Methods and pharmaceutical compositions for inducing immune tolerance by mucosal vaccination with fc-coupled antigens

Abstract

To provide methods for inducing immune tolerance by mucosal vaccination with Fc-coupled antigens.SOLUTION: The present invention provides a method for inducing tolerance to one antigen of interest in a subject in need thereof, comprising the mucosal administration to the subject of a therapeutically effective amount of a recombinant chimeric construct comprising an FcRn targeting moiety and an antigen-containing moiety.SELECTED DRAWING: None

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present invention relates to methods and pharmaceutical compositions for inducing immune tolerance by mucosal vaccination with Fc-binding antigens. [Background technology]

[0002] Background of the invention: Autoimmune diseases are a major health problem. For example, type 1 diabetes (T1D) is an autoimmune disease mediated by autoreactive T cells that recognize β-cell antigens (Ag), leading to the destruction of pancreatic islets. The main problem in T1D management is its late diagnosis. This typically occurs after a variable period of asymptomatic subclinical autoimmunity, after a significant proportion of β-cells have already been destroyed. The resulting insulin deficiency leads to hyperglycemia and clinical onset. Therefore, prevention and treatment of T1D should target the underlying autoimmune mechanisms, not the metabolic consequences, as is currently done with insulin replacement therapy. However, since T1D mainly affects children and young adults and is not short-term and fatal, immunotherapies aimed at blunting β-cell autoimmunity must have an excellent safety profile.

[0003] Therefore, β-cell Ag-specific therapies are attractive in terms of their selectivity and safety compared with treatments that broadly target T-cell subsets involved in the disease. 1 Such therapy is administered in the form of a vaccine containing a β-cell Ag whose immune recognition mediates islet destruction. These vaccines are formulated to achieve the opposite immune outcome to that pursued in classical vaccination (i.e., to neutralize rather than stimulate a T-cell response to the administered Ag, resulting in a condition known as immune tolerance). However, clinical trials have been deceptive. 2-4 Insulin and its precursor preproinsulin (PPI)-derived β-cell Ags are involved in the pathogenesis of nonobese diabetic (NOD) 5,6 In mice and possibly humans 2Because insulin is also a causative agent in T1D, several attempts have focused on tolerogenic vaccination with it. Despite evidence of successful induction of insulin-specific immune tolerance, a recent trial using intranasal administration of insulin to halt autoimmune β-cell destruction in newly diagnosed T1D patients with late-onset disease did not result in significant β-cell preservation. 7 These results suggest that earlier intervention may be necessary before significant β-cell loss and before autoimmune progression. While PPI recognition initiates the autoimmune cascade, subsequent β-cell destruction releases additional Ags that are further recognized (a phenomenon known as Ag spreading), rendering tolerance restoration to the sole PPI insufficient.

[0004] The same problem arises in preventive trials using insulin vaccination, where safety issues are even more important for the treatment of at-risk subjects who are not yet diabetic. Despite the absence of clinical disease, the selection of at-risk subjects is based on the positivity of multiple autoantibodies (autoAbs), which attest to an autoimmune response already involving several Ags. 3,8,9 A recent study further suggests that β-cell autoimmunity begins very early, as the mean age at autoAb seroconversion was 9–18 months in a large prospective cohort of genetically at-risk children. 10,11 Therefore, in at-risk children with a high HLA-associated genetic risk for the disease but without signs of active autoimmunity (i.e., autoAbs), insulin Ag-specific prophylactic strategies should be implemented at an earlier stage. 12,13 .

[0005] The perinatal period offers such an opportunity, not only in terms of timing, but also because it is characterized by an immune response to the introduced Ag that is biased toward a tolerogenic outcome. Indeed, Ag introduction during the fetal and neonatal periods results in Ag-specific immune tolerance that persists in adulthood. 14-16Central tolerance, a process occurring in the thymus where developing T cells are challenged with ectopically expressed self-tissue Ags, such as PPIs, plays a key role in this process. Their recognition leads to the elimination of autoreactive pathogenic T effector cells (Teff) and the positive selection of T regulatory cells (Treg). 17 This process is highly active during the perinatal period and defines the immunological self that subsequently imprints peripheral immune responses. However, the “immune self-image” presented in the thymus is incomplete, since the self-Ag repertoire is only partially expressed. 17 Indeed, failure of central tolerance is the first checkpoint in T1D progression. Some autoreactive Teffs escape thymic selection and can subsequently become activated in the periphery and destroy pancreatic islets. In support of this notion, the NOD mouse model of T1D develops accelerated diabetes when PPI expression is abrogated in the thymus (Ins2- / -NOD mice). 18,19 Second, human INS polymorphic variants may predispose to T1D by reducing INS expression in the thymus. 20 .

[0006] However, this finding does not translate into therapeutic strategies aimed at enhancing central tolerance from the outset. All tolerogenic vaccination approaches explored so far using subcutaneous, intranasal or oral routes have targeted peripheral tolerance, aiming to blunt the potential pathogenicity of autoreactive Teff and / or enhance Treg activity. 1 If self-Ag, such as PPIs, could be introduced into the thymus during the perinatal period, it could promote the T cell selection process and intervene early in the progression of autoimmunity.

[0007] The present invention relates to the use of Ag fused with the Fc portion of IgG to induce immune tolerance by mucosal vaccination. Currently, Fc fusion proteins account for 20% of all FDA-approved Ab-based drugs and are being actively investigated in various settings, as the addition of an Fc portion increases the half-life of protein therapeutics. Upon binding of Fc domains to the neonatal Fc receptor (FcRn) expressed in endothelial cells, they are temporarily sequestered intracellularly and gradually released into the circulation. FcRn is a heterodimer composed of a major histocompatibility complex (MHC) class I-like heavy chain and β2-microglobulin. 21 The interaction between IgG and FcRn requires an acidic pH (<6.5) and is inefficient at physiological pH (7.4). 21 It occurs in a 1:2 stoichiometry, with one IgG binding to two FcRn molecules via the FcRn heavy chain and the CH2-CH3 portion of the IgG Fc domain. 22 .

[0008] In addition to endothelial cells, FcRn is expressed in several other tissues and cells, including mammalian placental syncytiotrophoblasts or yolk sac, liver, intestine, bronchi, kidney (proximal tubules), genital tract, epithelium of the eye and choroid plexus, renal podocytes; skin (hair follicles, sebaceous glands, epidermal keratinocytes and melanocytes); hematopoietic cells, such as dendritic cells, monocytes and macrophages (including macrophages in the lamina propria of the small intestine). 23 Expressed in 24 In the respiratory tract, FcRn is found primarily in the bronchial epithelium of the upper and central airways. In the human gastrointestinal tract, FcRn is expressed in epithelial cells of the stomach, small intestine (duodenum, jejunum, and ileum), and colon. 25-27 In the intestine, there is an increasing proximal-distal gradient of mucosal FcRn mRNA and protein expression, with expression lowest in the duodenum-jejunum and highest in the proximal colon. This expression gradient correlates with the efficiency of in vitro monoclonal Ab (mAb) transcytosis for these different intestinal regions, and systemic entry occurs via lymphatics. 27The same expression gradient was seen in cynomolgus monkeys, where serum mAb levels were greater after ileum-proximal colon injection than after duodenum-jejunum administration. 27 Taken together, FcRn expression and mAb uptake patterns suggest that the ileum-proximal colon is the region where the majority of mAbs are transcytosed via FcRn.

[0009] Thus, more recently, the application of Fc-binding agents has been expanded to strategies aiming to deliver bioactive molecules using minimally invasive administration routes (i.e., gastrointestinal or pulmonary routes). Typically, IgG is endocytosed on the apical membrane of epithelial cells and binds to FcRn at the acidic pH present in endosomes. The vesicles then fuse again with the basolateral membrane, where the extracellular neutral pH promotes the separation of IgG from FcRn. Importantly, FcRn-mediated transport of IgG is bidirectional (i.e., from both the apical and basolateral membranes of epithelial cells). 28 In in vitro transwell experiments, this occurs rapidly within 1 hour after addition of IgG. 26 Examples of Fc-binding agents that have been explored for oral delivery include Fc-binding follicle-stimulating hormone (FSH). 29 , IgG mAb 27 and Fc-coated nanoparticles containing a bioactive molecule such as insulin 30 Overall, the systemic bioavailability achieved by oral administration of Fc binding agents is relatively limited. Examples of Fc binding agents that have been considered for pulmonary delivery include Fc binding erythropoietin. 31-33 and Fc-bound FSH 29 Examples include:

[0010] Thus, Fc binders have been used to increase the half-life of systemically administered therapeutic proteins and to facilitate their systemic delivery via the intestinal or bronchial routes. Herein, we propose to use this strategy to induce immune tolerance. For this purpose, Ags may be modified by fusing them to the Fc portion of IgG1, allowing the resulting Ag-Fc protein to interact with FcRn and cross the mucosal barrier. This physiologically delivers maternal IgG to the fetus (via the placenta) and to the newborn (via the intestine during lactation) during the intrauterine period and 6 months after birth, when IgG production is not yet available. 21 , the same pathway that provides passive IgG protection. This concept was first validated using the transplacental delivery route, showing that Fc-fusion Ags administered intravenously to pregnant mice reach the fetal thymus in an FcRn-dependent manner and promote the generation of Ag-specific Tregs, resulting in Ag-specific tolerance. 34 When applied to a T1D mouse model, transplacental delivery of PPI-Fc protects offspring from developing diabetes later in life. 35,36 Pregnant PPI T cell receptor transgenic G9C8 NOD mice (all T cells are PPI B15-23 Epitope 37 A single dose of 100 μg of PPI-Fc administered intravenously to mice (which recognize the placenta) is transported transplacentally and protects offspring from diabetes without inducing metabolic or other adverse effects. This transport is Fc- and FcRn-dependent. Protection from diabetes is due to the expression of peripheral PPI-reactive CD8 + Teff and thymus-derived neuropilin-1 (NRP1 / CD304) secrete the regulatory cytokine transforming growth factor (TGF)-β + CD4 + PPI-Fc is associated with an increase in Tregs. lo CD11b + SIRPα +) are transported by dendritic cells (DCs) to the thymus, and when migration is inhibited by early administration of anti-vascular cell adhesion molecule (VCAM)-1 Ab, diabetic protection is lost. Importantly, diabetic protection is also effective in polyclonal NOD mice. 35 Although successful, for autoimmune diseases that cannot be reliably predicted early enough, especially prenatally (e.g., T1D), this strategy remains invasive. Such a strategy applied to pregnant women, in advance of what may or may not develop into a disease that is in most cases short-lived and non-fatal, may be considered to entail unacceptable risks for the fetus and mother. Summary of the Invention

[0011] The present invention relates to methods and pharmaceutical compositions for inducing immune tolerance by mucosal vaccination with Fc-binding Ags. In particular, the invention is defined by the claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0012] Detailed Description of the Invention We now have evidence that antigen-Fc administered orally directly to newborn mice induces significant T cell modifications suggestive of tolerance induction, and therefore we propose to explore its suitability for the prevention or treatment of diseases and conditions such as autoimmune diseases, allergic diseases, and immune responses to exogenously administered antigens.

[0013] Thus, one object of the present invention relates to a method for inducing tolerance to an antigen of interest in a subject in need thereof, comprising mucosally administering to the subject a therapeutically effective amount of a recombinant chimeric construct comprising an FcRn targeting portion and an antigen-containing portion.

[0014] The term "antigen" as used herein has its general meaning in the art and generally refers to a substance or fragment thereof that is recognized and selectively bound by an antibody or T cell antigen receptor, resulting in the induction of an immune response. Typically, antigens of the present invention are, but are not limited to, peptides and proteins. Antigens can be natural or synthetic and generally induce an immune response specific to that antigen. Specialized T cell subsets, such as natural killer T cells 38-40 and mucosal-associated invariant T cells 41-43 Other non-protein antigens recognized by may also be considered.

[0015] In some embodiments, the antigen is an autoantigen. The term "auto-antigen" as used herein refers to any self-antigen that originates from one's own body tissue and is mistakenly recognized as foreign by the immune system. Auto-antigens include, but are not limited to, cellular proteins, phosphoproteins, cell surface proteins, cellular lipids, nucleic acids, glycoproteins (including cell surface receptors).

[0016] In some embodiments, the antigen is an allergen. The term "allergen" as used herein generally refers to an antigen or antigenic portion of a molecule (usually a protein) that induces an allergic reaction when exposed to a subject. Typically, a subject is allergic to an allergen as shown, for example, by the wheal and flare test or any method known in the art. A molecule is said to be an allergen even if only a small proportion of subjects show an allergic immune response when exposed to the molecule.

[0017] In some embodiments, the antigen is a molecule that is exogenously administered for therapeutic or other purposes and may trigger an undesirable immune response. Although frequently neutralizing the biological activity that the molecule induces, such an immune response may have further harmful effects that are unrelated to the purpose for which the molecule was originally administered. Examples of this type include immune responses against therapeutic coagulation factor VIII in hemophilia A or factor IX in hemophilia B, immune responses against various enzymes in congenital enzymopathies and more generally in any type of replacement therapy in the context of genetic deficiencies. Similarly, allogeneic immune responses against antigens expressed by tissues or hematopoietic and / or blood cells transplanted into an individual are also conceivable.

[0018] The term "tolerance" as used herein refers to a defective or hyporesponsiveness to antigens (including self-antigens, allergens, and endogenously administered molecules). It may mean that a productive (immunogenic) response is not induced upon endogenous or exogenous exposure to said antigen. This response may be partially or completely replaced by a tolerogenic response (i.e., an active process that further limits the immunogenic response). Examples of tolerogenic responses include, but are not limited to, the generation of T regulatory cells, the elimination of effector (conventional) T cells by apoptosis or their neutralization by anergy, and the skewing of T cells and other immune cells to a phenotype that promotes a tolerant state (e.g., the production of regulatory cytokines and other anti-inflammatory mediators such as interleukin-10 and TGF-β, and downregulation of the expression of costimulatory molecules). These immunological concepts are well known to those skilled in the art.

[0019] In some embodiments, the subject is a human, although treatment of other animals is also encompassed. In some embodiments, the subject is an adult. In some embodiments, the subject is a pregnant woman. In some embodiments, the subject is a child. In some embodiments, the subject is a child under the age of 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1. In some embodiments, the subject is a newborn. In some embodiments, the subject is a newborn. As used herein, a "newborn" is a newborn less than about 28 days of age. In some embodiments, the subject is a pregnant woman who may give birth to a child at risk of developing a disease.

[0020] In some embodiments, the subject is likely to develop or is likely to develop, or has already developed or is developing, at least one symptom of a disease or condition caused by inappropriate or undesirable immune system activity against an antigen. The subject may be identified or diagnosed as having or likely to develop such a condition based on various factors, such as family history and / or genetic testing of the mother and / or father, siblings, other relatives (such as grandparents, aunts, uncles, and cousins), other disease biomarkers, such as the presence of (auto)antibodies against different (auto)antigens. In general, the subject is known to have a genetic predisposition to developing an autoimmune disease, allergy, or other undesirable immune response. "Known to have a genetic predisposition" means that one or both of the parents or siblings may have the disease or condition and / or are known to be carriers of a gene associated with the disease or condition, such that the statistical probability that the subject will have or develop the disease is at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90% or 100%. This determination may be based on observing or testing the health of the parents, siblings or the subject, and identifying one or more genes in a form known to be associated with or cause the disease or condition (e.g., having a particular sequence, such as an allele, mutation, insertion, deletion, etc.). The risk of the disease may or may not also be ascertained by genotyping the subject's cells and / or by assessing them with appropriate biomarkers, including non-genetic biomarkers (e.g., antibodies and other immunophenotypes or epigenetic modifications, etc.). Those skilled in the art will also recognize that such genetic traits may not be "all or nothing" in that gene dosages may be applied. Nonetheless, if the subject is considered to be at risk and if practice of the method could extend or improve the subject's lifespan, then the subject is a likely candidate for treatment.

[0021] In some embodiments, the subject is susceptible or believed to be susceptible to developing an autoimmune disease, or has already developed or is developing an autoimmune disease. As used herein, the term "autoimmune disease" refers to the presence of an autoimmune response (an immune response to an auto- or self-antigen) in a subject. Autoimmune diseases include diseases caused by a breakdown in self-tolerance, such that the adaptive immune system responds to self-antigens in coordination with cells of the innate immune system, mediating cell and tissue damage. In some embodiments, autoimmune diseases are characterized as being at least in part the result of a humoral immune response and / or a cellular immune response. Examples of autoimmune diseases include, but are not limited to, acute disseminated encephalomyelitis (ADEM), acute necrotizing hemorrhagic leukoencephalitis, Addison's disease, agammaglobulinemia, alopecia areata, amyloidosis, ankylosing spondylitis, anti-GBM / anti-TBM nephritis, antiphospholipid syndrome (APS), autoimmune angioedema, autoimmune aplastic anemia, autoimmune autonomic neuropathy, autoimmune hepatitis, autoimmune hyperlipidemia, autoimmune deficiency, autoimmune inner ear disease (AIED), autoimmune myocarditis, autoimmune pancreatitis, autoimmune retinopathy, autoimmune thrombocytopenic purpura (ATP), autoimmune thyroid disease, autoimmune urticaria, axonal and neuronal neuropathy, Behcet's disease, bullous pemphigoid, autoimmune cardiomyopathy, Castleman's disease, celiac disease, Chagas disease, chronic fatigue syndrome, chronic inflammatory demyelinating polyneuropathy (CIDP), chronic relapsing multifocal Critically ill osteomyelitis (CRMO), Churg-Strauss syndrome, cicatricial pemphigoid / benign mucous membrane pemphigoid, Crohn's disease, Cogan's syndrome, cold agglutinin disease, congenital heart block, Coxsackie myocarditis, CREST disease, essential mixed cryoglobulinemia, demyelinating neuropathy, dermatitis herpetiformis, dermatomyositis, Devic's disease (neuromyelitis optica), discoid lupus, Dressler's syndrome, endometriosis, eosinophilic fasciitis , erythema nodosum, experimental allergic encephalomyelitis, Evans syndrome, fibromyalgia, fibrosing alveolitis, giant cell arteritis (temporal arteritis), glomerulonephritis, Goodpasture's syndrome, granulomatosis with polyangiitis (GPA), Graves' disease, Guillain-Barré syndrome, Hashimoto's encephalitis, Hashimoto's thyroiditis, hemolytic anemia, Henoch-Schönlein purpura, herpes gestationis, hypogammaglobulinemia, hypergammaglobulinemia,Idiopathic thrombocytopenic purpura (ITP), IgA nephropathy, IgG4-related sclerosing disease, immunomodulatory lipoproteins, inclusion body myositis, inflammatory bowel disease, insulin-dependent diabetes mellitus (type 1), interstitial cystitis, juvenile arthritis, Kawasaki syndrome, Lambert-Eaton syndrome, leukocytoclastic vasculitis, lichen planus, lichen sclerosus, lignoconjunctivitis, linear immunoglobulin A disease (LAD), lupus (SLE), Lyme disease, Meniere's disease, microscopic polyangiitis, mixed connective tissue disease (MCTD), benign monoclonal gammopathy (MGUS), Mooren's ulcer, Mucha-Habermann disease, multiple sclerosis, myasthenia gravis, myositis, narcolepsy, neuromyelitis optica (Devic's disease), autoimmune neutropenia, ocular cicatricial pemphigoid, optic neuritis, relapsing rheumatism, PANDAS (Streptococcus associated autoimmune neuropsychiatric disorder of childhood), paraneoplastic cerebellar degeneration, paroxysmal nocturnal hemoglobinuria (PNH), Paley-Romberg syndrome, Parsonage-Turner syndrome, pars planitis (peripheral uveitis), pemphigus, peripheral neuropathy, perivenous encephalomyelitis, pernicious anemia, POEMS Syndrome, polyarteritis nodosa, autoimmune polyglandular syndrome types I, II and III, polymyalgia rheumatica, polymyositis, post-myocardial infarction syndrome, post-pericardiotomy syndrome, progesterone dermatitis, primary biliary cirrhosis, primary sclerosing cholangitis, psoriasis, psoriatic arthritis, idiopathic pulmonary fibrosis, pyoderma gangrenosum, pure red cell aplasia, Raynaud's phenomenon, reflex sympathetic dystrophy, Reiter's syndrome, relapsing polychondritis, restless legs syndrome, retroperitoneal fibrosis, rheumatic fever, rheumatoid arthritis, sarcoidosis, Schmidt's syndrome, scleritis, sclerosing legs syndrome ... In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, Sjogren's syndrome, sperm and testicular autoimmunity, stiff-body syndrome, subacute bacterial endocarditis (SBE), Susac's syndrome, sympathetic ophthalmia, Takayasu's arteritis, temporal arteritis / giant cell arteritis, thrombocytopenic purpura (TTP), Tolosa-Hunt syndrome, transverse myelitis, ulcerative colitis, undifferentiated connective tissue disease (UCTD), uveitis, vasculitis, vesicular bullous dermatoses, vitiligo, Waldenstrom's macroglobulinemia (WM), and Wegener's granulomatosis (granulomatosis with polyangiitis (GPA)). In some embodiments, the autoimmune disease is selected from the group consisting of rheumatoid arthritis, type 1 diabetes, systemic lupus erythematosus (lupus or SLE), myasthenia gravis, multiple sclerosis, scleroderma, Addison's disease, bullous pemphigoid, pemphigus vulgaris, Guillain-Barre syndrome,The autoimmune disease is selected from the group consisting of Sjogren's syndrome, dermatomyositis, thrombotic thrombocytopenic purpura, hypergammaglobulinemia, monoclonal gammopathy (MGUS), Waldenstrom's macroglobulinemia (WM), chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), Hashimoto's encephalopathy (HE), Hashimoto's thyroiditis, Graves' disease, and Wegener's granulomatosis (granulomatosis with polyangiitis). In some embodiments, the autoimmune disease is type 1 diabetes.

[0022] In some embodiments, the subject is susceptible or believed to be susceptible to developing an allergy, or has already developed or is developing an allergy. The term "allergy" as used herein generally refers to an inappropriate immune response characterized by inflammation, including but not limited to food allergies, respiratory allergies, and other allergies that cause or may cause a systemic response (e.g., Quincy's edema and anaphylaxis). The term encompasses allergies, allergic diseases, hypersensitivity-related diseases, or respiratory diseases associated with airway inflammation, such as asthma or allergic rhinitis. In some embodiments, the methods of the invention are effective in preventing, treating or alleviating one or more symptoms associated with anaphylaxis, drug hypersensitivity, skin allergies, eczema, allergic rhinitis, urticaria, atopic dermatitis, dry eye disease, allergic contact allergies, food hypersensitivity, allergic conjunctivitis, insect venom allergy, bronchial asthma allergic asthma, intrinsic asthma, occupational asthma, atopic asthma, acute respiratory distress syndrome (ARDS), and chronic obstructive pulmonary disease (COPD). Hypersensitivity-related diseases or disorders that can be treated by the method of the present invention include, but are not limited to, anaphylaxis, drug reactions, skin allergies, eczema, allergic rhinitis, urticaria, atopic dermatitis, dry eye disease (also called keratoconjunctivitis sicca, also called xerophthalmia), contact allergies, food allergies, allergic conjunctivitis, insect venom allergies, and respiratory diseases associated with airway inflammation, such as IgE-mediated asthma and non-IgE-mediated asthma.Respiratory diseases associated with airway inflammation can include, but are not limited to, rhinitis, allergic rhinitis, bronchial asthma, allergic (extrinsic) asthma, non-allergic (intrinsic) asthma, occupational asthma, atopic asthma, exercise-induced asthma, cough-induced asthma, acute respiratory distress syndrome (ARDS) and chronic obstructive pulmonary disease (COPD).

[0023] In some embodiments, the subject is susceptible, or is believed to be susceptible to, or has already developed, an immune response to a molecule administered exogenously, therapeutically or otherwise, that may trigger an undesired immune response. Non-limiting examples of this type include, but are not limited to, hemophilia A, hemophilia B, congenital deficiencies of other clotting factors, such as factor II, prothrombin and fibrinogen, primary immune deficiencies (e.g., severe combined immunodeficiency, X-linked agammaglobulinemia, IgA deficiency), major hormone deficiencies, such as growth hormone deficiency and leptin deficiency, congenital enzymopathies and metabolic disorders, such as disorders of carbohydrate metabolism (e.g., sucrose isomaltase deficiency, glycogen storage disease), disorders of amino acid metabolism (e.g., phenylketonuria, maple syrup urine disease, glutaric acidemia type 1), urea cycle disorders (e.g., carbamoyl phosphate synthetase I deficiency), disorders of organic acid metabolism (e.g., erythrocyte sedimentation syndrome ... For example, immune responses to replacement therapy drugs in the context of genetic deficiencies, including disorders of fatty acid oxidation and mitochondrial metabolism (e.g., medium-chain acyl-coenzyme A dehydrogenase deficiency), disorders of porphyrin metabolism (e.g., porphyrias), disorders of purine or pyrimidine metabolism (e.g., Lesch-Nyhan syndrome), disorders of steroid metabolism (e.g., lipogenic congenital adrenal hyperplasia, congenital adrenal hyperplasia), disorders of mitochondrial function (e.g., Kearns-Sayre syndrome), disorders of peroxisome function (e.g., Zellweger syndrome), and lysosomal storage disorders (e.g., Gaucher disease, Niemann-Pick disease). In the case of genetic deficiencies, the proposed method may not only restore immune tolerance to replacement therapy drugs used to treat the disease, but may also restore the biological activity for which the therapeutic agent is administered. Other therapeutic agents for which the methods may be suitable for limiting unwanted immune responses include, by way of example, other biological agents such as cytokines, monoclonal antibodies, receptor antagonists, soluble receptors, hormones or hormone analogues, clotting factors, enzymes, bacterial proteins or viral proteins.For example, children with hemophilia may be prophylactically treated with regular clotting factor (e.g., factor VIII) replacement therapy, which reduces the chance of fatal bleeding due to injury. In addition to the cost and inconvenience of such treatment, repeated administration leads in some patients to the formation of inhibitory antibodies against the clotting factor. If the antibodies in these patients are low titer antibodies, the patients are treated with higher doses of blood clotting factor. If the antibodies are high titer antibodies, the treatment regimen for these patients becomes more complicated and expensive. In some embodiments, the therapeutic protein is produced in the subject after gene therapy, e.g., suitable for the treatment of congenital defects. Gene therapy typically involves genetic manipulation of genes involved in the disease. One possible approach for patients, such as hemophiliacs, who are deficient in a single functional protein, is the transfer of genetic material encoding a protein of therapeutic interest. However, repeated administration of gene therapy vectors, such as viral vectors, may also trigger undesirable immune responses against the therapeutic protein introduced into the vector and / or against other components of the vector. Thus, the methods of the present invention may be suitable for overcoming the body's immune response to gene therapy vectors, such as viral vectors. In fact, viral vectors, especially those like adenoviruses and adeno-associated viruses (AAVs) that express immunogenic epitopes in the organism, are most likely to induce immune responses.Various viral vectors are used in gene therapy, including but not limited to retroviruses for X-linked severe combined immunodeficiency (X-SCID); adenoviruses for various cancers; adeno-associated viruses (AAVs) for treating muscle and eye diseases; lentiviruses, herpes simplex viruses, and other suitable means known in the art.

[0024] In some embodiments, the subject is susceptible or believed to be susceptible to developing, or has already developed or is developing, an immune response against transplanted tissue or transplanted hematopoietic cells or transplanted blood cells. Typically, the subject may have received a transplant selected from the group consisting of heart, kidney, lung, liver, pancreas, pancreatic islets, brain tissue, stomach, large intestine, small intestine, cornea, skin, trachea, bone, bone marrow, muscle, or bladder. The method of the invention is also particularly suitable for preventing or suppressing immune responses associated with the rejection of donor tissue, cell, graft, or organ transplant by the recipient subject. Graft-related diseases or disorders include graft-versus-host disease (GVHD), e.g., associated with bone marrow transplantation, and immune disorders resulting from or associated with the rejection of organ, tissue, or cell graft transplants (e.g., tissue or cell allografts or xenografts), including, e.g., skin, muscle, neuron, pancreatic islets, organ, liver parenchymal cell grafts, and the like. Thus, the methods of the invention are useful for preventing host-versus-graft disease (HVGD) and graft-versus-host disease (GVHD). The chimeric construct may be administered to the subject before, during, and / or after transplantation (e.g., at least one day before transplantation, at least one day after transplantation, and / or during the transplantation procedure itself). In some embodiments, the chimeric construct may be administered to the subject periodically before and / or after transplantation.

[0025] In some embodiments, the methods of the present invention are particularly suitable for the treatment of autoimmune diseases, allergic diseases and birth defects.

[0026] The term "treatment" or "treating" as used herein refers to both prophylactic or preventative treatments, including treatment of patients at risk of or suspected of having a disease, as well as patients who are ill or have been diagnosed with a disease or medical condition, and includes the prevention of clinical recurrence. Treatment may be administered to a subject who has a medical disorder or who may eventually develop a disorder to prevent, cure, delay the onset, reduce or ameliorate the severity of one or more symptoms of the disorder or recurrent disorder, or to extend the subject's survival beyond that expected in the absence of such treatment. "Therapeutic regimen" refers to a pattern of treatment of a disease, such as a dosing pattern used during treatment. The therapeutic regimen may include induction regimens and maintenance regimens. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or a portion of a therapeutic regimen) used for the initial treatment of a disease. The general purpose of an induction regimen is to provide a high level of drug to the patient during the initial period of the treatment regimen. The induction regimen may use (partially or entirely) a "loading regimen" which may include administering a higher dose of drug than the physician uses during the maintenance regimen, administering a drug more frequently than the physician uses during the maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a treatment regimen (or a portion of a treatment regimen) used to maintain a patient during disease treatment, for example, to maintain a patient in remission for an extended period of time (months or years). A maintenance regimen may use continuous therapy (e.g., administering a drug at regular intervals, for example, daily, weekly, monthly, yearly) or intermittent therapy (e.g., intermittent treatment, intermittent treatment, treatment upon relapse, or treatment upon achievement of a certain predetermined criterion (e.g., disease manifestation, etc.).

[0027] According to the invention, the chimeric construct comprises an antigen-containing portion. This portion comprises an antigen molecule or a portion of an antigen molecule for which it is desired to generate immune tolerance, and may comprise multiple antigens or portions of antigens. Preferably, the antigen is a protein, polypeptide or peptide. In some embodiments, the antigen-containing portion comprises one or more known epitopes of interest, e.g., regions or residues of an antigen known to elicit an immune response. Alternatively, putative epitopes and antigenic regions may be selected based on antigenic potential due to accessibility, surface exposure, charge, amino acid sequence, etc., using, for example, predictive software programs well known in the art. Typically, the antigen-containing portion comprises a contiguous sequence of the primary sequence of the antigen. Alternatively, sufficient residues of the antigen may be present such that the secondary and tertiary structure is at least partially preserved, and there are antigenic regions that are not necessarily contiguous in the primary sequence but are adjacent after folding of the molecule, or are generated by fusion of different non-adjacent antigen sequences as described for "hybrid" epitopes. The antigen portion of the construct may contain one epitope or multiple epitopes derived from one or more antigens of interest. The epitopes may be contiguous in the construct sequence or may be separated by a suitable linker. The epitopes may be the same, e.g., multiple copies of the same epitope may be present; or the epitopes may be different, e.g., single or multiple copies of two or more different epitopes may be present. The epitopes may be different from each other (different), e.g., by originating from different antigen molecules, or from different parts of the same antigen molecule, or both, e.g., the same region of an antigen from several different variants may be used. Combinations of the above may also be present. Post-translationally modified epitopes or epitopes generated by alternative splicing may also be included. The amino acid sequence of the antigen-containing portion may have 100% identity with the amino acid sequence of the protein, polypeptide or peptide that is the natural antigen.Alternatively, the antigenic portion may comprise a portion (e.g., at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more) of the residues of the native sequence to which immune tolerance is desired. Furthermore, the amino acid sequence in the antigenic portion may be the same primary sequence as that of the native protein antigen, or may be a variant thereof having at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95% or more identity or homology to the native protein sequence or to the portion of the native sequence on which it is based.

[0028] In some embodiments, the antigen-containing moiety is selected from the group consisting of preproinsulin (PPI), glutamic acid decarboxylase (GAD), insulinoma-associated protein 2 (IA-2), islet-specific glucose-6-phosphatase catalytic subunit-associated protein (IGRP), zinc transporter 8 (ZnT8), preproislet amyloid polypeptide (ppIAPP), 78 kDa glucose-regulated protein (GRP78 and its precursor; also known as heat shock 70 kDa protein 5 (HSPA5)), and in T1D, myotonic Dystrophy kinase (DMK) and chromogranin A; myeloperoxidase and proteinase 3 in granulomatosis with polyangiitis; myelin oligodendrocyte glycoprotein (MOG), myelin basic protein (MBP) and proteolipid protein (PLP) in multiple sclerosis; various synovial antigens such as vimentin, nuclear ribonucleoprotein-A2 (RA33), fibrinogen, alpha-enolase in rheumatoid arthritis; tissue transglutaminase and gliadin in celiac disease. Post-translationally modified epitopes, alternative splicing isoforms and hybrid peptides derived from said antigens - alone or in combination with other antigens - are also included. Canonical antigens or antigen isoforms that are not adequately expressed in the thymus may be particularly suitable for this purpose. Examples of antigen moieties are generated from said proteins after they have been processed by antigen-presenting cells (including dendritic cells) and presented in the context of different human leukocyte antigen (HLA) class I or class II molecules. Thus, the peptide antigens differ not only according to the antigen from which they are sourced, but also according to the HLA molecule that presents them. For example, a list of both mouse and human T1D-associated peptide antigens is provided by DiLorenzo et al., Clin. Exp. Immunol. 148:1, 2007. 44 can be seen in.

[0029] In some embodiments, the antigen-containing moiety is derived from an allergen, including but not limited to phospholipase A2 (APIml) associated with severe reactions to wasps, Derp-2, Der p 2, Der f, Der p 5 and Der p 7 associated with reactions to house dust mites (Dermatophagoides pteronyssinus), cockroach allergen Bla g 2, and the major birch pollen allergen Bet v 1.

[0030] In some embodiments, the antigen-containing moiety is derived from a therapeutic protein. As used herein, the term "therapeutic protein" refers to any protein or peptide compound of any amino acid length that is or will be administered in vivo to achieve a therapeutic effect. Examples of such therapeutic proteins are, but are not limited to, antibodies of different species (in their native form or partially / fully humanized), cytokines, receptor antagonists, soluble receptors, hormones or hormone analogs, clotting factors, enzymes, bacterial proteins or viral proteins. Examples of therapeutic applications for which therapeutic proteins may be appropriate include, but are not limited to, cytokine and antibody-based immunotherapy, hormone replacement therapy, and replacement therapy of clotting factors (e.g., factor VIII in hemophilia A) or enzyme deficiencies (e.g., β-glucuronidase in mucopolysaccharidosis VII). In all these situations, mounting an immunogenic response against the administered protein is undesirable, as it would be counterproductive to achieving the desired therapeutic effect (e.g., side effects such as cytokine release syndrome; or neutralization / degradation of the therapeutic protein).

[0031] The term "neonatal Fc receptor" or "FcRn" as used herein has its general meaning in the art and refers to a neonatal Fc receptor that is an Fc receptor. Unlike FcγR, which belongs to the immunoglobulin superfamily, human FcRn is structurally similar to polypeptides of the major histocompatibility complex (MHC) class I. 45Typically, FcRn is expressed as a heterodimer consisting of a transmembrane α or heavy chain complexed with a soluble β or light chain (β2 microglobulin). FcRn shares 22-29% sequence identity with class I MHC molecules and possesses a non-functional MHC peptide-binding groove. 46 Like MHC, the α chain of FcRn consists of three extracellular domains (α1, α2, α3) and a short cytoplasmic tail that anchors the protein to the cell surface. The α1 and α2 domains interact with the FcR binding site in the Fc region of antibodies. 47 .

[0032] In some embodiments, the FcRn targeting moiety is typically a protein or polypeptide that can bind to the entire construct and mediate its uptake by the FcRn receptor. In general, the FcRn targeting moiety is the Fc of an IgG antibody, preferably an IgG1 or IgG4 antibody, particularly more preferably an IgG1 antibody, or a sufficient portion of said Fc to allow binding and uptake of the construct. The term "Fc region" as used herein includes the amino acid sequence derived from the constant region of an antibody heavy chain. The Fc region is a portion of the heavy chain constant region of an antibody that includes the hinge, CH2 and CH3 domains, starting from the N-terminus of the hinge region at the papain cleavage site (approximately position 216 of the EU index). Exemplary Fc regions or portions thereof that can be used in the practice of the present invention are well known in the art.

[0033] In some embodiments, a recombinant chimeric construct of the invention is a fusion protein comprising an amino acid sequence consisting of a portion of an Fc region (e.g., a portion of an Fc region that confers binding to FcRn) and an amino acid sequence of a non-immunoglobulin polypeptide comprising an antigenic portion of an antigen.

[0034] The term "fusion protein" as used herein refers to a chimeric polypeptide comprising a first amino acid sequence linked to a second amino acid sequence to which it is not naturally linked in nature. As used herein, the terms "linked", "fused" or "fusion" are used interchangeably. These terms refer to the connection of two or more elements or components to each other by any means, including chemical conjugation or recombinant means. An "in-frame" or "operably linked" fusion refers to the connection of two or more open reading frames (ORFs) to form a continuous, longer ORF in a manner that maintains the correct reading frame of the original ORFs. The resulting recombinant fusion protein is thus a single protein that contains two or more segments that correspond to the polypeptides encoded by the original ORFs (which segments are not normally so connected in nature). Thus, the reading frame will be continuous throughout the fusion segment, although the segments may be physically or spatially separated, for example, by an in-frame linker sequence. A detectable label may further be included to facilitate the production of the fusion construct or its detection after in vivo administration. In addition, various other functional groups can be included in the construct. Examples of such functional groups include, but are not limited to, domains of antigenic proteins that are required to exert one or more desired biological activities (e.g., binding to a receptor or avoiding such binding) or that have been modified to increase such biological activity. Fusion proteins of the invention can be produced by any method known in the art, such as by chemical synthesis, or by creating and translating a polynucleotide that encodes peptide regions in the desired relationship.

[0035] In some embodiments, the Fc region of the fusion protein comprises the entire Fc region of an antibody, starting from the hinge region immediately upstream of the papain cleavage site that chemically defines an IgG Fc (approximately residue 216 in the EU numbering system, assuming the first residue of the heavy chain constant region is 114) and ending at its C-terminus. The exact site at which the fusion is made is not critical; specific sites are well known and may be selected to optimize the biological activity, secretion or binding properties of the molecule. Methods for making fusion proteins are known in the art. The term "hinge region" as used herein includes the portion of the heavy chain molecule that connects the CH1 domain to the CH2 domain, e.g., approximately positions 216-230 in the EU numbering system. This hinge region comprises approximately 25 residues and is flexible, allowing the two N-terminal antigen binding regions to move independently. The hinge region may be subdivided into three separate domains: the upper, middle and lower hinge regions. 48 The term "CH2 domain" as used herein includes, for example, a portion of a heavy chain molecule extending from about EU positions 231-340. The CH2 domain is unique in that it is not closely paired with another domain. Rather, two N-linked branched carbohydrate chains are interposed between the two CH2 domains in an intact, native IgG molecule. The term "CH3 domain" as used herein includes a portion of a heavy chain molecule extending from the N-terminus of the CH2 domain to about 110 residues (e.g., about residues 341-446 in the EU numbering system). Typically, the CH3 domain forms the C-terminal portion of the antibody. However, in some immunoglobulins, additional domains may extend from the CH3 domain to form the C-terminal portion of the molecule (e.g., the CH4 domain of the IgM chain and the E chain of IgE).

[0036] In some embodiments, the Fc region of the fusion protein does not include the hinge region, but comprises the CH2 and CH3 domains fused to an amino acid sequence comprising an antigenic portion of an antigen.

[0037] Additional methods of reducing the size of the construct may also be used, such as those described in US Patent Application Publication Nos. 2002 / 0155537, 2007 / 0014794, and 2010 / 0254986 (Carter et al., respectively), and US Patent Application Publication No. 2014 / 0294821 (Dumont et al.). For example, the formation of Fc-Fc and antigen-Fc / antigen-Fc dimers may be prevented.

[0038] In some embodiments, the Fc region can be mutated to increase the binding affinity or specificity of FcRn. Examples of such mutations are given in recent reviews. 23 These include, but are not limited to, H435A 49 , N434A 50 and M428L modification 51 FcRn-binding peptides that achieve similar or superior effects compared to native Fc domains have also been described. 52 can be used to generate the fusion protein with an antigen of interest, further allowing for a reduction in the relative molecular weight of the added FcRn binding moiety, hi some embodiments, the Fc region can be mutated to limit enzymatic degradation, for example by pepsin.

[0039] In some embodiments, the FcRn binding moiety is coupled to the surface of a nanoparticle and the antigen of interest is encapsulated in such a nanoparticle. Examples of nanoparticles include, but are not limited to, biodegradable and biocompatible poly(lactic acid)-b-poly(ethylene glycol) (PLA-PEG) block copolymers. 30 The FcRn binding moiety can be coupled to the nanoparticle surface by suitable methods, for example using ring-opening polymerization with a maleimide group at the free end. This strategy may have the advantage of protecting the antigen from degradation. Procedures for producing suitable nanoparticles are known in the art, examples of which are described in 30 can be seen in.

[0040] The above-mentioned "therapeutically effective amount" of the chimeric construct of the present invention means an amount of said construct sufficient to achieve a therapeutic effect. However, it will be understood that the total daily usage of the compounds and compositions of the present invention will be determined by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend on a variety of factors, including the disorder being treated and the severity of the disorder; the activity of the specific compound used; the specific composition used, the age, weight, general health, sex and diet of the subject; the administration time, route of administration and excretion rate of the specific compound used; the duration of treatment; drugs used in combination or concomitantly with the specific inhibitor used; and similar factors well known in the medical art. For example, it is well within the skill of the art to begin administering the compound at a level lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the product may vary over a wide range, from 0.01 to 1,000 mg / adult / day. Typically, for symptomatic adjustment of the dosage to the subject to be treated, the composition contains 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of active ingredient. The medicament typically contains from about 0.01 mg to about 500 mg of active ingredient, preferably from 1 mg to about 100 mg of active ingredient. An effective amount of drug is usually supplied at a dosage level of from 0.0002 mg / kg to about 20 mg / kg body weight / day, particularly from about 0.001 mg / kg to 7 mg / kg body weight / day.

[0041] The term "mucosal administration" as used herein includes any form of administration of a recombinant chimeric construct of the present invention via a mucosal surface expressing a neonatal Fc receptor. The term "mucosa" refers to tissues including mucosa, such as, but not limited to, nasal mucosa, pulmonary mucosa, oral mucosa (including sublingual, oral, buccal, intestinal, intestinal, rectal or gastric), or vaginal mucosa. In particular, the term encompasses pulmonary administration and oral administration. In some embodiments, a recombinant chimeric construct of the present invention is delivered via the oral cavity. In some embodiments, a recombinant chimeric construct is delivered via the respiratory tract, for example, by intranasal delivery, inhalation, and any method known in the art.

[0042] The recombinant chimeric construct of the present invention is typically administered to a subject in the form of any pharmaceutical composition compatible with a mucosal administration route. For example, the recombinant chimeric construct of the present invention can be administered as a solution or suspension together with a pharma- ceutically acceptable carrier. Such a pharma- ceutically acceptable carrier can be, for example, water, phosphate buffered saline, saline or other physiologically buffered saline, or a vehicle such as glycol, glycerol and oil, such as olive oil or an injectable organic ester. The pharma- ceutically acceptable carrier can also contain liposomes or micelles prepared by mixing the recombinant chimeric construct of the present invention with surfactants and glycosides, such as Quil A. In some embodiments, the recombinant chimeric construct of the present invention is combined with the carrier in any convenient and practical manner, for example, by solution, suspension, emulsification, mixing, encapsulation, absorption. Such procedures are routine to those skilled in the art. In some embodiments, the recombinant chimeric construct of the present invention is thoroughly mixed or combined with a semi-solid or solid carrier. Mixing may be performed in any convenient manner, such as by grinding. Stabilizers may also be added in the mixing process to protect the composition from loss of therapeutic activity, e.g., denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids, such as glycine and lysine, carbohydrates, such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, protease inhibitors, and the like. The composition for oral administration may further be formulated into hard or soft shell gelatin capsules, tablets, or pills. More preferably, the gelatin capsules, tablets, or pills are enteric coated. The enteric coating prevents denaturation of the composition in the stomach or upper intestine, where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001. Upon reaching the small intestine, the basic pH there dissolves the coating, allowing the release of the recombinant chimeric construct of the present invention and absorption by specialized cells (e.g., epithelial intestinal cells).

[0043] In some embodiments, the chimeric construct may be further protected from degradation by expressing it in biologically contained Lactococcus lactis or other suitable bacteria, either alone or in combination with immunomodulatory molecules, including but not limited to interleukin-10 and transforming growth factor beta. Such formulations may have the added advantage of avoiding the need to synthesize the necessary chimeric construct as a suitable clinical grade recombinant protein, and achieving stable low dose delivery of the antigen-Fc construct, which may be effective in restoring long term tolerance. 53 This method is known in the art and examples are 53-55 The recombinant bacteria of the present invention may be found in the following: Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Escherichia, Streptococcus, Agrobacterium, Bacillus, Corynebacterium, Clostridium, Gluconobacter, Citrobacter, Enterobacter, Klebsiella, and Pseudomonas. Thus, the methods of the present invention encompass the use of a recombinant bacterium, in whose genome a nucleic acid encoding a recombinant chimeric construct of the present invention has been inserted. The recombinant bacterium of the present invention may be selected from the group consisting of Lactobacillus, Leuconostoc, Pediococcus, Lactococcus, Streptococcus, Escherichia, Streptococcus, Agrobacterium, Bacillus, Corynebacterium, Clostridium, Gluconobacter, Citrobacter, Enterobacter, Klebsiella, and Pseudomonas. In some embodiments, the recombinant bacterium of the present invention is a probiotic lactic acid bacterium, in particular of the genus Lactococcus, more particularly of the species Lactococcus lactis.

[0044] In some embodiments, the chimeric constructs of the invention are administered via the airways, for example to the nasal cavity, trachea or lungs. Typically, the chimeric constructs of the invention are delivered by any device adapted to introduce a therapeutic composition into the upper and / or lower airways. In some embodiments, the device of the invention can be a metered dose inhaler. The device can be adapted to deliver the therapeutic composition of the invention in the form of a finely dispersed mist of liquid, foam or powder. The device can use piezoelectric effect or ultrasonic vibration to remove powder deposited on a surface such as a tape to generate a mist suitable for inhalation. The device can use any propulsion system known to those skilled in the art, including but not limited to pumps, liquefied gas, compressed gas, and the like. The device typically includes a container having one or more valves through which the flow of the therapeutic composition passes and an actuator to control the flow. In particular, devices suitable for administering the constructs of the invention include inhalers and nebulizers, such as those typically used to deliver steroids to asthmatics. In some cases, for example, when the subject is a child, a spacer can be used to facilitate effective administration from the inhaler. Inhalers of various designs are commercially available and may be used to deliver the medicament of the present invention. Without being bound by theory, these include Accuhaler, Aerohaler, Aerolizer, Airmax, Akita Jet, Autohaler, Clickhaler, Diskhaler, Easi breathing inhaler, Fisonair, Integra, Jet inhaler, Miat-haler, Novolizer inhaler, Pari Boy, Pulvinal inhaler, Rotahaler, Spacehaler, Spinhaler, Syncroner inhaler and Turbohaler devices.

[0045] The present invention is further illustrated by the following figures and examples, which, however, should not be construed as limiting the scope of the present invention in any way. [Brief description of the drawings]

[0046] [Figure 1] Schematic of the delivery strategy, exploiting the physiological pathway that transports maternal breast milk IgG to the neonate: this pathway is FcRn-dependent, and involves fusion of preproinsulin (PPI) to the IgG Fc fragment, allowing access to the bloodstream through the intestinal epithelium. [Diagram 2] In the absence of FcRn, orally administered PPI-Fc remains localized to the intestine. 50 μg of Alexa-labeled PPI-Fc was force-fed to 1-day-old C56BL / 6 wild-type (FcRn wt) and FcRn − / − mice and imaged 72 h later. PPI-Fc fluorescence remains detectable in the intestine of FcRn − / − but not in FcRn wt mice. [Diagram 3] Systemic and thymic delivery of PPI-Fc by oral administration. 50 μg of Alexa-labeled PPI-Fc or PPI was force-fed to 1-day-old PPI T cell receptor (TCR) transgenic G9C8 NOD neonatal mice and imaged 72 hours later. Accumulation of PPI-Fc, but not PPI, is detected at the systemic level (top) and in the thymus (bottom). Results are for a representative experiment out of two performed. [Figure 4] Serum concentrations of PPI-Fc and PPI upon oral dosing. ELISA quantification of serum samples taken at the indicated times after gavage on day 1. Data are presented as mean + SEM. [Figure 5A] Oral administration of PPI-Fc induces immune tolerance. A. Percentage of splenic CD8+CD3+ and CD4+CD3+ T cells in 4-week-old G9C8 mice treated on postnatal day 1 as above. B. Percentage of splenic CD4+ T cell subsets in the same mice. Bars represent median and interquartile range. ***, p<0.001; **, p<0.01; *, p<0.05. [Figure 5B]Oral administration of PPI-Fc induces immune tolerance. A. Percentage of splenic CD8+CD3+ and CD4+CD3+ T cells in 4-week-old G9C8 mice treated on postnatal day 1 as above. B. Percentage of splenic CD4+ T cell subsets in the same mice. Bars represent median and interquartile range. ***, p<0.001; **, p<0.01; *, p<0.05. [Figure 6] Gating strategy for the analysis of antigen-presenting cells. Relevant gates are numbered as follows: 1, macrophages; 2, B cells; 3, plasmacytoid dendritic cells (DCs); 4, migratory CD103+CD11b-cDCs; 5, resident CD103-CD11b-SIRPα-cDCs; 6, resident CD103-CD11b-SIRPα+cDCs; 7, migratory CD103+CD11b+cDCs; 8, resident CD103-CD11b+cDCs. Representative results from the spleen of a 2-week-old G9C8 mouse are shown. [Figure 7] Gating strategy for analysis of T cell subsets. After gating on viable CD3+ cells (not shown), CD8+ (panel A) and CD4+ T cells (panel F) were analyzed for the expression of different markers. Relevant gates are numbered as follows: 1, CD8+ T cells; 2, activated / memory CD8+ T cells; 3, naive CD8+ T cells; 4, CCR9+ activated CD8+ T cells; 5, α4β7+ activated CD8+ T cells; 6, CCR9+ naive CD8+ T cells; 7, α4β7+ naive CD8+ T cells; 8, CD4+ T cells; 9, activated / memory CD4+ T cells; 10, naive CD4+ T cells; 11, CCR9+ activated CD4+ T cells; 12, α4β7+ activated CD4+ T cells; 13, CCR9+ naive CD4+ T cells; 14, α4β7+ naive CD4+ T cells; 15, thymus-derived Tregs; 16, peripheral Tregs; 17, Th3 cells; 18, Tr1 cells. Representative results from the spleens of 2-week-old G9C8 mice are shown. [Figure 8]CCR9hiCD8+ T cells in PP1-Fc vs. IgG1 treated G9C8 mice. A-B. Percentage (A) and number (B) of activated (CD44hiCD62L-) CCR9hiCD8+ T cells in 2-week-old G9C8 mice treated on postnatal day 1 as above. C-D. Percentage (C) and number (D) of naive (CD44-CD62L+) CCR9hiCD8+ T cells in the same mice. Bars represent median values. *, p<0.05. [Figure 9] α4β7+CD4+ T cells in PP1-Fc-treated G9C8 mice versus IgG1-treated G9C8 mice. A-B. Percentage (A) and number (B) of activated (CD44hiCD62L-) α4β7+CD4+ T cells in 2-week-old G9C8 mice treated on postnatal day 1 as above. C-D. Percentage (C) and number (D) of naive (CD44-CD62L+) α4β7+CD4+ T cells in the same mice. Bars represent median values. *, p<0.05. [Figure 10] Peripheral and thymic Tregs in PP1-Fc-treated G9C8 mice versus IgG1-treated G9C8 mice. A-B. Percentage (A) and number (B) of peripheral (NRP1-) Foxp3+CD4+ Tregs in 2-week-old G9C8 mice treated on postnatal day 1 as above. C-D. Percentage (C) and number (D) of thymic (NRP1+) Foxp3+CD4+ Tregs in the same mice. Bars represent median values. *, p<0.05. [Figure 11] Orally delivered PPI-Fc protects against diabetes. Diabetes incidence in G9C8 mice force-fed with 50 μg PPI-Fc (solid line), equimolar amounts of PPI (dashed line) or IgG1 (dotted line) on day 1. Diabetes was subsequently induced by immunization with PPIB15-23 and CpG at 4 and 6 weeks of age. **P<0.01 by Mann-Whitney U test. EXAMPLES

[0047] Materials and Methods Preparation of mouse PPI1-Fc and PPI2-Fc fusion proteins The sequences encoding PPI1 and PPI2 were PCR amplified from pancreatic and thymus cDNA, respectively, and expressed in pCR4-TOPO plasmid (Invitrogen). 35 After digestion with the appropriate restriction enzymes, the PPI1 / 2 sequences were inserted into the EcoRV / BglII sites by sticky end ligation into the pFUSE-hIgG1-Fc2 expression vector (InvivoGen) downstream of the IL-2 signal peptide and upstream of the human Fcγ1 sequence. The PPI1-Fc and PPI2-Fc sequences were then reamplified by PCR and ligated into the pFastBac1 expression vector (Invitrogen) at the XbaI / XhoI sites. These constructs were inserted into the Bac-to-Bac baculovirus expression system (Invitrogen), expressed in Hi5 insect cells, and the protein products were purified by Sepharose-bound protein G (GE Healthcare) and the protein identities were confirmed by reducing SDS-PAGE and Western blot using rabbit anti-insulin polyclonal antibody (H-86, Santa Cruz) and mouse anti-human Fc monoclonal Ab (Southern Biotech) as previously described. 56 In the experiments shown, PPI1 and PPI2 were purified from Hi5 insect cell pellets. PPI1-Fc (hereafter referred to as PPI-Fc) was used.

[0048] mouse C56BL / 6 wild-type and C56BL / 6 FcRn- / - mice were obtained from Janvier Labs and Jackson Laboratory, respectively. - / - NOD mice are susceptible to PPI B15-23 Transgenic for TCR and previously described and characterized 35,37 .

[0049] In vivo PPI-Fc imaging PPI-Fc and PPI protein were conjugated with Alexa Fluor (AF) 680 using the SAIVI Rapid Antibody / Protein labeling kit (Invitrogen). 50 μg of PPI-Fc or an equimolar amount of PPI was force-fed to 1-day-old neonatal mice. Fluorescence was detected using a Fluobeam imaging system (Fluoptics) with an excitation wavelength of 690 nm and an emission wavelength of >700 nm, and an exposure time of 50–100 ms.

[0050] ELISA quantification of serum PPI-Fc and PPI concentrations After gavage as described above, blood was collected at the indicated time points for ELISA quantification, and standard curves were generated by serial dilutions of PPI-Fc and PPI protein. Plate-coated H-86 anti-insulin Ab (Santa Cruz) was used to capture both PPI-Fc and PPI. Horseradish peroxidase-labeled goat anti-human Fc antibody (Southern Biotech) was used to detect PPI-Fc. Anti-proinsulin monoclonal Ab (KL-1; kindly provided by Dr. L. Harrison, Walter and Eliza Hall Institute, Parkville, Australia) was used to visualize PPI.

[0051] Phenotyping of splenic T cells The following monoclonal antibodies were used on splenocytes from treated mice: PE-conjugated anti-Foxp3, APC-eFluor780-conjugated anti-CD3ε (eBioscience); APC-conjugated anti-neuropilin-1 (NRP1; R&D); Brilliant Violet (BV)605-conjugated anti-CD4, and BV711-conjugated anti-CD8a (BioLegend). Cells were further stained with Live / Dead Red (Invitrogen).

[0052] K d Preparation of multimers Monomers composed of mouse MHC class I heavy chain H-2Kd, human β2-microglobulin, and PPI B15-23 peptide (LYLVCGERL) were synthesized as previously described. 35 The resulting multimers (MMr) were stored at 4° C., protected from light, and used the same day.

[0053] Phenotyping of antigen-presenting cells and T cell subsets in the spleen and lymph nodes. All cells were washed and resuspended in PBS 1x before being transferred to a 96-well V-bottom plate (200 μl / well) for FACS staining. BD CompBeads (10 μl / well, 1 drop in 500 μl PBS) were added to each well before staining to normalize cell numbers. Two antibody panels were used as follows:

[0054] T cell panel. Dasatinib (50 μM, 100 μl / well) was added to each cell pellet for 30 min at 37°C. After centrifugation, cells were resuspended in 18 μl of MMr solution containing a 20% dilution of 50 μM dasatinib for 20 min at room temperature, followed by incubation with 18 μl of the following antibody premixes for 20 min at 4° C.: Live / DEAD Aqua (Invitrogen, 1 / 1,000), CD3-APC-eFluor780 (clone 145-2C11, eBioscience, 1 / 100), CD4-BV711 (clone RM4-5, BD Biosciences, 1 / 200), CD8-AF700 (clone 53-6.7, BD Biosciences, 1 / 150), CCR9-PE-Cy7 (clone CW-1.2, BioLegend, 1 / 100); NRP1-APC (clone 3E12, BioLegend, 1 / 50), LPAM-1 (α4β7)-PE-CF594 (clone ATK32, BD Biosciences, 1 / 100), latency associated peptide (LAP)-BV421 (clone TW7-16B4, BioLegend, 1 / 200), CD62L-BV605 (clone MEL-14, BD Biosciences, 1 / 200), CD44-BV786 (clone IM7, BD Biosciences, 1 / 100). Then 10 μL of the second antibody mix was added for 15 min at 37 °C: CD49b-FITC (clone HMα2, BioLegend, 1 / 200), LAG3-PerCP-Cy5.5 (clone C9B7W, BD Biosciences, 1 / 200). Cells were then washed with PBS, fixed, permeabilized with Foxp3 Fix / Perm Buffer Set (BioLegend) and incubated with 30 μl of anti-Foxp3 antibody (Foxp3-PE, clone FJK-16s, eBioscience, 1 / 50 in FoxP3 / Perm buffer). After the final wash, cells were resuspended in 200 μl PBS 1× and kept at 4°C before flow cytometry acquisition.

[0055] Antigen presenting cell (APC) panel. The same staining protocol was applied without fixation and permeabilization, followed by incubation for 20 min at 4°C with 30 μl of the following antibody mix: Live / DEAD Aqua (Invitrogen, 1 / 1,000), NK1.1-BV510 (clone PK136, BD Biosciences, 1 / 100), CD3-BV510 (clone 145-2C11, BD Biosciences, 1 / 100), F4 / 80-BV711 (clone BM8, BioLegend, 1 / 50), CD8-AF700 (clone 53-6.7, BD Biosciences, 1 / 150), B220-PE-Cy7 (clone RA3-6B2, BD Biosciences, 1 / 150), SIRPα-PerCP-eFl710 (clone P84, eBioscience, 1 / 50), PDCA-1-Pacific Blue (clone 927, BioLegend, 1 / 200), CD11b-BV650 (clone M1 / 70, BD Biosciences, 1 / 150), CD11c-BV605 (clone N418, BioLegend, 1 / 50), and CD103-BV786 (clone M290, BD Biosciences, 1 / 100).

[0056] PPI-Fc treatment, diabetes induction and follow-up G9Cα- / -.NOD mice were force-fed 50 μg of PPI-Fc or control protein (i.e., equimolar amounts of Fc-deficient PPI or Herceptin IgG1) on postnatal day 1. For diabetes induction, 4-week-old mice were force-fed PPI-Fc 4 days after weaning. B15-23 Priming with 50 μg peptide and 100 μg CpG was followed by a second identical immunization 15 days later. The onset of diabetes was monitored by testing for glucosuria, and confirmed by blood glucose if positive. Diabetic mice were sacrificed by cervical dislocation.

[0057] result Intestinal transport of orally administered PPI-Fc is FcRn- and Fc-dependent A schematic of the strategy used is shown in FIG. 1. We exploited the intestinal FcRn pathway that physiologically delivers breast milk IgG to the newborn. To this end, we fused PPI1 or PPI2 proteins to the N-terminus of the CH2-CH3 Fc domain from human IgG1 to obtain PPI1-Fc and PPI2-Fc fusion proteins. Fc-deficient PPI1 / 2 cannot cross the intestinal epithelium, but the addition of the Fc part should facilitate this transport. We investigated this strategy using the PPI1-Fc construct (hereafter referred to as PPI-Fc).

[0058] First, we performed ex vivo imaging on 1-day-old FcRn- / - and wild-type C56BL / 6 mice force-fed with fluorescently labeled PPI1-Fc and sacrificed 72 h later (Figure 2). PPI-Fc fluorescence was still detectable in the intestines of FcRn- / - mice but not in the intestines of wild-type mice, suggesting no transport in the absence of FcRn.

[0059] To verify the occurrence of such transport, fluorescently labeled PPI-Fc or Fc-deficient PPI was force-fed to 1-day-old G9C8 neonatal mice (Figure 3). After 72 h, systemic accumulation of PPI-Fc was rapidly visualized, whereas this was not the case for PPI. PPI-Fc was also visualized in the thymus, whereas PPI was not.

[0060] Serum concentrations of PPI1-Fc (Figure 4) were approximately 1 μg / ml 24 hours after dosing (day 2) and remained relatively stable up to 72 hours (day 3; approximately 0.75 μg / ml). PPI1 was not detectable at any of these time points.

[0061] Taken together, these results indicate that a single oral dose of 50 μg PPI-Fc, but not a single oral dose of 50 μg PPI, results in intestinal transport, systemic antigen bioavailability and thymic delivery that is FcRn- and Fc-dependent.

[0062] Oral vaccination with PPI-Fc induces tolerogenic T cell modifications. Next, we assessed whether thymic delivery of PPI-Fc induced T cell modification compatible with immune tolerance (Figure 5A). In PPI-Fc-treated neonates, splenocytes obtained at 4 weeks of age after the above postnatal day 1 treatment were CD8 + There was a significant decrease in the number of effector T cells. + No significant differences were observed in T cell counts. However, CD4 + When T cell subsets were analyzed, significant differences were detected (Figure 5B). Compared to PBS-treated mice, PPI1-Fc-treated animals showed significantly higher CD4 + Effector T cells (Foxp3 - ) and Foxp3 + Increased numbers of regulatory T cells (Tregs), both of which are thymic-derived (NRP1 / CD304 + ) and peripherally inducible (NRP1 / CD304 - ) was shown.

[0063] To distinguish between effects induced by the Fc portion and those that are dependent on the antigenic portion of the PPI, a second set of experiments was performed by comparing G9C8 mice force-fed with either PPI-Fc or IgG1. These mice were analyzed at 2 weeks of age (i.e. closer to postnatal day 1 when orally vaccinated). Both APC and T cell subsets were analyzed in the spleen, mesenteric lymph nodes (MLN) and pancreatic lymph nodes (PLN).

[0064] The APC gating strategy is shown in Figure 6. After gating on forward and side scatter and live cells (not shown), CD3 - NK1.1 - (lineage negative) cells were selected to exclude T cells and NK cells, respectively (Figure 6A). Three gates were then selected according to two markers: F4 / 80 + B220 - , F4 / 80 - B220+ and F4 / 80 - B220 - (Figure 6B). Then, F4 / 80 was analyzed for CD11c and CD11b expression. + B220 - Plot the cells and measure CD11c - CD11b + Macrophages were identified (FIG. 6C, gate 1). Similarly, F4 / 80 for CD11c and CD11b markers. - B220 + Plot the cells and measure CD11c - CD11b - B cells (Figure 6D, gate 2) and CD11b - CD11c + Cells were gated on F4 / 80, which upon further gating for the PDCA-1 marker (FIG. 6E, gate 3) and the migration marker CD103 revealed a plasmacytoid dendritic cell (pDC) population. - B220 - Among the cells, two gates were selected according to the CD11c and CD11b markers (Figure 6F). + The fraction consisted of conventional dendritic cells (cDCs), which are migratory (CD103 + , gate 4) or resident (CD103 - , Gate 5, Figure 6G) - cDC; and migratory (CD103 + , gate 7) or resident (CD103 - , Gate 8, Figure 6H) + cDCs) are further divided into CD103 - SIRPα + CD11b - The cDC population was also visualized (gate 6, FIG. 6G). No differences in the frequency or number of these different APC populations were observed when comparing 2-week-old mice fed PPI-Fc and IgG1 (not shown). Taken together, these results indicate that APC composition is not affected by previous oral vaccination, at least at the 2-week time point analyzed.

[0065] The T cell gating strategy is shown in Figure 7. - CD3 + Among the cells, CD8 + T cells were gated (Figure 7A, gate 1). CD8 + Among the cells, MMr + Select cells and perform PPI B15-23 The specificity of these peptides was verified (Figure 7B). + T cells were further gated. CD44 hi CD62L - Subset is activated / memory CD8 + On the other hand, CD44 (Gate 2) corresponds to T cells. - CD62L + are naive cells (gate 3, Figure 7C). Finally, activated CD8 + T cells and naive CD8 + Both T cells were analyzed (gates 4-5, Figure 7D; and gates 6-7, Figure 7E). + For T cells (Figure 7F), the same phenotypic markers (CD44 and CD62L, Figure 7G; and CCR9 and α4β7, Figures 7H-I) were used. In addition, three Treg populations were analyzed: thymic-derived (Foxp3 + NPR1 + ; Gate 15) or peripherally inducible (Foxp3 + NRP1 - ; gate 16, Fig. 7J) classical Treg; T helper (Th) 3 cells (LAP + FoxP3 - ; gate 17, FIG. 7K) and T regulatory 1 (Tr1) cells (Foxp3 - CD49b + LAG3 + ; Gate 18, Figure 7L). Using this gating strategy, subtle differences between PPI-Fc- and IgG1-fed animals were already seen at 2 weeks of age. First, in PPI-Fc-fed mice, there was a higher prevalence of splenic CCR9 expression, which may be derived from the gut. hi CD8 +The proportion of T cells was increased (Figure 8). hi CD8 + T cells (Figure 8A-B) and naive CD8 + Although this was not observed in a subset of T cells (Figure 8C-D), this further suggests that T cells migrate from the intestine to the spleen upon encountering their PPI B15-23 alloantigen, which is recognized by most T cells in this G9C8 TCR-transgenic model. Second, splenic α4β7 + CD4 + A similar increase was observed in T cells (another population reported to be derived from the gut) in the PPI-Fc group (Figure 9). Again, this increase was observed only in the activated subset of these cells (Figure 9A-B) and not in the naive subset (Figure 9C-D), likely reflecting prior allogeneic PPI-Fc priming in the gut. Third, while a slight increase in splenic peripheral Tregs was observed with PPI-Fc treatment (Figure 10A-B), splenic thymus-derived Tregs were not increased at this time point (Figure 10C-D).

[0066] Taken together, these results demonstrate that oral vaccination with PPI-Fc induces T cell alterations characteristic of oral tolerance (i.e., gut-derived activated CD8 + and CD4 + and an increase in T cells and peripheral Tregs; and modifications suggestive of deleterious and regulatory tolerance mechanisms (i.e., CD8 + and CD4 + Reduction of effector T cells and CD4 + It has been shown that oral vaccination with PPI-Fc induces a novel mechanism of action of oral vaccination (increasing Tregs). More notably, the putative mechanism of action of oral vaccination with PPI-Fc differs from classical oral tolerance with Fc-deficient antigens. Systemic and thymic bioavailability of antigen-Fc is achieved here, enhancing both peripheral and central tolerance mechanisms, as evidenced by an increase in the numbers of both thymus-derived and peripherally-derived Tregs.

[0067] Neonatal oral vaccination with PPI-Fc protects G9C8 mice from the development of diabetes Finally, we tested whether oral vaccination with PPI-Fc and associated T cell engineering could provide diabetes protection in later life. To this end, we orally vaccinated 1-day-old neonatal G9C8 mice with 50 μg of PPI-Fc. They were then immunized with PPI B15-23 peptide and CpG at 4 and 6 weeks of age to induce diabetes and followed prospectively. As controls, equimolar amounts of recombinant IgG1 (i.e., an unrelated protein with preserved FcRn binding) and PPI (i.e., alloantigen without FcRn binding) were administered. In IgG1-fed mice, diabetes onset was rapid and occurred simultaneously with prime-boost immunization, with 93% of mice becoming ill. Conversely, when fed PPI-Fc, only 44% of mice developed diabetes (p<0.01). As expected, PPI conferred modest protection, with 72% of mice eventually developing diabetes. Taken together, these results demonstrate that oral vaccination with PPI-Fc protects G9C8 mice from diabetes more efficiently than Fc-deficient PPI.

[0068] References: Throughout this application various references describe the state of the art to which this invention pertains, the disclosures of which are incorporated by reference into this disclosure. [Table 1] TIFF2025020198000002.tif248168 TIFF2025020198000003.tif242168 TIFF2025020198000004.tif249168 TIFF2025020198000005.tif249168 TIFF2025020198000006.tif89168

Claims

1. A method for inducing tolerance to an antigen of interest in a subject in need thereof, comprising mucosally administering to the subject a therapeutically effective amount of a recombinant chimeric construct comprising an FcRn targeting portion and an antigen-containing portion.

2. 2. The method of claim 1, wherein the antigen is a self-antigen, the antigen is an allergen, or the antigen is a molecule that is exogenously administered for therapeutic purposes.

3. The method of claim 1, wherein the subject is an adult, a pregnant woman, or a child.

4. The method of claim 1 , wherein the subject is a newborn or neonatal child.

5. 10. The method of claim 1, wherein the subject is susceptible or thought to be susceptible to developing, or has already developed or is developing, an autoimmune disease.

6. 2. The method of claim 1, wherein the subject is susceptible or thought to be susceptible to developing an allergy, or has already developed or is developing an allergy.

7. 2. The method of claim 1, wherein the subject is susceptible to, or believed to be susceptible to, or has already developed or is developing an immune response to a molecule exogenously administered for therapeutic or other purposes.

8. The method of claim 1, wherein the subject is susceptible to developing or is thought to be susceptible to developing, or has already developed or is developing, an immune response against transplanted tissue or transplanted hematopoietic cells or transplanted blood cells.

9. 2. The method of claim 1, wherein the FcRn targeting moiety is the Fc or a part of the Fc of an IgG antibody, preferably of an IgG1 or IgG4 antibody, particularly preferably of an IgG1 antibody.

10. The method according to claim 1, wherein the recombinant chimeric construct of the present invention is a fusion protein comprising an amino acid sequence consisting of a part of an Fc region and an amino acid sequence comprising an antigenic portion of an antigen.

11. The method of claim 1, wherein the recombinant chimeric construct is administered to the subject by a recombinant bacterium expressing the construct.

12. The method of claim 1 , wherein the recombinant chimeric construct is delivered via the oral cavity.

13. The method of claim 1 , wherein the recombinant chimeric construct is delivered via the airways.

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