath and solid calcitriol (83 mg, 0.20 mmol) and DMAP (29 mg, 0.24 mmol, 1.20 equiv) were added. The reaction mixture was warmed for 14 h, diluted with EtOAc, stirred for 10 min, then filtered through Celite®. The filtrate was concentrated to give the crude product as a pale yellow oil, which was then purified by flash column chromatography (SiO, 95:5→90:10→70:30 hexanes / EtOAc) to give a ca. 1:1 mixture of 1- and 3-acetylated conjugates (75 mg, 39% yield) as a clear, colorless oil. R f = 0.26 (SiO2, 70:30 hexane / EtOAc);

number

[0264] Example W: Synthesis of disubstituted calcitriol, INT-D087

[0265] An example synthetic scheme for preparing a calcitriol lipid conjugate disubstituted with two lipid moieties is provided below: [ka]

[0266] Preparation of lipid nanoparticles (LNPs)

[0267] The lipids 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC), cholesterol, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol) (PEG-DSPE) or 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG-DMG) were dissolved in ethanol. DSPC, DMPC, PEG-DSPE and PEG-DMG were purchased from Avanti Polar Lipids (Alabaster, AL), and cholesterol was obtained from Sigma (St Louis, MO).

[0268] Lipid conjugates (referred to as "prodrugs" in the examples below) containing immunomodulatory agents (see Figures 10A-10M) were synthesized as described above and previously (see, e.g., WO2020 / 191477, which is incorporated by reference in its entirety).

[0269] Lipid conjugates (prodrugs) were dissolved in ethanol, isopropanol, DMSO or THF. LNPs were prepared by rapidly mixing DSPC or DMPC, cholesterol, prodrug, and PEG-DSPE (49 / 40 / 10 / 1 molar ratio) with phosphate-buffered saline (PBS) using a cross-junction mixer. The formulation was dialyzed against PBS to remove residual ethanol. When a peptide antigen, such as ovalbumin peptide 323-329 (OVA), was co-formulated into the LNPs, the peptide was dissolved in PBS and rapidly mixed with the lipid phase. Dialysis or tangential flow filtration was used to remove all unentrapped peptide.

[0270] To prepare LNPs containing mRNA encoding the antigen, an ionizable lipid such as INT-A002 (co-owned WO2021 / 026647, p. 32; application no. PCT / CA2020 / 051098, incorporated herein by reference), DSPC, cholesterol and PEG-DMG were dissolved in ethanol. Prodrugs were dissolved in ethanol, isopropanol, DMSO or THF. mRNA was dissolved in 10 mM citrate or 25 mM acetate buffer at pH 4.0. LNPs were prepared by rapidly mixing the lipid components in ethanol (45 / 8.5 / 35 / 1.5 / 10 molar ratio of INT-A002 / DSPC / cholesterol / PEG-DMG / prodrug) with the nucleic acid in buffered aqueous solution at room temperature with a volumetric flow ratio of 1:3 (ethanol to aqueous solution, combined flow rate 28 ml / min). The product was then dialyzed against 1× phosphate buffered saline (PBS) at pH 7.4 for 24 h to remove residual ethanol and increase the pH.

[0271] The physicochemical properties of the LNPs prepared as described above were then characterized. Particle size was determined by dynamic light scattering using a Malvern Zetasizer Nano ZS (Malvern, UK) after buffer exchange into phosphate buffered saline. Number-weighted size and distribution data were used. Lipid concentration was determined by measuring total cholesterol using a cholesterol E enzymatic assay kit from Wako Chemicals USA (Richmond, VA). mRNA capture was determined using a modified Quanti-iT Ribogreen assay (ThermoFisher, Waltham, MA). The LNP-mRNA system was incubated in the presence or absence of 1% Triton X-100 (Sigma-Aldrich, St. Louis, MO). Fluorescence intensity (Ex / Em: 480 / 520 nm) was compared to determine the % mRNA capture.

[0272] Example 1: LNPs efficiently accumulate in APCs in pancreatic islets, pancreatic lymph nodes, and spleen

[0273] This example demonstrates that LNPs provide a potential delivery platform for effectively delivering drugs and antigens to APCs located in a subject, which may have the added benefit of limiting side effects and reducing dose requirements by avoiding drug accumulation in other cell types and providing effective delivery to the APC population.

[0274] To determine LNP accumulation in pancreatic APCs, mice received two injections, 24 hours apart, of LNPs containing the fluorescent marker DiO, injected ip at a dose of 600 mg / kg. 48 hours after the first injection, animals were euthanized and islets and lymph nodes were harvested. Islets were hand-picked to 99% purity. Islets and lymph nodes were dispersed into single cell suspensions and stained for viability, CD45 (a pan-immune cell marker) and CD11b and CD11c (APC markers), and the number of DiO-positive cells was quantified by flow cytometry.

[0275] Figures 1A-1D show that upon simple injection, LNPs are taken up by APCs in both pancreatic islets and pancreatic lymph nodes. Furthermore, as shown in both lymph nodes and islets, there is limited uptake of LNPs by other cell types, including endocrine cells in pancreatic islets and non-APC immune cells (e.g., T cells) in lymph nodes (Table 1).

[0276] [Table 1]

[0277] To compare the effect of lipid composition of LNPs on targeting islet APCs, C57Bl / 6J male mice were injected with ionizable LNPs containing 150 mg / kg DSPC / cholesterol or DiO 24 hours prior to isolation of islets, pancreatic lymph nodes, and splenocytes. Islets were hand-picked to 99% purity. Tissues were dispersed into single cell suspensions and stained for viability, CD45 (a pan-immune cell marker) and CD11b and CD11c (APC markers), and the number of DiO-positive cells was quantified by flow cytometry.

[0278] Significant accumulation in APCs is observed for both liposomes and LNPs (DSPC / cholesterol or ionizable) in pancreatic islets, pancreatic lymph nodes and spleen (Figure 2). Example 2: LNPs can efficiently co-encapsulate and stably retain lipid conjugates (prodrugs) of tolerizing agents As shown in Example 1, both liposomes and LNPs (DSPC / cholesterol and ionizable) can accumulate in APCs in vivo. Another non-limiting embodiment of the present disclosure provides LNPs with two or more different immunomodulatory agents co-formulated therein. Prodrugs are uniquely suited for co-formulation due to their lipophilic nature. The results not only show that more than one prodrug can be formulated into LNPs with high encapsulation efficiency, but also show that the prodrugs can be stably retained in the LNPs. Moreover, the results herein show that the prodrug strategy is particularly suited for the delivery of a combination of two or more immunomodulatory agents.

[0279] As shown in Figure 3, both dexamethasone (D045) and calcitriol (D053, D068, D083) lipid prodrugs are stably retained within the LNPs after 2 hours of incubation in human plasma. While this example demonstrates the ability to prepare LNPs containing up to 20 mol% dexamethasone and calcitriol prodrugs, it has been shown that the LNPs of the present disclosure can incorporate up to 99 mol% of the prodrugs. Thus, using the prodrug strategy of the present disclosure, co-formulation of additional immunomodulatory agents (such as, but not limited to, acetylsalicylic acid, mycophenolic acid, sirolimus, and tacrolimus) can be achieved in the LNP formulations disclosed herein.

[0280] Results showing efficient entrapment of different lipid prodrugs in LNPs are shown in Table 2. Tables 3, 4 and 5 below demonstrate that different ratios of these prodrugs can be achieved without affecting entrapment efficiency.

[0281] [Table 2] For structures of dexamethasone and calcitriol lipid prodrugs, see WO2020 / 191477 (PCT / CA2020 / 000039; incorporated herein by reference) and Figures 10A-G herein.

[0282] [Table 3]

[0283] [Table 4]

[0284] [Table 5]

[0285] Example 3: Tolerization of APCs with calcitriol and dexamethasone prodrugs encapsulated in LNPs

[0286] This example demonstrates that the prodrug LNPs of the present disclosure are capable of tolerizing APCs ex vivo.

[0287] Bone marrow-derived dendritic cells (BMDCs) were treated with LNPs containing various calcitriol and dexamethasone prodrugs (alone or in combination) for 48 hours, after which BMDCs were challenged with lipopolysaccharide (LPS) stimulation for 24 hours to determine whether the prodrug formulations could prevent LPS-mediated activation (i.e., tolerize BMDCs).

[0288] In particular, mouse bone marrow cells were differentiated into APCs (dendritic cells in this case) according to standard procedures. Briefly, bone marrow-derived dendritic cells (BMDCs) were generated from bone marrow isolated from C57Bl / 6 male mice (Charles River) by culturing in RPMI1640 medium (supplemented with 10% FBS, 10 mM HEPES, 50 μM β-mercaptoethanol, 1×GlutaMAX, 0.1 mM non-essential amino acids, 1% Pen / Strep, 30 ng / mL GM-CSF, and 30 ng / mL IL-4). After 5 days of differentiation, BMDCs were treated with LNP-encapsulated prodrug or empty LNPs (LNP control) or vehicle for 48 hours, and then activated by addition of lipopolysaccharide (LPS, 10 ng / mL) for 24 hours. BMDCs were then harvested for downstream analysis.

[0289] To characterize cell surface markers on BMDCs (above), floating and loosely attached cells were harvested and washed twice with PBS. 500,000 cells were incubated with 1 μg / sample of Fc blocker (Invitrogen CD16, CD32 Cat. No. 14-016186) for 10 min at room temperature. CD80-PE (BioLegend Cat. No. 104707), CD86-PE-Cy7 (BioLegend Cat. No. 105013), MHC II-APC (BioLegend Cat. No. 107613), Fixable Viability-eF780 (Invitrogen Cat. No. 65-0865-14), and CD11c-BV510 (BioLegend Cat. No. 117337) were added to the samples according to the manufacturer's recommendations and incubated for 20 min at room temperature. Samples were then washed twice with FACS buffer (PBS, 2% FBS) before analysis by flow cytometry.

[0290] As shown in Figure 4, prodrug treatment effectively tolerized BMDCs ex vivo. Various tolerizing prodrugs and prodrug combinations inhibited LPS-induced expression of B7 molecules (CD80 and CD86) on the cell surface of DCs, consistent with induction of a tolerogenic or immature phenotype. Activated DCs have high expression of MHCII and B7 molecules, while tolerogenic / immature DCs may have reduced expression of B7. Furthermore, as shown in Figure 5, single prodrug treatments induced functional tolerogenic DCs in allogeneic mixed leukocyte reactions, with complete prevention of proliferation observed with some formulations. Notably, this is a very robust type of immune response (similar to organ transplant rejection), supporting the efficacy of these tolerizing prodrugs. Greater tolerance or suppression of T cell proliferation was observed when combinations of these immunomodulators were used (Figures 6 and 7).

[0291] Example 4: Prodrug-LNPs can reduce the proliferation of CD4+ T cells in an antigen-specific mouse model

[0292] Previous demonstrations of tolerizing APCs have been successful in allogeneic mouse models in which APCs and T cells are derived from MHC-mismatched donors. However, to demonstrate the ability of prodrugs to reduce antigen-specific stimulation, an antigen-specific mouse model was used. CD4 T cells were harvested from OT-II mice. These mice have T cell receptors on their CD4+ T cells engineered to bind and respond to ovalbumin 323-339 peptide fragment (OVA) when presented on the I-Ab MHC class II. When APCs are loaded with OVA and mixed with these T cells, robust proliferation of T cells is observed (similar to having an antigen-specific immune disorder).

[0293] For antigen-specific models, splenocytes were isolated from OT-II mice (B6.Cg-Tg(TcraTcrb)425Cbn / J; Jackson Laboratories). CD4+ T cells were purified by CD4 positive selection (EasySep™ Mouse CD4+ T Cell Isolation Kit, Catalog No. 19852, Stem Cell Technologies), labeled with 10 μM CFSE, and seeded at a density of 100,000 cells / well in 96-well U-bottom plates. On the same day, prodrug-treated C57BL / 6 BMDCs (above) were harvested, pulsed with / without OVA peptide, irradiated at 30 Gy, and co-cultured with CD4+ cells at T:DC ratios of 50:1, 10:1, and 5:1 in RPMI1640 medium (supplemented with 10% FBS, 10 mM HEPES, 50 μM β-mercaptoethanol, 10 mM sodium pyruvate, 1× GlutaMAX, and 1% Pen / Strep) for 3 days. Cells were then stained for viability (eBioscience Fixable Viability Dye eFlour780, Cat. No. 65-0865) and CD4 (eBioscience CD4 monoclonal antibody (RM4-5), Cat. No. 48-0042-82), and T cell proliferation was quantified by CFSE dilution by flow cytometry.

[0294] As shown in Figure 8, D053 significantly reduces the amount of T cell proliferation induced by OVA-loaded BMDCs. Furthermore, this response is preserved when the OVA peptide is co-formulated into LNPs containing the tolerizing prodrug (Figure 9).

[0295] Example 5: Decreased proliferation of CD4+ T cells is observed during co-treatment with prodrug-LNP and LNP-mRNA

[0296] In the previous examples, it was shown that immunomodulator-lipid conjugates (either in separate LNPs or co-loaded in the same LNPs as peptide antigens) can suppress the proliferation of Ova-specific CD4+ T cells. In addition to peptides or proteins, mRNA that is translated into its encoded protein and then processed along the antigen presentation pathway can be used as a source of antigen.

[0297] The mRNA used in this example was modified to minimize immune stimulation and encodes the full-length ovalbumin (Ova) protein (Trilink, L-7210). Bone marrow-derived dendritic cells (BMDCs) were prepared as described above and co-cultured with isolated CD4+ T cells (OT-II T cells) that specifically recognize the Ova323-339 epitope loaded on the H-2b MHC class II (C57BL / 6 haplotype). Proliferation was assessed based on CFSE dilution by flow cytometry. BMDCs pulsed with free Ova323-339 peptide (0.1 μg / mL) for 4 hours served as antigen-loaded control (free Ova peptide), and untreated BMDCs served as no antigen control. 48 hours prior to co-culture, BMDCs were treated with LNP-mRNA alone or LNPs containing LNP-mRNA and dexamethasone (D034) or calcitriol (D053) prodrugs.

[0298] As shown in Figure 11, LNP-mRNA induced similar levels of T cell proliferation as free peptide antigen. When BMDCs were co-treated with LNPs containing lipid conjugates D034 or D053, the level of proliferation was suppressed. This further demonstrates that prodrug-LNPs (i.e., lipid conjugate-LNPs) can be used to reduce the proliferation of antigen-specific T cells, regardless of whether the antigen is a peptide (Figure 8) or mRNA (Figure 11).

[0299] Example 6: LNPs can efficiently co-encapsulate mRNA and a prodrug of a tolerizing agent

[0300] In this example, the ability to co-load mRNA and immunomodulator-lipid conjugates into the same LNP is demonstrated. Up to three different prodrugs and up to 10 mol% are shown in Table 6. There was little effect on particle size (Z-Ave), polydispersity (PDI) or mRNA entrapment. Entrapment of the prodrug was also unaffected (Table 7).

[0301] [Table 6]

[0302] [Table 7]

[0303] Example 7: LNPs co-loaded with mRNA antigens and immunomodulatory agent-lipid conjugates induce distinct tolerogenic mechanisms in CD4 T cells

[0304] Upon recognition of antigens presented by antigen-presenting cells, several responses in T cells may contribute to antigen-specific immune tolerance, including alterations in T cell function (e.g., anergy or exhaustion), suppressed proliferation or loss of antigen-specific effector T cells, and proliferation of antigen-specific regulatory T cells, including Foxp3+Treg and IL-10-producing Tr1. To determine whether treatment of antigen-presenting cells with LNPs co-loaded with antigen-encoding mRNA and prodrugs can induce tolerogenic responses in antigen-specific T cells, the phenotype of Ova-reactive OT-II CD4+T cells was studied in response to co-culture with antigen-presenting cells (BMDCs) pretreated with LNPs co-loaded with Ova mRNA and various lipid conjugates.

[0305] BMDCs were generated from C57BL / 6 donor mice, treated (or not) with the LNP formulation for 48 hours, and matured with 10 ng / mL LPS for the final 24 hours. The mRNA encoding full-length Ova in the LNP formulation of this example was modified to minimize immune stimulation (L-7210, TriLink), and BMDCs pulsed with free Ova323-339 peptide (0.1 μg / mL) for 4 hours served as antigen-loaded controls. Splenic CD4+ T cells were isolated from OT-II mice (I-Ab:Ova323-339-specific TCR transgenic mice, Jackson Laboratory) using the CD4 Easy Sep Kit (Stem Cell Technologies) and labeled with CFSE. On the day of co-culture, BMDCs were washed twice and co-cultured with CD4 T cells at a density of 100,000 CD4 cells per well, at a ratio of 1:5 BMDC:CD4 cells. T cells were stained after 3 days of co-culture with various markers (fixable viability dye, CD4, PD1, CTLA4, CD25, Foxp3, CD49b, and LAG3) and assessed by flow cytometry.

[0306] As shown in Figure 12, OT-II CD4+ T cells proliferated significantly in response to BMDCs pretreated with Ova mRNA-only LNPs (comparable to induction by BMDCs loaded with free Ova323-339 peptide), whereas OT-II CD4+ T cells minimally proliferated in response to untreated (no antigen) BMDCs. Furthermore, in the absence of immunomodulator-lipid conjugates, there was no change in the frequency of CD49b+LAG3+ (Tr1 marker) cells, CD25+Foxp3+ (Treg marker) cells, and no induction of PD1+ and CTLA-4+ cells among OT-II CD4+ cells stimulated with BMDCs presenting Ova antigen. In contrast, BMDCs pretreated with LNPs co-loaded with Ova mRNA and lipid conjugates induced various changes in CD4+ T cell responses; some LNP formulations reduced antigen-specific proliferation indicating a decrease in the proliferation of effector CD4+ T cells (top panel); other LNP formulations increased the frequency of CD4+ cells expressing CTLA-4 and PD1 (second and third panels); whereas other formulations significantly induced the frequency of Tregs (CD25+Foxp3+, fourth panel) or Tr1s (CD49b+LAG3+, last panel). Thus, LNPs co-loaded with both modified mRNA-encoded antigen and immunomodulatory lipid conjugates can induce BMDCs to induce distinct tolerogenic responses (anergy, regulatory cell proliferation, limited effector cell proliferation) in antigen-specific CD4+ T cells.

[0307] Figures 13A and 13B show that delivery of an mRNA-encoded antigen alone in LNPs (albeit modified to minimize the innate immune response to the mRNA) resulted in the induction of Th1 and Th2 cytokine secretion, whereas co-delivery of various immunomodulatory prodrugs inhibited this response, and in many cases even suppressed it below baseline levels (as in the case of IFNγ and TNFα). Thus, co-delivery of immunomodulatory prodrugs with modified mRNA-encoded antigens to BMDCs reduces antigen-specific Th1 and Th2 responses.

[0308] Example 8: LNPs co-loaded with mRNA-encoded antigen and immunomodulatory lipid conjugates can induce antigen-specific tolerance in vivo.

[0309] This example demonstrates that LNPs co-loaded with modified mRNA-encoded antigen and immunomodulatory lipid conjugates are capable of inducing antigen-specific tolerance in vivo.

[0310] Female C57BL / 6J mice were injected with LNPs loaded with modified Ova mRNA (L-7210, TriLink) with or without D034 co-loaded on the same particle. Mice were injected ip once every other week with LNPs (administered as 10 μg mRNA / mouse) or buffer alone for a total of three injections. Ova-specific IgG1 antibodies were measured from serum collected 2 weeks after the last injection by mouse anti-Ova IgG1 ELISA (Cayman Chemical, catalog no. 500830-96).

[0311] As shown in FIG. 14, injection of LNPs with Ova mRNA into mice induces high levels of anti-Ova antibodies in serum, indicating an antigen-specific immune response to the Ova antigen. In contrast, mice receiving LNPs co-loaded with Ova mRNA and lipid conjugate D034 robustly suppressed anti-Ova IgG1 levels. Thus, humoral immune responses to the Ova antigen encoded by the modified mRNA are reduced by co-loading LNPs with immunomodulatory lipid conjugates. Similar results (not shown) were observed for LNPs co-formulated with D034 in combination with additional lipid conjugates (D034+D053, and D034+D053+D097).

[0312] Although the invention has been described and illustrated with reference to the foregoing examples, it will be apparent that various modifications and changes can be made without departing from the invention.

Claims

1. a lipid conjugate comprising an immunomodulatory agent covalently linked to a lipophilic moiety by a cleavable linkage or via a cleavable linker; and Antigens and / or one or more nucleic acids encoding the antigens, wherein the antigens are proteins, polypeptides, peptides, lipoproteins, glycolipids, polynucleotides, or polysaccharides. an immunomodulatory combination comprising: An immunomodulatory combination wherein the lipid conjugate and the antigen and / or one or more nucleic acids encoding the antigen are formulated in separate delivery vehicles or are co-formulated in the same delivery vehicle.

2. the delivery vehicle is a lipid nanoparticle and / or a liposome; Optionally, the delivery vehicle is a lipid nanoparticle that delivers to antigen-presenting cells (APCs). The immunomodulatory combination of claim 1.

3. the antigen or one or more nucleic acids encoding the antigen are entrapped within a lipid nanoparticle or liposome and have a net charge opposite to the net charge of the lipid in the lipid nanoparticle; or the antigen is lipophilic and is incorporated into the lipid compartment of a lipid nanoparticle or liposome, or The antigen is hydrophilic and is entrapped in liposomes that contain an aqueous core; The immunomodulatory combination according to claim 2.

4. 3. The immunomodulatory combination of claim 1 or 2, wherein the lipid conjugate is co-formulated in the same delivery vehicle as the antigen or one or more nucleic acids encoding the antigen.

5. 10. The immunomodulatory combination of claim 1, wherein the lipid conjugate is a first lipid conjugate, and the immunomodulatory combination further comprises a second lipid conjugate, wherein the second lipid conjugate comprises an immunomodulatory agent covalently linked to a lipophilic moiety by a cleavable linkage or via a cleavable linker, wherein the immunomodulatory agent of the second lipid conjugate is different from the immunomodulatory agent of the first lipid conjugate, and wherein the first lipid conjugate and the second lipid conjugate are formulated in separate delivery vehicles or are co-formulated in the same delivery vehicle; In some cases, the immunomodulatory combination comprises a plurality of lipid conjugates, wherein the plurality of lipid conjugates comprises 3, 4, 5, 6, 7, 8, 9, or 10 immunomodulatory agents, wherein each immunomodulatory agent of the plurality is different, and wherein each lipid conjugate of the plurality is formulated independently in a delivery vehicle separate from the other components of the immunomodulatory combination or is formulated with one or more of the other components of the immunomodulatory combination.

6. 6. The immunomodulatory combination of claim 5, wherein the immunomodulatory agent of the second lipid-conjugate targets a different immune pathway than the immunomodulatory agent of the first lipid-conjugate.

7. 7. The immunomodulatory combination of claim 5 or 6, wherein the first lipid conjugate and the second lipid conjugate are co-formulated in the same delivery vehicle.

8. 7. The immunomodulatory combination of claim 1, 2, 5 or 6, wherein each immunomodulatory agent is a tolerogenic agent or an anti-inflammatory agent.

9. 7. The immunomodulatory combination of claim 1, 2, 5 or 6, wherein each immunomodulatory agent is an immunostimulant or an immunosuppressant.

10. 7. The immunomodulatory combination of claim 1, 2, 5, or 6, wherein each immunomodulatory agent is independently a nonsteroidal anti-inflammatory drug (NSAID), an inflammasome inhibitor, a Janus kinase (JAK) inhibitor, a corticosteroid, an mTOR inhibitor, a DMARD (disease-modifying antirheumatic drug), a calcineurin inhibitor, or a vitamin D receptor agonist.

11. Each immunomodulatory agent may independently be selected from the group consisting of prednisone, budesonide, prednisolone, methylprednisolone, hydrocortisone, cortisone, betamethasone, budesonide, triamcinolone, flunisolide, beclomethasone, fluticasone, mometasone, fludrocortisone, flumethasone, triamcinolone acetonide, isoflupredone, corticosterone, desoxycortone acetate, desoxycortone enanthate, 11-deoxycorticosterone, 11-deoxycortisol, aldosterone, dexamethasone, calcitriol, acetophenone, acetophenone, acetophenone-3, acetophenone-4, acetophenone-5, acetophenone-6, acetophenone-7, acetophenone-8, acetophenone-9, acetophenone-10, acetophenone-11, acetophenone-12, acetophenone-13, acetophenone-14, acetophenone-15, acetophenone-16, acetophenone-17, acetophenone-18, acetophenone-19, acetophenone-20, acetophenone-21, acetophenone-22, acetophenone-23, acetophenone-24, acetophenone-25, acetophenone-26, acetophenone-27, acetophenone-28, acetophenone-29, acetophenone-30, acetophenone-31, acetophenone-32, acetophenone-33, acetophenone-34, acetophenone-35, acetophenone-36, acetophenone-37, acetophenone-38, acetophen Tyrosalicylic acid, salicylate, mycophenolic acid, sirolimus, tacrolimus, cholecalciferol, calcifediol, alfacalcidol, calcipotriol, falecalcitriol, maxacalcitol, paricalcitol, doxercalciferol, 22-oxacalcitriol, tacalcitol, eldecalcitol, elocalcitol, inecalcitol, becocalcidiol, seocalcital, ergocalciferol, lexacalcitol, retinoic acid, cyclophosphamide Nitrogen mustard, filgotinib, baricitinib, tofacitinib, ruxolitinib, upadacitinib, oclacitinib, peficitinib, fedratinib, delgocitinib, deuclavacitinib, abrocitinib, auranofin, apremilast, azathioprine, chloroquine, hydroxychloroquine, cyclosporine, leflunomide, methotrexate, minocycline, sulfasalazine, salicylic acid, diflunisal, salsalate, naproxen, ibuprofen, oxaprozin, loxoprofen, and zalto Profen, indomethacin, tolmetin, sulindac, etodolac, ketorolac, diclofenac, aceclofenac, bromfenac, nabumetone, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, phenylbutazone, celecoxib, firocoxib, parecoxib, etoricoxib, clonixin, licofelone, MCC950, glyburide, CY-09, tranilast, oridonin, BOT-4-one (2-cyclohexylimino-6-methyl-6,7-dihydro-5H-benzo[1,3] oxathiol-4-one), INF39 (ethyl 2-(2-chlorobenzyl)acrylate), MNS (3,4-methylenedioxy-β-nitrostyrene), fenamic acid, beta-hydroxybutyric acid, quercetin, JC-171, ibrutinib, OLT1177, FC11A-2, INF58, JC124, or ethyl 2-((2-chlorophenyl)(hydroxy)methyl)acrylate.

12. 10. The immunomodulatory combination of claim 1, wherein the antigen is a first antigen, and the immunomodulatory combination further comprises a second antigen or one or more nucleic acids encoding the second antigen, wherein the first antigen is different from the second antigen; the second antigen or one or more nucleic acids encoding the second antigen are formulated in a delivery vehicle separate from the other components of the immunomodulatory combination or are formulated together with one or more of the other components of the immunomodulatory combination; In some cases, the immunomodulatory combination comprises multiple antigens, or one or more nucleic acids encoding multiple antigens, or a combination of antigens and nucleic acid(s) encoding the antigens to provide the multiple antigens, wherein the multiple antigens include 3, 4, 5, 6, 7, 8, 9, or 10 antigens, and each antigen or nucleic acid encoding an antigen is formulated independently in a delivery vehicle separate from the other components of the immunomodulatory combination or is formulated with one or more of the other components of the immunomodulatory combination.

13. 13. The immunomodulatory combination of claim 12, wherein the one or more nucleic acids encoding the first antigen and the one or more nucleic acids encoding the second antigen are comprised within a single nucleic acid, and the first antigen and the second antigen are co-formulated within the same delivery vehicle.

14. 14. The immunomodulatory combination of claim 1, 2, 5, 6, 12 or 13 for use in treating a subject with an antigen-induced disorder or an unwanted antigen-driven immune response, or for use in the manufacture of a medicament for treating a subject, Optionally, the antigen-induced disorder or unwanted antigen-driven immune response is selected from autoimmune diseases (T cell and / or antibody responses against self-antigens), allergic diseases (T cell and IgE responses against environmental or food antigens), transplantation (T cell responses against major and minor histocompatibility antigens in donor tissues / organs / cells), anti-drug antibody responses (antibody responses that reduce the effectiveness of therapeutic drugs), gene / protein replacement therapy (T cell / antibody responses against therapeutically replaced proteins in genetic protein deficiencies), In some cases, the antigen-induced disorder or unwanted antigen-driven immune response is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, myelin oligodendrocyte glycoprotein antibody disorder, vitiligo, type 1 diabetes, primary biliary cholangitis, anti-GBM nephritis / Goodpasture's disease, celiac disease, psoriasis, myasthenia gravis, immune thrombocytopenic purpura, Graves' disease, neuromyelitis optica, pemphigus vulgaris, bullous pemphigoid, cicatricial pemphigoid, systemic lupus erythematosus (SLE), lupus, including SLE, autoimmune liver disease, myositis, Evans syndrome, transverse myelitis, Guillain-Barré syndrome, warm autoimmune hemolytic anemia, chronic inflammatory demyelinating polyposis, and the like. Immunomodulatory combinations selected from neuropathy, autoimmune autonomic neuropathy, autoimmune angioedema, Hashimoto's thyroiditis, Lambert-Eaton syndrome, peanut / regum allergy, tree nut allergy (antigens derived from any of cashews, pistachios, hazelnuts, walnuts, and almonds), egg allergy, milk allergy, soy allergy, fish allergy, shellfish allergy, sesame allergy, wheat allergy, allergic airway disease, and allergies caused by environmental allergens (antigens derived from pollen, dust, pet dander, mold, and cockroaches).

15. 10. A method for treating a subject having an antigen-induced disorder or an unwanted antigen-driven immune response comprising administering to the subject the immunomodulatory combination of claim 1, wherein the antigen, or one or more nucleic acids encoding the antigen, and the lipid conjugate are administered together or sequentially; optionally, the antigen-induced disorder or unwanted antigen-driven immune response is selected from autoimmune disease (T cell and / or antibody responses against self-antigens), allergic disease (T cell and IgE responses against environmental or food antigens), transplantation (T cell responses against major and minor histocompatibility antigens in donor tissues / organs / cells), anti-drug antibody responses (antibody responses that reduce the effectiveness of therapeutic drugs), or gene / protein replacement therapy (T cell / antibody responses against therapeutically replaced proteins in genetic protein deficiencies); Optionally, the antigen-induced disorder is selected from the group consisting of multiple sclerosis, rheumatoid arthritis, myelin oligodendrocyte glycoprotein antibody disorder, vitiligo, type 1 diabetes, primary biliary cholangitis, anti-GBM nephritis / Goodpasture's disease, celiac disease, psoriasis, myasthenia gravis, immune thrombocytopenic purpura, Graves' disease, neuromyelitis optica, pemphigus vulgaris, bullous pemphigoid, cicatricial pemphigoid, lupus including systemic lupus erythematosus (SLE), autoimmune liver disease, myositis, Evans syndrome, transverse myelitis, Guillain-Barre syndrome, warm autoimmune hemolytic anemia, chronic inflammatory demyelinating polyneuropathy, The method is selected from allergies caused by autoimmune dysautonomia, autoimmune angioedema, Hashimoto's thyroiditis, Lambert-Eaton syndrome, peanut / regum allergy, tree nut allergy (antigens derived from any of cashews, pistachios, hazelnuts, walnuts, and almonds), egg allergy, milk allergy, soy allergy, fish allergy, shellfish allergy, sesame allergy, wheat allergy, allergic airway disease, or environmental allergens (antigens derived from pollen, dust, pet dander, mold, and cockroaches).

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