CN116472040A

CN116472040A - Methods, compositions and therapeutic vaccines for the treatment of autoimmune diseases and allergies

Info

Publication number: CN116472040A
Application number: CN202180073143.3A
Authority: CN
Inventors: 王天欣
Original assignee: Individual
Current assignee: Individual
Priority date: 2020-11-07
Filing date: 2021-10-06
Publication date: 2023-07-21

Abstract

Compositions, agents, formulations, and methods for treating diseases, including autoimmune diseases and allergies are described. The composition comprises an antigen that causes immune intolerance and an immunosuppressant in a slow release formulation. Methods, compositions, formulations and agents for treating allergy are described, as are combinations of allergens and immune activity enhancers in sustained release formulations for administration to a subject in need thereof.

Description

Methods, compositions and therapeutic vaccines for the treatment of autoimmune diseases and allergies

Cross Reference to Related Applications

The present application claims priority for U.S. provisional patent application 63/111018, U.S. provisional patent application 63/121974, U.S. provisional patent application 63/130396, U.S. provisional patent application 12/23, 2020. It is also part of U.S. application Ser. Nos. 15/723,173, 16/380,951, 16/029,594, 17/344,932, 16/566,716, 16/819,168 and 17/385,908. The entire disclosure of the prior application is considered to be part of the disclosure of the present application and is incorporated herein by reference.

Background

Technical Field

The present invention relates to compositions, formulations and agents for the treatment of diseases including autoimmune diseases and allergies. Methods of treating autoimmune diseases and allergies are also disclosed. Compositions for the treatment of autoimmune diseases and allergies involve the combination of a disease-associated antigen and an immunosuppressant in a slow-release formulation. Methods, compositions, formulations, and agents for treating allergy also involve administering a combination of an allergen and an immune activity enhancer in a sustained release formulation to a subject in need thereof.

Background informationAn immune response is necessary to prevent potentially pathogenic microorganisms. However, unwanted immune activation can cause deleterious processes, resulting in damage or destruction of the own tissues. Unwanted immune activation occurs, for example, in autoimmune diseases, wherein antibodies and/or T lymphocytes react with self-antigens to damage body tissue. The same is true of allergic reactions, which are characterized by an excessive immune response to certain environmental substances, possibly leading to inflammatory reactions and thus tissue destruction. This is also the case in transplanted organ rejection, which is mediated by alloreactive T cells that recognize donor alloantigens or xenogeneic antigens present in the host. Immune tolerance is the lack of availability of specific immune responses to antigens where immune responses typically occur. Typically, to induce tolerance, exposure to tolerance antigens is necessary, which can lead to death or functional inactivation of certain lymphocytes. This process generally accounts for tolerance or self-tolerance to autoantigens. Immunosuppressants may be used to prevent or reduce unwanted immune responses, for example, in treating patients suffering from autoimmune diseases or receiving allografts. Generation and failure Conventional strategies for immune response-related immunosuppression are based on broad-spectrum immunosuppressive drugs. Unfortunately, the use of broad-spectrum immunosuppressants carries the risk of serious side effects such as tumors, infections, nephrotoxicity and metabolic disorders. Thus, new immunosuppressive therapies would be beneficial.

Disclosure of Invention

The inventors' prior U.S. application Ser. Nos. 15/723,173, 16/380,951, 16/029,594, 16/566,716, 16/819,168, 17/344,932 and 17/385,908 disclose methods, agents, devices and compositions for treating autoimmune diseases and allergies that prevent the production of antigen-specific antibodies, including anti-drug antibodies. Agents in the previous U.S. application include antigen-drug conjugates, such as antigen-immunosuppressive molecule conjugates. The agents and compositions may be mixtures of antigens and immunosuppressive molecules or conjugates thereof. They may be in the form of linear polymers, microparticles, nanoparticles, liposomes, implants or transdermal drug delivery systems such as transdermal patches. Examples of antigens include B cell antigens, T cell antigens in the form of MHC-peptide complexes or antigenic peptides (or derivatives thereof) that can bind to MHC. The carrier system can be used in previous and current applications to construct conjugates. For example, liposomes or microparticles or nanoparticles can be used as carriers. The antigen may be immobilized on the surface of a liposome or particle, and the effector molecule (e.g., α -gal, rhamnose, immunosuppressive cytokine, tregitope peptide, toxin, siRNA or miRNA, etc., immunosuppressant, antisense molecule) may be encapsulated inside a liposome or particle or co-immobilized on the surface of a liposome or particle. The carrier may also be a linear or branched polymer such as dextran, hyaluronic acid, heparin, chondroitin sulfate and polypeptides. Both antigens and effector molecules (e.g., immunosuppressants) may be bound to the polymer. They may be administered to a subject in need of treatment for autoimmune diseases and allergy or inhibition of anti-drug antibody production or induction of antigen-specific immune tolerance by administering the conjugates to the subject (e.g., subcutaneous or intralymphatic injection or application to the skin such as upper arm skin). Further details can be found in the foregoing disclosure.

In one aspect, compositions and formulations are disclosed that comprise one or more disease-causing antigens and one or more immunosuppressants in a sustained (extended) release (sustained release) system, such as an in situ gelling system or implant, to treat a disease selected from the group consisting of autoimmune diseases, allergies and anti-drug antibodies. The invention also discloses a method of treating autoimmune diseases or allergies or inhibiting the production of anti-drug antibodies or inducing antigen-specific immune tolerance by administering the above compositions and formulations as an injection to a subject.

In one aspect, compositions and formulations are disclosed that comprise one or more disease-causing antigens and vaccine adjuvants (e.g., TLR receptor agonists, STING receptor agonists) in a sustained (prolonged) release system, such as an in situ gelling system or implant, to treat the disease condition selected from autoimmune diseases, allergies, and anti-drug antibodies. The invention also discloses a method of treating autoimmune diseases or allergies or inhibiting the production of anti-drug antibodies or inducing antigen-specific immune tolerance by administering the above compositions and formulations as an injection to a subject.

In one aspect, compositions and formulations are disclosed that comprise one or more disease-causing antigens in a sustained (extended) release system (e.g., an in situ gelling system or implant) to treat a symptom selected from the group consisting of autoimmune disease, allergy, and anti-drug antibodies. The compositions and formulations are free of vaccine adjuvant type agents and immunosuppressants. The invention also discloses a method of treating autoimmune diseases or allergies or inhibiting the production of anti-drug antibodies or inducing antigen-specific immune tolerance by administering the above compositions and formulations as an injection to a subject.

The compositions and formulations of the present invention may be in gel form or in high viscosity liquid or solid form or in the form of implants. Gel, as used herein, refers to a solid, jelly-like material that can have characteristics ranging from soft, weak to hard, and tough. As is well known in the art, a gel is a non-fluid colloidal network or polymer network that expands throughout its volume by a fluid. Hydrogels are gels that contain a network of hydrophilic polymer chains, sometimes in the form of a colloidal gel with water as the dispersing medium. Hydrogels are highly absorbent and may contain a high degree of water, for example greater than 90% water. In some embodiments, the gels described herein comprise a natural or synthetic polymer network. In some embodiments, the gel comprises a hydrophilic polymer matrix. In other embodiments, the gel comprises a hydrophobic polymer matrix. In some embodiments, the gel has flexibility very similar to natural tissue. In certain embodiments, the gel is biocompatible and absorbable. In certain embodiments, the gel forms after administration to a patient.

The compositions and formulations may contain a viscosity increasing agent to increase its viscosity, which is used as a slow release formulation. In certain embodiments, the formulation is a viscous liquid. In certain embodiments, the injectable formulation has a viscosity of greater than 5,000cps at room temperature. In certain embodiments, the injectate has a viscosity of greater than 50,000cps at room temperature. In certain embodiments, the injectate has a viscosity of greater than 500,000cps at room temperature. In certain embodiments, the injection has a viscosity of 5,000,000cps at room temperature. Examples of viscosity enhancers can be readily found from known pharmaceutically acceptable excipients, such as hyaluronic acid (linear or cross-linked), HPMC, MC, CMC, starch and carbomers. In some embodiments, the viscosity enhancing agent is biodegradable.

In some preferred embodiments, the compositions of the invention are in a sustained release system to release the active agent over an extended period of time, e.g., 50% of the agent (e.g., antigen, immunosuppressant, TLR agonist) is released over days to weeks. The formulation is an extended (sustained) release formulation. In some preferred embodiments, the composition of the invention is in an in situ gelling system and the formulation is the drug-loaded in situ gelling formulation. In situ gelling systems are typically polymeric formulations that are in liquid form prior to entry into the body, but which convert to gel form under physiological conditions. The sol-gel transition depends on a combination of one or more different stimuli, such as pH change, temperature adjustment, solvent exchange, uv irradiation and the presence of specific ions or molecules. Medicaments having such properties The delivery system can be widely used for preparing sustained delivery carriers of bioactive molecules. Some important advantages of these intelligent systems are ease of application and reduced frequency of administration, as well as protection of the drug from changes in environmental conditions. A variety of natural and synthetic polymers may undergo in situ gel formation and may be used. Pectin, xyloglucan, gellan gum, chitosan and alginic acid are some of these natural polymers. Pectin gelation occurs in the presence of calcium ions. Xyloglucan exhibits thermoreversible gelation with body temperature. Aqueous solutions of alginates are useful in the addition of divalent and trivalent metal ions (e.g., ca in body fluids ²⁺ ) After which a firm gel is formed. Examples of alginates that may be used include sodium alginate, potassium alginate, ammonium alginate and other pharmaceutically acceptable amine salts of alginic acid. For example, sodium alginate and hydroxypropyl methylcellulose may be used in the in situ gel formulation. In situ gel formation of gellan gum occurs due to temperature regulated or induced cations. Temperature and ionic conditions (Ca in body fluids ²⁺ ) Causing the gellan gum aqueous solution to transition to the gel state. Carbopol (polyacrylic acid, carbomer) is a well known pH dependent polymer that remains in solution at acidic pH values but forms a low viscosity gel at alkaline pH values. In situ gels may be formulated using carbomers and hydroxypropyl methylcellulose (HPMC). The latter is used to impart viscosity to a carbopol (also known as carbomer) solution while reducing its acidity. The aqueous solution of Carbopol-HPMC system is also an in situ gelling system. Pluronic F-127 is a triblock copolymer with nonionic properties that can be gelled in situ by temperature changes. In situ gels can be prepared with Carbopol934, HPMC. The aqueous chitosan solution forms a hydrogel-like precipitate at a pH exceeding 6.2. The addition of a multi-alkoxide with a single anionic head, such as glucose phosphate, to an aqueous chitosan solution can convert the cationic polysaccharide solution into a thermosensitive pH-dependent gel. The liquid form of this formulation (at room temperature) becomes a gel implant when injected in vivo. Examples of these may be found in PMID:25237648, and can be readily used in the present invention. In some embodiments, the gel is made of a hyaluronic acid gel with an optional calcium salt or iron salt, e.g., calcium ions And the hyaluronic acid gel material is characterized in that: comprises hyaluronic acid and CaCl ₂ Or FeCl ₃ And deionized water 0.01-10 weight percent: 0.01 to 10:100. examples of in situ gelling polymers used in the in situ gelling system include chitosan, alginic acid, xyloglucan, gellan gum, sodium hyaluronate, pectin, hydroxypropyl methylcellulose (HPMC), methylcellulose (MC), carboxymethylcellulose, cellulose Acetate Phthalate (CAP), PGA, prolifeprospan, carbopol, pluronics, poly (lactide-co-glycolide) (PLGA), poly (D, L-lactide-co-hydroxymethylglycolide) (PLHMGA).

Drug loaded in situ gelling systems may use pH triggered in situ gelling polymers: the pH triggered in situ gelling system is a solution that converts to a gel phase when exposed to the pH of body fluids, such as carboxymethyl cellulose, hyaluronate, cellulose acetate phthalate, and carbomers. The cellulose acetate phthalate latex remains a free flowing solution at an acidic pH (e.g., about pH 4) and converts to a gel at a neutral pH (about pH 7). Polyacrylic acid, commercially known as Carbopol, is a widely used polymer that undergoes a sol-to-gel transition in aqueous solution when the pH is raised above its pKa of about 5.5. The formulation of these types of systems can have a low pH (4-5) to maintain the solution state and change to gel as a result of pH changes into the body. Polyacrylic acid (e.g 934 HPMC (e.g., methocel k 4M) may be used as a gelling agent and HPMC (e.g., methocel k 4M) may be used as a viscosifier. Polyacrylic acid (Carbopol) may be used as a gelling agent in combination with chitosan (as a viscosity increasing agent). In situ gelling systems based on 0.4% w/v Carbopol/0.5% w/v chitosan are liquid at room temperature and formulation pH values such as pH6.0 and rapidly transition to a viscous gel phase at in vivo pH 7.4.

Drug loaded in situ gelling systems may use temperature triggered in situ gelling polymers: the temperature triggered in situ gelling polymer remains liquid at low temperatures (below 20 ℃) and gels at physiological temperatures (35-37 ℃). The following are some of the temperature-triggered in-situ gel polymer systemsExamples: poloxamer: poloxamers, commercially known asIs a thermally reversible polymer commonly used to form thermosensitive in situ gelling systems. When heated from 4 ℃ to 23 ℃ or higher, an aqueous solution of Pluronic F127 or Poloxamer 407 having a concentration of 15% or more is converted from a low viscosity solution to a semi-solid gel. For example, the system may comprise 20% w/w poloxamer 407 and 10% w/w poloxamer P188. The low viscosity aqueous solution of poloxamer 407 (P407), at a concentration of more than or equal to 18% w/w (PEO to PPO ratio of 7:3), can delay the gelation temperature by several degrees Celsius at ambient temperature and by adding hyaluronic acid (HA in the poloxamer mixture), and at a specific concentration of poloxamer/HA, a thermoreversible gel with gelation temperature close to body temperature can be obtained. Viscosity enhancers (HPMC, MC and CMCNa) can be added to 15% w/w PF-127 to form hydrogels, e.g., a 15% PF-127 formulation containing 3% methylcellulose can be used as a temperature triggered in situ gelling system loading drug.

Poloxamine is another temperature-initiated in situ gelling system commonly known as Tetronics (tetrafunctional block copolymers of ethylene oxide and propylene oxide), such as tetra-oligomeric lactide copolymers (from1307 and pure L-lactide). Another temperature at which the in situ gelling system is initiated is the cellulose derivative: ethyl (hydroxyethyl) cellulose, methylcellulose and HPMC are some cellulose derivatives used as in situ gelling polymers. Aqueous solutions of ethyl (hydroxyethyl) cellulose (EHEC) exhibit thermal gelation. Sodium dodecyl sulfate or cetyltriammonium bromide is added and the EHEC (1% -4% w/w) solution undergoes sol-to-gel phase change when heated to 30-40℃ and forms a hard and transparent gel. Some cellulose derivatives remain liquid at low temperatures and become gels upon heating, for example aqueous solutions of methylcellulose and HPMC undergo a phase transition between 40-50℃and 75-90℃respectively, to become gels. However, the phase transition temperature of methylcellulose and HPMC is higher than physiological temperature, but can be increased by polymerizationThe chemical or physical changes in the material are reduced. For example, the addition of NaCl to methylcellulose or the reduction of the molar substitution of HPMC with hydroxypropyl groups, the phase transition temperatures of these polymers can be reduced to 32-34℃and 40℃respectively.

By adding different proportions of fructose and trisodium citrate dihydrate (SC), the gelation temperature of the 1% methylcellulose solution can be reduced to a physiological temperature, i.e. 37 ℃. Methylcellulose (1%) and fructose (10%) may be added with 1-5% SC to become a temperature triggered in situ gelling system.

Xyloglucan, a polysaccharide extracted from tamarind seeds, has been approved for use as a food additive. Beta-galactosidase partially degrades xyloglucan to a galactose removal rate of >35%, exhibiting thermo-reversible gelation in dilute aqueous solutions. The sol-gel transition temperature of xyloglucan varies with the degree of galactose elimination and polymer concentration and is inversely related, for example, when galactose removal rate increases from 35% to 58%, a decrease in the sol-gel transition temperature of xyloglucan from 40 ℃ to 5 ℃ is observed. Xyloglucan forms a gel by lateral stacking of rod-like chains. In situ gelling formulations based on 1.5% w/w xyloglucan showed a similar reaction to 25% w/w Pluronic F127 gel.

The well-known thermosensitive polymer poly (N-isopropylacrylamide) (PNIPAAm) has a thermally reversible phase transition temperature of 32 ℃. Such in situ gel forming polymers have been utilized because of their phase transition temperature being closer to the surface temperature of the human body.

Methylcellulose, HPMC, CMC, mannitol and sorbitol may be used as viscosity enhancers added to the in situ gelling polymer. A temperature-triggered in situ gelling system based on a heat-sensitive neutral solution of chitosan/polyol combination (DOI: 10.1016/s0142-9612 (00) 00116-2) is also one that can be used.

Drug loaded in situ gelling systems may use ion triggered in situ gelling polymers. These include polymers whose solution viscosity increases upon exposure to the ionic concentration of body fluids such as tears. It is also known as osmotically induced gelation. The ion-sensitive polymer can be crosslinked with cations (monovalent and divalent) existing in the tears of the ocular surface to improve the stagnation of the drugAnd (5) a time is reserved. The ion-triggered in situ gel polymer system includes gellan gum, which is commercially known asAnd alginic acid/sodium alginate: sodium alginate is a natural hydrophilic polysaccharide approved by the FDA for use as a material and food additive in human wound dressings, consisting of units of different compositions and sequences of (1→4) linked β -D-mannuronic acid (M) and α -L guluronic acid (G). Alginate is exposed to divalent cations in vivo (e.g., ca ²⁺ ) Which will turn into a stable gel. The percentage of guluronic acid in the polymer backbone plays a major role in alginate gelation and drug release. Guluronic acid content >65% of the alginate gelled instantaneously, while alginate with low guluronic acid content gelled slowly and formed a weak gel. Combinations of ion activated in situ gelling of sodium alginate with other viscosity enhancing agents such as HPMC may be used. The preparation can be preloaded with low concentration of calcium salt before injection, which does not cause gelation in vitro, but helps gelation in vivo, such as 0.1% -0.6% calcium gluconate solution. The amount of calcium salt to be added to aid in gelling in vivo but not to cause gelling in vitro depends on the concentration of alginate in the formulation. Higher concentrations of alginate require lower concentrations of calcium salt, and lower concentrations of alginate can tolerate higher concentrations of calcium salt while remaining in a non-gel state in vitro. By adding different amounts of calcium salt to the alginate containing formulation and selecting the highest amount of calcium salt that does not produce an injectable gel in vitro, the appropriate amount of calcium salt preloaded into the formulation can be readily determined experimentally. In some examples, the in situ gelling system matrix is 1% w/v sodium alginate (e.g., VLVG, novaMatrix, FMC Biopolymers, dragmen, norway) and 0.3% w/v D-calcium gluconate in the final drug-containing formulation of the present invention.

Combinations of gel enhancers including polymers with different gelling mechanisms may also be used. In order to reduce the amount of polymer required for gelation and to obtain a better gel with improved gelation properties, combinations of two or more polymers with different gelation mechanisms may be used to develop in situ drug delivery systems. Example(s) For example, a combination of thermosensitive polymer, methylcellulose or HPMC and pH-triggered polymer Carbopol may be used. The former polymer exhibits thermal gelation, while the latter exhibits pH-dependent gelation. The final formulation forms a flowable formulation that gels reversibly upon a sol-gel transition between 25 ℃ and 37 ℃ and an increase in pH from 4.0 to 7.4. In some examples, 25% (w/v) Pluronics and 30% (w/v) CAP are used. In one example, an in situ gelling system based on poloxamer+chitosan may be used. The optimal gelation temperature for the poloxamer-chitosan (16:1) system was 32 ℃. In one example, the combination of pH and ion trigger polymer based on an in situ gelling system can be prepared by mixing three different polymers such as Carbopol940, sodium alginate and guar gum. In one example, a formulation may consist of 15% Pluronic F127 and 0.1% low molecular weight chitosan. Carbopol and Pluronic at 0.3% and 14% (w/w) concentrations can be used to prepare in situ gel formulations. In another example, poloxamers 407 and 188 are used as thermosensitive polymers and Carbopol 1342P NF is used as a pH sensitive polymer, and the combined solutions form a gel under physiological conditions. In one example, about 15% Pluronic F127 is used in combination with polymers such as HPMC as a viscosity increasing agent, or with polymers such as Carbopol940, xanthan gum, and sodium alginate (high glucuronic acid content) for pH and cation triggered sol-gel transition matrices. In some examples, methylcellulose or HPMC is combined with Carbopol. In one example, the sodium alginate solution used for in situ gelation has a concentration of 2% w/w and for Pluronic F127 its concentration is 14% (w/w). In some examples, the Triblock (TB) polycaprolactone-polyethylene glycol-polycaprolactone [ (PCL-PEG-PCL), BAB ]And pentablock copolymer (PBC) polylactic acid (PLA) [ (PLA-PCL-PEG-PCL-PLA), CBABC]And [ (PEG-PCL-PLA-PCL-PEG), ABCBA]May be used. In one example, the in situ gelling system is sodium alginate as the ion-sensitive polymer and methylcellulose as the viscosity enhancing agent. In some examples, polyacrylic acid (Carbopol 940) or hyaluronic acid, pluronicF127, and gellan gum are used for pH triggered in situ gelation, thermoreversible gelation, and ion activation systems, respectively. HPMC is added as a co-agent with Carbopol or hyaluronic acidViscosity enhancing agents are used in combination with Pluronic F127 to reduce the concentration of Pluronic F127.(0.6%) can be used for cation-induced gelation. In some embodiments, drug-loaded thermosensitive PEG-PCL-PEG (PECE) hydrogels that synthesize PECE block polymers by coupling MPEG-PCL copolymers using IPDI reagents with sol-gel transitions that vary with temperature can be used. Formulations containing PECE (30% w/v) in water showed sol-gel transition at 35 ℃.

In addition, drug-loaded liposomes, nanoemulsions, suspensions, cyclodextrins, micelles, nanoparticles or microparticles may also be placed within the in situ gel. The drug-loaded in situ gel system may also employ a reactive in situ gel, wherein the two reactive components are mixed together and crosslinked to form a hydrogel. In some embodiments, the hydrogels are prepared by simple mixing of glycol chitosan and an aqueous solution of oxidized alginate, and remain injectable for a short period of time after mixing together due to the time required for the complete cross-linking reaction. Polymers (e.g. hyaluronic acid) and cross-linking agents (e.g. H ₂ O ₂ Pentasodium tripolyphosphate) may also be co-injected (e.g., using a dual syringe device) into the body to allow crosslinking to occur in the body. In some embodiments, PEG hydrogels are prepared by thiol-maleimide reactions using 4-arm PEG-Mal and 4-arm PEG-SH. In some embodiments, the in situ gelling drug delivery system is thiolated polyaspartic acid (thiopascp). In some embodiments, the hydrogel consists of maleimide modified c-polyglutamic acid (c-PGA-MA) and thiol-end functionalized 4-arm polyethylene glycol (4-arm PEG-SH), such as those described in Acta Biomaterialia (2019) 280-290.

Another type of reactive in situ gelling system matrix is an Injectable Drug Eluting Elastomeric Polymer (iDEEP), such as those described in doi 10.1016/j.gie.2011.12.009. For example, polyethylene glycol maleate citrate (PEGMC) was dissolved in deionized water (20%) and combined with polyethylene glycol diacrylate (12%) and tetramethyl ethylenediamine (0.5%) as part of iDEEP A, to which a suitable amount of drug was also added. The iDEEP B component (iDEEP-B) was prepared by dissolving ammonium persulfate redox initiator (0.25 wt%) in deionized water. And mixing the solutions of the part A and the part B in a ratio of 2:1 respectively to obtain the iDEEP gel.

Photocrosslinkers can also be used to form in situ gels, which are also a reactive matrix, which are brought about by irradiation with light to initiate the gelling reaction. Examples of photocrosslinkable include polyethylene glycol diacrylate (PEGDA) and photocrosslinkable chitosan hydrogels such as those described in (DOI): 10.1055/s-0028-1103483. PEGDA rapidly gels at room temperature with a photoinitiator and light such as ultraviolet light).

In some embodiments, a drug-loaded in situ gelling implant/insert may be used. For example, a combination of sodium carboxymethylcellulose (CMC) and sodium Alginate (ALG) may be used as the matrix. In some embodiments, the in situ gel loaded drug is in a chitosan/HPMC based polymer matrix. In some embodiments, the drug-loaded injectable gel or nano/micro particles are in a matrix of a polymer based on a biochromer polymer (tri (ethylene glycol) poly (orthoester), TEG-POE). For example, injectable gels are 80% TEG-POE (MW 6 kDa), about 9% methoxypoly (ethylene glycol) (MW 550 Da), and 0.1-1% by weight of drug.

In some embodiments, the drug-loaded in situ gelation is a polymer matrix based on chitosan-calcium alginate gel microspheres, such as those described in patent number CN1628861 a. For example, the matrix may be a chitosan-calcium alginate gel microsphere material consisting of calcium alginate gel microspheres optionally covered with chitosan in 0.5-4.0% sodium alginate solution. The particle size of the calcium alginate gel microsphere is between 1 and 200 mu m; the volume ratio of the chitosan-calcium alginate gel microspheres to the sodium alginate solution is 10:1-10:30. The drug may be encapsulated in microspheres or alginate solution phase or both.

Another in situ gelling material that may be used in the formulation is a biodegradable water insoluble polymer such as poly (D, L-lactide-co-hydroxymethylglycolide) (PLHMGA), PLA, PLGA, PCL, PGA, prolifeprospan such as pro-prospan 20 or PHB. Soluble in biological phasesA water-soluble organic solvent such as N-methylpyrrolidone or DMSO is used as a matrix for drug delivery, and the drug may be dissolved/dispersed in PGA or PLGA solution (e.g., 10% -50% PLGA in N-methylpyrrolidone) or the two components may be mixed immediately prior to injection. In some embodiments, 50:50 lactide/glycolide PLGA or PLGA with a lower lactide content, such as 10:90 lactide/glycolide PLGA, may be used. When the formulation is injected into the body, the water-miscible organic solvent dissipates and water permeates into the organic phase. This results in phase separation and precipitation of the polymer, forming a reservoir at the injection site as a slow release implantable material. Although it is not a classical hydrogel gelling system, it will be referred to as gelling in the present invention for the sake of illustration. Can be obtained in the Atrigel ^TM Examples are found in delivery systems and doi 10.1016/j.jcon.2014.05.057.

Other gels or high viscosity materials useful in the present invention include: RAD16 peptide, collagen, PNIPAAm-g-MC, polymer of patent No. CN102344559A, modified sodium hyaluronate gel of patent No. CN104086788B, hyaluronic acid/polyethylene glycol hydrogel for injection of patent No. CN106519072A, sodium hyaluronate collagen hydrogel of patent No. CN107189119A, F127 and->F68, PNIPAAm, poly (lactic-co-glycolic acid) -Poly (ethylene glycol) -Poly (lactic-co-glycolic acid) PLGA-PEG-PLGA) hydrogels (such as those described in International journal of pharmacy 490 (2015) 375-383), thermosensitive triblock polymers poly- (DL-lactic-co-glycolic acid) (PLGA) -polyethylene glycol PEG-PLGA (such as described in DOI 10.3109/03639041003680826), systems containing poloxamer 188/poloxamer 407/carbopol 934/HPMC (such as described in dx.doi. Org/10.1155/2014/280928), injectable bioresponsive gel depots (such as those described in DOI: 10.1002/adma.20180527), PVA-tsba hydrogels (e.g., described in sci.Transl.med.10, ean 3682, 2018), fibrin hydrogels (e.g., described in patent No. CN110393699 a)) Thermal gel polyurethane/PEG block copolymers (e.g., amine functionalized ABA block copolymers, composed of hydrophobic blocks (B): poly (serinol hexamethylenecarbamate) and hydrophilic block (a): polyethylene glycol-polygalamine hexamethylenecarbamates composed of poly (ethylene glycol) (such as those disclosed in DOI:10.1016/j. Biological materials.2010.09.044), injectable self-healing polymer-nanoparticle (PNP) hydrogel dodecyl-modified hydroxypropyl methylcellulose (HPMC-C12) conjugated with polyethylene glycol-b-polylactic acid (PEG-PLA) Nanoparticles (NPs), 2wt% HPMC-C12+10wt% NPs (such as those in DOI. Org/10.1021/acmentsci.0c00132), vaccine self-assembled immune matrices made of (RADA) 4 synthetic oligopeptides (such as those described in DOI: 10.1128/CVI.00914-14), propylene with N- [ (2-hydro-3-trimethylum) propyl ]Thermosensitive hydrogels formulated with chitosan chloride (HTCC) and α, β -glycerophosphate (α, β -GP) (e.g., as described in doi.org/10.1016/j.biological materials.2011.11.068), poly (d, L-lactide) -poly (d, L-lactide) (PDLLA-PEG-PDLLA, PLEL) (e.g., as described in doi.org/10.1016/j.apmt.2020.100608), gel systems in patent application No. WO2014006215A1, injectable PEG-b-poly (L-alanine) hydrogels (e.g., as described in doi: 10.7150/thno.30577), injectable chitosan-alginate porous gels as described in doi10.1002/mabi.201800242, and injectable chitosan-alginate porous gels with low viscosity at high shear rates (e.g., injection procedure 100S) ^–1 ) And at low shear rates), e.g. post injection conditions, has a high viscosity, preferably>More than 10 times) of the reagent.

Other agents having a low viscosity at high shear rates and a high viscosity at low shear rates may also be used as matrices in the formulations of the present invention, alone or in combination with other in situ gelling matrices. Examples thereof include materials having a pseudoplastic viscosity, such as those disclosed in WO2013077357A1, for example xanthan gum, carrageenan, gellan gum, guar gum, locust bean gum, sacran or salts thereof. Suitable concentrations of these polysaccharides are 0.5 to 5w/v% and the pH of the formulation is between 3 and 8. In one example, 1-2% xanthan gum (KELTROL, CGT, CP Kelco) is used as a pseudoplastic tackifier alone or in combination with 2% sodium alginate as an in situ gelling matrix in the formulation.

Other examples and procedures for making these in situ gelling matrices can be found from the following sources: DOI:10.15406/japlr.2016.02.00022, dx.doi.org/10.1016/j.drusis.2013.10.001, doi.org/10.1016/S0920-4105 (00) 00034-6. These documents and their related references, as well as other references listed in the present invention, can be readily used in the present invention.

Liquid solutions as used herein refer to solutions, suspensions, emulsions, drops, ointments, lotions, sprays, liposomes as are well known in the art. In some embodiments, the liquid solution comprises an aqueous pH buffer that resists pH changes when small amounts of acid or base are added. In some embodiments, the liquid solution has an osmotic pressure approaching the physiological osmotic pressure value, which may be achieved by adding an appropriate amount of pharmaceutically acceptable excipients to the formulation.

In certain aspects, the present invention and the inventors' prior applications disclose methods, compositions and agents/drugs for treating autoimmune diseases and allergies or inhibiting the production of anti-drug antibodies or inducing antigen-specific immune tolerance by using a combination of antigen and immunosuppressant as physical mixtures or as synthetic conjugates or as nano/micro or larger particles or implants or liposomes in sustained (prolonged) release systems (e.g., in situ gelling systems or high viscosity formulations) for administration to a subject/patient in need thereof. The term nano/micro particles means that the size (diameter) of the particles is in the nano or micro range. For example, the nano/micro particles may be in the size range of 50nm to 100 μm. The large particles may be in the size range of 100 μm to 10 mm. The particles may be made of biodegradable materials such as PLGA or polysaccharides (e.g. alginate). By physical mixture is meant that the mixture of antigen and immunosuppressant is simply mechanically mixed together (e.g. by stirring or mixing) in its original form (e.g. in liquid or solid form, such as powder or particles, not encapsulated in other nano-or microparticles) without any additional process, e.g. mixing them together in their original form, or applying a further size reduction process (e.g. crushing, milling, grinding or homogenising) before or after mechanical mixing, or dispersing or dissolving in the same or different types of liquid, respectively, and then mixing, or co-dispersing in a liquid, or co-dissolving in a solvent (e.g. water), and an optional drying process (e.g. spray drying or freeze drying) may be applied together with an optional further size reduction process. By physical mixture is meant that the antigen and immunosuppressant are not encapsulated or conjugated together or encapsulated in a nano-or microparticle. An exemplary list of immunosuppressive drugs can be found in the wikipedia "immunosuppressive drugs" article page. Immunosuppressants/drugs (immunosuppressants) suitable for current use include, but are not limited to, statins; mTOR inhibitors, such as rapamycin or rapamycin analogues (e.g. everolimus, ridaforolimus/deforolimus and temsirolimus) or second generation mTOR inhibitors known as ATP-competitive mTOR kinase inhibitors; anti-inflammatory corticosteroids; TGF-beta signaling agents; TGF-beta receptor agonists; TLR (Toll-like receptor) inhibitors; pattern recognition receptor inhibitors; NOD-like receptor (NLR) inhibitors;

RIG-I like receptor inhibitors; NOD2 inhibitors; histone deacetylase inhibitors such as trichostatin a; corticosteroids; mitochondrial function inhibitors such as rotenone; a P38 inhibitor; NF- κβ inhibitors such as 6Bio, dexamethasone, TCPA-1, IKK VII; adenosine receptor agonists; prostaglandin E2 agonists (PGE 2), such as misoprostol; phosphodiesterase inhibitors, including phosphodiesterase 4 inhibitors (PDE 4), such as rolipram; a proteasome inhibitor; a kinase inhibitor; a G protein-coupled receptor agonist; g protein-coupled receptor antagonists; glucocorticoids; tretinoin; a cytokine inhibitor; cytokine receptor inhibitors; cytokine receptor activators; peroxisome proliferator activated receptor antagonists; peroxisome proliferator activated receptor agonists; histone deacetylase inhibitors; calcineurin inhibitors; a phosphatase inhibitor;

PI3 KB inhibitors, such as TGX-221; autophagy inhibitors such as 3-methyladenine; an aromatic hydrocarbon receptor inhibitor; proteasome inhibitor I (PSI); oxidizing ATP; P2X receptor blockers. Immunosuppressants also include IDO, vitamin D3, cyclosporins such as cyclosporin a, aromatic receptor inhibitors, resveratrol, azathioprine (Aza), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), FK506, sanglifehrin a, salmeterol, mycophenolate Mofetil (MMF), aspirin and other COX inhibitors, niflumic acid, estriol and triptolide, siglec ligands such as sialic acid and derivatives thereof including polysialic acid-lipid conjugates. In embodiments, an immunosuppressant can comprise any of the agents provided herein. The immunosuppressant may be a compound that directly provides an immunosuppressive (e.g., tolerogenic) effect on the APC, or it may be a compound that indirectly provides an immunosuppressive (e.g., tolerogenic) effect (i.e., after being processed in some manner after administration). Thus, immunosuppressants include prodrug forms of any of the compounds provided herein. In some preferred embodiments, the immunosuppressant used is an mTOR inhibitor (e.g., rapamycin or rapamycin analog) or methotrexate.

Immunosuppressants also include heme oxygenase-1 (HO-1) inducers such as cobalt protoporphyrin (CoPP), protoporphyrin IX containing ferric ions (heme B) and chloroligands (heme), heme, iron protoporphyrin or heme degradation products such as those described in PCT/EP 2015/074819. Siglecs (sialic acid binding immunoglobulin-type lectin) ligands such as sialic acid or derivatives thereof are also another class of immunosuppressants useful in the present invention. PD-L1 is also another class of immunosuppressants useful in the present invention. PD-L1 is effective in inhibiting cytotoxic T cells. Fragments or mimetics or derivatives of PD-L1 that bind to PD-1 may also be used instead. Other inhibitory ligands (e.g., A2AR, BTLA, CTLA-4, CD47, KIR, LAG3, TIM-3, VISTA, etc.) such as B7-H3, B7-H4, may also be substituted for PD-L1. Molecules that promote T/B reg expansion (e.g., cytokines such as IL-2 and TGF-beta that stimulate T/B reg expansion) are also another class of immunosuppressants. Different immunosuppressants may be used as mixtures and in combination in the present invention.

Immunosuppressants also include nucleic acids encoding peptides, polypeptides, or proteins provided herein that result in an immunosuppressive (e.g., tolerogenic) immune response. Thus, in some embodiments, an immunosuppressant is a nucleic acid encoding a peptide, polypeptide, or protein that results in an immunosuppressive (e.g., tolerogenic) immune response. The nucleic acid may be coupled to a synthetic nanocarrier. The nucleic acid may be DNA or RNA, such as mRNA. In embodiments, the compositions of the invention comprise a complement of any nucleic acid provided herein, e.g., a full-length complement or degeneracy (due to the degeneracy of the genetic code). In some embodiments, the nucleic acid is an expression vector that can be transcribed when transfected into a cell line. In embodiments, the expression vector may comprise a plasmid, retrovirus, adenovirus, or the like. Nucleic acids can be isolated or synthesized using standard molecular biology methods, for example, by using the polymerase chain reaction to generate nucleic acid fragments, which are then purified and cloned into expression vectors.

In some embodiments, the immunosuppressants provided herein are conjugated or fused to affinity ligands. Where both the immunosuppressant and affinity ligand are peptides/proteins, they may be constructed as fusion proteins by genetic engineering and expression, one may be linked to the N or C terminus of the other by an optional linker sequence. The affinity ligand may target or bind to an organ or tissue or cell or protein or antigen that causes or is affected by an autoimmune disease. Affinity ligands may be whole antibodies, antibody fragments, antibody mimics or derivatives thereof, or may be non-protein molecules such as aptamers, examples of which are disclosed in the inventors' prior U.S. patent application. The term antibody in this application includes full length antibodies, antibody fragments, nanobodies and derivatives thereof. The resulting conjugate or fusion protein may be used to treat an associated autoimmune disease or allergy by administering it to a subject in need thereof in a therapeutically effective amount (e.g., by injection). The resulting organ/tissue/cell/protein/antigen targeting ligand-immunosuppressant conjugate or fusion can protect diseased cells and induce tolerance. For example, collagen II is abundant in cartilage, and anti-collagen II scFv-PD-L1 fusion proteins or other anti-collagen II Fab-PD-1 agonist fusion or conjugates can be used to treat rheumatoid arthritis, which would allow the attached PD-L1 to encapsulate cartilage and chondrocytes and the like, protect them from T-cell attack and induce tolerance. In some embodiments, the affinity ligand is an antibody. In some embodiments, the antibody is IgG4 or its Fc is engineered to reduce its ADCC and CDC effects. In some embodiments, the antibodies are engineered to have enhanced ADCP effects, such as those described in the inventors' prior U.S. patent application. In some embodiments, mTOR inhibitors (e.g., rapamycin, everolimus, ridaforolimus/deforolimus, and temsirolimus) are conjugated to the antibody. A protocol for preparing mTOR inhibitor-antibody conjugates can be found in patent application WO2018227018A1 and is readily employed by the present invention. Other immunosuppressants which can be used in the present invention can also be found in patent application WO2018227018 A1. In some embodiments, a calcineurin inhibitor (e.g., cyclosporine or tacrolimus) is conjugated to the antibody. In some embodiments, an anti-inflammatory corticosteroid (e.g., dexamethasone or betamethasone) is conjugated to the antibody. In some embodiments, PD-L1 is fused to an antibody. In some embodiments, anti-inflammatory cytokines or derivatives thereof (e.g., IL-2-anti-IL-2 antibody complex, IL-10, TGF-beta) are fused to the antibody. In some embodiments, antibodies thereof may bind to autoantigens expressed by cells, such as those described later in this application (e.g., insulin, islet cell autoantigen-2, GAD, IGRP, for use in diabetes treatment). In some embodiments, antibodies thereto may bind to tissues or cells or organs affected by the disease through surface markers that bind to non-self antigens of these organs/tissues/cells.

When an antibody is to bind IgE, it preferably has only one antigen binding portion, e.g. the antibody fragment has one Fab, e.g. the Fab of Omalizumab), a single chain variable fragment (scFv), the scFv-Fc fusion to avoid IgE aggregation at mast cells. It may be combined with immunosuppressants or cytotoxic drugs, such as those ADC's for the treatment of cancer. The resulting conjugates are useful for treating allergies.

In some embodiments, the immunosuppressants provided herein are coupled to synthetic nanocarriers or microcarriers. In a preferred embodiment, the immunosuppressant is an element other than the material comprising the synthetic nanocarrier or microcarrier structure. For example, in one embodiment, when the synthetic nanocarrier or microcarrier is comprised of one or more polymers, the immunosuppressant is a compound that is otherwise coupled to the one or more polymers. As another example, in one embodiment, when the synthetic nanocarrier or microcarrier is comprised of one or more lipids, the immunosuppressant is again attached and coupled to the one or more lipids. In embodiments, for example, where the material of the synthetic nanocarrier or microcarrier also results in an immunosuppressive (e.g., tolerogenic) effect, the immunosuppressive agent is an element that is present in addition to the material of the synthetic nanocarrier or microcarrier that results in an immunosuppressive (e.g., tolerogenic) effect.

Other exemplary immunosuppressants include, but are not limited to, small molecule drugs, natural products, antibodies (e.g., antibodies to CD20, CD3, CD 4), biological agent-based drugs, carbohydrate-based drugs, nanoparticles, liposomes, RNAi, antisense nucleic acids, aptamers, methotrexate, non-steroidal anti-inflammatory drugs; fingolimod; natalizumab; alemtuzumab; anti-CD 16, anti-CD 3; other immunosuppressants such as tacrolimus (FK 506) are well known to those skilled in the art, and the present invention is not limited thereto. Other immunosuppressants can be found in related patents and patent applications, such as U.S. Pat. No. 13/880,778, U.S. Pat. No. 14/934,135, CA2910579, U.S. Pat. No. 13/084,662, U.S. Pat. No. 14/269,048,US8652487,WO2012054920A2,WO2016073799A1,WO2012149393 A3,WO2014179771A1,PCT/U.S. Pat. No. 2012/035405, U.S. Pat. No. 5,20110262491, U.S. Pat. No. 5,8652487 and other Selecta Biosciences filed patent applications.

The publication of Selecta Bio discloses methods and related compositions for synthesizing nanocarriers, including B-cell and/or MHC class II restricted epitopes and immunosuppressants, to generate tolerogenic immune responses. In their disclosure, an antigen/epitope is coupled to a nanocarrier and an immunosuppressant is coupled to the nanocarrier. Another method and composition is to use nanoparticles/microparticles having antigens/epitopes non-covalently adsorbed to their surface and immunosuppressants encapsulated in an in situ gelling system or high viscosity formulation. The nanoparticles/microparticles may be made of biodegradable materials, such as PLGA. These nano/micro particles (e.g., 10nm-10 μm in diameter) may be administered as an injection or inhalant to a patient in need thereof to induce immune tolerance. Encapsulation of immunosuppressants is well known to those skilled in the art and can be readily employed from the relevant publications. The surface of the nano-particle/micro-particle can be provided with amino or carboxyl and other charged groups so as to increase the combination of the antigen/epitope and the surface of the nano-particle/micro-particle; it may also have a hydrophobic surface to allow binding of antigens/epitopes by hydrophobic interactions; or a combination thereof. Introduction of charged groups to the surface nanoparticles/microparticles can be prepared by using surface modification or using molecules containing amine or carboxyl groups. The antigen/epitope may also be conjugated to a lipophilic moiety such as fatty acid or cholesterol, etc. lipid molecules to increase its binding to the nanoparticle/microparticle. Adsorption of the antigen/epitope to the nanoparticle/microparticle surface can be accomplished by incubating the antigen/epitope with the nanoparticle/microparticle (e.g., overnight at 4 ℃ in an aqueous buffer such as 1 xPBS) and then removing unbound antigen/epitope (e.g., washing the microparticles several times with aqueous buffer, similar to the ELISA plate coating procedure). In one embodiment, 50nm to 200nm sized PLGA nanoparticles are prepared according to the literature, encapsulated with 10% by weight rapamycin. Next, PLGA nanoparticles were mixed with OVA (10 mg/mL) overnight at 4℃to yield OVA (ovalbumin) -coated particles. The particles were washed 3 times with PBS to remove unbound OVA. In another example, rapamycin is dissolved in DMSO at 50 mg/ml. A total of 50. Mu.L of rapamycin was added to 1ml PLGA solution (5 mg/ml) dissolved in methylene chloride. Next, the mixture was homogenized with 0.4ml of 5% OVA solution using ultrasonic waves for 10 minutes. The o/w emulsion was added to 2.1ml of a 5% w/v PVA solution to evaporate the organic solvent at room temperature for 4 hours. After centrifugation at 3,500g for 20 minutes, OVA-coated nanoparticles containing rapamycin were obtained. Additional washing steps may be performed to obtain particles free of unbound OVA. Such OVA coated particles in 2-6% sodium alginate or 0.5-2% cross-linked hyaluronic acid may be administered to a subject in need of induction of OVA immune tolerance using similar protocols as described in publications (e.g. those from Selecta Bio). For example, 5mg to 50mg of particles dissolved in 3% sodium alginate or 1% crosslinked hyaluronic acid may be injected 3 times per week to an OVA intolerant patient to induce OVA tolerance by subcutaneous or intramuscular injection or intralymphatic injection or injection near lymph nodes. OVA can be replaced by other antigen/epitope molecules to induce corresponding immune tolerance. In another sample, PLGA particles with surface charges can be prepared by adding lipophilic carboxylic acid or lipophilic amine or anionic detergent or cationic detergent (such as fatty acid (octanoic acid, lauric acid, etc.), or cationic lipid (DOTMA, DOTAP, cholesterol amine, etc.) into PLGA. In one example, rapamycin is dissolved at 50mg/mL in DMSO containing 10mg/mL lauric acid. A total of 50. Mu.L of rapamycin/lauric acid was added to 1ml PLGA (5 mg/ml PLGA) dissolved in methylene chloride. Next, the mixture was homogenized with 0.1ml of 2% octanoic acid solution using ultrasonic waves for 10 minutes. The o/w emulsion was evaporated at room temperature for 4 hours to remove the organic solvent. The resulting PLGA particles were washed 3 times with PBS and then incubated with OVA to prepare OVA-conjugated particles. In one example, 10mg to 100mg of the particles in 2% sodium alginate and 1% hpmc may be injected 3 times per month to OVA intolerant patients, inducing OVA tolerance by subcutaneous or intramuscular injection or intralymphatic injection. On the other hand, PLGA rapamycin microparticles were synthesized using a single emulsion method using PLGA polymers (PLGA, 50:50 or 65:35, molecular weight 10,000-85,000 Da). Briefly, 100mg of PLGA was dissolved in 2mL of Dichloromethane (DCM) containing 10mg of rapamycin and homogenized in 1% polyvinyl alcohol (10 mL,87-89% hydrolyzed, MW 13,000-23,000kDa, sigma # 363170) at 2000 rpm. The solution was added to 100mL of 1% PVA and stirred continuously for 3-4 hours to completely evaporate the DCM. The solution was then centrifuged at 11,000g and washed twice with deionized water to wash out excess PVA. The microparticles were then resuspended in deionized water and flash frozen at-80 ℃ and then lyophilized. The packaging efficiency can reach 30-50%. Sterile suspensions injected with 10mg/mL microparticles mixed with an appropriate amount of disease-associated antigen (e.g., 1mg/mL OVA as the final concentration) in 1XPBS pH7 or 3% sodium alginate or 15-25% Pluronic F127 or Poloxamer 407 can be used to treat the associated antigen intolerant disease.

In addition, the antigen/epitope may be encapsulated within the nanoparticle/microparticle in addition to being conjugated or adsorbed to its surface. The preparation of antigen/epitope encapsulation is well known to those skilled in the art and can be readily employed from related publications, for example using double emulsion water/oil/water systems. In one example, 10g of DL-PLGA (80:20, MW 50,000) is dissolved in 50g of a mixed solvent consisting of 35wt% acetone and 65wt% chloroform. 200-500 mg rapamycin or rapamycin analogue and 10-50 mg peptide antigen are added and vigorously stirred for 30 minutes. The organic phase is then slowly added to 500g of 5wt% aqueous poly (vinyl alcohol). During the addition of the organic phase, the PVA solution was stirred at 800rpm to form a stable oil-in-water emulsion. After stirring the emulsion for 10 minutes, a vacuum was applied and the stirring rate was reduced to 600rpm for 20 hours to remove volatile solvents. After centrifugation, the resulting microcapsule precipitate was thoroughly washed with deionized water, and the microcapsules were wet-sieved to collect particles having a diameter of 30-80 μm. And then dried in a vacuum chamber. Kept at room temperature. The size of the microcapsules can be adjusted by using sieves of different mesh numbers. 50mg/mL microparticle sterile suspensions injected in ph=71 x PBS or 3% sodium alginate or 15-25% Pluronic F127 or poloxamer 407 to treat the relevant peptide antigen intolerant diseases. Larger sized particles have longer in vivo drug release times.

Rapamycin containing microparticles and optionally an anti-inflammatory steroid may also be used in the present invention. These compositions/formulations may also be in a slow release system, such as the in situ gelling systems disclosed herein.

U.S. patent application No. US20130287729 discloses antigen-specific tolerance-inducing microparticles and uses thereof. It discloses a microparticle (0.5 μm-10.0 μm in size) for targeting an antigen presenting immune cell of interest and for inducing antigen specific immune tolerance, wherein the microparticle comprises an antigen and a therapeutic agent, wherein the therapeutic agent is an immunomodulator, an immunosuppressant tolerogenic agent, or an agent that recruits an antigen presenting immune cell of interest, wherein the surface of the microparticle comprises a ligand that targets the antigen presenting immune cell of interest, and the microparticle is made of a biodegradable material. A further improvement of the method and composition is the use of nanoparticles/microparticles, preferably made of biodegradable material, with a size of 50nm-5 μm or those disclosed in application US20130287729, in a slow release formulation such as an in situ gelling system or a high viscosity formulation. In some embodiments, the surface of the nanoparticle/microparticle is coated with the Fc portion of the antibody or the whole antibody with the Fc portion facing outward. This will bind to FcR to promote APC uptake. In other embodiments, the surface of the nanoparticle/microparticle need not have ligands that target antigen presenting immune cells. In some embodiments, it may have an antigen/epitope coated on its surface. The interior of the nanoparticle/microparticle comprises the immunosuppressants listed in this application and optionally an antigen/epitope, for example by encapsulation. This preparation method is well known to those skilled in the art and can be readily employed from the relevant publications. For example, 0.5mg to 50mg of the above particles containing gluten and rapamycin (5-25% of the formulation) may be injected 3 times per month into a gluten intolerant patient in 3% sodium alginate and optionally 0.5-2% HPMC, or in 15-25% Pluronic F127 or 15-25% poloxamer 407, to induce gluten tolerance by subcutaneous or intralymphatic injection.

U.S. patent application 20160338953 discloses a liposome-based immunotherapy. It provides a liposome encapsulating an autoantigen, wherein the liposome has a size of 500 to 15000nm and the liposome membrane comprises Phosphodiserine (PS) in an amount of 10 to 40% by weight relative to the total membrane liposome composition. Also provided are pharmaceutical or veterinary compositions comprising a therapeutically effective amount of the liposomes. Furthermore, it provides a liposome as defined above for use as a medicament and a pharmaceutical or veterinary composition, in particular for use in the treatment of autoimmune diseases. Finally, it provides a liposome and a pharmaceutical or veterinary composition as defined above for restoring tolerance to itself in patients suffering from autoimmune diseases. The invention also discloses antigen-specific tolerance-inducing liposome and application thereof. The liposomes are internally encapsulated with the immunosuppressants (and optionally antigen/epitope molecules) listed herein. Optionally, the surface of the liposome may also be coated with an antigen/epitope. Can be administered as an injection to a patient in need thereof to induce immune tolerance. Lipids used in liposomes can include, but are not limited to, phosphatidyldiserine at 10% to 40% by weight of the membrane. Membranes can also be made with non-phosphatidyl-di-serine lipids. The antigen/epitope may also be conjugated with a lipid-type molecule such as a fatty acid or a phospholipid or cholesterol derivative to allow insertion into the liposome membrane. Suitable liposomes can have a size between 50nm and 20 μm. Methods for their preparation and their use schemes are well known to those skilled in the art and can be readily adopted from related publications such as those in US 20160338953. Examples of lipid molecules suitable for use in preparing liposomes according to the invention include, but are not limited to, phospholipids, glycerolipids, glycerophospholipids, sphingolipids, ceramides, glycerophospholipids ethanolamine, sterols, or steroids. These lipid molecules can also be used to prepare antigen/epitope-lipid conjugates. Instead of antigen/epitope-lipid conjugates, membrane anchored peptide-antigen/epitope conjugates can also be used. In addition, other molecules that may promote T/B reg amplification (e.g., IL-2 and/or TGF-. Beta.and PD-L1) may also be coated/coupled and/or encapsulated within liposomes and nanoparticles/microparticles. These liposomes, as well as the liposomes of patent application US20160338953, can be formulated as sustained release formulations, such as in situ gelling systems or high viscosity formulations. For example, the liposome containing ovalbumin and rapamycin (5-25% of the preparation) of 0.5-50 mg of ovalbumin and rapamycin can be injected into patients with egg white intolerance 3 times per month in 3% sodium alginate, optionally 0.5-2% HPMC, or 15-25% pluronic F127 or 15-25% poloxamer 407, and can induce egg white tolerance by subcutaneous or intramuscular injection or inguinal lymph node intralymphatic injection.

Novel agents and compositions are disclosed that comprise an antigen and an immunosuppressant in a sustained release formulation such as an in situ gelling system or a high viscosity formulation. Those novel agents and formulations may be administered as subcutaneous or intramuscular or intradermal injections in pharmaceutically effective amounts to treat autoimmune diseases or allergies or to inhibit the production of anti-drug antibodies or to induce antigen-specific immune tolerance in a subject. In addition, these agents and compositions may be injected into lymph nodes (e.g., inguinal lymph nodes) for the same purpose. Intralymphatic allergen administration is known and the same procedure can be readily employed for the present invention. The agents and formulations disclosed by the inventors in said prior application may also be used as intralymphatic injections. Molecules that promote T/B reg expansion and/or inhibit unwanted autoreactive T/B cells (e.g., IL-2, TGF-beta, PD-L1, IL-15, IL-10, IL-21, IL-27, IL-2/anti-IL-2 antibody complexes or mimics or derivatives thereof such as pegylated IL-2 NKTR-358) may also be co-injected or included in the formulation to be intralymphatically injected. The reagents and formulations of the present inventors in the above applications and the present invention contain disease specific antigens such as B cell antigens, T cell antigens in the form of MHC-peptide complexes or T cell epitopes, mimotopes, peptides (or derivatives thereof) which bind to MHC to form cell antigens of MHC-peptide complexes. In addition to the direct use of antigens in the agent, composition or formulation, nucleic acids encoding these antigens/epitopes may also be used instead, for example mRNA encoding them. The mRNA can be modified in a delivery system, such as a liposome or lipid carrier, or using well known methods and protocols to enhance expression of interest. In some embodiments, the amount of agent or composition infused into the lymph node is 0.01mg to 50mg of drug, each lymph node is injected in a volume of 0.1ml to 1ml, e.g., 1mg per month or 1mg every two weeks, for 3 months to induce antigen-specific immune tolerance.

The immunosuppressant may be in the form of an active agent, a prodrug, a microparticle or nanoparticle or in the form of a liposome. The antigen may be a B cell antigen/epitope or a T cell antigen/epitope (e.g., an MHC-peptide complex or conjugate; or a peptide antigen that can bind to MHC) or a combination thereof. The combination may be a B cell antigen/epitope and a T cell antigen/epitope; or a combination of several different B cell antigens/epitopes and/or several different T cell antigens/epitopes for the same disease or different diseases. The use of peptide antigens (T cell epitopes) that can bind to MHC to form MHC-peptide complexes (T cell antigens) in vivo in place of peptide-MHC complexes to reduce size and molecular weight can improve transdermal delivery. Examples of which can be found in the prior and current applications and related publications.

Human MHC class I and class II are also known as Human Leukocyte Antigens (HLA). The most studied HLA genes are nine classical MHC genes: HLA-A, HLA-B, HLA-C, HLA-DPA1, HLA-DPB1, HLA-DQA1, HLA-DQB1, HLA-DRA and HLA-DRB1. In humans, MHC gene clusters are divided into three regions: I. class II and III. A. The B and C genes belong to the mhc class and the six D genes belong to class II. Humans also have non-classical MHC. Peptides or peptide MHC complexes (peptide-MHC), pMHC for short, suitable for use in the present invention can be easily found from the prior art and publications. The peptide and MHC in the peptide MHC complex may be covalently conjugated (or expressed) together or bound together to form a non-covalent complex. Many peptide MHC complexes associated with autoimmune diseases are being identified in humans or animals. For example, patent applications US20170095544, US20180127481, US20090155292 and US20150125536 disclose disease-specific peptide MHC complexes, which may be employed in the present application. The MHC class I component may comprise all or part of an HLA-A, HLa-B, HLA-C, HLA-E, HLA-F, HLA-G molecule, in particular all or part of an HLA-A molecule, e.g. an HLA-A x 0201MHC class I molecule. The non-classical MHC class I component may comprise a CD 1-like molecule. The MHC class II component may comprise all or part of HLA-DR, HLA-DQ or HLA-DP. In certain aspects of treating autoimmune diseases and allergies, the antigen/MHC complex is coupled or attached to a substrate (antigen/MHC/particle complex or antigen/MHC/linear polymer) covalently or non-covalently. As used herein, unless specifically indicated, the term MHC in the context of pMHC complexes means classical or non-classical MHC class I proteins and/or classical or non-classical MHC class II proteins, any locus of HLA DR, HLA DQ, HLA DP, HLA-a, HLA-B, HLA-C, HLA-E, CD1d or fragments or bioequivalents thereof, double-or single-stranded constructs, dimers (Fc fusions). In certain embodiments, the MHC class 1 component may comprise HLA-A, HLA-B, HLA-C, HLA-E, HLA-F, HLA-G, or CD-1 molecules. In embodiments wherein the MHC component is an MHC class II component, the MHC class II component may comprise, consist essentially of, or alternatively consist further of all or part of HLA-DR, HLA-DQ, or HLA-DP. In certain embodiments, MHC may comprise HLA DRB1, HLA DRB3, HLA DRB4, HLA DRB5, HLA DQB1, HLA DQA1, IAg, I-Ab, I-Ad, HLA-DQ, HLA-DP, HLA-A, HLA-B, HLA-C, HLA-E, or CD1d. Non-classical MHC molecules may also be used in MHC complexes of the present disclosure. In some embodiments, the non-classical MHC molecules are non-polymorphic, are conserved across species, and have narrow, deep, hydrophobic ligand binding pockets. These binding pockets are capable of presenting glycolipids and phospholipids to Natural Killer T (NKT) cells. NKT cells represent a unique lymphocyte population that co-expresses NK cell markers and a semi-constant T Cell Receptor (TCR). They are involved in the modulation of immune responses associated with a variety of diseases.

T cells recognize T cell antigens via their TCR receptors. T cell antigens are typically present in the form of MHC epitope binding complexes. Epitopes are typically peptides (sometimes other molecules such as carbohydrates) processed by APCs. In the present invention, the antigen of the T cell may be an MHC-epitope complex formed or a fragment/derivative/mimetic thereof, which has a higher specific affinity for the TCR than the epitope alone. It may be in monomeric or oligomeric (dimer, trimer, tetramer, pentamer or even higher multimer) form, such as the MHC tetramer currently being studied for labeling immune cells. For example, HLA-A2insB10-18 tetramers (e.g., those described in doi: 10.1073/pnas.0508621102) can be conjugated to a cell inactivating agent with an optional linker to treat type 1 diabetes in humans by inactivating autoimmune T cells. Epitopes (e.g. peptides) can be covalently conjugated to MHC to increase their stability by well known means disclosed in well known publications. Similarly, antigens for B cells in the present invention may also be in oligomeric or multimeric form. However, antigens sometimes used for B cell inactivation do not require MHC components.

In some embodiments, the autoimmune disease-associated antigen is:

one or more diabetes-related antigens and derived from one or more antigens selected from the group consisting of: preproinsulin (PPI), islet-specific glucose 6-phosphatase (IGRP), glutamate decarboxylase (GAD), islet cell autoantigen 2 (ICA 2), insulin, proinsulin or fragments or equivalents thereof, and combinations thereof;

one or more antigens associated with multiple sclerosis and derived from one or more antigens selected from the group consisting of: myelin basic protein, myelin-associated glycoprotein, myelin oligodendrocyte protein, proteolipid protein, oligodendrocyte myelin oligoprotein, myelin-associated oligodendrocyte basic protein, oligodendrocyte-specific protein, heat shock protein, oligodendrocyte-specific protein, NOGO a, glycoprotein Po, outer Zhou Suiqiao protein 22, 2'3' -cyclic nucleotide 3' -phosphodiesterase,

or a fragment or equivalent of each thereof, and combinations thereof;

one or more celiac-related antigens and derived from gliadin or fragments or equivalents thereof, and combinations thereof;

One or more primary biliary cirrhosis-associated antigens and derived from PDC-E2 or fragments or equivalents thereof, and combinations thereof;

one or more follicular pemphigus-related antigens and/or pemphigus vulgaris-related antigens, and derived from an antigen selected from one or more of the following groups: DG1, DG3 or respective fragments or equivalents thereof, and combinations thereof;

one or more neuromyelitis optica lineage disorder associated antigens and derived from AQP4 or fragments or equivalents thereof, and combinations thereof;

one or more arthritis-related antigens and derived from an antigen selected from one or more of the following groups: heat shock proteins, immunoglobulin binding proteins, heteronuclear RNPs, annexin V, calpain inhibitor proteins, type II collagen, glucose-6-phosphate isomerase, elongation factor human cartilage gp39, mannose binding lectin, citrulline vimentin, type II collagen, fibrinogen, alpha enolase, anti-carbamoylation protein (anti-CarP), type 4 peptide arginine deiminase (PAD 4), BRAF, fibrinogen gamma chain, meta alpha-trypsin inhibitor heavy chain H1, alpha-1-antitrypsin, plasma protease C1 inhibitor, gelsolin, alpha 1-B glycoprotein, plasma ceruloplasmin, meta alpha-trypsin inhibitor heavy chain H4, complement factor H, alpha 2 macroglobulin, serum amyloid, C-reactive protein, serum albumin, fibrinogen beta chain, serum transferrin, alpha 2HS glycoprotein, vimentin, complement C3 or fragments or equivalents thereof, respectively, and combinations thereof;

One or more allergic asthma-associated antigens, and derived from an antigen selected from one or more of the following groups: DERP1, DERP2, or fragments or equivalents thereof, respectively, and combinations thereof;

one or more inflammatory bowel disease-related antigens and derived from an antigen selected from one or more of the following groups: flagellin, fla-2, fla-X, YIDX, bacteroides integrase or a fragment or equivalent of each thereof, and combinations thereof;

one or more systemic lupus erythematosus-associated antigens and derived from one or more antigens selected from the group consisting of: double stranded (ds) DNA, ribonucleoprotein (RNP), smith (Sm), sjogren's syndrome associated antigen se:Sup>A (SS-se:Sup>A)/Ro, sjogren's syndrome associated antigen B (SS-B)/lse:Sup>A, ro60, ro52, histone or fragments or equivalents thereof, respectively, and combinations thereof; one or more atherosclerosis-related antigens and derived from an antigen selected from one or more of the following groups: apoB, apoE or fragments or equivalents thereof, respectively, and combinations thereof;

one or more COPD-associated antigens and/or emphysema-associated antigens and derived from elastin or a fragment or equivalent thereof, and combinations thereof;

One or more psoriasis-associated antigens, and derived from an antigen selected from one or more of the following: cap18, ADTSL 5, ATL5 or fragments or equivalents thereof, respectively, and combinations thereof;

one or more autoimmune hepatitis-associated antigens, and derived from an antigen selected from one or more of the following groups: CYP2D6, SLA, or a fragment or equivalent of each thereof; and combinations thereof;

one or more sjogren syndrome associated antigens, and derived from an antigen selected from one or more of the following groups: (SS-se:Sup>A)/Ro, (SS-B)/lse:Sup>A, MR3, ro60, ro52, or fragments or equivalents of each; and combinations thereof;

one or more scleroderma-related antigens and derived from an antigen selected from one or more of the group consisting of: CENP-C, TOP 1, RNA polymerase III, or fragments or equivalents of each thereof, and combinations thereof;

one or more antiphospholipid syndrome associated antigens derived from APOH or fragments or equivalents thereof, and combinations thereof;

one or more ANCA-associated vasculitis-associated antigens, and derived from an antigen selected from one or more of the following groups: MPO, PRTN3, or fragments or equivalents thereof, respectively, and combinations thereof;

One or more stiff human syndrome-associated antigens and derived from GAD or a fragment or equivalent thereof.

Examples of such antigenic peptide sequences can be readily found in patent applications US20170095544, US20090155292 and other prior art. For example, diabetes-related antigens include, but are not limited to, antigens derived from PPI, IGRP, GAD, islet cell autoantigen-2 (ICA 2), and/or insulin. Apart from the peptides and proteins disclosed in US patent 10124045B2, and their respective equivalents and/or combinations, autoreactive diabetes-related antigenic peptides include, but are not limited to, those listed below. For example, they may be the following antigens disclosed in US patent 10124045B 2: GAD65114-123, GAD65536-545, GFAP143-151, GFAP214-222, IA-2172-180, IA-2482-490, IA-2805-813, ppIAPPs5I3, ppIAPP9-17, IGRP152-160, IGRP211-215, IGRP215 223, IGRP222-230, IGRP228-236, IGRP265-273, IGRP293-301, proinsulin L2-10, proinsulin L3-11, proinsulin L6-14, proinsulin B5-14, proinsulin B10-18, proinsulin B14-22, proinsulin B15-24, proinsulin B17-25, proinsulin B18-27, proinsulin B20-27 proinsulin B21-29, proinsulin B25-C1, proinsulin B27-C5, proinsulin C20-28, proinsulin C25-33, proinsulin C29-A5, proinsulin A1-10, proinsulin A2-10, proinsulin A12-20, hInsB10-18, hIGRP228-236, hIGRP265-273, IGRP206-214, hIGRP206-214, NRP-A7, NRP-I4, NRP-V7, YAI/Db, INS B15-23, PPI76-90 (K88S), IGRP13-25, GAD555-567 (557I), IGRP23-35, B24-C36, PPI76-90, INS-I9, TUM, G6Pase.

In certain aspects, the human diseases and disease-related pMHC complexes for treatment may be:

type I diabetes and pMHC complex are selected from: insB10-18-HLA-A2, PPI76-90 (K88S) -HLA-DRB1 x 0401/DRA, IGRP13-25-HLA-DRB1 x 0301/DRA, GAD 555-567-HLA-DRB 1 x 0401/DRA, GAD555-567 (557I) -HLA-DRB1 x 0401/DRA, IGRP23-35-HLA-DRB1 x 0401/DRA, B24-C36-HLA-DRB1 x 0301/DRA, or PPI76-90-HLA-DRB1 x 0401/DRA;

the multiple sclerosis and pMHC complex is selected from: MBP86-98-HLA-DRB1 x 1501/DRA, MBP89-101-HLA-DRB5 x 0101/DRA, MOG38-52-HLA-DRB4 x 0101/DRA, MOG 97-109 (E107S) -HLA-DRB1 x 0401/DRA, MOG203-217-HLA-DRB3 x 0101/DRA, PLP54-68-HLA-DRB3 x 0101/DRA, PLP94-108-HLA-DRB1 x 0301/DRA, PLP250-264-HLA-DRB4 x 0101/DRA, MPB13-32-HLA-DRB5 x 0101/DRA, MPB83-99-HLA-DRB5 x 0101/DRA, MPB111-129-HLA-DRB5 x 0101/DRA, MPB 146-DRB 5-HLA 146-DRB 1/DRB 95 x 0101/DRA, or MPB 95-HLA 5-HLA 4 x 0101/DRB 4 x 0101/DRA, MPB 35-HLA 4-HLA 5/DRB 4 x 0101/DRB 4, MPB 3-HLA 5/DRB 5 x 0101/DRB 4 x 0101/DRB 4, or MPB 4-HLA 5-DRB 1/DRB 4/DRB 1/DRB 4;

celiac disease and pMHC complex is selected from: aGlia 57-68-HLA-DQB 1 x 0501/HLA-DQB1 x 0201, aGlia 62-72-HLA-DQB 1 x 0501/HLA-DQB1 x 0201, aGlia217-229-HLA-DQA1 x 0501/HLA-DQB1 x 0302, or aGlia217-229-HLA-DQA1 x 03/HLA-DQB1 x 0302;

The primary biliary cirrhosis and pMHC complex is selected from: PDC-E2122-135-HLA-DRB4 0101/DRA, PDC-E2249-262-HLA-DRB4 0101/DRA, PDC-E2249-263-HLA-DRB1 0801/DRA, PDC-E2629-643-HLA-DRB1 0801/DRA, PDC-E272-86-HLA-DRB3 0202/DRA, PDC-E2353-367-HLA-DRB3 x 0202/DRA, PDC-E2422-436-HLA-DRB3 x 0202/DRA, PDC-E2629-643-HLA-DRB4 x 0101/DRA, PDC-E280-94-HLA-DRB5 x 0101/DRA, PDC-E2353-367-HLA-DRB5 x 0101/DRA, or PDC-E2535-549-HLA-DRB5 x 0101/DRA, mPDC-E2166-181-I-Ag7, or mPDC-E282-96-I-Ag7;

the neuromyelitis spectrum disorder and pMHC complex are selected from: AQP4284-298-HLA-DRB1 x 0301/DRA, AQP463-76-HLA-DRB1 x 0301/DRA, AQP4129-143-HLA-DRB1 x 0401/DRA, or AQP439-53-HLA-DRB1 x 1501/DRA;

allergic asthma and pMHC complexes are selected from: DERP-116-30-HLA-DRB1 x 0101/DRA, DERP-116-30-HLA-DRB1 x 1501/DRA, DERP1171-185-HLA-DRB 1 x 1501/DRA, DERP-1110-124-HLA-DPB1 x 0401/DRA, DERP-226-40-HLA-DRB1 x 0101/DRA; DERP-226-40-HLA-DRB1 x 1501/DRA, or DERP-2107-121-HLA-DRB1 x 0301/DRA.

The liquid and the solution in the invention are aqueous solutions unless otherwise specified. The drug (e.g., TLR agonist, antigen, immunosuppressant) in the liquid formulation may be in a dissolved drug form or in an insoluble form such as aggregates, particles including crystals and precipitates. In some embodiments, the drug in liquid form is present as a suspension. Some drugs such as imiquimod, rapamycin have low water solubility and they may be present in liquid form as fine particle suspensions. Additional water-solubility enhancing adjuvants may be added to the formulation to enhance the solubility of poorly water-soluble drugs, such as suitable amounts of surfactants (e.g., 0.05% -0.5% tween-20, tween-60, tween-80, lecithin, span, fatty acid esters of glycerol, alkyl polyglucosides), polymers (e.g., 0.2-2% PVA,1% -10% PEG), organic solvents as co-solvents (e.g., 2-20% ethanol, DMSO, propylene glycol).

Biodegradable implants that encapsulate antigens and immunosuppressants, or that encapsulate antigens and vaccine adjuvants (e.g., TLR agonists), or that encapsulate antigens alone, can also be used to induce tolerance to antigens to treat antigen-related diseases such as allergy. If the implant is a large particle, the size of the implant may be greater than 10 μm in diameter, preferably > 100 μm. For example, a 2mm (length) x 0.3mm (diameter) stick made of PLGA containing 0.5 μg to 0.5mg of prolamin and optionally 1 to 3mg of rapamycin (or a TLR agonist, such as 0.1 to 1mg imiquimod or 0.5 to 5mg poly IC or 0.5 to 5mg CPG ODN) may be used as a subcutaneous implant to treat gluten intolerance. Other implant forms, such as NanoPortal capsule from Nanoprecision Medical, may also be used ^TM And Medici Drug Delivery System from Intarcia ^TM As long as they are capable of delivering antigen and optionally immunosuppressant or optionally adjuvant type agents simultaneously and continuously over time as a sustained delivery system. Macroscopic drug delivery systems such as mesoporous silica nanorod stents can also be used as sustained release systems. Other therapeutically safe and effective amounts of allergies Precursors such as pollen extracts, dust mite extracts, and other food allergens can also be used in place of prolamin to treat the corresponding disease.

https://patents.google.com/patent/WO2022098467A1/enoq＝PCTus2021％2f053823#

In one example, the composition in a 2-5% sodium alginate liquid formulation at ph=7 contains 0.5ug-0.5mg gluten/mL (e.g. G5004 gluten from wheat, sigma) for gluten (also known as gluten protein) intolerance treatment. In one example, the compositions and liquid formulations contain 0.5ug-0.5mg gluten/mL (e.g., G5004 gluten from wheat, sigma) and 0.05mg to 5mg/mL rapamycin or 0.1mg-10mg/mL methotrexate in 2-5% sodium alginate, for gluten intolerance treatment, optional solubility enhancing excipients such as 0.1% tween 20 or 5% propylene glycol may also be added to the formulation. It may also comprise 200-2000IU/mL IL-2/anti-IL-2 antibody complex or a mimetic or derivative thereof, e.g. PEGylated IL-2NKTR-358. The initial injection dose may be the maximum tolerated dose, for example, 10 μg/mL gluten in a 0.5mL formulation may be injected into a person who may tolerate 5 μg gluten. That is, the treatment involves a series of doses or formulations, the first dose or formulation containing the lowest amount of allergen and gradually increasing over time in the subsequent doses or formulations, while the amount of other drugs (e.g., immunosuppressants or immunopotentiators) may remain unchanged. The amount of allergen in the first formulation may be the highest amount of allergen that the patient can tolerate without eliciting a severe allergen response. When tolerance is enhanced, the amount injected and/or antigen concentration may be increased, similar to standard allergen desensitization treatment methods. The antigen (e.g., gluten) and immunosuppressant (e.g., rapamycin and/or methotrexate) in the in situ gelling formulation may be in dry form, such as lyophilized powder/lyophilized cake with optional bulking agent or lyoprotectant (e.g., 2-5% sucrose), rather than liquid, these components may simply be physically mixed together, they may also be co-dissolved, then dried, and then placed into a vial. In one example, 1-1000 μg gluten and 1.7-2g poloxamer 407 are vigorously mixed in 10mL water for 10 minutes, then lyophilized, and then the dry mixture can be reconstituted with water prior to injection. In another example, 1-1000 μg gluten and 1mg rapamycin, 1.7-2g poloxamer 407 are vigorously mixed in 10mL water for 10 minutes, then lyophilized, and then the dry mixture can be reconstituted with 10mL water prior to injection. 1mg of rapamycin in the present formulation may be replaced with 1mg of polyIC or 0.25mg of imiquimod to treat allergy. In another example, the liquid formulation contains 5mg/mL methotrexate or 2mg/mL rapamycin, 5-50 μg/mL gluten such as G5004 gluten from wheat, sigma) or PLGA solution (e.g., 30% -50% PLGA in N-methylpyrrolidone or DMSO). This can be injected into patients to induce gluten tolerance and treat gluten intolerance. Gluten may be replaced with prolamine.

For the treatment of egg allergy, gluten in the above examples may be replaced with egg white proteins, such as egg mucin (Gald 1) or 0.1 μg-0.5mg/mL ovalbumin (Gald 2) or a combination thereof with optionally 0.1ug-0.5mg/mL egg transferrin (Gald 3) and 5ug-5mg/mL lysozyme (Gald 4) in a final concentration of 0.1 μg-0.5mg/mL for the treatment of egg white allergy. In one example, the antigen is the peanut antigen ara h2 at 2 μg/mL in 2-5% sodium alginate at pH7 or 17-20% poloxamer 407 solution at pH7 to treat peanut allergy as an injection. In another example, the antigen is the peanut antigen ara h2 at 2 μg/mL, and rapamycin at 0.2-1mg/mL is used as an injection in 2-5% alginate or 17-20% poloxamer 407 solution to treat peanut allergy. In one embodiment, the peanut antigen ara h2 0.1ug-10ug,0.1-2mg rapamycin or 0.1-1mg imiquimod or 0.1-1mg poly IC in 1mL 50% PLGA N-methylpyrrolidone or DMSO solution is used as an injection for the treatment of peanut allergy. Peanut antigen ara h2 may also be replaced by peanut protein extracts containing a mixture of proteins, such as defatted peanut flour. As another example, an injection for treating lupus contains DNA antigen, as shown in FIG. 3 of U.S. patent application Ser. No. 16/029,594, double stranded DNA (1 mg-10 mg/mL) and 0.3-3mg/mL rapamycin or Fuji mycin or temsirolimus in a solution of 2-3.5% sodium alginate or 25% Pluronic F127 or 20% poloxamer 407, or 45%50:50 lactide/glycolide in N-methylpyrrolidone or DMSO.

Other pharmaceutically acceptable amounts of antigen and immunosuppressant (or vaccine adjuvant such as TLR agonist) may also be used in the formulation, as long as satisfactory biological and therapeutic (e.g. tolerogenic) effects are produced, which can be determined experimentally by screening and testing according to well-known protocols and methods. If the antigen is an allergen, such as pollen extract, dust mite extract, animal hair extract or nut protein, the amount of antigen in the desired formulation can be determined experimentally using an allergic skin test, and the highest allergen content tolerated in the formulation can be used in the formulation.

Sustained delivery of antigen and immunosuppressive drugs will be taken up by APCs, induce/activate tolerogenic dendritic cells and tregs/bregs, suppress B cell activation/antibody production, germinal center formation and antigen-specific hypersensitivity reactions, resulting in long-term antigen-specific immune tolerance.

Methods and agents for treating autoimmune diseases and allergies or inhibiting the production of anti-drug antibodies or inducing antigen-specific immune tolerance by administering a mixture of the antigen and the immunosuppressant/drug in a sustained release formulation as an injection or implant to a subject/patient in need thereof. Also disclosed are methods and agents for treating allergy by administering the mixture of antigen and vaccine adjuvant agents as an injection or implant to a subject/patient in need thereof in a sustained release formulation, where the methods and mixtures are not intended to treat autoimmune diseases and are not intended to inhibit drug-resistant antibodies. The injection may be administered by subcutaneous injection or intramuscular injection or intradermal injection or intralymphatic injection. The injection may contain a viscosity increasing agent to increase its viscosity or become a gel after injection as a sustained release preparation of antigen and immunosuppressant. Molecules that promote T/Breg amplification (e.g., IL-2 and/or TGF-. Beta.and/or PD-L1) may also be added to the injection in combination with other immunosuppressants. The antigen and immunosuppressant may be in free molecular form, or may be in nanoparticle/microparticle form, including liposomal form. In certain embodiments, the injectate has a viscosity of greater than 10,000cps at room temperature. In certain embodiments, the injectate has a viscosity of greater than 100,000cps at room temperature. In certain embodiments, the injectate has a viscosity of greater than 1,000,000cps at room temperature. In certain embodiments, the injection has a viscosity of 10,000,000cps at room temperature. Examples of viscosity enhancing agents can be readily found from known pharmaceutically acceptable excipients such as hyaluronic acid, starch and carbomers. In some embodiments, the viscosity enhancing agent is biodegradable. In one example, the viscous injection contains 0.1-100 μg/mL gluten (e.g., G5004 gluten from wheat, sigma) and 0.2-2mg/mL rapamycin or 0.5-5mg/mL methotrexate and an appropriate amount of hyaluronic acid (e.g., 20-50 mg/mL) to achieve a viscosity of 300,000cps, optionally containing 1mg/mL IL-2. Hyaluronic acid may be crosslinked to extend its in vivo half-life. The injectable formulation may also be a thermal phase change formulation. A thermal phase change formulation refers to a formulation whose phase changes from a liquid at low or room temperature (25 ℃) to a semi-solid/gel when the temperature is raised to body temperature (37 ℃), which may use a temperature triggered in situ gelling system such as poloxamer as an adjuvant. The hot phase change injectable formulation comprising an antigen and an immunosuppressant may be administered in the form of subcutaneous injection or intramuscular injection or intradermal injection to induce antigen specific immune tolerance and treat the corresponding autoimmune disease or allergy. It has a low viscosity at low or room temperature but a high viscosity at body temperature. The preparation of such thermal phase change injections can be readily adopted by those skilled in the art from the relevant publications. For example, a composition of the thermal phase change injection formulation is 15 μg/mL gluten (e.g., G5004 gluten from wheat, sigma) and 3mg/mL of a 25% (w/w) Poloxamer-407 solution at pH7, which can be injected 0.5-1mL, once every two weeks, 3 times total, to induce gluten tolerance by subcutaneous or intralymphatic injection.

Immunosuppressants can also be conjugated to carbohydrate polymers or other biocompatible polymers (e.g., dextran or heparin or hyaluronic acid or polypeptides) to form prodrugs, as described in U.S. patent applications 15/723,173, 16/380,951 and 16/029,594. The novel prodrug may be in the form of a carbohydrate (or other polymer) drug conjugate in which the drug is conjugated to the carbohydrate (or other polymer) via a cleavable bond. More than one drug may be bonded to the polymer backbone. Suitable carbohydrates include sialic acid containing polymers, hyaluronic acid, chondroitin sulfate, dextran, carboxydextran, cellulose, carboxycellulose and derivatives thereof. It may also be a linear polymer backbone (e.g. dextran or synthetic polymers such as PVA, PAA). In addition, immunosuppressive drugs can also be used in formulations directly coupled to antigens or coupled to antigens via linkers or carriers. The carrier may be a polymer. For example, polysialic acid-rapamycin of U.S. patent application 15/723,173 in fig. 8 can be used to conjugate with protein lysine (e.g., gluten or antibody drugs or prolamin or peanut antigen protein ara h 2) by EDC coupling and to replace a mixture of antigen and drug in a formulation, e.g., 100 μg-15 mg.

The formulations or implants of the present inventors and of the previous applications may comprise antigen + drug or antigen-drug conjugate or encapsulated antigen/drug (e.g. in microspheres or nanoparticles or liposomes) or a combination thereof. The antigen may be a crude antigen (e.g. peanut extract, gluten, egg white powder, pollen extract, dust mite extract) or a purified antigen (e.g. peanut antigen protein ara h2, prolamin) or an antigen-drug conjugate or in the form of an encapsulated antigen (e.g. in microspheres or liposomes) or a mixture thereof.

When liposomes are used, both the drug or antigen and the immunosuppressive drug can be entrapped in the liposomes. Dendritic Cells (DCs) are abundant in the skin and the addition of DC-modulating drugs with antigens/allergens to the formulation is effective in inducing tolerance. When liposomes that express both antigen and siglec ligand are used (e.g., those described in the present invention, J Clin invest.2013Jul;123 (7): 3074-83,J Immunol.2013Aug 15;191 (4): 1724-31 and those described in U.S. Pat. No. 3, 9552183), the liposomes may also encapsulate immunosuppressive drugs such as rapamycin. For example, each liposome particle can contain a pharmaceutically effective amount of rapamycin (e.g., 1% to 50% by weight of the liposome of rapamycin). This will further increase the efficacy of inducing immune tolerance and treating autoimmune diseases/allergies.

Another form suitable for the current application is the use of microspheres (also known as microparticles). The term microsphere includes particles ranging from nano-sized to micro-sized (e.g., 50nm-50 μm in diameter). Preferably, the microspheres are biodegradable (e.g., made of biodegradable polymers such as PLGA). For example, the microspheres are made of biodegradable synthetic polymers such as PLGA and encapsulate immunosuppressive drugs such as rapamycin (e.g., 1% to 80% by weight of the microspheres). The size of the microspheres is 3 μm or 300nm. Antigens may also be bound to the microsphere surface either directly or through linkers. Alternatively, the antigen is encapsulated in a microsphere. Alternatively, the drug (immunosuppressant) may be bound to the microsphere surface rather than being encapsulated. Examples of microspheres or antigen-immunosuppressant conjugates suitable for use in the present application may be readily employed from the disclosures in publications such as those of patent applications US13/880,778, US14/934,135, CA2910579, US13/084,662 and US8652487 and other patent applications filed at Selecta Biosciences. It can be used for treating autoimmune diseases or allergies or inducing immune tolerance, and can be injected or implanted (encapsulated in an implant) or topically applied to a patient. Pharmaceutically acceptable sustained release formulations such as microspheres or conjugates in an in situ gelling matrix may be used provided that they produce a satisfactory therapeutic (e.g., immune tolerance) effect, which can be determined by screening and testing by well known experimental procedures.

Sustained delivery formulations comprising antigen and immunosuppressant combinations are useful for allergy, autoimmune diseases and anti-drug antibody treatment. When the immunosuppressant in the examples and methods of the present application is replaced with an immunopotentiator (e.g., a vaccine adjuvant such as a TLR agonist or STING agonist) and the antigen is a pathogen antigen or tumor antigen, the transdermal delivery system becomes a vaccine or booster vaccine for the pathogen or tumor of interest. For example, sustained delivery formulations are in situ gelling formulations containing a compounded immunopotentiator and pathogen antigen. It may also be in the form of an implant as previously described. Vaccine adjuvant molecules such as TLR agonists may be used in the present invention, such as MPLA, cpG ODN, imiquimod, poly IC, raschimod, adequimod, R848 and 3M-052. Examples of pathogen antigens may be synthetic or purified or mixtures of pathogen antigens. For example, it may be HIV gp-120, it may be influenza neuraminidase, it may be influenza virus lysate, it may be HBV surface antigen, it may be tumor cell lysate. The use of these antigens will generate an immune response against the pathogen or tumor as a vaccine or booster. In some embodiments, the sustained release vaccine formulation is a liquid injection containing 10 μg to 1mg antigen, such as a pathogen antigen or tumor antigen, 50 μg to 5mg TLR agonist per dose or per ml of injection in a sustained release matrix as described, for example, in situ gelling systems as previously described. For example, an in situ gelling system may be 2-9% sodium alginate and optionally a gelling enhancer such as 1% HPMC and optionally a solubility enhancing adjuvant such as 0.1% tween-20 or 5-15% DMSO may also be added to the formulation; or with a temperature triggered in situ gelling polymer, such as 17-25% Pluronic F127 or Poloxamer 407; or 40-50% PLGA in DMSO or N-methylpyrrolidone. In one example, the formulation comprises 100 μg-1mg/mL influenza virus lysate, 0.2-2mg/mL imiquimod or 0.2-2mg/mL polyIC and 1mg/mL cetirizine, containing 3.5% sodium alginate and optionally 1-2% HPMC. In another example, the vaccine formulation contains 20-100 μg/mL HBV surface antigen, 0.5-10mg/mL CPGODN1018 adjuvant in 20% poloxamer 407. It can be used for generating immunity against HBV by subcutaneous injection or intramuscular injection or intradermal injection or injection, and also can be used for intralymphatic injection. In another example, the vaccine formulation contains 100 μg/mL pathogen antigen, 2-10mg/mL poly IC, 1-5mg/mL imiquimod, optionally 1-5mg/mL cetirizine, and 45% PLGA in N-methylpyrrolidone or DMSO solution containing 7-17kDa 50:50 lactide/glycolide. The pathogen or tumor antigen may also be an antigenic peptide capable of binding to MHC to form an MHC-peptide complex.

In one aspect, the invention discloses compositions and formulations for treating allergies comprising an antigen and an immune activity enhancer that cause the disorder in a sustained release formulation or implant. The antigen may be an allergen, a B-cell antigen in the form of an allergen or fragment thereof, a T-cell antigen in the form of an MHC-peptide complex or an antigenic peptide of a T-cell antigen (or derivative thereof) which can bind to MHC to form an MHC-peptide complex. Examples of immune activity (or called immune function) enhancers may be selected from TLR agonists such as imiquimod, poly IC and CPGODN. Also disclosed is a method of treating allergy or inhibiting IgE-induced responses by inducing antigen-specific immune tolerance and/or inducing production of competitive IgG against an antigen in a subject by administering the composition/formulation as a subcutaneous injection to the subject, but also intramuscularly or intradermally or intralymphatically or as an implant.

The invention further discloses methods and agents for treating allergy by applying the combination/composition of an allergy-causing antigen and an immune activity enhancer/drug to a subject/patient as a physical mixture or as a synthetic conjugate or as nanoparticles/microparticles or liposomes in a therapeutically effective amount. The combination/composition may be in a sustained (extended) release system, such as an in situ gelling system or implant.

Examples of suitable immune activity (function) enhancers include Pattern Recognition Receptor (PRR) ligands, RIG-I like receptor (RLR) ligands, nod Like Receptor (NLR) ligands, C Lectin Receptor (CLR) ligands, STING Toll-like receptor ligands, such as TLR3 ligands, TLR4 ligands, TLR5 ligands, TLR7/8 ligands, TLR9 ligands, or combinations thereof. The immune function enhancer may be a vaccine adjuvant. Examples of suitable vaccine adjuvants may be saponins, such AS Matrix-M adjuvants (quillaja saponins formulated with cholesterol and phospholipids AS nanoparticles), squalene, such AS MF59 (oil-in-water emulsion of squalene oil) and AS03 adjuvants (vitamin E and oil-in-water emulsion of squalene), MPL, such AS01B, QS-21, purification of saponaria from quillaja bark, AS04 is a combination of aluminium hydroxide and monophosphoryl lipid a (MPL), aluminium salts such AS aluminium hydroxide, aluminium phosphate, alum (potassium aluminium sulphate) or mixed aluminium salts. The concentration of these vaccine adjuvants may be the same as or up to 20 times higher than those currently used. Preferably, the Toll-like receptor ligand is a Toll-like receptor (TLR) agonist. Examples include the imidazoquinoline family of TLR7/8 ligands (e.g., imiquimod (R837), jidiaquinimod, remiquinimod (R848), 3M-052, 3M-852, 3M-S-34240, mo Tuo mod/VTX-2337, NKTR-262; cpg ODNs, such as SD-101, ODN 1826 and ODN 2216, TLR agonists, including TLR peptide agonists disclosed in patent applications WO2018055060A1, WO2013120073A1, WO2016146143A1 and US20180133295A1 and references thereto, synthetic analogs of dsRNA, such as poly ICs (e.g., poly ICLC, poly IC-kanamycin, polyI: polyC 12U), TLR4/5 ligands, such as bacterial lipopolysaccharide (LPS, e.g., monophosphoryl lipid A), bacterial flagellin (e.g., vibrio vulnificus flagellin B), glucopyranosyl Lipid A (GLA), the TLR7 agonist loxoribine or derivatives/analogues thereof, or others, which may be in the form of an active drug, prodrug, liposome, emulsion, micelle, insoluble precipitate (e.g., complexed with a precipitant), combined with a polymeric drug carrier (e.g., dextran) or encapsulated in biodegradable microparticles/nanoparticles polyacrylic acid containing polymers such as carbomers are also useful immune function enhancers in addition to TLR agonists and STING agonists, other molecules that activate/enhance immune system and immune cell function such as APC, B cells and T cells may also be incorporated into the formulation, examples of suitable immune function activating/enhancing molecules and other suitable immune function enhancing agents can be found in US16271877 and US 16924184. They may be added to the formulations described herein in therapeutically effective amounts.

In some embodiments, the immune activity enhancer may comprise any of the agents provided herein. The immune activity enhancer may be a compound that directly provides an immune enhancing (e.g., activating) effect on APC, or it may be a compound that indirectly provides an immune enhancing effect (i.e., after being processed in some way after administration). Thus, an immune activity enhancer includes a prodrug form of any of the compounds provided herein. Different immune activity enhancers may be used as a mixture and combined in the present invention. An immune activity enhancer also includes nucleic acids encoding a peptide, polypeptide, or protein provided herein that results in an immune enhancing (e.g., activating) immune response. Thus, in embodiments, an immune activity enhancer is a nucleic acid encoding a peptide, polypeptide, or protein that results in an immune enhancing (e.g., activating) immune response. The nucleic acid may be coupled to a synthetic nanocarrier. The nucleic acid may be DNA or RNA, such as mRNA. In embodiments, the compositions of the invention comprise a complement of any nucleic acid provided herein, e.g., a full-length complement or degeneracy (due to the degeneracy of the genetic code). In embodiments, the nucleic acid is an expression vector that can be transcribed when transfected into a cell line. In embodiments, the expression vector may comprise a plasmid, retrovirus, adenovirus, or the like. Nucleic acids can be isolated or synthesized using standard molecular biology methods, for example, by using the polymerase chain reaction to generate nucleic acid fragments, which are then purified and cloned into expression vectors.

Publications listed before Selecta disclose methods of synthesizing nanocarriers and related compositions, including B-cell and/or MHC class II restriction epitopes and an immune activity inhibitor, to generate immune responses. In their disclosure, an antigen/epitope is coupled to a nanocarrier and an immunosuppressant is coupled to the nanocarrier. Another method and composition is the use of nanoparticles/microparticles having an allergy-inducing antigen/epitope encapsulated or non-covalently adsorbed to their surface and an immune activity enhancer encapsulated therein. The nano/microparticles may be made of biodegradable material, such as PLGA. These nanoparticles/microparticles (e.g., 10nm-10 μm in diameter) can be injected or inhaled or administered orally or topically to a patient in need thereof to achieve an antiallergic effect. Encapsulation of immune activity enhancers and antigens is well known to those skilled in the art and can be readily employed from related publications. The surface of the nano-particles/microparticles can be provided with amino or carboxyl and other charged groups so as to increase the binding of the allergy-causing antigen/epitope on the surface; it may also have a hydrophobic surface to allow binding of antigens/epitopes by hydrophobic interactions; or a combination thereof. Introduction of charged groups to the surface nanoparticles/microparticles may be prepared by using surface modification or using molecules containing amine or carboxyl groups. Allergy-causing antigens/epitopes may also be conjugated with lipophilic moieties such as lipid molecules like fatty acids or cholesterol to increase their binding to nanoparticles/microparticles. Adsorption of the allergy-causing antigen/epitope to the nanoparticle/microparticle surface can be achieved by incubating the antigen/epitope with the nanoparticle/microparticle (e.g. overnight at 4 ℃ in an aqueous buffer such as 1 xPBS) and then removing unbound antigen/epitope (e.g. washing the nanoparticle/microparticle several times with an aqueous buffer, similar to the ELISA plate coating procedure). In one embodiment, 50nm to 200nm sized PLGA nanoparticles are prepared coated with 10% imiquimod by weight. Next, PLGA nanoparticles were mixed with allergen OVA (10 mg/mL) overnight at 4℃to yield OVA (ovalbumin) coated particles. The particles were washed 3 times with PBS to remove unbound OVA. In another example, imiquimod is dissolved in DMSO at a concentration of 50 mg/ml. A total of 50. Mu.L imiquimod was added to 1ml PLGA (5 mg/ml) dissolved in methylene chloride. Next, the mixture was homogenized with 0.4ml of 5% ova solution using ultrasonic waves for 10 minutes. The o/w emulsion produced was added to 2.1ml of a 5% w/v PVA solution and the organic solvent was evaporated at room temperature for 4 hours. After centrifugation at 3,500g for 20 minutes, OVA-coated nanoparticles containing imiquimod were obtained. Additional washing steps may be performed to obtain particles free of unbound OVA. Such OVA-coated particles in an in situ gelling system such as a pH7 2-5% alginate or a pH717-20% poloxamer 407 solution may be administered to a subject in need of induction of OVA immune tolerance treatment for allergy to IgE, subcutaneously or intramuscularly or intradermally or intralymphatically to treat allergy to OVA. OVA can be replaced with other allergen/epitope molecules to treat the corresponding allergy. In another sample, PLGA particles having a surface charge can be prepared by adding a lipophilic carboxylic acid or lipophilic amine or an anionic detergent or a cationic detergent (e.g., fatty acids such as octanoic acid, lauric acid; or cationic lipids such as DOTMA, DOTAP, cholesterol amine) to PLGA. In one example, imiquimod is dissolved in DMSO containing 10mg/ml lauric acid at a concentration of 50 mg/ml. A total of 50. Mu.L imiquimod/lauric acid was added to 1ml PLGA (5 mg/ml PLGA) dissolved in methylene chloride. Next, the mixture was homogenized with 0.1ml of 2% octanoic acid solution using ultrasonic waves for 10 minutes. The o/w emulsion was evaporated at room temperature for 4 hours to remove the organic solvent. The resulting PLGA particles were washed 3 times with PBS and then incubated with OVA to prepare OVA-conjugated particles. It can be added to an in situ gelling matrix and then used to treat allergies to OVA. In one example, 1mg to 10mg of the particles in 3.5% sodium alginate and 1% HPMC can be injected 3 times per month to OVA intolerant patients, inducing OVA tolerance by subcutaneous or intralymphatic injection.

In addition, the allergen (antigen)/epitope causing allergy may be encapsulated within the nanoparticle/microparticle in addition to being conjugated or adsorbed to its surface. The preparation of antigen/epitope encapsulation is well known to those skilled in the art and can be readily adopted from the relevant publications, for example using a double emulsion water/oil/water system.

Patent application US20130287729 discloses antigen-specific, tolerance-inducing microparticles and uses thereof. It discloses a microparticle (0.5 μm-10.0 μm in size) for targeting an antigen presenting immune cell of interest and for inducing antigen specific immune tolerance, wherein the microparticle comprises an antigen and a therapeutic agent, wherein the therapeutic agent is an immunomodulator, an immunosuppressant tolerogenic agent, or an agent that recruits an antigen presenting immune cell of interest, wherein the surface of the microparticle comprises a ligand that targets the antigen presenting immune cell of interest, and the microparticle is made of a biodegradable material. A further improvement of the method and composition for treating allergy is the use of microparticles or nanoparticles, preferably made of biodegradable material, with a size of 50nm-5 μm and the use of an immune activity enhancer instead of an immunosuppressant, preferably in a slow release formulation, such as an in situ gelling system or a high viscosity formulation. The particles comprise an antigen that causes allergy by encapsulation or coating or encapsulation + coating. In some embodiments, the surface of the nanoparticle/microparticle is coated with the Fc portion of the antibody or the whole antibody with the Fc portion facing outward. This will bind to FcR to promote APC uptake. In other embodiments, the surface of the nanoparticle/microparticle need not have ligands that target antigen presenting immune cells. In some embodiments, it may have an allergy-inducing antigen/epitope coated on its surface. The interior of the nanoparticle/microparticle comprises the immune activity enhancer listed in the present application and optionally the allergy-causing antigen/epitope, for example by encapsulation. The preparation methods are well known to those skilled in the art and can be readily adopted from the relevant publications. For example, 5mg to 50mg of the above gluten and poly IC containing particles may be injected into a gluten intolerant patient to induce gluten tolerance by subcutaneous or intramuscular or intralymphatic injection in 3% sodium alginate and optionally 0.5 to 2% HPMC, or in 15 to 25% Pluronic F127 or 15 to 25% Poloxamer 407.

U.S. patent application 20160338953 A1 discloses a liposome-based immunotherapy. It provides a liposome encapsulating an autoantigen, wherein the liposome has a size of 500 to 15000nm and the liposome membrane comprises Phosphodiserine (PS) in an amount of 10 to 40% by weight relative to the total membrane liposome composition. Also provided are pharmaceutical or veterinary compositions comprising a therapeutically effective amount of the liposomes. Furthermore, it provides a liposome as defined above for use as a medicament and a pharmaceutical or veterinary composition, in particular for use in the treatment of autoimmune diseases. The invention also discloses antigen-specific liposomes for the treatment of allergies and uses thereof. The liposomes are internally encapsulated with an immune activity enhancer (and optionally an allergy-causing antigen/epitope molecule) as set forth herein. Optionally, the surface of the liposome may also have an allergen/epitope coating that causes allergy. Can be administered as injection to patients in need thereof to induce immune tolerance to allergen and treat allergy. Allergy-causing antigens/epitopes may also be conjugated to lipid molecules such as fatty acids or phospholipids or cholesterol derivatives to allow their insertion into the liposome membrane. Suitable liposomes can have a size between 50nm and 20 μm. Methods for their preparation and their use are well known to the person skilled in the art and can be readily adopted from related publications such as those in US 20160338953. Examples of lipid molecules suitable for use in preparing liposomes according to the present invention include, but are not limited to, phosphoglycerides, glycerophospholipids, sphingolipids, ceramides, glycerophospholipids ethanolamine, sterols, or steroids. These lipid molecules can also be used to prepare allergy-inducing antigen/epitope-lipid conjugates. Instead of antigen/epitope-lipid conjugates, membrane anchored peptide-antigen/epitope conjugates can also be used. These liposomes can be in sustained release formulations, such as in situ gelling systems or high viscosity formulations. For example, 5mg-50mg of these liposomes (5-25% of the formulation), containing egg white antigen such as ovomucoid, and rapamycin, can be injected 3 times per month into egg white intolerant patients, either subcutaneously or intramuscularly or intrainguinal lymph node injection, in 3% sodium alginate and optionally 0.5-2% HPMC, or in 15-25% Pluronic F127 or 15-25% poloxamer 407.

Methods, agents, compositions and formulations for treating allergy by injecting a mixture of an allergy-inducing antigen and an immune activity enhancer into a sustained release formulation, such as an in situ gelling system or implant, of a subject/patient in need thereof are disclosed. It may also contain antiallergic agents such as antihistamines, corticosteroids, mast cell stabilizers and leukotriene inhibitors. The addition of these antiallergic agents can prevent allergic reactions in patients caused by administration of allergens to patients. The method and the composition/formulation are useful for inducing the production of anti-allergen IgG antibodies to compete for endogenous IgE production of an allergic reaction; thus, it induces immune tolerance to allergens. The immune activity enhancer may be in the form of an active agent, a prodrug, a microparticle or nanoparticle or a liposome. The allergy-inducing antigen may be a B cell antigen/epitope or a T cell antigen/epitope (e.g. an MHC-peptide complex or conjugate; or an antigenic fragment, e.g. a peptide which can bind to MHC), or a combination thereof. The combination may be a B cell antigen/epitope and a T cell antigen/epitope; or a combination of several different B cell antigens/epitopes and/or several different T cell antigens/epitopes for the same disease or different diseases. The use of peptide antigens (T cell epitopes) that bind to MHC to form MHC-peptide complexes (T cell antigens) in vivo in place of the peptide-MHC complexes reduces size and molecular weight and may be used instead. The use of peptide antigens with a single epitope domain may reduce the risk of activating mast cells by not cross-linking with IgE on the cell surface, thus providing better safety and still being able to induce immune tolerance. The allergen (allergen) that causes allergy used in the present invention may be an intact allergen or a fragment thereof such as an epitope thereof, or a combination thereof. Examples of which can be found in this application and related publications and patent applications.

The mixture of the allergy inducing antigen and the immune activity enhancer may be a physical mixture. By physical mixture is meant that the mixture of antigen and immune activity enhancer is simply mechanically mixed together (e.g. by stirring or mixing) in its original form (e.g. liquid or solid form, such as powder or particles) without any additional process (e.g. by mixing them together in their original form), or that a further size reduction process is applied before or after mechanical mixing (e.g. comminution, grinding, or homogenization), or dispersed or dissolved in the same or different types of liquid, respectively, and then mixed, or co-dispersed in a liquid, or co-dissolved in a solvent (e.g. water), and that an optional drying process (e.g. spray drying or freeze drying) can be applied together with an optional further size reduction process.

In some embodiments, the method is to use an in situ gelling liquid containing an allergen or fragment thereof and an immunopotentiator drug (the drug listed above, such as imiquimod or poly IC). It may also contain antiallergic agents such as antihistamines, corticosteroids, mast cell stabilizers and leukotriene inhibitors. Examples of allergens may be pollen extracts, dust mite extracts, animal hair extracts or food allergens such as nut/peanut/milk/egg proteins. Alternatively, biodegradable implants encapsulated with an allergy-inducing antigen and an immune activity enhancer can be used to treat and prevent allergy. If the implant is a large particle, the size of the implant may be greater than 10 μm in diameter, preferably > 100 μm. The large particles may be in the size range of 100 μm to 10 mm. The particles may be made of biodegradable material, such as PLGA. The implant may also be in the shape of an aspheric body. For example, a 2mm (length) by 0.3mm (diameter) rod made of PLGA containing 3mg imiquimod and 0.5mg prolamin or a 5mm (length) by 2mm (diameter) rod made of PLGA containing 1mg imiquimod and 5mg prolamin can be used as a subcutaneous implant to treat gluten intolerance. Other implant forms may also be used, including non-degradable devices such as NanoPortal Capsule of Nanoprecision Medical ^TM And Intarcia Medici Drug Delivery System ^TM So long as the antigen and the immune activity enhancer can be delivered simultaneously. In some embodiments, a sustained release implant such as NanoPortal Capsule ^TM And Medici Drug Delivery System ^TM Contains only allergen and does not needImmunopotentiators. Current allergen injections for treating allergies require very frequent injections, and the use of implants to provide sustained release of the allergen will reduce the frequency of implantation (e.g. once a month or every 3 months of the implant) and be more patient friendly. The allergen dose of the first implantation is low, and the later period is gradually increased so as to ensure safety. The implant may also contain therapeutically effective amounts (e.g., clinically useful doses) of antiallergic agents, such as antihistamines, corticosteroids, mast cell stabilizers, and leukotriene inhibitors. In some embodiments, the implant may release the same daily dose of antigen as the currently used antigen injection dose for allergy prevention/treatment, and the release may last from weeks to months. Preferably, the daily dose released is capable of inducing tolerance to an allergen but does not cause a severe allergic reaction, such as a severe immediate hypersensitivity reaction. For example, the implant contains 0.3-3mg allergen, which is allowed to release 0.1ug-0.1mg allergen per day for 30 days, and the allergen may be peanut protein or egg white protein or pollen extract. If severe allergic reactions are observed, the implant may be made of materials and configurations that allow it to be removed from the patient to increase its safety. For example, it may be a small cartridge that can be removed from the patient, which contains the allergen and optionally the TLR agonist or immunosuppressant in a sustained release system.

In one example, the compositions and liquid formulations contain 0.5ug-100 μg/mL gluten (e.g., G5004 gluten from wheat, sigma) and 0.2mg-2/mg/mL imiquimod or 0.2-2mg/mL poly IC or a combination thereof in 2-5% sodium alginate ph=7 and optionally 0.1% Tween-60 or optionally 5% propylene glycol to increase drug solubility as an injection to treat gluten intolerance. It may be lyophilized with optional bulking agents and reconstituted prior to use. Other allergens such as pollen extract, dust mite extract, animal hair extract or nut proteins may be used instead, the concentration in the formulation may be the maximum tolerated concentration determined experimentally, for example by subcutaneous injection allergen experiments.

In another example, the compositions and liquid formulations contain 0.5-25 μg/ml gluten and 0.5-5mg/ml poly IC, and optionally 2mg/ml cetirizine in 2-5% ph=7 sodium alginate. In another example, the compositions and liquid formulations are those containing 5-50 micrograms/milliliter gluten, 0.5-2 milligrams/milliliter STING agonist MK-1454, or 0.5-5 milligrams/milliliter CpG ODN 1826 in 20% poloxamer 407. In another example, 1-100 μg/ml gluten and 0.1-1mg/ml imiquimod are mixed with 1mL 50% PLGA N-methylpyrrolidone or DMSO solution as an in situ gelling formulation. In another example, the compositions and liquid formulations contain 0.1mg/ml gluten (e.g., G5004 gluten from wheat, sigma) and 0.5mg/ml imiquimod or 20 μg/ml 3M-052 in ph=7.5% sodium alginate and 1% HPMC. In another example, the compositions and liquid formulations contain 5-500 μg/ml gluten (e.g., G5004 gluten from wheat, sigma) and 1-5mg/ml poly IC in 19% poloxamer 407 and 0.5% hyaluronic acid. These formulations can be used to induce gluten tolerance and to treat gluten intolerance, such as subcutaneous or intramuscular injection or intradermal or intralymphatic injection. Gluten may be replaced with prolamines, such as deamidated prolamines. In some embodiments, a formulation containing gluten or deamidated prolamin peptide may be injected into a patient at a maximum tolerated dose for the patient. Gluten in the above examples may be replaced with egg white proteins, for example 2-100 μg/ml ovomucoid (Gal d 1) or 5-100 μg/ml ovalbumin (Gal d 2) or a combination thereof with optional 2-100 μg/ml ovotransferrin (Gal d 3) and 2-100 μg/ml lysozyme (Gal d 4) to treat egg white allergy. In another example, a composition and liquid formulation contains peanut antigen ara h2 0.01-1 μg/ml and 1mg/ml sodium alginate 3.5% pH7 to treat peanut allergy. In one example, the compositions and liquid formulations comprise the peanut antigen ara h2 0.5 μg/ml, imiquimod 0.5mg/ml, in 20% poloxamer 407, as an injection to treat peanut allergy. In one example, a composition and liquid formulation is prepared with 45%50:50 lactide/glycolide in N-methylpyrrolidone or DMSO, and 0.2 μg/ml peanut antigen ara h2, 0.2mg/ml imiquimod to treat peanut allergy. The immune activity enhancer drug or immune activity enhancer drug and allergen may be in the form of a powder or gel or semi-liquid, or in the form of liposomes (e.g. 100nm-5 μm diameter), or in the form of nanoparticles/microparticles (e.g. 100nm-1 μm) or coupled with dendrimers or linear polymers (e.g. polyacrylic acid or polysialic acid coupled via ester linkages to form polymer-based prodrugs of mw=5K-500K daltons), and then formulated into an in situ gelling system or high viscosity liquid. Other pharmaceutically acceptable amounts of allergy-causing antigens and immune activity enhancers may also be used, provided that they produce satisfactory biological and therapeutic (e.g., immune tolerance) effects, as determined experimentally by well-known methodology protocols screening and testing.

Other allergens such as pollen extract, dust mite extract, animal hair extract and food allergens such as nut/peanut/milk/egg extract may be substituted in the above formulations to treat related allergies. Examples of the concentrations of these allergens may be between 0.1 μg/mL and 1mg/mL, provided they are tolerated by the subject in need thereof. During the course of treatment, as the patient's tolerance increases, the concentration of allergen in the formulation increases, which is a standard practice for allergic immunotherapy.

The amount of allergen corresponding to a given level of efficacy varies greatly depending on the allergen species. In another embodiment of the invention, the concentration of the major allergen in a single dose may be 0.05 to 500 μg, for example 0.1 μg to 100 μg in an injectable formulation. In the field of allergy extracts, there is currently no internationally accepted standardized method. There are many different extraction intensity units, i.e. bioefficacy. The methods and units used generally measure allergen content and biological activity. Such as SQ-Units standardized mass Units), BAU bio-allergen Units), BU bio-Units), UM mass Units), IU international Units) and IR reactivity index). Thus, if extracts from sources other than those disclosed herein are used, they need to be standardized against the extracts disclosed herein to determine their efficacy in SQ units or any of the above units. The biological efficacy of a given extract, i.e., the in vivo allergen activity, depends on many factors, most importantly the content of major allergens in the extract, which varies with the composition of the biogenic material. The amount of allergen extract (in grams) used to obtain the desired bioefficacy varies depending on the type of extract in question, and for a given type of extract, the amount of allergen extract varies depending on the actual bioefficacy. Is an extract of (a) a plant. For a given batch of extract, the grams of allergen extract used to obtain the desired biological efficacy can be determined using the procedure described in U.S. patent No. US9248097B 2.

SQ-Unit is determined according to the SQ potency normalization method, where 100,000SQ units are equal to the standard subcutaneous maintenance dose. Typically 1 mg of the extract contains 100,000 to 1,000,000SQ units, depending on their allergen source and the manufacturing process used. The precise allergen amount can be determined by immunoassay, i.e. total major allergen content and total allergen activity. BAU is a bioefficacy unit determined by FDA requirements for allergen products. According to the above method, the dosage of 100,000SQ units containing grass extract is equal to the content of 2600-4700 BAU. Also, other extracts can be evaluated according to the methods described above.

Also disclosed are methods and agents for treating or preventing allergy by injecting a mixture of an allergen and the immune activity enhancer/drug into a subject/patient in need thereof. The injection may be administered subcutaneously or intramuscularly or intradermally. The injection may contain a viscosity enhancing agent to increase its viscosity upon injection as a slow release formulation of antigen and immune activity enhancing agent. The allergy inducing antigens and immune activity enhancers may be in free molecular form or from nanoparticles/microparticles including liposomal forms. In certain embodiments, the injectate has a viscosity of greater than 5,000cps at room temperature. In certain embodiments, the injectate has a viscosity of greater than 50,000cps at room temperature. In certain embodiments, the injectate has a viscosity of greater than 500,000cps at room temperature. In certain embodiments, the injection has a viscosity of 1,000,000cps at room temperature. Examples of viscosity enhancing agents can be readily found from known pharmaceutically acceptable excipients, such as hyaluronic acid (linear or cross-linked), starch and carbomers. In some embodiments, the viscosity enhancing agent is biodegradable. In one example, the viscous injection contains 0.1-500ug/mL gluten (e.g., G5004 gluten from wheat, sigma) and 0.1-0.5mg/mL imiquimod or 100ug-0.5mg/mL poly IC and an appropriate amount of hyaluronic acid (e.g., 10-50mg/mL linear or cross-linked hyaluronic acid) to achieve a viscosity of 300,000 cps. In one example, the viscous injection contains 0.1-10 micrograms/ml gluten (e.g., G5004 gluten from wheat, sigma) and 50 micrograms/ml imiquimod or 100 micrograms/ml poly IC and an appropriate amount of hyaluronic acid (e.g., 20-50 milligrams/ml cross-linked hyaluronic acid) to achieve a viscosity of 500,000cps, and optionally 2mg/ml cetirizine.

The injectable formulation may also be a thermal phase change formulation as previously described. The thermal phase change injectable formulation comprising an antigen and an immune activity enhancer may be administered in the form of subcutaneous injection or intramuscular injection or intradermal injection to induce antigen specific immune tolerance and treat the corresponding autoimmune disease or allergy. For example, a thermal phase change injection having a composition of 0.01-0.1mg/mL gluten (e.g., G5004 gluten from wheat, sigma) and 0.2-1mg/mL imiquimod in 25% (w/w) Poloxamer-407 ph=7 solution, and optionally 2mg/mL cetirizine, may be injected 0.1-1mL to a gluten intolerant patient to treat gluten allergy, subcutaneously or intralymphatically. Gluten in the above examples may be replaced with egg white proteins, for example 5-100 μg/ml ovomucoid (Gal d 1) or 5-100 μg/ml ovalbumin (Gal d 2) or a combination thereof with optional 5-100 μg/ml ovotransferrin (Gal d 3) and 5-100 μg/ml lysozyme (Gal d 4) for the treatment of egg allergy.

The formulation of the invention comprising an implant for treating allergy may also comprise an antiallergic drug as described previously. The amount of antiallergic agent added may be the same as that used in current antiallergic treatments. The addition of these antiallergic agents can prevent allergic reactions in patients caused by administration of allergens to patients. In addition, these agents and formulations can also be injected into lymph nodes for allergy treatment. Intralymphatic allergen administration is known and the same procedure can be readily employed for the present invention. In some embodiments, the amount of agent or formulation injected into the lymph nodes is between 0.1ug and 0.1mg allergen, and the injection volume per lymph node is between 0.1ml and 1ml for 3 months.

The immune activity enhancer may also be conjugated with a carbohydrate polymer or other biocompatible polymer (e.g., dextran or heparin or hyaluronic acid or polypeptide) to form a prodrug, as described in U.S. patent application nos. 15/723,173;16/380,951 and 16/029,594. The prodrug may be in the form of a carbohydrate (or other polymer) drug conjugate, wherein the drug may be conjugated to the carbohydrate (or other polymer) through a cleavable bond. More than one drug may be bonded to the polymer backbone. Suitable carbohydrates include sialic acid containing polymers, hyaluronic acid, chondroitin sulfate, dextran, carboxydextran, cellulose, carboxycellulose and derivatives thereof. It may also be a linear polymer backbone (e.g. dextran or synthetic polymers such as PVA, PAA). In addition, the immunopotentiator may be coupled to the antigen directly or via a linker or carrier. The carrier may be a polymer. The allergy-causing antigen may be conjugated to a carrier to form a multimer. Allergen antigens and immunopotentiators may also be conjugated together. They may be in the form of linear polymers, microparticles, nanoparticles, liposomes or implants. The carrier system can be used in previous and current applications to construct conjugates. For example, liposomes or microparticles or nanoparticles can be used as carriers. The antigen may be immobilized on the surface of the liposome or particle and the immunopotentiator may be encapsulated within the liposome or particle or co-immobilized on the surface of the liposome or particle. The carrier may also be a linear or branched polymer such as dextran, hyaluronic acid, heparin, chondroitin sulfate and polypeptides. Both allergen antigens and immunopotentiators may be conjugated to the polymer. The conjugates can be administered to a subject in need of allergy treatment by administering them to the subject (e.g., subcutaneous injection). Further details can be found in the foregoing disclosure.

When liposomes are used, both the immunopotentiator or the antigen + drug may be entrapped in the liposomes. The injectable formulation or implant may comprise an antigen+drug or antigen-drug conjugate or encapsulated antigen/drug (e.g., in a microsphere or liposome) or a combination thereof. The antigen may be a crude antigen (e.g. peanut extract, gluten, pollen extract, dust mite extract) or a purified antigen (e.g. peanut antigen protein ara h2, prolamin) or an antigen-drug conjugate or an encapsulated antigen (e.g. in microspheres or liposomes) or a mixture thereof.

Microspheres are used in slow release formulations such as in situ gelling systems. The term microsphere includes particles ranging from nano-sized to micro-sized (e.g., 50nm-50 μm in diameter). Preferably, the microspheres are biodegradable (e.g., made of biodegradable polymers such as poly (lactide), PLGA), and the microspheres may also encapsulate an immunosuppressive drug such as imiquimod (e.g., 1% to 80% by weight of the microspheres). For example, the microspheres may be biodegradable synthetic polymers, such as PLGA. Encapsulating imiquimod and other immunopotentiating drugs (for example, accounting for 1 to 80 percent of the weight of the microsphere). The size of the microspheres is 3 μm or 300nm. Antigens may also be bound to the microsphere surface either directly or through linkers. Antigens may also be encapsulated in microspheres. Alternatively, the drug (immune activity enhancer) may be conjugated to the microsphere surface instead of being encapsulated. Examples of microspheres suitable for current use can be readily taken from the disclosures in publications such as patent application Ser. No. 13/880,778, U.S. Pat. No. 14/934,135, CA 2910579, U.S. Pat. No. 13/084,662 and U.S. Pat. No. 8652487 and those other patent applications filed by Selecta Biosciences. It can be formulated as an in situ gel formulation or a high viscosity liquid for the treatment of allergies, and can be injected or implanted (the formulation is encapsulated within an implant) into a patient.

Another form suitable for current use is the use of a polymeric carrier conjugated to an allergen and an immune activity enhancer, such as the in situ gelling matrix, in a sustained release formulation. The polymer is conjugated to multiple antigens (e.g., 1-100) and multiple copies of an immune activity enhancer (e.g., 5-500 molecules).

The formulation/composition of the invention may contain an increased dose of allergen at the end of the treatment, similar to the dosing regimen used in current treatment regimens (oral or topical or injection) using allergens. That is, the treatment involves a series of formulations, the first formulation containing the lowest amount of allergen that is tolerated and increasing gradually over time in the subsequent formulations, while the amount of the drug may remain unchanged if other drugs (e.g., immunopotentiators or immunosuppressants) are present. The amount of allergen in the first formulation may be the highest amount of allergen that the patient can tolerate without eliciting a severe allergen response. In one example, the patient began using a first single dose injectable formulation containing 0.1ug of albumin, and after the initial dose in PBS, the subject received approximately twice the dose of albumin extract every 30 minutes until the highest tolerated single dose was reached. Based on the highest tolerised single dose, subjects began weekly using 0.5ml of 3.5% sodium alginate formulation containing the highest tolerised dose of ovalbumin and 0.2mg imiquimod for 2 weeks with 1 dose per week. Egg white in 0.5ml of 3.5% sodium alginate containing 0.2mg imiquimod in the formulation increases every 2 weeks until 200ug is reached, as long as the subject tolerates the current dose. Once subjects reached a dose of 200ug, they would be instructed to use the dose every 6 months for 2 years. Likewise, imiquimod may be replaced with 0.1-0.5mg rapamycin or methotrexate. Alternatively, TLR agonists and immunosuppressants may not be included in the antigen-containing sustained release formulation. Other allergens such as pollen extract, dust mite extract, animal hair extract and food allergens such as nut/peanut/milk/egg extract may be substituted in the above formulations to treat related allergies. Examples of the concentration of these allergens may be between 0.1 μg/mL and 1mg/mL, depending on the species and patient tolerance, as long as they are tolerated by the subject in need thereof. During the course of treatment, as the patient's tolerance increases, the concentration of allergen in the formulation increases, which is a standard practice for allergic immunotherapy.

These formulations and compositions containing allergen and adjuvant-type drugs (e.g., TLR agonists or STING agonists) are primarily used to treat allergies by producing allergen-specific IgG-competitive endogenous IgE that causes the allergic reaction. They are not used for the treatment of autoimmune diseases against autoantigens, nor for the prevention of drug-resistant antibodies. Preferably, the adjuvant-type agent is a Thl-biased immunostimulant, including STING agonists, natural or synthetic agonists acting on Toll-like receptors (TLR) (e.g. TLR-1, TLR-2, TLR-3, TLR-4, TLR-5, TLR-6, TLR-7, TLR-8, TLR-9, TLR-10and TLR-11 agonists), and also imidazoquinolinamines, such as 2-bridged imidazoquinolinamines, imidazopyridinamines, cycloalkylimidazopyridinamines, cpG, immunostimulatory RNAs, lipopolysaccharides, VSV-G or HMGB-1 such as imiquimod, R848, 3M-052 and polyIC. In some preferred embodiments, the TLR agonist used is selected from imiquimod and poly IC.

In one aspect, compositions and formulations are disclosed that comprise one or more allergy-causing antigens and optionally a vaccine adjuvant type agent (e.g., TLR agonist, STING agonist), in a sustained release (extended) release system (e.g., in situ gelling system or implant) or in a high viscosity liquid to treat allergy. Also disclosed is a method of treating allergy in a subject by administering these compositions and formulations to the subject as an injection or implant. In some embodiments, the formulation is prepared in situ prior to use by mixing a ready-to-use allergen-containing product for desensitizing a patient (e.g., those allergen extract products for clinical use, such AS those subcutaneous allergy immunotherapy (SCIT) injections from Alk Abello AS) with a sustained release system matrix, and then injecting the mixture AS a final formulation into the patient. The slow release system matrix is a composition/formulation containing an appropriate amount of slow release system material, such as an in situ gelling material or a high viscosity liquid, and optionally an auxiliary agent (e.g., TLR agonist, STING agonist). The concentration of in situ gelling material in the matrix of the slow release system needs to be high enough to provide the desired gelling effect after mixing with the allergen extract product, which can be determined according to the dilution factor caused by the addition of the allergen extract product. Preferably, the pH of the final injectable formulation after mixing together is between 6 and 8 and the osmotic pressure approaches physiological osmotic pressure, which can be controlled by adjusting the buffer capacity, pH, osmotic pressure of the matrix of the slow release system based on the mixing ratio. In some embodiments, the composition/formulation is a liquid or lyophilized form thereof containing an optional adjuvant-type agent and an appropriate amount of the presently disclosed self-gelling polymer, optionally with the addition of bulking agents/lyoprotectants prior to lyophilization, in an amount necessary to desensitize the patient Is sufficient to form a gel in vivo after mixing. In some embodiments, the composition/formulation is 2-50% sodium alginate in water or saline and the pH is brought to 5-8 by the addition of a base or acid such as NaOH or HCl. In some embodiments, the compositions and formulations are 2-20% sodium alginate in water or saline, and 2M NaOH or 2M HCl is added to bring the pH to 7-8. In some embodiments, the osmolality of the formulation is adjusted by physiologically acceptable excipients to have an osmolality similar to physiological conditions. In some embodiments, the formulation has a low osmolality and low pH buffering capacity so that the osmolality and pH of the allergen-containing formulation are not affected after mixing together, particularly for those pre-formulated allergen-containing pharmaceutical products of the solid type (e.g. lyophilized form); for example, the osmotic pressure and pH buffering capacity of the formulation is below 0.5xPBS. The calcium salt or other divalent cation salt may be incorporated into the formulation in a low concentration that does not cause gelling in vitro. For example, a low concentration of Ca of 2-20 mg per 1 g alginate may be used ²⁺ The salt is favorable for in vivo gelation, such as 0.05 to 0.5 percent of calcium gluconate is added into the preparation. The formulation may also comprise a gel-enhancing polymer as described above, for example, 0.1 to 1% HA, CMC, HPMC, carbomer, MC, chitosan; 10-30% poloxamer or a combination thereof. After mixing, the final solution preferably contains 2-6% alginate. In some embodiments, a therapeutically effective amount of a TLR agonist (e.g., poly IC or imiquimod or R848 or 3M-052 or CPG ODN such as CPG ODN 1018 or a combination thereof) or a STING agonist or a combination thereof may be further incorporated into the alginate-containing matrix formulation. In one example, the matrix formulation is 5% sodium alginate, optionally 1% HPMC and optionally 0.2-2mg/mL poly IC and optionally 0.2-2mg/mL CPG ODN 1018 in 1 XPBS at pH 7; 1mL of the extract was mixed with 1mL of Alutard SQ dust mite extract (1-10 allergy units/mL dilution), and the resulting final preparation was injected into patients to treat dust mite allergy. Other allergen extracts such as cat hair allergen extract, pollen allergen extract, and mixed wasp venom proteins can also be used in place of dust mite extract for treating related allergies. Example(s)For example, the matrix formulation is a 0.5% aqueous NaCl solution containing 6% sodium alginate and optionally 0.2-2mg/mL imiquimod and optionally 5mg/mL diphenhydramine hydrochloride as antihistamine, pH 7, and the final formulation is produced by mixing with the appropriate concentration of the extract of the pollen of the BAIYUDACAO in a ratio of 1:1, and is injected subcutaneously or intramuscularly or intraply or near lymph node into the patient in a volume of 0.2-0.5 mL. In some embodiments, the alginate may be replaced with 3-6% hyaluronic acid to form a viscous solution. In one example, the matrix formulation is in 5% aqueous hyaluronic acid having a pH of 7 and optionally 5mg/mL poly IC;1mL of this formulation was diluted and mixed with 0.5mL of Alutard SQ dust mite extract (1-10 allergy units/mL), and the resulting final formulation was injected into patients to treat dust mite allergy. In some embodiments, alginate may be replaced with 30-50% Pluronic F127 or poloxamer 407 in the matrix formulation, and the final concentration of Pluronic F127 or poloxamer 407 after mixing should be greater than its in situ gelling concentration (e.g. >17%) in one example, the matrix formulation is 30% poloxamer 407 and optionally 0.2-2mg/mL aqueous poly IC solution, pH 7;1mL of this solution was mixed with 0.5mL of Alutard SQ dust mite extract (1-10 allergy units/mL), and the resulting final formulation was injected into patients to treat dust mite allergy. In some embodiments, the PLGA organic solvent solution may be used as an in situ gelling agent in a matrix formulation, and the final formulation should not cause gelling in vitro but still form a gel in vivo after mixing, which can be controlled by adjusting the ratio of allergen extract product and PLGA solution, as well as the concentration of PLGA. In one example, the matrix formulation is 60%50:50 lactide/glycolide PLGA and optionally 0.1-1mg/mL imiquimod and optionally 5mg/mL diphenhydramine in N-methylpyrrolidone or DMSO; 1mL of this solution was mixed with 0.1mL of Alutard SQ dust mite extract (10-50 allergy units/mL dilution), and the resulting final formulation was injected into patients to treat dust mite allergy. Alternatively, the adjuvant-like agent in the matrix formulation described above may be replaced with a therapeutically effective amount of an immunosuppressant as previously described, e.g., an mTOR inhibitor (e.g., 0.1-2mg/mL rapamycin or an analogue thereof or methotrexate), to treat allergies. In one example, the matrix formulation is 5% sodium alginate, optionally 1% HPMC and 0.2mg/mL rapamycin and optionally Tween-20 as dissolution enhancer in 1x PBS at pH 7; 1mL of the extract was mixed with 1mL of Alutard SQ dust mite extract (1-10 allergy units/mL), and the resulting final preparation was injected into patients to treat dust mite allergy. Other allergen extracts such as cat hair allergen extract, pollen allergen extract, and mixed wasp venom proteins can also be used in place of dust mite extract for treating related allergies. For example, the matrix formulation is an aqueous solution containing 6% sodium alginate and optionally 0.1-1mg/mL rapamycin and optionally 5mg/mL diphenhydramine hydrochloride as antihistamine, in 0.5% NaCl at pH 7; mixing with the proper concentration of the extract of the pollen of the Bituminous plant in a ratio of 1:1 to produce the final preparation, and administering the final preparation to the patient by subcutaneous or intramuscular injection or intralymphatic injection or injection near lymph node in a volume of 0.2-0.5 ml. In one example, the matrix formulation is 3% crosslinked sodium hyaluronate and optionally 1mg/mL rapamycin in water at a pH of 7; 1mL of the extract was mixed with 0.5mL of Alutard SQ dust mite extract (1-10 allergy units/mL), and the resulting final preparation was injected into patients to treat dust mite allergy. In some embodiments, the alginate may be replaced with 30-50% Pluronic F127 or poloxamer 407 in the matrix formulation, and the final concentration of Pluronic F127 or poloxamer 407 after mixing should be greater than its in situ gelling concentration (e.g. >17%). In one example, the matrix formulation is 30% poloxamer 407 and optionally 0.3mg/mL rapamycin or 0.3mg/mL methotrexate in water at a pH of 7;1mL of this solution was mixed with 0.5mL of Alutard SQ dust mite extract (1-10 allergy units/mL), and the resulting final formulation was injected into patients to treat dust mite allergy. In some embodiments, the PLGA organic solvent solution may be used as an in situ gelling agent in a matrix formulation, and the final formulation should not cause gelling in vitro but still form a gel in vivo after mixing, which can be controlled by adjusting the ratio of PLGA solution to allergen extract product and the concentration of PLGA. In one example, the matrix formulation is 60%50:50 lactide/glycolide PLGA in N-methylpyrrolidone or DMSO and contains 0.1-1mg/mL rapamycin and optionally 5mg/mL diphenhydramine, 1mL of this solution is mixed with 0.1mL Alutard SQ dust mite extract (10-50 allergen units/mL), and the resulting final formulation can be injected into patients to treat dust mite allergy. Thus, one isIn aspects, compositions and formulations are disclosed that comprise one or more allergy-inducing antigens and an immunosuppressant in a sustained (prolonged) release system (e.g., an in situ gelling system or implant) to treat allergy. Also disclosed is a method of treating allergy in a subject by administering the composition and formulation to the subject as an injection. The formulations are prepared by mixing a ready-to-use allergen-containing product (such as those used clinically) for desensitizing a patient prior to use with an immunosuppressant-containing sustained release system matrix in situ, and then injecting the final formulation into the patient. The slow release system matrix is a composition/preparation containing a proper amount of slow release system material and an immunosuppressant, and is an in-situ gel material or a high-viscosity liquid.

The other two ready-to-use products for Allergy Immunotherapy (AIT) treatment are sublingual drops or tablets. Sublingual allergy immunotherapy (SLIT) drops are administered sublingually. The patient uses the drops at home himself without having to regularly visit the doctor. Tablets are taken by the patient at home, typically sublingual buccal tablets. Many tablets have been marketed for allergy to house dust mites, grasses, ragweed and japanese cedar. The above-described sustained-release system matrix may also be used in combination with sublingual drops or tablets, which are placed under the tongue simultaneously. As mentioned above, the sustained release system matrix may contain an immunosuppressant such as rapamycin or methotrexate, or an adjuvant such as imiquimod or polyIC, or no active drug at all. It may be the aforementioned in situ gelling formulation or the aforementioned high viscosity liquid. For example, a matrix formulation is a saline solution containing 2% sodium alginate and optionally 1mg/mL rapamycin and optionally 5mg/mL diphenhydramine hydrochloride as antihistamine, in 0.5% NaCl at pH 7, and the patient can add 1 or 2 drops of this matrix and commercial SLIT drops or tablets while treating allergy sublingually; or they may be mixed together and then the mixture placed under the tongue. In another example, 2% of sodium alginate is replaced by 1% HPMC, so the matrix is a high viscosity solution. In another case, the matrix formulation is a saline solution containing 2% sodium alginate and optionally 1mg/mL poly IC or 0.5mg/mL imiquimod and optionally 5mg/mL diphenhydramine hydrochloride as antihistamine, in 0.5% NaCl at pH 7, the patient can add 1 or 2 drops of this matrix together with commercial SLIT drops or tablets under the tongue daily or weekly to treat allergy. In some embodiments, the matrix is not a sustained delivery system, and thus, a viscosity enhancing agent or gelling agent in the matrix is not required. The matrix is simply a solution containing an effective amount of an immunosuppressant such as rapamycin or methotrexate, or an adjuvant such as imiquimod or polyIC. In some embodiments, the concentration of rapamycin or methotrexate or imiquimod or polyIC is between 0.1-10 mg/mL. The solution may also incorporate optional transdermal enhancers and dissolution enhancers, such as tween-80, DMSO, transcutol. In one example, the solution is 1mg/ml rapamycin in 1 XPBS and 0.1% Tween 80. Patients can be treated for allergy by sublingual addition of 1 or 2 drops of this solution together with commercially available SLIT drops or tablets. In another example, the solution is 1mg/mL polyIC in 1 XPBS. Patients may add one or two drops of this solution sublingually with commercial SLIT drops or tablets daily or weekly for use in treating the associated allergy.

In some embodiments, instead of the liquid matrix described above, a tablet or film containing an effective amount of an immunosuppressant such as rapamycin or methotrexate, or a vaccine adjuvant drug such as imiquimod or polyic is used. By placing the tablet/film together with SLIT drops or SLIT tablets under the tongue or in the mouth, the tablet or film can be used in combination with SLIT drops or SLIT tablets to treat the associated allergy. In some embodiments, the concentration of rapamycin or methotrexate or imiquimod or polyIC is between 0.05-0.5mg per tablet/membrane. These tablets/films are sublingual dissolvable tablets/films in nature. Optional transdermal enhancers such as tween 80, DMSO, transcutol may also be incorporated into the tablet or film. In one example, a 5mm diameter tablet comprises 0.25mg rapamycin or 1mg poly IC or 0.2mg imiquimod or 0.5mg methotrexate, 40% mannitol, 40% lactose, 1% sodium cyclamate, 1% pvp k30 and 1% magnesium stearate. The patient may use one such tablet and a commercial SLIT drop/tablet simultaneously, placed sublingually next to each other, daily to weekly for allergy treatment. In one example, a tablet with a diameter of 3 mm contains 0.1 mg rapamycin or 0.5mg poly IC, 2mg antihistamine, 20% sucrose, 50% lactose, 25% polyethylene glycol 6000 and 1% PVP K30. Patients may use one such tablet and commercial SLIT drops/tablets simultaneously in sublingual locations each other daily to weekly to treat allergies. The tablet may also be used with other oral allergy immunotherapy drugs such as those described in patent numbers US9271899B2 and CN103025303 a. Alternatively, the allergen and immunosuppressant, or allergen and vaccine adjuvant, may be combined in one tablet for SLIT treatment. These tablets may be substantially identical to the SLIT tablets currently in use and incorporate additional immunosuppressant or additional adjuvant-type drugs into the tablets. For example, a SLIT tablet of the present invention may have the same or similar composition as Oralair tablets (Stallergenes) and may contain an additional 0.1-0.2mg rapamycin or 0.1-0.2mg methotrexate per tablet to treat grass pollen allergy symptoms. In another example, the SLIT tablet of the present invention has the same or similar ingredients as the Acarinzax/Odactra tablet except that it contains an additional amount of rapamycin or 0.1-0.2mg of methotrexate and 1mg of cetirizine or 1mg of a corticosteroid such as fluticasone or budesonide per tablet to treat allergic symptoms of dust mites. In another example, the SLIT tablets of the present invention have the same or similar ingredients as the agarizax/Odactra tablets except that they contain an additional 0.1-0.2mg imiquimod or 0.1-0.2poly IC and 1mg diphenhydramine hydrochloride to treat allergic symptoms to dust mites. When the allergen containing drug is orally taken into the digestive system rather than sublingually, such as PALFORZIA peanut allergen powder from Aimmune Therapeutics, additional immunosuppressant or additional adjuvant type agents may be added to the tablet or capsule. For example, enteric-coated capsules or tablets containing 1-100mg defatted peanut flour and 0.1-1mg rapamycin or 0.1-1mg methotrexate or 100-500mg sialic acid or polysialic acid can be used orally to treat peanut allergies.

In one aspect, compositions and formulations are disclosed that comprise a drug and an immunosuppressant that can produce an anti-drug antibody in a sustained release (extended) release system (e.g., an in situ gelling system or implant) or a high viscosity liquid (e.g., those described) to treat the anti-drug antibody. Also disclosed are methods of treating or inhibiting anti-drug antibody production in a subject by administering the compositions and formulations to the subject as an injection. In some embodiments, the formulation is prepared by mixing in situ a drug that will produce ADA (anti-drug antibody) prior to use, such as those protein drugs that include antibodies that are clinically used, with a slow release system matrix, and then using the mixture as the final formulation. The final formulation is then administered to the patient as an injection. The sustained release system matrix is a composition/formulation or methotrexate containing an appropriate amount of sustained release system material as an in situ gelling material or high viscosity liquid and a therapeutically effective amount of the previously described immunosuppressant such as an mTOR inhibitor (e.g. 0.1-5mg/mL rapamycin).

The concentration of in situ gelling material in the matrix of the sustained release system needs to be high enough to provide the desired gelling effect after mixing with the ADA-producing drug product, which can be determined by the dilution factor caused by the addition of the ADA-producing drug. Preferably, the pH of the final injectable formulation after mixing is between 6 and 8 and the osmotic pressure approaches physiological osmotic pressure, which can be achieved by adjusting the pH buffering capacity, pH, osmotic pressure of the matrix of the sustained release system based on the mixing ratio. In some embodiments, the composition/formulation is a liquid or lyophilized form thereof comprising an immunosuppressant and an amount of a self-gelling polymer disclosed herein, which is required to form a gel in vivo after mixing with the ADA-producing drug, and an optional bulking agent/lyoprotectant is added prior to lyophilization. In some embodiments, the composition/formulation is 2-20% sodium alginate and 0.1-2mg/mL rapamycin in water or saline and is brought to a pH of 5-8 by the addition of a base or acid such as NaOH or HCl. In some embodiments, the compositions and formulations are 2-20% sodium alginate and 1-5mg/mL rapamycin in water or saline, and 2M NaOH or 2M HCl is added at a pH of 7-8. In some embodiments, the osmolality of the formulation is adjusted with physiologically acceptable excipients to have osmolality similar to physiological conditions And the osmotic pressure concentration. In some embodiments, the formulation has a low osmolality and low pH buffering capacity so that when mixed together does not affect the osmolality and pH of a pharmaceutical formulation that would produce ADA, particularly for those solid pre-formulated drugs that can produce ADA, such as lyophilized products, e.g., the formulation has an osmolality and pH buffering capacity below 0.5xPBS. The calcium salt or other divalent cation salt may be incorporated into the formulation in a low concentration that does not cause gelling in vitro. For example, 2-20 mg Ca can be used per 1 g alginate ²⁺ To facilitate in vivo gelation, such as adding 0.05% -0.5% calcium gluconate into the preparation. The formulation may also comprise a gel-enhancing polymer as described above, for example, 0.1 to 1% HA, CMC, HPMC, carbomer, MC, chitosan; 10-30% poloxamer or a combination thereof. After mixing, the final solution preferably contains 2-6% alginate. In some embodiments, a therapeutically effective amount of an immunosuppressant other than an mTOR inhibitor, such as IL-2, TGF-beta, PD-L1, IL-15, IFN-gamma, IL-10, IL-21, IL-27, IL-2 anti-IL-2 antibody complex or a mimetic or derivative thereof, such as pegylated IL-2NKTR-358 or a combination thereof, may be further added to the alginate-containing matrix formulation. In one example, the matrix formulation is 5% sodium alginate, optionally 1% HPMC and 0.2mg/mL rapamycin and optionally 200-2000IU/mL IL-2/anti-IL-2 antibody complex in 1 XPBS at pH 7; 1mL of this was admixed with 1mL of Humira (20 mg/0.4mL of the pharmaceutical product) and the resulting final formulation was injectable into patients to treat or prevent the production of ADA for Humira. Other drugs such as antibodies or growth hormone and other recombinant proteins or viral vectors may be substituted for Humira to treat or prevent the associated ADA. For example, the matrix formulation is a 0.5% aqueous NaCl solution at pH 7 containing 6% sodium alginate and 0.2mg/mL rapamycin and optionally 0.1% Tween-20, mixed with a pharmaceutical product containing an appropriate amount of AAV virus (e.g., 10≡10 copies/mL) at a ratio of 1:1 to form the final formulation, as a subcutaneous or intramuscular injection or an intralymphatic injection or injected near the lymph node at a volume of 0.2-0.5mL to prevent T-cell and B-cell immunity against AAV. In some embodiments, alginate may be used in matrix formulations 30-50% of Pluronic F127 or poloxamer 407 is replaced, and the final concentration of Pluronic F127 or poloxamer 407 after mixing should be greater than its in situ gelling concentration (e.g.>17%). In one example, the matrix formulation is 30% poloxamer 407 and 0.5mg/mL rapamycin in water at pH 7; 1mL of the same was mixed with 0.5mL of Humira, and the resulting final formulation was injected into a patient to treat or prevent Humira ADA. In some embodiments, the PLGA organic solvent solution may be used as an in situ gelling agent in a matrix formulation, and the final formulation should not cause gelling in vitro but still form a gel in vivo after mixing, which can be controlled by adjusting the ratio of PLGA solution to allergen extract product and the concentration of PLGA. In one example, the matrix formulation is 0.5mg/mL rapamycin in a 60%50:50 solution of lactide/glycolide PLGA in N-methylpyrrolidone or DMSO. 1mL of this matrix formulation was mixed with 0.1mL of Humira (40 mg/0.4mL of drug product) and the resulting final formulation was injected into patients to treat or prevent Humira ADA.

In some embodiments of the invention, in situ gelling matrices such as pectin, alginate, hyaluronic acid and gellan gum as previously described rely on gelling formation in the presence of divalent or trivalent cation or polycationic molecules. In some embodiments, low water solubility divalent/trivalent/polycationic compounds (e.g., low water solubility divalent cation salts such as calcium carbonate, calcium phosphate, dicalcium phosphate, calcium silicate, caSO ₄ 、ZnCO ₃ 、BaCO ₃ 、BaSO ₄ Or a combination thereof) may be added to these types of in situ gelling formulations prior to injection, and the gel may be slowly formed in the body by injection while it is still in a low viscosity state. In some embodiments, the final concentration of the low water-soluble calcium or salt zinc or barium salt in the final drug-loaded formulation to be injected is between 0.3-10%. Sustained release formulations that can slowly release these cations, such as calcium or zinc ions, or combinations thereof, are also considered low water-soluble cationic compounds that release their encapsulated calcium nanoparticles or microparticles in 15 minutes to 1 hour when contacted with water, for example. Thus, the present invention also provides a kit for treating allergy, ADA or autoimmune diseases comprisingThe two separate components of the device are mixed together prior to injection. If both components are in solid form (e.g., both are dry, such as lyophilized), they may also be placed in a container. A component comprising a low water solubility di/tri/polycationic compound in solid or liquid form may enhance the gelling of the second component. The second component is an optional drug-loaded in situ gelling formulation, as previously described, or a similar formulation with a higher concentration of in situ gelling agent and optional drug, which will compensate for the dilution factor when mixed and provide the same concentration of drug/gelling agent as those in the previously described formulation after it is mixed with the first component. In one example, the kit comprises two components, one of which is 6% calcium carbonate or CaSO ₄ Or ZnCO ₃ Or an aqueous suspension of calcium phosphate or dibasic calcium phosphate, and the other is a previously selected drug-loaded formulation, such as an aqueous saline solution containing 4% sodium alginate, an optional immunosuppressant (e.g., 0.5-1mg/mL rapamycin or methotrexate) and an optional antigen. For example, the two components may also be mixed together with an antigen solution such as a commercially available allergen injection or a protein drug solution having ADA-producing activity in a ratio of 1:1:0.5 and then injected to treat the antigen-related disorder. Additional tackifying polymers such as starch, cellulose, methylcellulose, HPMC may also be incorporated into the first component at suitable concentrations such as 0.1 to 5% w/w. Alternatively, one of the components may contain the antigen of interest and optionally an immunosuppressant, such as an allergen or a protein drug with which ADA is produced, so that only component one and component two are mixed for injection. If the formulation is for allergy treatment, an adjuvant type agent such as 1-2mg/mL poly IC or 0.5-1mg/mL imiquimod may be used in place of the immunosuppressant. In some embodiments, the immunosuppressant or disease-associated antigen or both may be in component 1, while component 2 contains only the gelling polymer. Immunosuppressants may be in component 1 or 2. The antigen may also be in component 1 or 2, or in a commercially available product, in need of mixing with a commercially available product containing the antigen.

In some embodiments, water-soluble (e.g., solubility at room temperature>0.5%) of divalent/trivalent/polycationic compounds (e.gCaCl ₂ Calcium gluconate, ca-EDTA, zinc chloride or gluconic acid, ferrous chloride FeCl ₂ Ferrous gluconate, feCl ₃ Iron gluconate and BaCl ₂ Barium gluconate, ornithine or a derivative thereof, lysine or a derivative thereof such as ethyl lysine, arginine or a derivative thereof such as ethyl arginine, chitosan, polylysine, polyarginine, polyornithine or a combination thereof) may be injected to the same injection site before or after the injection of the in situ gelling formulation of the antigen/immunosuppressant to enhance the gelling effect. They may also be co-injected using a dual syringe system. In some embodiments, the concentration of the water-soluble divalent/trivalent/polycationic compound in the solution is between 0.2 and 10%. In some embodiments, the concentration of the water-soluble calcium salt in the solution is between 0.2-6%. When injected prior to injection of the formulation containing the in situ gelling agent, it will perfuse the injection site with a higher concentration of calcium ions than physiological calcium ions to provide better in situ gelling. Thus, the present invention also provides a kit for treating allergy, ADA or autoimmune diseases comprising two separate components in different containers, which can be sequentially injected into a site or co-injected into a site using a dual syringe system. One component contains a water-soluble di/tri/polycationic compound that enhances the gelling of the second component, whether in solid or liquid form. The second component is an in situ gelling formulation, such as those previously described, or a similar formulation with a higher concentration of in situ gelling agent and drug, which will compensate for the dilution factor and provide the same concentration of drug/gelling agent as in the previously described formulation after mixing with the first component in vivo. In one example, the kit comprises two components, the first being 0.5-5% CaCl ₂ Or calcium gluconate or chitosan or lysine or arginine in water at a pH between 5 and 8, the second being an in situ gelling formulation, a saline solution containing 4% sodium alginate, optionally an immunosuppressant (e.g. 0.5-1mg/mL rapamycin or methotrexate) and optionally an antigen. Additional tackifying polymers such as starch, cellulose, methylcellulose, HPMC may also be incorporated into the first component at suitable concentrations such as 0.1 to 5% w/wIs a kind of medium. In one embodiment, component 1 is a composition comprising 0.5% HPMC, 1% CaCl ₂ Or pH 7 solution of 2% calcium gluconate or 1.5% lysine or 1% chitosan, regulating osmotic pressure to be close to physiological value with NaCl, and injecting into target site. Next, solution component 2 containing alginate loaded with antigen and immunosuppressant is injected into the same site to treat a subject in need thereof. Component 1 may also be injected after injection of component 2. Alternatively, the immunosuppressant or disease-associated antigen or both may be in component 1 and component 2 contains only gelling polymers. Immunosuppressants may be in component 1 or 2. The antigen may also be in component 1 or 2, or in a commercially available product, to be mixed with a commercially available product containing the antigen. If the formulation is for allergy treatment, an adjuvant type agent such as 1-2mg/mL poly IC or 0.5-1mg/mL imiquimod may be used in place of the immunosuppressant.

Components 1 and 2 may also be injected in a syringe, separated by a biologically and pharmaceutically acceptable liquid solution as a buffer layer to prevent them from mixing together within the syringe. In some embodiments, the buffer layer liquid may be a liquid having a high viscosity (e.g., >500cps, or in other embodiments > 2000) to reduce permeation and contamination between components 1 and 2. For example, it may be glycerol, or a solution at pH 7 containing a tackifying polymer such as starch, cellulose, methylcellulose, HPMC, HA at a concentration of 0.3-3% w/w, with osmotic pressure adjusted to 250-350mOsm/kg with NaCl. In one example, the syringe contains 0.5ml of component 1 and 0.5ml of component 2, with 0.3ml of buffer in between for injection. Thus, a kit for treating allergy, ADA or autoimmune disease may further comprise a third component, namely a buffer layer liquid as described above. In some embodiments, a solution of a water-soluble (e.g., solubility > 0.5% at room temperature) divalent/trivalent/polycationic compound may also be first mixed with the alginate-containing formulation and then injected. Viscosity enhancing polymers such as starch, cellulose, methylcellulose, HPMC may also be incorporated into either component at suitable concentrations such as 0.1-5% w/w to prevent gelling prior to injection. Another approach to slow down and control gelation is to use a buffer containing phosphate (e.g., sodium hexametaphosphate) because the phosphate groups in the buffer compete with the carboxylate groups of the alginate in reaction with calcium ions and act as sequestering agents to slow down gelation. Typical sequestering agents that may be used include various inorganic phosphates such as sodium hexametaphosphate, tetrasodium pyrophosphate, disodium orthophosphate, and sodium tripolyphosphate. Sodium citrate may also be used.

In some embodiments, prior to administration of the formulation to a subject in need thereof, a low water solubility divalent cation salt (e.g., calcium carbonate, calcium phosphate, dicalcium phosphate, calcium silicate, caSO ₄ 、ZnCO ₃ 、BaCO ₃ 、BaSO ₄ Or a combination thereof) or Ca-EDTA-containing formulations may be further mixed with agents that may slowly release these cations from the low solubility salts or Ca-EDTA complexes, which will result in slow gelling in vivo. Examples of such agents may be selected from the group consisting of D-glucono-delta-lactone (GDL), L-glucono-delta-lactone, D-erythronolide, L-erythronolide, D-glucuronolactone, L-glucuronolactone, D-galacto-gamma-lactone, L-galacto-gamma-lactone, D-xylo-gamma-lactone, L-xylo-gamma-lactone, D-gulono-gamma-lactone, L-gulono-gamma-lactone 3, D-glucono-gamma-lactone and L-glucono-gamma-lactone. These lactones slowly hydrolyze in water releasing acids which release free divalent cations into the water causing gelation. Higher pH increases the rate of hydrolysis and lower pH decreases the rate of hydrolysis, which in turn affects the gelation time. The pH of the formulation (e.g., pH 5-8) may be adjusted accordingly to achieve the desired gel time. The ratio of these reagents to divalent cation salt may be between 1:5 and 5:1 molar ratio. For example, when GDL and CaCO are used in the formulation ₃ Or CaCO (CaCO) ₃ With CaSO ₄ The molar ratio (GDL: calcium salt) of them may be 1:2 or 1:1 or 2:1. In one example, 5mL of a drug-loaded formulation containing 2% sodium alginate is combined with 0.1g CaCO ₃ The powder was mixed and then mixed with 0.05g of GDL powder. After stirring, the final formulation is injected to form a gel in vivo.

In one example, the first formulation is an in situ gelling formulation, is an aqueous solution comprising 4% sodium alginate, and optionally comprisesImmunosuppressants (e.g., 0.5-1mg/mL rapamycin or methotrexate) and appropriate amounts of disease-associated antigens (e.g., allergens or autoantigens that cause autoimmune diseases or ADA-producing drugs); additional tackifying polymers such as starch, cellulose, methylcellulose, HPMC may also be incorporated into the first component formulation at suitable concentrations such as 0.1-5% w/w. 5mL of the first formulation containing 2% sodium alginate was combined with 0.1g CaCO ₃ The powder was mixed with 0.05g GDL powder. After stirring, the final formulation is injected into a subject in need thereof by subcutaneous injection or intramuscular injection or intralymphatic injection to form a gel.

In another example, the first formulation (component) is a 2.5mL saline solution containing 4% sodium alginate and optionally 2mg/mL poly IC or 0.mg/mL rapamycin, and the second formulation (component) is 0.05g CaCO ₃ (or ZnCO) ₃ )、0.05g CaSO ₄ And 0.05g of GDL (or L-gulonic acid-gamma-lactone). Additional viscosity enhancing agents may be incorporated into either the first or second or both formulations, which will reduce the gelation rate. A suitable concentration of 0.5mL of alugard SQ dust mite extract (e.g., 10 allergy units/mL of diluent) is mixed with the first and second formulations to produce a mixture, and the resulting final formulation can be injected into a patient to treat dust mite allergy. The first formulation may also be in dry form, e.g. lyophilized form, with a suitable bulking agent/lyoprotectant, and the dried formulation 1 is placed in the same vial as the formulation 2 powder. The user simply adds the aluard SQ dust mite extract solution to the vial to form the final injection formulation. In another example, the first formulation is 2.5mL saline solution containing 4% sodium alginate, 5mg/mL adalimumab and 1mg/mL rapamycin, and the second formulation is 0.05g CaCO3 (or ZnCO 3), 0.05g CaSO4 and 0.05g GDL (or L-gulono-gamma-lactone). Additional viscosity enhancing agents may be incorporated into either the first or second or both formulations, which will reduce the gelation rate. The first and second formulations are mixed to produce a mixture and the resulting final formulation can be injected into a patient to prevent or treat ADA against adalimumab. The first formulation may also be in dry form, e.g. lyophilized form, and suitable bulking agent/lyoprotection Agent, and placing dried formulation 1 in the same vial with formulation 2 powder. The user simply adds a diluent (e.g., PBS or water) to the vial to form the final injection dosage form.

The antigen/drug loaded sustained release formulation in the present invention may be an in situ gelling formulation or a nanoparticle/microparticle based formulation or a combination thereof. The drug (e.g., immunosuppressant or adjuvant drug) and antigen may be encapsulated in nanoparticles or microparticles as a sustained release form to be injected. In some embodiments, the drug/antigen encapsulated nanoparticle or microparticle is a polysaccharide, such as alginate-based particles, which uses a polysaccharide, such as alginate, to form a particle matrix. For example, it may be chitosan-calcium alginate gel nano/microspheres as described in patent number CN1628861a, or chitosan-calcium alginate nano/microspheres, or calcium alginate nano/microspheres as described in patent application number CN107057085A, or sodium alginate-calcium carbonate hybrid microparticles, such as those described in patent application number CN102286155A, or calcium phosphate/calcium alginate hybrid microspheres, such as those described in patent application number CN101081911 a. By employing or modifying the protocols described in the previous publications, these antigen/drug loaded particles can be readily prepared using alginate solutions containing the antigen/drug.

For example, 100mg sodium alginate was dissolved in 10ml distilled water, heated in a 40 ℃ water bath, 300mg sodium phosphate solution was added, stirred in a water bath for 30 minutes, 5mg rapamycin, 50mg adalimumab was added, then 5ml 1% calcium chloride solution was slowly added, stirred for 1 hour, and centrifuged to obtain antigen/drug-loaded alginic acid-calcium phosphate microparticles, which were used to inhibit adalimumab ADA. In another example, drug-loaded alginate-Ca particles are prepared by adding a 2.0% (w/v) sodium alginate solution containing the drug (e.g., 5mg/mL poly IC or 1mg/mL imiquimod or 1mg/mL rapamycin and 10mg/mL gluten or albumin) in an alginate solution using electrostatic dropping to 1% CaCl ₂ The solution is gel bath, and is vigorously stirred to obtain calcium alginate gel microspheres, then the calcium alginate gel microspheres loaded with antigen/drug are coated with 0.7% (w/v) chitosan solution, and the two are mixed in a ratio of 1:10v/v. And then centrifuged to obtain an antigen/drug loaded seaCalcium alginate chitosan particles.

Skin patches containing allergens, such as those developed by DBV Technologies, are used to treat allergies by inducing tolerance to antigens (allergens). Such patches may be readily adapted for current applications. For example, the topical formulations described in U.S. Pat. nos. 6676961, 8932596B2 and 8202533B2, as well as those commercially available patches (e.g., VIASKINR MILK and via kit nr peanout) may be used for current applications by adding additional immunosuppressive drugs thereto (e.g., 0.05mg-5mg rapamycin or Fuji mycin or 0.1mg-10mg methotrexate or derivatives or prodrugs thereof). The method of administration may be substantially the same as in the prior art, except that the patch contains an immunosuppressant. Similar patches are also described in U.S. application Ser. Nos. 15/723,173, 16/380,951, 16/029,594, 17/344,932, 16/566,716, 16/819,168 and 17/385,908. Additional dermal penetration enhancers (e.g., DMSO, azone, fatty acids, hyaluronic acid, etc., which and their appropriate amounts can be readily found in publications) can be added to the patch or applied to the skin prior to application of the patch. Examples of transdermal enhancers that may be added include DMSO (e.g., 10-300 mg/patch), azone (e.g., 1% -10% of total drug weight), surfactants, fatty acids (e.g., 1% -10% oleic acid). The skin stratum corneum may also be removed by exfoliation or other means to enhance transdermal delivery. In one example, the patch contains 500 μg-10mg gluten (e.g., G5004 gluten from wheat, sigma) and 0.1mg-2mg rapamycin or 0.1mg-5mg methotrexate. For example, antigens such as gluten and immunosuppressants such as rapamycin and/or methotrexate may be in powder form, they may simply be physically mixed together, they may also be co-dissolved and then dried and then placed in a patch. In another example, the patch contains 5 milligrams of gluten (e.g., G5004 gluten from wheat, sigma) and 0.5 milligrams of rapamycin or 0.5 milligrams of methotrexate and optionally an additional 30 milligrams of azone. In another example, the patch contains 5 milligrams of gluten (e.g., G5004 gluten from wheat, sigma) and 100 milligrams of sialic acid or sialic acid-cholesterol conjugate or 2 milligrams of methotrexate. This can be used to induce gluten tolerance and to treat gluten intolerance. Gluten may be replaced with deamidated prolamine. In an embodiment, the patch may be used daily for 1-5 weeks. In another example, the antigen is peanut antigen ara h2200 μg, and 0.2mg rapamycin is included in the patch to treat peanut allergy. In one example, the peanut antigen ara h2200 μg, 0.2mg rapamycin and 50mg sucrose were dissolved in water, then lyophilized and then placed into a patch. In one example, the peanut antigen ara h2200 μg, 0.5mg rapamycin, 50mg sds and 50mg sucrose were dissolved in water, then lyophilized and then placed into a patch. In one example, the peanut antigen ara h2200 g, 0.2mg rapamycin, 100mg DMSO and 50mg sucrose were dissolved in water, then lyophilized and then placed into a patch. For another example, the antigen is double-stranded DNA (1 mg-10 mg) for treating lupus in the previous figures, and the drug is rapamycin or Fuji mycin or temsirolimus at 1 mg. In another example, the nasal spray contains 1mg of gluten (e.g., G5004 from Sigma, gluten from wheat) and 0.5mg of rapamycin or 1mg of methotrexate per spray dose in a suitable spray form. In another example, the sublingual tablet contains 50mg of gluten (e.g. G5004 from Sigma, gluten from wheat) and 0.1-0.5 mg rapamycin or 0.1-0.5 mg methotrexate. In another example, the gel contains 50mg of gluten, e.g., G5004 gluten from wheat, sigma) and 0.2mg rapamycin or 1mg methotrexate per 1ml of gel. The immunosuppressive drug or drugs and antigen may be in powder or gel or semi-liquid form, or in liposome form (e.g. 100nm-5 μm in diameter) or nanoparticles/microparticles (e.g. 100nm-1 μm) or conjugated to dendrimers or linear polymers (e.g. coupled to polyacrylic acid or polysialic acid via ester linkages to form polymer-based prodrugs, mw=5 KD-500 KD). Other pharmaceutically acceptable amounts of antigen and immunosuppressant may also be used in the patch, provided that it produces satisfactory biological and therapeutic (e.g., immune tolerance) effects, as determined experimentally by screening and testing using well known protocols and methods. Other allergens such as pollen extract, dust mite extract, animal hair extract and food allergens such as nut/peanut/milk/egg extract may be substituted in the above formulations to treat related allergies. Examples of the concentration of these allergens may be between 0.1mg and 1mg per patch, provided that the subject in need thereof is tolerated. During the course of treatment, as the patient's tolerance increases, the concentration of allergen in the formulation increases, which is a standard practice for allergic immunotherapy.

Pharmaceutically acceptable amounts of the allergy-inducing antigen and the immune activity enhancer may be used in the patch instead of using the antigen and the immune inhibitor in the patch. In some embodiments, the method is using a patch comprising a mixture of an allergen or fragment thereof and the previously described immunopotentiator (e.g., imiquimod or poly IC). It may also contain antiallergic agents such as antihistamines, corticosteroids, mast cell stabilizers and leukotriene inhibitors. The mixture of the allergy inducing antigen and the immune activity enhancer may be a physical mixture. By physical mixture is meant that the mixture of antigen and immune activity enhancer is simply mechanically mixed together (e.g. by stirring or mixing) in its original form (e.g. liquid or solid form, such as powder or particles) without any additional process (e.g. by mixing them together in their original form), or that a further size reduction process is applied after mechanical mixing (e.g. comminuting, grinding, milling or homogenizing), or dispersed or dissolved in the same or different types of liquid, respectively, and then mixed, or co-dispersed in a liquid, or co-dissolved in a solvent (e.g. water), and that an optional drying process (e.g. spray drying or freeze drying) can be applied together with an optional further size reduction process.

For example, the topical formulations described in U.S. Pat. Nos. 6676961, 8932596B2 and 8202533B2 or commercially available patches (e.g., VIASKINR MILK and VIASKINR PEANUT) may be used for the present application by adding additional immunopotentiating drugs thereto (e.g., imiquimod or poly IC or derivatives or prodrugs thereof in an amount of 0.1mg-20 mg)Such as 10-300 mg/patch), azone (e.g., 1% -10% of total drug weight), surfactant, fatty acid (e.g., 1% -10% oleic acid). In one example, the patch contains 500 μg-10mg gluten (e.g., G5004 gluten from wheat, sigma) and 0.05mg-5mg imiquimod or 0.05mg-5mg r848. For example, the allergy-inducing antigen such as gluten and the immune activity enhancer such as imiquimod and/or poly IC may be in powder form, they may be simply physically mixed together, or they may be co-dissolved, then dried, and then put into a patch. For example, 10mg of gluten powder and 0.25mg of imiquimod powder are mixed, then homogenized with a grinder, and then applied to the skin contact surface of a 5X5cm 2 skin patch. In another example, 10mg of gluten and 10mg of poly IC are vigorously mixed in 10mL of water containing 30mg of sucrose for 10 minutes, then lyophilized, and then the dried mixture is spread over 5X5cm ² The patch surface of the skin contact surface. As another example, 10mg of gluten, 5mg of STING agonist MK-1454 and 5mg of CpG ODN were dissolved in 5mL of 25% EtOH in water, then dried under vacuum, and the dried mixture was placed on the skin contact surface 3X3cm ² One side of the skin patch. In another example, 10mg of gluten and 0.5mg of imiquimod are dissolved in 5mL of 1% SDS aqueous solution, followed by vacuum drying, and then the dried mixture is placed at 3X2cm ² The surface of the skin contact surface of the skin patch. In another example, 10x10cm ² The patch contains 5mg of gluten (e.g. G5004 gluten from wheat, sigma) and 0.5mg of imiquimod or 0.1 mg of 3M-052 and optionally an additional 30 mg of azone. In another example, the patch contains 5 milligrams of gluten (e.g., G5004 gluten from wheat, sigma) and 100 grams of sialic acid or sialic acid-cholesterol conjugate and 1 milligram of poly IC. This can be used to induce gluten tolerance and to treat gluten intolerance. Gluten may be replaced with prolamine. In an embodiment, a patch containing gluten or gliadin may be applied daily to the forearm for 8 hours per day for 1-5 weeks. Gluten in the above examples may be replaced with egg white proteins, for example 5-10mg of ovomucoid (Gal d 1) or 5-10mg of ovalbumin (Gal d 2) or their combination with optional 5-10mg of ovotransferrin (Gal d 3) and 5-10mg of lysozyme (Gal d 4) for the treatment of egg white Allergy. In another example, the antigen is peanut antigen ara h2 200 μg, and the patch contains 0.5mg imiquimod to treat peanut allergy. In one example, the peanut antigen ara h2 200 μg, 0.2mg imiquimod and 50mg sucrose are dissolved in water, then lyophilized and then placed into a patch. In one example, the peanut antigen ara h2 200 μg, 0.2mg imiquimod, 50mg SDS and 50mg sucrose are dissolved in water, then lyophilized and then placed at 5x5cm ² Is a patch of (a). In one example, the peanut antigen ara h2 200 μg, 0.2mg imiquimod, 100mg DMSO and 50mg sucrose are dissolved in water, then lyophilized and then placed into a patch. In another example, a nasal spray or nasal drops contains 1mg of gluten (e.g., G5004 from Sigma, gluten from wheat) and 0.05mg of imiquimod or 0.5mg of polyIC in suitable form per spray or 3 drops, and a viscosity enhancing agent such as hyaluronic acid or carbomer may be added. In another example, the sublingual tablet contains 50mg of gluten (e.g. G5004 from Sigma, gluten from wheat) and 0.1 mg imiquimod or 2mg poly IC. In another example, the gel contains 50mg gluten (e.g., G5004 gluten from wheat, sigma) and 0.2mg imiquimod or 2mg poly IC per 1ml gel. The immune activity enhancer drug or immune activity enhancer drug and allergen may be in the form of a powder or gel or semi-liquid, or in the form of liposomes (e.g. 100nm to 5 μm diameter), or in the form of nanoparticles/microparticles (e.g. 100nm-1 μm) or coupled with dendrimers or linear polymers (e.g. polyacrylic acid or polysialic acid coupled via ester linkages to form polymer-based prodrugs with mw=5 KD-500 KD).

In some embodiments, the topical formulation contains 0.1-10mg allergen, 0.01-5mg TLR7/8 ligand (e.g. imiquimod or jidiaquimod or resiquimod), 0.1-5mg TLR3/RLR ligand (e.g. dsRNA, e.g. polyIC or polyICLC), 0.1-5mg TLR9 ligand (e.g. CpG ODN, e.g. ODN 1826 or ODN 2216) per ml gel or emulsion or liquid. Transdermal enhancers such as DMSO, azone (e.g., 1% to 10%), surfactants, fatty acids (e.g., 1% to 10% oleic acid) may also be added. In one example, the formulation contains 5mg/mL gluten, 0.5mg/mL imiquimod, 1mg/mL poly IC, 1mg/mL A class CpG ODN 2216, 20mg/mL SDS, and 5% sucrose in 1 XPBS solution, and then is lyophilized. In another example, 10-100mg egg white powder, 0.1-0.5mg imiquimod, 1-5mg poly IC and 5-50mg azone are mixed together and added to a Viaskin type skin patch. Can be applied to skin 2 times per week for 2 weeks, each time for 2 days, 1 month and 3 months, and further for 2 days as a booster to produce egg white tolerance. In another example, 10mg peanut protein, 0.5mg imiquimod, 2mg poly IC and 100mg DMSO were mixed together and added to the viasky type patch. Can be applied to skin 2 times per week for 2 weeks, 1 time each, 1 month, and 3 months for 2 days to produce peanut tolerance. In some embodiments, the topical formulation contains 0.1-100mg of antigen, 0.05-5mg TLR agonist per mL gel or lotion or liquid; transdermal enhancers such as DMSO, azone (e.g., 1% -10%), surfactants, fatty acids (e.g., 1% -10% oleic acid) may also be added.

The formulation may also be an oral formulation, such as a tablet or capsule containing a mixture of allergen and immunopotentiator. It may be the same as that used for oral formulations of aimmine (e.g. AR101 for peanut allergy) except that the immunopotentiator is additionally added. Viscosity enhancers may also be incorporated similar to those described above. It may also contain therapeutically effective amounts (e.g., clinically useful doses) of antiallergic agents such as the antihistamines, corticosteroids, mast cell stabilizers and leukotriene inhibitors described above. The addition of these antiallergic agents can prevent allergic reactions in patients caused by administration of allergens to patients. In one embodiment, the formulation is an enteric capsule containing 1mg-100mg peanut protein, 1-10mg imiquimod, 10mg carbomer 940 and 10mg cetirizine. In the post-treatment, peanut proteins were used in the same amounts as AR 101. In some embodiments, the immunopotentiator may be replaced with silicic acid or polysialic acid or sialic acid polymer or siglec ligand or derivative thereof (e.g. 50-500 mg/capsule).

In some embodiments, the formulation is applied to the oral mucosa. It can be in the form of suppositories, troches, tablets, films for sublingual and buccal mucosal administration. Formulations currently used for sublingual delivery and oral mucosal delivery of allergens (e.g. pollen extract, dust mite extract) can be used with additional immune activity enhancers added to the formulation. Viscosity enhancers and/or mucoadhesives may also be incorporated as described above. It may also contain therapeutically effective amounts (e.g., clinically useful doses) of antiallergic agents such as the antihistamines, corticosteroids, mast cell stabilizers and leukotriene inhibitors described above. It may also be a non-biodegradable container (e.g. a tablet made of plastic or metal) with small holes, and once placed in the mouth, the blocked allergen/drug will be released to the oral mucosa instead of being in the form of an orally soluble tablet/lozenge. Devices that can prevent the formulation from being swallowed may be incorporated into the delivery system, such as ropes, belts, rollers, or dental fixtures. This will allow the formulation to be easily removed from the mouth when severe allergic reactions are exhibited. For example, it may be in the form of a lollipop, which contains allergens and other drugs. In one embodiment, the formulation is a mucoadhesive tablet containing 1mg to 100mg peanut protein, 0.1 to 1mg imiquimod or polyIC and 3mg cetirizine with a wood stalk attached in the form of a lollipop.

For transdermal/transmucosal delivery or implantable or oral or sustained release formulations, the initial amount of allergy-inducing antigen and the amount of immune activity enhancer may be between 0.1 and 100 mg. When injectable or non-sustained release oral dosage forms are used, the initial amount of allergen may be between 50 μg and 5mg and the amount of the immune activity enhancer may be between 0.05 and 5 mg.

The formulation/composition may comprise an increased dose of allergen at a later stage, similar to the dosing regimen used in current treatment regimens (oral or topical or injection) using allergens. That is, the treatment involves a series of formulations, the first formulation containing the lowest amount of allergen and the dosage being gradually increased over time in the latter formulation, while the amount of other drugs (e.g., immunopotentiators) may be unchanged. The amount of allergen in the first formulation may be the highest amount of allergen that the patient can tolerate without eliciting a severe allergen response. In one example, the patient begins taking a first single dose oral mucosal tablet containing 0.1mg egg white powder and 0.2-2mg imiquimod, and after the initial dose, the subject receives about twice the dose of egg white every 30 minutes, but the amount of imiquimod is the same until the highest tolerated single dose is determined as shown in table 1 of the previous application. Based on the highest tolerised single dose, the subject began to use a formulation containing this dose of egg white powder and 0.2-2mg imiquimod daily, 1 dose per day for 2 weeks. As long as the subject tolerates the current dose, the amount of egg white powder containing 5mg imiquimod in the formulation increases by 25mg every 2 weeks until 150mg is reached, then 50mg every 2mg until 300mg is reached. Once subjects reached a daily dose of 300mg, they would be instructed to take a daily dose of 2 years without imiquimod.

The invention also discloses an autoimmune cell treatment method, which is used for treating autoimmune diseases, allergy, inhibiting the generation of drug-resistant antibodies or inducing antigen-specific immune tolerance. It comprises the following steps: the autoimmune cells are collected and isolated from a subject in need thereof, stimulated with disease-associated antigens and immunosuppressants to expand antigen-specific regulatory immune cells and/or suppressive immune cells, including tolerogenic DC cells, in vitro to achieve a desired number of target cells, and the expanded autoimmune cells are then returned to the subject to achieve a desired therapeutic effect. The source of the autoimmune cells collected and isolated from the subject may be bone marrow or lymph node extracts or blood fractions or combinations thereof from the subject. In some embodiments, lymphocytes may be isolated from the blood of a subject in need thereof using a blood cell separator and/or a leukapheresis procedure. For example, 200ml of blood is withdrawn from a patient, and lymphocytes are collected on this 200ml of blood using a blood cell separator. Procedures for harvesting lymphocytes from blood are well known to those skilled in the art. It can be performed using a commercial blood cell separator. The lymphocytes produced comprise B cells and T cells, possibly together with other leukocytes. Optionally, B cells can be further removed, for example using a cell sorter such as FACS or magnetic particles coated with antibodies specific for B cell surface markers, there are a number of commercial kits and instruments available for this purpose and the process is art well known to those skilled in the art. However, in other embodiments, it is desirable that B cells remain to transform them into Breg cells. In the present invention, an inhibitory immune cell that can inhibit immune function is considered to be a regulatory immune cell, and thus a regulatory immune cell includes an antigen-specific regulatory immune cell and a non-antigen-specific inhibitory immune cell.

In some embodiments, the collected immune cells comprise DC cells, T cells, and B cells. In some embodiments, the collected immune cells comprise DC cells and T cells but no B cells. In some embodiments, the collected immune cells comprise DC cells and are T cell/B cell free.

The medium contains disease-associated antigens and immunosuppressants (e.g., rapamycin, IL-10, IL-2/anti-IL-2 mAb, PD-L1, which can be found in the inventors' U.S. application Ser. No. 16/566,716). Preferably, the antigen is in its native form or a peptide fragment thereof rather than in the form of an MHC binding complex. For example, it may be a dust mite extract or pollen extract or a food allergen (e.g. peanut proteins, gluten) to treat related allergies. Also useful as a protein drug for the treatment of ADA. It may also be double-stranded DNA for the treatment of lupus. Can also be antigen protein of autoimmune diseases (such as PPI, IGRP, GAD, islet cell autoantigen 2, insulin receptor for treating diabetes, collagen for treating rheumatoid arthritis) to treat related diseases. Cell culture protocols can be readily adopted from prior U.S. application Ser. No. 16/566,716 and well known publications. For example, the procedure for the in vitro culture of DC cells to treat cancer is well established, and similar procedures and protocols can be used to replace tumor antigens with autoantigens associated with the disease and to add tolerogenic immune cells to the culture medium to induce immunosuppressants. TLR agonists may also be added to the medium to stimulate for more than 24 hours, inducing DC depletion.

For example, the concentration of antigen (e.g., peanut protein or type II collagen) in the medium may be between 0.1 μg/ml and 10 mg/ml. In one example of this, in one instance,DC cells collected from subjects were cultured in complete medium consisting of 10% heat-inactivated fetal bovine serum (Biosource International), non-essential amino acids, 0.5mM sodium pyruvate, 5mM Hepes, 1mM glutaMax I (all from Invitrogen) with 50 μg/ml gluten as antigen in DMEM matrix. Cultures were monitored daily and maintained at 0.7-1X 10 by dilution with complete medium for 8-12 days or until the desired number of target cells was obtained ⁶ /ml. The medium contains 200-2000IU/mL IL-2/anti-IL-2 mAb (e.g., those described in DOI: 10.4049/jimmimunol. 1402540). The culture medium also contains 2-20ng/mL rapamycin. Alternatively, allogeneic cells may be used instead. DC cells from healthy donors are cultured in the same manner and then transferred to subjects in need thereof.

The compounds and compositions described herein (e.g., compositions, conjugates, polymers, and nanoparticles/microparticles disclosed in the present invention) can be administered with a drug or medicament formulated as a pharmaceutically acceptable adjuvant, carrier. The pharmaceutical compositions of the present invention may be formulated as solutions or lyophilized powders for parenteral administration. The powder may be reconstituted by adding a suitable diluent or other pharmaceutically acceptable carrier prior to use. The liquid formulation may be a buffered, isotonic aqueous solution. The powder may also be sprayed in dry form. Examples of suitable diluents are isotonic physiological saline, standard 5% dextrose in water or buffered sodium or ammonium acetate solutions. As used herein, the term "pharmaceutically acceptable adjuvant, carrier" refers to a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting any supplement or composition or component from one part of the body to another part of the body, or delivering an agent to a desired tissue or tissue adjacent to a desired tissue. Pharmaceutically acceptable carriers known to those of ordinary skill in the art may be used, including water or saline. As known in the art, the components and their relative amounts are determined by the intended use and method of delivery. The diluents or carriers used in the compositions can be selected so that they do not detract from the intended effect of the composition. Examples of suitable compositions include aqueous solutions, such as saline solution, 5% dextrose. Other well known pharmaceutically acceptable liquid carriers may be used, such as alcohols, glycols, esters and amides. In certain embodiments, the composition further comprises one or more excipients, such as, but not limited to, ionic strength modifiers, dissolution enhancers, sugars such as mannitol or sorbitol, pH buffers, surfactants, stabilizing polymers, preservatives, and/or co-solvents. In certain embodiments, the polymeric material is used as a pharmaceutically acceptable carrier. Polymeric materials described herein may include natural or non-natural polymers such as sugars, peptides, proteins, laminin, collagen, hyaluronic acid, ionic and nonionic water soluble polymers; an acrylic polymer; hydrophilic polymers such as polyethylene oxide, polyoxyethylene-polyoxypropylene copolymer, and polyvinyl alcohol; cellulose-based polymers such as hydroxypropyl cellulose, hydroxyethyl cellulose, hydroxypropyl methylcellulose phthalate, methylcellulose, carboxymethyl cellulose, and etherified cellulose, and cellulose-based polymer derivatives. Poly (lactic acid), poly (glycolic acid), copolymers of lactic acid and glycolic acid, or other natural and synthetic polymeric agents. In certain embodiments, the compositions provided herein may be formulated as films, gels, foams, or other dosage forms. Suitable ionic strength modifiers include, for example, glycerol, propylene glycol, mannitol, glucose, dextrose, sorbitol, sodium chloride, potassium chloride, and other electrolytes. Suitable pH buffers for use in the compositions herein include, for example, acetate, borate, carbonate, citrate and phosphate buffers, as well as hydrochloric acid, sodium hydroxide, magnesium oxide, monobasic potassium phosphate, bicarbonate, ammonia, carbonic acid, hydrochloric acid, sodium citrate, citric acid, acetic acid, dibasic sodium phosphate, borax, boric acid, sodium hydroxide, diethylbarbituric acid and proteins, and various biological buffers, for example, TAPS, bicine, tris, tricine, HEPES, TES, MOPS, PIPES, dimethylarsinate or MES. In certain embodiments, a pH adjuster (e.g., HCl, hePO4, naOH) and/or a pH buffer system (e.g., sodium phosphate, sodium acetate, sodium citrate, sodium borate, or boric acid) is added to maintain the pH within the following ranges: from about pH 4 to about pH 8, or about pH 5 to about pH 8, or about pH 6 to about pH 8, or about pH 7 to about pH 8.

As used herein, the phrase "effective amount" refers to a dosage sufficient to provide a sufficiently high concentration to impart a beneficial effect to its recipient. The specific therapeutically effective dosage level of any particular subject will depend upon a variety of factors including the disorder being treated, the severity of the disorder, the activity of the particular compound, the route of administration, the clearance of the compound, the duration of treatment, the drugs used in combination or concurrently with the compound, the age, weight, sex, diet and general health of the subject, and like factors well known in medicine and science. Various general considerations that are considered in determining a "therapeutically effective amount" are known to those skilled in the art and are described. The exact formulation, route of administration and dosage may be selected by the individual physician according to the patient's condition.

In this application, "/" means "and/or" and/or combinations thereof. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All patents and publications mentioned in the specification are indicative of the levels of those skilled in the art to which the invention pertains. All patents and publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference. The above-described invention is directed to many well-known chemistries, instruments, methods and techniques. The skilled person can easily find corresponding knowledge from textbooks such as chemical textbooks, scientific journal papers and other well known reference resources.

Claims

1. A composition for inducing immune tolerance to an antigen comprising an antigen that causes immune intolerance and an immunosuppressant in a self-gelling formulation.

2. The composition of claim 1, wherein the immunosuppressant is rapamycin.

3. The composition of claim 1, wherein the antigen is an allergen.

4. The composition of claim 1, wherein the self-gelling formulation comprises alginate and calcium ions.

5. The composition of claim 1, wherein the self-gelling formulation comprises PLGA and N-methylpyrrolidone.

6. A composition for inducing immune tolerance to an antigen comprising an antigen that causes immune intolerance and an immunosuppressant in a sublingual formulation.

7. The composition of claim 6, wherein the immunosuppressant is rapamycin.

8. The composition of claim 6, wherein the antigen is an allergen.

9. The composition of claim 6, wherein the sublingual formulation is a sublingual drop.

10. The composition of claim 6, wherein the sublingual formulation is a sublingual tablet.