WO2010056143A1 - The use of adjuvant to facilitate the induction of immune tolerance - Google Patents

Abstract

The disclosure provides a method for inducing immune tolerance, using the combination of an antigen, an adjuvant and a tolerogenic agent. The effect is antigen-specific. Tolerance induction is effective in pre-sensitized subjects. The invention is useful for enhancing peptide therapy and gene therapy involving immunogenic peptides or other antigens.

Description

THE USE OFADJUVANT TO FACILITATE THE INDUCTION OF

IMMUNE TOLERANCE

Reference to Related Applications

This application claims the benefit of and priority under 35 U.S.C. § 119(e) to U.S. Provisional Application Serial No. 61/114,373, filed on November 13, 2008, which is incorporated by reference in its entirety, including all drawings and ail parts of the specification.

Field of the Disclosure

This disclosure relates to the field of immunology. More specifically, the disclosure is drawn to the use of an adjuvant to promote tolerance induction.

Background of the Disclosure

Immune tolerance (or "immunological tolerance") is vital to the function of the immune system. Antigen-specific immune tolerance is characterized by a decrease in responsiveness to an antigen, which is induced by previous exposure to that antigen. When specific lymphocytes encounter antigens, the lymphocytes may be activated, leading to an antigen-specific immune response, or the cells may be inactivated or eliminated, leading instead to antigen-specific immune tolerance. The same antigen may induce an antigen-specific immune response or tolerance, depending on the circumstances of the antigen's presentation to the immune system. Factors or reagents that act in the presence of an antigen to induce tolerance are said to be tolerogenic agents. (Cellular and Molecular Immunology, 6^th Ed. Abbas et al. Eds., Saunders Elsevier, Philadelphia, 2007).

Tolerance to self-antigens, also called self-tolerance, is a fundamental property of the normal immune system. Normal individuals are tolerant of their own (i.e., self) antigens because the lymphocytes that recognize self antigens are killed or inactivated, or change their specificity. If the body fails to properly distinguish self versus foreign antigens, it may lead to immune disorders such as autoimmune diseases, where the immune system attacks its own antigens as if they are foreign.

In general, it has been believed that protein antigens administered subcutaneously or intradermally with adjuvants favor a robust immune response to the antigen and 'immunity', whereas high doses of antigens administered systematically without adjuvants tend to induce tolerance. Published studies show that induction of T cell tolerance is observed when an aqueous antigen, but not an adjuvant-associated antigen, is given to an animal. For example, Pape et al. demonstrated that mice exposed to an antigenic peptide in an immunogenic form (with adjuvant) showed great expansion of T cells, but not mice exposed to the same peptide in a tolerogenic form (large dose of aqueous peptide without adjuvant) (Pape KA et al., Immunological Reviews 156:67-78, 1997). The mechanism underlying the observed effect of adjuvants was believed to be that adjuvants stimulate release of cytokines and expression of costimulators on antigen presenting cells (APCs), resulting in immunity being favored over tolerance.

Summary of the Disclosure

The present invention is based at least in part on the surprising discovery made by the inventors that adjuvants can be used to promote the induction of antigen-specific immune tolerance in vivo. As alluded to above, it has been generally accepted that pro-inflammatory signals create a barrier for the induction of immune tolerance. As a consequence, most protocols attempted tolerance induction in the absence of inflammatory "danger" signals or, at least, where these signals are minimized as much as possible. Surprisingly, it has been discovered that the combination of adjuvants, including pro-inflammatory adjuvants, with antigens facilitates tolerance induction to those antigens. Using the method described herein, it is possible to induce a state of tolerance that decreases or prevents the generation of an antigen- specific immune response by administering to a subject an antigen of interest, an adjuvant and a tolerogenic agent. The subject remains immunocompetent to mount protective immune responses against unrelated antigens.

Thus, methods of the invention described herein are useful for inducing immune tolerance in a subject. According to the invention, the method involves administering to a subject an isolated antigen, an adjuvant and a tolerogenic agent to induce antigen-specific immune tolerance in a subject. In any of the embodiments described herein, the subject is in need of (e.g., is likely to benefit from) increased tolerance to the antigen.

In any of the embodiments described herein, the subject may be a mammal, and in any of the embodiments described herein, the subject may be a human subject.

In this and other aspects and embodiments of the invention, the subject may have an allergy and may be previously sensitized to the isolated antigen.

For example, the subject may be allergic to an antigen derived from: mite, venoms, insects, animal particles (epithelia, dander, hair and feathers), fungi (spores), smuts, pollen, foods, dust and/or drugs (e.g. penicillin). More specifically, the allergen may be derived from one or more of the following genuses: Canine (Canis familiaris); Dermatophagoides (e.g. Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata); Alder; Alnus (Alnus gultinoasa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago lanceolata); Parietaria (e.g. Parietaria officinalis or Parietaria judaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apis multiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana); Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis glomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalaris arundinacea); Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghum halepensis); and Bromus (e.g. Bromus inermis).

In any of the embodiments embraced by the instant disclosure, the adjuvant may be selected from the group consisting of: alum (aluminum hydroxide, aluminum phosphate); mineral oil, non-mineral oil, water-in-oil emulsions, oil-in-water emulsions, Seppic ISA series of Montanide adjuvants; MF-59, PROVAX; saponins (e.g., QS21); poly[di(carboxylatophenoxy)phosphazene (PCPP), monophosphorlyl lipid (MPL), muramyl dipeptide (MDP) and threonyl muramyl dipeptide (tMDP); OM- 174; and Leishmania elongation factor; ISCOMS; SB-AS2; SB-AS4; CRL 1005; Syntex Adjuvant Formulation and an immunostimulatory nucleic acid molecule. In some embodiments, the adjuvant is not an immunostimulatory nucleic acid. In some embodiments, the adjuvant is a pro-inflammatory adjuvant. In some embodiments, the adjuvant is alum. In some embodiments, the adjuvant is a saponin. In any of the embodiments, the adjuvant may be mixed together with the antigen. In any of the foregoing embodiments, the adjuvant and the antigen may be administered as described in more detail below, and in some embodiments the adjuvant and the antigen may be administered subcutaneously, intradermally, intramuscularly or intravenously.

In any of the embodiments described herein, the tolerogenic agent can be an agent that interferes with T-cell activation. In some embodiments, the tolerogenic agent is an agent that blocks a co-stimulation signal. In some embodiments, the tolerogenic agent is an agent that binds to a cell surface molecule expressed on a T cell or on an antigen presenting cell.

In any of the embodiments embraced by the invention, the tolerogenic agent may be selected from the group consisting of: anti-CD4, anti-CD3_> anti-CD25, anti-CD28, anti-PDl, anti-BTLA, anti-B7 (anti-B7-l and anti-B7-2), anti-ICOS, anti-CTLA, anti-CD40, anti- CD40L, anti-CD99, anti-CD2, anti-LFA3, anti-CD27, anti-CD70, anti-DC8, anti-OX40, anti- OX40L, anti-LFA-l, anti-CDlla, anti-ICAMl, anti-CD26, anti-CD44, anti-CD137 (anti-4- IBBL), CTLA4-Ig, anti-MHC class II and anti-MHC class I.

In some embodiments of the invention, the subject is a candidate for an exogenous peptide therapy. In some embodiments, the subject has a peptide deficiency. In some embodiments, the subject has received or is going to receive an exogenous antigen administered as a therapy, and the exogenous antigen comprises the isolated antigen. In other embodiments, the subject has received or is going to receive an exogenous peptide, and the exogenous peptide comprises the isolated antigen. In other embodiments, the subject has received or is going to receive gene therapy, and an expression product of the gene therapy comprises the isolated antigen. In still other embodiments, the subject has received or is going to receive gene therapy, and the gene therapy comprises the isolated antigen.

The peptide used for the exogenous peptide therapy may be selected from the group consisting of: Factor VIII, Factor IX, Von Willebrand Factor (VWF), Factor XI, Factor VII, T4 endonuclease V, erythropoiesis stimulating protein, insulin, Protein C, neuregulin 1 , Laminin- 111, p53, Erythropoietin (EPO), alpha-1 antitrypsin, Rod derived Cone Viability Factor (RdDVF), estrogen, estradiol, testosterone, Dehydroepiandrosterone, Phenylalanine hydroxylase, DNase I, Glucocerebrosidase, Growth hormone (GH), Alpha interferon, Gamma- Ib interferon, Interleukin-2 (IL-2), Interleukin-3 (IL-3), Interleukin-4 (IL-4), Tissue plasminogen activator tPA), monoclonal antibodies, adenosine deaminase, Nutropin (somatropin), Neupogen and Leukine (G-CSF, GM-CSF).

In any of the embodiments, the subject may or may not be previously sensitized to the peptide.

In some embodiments of the invention, the subject has a disease or disorder selected from the group consisting of: hemophilia (A and B), Type I diabetes, DNA repair disorder, Xeroderma Prgmentosum (XP), Cockayne Syndrome (CS), Trichothiodystrophy (TTD), severe protein C deficiency, purpura fulminans (PF), disseminated intravascular coagulation (DIC), venous thromboembolism (VTE), chronic granulomatous disease, cystic fibrosis (CF), Gaucher 's disease, Duchenne muscular dystrophy (DMD), Alpha-1 antitrypsin chronic deficiency, multiple sclerosis (MS), retinitis pigmentosa (RP), blood clots, pulmonary embolism, myocardial infarction, stroke, hormone deficiency, phenylketonuria, anemia, chronic renal disease, lysossomal storage diseases (e.g., Fabry Disease), Nanism hypophyseal, leukemia, Kaposi's sarcoma, hepatitis B, hepatitis C, Chronic granulomatous disease, cancer, Acute myocardial infarction, massive pulmonary embolism, rheumatoid arthritis, SLE, multiple sclerosis, inflammatory bowel disease and severe combined immune deficiency (SCID).

Each of the limitations of the invention involving any one element or combinations of elements can be included in each aspect of the invention. This invention is not limited in its application to the details of construction and the arrangement of components set forth in the following description or illustrated in the drawings. The invention is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing," "involving," and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

Brief Description of the Drawings

Figure 1. Prevention of the generation ofOVA-speciβc immunoglobulions with tolerogenic MAbs administered together with OVA-alum. (A) Female BALB/c mice were sensitized with 20 μg OVA-alum i.p. and challenged with 50 μg OVA in saline i.n. on the indicated days. Some animals were treated with 1 mg of anti-CD4 or an isotype control i.p. on the indicated days. (B) Quantification of OVA-specific IgE and IgGl in the serum was performed by ELISA. Anti-CD4 MAb treated mice show a significant reduction of the allergen-specific Th2 -driven immunoglobulins (PO.001). (C) Quantification of OVA-specific IgG 1 and total IgE in the serum of animals treated with anti-CD40L MAb. A reduction of immunoglobulins was also observed in animals treated with the tolerogenic MAb. (D) Serum immunoglobulins in animals treated with anti-OX40L. No changes were observed in ThI- driven IgG2a in any experiment (below detection in all groups of animals, not shown). Data are representative of three independent experiments.

Figure 2. Tolerance induction is antigen-specific and prevents subsequent immunization. (A) Treatment protocol, using the same doses of MAb as described previously. Mice were initially tolerized to OVA or β-LG, and 50 and 64 days latter immunized with the same or the third-party allergen. All animals were challenged i.n. with the same allergen used for sensitization at day 50 and 64. (B) Serum concentration of OVA-specific and β-LG-specific IgGl. Tolerance induction to OVA prevents subsequent production of OVA-specific but not β- LG-specific IgGl. Conversely, tolerance to β-LG does not hamper the generation of OVA- specific IgGl .

Figure 3. Prevention of allergic sensitization to OVA-alum. (A) Female BALB/c mice were sensitized with 20 μg OVA-alum i.p. and challenged with 50 μg OVA in saline i.n. on the indicated days. Some animals were treated with 1 mg of anti-CD4 or an isotype control i.p.. (B) Cellular composition of the BAL. Animals treated with anti-CD4 have -100-fold less eosinophils and lymphocytes in the BAL (n=6, P<0.001). (C) Histological sections of lung tissue were stained with hematoxilin/eosin (left) and PAS (right). Tissue from anti-CD4 treated mice show a reduction in the inflammatory infiltrate to levels similar to the healthy unmanipulated controls. The goblet cell hyperplasia is also absent in the MAb treated animals. (D) No changes were observed in the levels of IFN-γ, TNF-α, IL- 17, and IL-10 in lung homogenates from sensitized animals treated with the isotype control MAb (black), mice treated with anti-CD4 (grey), or naive healthy controls (white). The Th2 cytokines IL- 13 (n=6, P<0.01), IL-4 and IL-5 (PO.05) were down to basal levels in anti-CD4 treated mice. (E) Invasive measurement of respiratory mechanics shows that animals treated with anti-CD4 MAbs have reduced airway resistance, tissue elastance and tissue damping in response to increasing doses of MCh, when compared with sensitized control animals (n=8, PO.01 for [MCh] >10 mg/ml). Data are representative of three independent experiments.

Figure 4. Tolerance induction is effective in sensitized animals in an antigen-specific way. (A) Treatment protocol, using the same doses of MAb as described previously. Mice were initially sensitized with OVA-alum or β-LG-alum. On day 50 the animals were tolerized with anti-CD4 to the same or the third-party antigen adsorbed in alum. All mice were challenged i.n. with the same antigen used for initial sensitization. (B) Cellular composition of the BAL. Only animals treated with anti-CD4 together with the antigen used for sensitization (OVA >tOVA and β-LG >tβ-LG) show reduced numbers of BAL eosinophils and lymphocytes (n=6, PO.001). Mice treated with the third-party antigen together with anti-CD4 (β-LG >tOVA and OVA >tβ-LG) are similar to animals that were not treated with anti-CD4 (isotype). (C) Tolerance induction in sensitized mice abrogates AHR. Mice treated with anti-CD4 50 days following sensitization have changes in airway resistance in response to inhaled MCh similar to naive healthy controls, and significantly lower than animals treated with the isotype (n=8, /*<0.001 for [MCh] = 20 mg/ml). Data are representative of two independent experiments.

Figure 5. Tolerance induction to OVA-alum is robust enough to prevent allergic airways disease in TCR transgenic mice. (A) OVA-specific DOl 1.10 mice were sensitized with OVA-alum i.p. in the presence of anti-CD4 or an isotype control MAb as indicated. All animals were challenged with OVA i.n. (B) The production of OVA-specific IgGl and IgE does not occur in anti-CD4 treated mice (n=6, P<0.00l). (C) Mice treated with anti-CD4 have reduced numbers of eosinophils in the BAL (n=6, /M)₁OOl). (D) Histological sections of lung tissue were stained with hematoxilin/eosin (left) and PAS (right). Tissue from anti-CD4 treated mice show a reduction in the inflammatory infiltrate and an absence of goblet cell hyperplasia. (E) The concentration of several cytokines was determined in lung homogenates. IL-4, IL- 13 and IL-10 are markedly reduced in anti-CD4 treated mice (n=6, i^><0.01 or PO.001 as indicated). (F) Following i.n. challenge with OVA, OVA-specific T cells (identified with KJl -26) from the MeLN were stained for several cytokines and Foxp3 and analyzed by flow cytometry. Animals treated with anti-CD4 show a reduction in the frequency and absolute number (not shown) of OVA-specific T cells containing the Th2 cytokines IL-4, IL-5, and IL-13 (n=6, P<0.01 or PO.001 as indicated). No marked differences were observed for IFN-γ or IL-10. The number and frequency of OVA-specific Foxp3⁺ Treg cells remained unchanged in anti-CD4 treated mice (not shown). Data are representative of two independent experiments.

Figure 6. Tolerance induction with anti-CD4 leads to AICD of antigen-specific T cells. Splenic and LN T cells from DOl 1.lO.Rag^'7" mice were stained with CFSE and injected i.v. into sex-matched BALB/c hosts. Mice were then treated with OVA-alum i.p. in the presence or absence of anti-CD4. Some animals were not exposed to OVA. (A) Analysis of MeLN at day 4. The dot plots are gated on CD3⁺ T cells. The numbers represent the cell frequency within the TCR transgenic T cell population. (B) Absolute number of DO 11.10 cells recovered from MeLN contained in each of the CFSE regions displayed above. The difference in cell numbers between undivided cells from mice not injected with OVA is significantly higher than T cells from mice treated with OVA-alum or OVA-alurn+anti-CD4 (n=4, P<0.05). (C) Frequency of apoptotic cells (positive for annexin V) within cells that did not divide or cells that have divided. The frequency of apoptotic cells is significantly higher within the T cells undergoing cell divisions from anti-CD4 treated animals (n=4, PO.05). Figure 7. Anti-CD4 treatment does not deplete DOlLlO T cells in the absence of OVA. DOl l.lO.Rag-/- mice were treated with two shots of 1 mg anti-CD4 in alternate days. Spleen and LN were collected at day 7 and stained with the TCR-specific MAb (KJ 1-26) and Foxp3. No significant difference was observed in the frequency or number of T cells from MAb treated animals.

Figure 8. Tolerance induction to hFVIIL (A) BALB/c mice were treated with IU hFVIII or hFVΪII-alum i.p. on days 1 and 10, and anti-CD4 MAb i.p. on days 0, 2, 9, and 11. All animals were injected i.p. with a further dose of IU hFVIII at day 30. (B) Serum samples were collected from all mice at day 60, and the serum concentration of hFVIII-specific immunoglobulins determined by ELISA. Animals treated with hFVIII-alum + anti-CD4, but not hFVIII + anti-CD4, have significantly lower concentrations of serum anti-hFVHI IgGl (n=5, P<0.01).

Figure 9. Prevention of the generation of HDM-specific immunoglobulions with tolerogenic MAbs administered together with HDM-alum. (A) Female BALB/c mice were sensitized with 20 μg HDM-alum i.p. and challenged with 50 μg HDM in saline i.n. on the indicated days. Some animals were treated with 1 mg of anti-CD4 or an isotype control i.p. on the indicated days. (B) Total number of eosinophilis in the BAL of the different groups of mice. (C) Measurement of airway resistance. Quantification of HDM-specific IgGl (D) and IgE (E) in the serum was performed by ELISA.

Figure 10. Tolerance induction is antigen-specific and prevents subsequent immunization. (A) Female BALB/c mice were sensitized with 20 μg HDM-alum i.p. and challenged with 50 μg HDM in saline i.n. on the indicated days. Some animals were treated with 1 mg of anti-CD4 or an isotype control i.p. on the indicated days. (B) Total number of eosinophilis in the BAL of the different groups of mice. (C) Measurement of airway resistance. Quantification of HDM-specific IgGl (D) and IgE (E) in the serum was performed by ELISA.

Figure 11. Tolerance induction is effective in sensitized animals in an antigen-specific way. (A) Treatment protocol, using the same doses of MAb as described previously. Mice were initially sensitized with OVA or HDM. On day 50 the animals were tolerized with anti-CD4 to the same antigen adsorbed in alum. All mice were challenged i.n. with the same antigen used for initial sensitization. (B) Total cell number of the BAL. (C) Tolerance induction in sensitized mice abrogates AHR. Mice treated with anti-CD4 50 days following sensitization have changes in airway resistance in response to inhaled MCh similar to naive healthy controls, and significantly lower than animals treated with the isotype. (D) HDM specific IgGl titer; and (E) IgE titer.

Detailed Description of the Disclosure

Previous studies have shown that antigens may be administered in ways that inhibit immune responses by inducing antigen-specific tolerance in lymphocytes. On the other hand, vaccines (e.g., immunization) generally attempt the opposite effect - that is, vaccines are designed to enhance the immunogenicity of antigens by administering them in ways that promote lymphocyte activation, promote a robust antigen-specific immune response and prevent tolerance induction. It has been widely accepted that adjuvants used in vaccines formulations facilitate lymphocyte activation and promotion of a robust antigen-specific immune response, thereby favoring immunity, not tolerance. Conversely, antigen formulations in an aqueous form but without adjuvant have been used in situations where immune tolerance induction is desirable. In general, it has been widely believed that protein antigens administered subcutaneously or intradermally with adjuvants favor immunity, whereas high doses of antigens administered systematically without adjuvants tend to induce tolerance.

As described in more detail herein, the present invention provides methods for inducing immune tolerance in a subject, where the subject is in need of, or is likely to benefit from, increased tolerance to a particular antigen. The methods described herein involve administration of an isolated antigen, an adjuvant, and a tolerogenic agent. The methods can be used to facilitate the induction of antigen-specific tolerance in the subject to lessen or prevent the induction of an undesirable immune response or decrease an existing immune response to the antigen. The invention is particularly useful in inducing tolerance against antigens such as peptides administered as therapy or against antigens that are allergens.

As used herein, a "subject" shall refer to any vertebrate, typically a mammal. Preferably, the subject is a human subject. In any of the embodiments embraced by this invention, a "subject in need of increased tolerance" means that the subject will benefit from increased immune tolerance to an antigen. The subject who benefits from increased tolerance can be a subject who, at the time of treatment, is naive to the antigen or a subject with a preexisting unwanted immune sensitivity to the particular antigen . As used herein, an "antigen" shall refer to a substance that is immunogenic (i.e.,, triggers an immune response) in a subject. An "isolated antigen" is a substance in a pure or substantially pure form, separated from other unrelated antigens, such that the antigen can be suitable for therapeutic use to provoke an antigen specific immune response. Antigens suitable for use in the methods described herein may be foreign to the subject, that is, that they are not naturally produced in the body of the subject. These antigens are "foreign" antigens. Other antigens suitable for use in the methods described herein may be native to the subject, that is, that they are naturally produced in the body of the subject but are not properly recognized as self by the immune system of the subject, triggering unwanted immune responses. Such antigens can be the basis for various autoimmune disorders. These antigens are "self- antigens." Antigens made outside the body of a subject and introduced into the subject are "exogenous" antigens.

The antigen typically is a peptide, such as a whole protein, a part of a whole protein or an important immunogenic portion or epitope of a protein. The antigen may be in certain instances attached to a carrier substance known in the art for assisting induction of an immune response to the antigen, typically employed in the case of very small antigens. The antigen also may be a, polysaccharide, lipid, glycoprotein, glycolipid, nucleic acid, or carbohydrate. The antigen also may be a particular cell type or cell extract. Polypeptide/peptide antigens, including peptide mimics of polysaccharides, may be encoded by nucleic acids and administered in vectors or as naked DNA according to methods known in the art.

A numbers of clinical indications have been targeted for gene therapy, including various cancers, immune diseases, cardiovascular diseases, infectious diseases, monogenic diseases, neurological diseases, ocular diseases. Some of the targets actively pursued for gene therapy for which a clinical trial has been completed, in progress or being considered include: Severe Combined Immunodeficiency Syndrome, Hepatocellular Carcinoma, transplantation (e.g., liver transplantation), Chronic Granulomatous Disease, Communicable Disease, Chronic Granulomatous Disease, Advanced Malignant Thyroid Tumors, Gyrate Atrophy, Brain and Central Nervous System Tumors, Arthritis, Rheumatoid, Arthritis, Psoriatic; Ankylosing Spondylitis, Glioma, Alzheimer's Disease, HIV infections, Fallopian Tube Cancer, Ovarian Cancer, Peritoneal Cavity Cancer, Retinal Degeneration, Angina Pectoris, Myocardial Infarction, Ischemic Heart Disease, pregnancy, Prostate cancer, Prostatic neoplasms, local neoplasm recurrence, Leukocyte Adhesion Deficiency Syndrome, Breast cancer, Familial Hypercholesterolemia, lymphoma, wound healing, ulcers, bone marrow suppression, Fanconi's Anemia, Pancytopenia, hemophilia, Muscular Dystrophies, Glioblastoma Multiforme, Anaplastic Astrocytoma, Alpha 1 -Antitrypsin Deficiency, Chronic Granulomatous Disease, Pompe disease, head and neck cancer, Hepatocellular carcinoma, Melanoma, Leber Congenital Amaurosis, Amaurosis, Retinal Diseases, Pleural Mesothelioma, Metastatic Pleural Effusions, Parkinson's disease, Brain and central nervous system tumors, Becker Muscular Dystrophy, Limb-Girdle Muscular Dystrophy, Neuroblastoma, Mucopolysaccharidosis II, Sickle Cell Anemia, Gaucher's Disease, Amino Acid Metabolism, Inborn Errors, Angina Pectoris, Painful Diabetic Neuropathy, Thalassemia, Advanced Heart Failure; Patients That Have Received a Left Ventricular Assist Device, Critical Limb Ischemia, Ornithine Transcarbamylase Deficiency Disease, Cystic Fibrosis, Chronic Myeloid Leukemia, Glioblastoma Multiforme; Anaplastic Astrocytoma, Osteoarthritis, Von Willebrand Disease, Beta Thalassemia Major, Congenital Anemias, HPV 16+, Cervical Intraepithelial Neoplasia (CIN 2/3), Duchenne Muscular Dystrophy, Peripheral Arterial Disease (PAD), Claudication, Familial Lipoprotein Lipase Deficiency, Sarcoma, Diabetic Neuropathy, Tay Sachs Disease, Bone Marrow Transplantation, Epstein-Barr Virus Infections, Atherosclerosis, Hypercholesterolemia, Fibromyalgia, Lymphohistiocytosis, Hemophagocytic, Common Variable Immunodeficiency, Lymphoproliferative Disorders, Aphthous Stomatitis; Burning Mouth Syndrome, Lichen Planus; Mouth Disease, Temporomandibular Joint Disorder, Fabry Disease, Multiple Sclerosis, Stem Cell Transplantation, Cytomegalovirus Infections, Systemic Sclerosis, Aplastic Anemia, Hemoglobinopathy, Vitiligo and Gastroesophageal Reflux. Some of these applications are described in more detail in U.S. Patents including: 7,592,320, 7,592,321, 7,582,602, 7,544,791, 7,521,043, 7,459,153, 7,410,799, 7,378,089, 7,361,639, 7,351,697, 7,217,571, 7,198,909, 7,175,840, 7,157,079, 7,115,258, 7,037,716, 7,022,319, 6,808,905 and 6,093,392, the entire contents of which are incorporated herein by reference. Some of these applications are reviewed, for example, in: Dzau VJ et al, Am J Cardio. 2003, 7;92(9B): 18N-23N; Ostenfeld T & Svendsen CN, Adv Tech Stand Neurosurg. 2003, 28:3-89; Korczyn AD & Nussbaum M, Drugs 2002; 62(5):775-86; Moullier P et al., Nephron. 1997;77(2): 139-51; Vanholder R et al., Artif Organs. 1995;19(11): 1120-5; Tani K & Asano S, Gan To Kagasu Ryoho. 1993;20(2):181- 8; Farrell PM & Sischler EH. Addv Pediatr 1992;39:35-70; Farre; PM et al., Pediatr Pulmonol Suppl. 1991;7:11-18; all of which are incorporated herein by reference.

Recombinant vectors (e.g., encoding an allergen or a therapeutic protein) may themselves be an antigen. The antigens are used in the methods described herein to induce immune tolerance. The induction of immune tolerance in one aspect is in the context of peptide therapy. In some embodiments of the invention, the subject has received, is receiving or will receive an exogenous antigen as a therapy, where the antigen may be immunogenic. As used herein, an "exogenous antigen administered as a therapy" refers to any known antigen that is deliberately introduced (e.g., administered) into the body of a patient for therapeutic purposes. Exogenous administration of an antigen may be systematic or local. In some embodiments, exogenous therapeutic antigens are isolated polypeptides. Isolated polypeptides that may be used as a therapy include isolated proteins (such as recombinant proteins), hormones, cytokines, and functionally equivalent derivatives thereof. Minor modifications of the primary amino acid sequences of polypeptide antigens may also result in a polypeptide which has altered antigenic activity as compared to the unmodified counterpart polypeptide. Such modifications may be deliberate, as by site-directed mutagenesis, or may be spontaneous. AU of the polypeptides produced by these modifications are included herein as long as immunogenicity still exists.

In some embodiments, the peptide therapy may be given to a subject who will benefit from increased in vivo availability of the particular peptide or derivative thereof, which may include proteins, cytokines and hormones. For example, in some cases, the subject has a deficiency in one or more peptides that are normally produced in the body. Examples include, but are not limited to, enzyme deficiencies and hormone deficiencies. In these subjects, it is desirable to supplement or replace these peptides in vivo.

In some circumstances, the deficiency is a result of a genetic disorder, such that a certain gene is lacking or there is impaired function with the corresponding gene product due to mutation(s). For example, certain blood clotting disorders and severe immunodeficiency disorders are known to be caused by one or more genetic mutations, and affected individuals may completely lack a functional protein, or the level of the protein or activity may be significantly reduced. In forms of hemophilia, for example, patients lack certain clotting factors that are necessary to control coagulation. Hemophilia A (clotting factor VIII deficiency) is the most common form of the disorder, occurring at about 1 in 5,000-10,000 male births. Hemophilia B (factor IX deficiency) occurs at about 1 in about 20,000-34,000 male births. Affected subjects may rely on exogenously administered factors to overcome the deficiency as treatment. However, adverse reactions to such therapy are relatively common. For example, the protein administered as therapy may be recognized as "foreign" by the immune system of the patient, and as a result renders factor replacement less effective or even harmful. In other circumstances, the deficiency is due to change in gene expression triggered, for instance, by aging, pregnancy, trauma, and medical condition and/or treatment. In these situations, the subject may benefit from exogenous therapy and increased immune tolerance to the therapy. Hormone replacement therapy is one such example.

There are a number of therapeutic peptides (e.g. enzymes, hormones, cytokines, etc.) that may be useful for the treatment of a variety of diseases and disorders, where the subject will benefit from increased tolerance to the therapeutic peptide. Non-limiting examples of such peptides include: Factor VIII, Factor IX, Von Willebrand Factor (VWF), Factor XI, Factor VII, T4 endonuclease V, erythropoiesis stimulating protein, insulin, Protein C, neuregulin 1, Laminin-111 , p53, Erythropoietin (EPO), alpha- 1 antitrypsin, Rod derived Cone Viability Factor (RdDVF), estrogen, estradiol, testosterone, Dehydroepiandrosterone, Phenylalanine hydroxylase, DNase I, Glucocerebrosidase, Growth hormone (GH), Alpha interferon, Gamma- 1 b interferon, Interleukin-2 (IL-2), Interleukin-3 (IL-3), Interleukin-4 (IL-4), Tissue plasminogen activator tPA), monoclonal antibodies (e.g., monoclonal antibodies to cancer antigens), adenosine deaminase, Nutropin (somatropin), Neupogen and Leukine (e.g., G-CSF, GM-CSF).

Diseases and disorders that may be treated with a peptide therapy, and where the subject will benefit from increased tolerance to the peptide, include, without limitation: hemophilia (A and B), Type I diabetes, DNA repair disorder, Xeroderma Prgmentosum (XP), Cockayne Syndrome (CS), Trichothiodystrophy (TTD), severe protein C deficiency, purpura fulminans (PF), disseminated intravascular coagulation (DIC), venous thromboembolism (VTE), chronic granulomatous disease, cystic fibrosis (CF), Gaucher's disease, Duchenne muscular dystrophy (DMD), Alpha- 1 antitrypsin chronic deficiency, multiple sclerosis (MS), retinitis pigmentosa (RP), blood clots, pulmonary embolism, myocardial infarction, stroke, hormone deficiency, phenylketonuria, anemia, chronic renal disease, lysossomal storage diseases (e.g., Fabry Disease), Nanism hypophyseal, leukemia, Kaposi's sarcoma, hepatitis B, hepatitis C, Chronic granulomatous disease, cancer, Acute myocardial infarction, massive pulmonary embolism, rheumatoid arthritis, SLE, multiple sclerosis, inflammatory bowel disease and severe combined immune deficiency (SCID).

Peptide therapy is extremely costly, particularly for chronic conditions and genetic disorders, which may require a long-term treatment regimen. As an example, enzyme replacement therapy for adenosine deaminase deficiency (ADA), which typically involves the use of bovine ADA enzyme costs an estimated $50,000-500,000 per year. It is therefore helpful to minimize unwanted immune responses that destroy or interfere with the activity of therapeutic peptides, thereby diminishing the effectiveness of the treatment.

Tolerance induction may also be useful for preventing immune reactions to the newly expressed genes in gene therapy protocols, and such methods are specifically contemplated by this invention.

In yet other embodiments, peptide therapy is part of immune therapy treatment for a disease, such as cancer. While some therapeutic antibodies are humanized to reduce immunogenicity, they can still trigger an immune response, which causes subsequent inhibition of the therapy in some subjects. It is therefore desirable to induce immune tolerance in the subject who is receiving or will receive such treatment. For example, for humanized therapeutic antibodies, only a small fraction of the immunoglobulin within the variable domain is derived from a non-human species, typically of a murine origin. Therefore, it is possible to define or at least limit possible sites of immunogenicity and use the fragment as an antigen to induce tolerance, in the presence of an adjuvant and a tolerogenic agent. Non-limiting examples of immunotherapy include the following: Abciximab, Adalimumab, Alemtuzumab, Basiliximab, Bevacizumab, Cetuximab, Certolizumab pegol, Daclizumab, Eculizumab, Efalizumab, Gemtuzumab, Ibritumomab tiuxetan, Infliximab, Muromonab-CD3, Natalizumab, Omalizumab, Palivizumab, Panitumumab, Ranibizumab, Rituximab, Tositumomab and Trastuzumab.

In some embodiments, the subject has been previously sensitized to the antigen. In other embodiments, the subject is a naive subject, e.g., the subject has not been previously sensitized to the antigen. In some embodiments, the subject may have been undergoing treatment with the antigen for at least 1, 2, 3, 4, 5, 6, 7 days or more. In some embodiments, the subject may have been undergoing treatment with the antigen for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more. In some embodiments, the subject may have been undergoing treatment with the antigen for at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more.

Using no more than routine experimentation, it may be determined whether and to what extent the subject is eliciting an immune response to the antigen by measuring antigen-specific responses (e.g., antigen specific immunoglobulin levels, including IgG, IgE, etc. levels and/or antigen specific lymphocyte levels) in blood samples collected from the subject or by symptomatic measures. It also may be determined whether and to what extent a subject is exhibiting an allergic response to an antigen, as described above. Allergic symptomatic responses include sneezing, rashes, difficulty in breathing, watery eyes, itching, neutrophil accumulation in the lungs, etc. Immune response measures may be made in subjects naive to the antigen or previously sensitized to the antigen. Such measures may be made in subjects prior to and/or after the treatments according to the invention. Antigen-specific immune responses may be periodically monitored following tolerance induction using the methods described herein. Tolerance induction can be confirmed when the immune response is diminished in an antigen specific manner. Tolerance induction may be confirmed by showing a decrease in antigen specific antibody levels or antigen specific lymphocyte levels to administered antigen in a sensitized individual treated according to the invention, where the individual has pre-existing antibody or lymphocyte levels to the antigen. Where an individual is naive to the antigen prior to treatment according to the invention, tolerance induction may be confirmed by showing antigen specific antibody levels or antigen specific lymphocyte levels after treatment that are consistent with control levels expected in a subject with immune tolerance. Control levels can be established using methods well known to those of ordinary skill in the art. For example, T cells proliferation may be measured in response to the antigen in question. Immunoglobulin levels may be also measured to assess an immune response.

Antigens which are allergens can trigger an allergic response in a subject. An "allergy" refers to acquired hypersensitivity to a substance (allergen). Allergic conditions include but are not limited to eczema, allergic rhinitis or coryza, conjunctivitis, hay fever, bronchial asthma, urticaria (hives) and food allergies, and other atopic conditions. In some embodiments, the subject has an allergy, i.e., has had an allergic reaction in response to an allergen. Currently, allergic diseases are generally treated either symptomatically with antihistamines for example or immunotherapeutically by the injection of small doses of antigen followed by subsequent increasing dosage of antigen. Symptomatic treatment offers only temporary relief. Immunotherapy is believed to induce tolerance to the allergen to prevent further allergic reactions. This approach, however, can take several years to be effective and is associated with the risk of side effects such as anaphylactic response. The methods of the invention described herein may improve the efficacy of immunotherapy.

A number of allergens are known. Common sources of allergens include various plants (e.g., pollen), dust, mite, foods, animal particles (e.g., hair, dander, epithelia, feather, etc.), insects, venoms, fungi (e.g., fungal spores), drugs (e.g., penicillin) and other environmental particles and substances.

Non-limiting examples of allergens derived from mites include: House mite dust from Dermatophagoides farinae; House mite dust from Dermatophagoides pteronyssinus; Food/Storage mite Acarus siro; House mite dust Blomia tropicalis; Storage mite Chortoglyphus arcuatas; House mite dust from Euroglyphus maynei; Food/Storage mite Lepidoglyphus destructor; Food/Storage mite Tyrophagus putrescentiae; House mite dust from Glycyphagus domesticus.

Non-limiting examples of allergens derived from venoms include those of: European Hornet Vespa crabro; Honey Bee Apis mellifera; Mixed Hornet Dolichovespula spp. ; Mixed Paper Wasp Polistes spp.; Mixed Yellow Jacket Vespula spp.; White (bald)-faced Hornet Dolichovespula maculate; Yellow Hornet Dolichovespula arenaria.

Non-limiting examples of insect allergens include those derived from:

Carpenter ant Camponotus pennsylvanicus; Fire ant Solenopsis invicta; Fire ant Solenopsis richteri; American Cockroach Periplaneta Americana; German Cockroach Blattella germanica; Oriental Cockroach Blatta oήentalis; Horse Fly Tabanus spp.; House Fly Musca domestica; Mayfly Ephemeroptera spp.; Mosquito Culicidae sp.; Moth Heterocera spp.

Non-limiting examples of allergens animal particles (e.g., epithelia, dander, hair, feathers, etc.) include those derived from: Canary Feathers Serinus canaria; Cat Epithelia Felis catus (domesticus); Cattle Epithelia Bos Taurus; Chicken Feathers Gallus gallus (domesticus); Dog Epithelia, Mixed Breeds Canisfamiliaris; Duck Feathers Anas platyrhynchos; Gerbil Epithelia Meriones unguiculatus; Goat Epithelia Capra hircus; Goose Feathers Anser domesticus; Guinea Pig Epithelia Cavia porcellus (cobaya); Hamster Epithelia Mesocricetus auratus; Hog Epithelia Sus scrofa; Horse Epithelia Equus caballus; Mouse Epithelia Mus musculus; Parakeet Feathers Psittacidae; Pigeon Feathers Columbafasciata; Rabbit Epithelia Oryctolagus cuniculus; Rat Epithelia Rattus norvegicus; Wool, Sheep Ovis aries; Cat Dander/Antigen Felis catus (domesticus); Dog Dander, Mixed-Breed Canisfamiliaris; Poodle Dander Canisfamiliaris.

Non-limiting examples of fungal allergens include those derived from: Acremonium strictum Cephalosporium acremonium; Alternaria alternata Alternaria tenuis; Aspergillus amstelodami Aspergillus glaucus; Aspergillus flavus; Aspergillus fumigatus; Aspergillus nidulans; Aspergillus niger; Aspergillus terreus; Aspergillus versicolor; Aureobasidium pullulans Pullularia pullulans; Bipolaris sorokiniana Drechslera sorokiniana, Helminthosporium sativum; Botrytis cinerea; Candida albicans; Chaetomium globosum; Cladosporium herbarum; Cladosporium sphaerospermum Hormodendrum hordei; Drechslera spicifera Curvularia spicifera; Epicoccum nigrum Epicoccum purpurascens; Epidermophyton floccosum; Fusarium moniliforme; Fusarium solani; Geotrichum candidum Oospora lactis; Gliocladium viride Gliocladium deliquescens; Helminthosporium solani Spondylocladium atrovirens; Microsporum canis Microsporum lanosum; Mucor circinelloides f. circinelloides Mucor mucedo; Mucor circinelloides f. lusitanicus Mucor racemosus; Mucor plumbeus; Mycogone perniciosa; Neurospora intermedia Neurospora sitophila, Monilia sitophila; Nigrospora oryzae; Paecilomyces variotii; Penicillium brevi-compactum; Penicilliutn camembertii; Penicillium chrysogenum; Penicillium digitatum; Penicillium expansum; Penicillium notatum; Penicillium roquefortii; Phoma betae; Phoma herbarum Phoma pigmentivora; Rhizopus oryzae Rhizopus arrhizus; Rhizopus stolonifer Rhizopus nigricans; Rhodotorula mucilaginosa Rhodotorula rubra var. mucilaginosa; Saccharomyces cerevisiae; Scopulariopsis brevicaulis; Seφula lacrymans Merulius lacrymans; Setosphaeria rostrata Exserohilum rostratum, Helminthosporium hatodes; Stemphylium botryosum; Stemphylium solani; Trichoderma harzianum Trichoderma viride; Trichophyton mentagrophytes Trichophyton interdigitale; Trichophyton rubrum; and Trichothecium roseum Cephalothecium roseum.

Non-limiting examples of smuts allergens include those derived from: Barley Smut Ustilago nuda; Bermuda Grass Smut Ustilago cynodontis; Corn Smut Ustilago maydis; Johnson Grass Smut Sporisorium cruentum; Oat Smut Ustilago avenae; and Wheat Smut Ustilago tritici.

Non-limiting examples of pollen and other plant-originated allergens include those derived from: Bahia Paspalum notatum; Bermuda Cynodon dactylon; Blue, Canada Poa compressa; Brome, Smooth Bromus inermis; Canary Phalaris arundinacea; Corn Zea mays; Couch/Quack Elytrigia repens (Agropyron repens); Johnson Sorghum halepense; Kentucky Blue Poa pratensis; Meadow Fescue Festuca pratensis (elatior); Oat, Cultivated Avena sativa; Orchard Dactylis glomerata; Red Top Agrostis gigantea (alba); Rye, Cultivated Secale cereale; Rye, Giant Wild Leymus (Elymus) condensatus; Rye, Italian Lolium perenne ssp. multiflorum; Rye, Perennial Lolium perenne; Sweet Vernal Anthoxanthum odoratum; Timothy Phleum pretense; Velvet Holcus lanatus; Wheat, Cultivated Triticum aestivum; Wheatgrass, Western Elymus (Agropyron) smithii; Allscale Atriplex polycarpa; Baccharis Baccharis halimifolia; Baccharis Baccharis sarothroides; Burrobrush Hymenocleq salsola; Careless Weed Amaranthus hybridus; Cocklebur Xanthium strumarium (commune); Dock, Yellow Rumex crispus; Dog Fennel Eupatorium capillifolium; Goldenrod Solidago spp.; Hemp, Western Water Amaranthus tuberculatus (Acnida tamariscina); Iodine Bush Allenrolfea occidentalis; Jerusalem Oak Chenopodium botrys; Kochia/Firebush Kochia scoparia; Lambs Quarter Chenopodium album; Marsh Elder, Burweed Iva xanthifolia; Marsh Elder, Narrowleaf Iva angustifolia; Marsh Elder, Rough Iva annua (ciliata); Mexican Tea Chenopodium ambrosioides; Mugwort, Common Artemisia vulgaris; Mugwort, Darkleaved Artemisia ludoviciana; Nettle Urtica dioica; Palmer's Amaranth A maranthus palmer i; Pigweed, Redroot/Rough Amaranthus retroflexus; Pigweed, Spiny Amaranthus spinosus; Plantain, English Plantago lanceolata; Poverty Weed Iva axillaris; Quailbrush Atriplex lentiformis; Rabbit Bush Ambrosia deltoidea; Ragweed, Desert Ambrosia dumosa; Ragweed, False Ambrosia acanthicarpa; Ragweed, Giant Ambrosia trifida; Ragweed, Short Ambrosia artemisiifolia; Ragweed, Slender Ambrosia confertiflora; Ragweed, Southern Ambrosia bidentata; Ragweed,Western Ambrosia psilostachya; Russian Thistle Salsola kali (pestifer); Sage, Coastal Artemisia californica; Sage, Pasture Artemisia frigida; Sagebrush, Common Artemisia tridentate; Saltbush, Annual Atriplex wrightii; Shadscale Atriplex confertifolia; Sorrel, Red/Sheep Rumex acetosella; Wingscale Atriplex canescens; Wormwood, Annual Artemisia annua; Acacia Acacia spp.; Alder, European Alnus glutinosa; Alder, Red Alnus rubra; Alder, Tag Alnus incana ssp. rugosa; Alder, White Alnus rhombifolia; Ash, Arizona Fraxinus velutina; Ash, Green/Red Fraxinus pennsylvanica; Ash, Oregon Fraxinus latifolia; Ash, White Fraxinus Americana; Aspen Populus tremuloides; Bayberry Myrica cerifera; Beech, American Fagus grandifolia (americana); Beerwood/Australian Pine Casuarina equisetifolia; Birch, Black/Sweet Betula lenta; Birch, European White Betula pendula; Birch, Red/River Betula nigra; Birch, Spring Betula occidentalis (fontinalis); Birch, White Betula populifolia; Box Elder Acer negundo; Cedar, Japanese Cryptomeria japonica; Cedar, Mountain Juniperus ashei (sabinoides); Cedar, Red Juniperus virginiana; Cedar, Salt Tamarix gallica; Cottonwood, Black Populus balsamifera ssp. trichocarpa; Cottonwood, Eastern Populus deltoids; Cottonwood, Fremont Populus fremontii; Cottonwood, Rio Grande Populus wislizeni; Cottonwood, Western Populus monilifera (sargentii); Cypress, Arizona Cupressus arizonica; Cypress, Bald Taxodium distichum; Cypress, Italian Cupressus sempervirens; Elm, American Ulmus Americana; Elm, Cedar Ulmus crassifolia; Elm, Siberian Ulmus pumila; Eucalyptus Eucalyptus globulus; Hackberry Celtis occidentalis; Hazelnut Corylus Americana; Hazelnut, European Corylus avellana; Hickory, Pignut Carya glabra; Hickory, Shagbark Carya ovata; Hickory, Shellbark Carya laciniosa; Hickory, White Carya alba; Juniper, Oneseed Juniperus monosperma; Juniper, Pinchot Juniperus pinchotii; Juniper, Rocky Mountain Juniperus scopulorum; Juniper, Utah Juniperus osteosperma; Juniper, Western Juniperus occidentalis; Locust Blossom, Black Robinia pseudoacacia; Mango Blossom Mangifera indica; Maple, Coast Acer macrophyllum; Maple, Red Acer rubrum; Maple, Silver Acer saccharinum; Maple, Sugar Acer saccharum; Melaleuca Melaleuca quinquenervia (leucadendron); Mesquite Prosopis glandulosa (juliflora); Mulberry, Paper Broussonetia papyrifera; Mulberry, Red Morus rubra; Mulberry, White Morus alba; Oak, Arizona/Gambel Quercus gambelii; Oak, Black Quercus velutina; Oak, Bur Quercus macrocarpa; Oak, California Black Quercus kelloggii; Oak, California Live Quercus agrifolia; Oak, California White/Valley Quercus lobata; Oak, English Quercus robur; Oak, Holly Quercus ilex; Oak, Post Quercus stellata; Oak, Red Quercus rubra; Oak, Scrub Quercus dumosa; Oak, Virginia Live Quercus virginiana; Oak, Water Quercus nigra; Oak, Western White/Garry Quercus garryana; Oak, White Quercus alba; Olive Olea europaea; Olive, Russian Elaeagnus angustifolia; Orange Pollen Citrus sinensis; Palm, Queen Arecastrum romanzofflanum (Cocos plumosa); Pecan Carya illinoensis; Pepper Tree Schinus molle; Pepper Tree/Florida Holly Schinus terebinthifolius; Pine, Loblolly Pinus taeda; Pine, Eastern White Pinus strobus; Pine, Longleaf Pinus palustris; Pine, Ponderosa Pinus ponderosa; Pine, Slash Pinus elliottii; Pine, Virginia Pinus virginiana; Pine, Western White Pinus monticola; Pine, Yellow Pinus echinata; Poplar, Lombardy Populus nigra; Poplar, White Populus alba; Privet Ligustrum vulgare; Sweet Gum Liquidambar styraciflua; Sycamore, Eastern Platanus occidentalis; Sycamore, Oriental Platanus oήentalis; Sycamore, Western Platanus racemosa; Sycamore/London Plane Platanus acerifolia; Walnut, Black Juglans nigra; Walnut, California Black Juglans californica; Walnut, English Juglans regia; Willow, Arroyo Salix lasiolepis; Willow, Black Salix nigra; Willow, Pussy Salix discolor; Daisy, Ox-Eye Chrysanthemum leucanthemum; Dandelion Taraxacum officinale; Sunflower Helianthus annuus; Alfalfa Medicago sativa; Castor Bean Ricinus communis; Clover, Red Trifolium pretense; Mustard Brassica spp.; and Sugar Beet Beta vulgaris.

Non-limiting examples of food allergens include those derived from: Almond Prunus dulcis; Apple Malus pumila; Apricot Prunus armeniaca; Banana Musa paradisiaca (sapientum); Barley Hordeum vulgare; Bean, Lima Phaseolus lunatus; Bean, Navy Phaseolus vulgaris; Bean, Pinto Phaseolus sp.; Bean, Red Kidney Phaseolus sp.; Bean, String/Green Phaseolus vulgaris; Blackberry Rubus allegheniensis; Blueberry Vaccinium sp.; Broccoli Brassica oleracea var. botrytis; Buckwheat Fagopyrum esculentum; Cabbage Brassica oleracea var. capitata; Cacao Bean Theobroma cacao; Cantaloupe Cucumis melo; Carrot Daucus carota; Cauliflower Brassica oleracea var. botrytis; Celery Apium graveolens var. dulce; Cherry Prunus sp.; Cinnamon Cinnamomum verum; Coffee Coffea Arabica; Corn Zea mays; Cranberry Vaccinium macrocarpon; Cucumber Cucumis sativus; Garlic Allium sativum; Ginger Zingiber officinale; Grape Vitis sp.; Grapefruit Citrus paradise; Hops Humulus lupulus; Lemon Citrus limon; Lettuce Lactuca sativa; Malt; Mushroom Agaricus campestris; Mustard Brassica sp.; Nutmeg Myristicafragrans; Q&tAvena sativa; Olive, Green Olea europaea; Onion Allium cepa var. cepa; Orange Citrus sinensis; Pea, Blackeye Vigna unguiculata; Pea, Green (English) Pisum sativum; Peach Prunus persica; Pear Pyrus communis; Pepper, Black Piper nigrum; Pepper, Green Capsicum annuum var. annuum; Pineapple Ananas comosus; Potato, Sweet Ipomoea batatas; Potato, White Solanum tuberosum; Raspberry Rubus idaeus var. idaeus; Rice Oryza sativa; Rye Secale cereale; Sesame Seed Sesamum orientate (indicum); Soybean Glycine max; Spinach Spinacia oleracea; Squash, Yellow Cucurbita pepo var. melopepo; Strawberry Fragaria chiloensis; Tomato Lycopersicon esculentum (ϊycopersicum); Turnip Brassica rapa var. rapa; Vanilla Bean Vanilla planifolia; Watermelon Citrullus lanatus var. lanatus; Wheat, Whole Triticum aestivum; Bass, Black Micropterus sp.; Catfish Ictalurus punctatus; Clam Mercenaria mercenaria; Codfish Gadus morhua; Crab Callinectes sapidus; Flounder Platichthys sp.; Halibut Hippoglossus sp.; Lobster Homarus americanus; Mackerel Scomber scombrus; Oyster Crassostrea virginica; Perch Sebastes marinus; Salmon Salmo salar; Sardine Clupeiformes; Scallop Pecten magellanicus; Shrimp Penaeus sp.; Trout, Lake Salvelinus sp.; Tuna Fish Thunnus sp.; Beef Bos Taurus; Lamb Ovis aries; Pork Sus scrofa; Chicken Gallus gallus; Egg, Chicken, White Gallus gallus; Egg, Chicken, Yolk Gallus gallus; Turkey Meleagris gallopavo; Casein, bovine Bos Taurus; Milk, bovine Bos Taurus; Brazil Nut Bertholletia excelsa; Cashew Nut Anacardium occidentale; Coconut Cocos nucifera; Filbert/Hazelnut Corylus Americana; Peanut Arachis hypogaea; Pecan Carya illinoensis; Walnut, Black Juglans nigra; and Walnut, English Juglans regia.

Non-limiting examples of miscellaneous allergens (e.g., inhalants and environmental products) include those derived from: Cottonseed Gossypium hirsutum; Flaxseed Linum usitatissimum; Gum, Acacia/ Arabic Acacia Senegal; Gum, Karaya Sterculia urens; Gum, Tragacanth Astragalus gummifer; Kapok Seed Ceiba pentandra; Orris Root Iris germanica var. florentina; Pyrethrum Chrysanthemum cinerariifolium; Silkworm (Cocoon and Pupa) Bombyx mori; Tobacco Leaf Nicotiana tabacum; Barley Grain Dust; Corn Grain Dust; House Dust; Mattress Dust; Oat Grain Dust; Wheat Grain Dust; and Upholstery Dust.

According to the invention, the method of immune tolerance induction involves administration of an isolated antigen, such as those listed above, and an adjuvant, in conjunction with a tolerogenic agent to induce antigen-specific tolerance in the subject. A variety of adjuvants is known in the art and may be used in this invention.

Adjuvants, when used together with an antigen, can stimulate the humoral and/or cellular immune response. Non-nucleic acid adjuvants include, for instance, adjuvants that create a depo effect, adjuvants that are immune stimulating adjuvants, and adjuvants that both create a depo effect and stimulate the immune system. Adjuvants also include Toll Like Receptor agonists, which include immunostimulatory nucleic acid adjuvants, such as oligonucleotides containing at least one unmethylated CpG dinucleotide motif. Toll Like Receptor agonists also include well known classes of small organic molecules. So of these are described in, for example, U.S. Patents 6,194,388, 6,207,646, 6,284,806, 6,218,371, 6,239, 116, 6,339,068, 6,429,199, 6,653,292, 6,821,957, 6,943,240, 6,949,520, 7,271,156, 7,402,572, 7,569,553, 7,585,847, 7,605,138, 7,427,629, 7,387, 271, the contents of which are incorporated by reference.

An adjuvant that creates a depo effect is an adjuvant that causes the antigen to be slowly released in the body, thus prolonging the exposure of immune cells to the antigen. This class of adjuvants includes but is not limited to: alum (e.g., aluminum hydroxide, aluminum phosphate); emulsion-based formulations including mineral oil, non-mineral oil, water-in-oil or oil-in- water-in oil emulsion, oil-in- water emulsions such as Seppic ISA series of Montanide adjuvants (e.g., Montanide ISA 720, AirLiquide, Paris, France); MF-59 (a squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron Corporation, Emeryville, Calif.; and PROVAX (an oil-in-water emulsion containing a stabilizing detergent and a micelle-forming agent; IDEC, Pharmaceuticals Corporation, San Diego, Calif.).

An immune stimulating adjuvant is an adjuvant that causes activation of a cell of the immune system. It may, for instance, cause an immune cell to produce and secrete cytokines. This class of adjuvants includes but is not limited to saponins purified from the bark of the Q. saponaria tree, such as QS21 (a glycolipid that elutes in the 21.sup.st peak with HPLC fractionation; Aquila Biopharmaceuticals, Inc., Worcester, Mass.); poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research Institute, USA); derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi ImmunoChem Research, Inc., Hamilton, Mont.), muramyl dipeptide (MDP; Ribi) andthreonylmuramyl dipeptide (t-MDP; Ribi); OM- 174 (a glucosamine disaccharide related to lipid A; OM Pharma SA, Meyrin, Switzerland); and Leishmania elongation factor (a purified Leishmania protein; Corixa Corporation, Seattle, Wash.).

Adjuvants that create a depo effect and stimulate the immune system are those compounds which have both of the above-identified functions. This class of adjuvants includes but is not limited to alum, ISCOMS (Immunostimulating complexes which contain mixed saponins, lipids and form virus-sized particles with pores that can hold antigen; CSL, Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2 which is an oil-in- water emulsion containing MPL and QS21 : SmithKline Beecham Biologicals [SBB], Rixensart, Belgium); SB-AS4 (SmithKline Beecham adjuvant system #4 which contains alum and MPL; SBB, Belgium); non-ionic block copolymers that form micelles such as CRL 1005 (these contain a linear chain of hydrophobic polyoxpropylene flanked by chains of polyoxyethylene; Vaxcel, Inc., Norcross, Ga.); and Syntex Adjuvant Formulation (SAF, an oil- in-water emulsion containing Tween 80 and a nonionic block copolymer; Syntex Chemicals, Inc., Boulder, Colo.).

Preferred adjuvants useful for the methods described herein include alum.

An essential component of the tolerance inducing methods described herein is a tolerogenic agent. A tolerogenic agent, or a tolerizing agent, is an agent that induces or promotes immune tolerance. Many tolerogenic agents target molecules that are involved in T cell activation and/or interfere with the interaction between a T cell and an antigen presenting cell (APC), such as dendritic cells (DCs). One of ordinary skill in the art is familiar with the molecules involved in these processes. As part of peripheral T cell tolerance, it has been postulated that the same mechanisms by which mature T cells that recognize self antigens in peripheral tissues become incapable of subsequently responding to these antigens may regulate unresponsiveness to tolerogenic forms of foreign antigens by anergy, deletion or suppression of T cells. It has been suggested that exposure of CD4+ T cells to an antigen in the absence of costimulation or innate immunity may make the cells incapable of responding to that antigen. This is consistent with the notion that full activation of T cells requires the recognition of antigen by the TCR (signal one) as well as recognition of costimulators (signal two). It was previously reported that prolonged antigen recognition alone, in the absence of the second signal, may lead to anergy (i.e., functional unresponsiveness). As shown in the Examples, the data presented here indicate that T lymphocytes that recognize foreign antigens without costimulation, but in the presence of an adjuvant, may cause the T cells to die (deletion) by activation-induced cell death (AICD).

A number of molecules that are involved in T cell activation have been identified and can be targets of tolerizing inducing agents. These are predominantly cell-surface molecules that are expressed on T cells or APCs. The T cell activation molecules include but are not limited to the following: CD4, CD40, CD40L, OX40, OX40L, CD26, CD44, CD28 /PDl/ BTLA / B7-1 /ICOS/ CTLA / B7-2family, CD99, CD137 (4-1BBL), CD2, LFA3, CD27, CD70, CD3, CD8, ICAMl, LFAl, MHC class II and MHC class I molecules.

Thus, one or more of these molecules may be targeted and used as a tolerizing agent. A tolerizing agent therefore will bind to and block one or more molecules involved in T cell activation, such as those listed above. In some embodiments, a tolerizing agent is a monoclonal antibody that binds to and blocks the action of one or more molecules listed above. For example, a non-depleting monoclonal antibody that binds CD4 can be used as a tolerizing agent. In some embodiments, a tolerizing agent is a molecule that is a fusion protein derived from a protein that binds to one or more molecules listed above. For example, CTLA4-Ig (ORENCIA®; abatacept) is a B7-specific fusion protein known to block T cell co-stimulation. Molecules that may be used as tolerizing agents include, but are not limited to: anti-CD4, anti- CD3, anti-CD25, anti-CD28, anti-PDl, anti-BTLA, anti-B7 (anti-B7-l and anti-B7-2), anti- ICOS, anti-CTLA, anti-CD40, anti-CD40L, anti-CD99, anti-CD2, anti-LFA3, anti-CD27, anti- CD70, anti-DC8, anti-OX40, anti-OX40L, anti-LFA-1, anti-CDlla, anti-ICAMl, anti-CD26, anti-CD44, anti-CD137 (anti-4- IBBL), CTLA4-Ig, anti-MHC class II and anti-MHC class I. Of course, one of ordinary skill in the art will recognize that these agents include whole immunoglobulin molecules, and antigen-binding fragments thereof, as well as other molecules that bind to the antigen to the extent that they block T cell activation. Using the method described herein, it is possible to induce a state of tolerance that prevents the generation of antigen-specific immunoglobulins on subsequent immunizations by administering to a subject an antigen of interest, adjuvants and a tolerogenic agent. The subject remains immunocompetent to mount protective immune responses against third-party antigens. Accordingly, the present disclosure presents a strategy for tolerance induction applicable to immunogenic proteins including allergens, coagulation factors in hemophilia, and other proteins (including protein-based biological drugs).

In some embodiments, an antigen of interest is combined with aluminum hydroxide (alum) as adjuvant and CTLA4-Ig as tolerance inducing agent to induce antigen-specific tolerance in a subject.

In some embodiments, the subject suffers from a coagulation disorder such as hemophilia A and hemophilia B.

In some embodiments, the subject has a lysosomal storage-associated disease, such asGaucher disease and Fabry disease and in need of replacing or supplementing an essential enzyme (e.g., Glucocerebrosidase).

The antigen and the adjuvant may be administered in any conventional manner for inducing an immune response. Most typically, the antigen and the adjuvant will be administered at about the same time in the same location, such that the effects of the adjuvant are present when the immune system 'sees' the antigen. .

The antigen and the adjuvant may be administered together as a single formulation and administered subcutaneously, intradermally, intramuscularly or intravenously. More typically, the antigen and the adjuvant are administered together as a single formulation and administered subcutaneously, intradermally. For Alum, for example, typically 0.5-5 mg of the adjuvant is used by mixing with 5-100 μg of isolated antigen. Typical antigen-to-alum ratios used in animal experimental models range from about 1 :50 to about 1 :500 by weight. In some embodiments, an antigen-to-adjuvant ratio suitable for the methods described herein may be about 1:10, -1:20, -1:50, -1 :100, -1:200, -1:300, -1 :400, -1:500, -1:750, -1:1000, -1 :2000, ~1 :3000, ~1 :4000, -1 :5000, etc. Of course, depending on the particular antigen and/or the adjuvant, and their formulation, preparation, purity, as well as the recipient, the manner of administration etc., the amount should be optimized using routine measures.

A tolerogenic agent is administered to the subject in conjunction with the antigen and the adjuvant. "In conjunction with" means that the biological effects of these components are spatially and temporally overlapping in vivo. For example, the tolerogenic agent may be administered to the subject at the same time as or somewhat before and/or somewhat after the administration of the antigen/adjuvant. Typically, the tolerogenic agent is administered within 36 hours of the administration of the antigen/adjuvant, and in particular at the same or approximately the same time as the antigen/adjuvant, such as within 1 hour, within 2 hours within 4 hours, within 8 hours, within 12 hours, or within 1 day of one another. It should be recognized, however, that the tolerogenic agent and antigen/adjuvant administrations may be further apart, such as, for example, within 2 days, within 3 days, within 4 days, within 7 days, or within 14 days of one another, so long as the biological effects of these components are spatially and temporally overlapping in vivo. Most typically, the tolerogenic agent will be administered intraperitonialy, intradermaly, intramuscularly or intravenously. In some embodiments, the tolerogenic agent may be formulated in the same preparation with the antigen/adjuvant, or in separate formulation.

The amount of a treatment may be varied for example by increasing or decreasing the amount of one or more of the components, or pharmacological agent or a therapeutic composition, by changing the therapeutic composition administered, by changing the route of administration, by changing the dosage timing and so on. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration, and like factors are within the knowledge and expertise of the health practitioner. For example, an effective amount can depend upon the severity of the peptide deficiency that the subject is suffering from.

An effective amount is in a broad sense a dosage of the therapeutic agent sufficient to provide a medically desirable result. An effective amount may also, for example, depend upon the degree of hypersensitivity to a particular allergen, or the degree of immunogenicity of a peptide administered as a therapy. It should be understood that the methods described herein are used, for example, to treat or prevent complications (e.g., allergy or unwanted immune responses to an antigen that is wanted) in a subject who is likely to benefit from increased tolerance to the particular antigen. Thus, for example, an effective amount is that amount which can lower the risk of, slow or perhaps prevent altogether the development of an immune response to the antigen of interest.

The factors involved in determining an effective amount are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. In some embodiments, a maximum dose of the pharmacological agents of the invention (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reason(s).

The therapeutically effective amount of a pharmacological agent of the invention is that amount effective to increase the level of immune tolerance and/or decrease the level of immune responses to the antigen of interest in a subject. For example, the desired response may include suppressing allergic responses, and unresponsiveness to the therapeutic antigen with respect to immunity. This can be monitored by routine diagnostic methods known to those of ordinary skill in the art. The desired response to treatment for allergy may be a reduction in the level of IgE or alleviation of clinical symptoms. The desired response for a patient on a peptide therapy may be a reduced level of serum antigen-specific Ig and increased efficacy of the peptide therapy.

The pharmacological agents used in the methods of the invention are preferably sterile and contain an effective amount of an antigen, an adjuvant and a tolerogenic agent for producing the desired response in a unit of weight or volume suitable for administration to a subject. The doses of pharmacological agents administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. The dosage of a pharmacological agent may be adjusted by the individual physician or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 500 mg/kg, and most preferably from about 0.2 mg/kg to about 250 mg/kg, in one or more dose administrations daily, for one or more days.

Various modes of administration are known to those of ordinary skill in the art which effectively deliver the pharmacological agents of the invention to a desired tissue, cell, or bodily fluid. The administration methods are discussed elsewhere in the application. The invention is not limited by the particular modes of administration disclosed herein. Standard references in the art (e.g., Remingtons' Pharmaceutical Sciences, 20th Edition, Lippincott, Williams and Wilkins, Baltimore MD, 2001) provide modes of administration and formulations for delivery of various pharmaceutical preparations and formulations in pharmaceutical carriers. Other protocols which are useful for the administration of pharmacological agents of the invention will be known to one of ordinary skill in the art, in which the dose amount, schedule of administration, sites of administration, mode of administration and the like vary from those presented herein.

When administered, the pharmaceutical preparations used for the methods described in the invention are applied in pharmaceutically-acceptable amounts and in phaπnaceutically- acceptable compositions. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

A pharmacological agent or composition may be combined, if desired, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the pharmacological agents of the invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents, including: acetate, phosphate, citrate, glycine, borate, carbonate, bicarbonate, hydroxide (and other bases) and pharmaceutically acceptable salts of the foregoing compounds. The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride, chlorobutanol, parabens and thimerosal.

The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. AU methods include the step of bringing the active agent into association with a carrier, which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product. For example, one composition may comprise an antigen and an adjuvant, and a second composition may comprise a tolerogenic agent. Alternatively, each of the three components may be formulated into a separate preparation until use.

The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi- dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle (e.g., saline, buffer, or sterile pyrogen-free water) before use. The pharmaceutical composition or compositions comprising an antigen, adjuvant and a tolerogenic agent may be administered orally, sublingualis buccally, intranasally, intravenously, intramuscularly, intrathecally, intraperitoneally, or subcutaneously. In some embodiments, the pharmaceutical composition comprising an antigen and an adjuvant is administered to a subject by injection. Typically, the antigen/adjuvant composition is delivered intramuscularly or subcutaneously. For example, the antigen/adjuvant composition is formulated in a pharmaceutically acceptable formulation and may contain about 0.1 to 1,000 mg of a purified antigen in association with a suitable adjuvant described elsewhere herein. More typically, the antigen/adjuvant composition contains about 5 to 500 mg of the purified antigen. The amount will depend on a number of factors as described above. In some embodiments, the antigen/adjuvant composition may be administered to the subject via an intramuscular or subcutaneous injection. Generally, antigens delivered subcutaneously are released into the body more slowly that those delivered intramuscularly. In some cases, the subject may receive a single injection of the antigen/adjuvant composition. In some cases, the subject may receive multiple injections with an interval ranging from one day to several weeks.

For each of the antigen/adjuvant administration, that the subject may also receive a composition comprising a tolerogenic agent, such as those described above, so as to induce antigen-specific tolerance. In some embodiments, the tolerizing agent-containing composition is formulated in a pharmaceutically acceptable formulation suitable for an injection. In some embodiments, the composition contains about 1 to 1,000 mg of the tolerizing agent. More typically, the subject receives about 25 to 600 mg of the tolerizing agent. As an example, for Muromonab-CD3 (Orthoclone OKT3®), which is an anti-CD3 monoclonal antibody that is commonly used to suppress or prevent transplant rejection, patients typically receive an intravenous injection of Muromonab-CD3 at 5 mg per dose for adults, and at 0.1 mg/kg body weight for pediatric patients. Similar doses can be used for the methods described herein. As another example, CTLA4-Ig, which is a fusion protein that blocks CTLA-4 interaction with B7, may be used as a tolerizing agent at a dose of about 0.5 to about 5 mg/kg/day. More typically, however, a 1-2 mg/kg/day range is used.

The composition comprising a tolerizing agent may be administered to the subject via injection, typically via intravenous injection. In some embodiments, the subject receives a single injection of the composition comprising a tolerizing agent. In some embodiments, the subject receives multiple injections, In some embodiments, the subject is administered with the tolerizing agent slightly before and/or after the subject receives the administration of an antigen/adjuvant. For example, in some cases, the subject receives an antigen/adjuvant injection on day 1, and the subject may receive a tolerizing agent injection on day 2, day 3, day 4, day 5, day 6, day 7, and so on. The tolerizing agent is likely effective provided that the antigen/adjuvant remains available in vivo. However, it is preferable that the composition comprising the antigen/adjuvant and the composition comprising the tolerogenic agent are administered to the subject no more than 2 days apart. Generally, the two compositions are administered back to back (e.g., sequentially) on the same day, or, the two composition are administered within 24 to 48 hours of each other. In some embodiments, the injections of the antigen/adjuvant and the tolerizing agent may be repeated after several days, several weeks or several months, such as a second administration of antigen/adjuvant and tolerizing agent 7-14 days apart from the first administration. In some embodiments, the subject receives a first injection of a tolerizing agent, an injection of an antigen/adjuvant, followed by a third injection of a tolerizing agent.

The therapeutic agent(s) may be contained in controlled release systems. The term "controlled release" is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release formulations. The term "sustained release" (also referred to as "extended release") is used in its conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. The term "delayed release" is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug therefrom. "Delayed release" may or may not involve gradual release of drug over an extended period of time, and thus may or may not be "sustained release."

Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.

The invention also contemplates the use of kits. In some aspects of the invention, the kit can include a pharmaceutical preparation vial, a pharmaceutical preparation diluent vial, an isolated antigen, an adjuvant and a tolerogenic agent. The vial containing the diluent for the pharmaceutical preparation is optional. The diluent vial contains a diluent such as physiological saline for diluting what could be a concentrated solution or lyophilized powder of the antigen, the adjuvant and/or the tolerogenic agent. The instructions can include instructions for mixing a particular amount of the diluent with a particular amount of the concentrated pharmaceutical preparation, whereby a final formulation for injection or infusion is prepared. The instructions may include instructions for treating a subject with an effective amount of each of the components. It also will be understood that the containers containing the preparations, whether the container is a bottle, a vial with a septum, an ampoule with a septum, an infusion bag, and the like, can contain indicia such as conventional markings which change color when the preparation has been autoclaved or otherwise sterilized.

The present invention is further illustrated by the following Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

Examples Introduction

The control of deleterious immune responses causing diseases, such as allergy, autoimmunity and transplant rejection, as well as reducing the efficacy of immunogenic drugs has been one of the main objectives of immunologists.

Several strategies have been described to achieve tolerance in transplantation, autoimmunity and allergy (1-3). Tolerance to injected proteins has been harder to achieve. Although immunoglobulins can be tolerized with relative efficiency (4-6), other proteins such as human recombinant coagulation factors or even ovalbumin (OVA) are significantly harder to tolerize with the same protocols (7, 8). We hypothesized that the difference in outcome may be due to the fact that tissue antigens (in transplantation or autoimmunity) are usually present together with inflammatory signals produced by the tissues themselves, and immunoglobulins can be readily picked up by antigen presenting cells (APCs), while other proteins are poorly targeted to APCs in the absence of adjuvants. In addition, immunoglobulins (due to a specific renal recapture system) have considerable longer half-life in circulation than other proteins, except tissue antigens in transplantation or autoimmunity that are chronically produced. Again, an adjuvant, such as aluminum-hydroxide (alum), induces a sustained release of protein antigens that consequently remain available for tolerization for a longer period of time.

The use of alum and antigen is common practice in allergy studies, because the adjuvant is necessary to induce the inflammatory pathology (9). As a consequence, reports dealing with tolerance induction in allergic diseases have used allergen in alum together with the tolerance inducing drug (10-14). However, adjuvants have been considered obstacles for tolerance induction as it is generally accepted that tolerance is facilitated in the absence of proinflammatory "danger" signals. Alum has been used in tolerance induction studies in allergy because the allergic diseases cannot be observed in the absence of alum, but never considered as a facilitator of tolerance induction. For the reasons described in the paragraph above, we tested for the first time whether alum can facilitate tolerance induction to proteins.

It has been surprisingly discovered that CD4 blockade at the time of exposure with OVA-alum can induce antigen-specific tolerance by promoting activation-induced cell death (AlCD) of the T cell clones being activated at the time of treatment. Since CD4 blockade is achieved with a non-depleting monoclonal antibody (MAb), T cells not activated by the antigen remain unaffected to mount protective immune responses towards unrelated antigens at a later time. Therefore our strategy leads to antigen-specific tolerance without affecting protective immune responses to third-party antigens.

Tolerance induction is robust enough to be effective in pre-sensitized animals or antigen-specific TCR transgenic mice where most T cells are specific to the antigen. The tolerant mice show long term protection from further immunization with the antigen.

The tolerogenic procedure is effective to all tested proteins: OVA, β-lactoglobulin (β- LG), and human recombinant factor VIII (hFVIII). Similar results were obtained using a common allergen, House Dust Mite (HDM). We have also shown that tolerance is effective in different genetic backgrounds (BALB/c and C57B1/6), and when anti-CD4 MAb is replaced by other tolerogenic reagents (anti-CD40L and anti-OX40L MAbs).

When the tolerated protein is an allergen, the animals are protected from manifestations of allergic disease induced by that allergen: they do not develop airways eosinophilia, goblet cell hyperplasia, production of Th2 cytokines in the lung, production of allergen-specific IgE or IgGl, and, importantly, do not develop airway hyperreactivity (AHR) in response to inhaled methacholine (MCh) - the hallmark of asthma.

Finally, data show that activation-induced cell death is an important mechanism in tolerance induction by reducing the clonal size of the antigen-specific T cells.

Example I: Prevention of the generation of OVA-specific immunoglobulins with tolerogenic MAbs administered together with OVA-alum.

Female BALB/c mice were immunized with two intra-peritoneal injections of OVA- alum on days 1 and 14, and challenged with 50 μg OVA intranasal on days 20, 21 and 22 (Figure IA). For tolerance induction, some experimental groups were treated with 1 mg i.p. of non-depleting anti-CD4 or isotype control (YKIX302, anti-canine CD4) on the days before and after each administration of OVA-alum. Animals were sacrificed 24 hours following the last intranasal challenge.

Anti-CD4 treatment in the presence of OVA-alum prevents effective generation of Th2- driven OVA-specific IgGl and IgE (n=6, PO.001, Figure IB). We could not detect ThI -driven OVA-specific IgG2a in any animal, that was below detection levels (not shown). Similar results were obtained when the antigen used for tolerance induction was β-LG alum or hFVIII-alum. We have also replaced anti-CD4 with tolerogenic MAbs targeting OX40L and CD40L (co-stimulation blockade) with similar results (Figure 1C and D).

Example 2:. Tolerant mice remain immunocompetent and protected from subsequent immunization.

BALB/c mice were treated with 1 mg non-depleting anti-CD4 MAbs together with OVA-alum (tOVA) or β-LG-alum (tβ-LG) in order to establish immune tolerance to the antigens (Figure 2A). At day 50 the animals were immunized with the same antigen used at the time of tolerization (day 0) or with the third-party antigen. All mice were subsequently challenged i.n. with the same antigen used at day 50. All animals remain protected from generating immune responses towards the antigen used for tolerization, but fully competent to undergo an immune response to the third-party antigen: The animals can produce IgE and IgGl following immunization with a third-party antigen, but remain tolerant to the tolerated antigen as the immunoglobulin response does not occur (Figure 2B). As a consequence, the mice become specifically tolerant to the antigens provided and not immune suppressed: the tolerance state is antigen-specific.

Example 3: Prevention of allergic sensitization with OVA-alum.

BALB/c mice were sensitized with two intra-peritoneal injections of OVA-alum on days 1 and 14, and challenged with 50 μg OVA intranasal on days 20, 21 and 22 (Figure 3A). The experimental groups were treated with 1 mg i.p. of anti-CD4 or isotype control on the days before and after each administration of OVA-alum. Animals were sacrificed 24 hours following the last intranasal challenge.

Mice treated with anti-CD4 have a ~ 100-fold reduction in broncho-alveolar lavage (BAL) eosinophils and lymphocytes when compared with animals sensitized in the absence of anti-CD4 (n=6, note Logio scale, ?<0.001, Figure 3B). The absence of inflammatory infiltrate in the airways of anti-CD4 treated mice, as well as the absence of goblet cell hyperplasia, was confirmed by histology (Figure 3C). We have determined the concentration of several cytokines in lung homogenates (Figure ID). Animals treated with anti-CD4 show a marked reduction of Th2 cytokines IL-13, IL-4 and IL-5 when compared with animals treated with the isotype control (n=6, PO.01 for IL-13; PO.05 for IL-4 and IL-5). The concentration of these cytokines in the lung of anti-CD4 treated animals is similar to naive animals. Importantly, we have no evidence for ThI or Th 17 deviation with equivalent levels of IL- 17 (below detection), IFN-γ and TNF-α in all the experimental groups. The anti-CD4 treatment is also not associated with increased levels of the immune-regulatory cytokine IL-IO. Similar results were obtained in mice from a different genetic background (C57B1/6, not shown).

In order to study the functional impact of the treatment we assessed AHR in response to increasing doses of inhaled MCh. Our data show that anti-CD4 treatment leads to decreased AHR, to levels equivalent to healthy non-sensitized animals (Figure IE).

Example 4: Tolerance can be induced in sensitized mice.

To assess whether tolerance can be induced in pre-sensitized mice in an antigen-specific way, BALB/c mice sensitized with OVA-alum or β-LG-alum were treated with anti-CD4 MAbs 50 days following the initial intervention, in the presence of either the initial or the third-party antigen adsorbed in alum (Figure 4A). AU groups of animals were subsequently challenged i.n. with the same antigen used for initial sensitization. We were able to demonstrate that sensitized mice are protected from airway eosinophilic and lymphocytic inflammation when treated with anti-CD4 in the presence of the same antigen used for sensitization (OVA >tOVA and β-LG >tβ-LG, n=6, /*<0.001, Figure 4B). The protective effect is not due to the persistence of the therapeutic antibody in circulation at the time of intranasal exposure to the antigen since mice treated with a third-party antigen (and therefore with equivalent doses of circulating anti-CD4) are not protected (OVA >tβ-LG and β-LG >tOVA). These observations, that animals receiving the antibody treatment together with a third-party antigen adsorbed in alum develop inflammatory changes similar to untreated control animals, are consistent with the antigen- specificity of the tolerance state described above.

In addition, sensitized mice treated with anti-CD4 are protected from AHR in response to inhaled MCh, to levels similar to naive mice (Figure 4C). Example 5: Tolerance induction can be achieved in TCR-transgenic animals.

In order to assess the mechanism leading to antigen-specific tolerance, we investigated the fate of antigen-specific T cells using OVA-specific TCR transgenic DOl 1.10 mice (Figure 5). OVA-specific IgGl and IgE is significantly lower in DOl 1.10 mice treated with anti-CD4 together with OVA-alum (Figure 5B). In addition, the tolerogenic treatment with non-depleting anti-CD4 MAbs administered together with OVA-alum is robust enough to prevent allergic airways disease in DOl 1.10 mice where most T cells are specific to the allergen (Figure 5C). Treated DOl 1.10 mice are protected from airways eosinophilic infiltrate and goblet cell hyperplasia, and their lung tissue has reduced levels of Th2-type cytokines (IL-4, IL-5, and IL- 13) without an increase of ThI -type cytokines. IL-IO levels are higher in allergic animals than in mice treated with anti-CD4. Furthermore, given the high frequency of OVA-specific T cells it is possible to monitor changes in the frequency of OVA-specific T cells with different functional characteristics. Anti-CD4-treated animals show a reduced number and frequency of Th2-type OVA-specific cells in mediastinal lymph nodes (MeLN) (Figure 5F). The number or frequency of OVA-specific Foxp3⁺ regulatory T cells (Treg) do not change with the tolerogenic treatment. T cells isolated from the lung show a similar phenotype (not shown). In addition, DOl 1.10.Rag^"y" mice, usually devoid of Foxp3⁺ Treg cells (15), treated with the tolerogenic protocol described above do not develop detectable Foxp3⁺ T cells in the spleen or LN (not shown).

Example 6: Tolerance induction relies on AICD of allergen-specific T cells.

To investigate the impact of anti-CD4 treatment in the presence of antigen adsorbed in alum on T cell proliferation and survival, CFSE-labeled OVA-specific T cells from DOll.lO.Rag^"7" mice were adoptively transferred into BALB/c mice. The host animals were immunized with OVA-alum i.p. in the presence or absence of anti-CD4 treatment. MeLN, mesenteric LN, and spleen T cells were isolated at day 4 or 7 following treatment and analyzed by flow cytometry. Consistent with a recent report on T cell response to OVA-alum administered i.p. (9), MeLN displayed the highest number of T cells responding to the antigen. An observation of the CFSE dilution profile in OVA-specific T cells isolated from MeLN may suggest that anti-CD4 treatment prevents T cell proliferation, as the undivided cells are a greater proportion of the TCR-transgenic population unlike in mice injected with OVA alone (Figure 6A). But if anti-CD4 would target T cell proliferation one would expect the absolute number of TCR transgenic cells recovered from the undivided CFSE gate to be similar in mice treated with anti-CD4 and animals not exposed to OVA where cells do not divide at all. The enumeration of undivided and divided TCR transgenic cells from the different experimental groups does not support the hypothesis of inhibition of proliferation (Figure 6B). In fact, the number of undivided cells from anti-CD4 treated animals is remarkably similar to animals treated with OVA alone, where extensive T cell proliferation does occur, and significantly lower than animals not treated with OVA. The difference between OVA and OVA + anti-CD4 treated groups is not in the undivided T cells but rather on the accumulation of T cells that underwent cell divisions. Given these data, either the anti-CD4 treatment is targeting the deletion of undivided cells or inducing the cell death of T cells entering cell cycle. The analysis of TCR transgenic cells positive for the apoptosis marker annexin V shows that the frequency of apoptotic cells is similar between undivided cells from all experimental groups (Figure 6C). Only the cells undergoing cell divisions from anti-CD4 treated mice have a significant increase in the frequency of apoptotic cells. As a consequence, anti-CD4 treatment does not prevent cell division, as the number of cells that enter cell cycle (and consequently disappear from the undivided CFSE region) is identical in OVA and OVA+anti-CD4 treated mice. The tolerogenic treatment but preferentially induces the apoptosis of activated cells. We have confirmed that the anti-CD4 MAb does not directly kill non-activated DOl 1.10 T cells (Figure 7), in agreement with the long experience on the non-depleting properties of this reagent in transplantation studies (16).

Example 7: Tolerance induction to hFVIII is more efficient when the protein is adsorbed in alum.

BALB/c mice were treated with hFVIII or hFVIII-alum in the presence of anti-CD4 MAb. The recombinant protein (1 U per injection, approximately 40U/Kg) was injected i.p. on days 1 and 10, and 1 mg anti-CD4 on the days before and after each hFVIII administration (Figure 8A). All animals received a further injection of 1 U hFVIII at day 30, in the absence of any other treatment. At day 60 all animals were bled and serum anti-hFVIII inhibitory immunoglobulins were quantified.

Mice treated with hFVIII-alum + anti-CD4, but not hFVIII + anti-CD4, had a statistically significant reduction in the serum concentration of anti-hFVIII immunoglobulins compared with animals injected with hFVIII in the absence of anti-CD4 (n=5, /^><0.01, Figure 8B). The level of anti-hFVIII immunoglobulins in animals injected with hFVIII-alum in the absence of anti-CD4 was even greater than in animals treated with hFVIII alone, as expected given the adjuvant properties of alum (not shown). Example 9: Prevention of the generation of HDM-speciftc immunoglobulins with tolerogenic MAbs administered together with HDM-alum.

Female BALB/c mice were immunized with two intra-peritoneal injections of HDM- alum on days 1 and 14, and challenged with 50 μg HDM intranasal on days 20, 21 and 22 (Figure 9A). For tolerance induction, some experimental groups were treated with 1 mg i.p. of non-depleting anti-CD4 or isotype control (YKIX302, anti-canine CD4) on the days before and after each administration of HDM-alum. Animals were sacrificed 24 hours following the last intranasal challenge.

Anti-CD4 treatment in the presence of HDM-alum prevents effective generation of Th2-driven HDM-specific IgGl and IgE (n=6, /^><0.001, Figures 9D and 9E). We could not detect ThI -driven HDM-specific IgG2a in any animal that was below detection levels (not shown).

Example 10: Tolerant mice remain immunocompetent and protected from subsequent immunization.

BALB/c mice were treated with 1 mg non-depleting anti-CD4 MAbs together with HMD-alum in order to establish immune tolerance to the antigen (Figure 10A). At day 50 the animals were immunized with the same antigen used at the time of tolerization (day 0) or with the third-party antigen. All mice were subsequently challenged i.n. with the same antigen used at day 50. All animals remain protected from generating immune responses towards the antigen used for tolerization. As a consequence, the mice become specifically tolerant to the antigens provided and not immune suppressed: the tolerance state is antigen-specific.

Example 11: Tolerance can be induced in sensitized mice.

BALB/c mice sensitized with HDM-alum were treated with anti-CD4 MAbs 50 days following the initial intervention, in the presence of the same antigen adsorbed in alum (Figure 1 IA). All groups of animals were subsequently challenged i.n. with the same antigen used for initial sensitization. The observed protective effect is not due to the persistence of the therapeutic antibody in circulation at the time of intranasal exposure to the antigen since mice treated with a third-party antigen (and therefore with equivalent doses of circulating anti-CD4) are not protected. These observations are consistent with the antigen-specificity of the tolerance state described above.

In addition, sensitized mice treated with anti-CD4 are protected from AHR in response to inhaled MCh, to levels similar to naive mice (Figure HC). Discussion

At a time of great enthusiasm for potentially immunogenic biological therapies, our results have clinical implications for tolerance induction to immunogenic molecules. In fact, it is possible to combine any desired immunogenic molecule with an adjuvant, such as alum or any other with similar immune-stimulatory action, to facilitate tolerance induction. We have performed most our studies with anti-CD4 MAbs, however several other reagents have been described to be tolerogenic including many different MAbs targeting T cell co-stimulation, T cell co-receptors, and adhesion molecules (CD2, CD3, CD8, CD28, CD40L, CD45, CTLA4Ig, OX40L, LFA-I, ICAM-I, among others). We have tested other tolerogenic MAbs besides anti- CD4 and have found that the combination of antigen with alum facilitates tolerance induction with anti-CD40L and anti-OX40L. Therefore, the results we now describe appear to be applicable to other tolerogenic reagents. Since we can achieve similar results with three different proteins (OVA, β-LG and hFVIII), as well as a common allergen (HDM), it appears that the combination of alum with the antigen is generally applicable to any immunogenic molecule. We have also tested one single administration of the antigen-alum, under the cover of anti-CD4, as well as a reduction of the anti-CD4 dose to two injections of 0.5 mg per animal. Tolerance can still be achieved with those changes to the protocol in the majority of the animals, but a few mice did not become tolerant (not shown). It should be noted that tolerance induction to proteins have been tested with several of the MAbs listed above but, with the exception of immunoglobulins, it has been difficult to induce long term tolerance to proteins in the absence of alum (7, 8).

Although we cannot exclude, at present, the participation of a minor regulatory cell population in our experimental system, we have established AICD as a major mechanism leading to the observed antigen-specific tolerance. It is known that AICD can influence the outcome of immune responses, including in human allergy, as it was recently shown for atopic dermatitis (17). The observation of AICD as a major mechanism for tolerance induction is also clinically relevant as immune monitoring of the tolerance state is considerably easier when the mechanism is based on deletion of pathogenic clones rather than the emergence of a regulatory T cell population. Compare for instance the difficulty in immune monitoring of dominant transplantation tolerance mediated by Treg cells with transplantation tolerance achieved by hematopoietic chimerism where the mechanism is deletional (18).

Our data also show that long-term tolerance maintained even following immunizations at a later time, does not hamper protective immune responses to third-party antigens. Thus, the tolerance state is not associated with non-specific immune suppression. This outcome is consequence of the exquisite antigen-specificity of tolerance induction: only the antigens associated with alum that are present at the time of tolerance induction become long-term tolerized.

Furthermore, tolerance induction is sufficiently robust to prevent immune responses in TCR-transgenic animals where most T cells are specific for the antigen. In addition, when the antigen is an allergen, the tolerance state can prevent the manifestations of allergic airways disease following intranasal allergen challenge: Th2 and eosinophilic infiltrate of the airways, goblet cell hyperplasia, and AHR (the hallmark of allergic asthma). The tolerogenic treatment is not only effective in preventing the disease in naive animals, but also in mice previously sensitized with the allergen.

Taken together, our observations suggest that an effective way to achieve immune tolerance is by associating the antigen to an adjuvant that facilitates tolerization.

Methods

Animals

BALB/c, C57B1/6, DOI l.10, and DOl l.lO.Ragl^"7" mice were bred and maintained under specific pathogen-free (SPF) facilities at the Institute Gulbenkian de Ciencia. Experimental animals were sex-matched and between 6 and 8 weeks of age. Procedures were conducted in accordance with guidelines from the Animal User and Institutional Ethical Comities.

Tolerance induction and immunization

Animals were tolerized, at the times described in the text, by i.p. injection of 20 μg of human recombinant factor VIII (Kogenate™, Bayer), ovalbumin (OVA, grade V; Sigma, St Louis, USA) or beta-lactoglobulin (Sigma), previously run through a DetoxyGel column (Pierce, Rockford, USA) following manufacturer instructions, House Dust Mite crude extract (Greer, Lenoir, USA) and suspended in 2.0 mg of endotoxin-free aluminum hydroxide (AIu- gel-S, Serva, Heidelberg, Germany). The antigen-alum was administered under the cover of the tolerogenic MAb at the times described in the text. For immunization the animals were treated with the same antigen-alum doses in the absence of the tolerogenic MAb. Mice were intranasally challenged with 50 μg of OVA, β-LG or House Dust Mite extract in pyrogen-free saline and sacrificed 24 hours after the last challenge. Antibodies and reagents

Non-depleting anti-CD4 (YTS177) (19), anti-CD40L (MRl), anti-OX40L (0X139), and the isotype control rat anti-dog CD4 (YKIX302) (20) MAbs were produced in our laboratory using Integra CLlOOO flasks (IBS, Chur, Switzerland), and purified from culture supernatants by 50% ammonium sulfate precipitation, dialyzed against PBS, and the purity checked by native and SDS gel electrophoresis. The hybridomas were generously provided by Professor Herman Waldmann (Oxford, UK). CTLA4Ig is the commercial form Abatacept (Bristol-Meyers Squibb Pharma, New York, USA).

Bronchoalveolar Lavage (BAL)

The airways were washed through the trachea by slowly infusing and withdrawing 1 ml of cold PBS 10% BSA (Sigma) three times. The BAL was then centrifuged, the supernatant removed, and the pellet ressuspended in PBS. The cells were counted with a hemocytometer. Differential cell counts were performed on cytospin samples stained with Giemsa- Wright (Sigma). At least 200 cells from each sample were counted, using blinded slides, to determine the relative frequency of each cell type.

Quantification of immunoglobulins and cytokines

Serum titers of antigen-specific IgGl, IgG2a, and IgE were measured by ELISA using the following kits: IgGl and IgG2a (SouthernBiotech, Birmingham, USA) with anti-OVA IgGl standard from Serotec, Oxford, UK; IgE (BD-Pharmingen) with anti-OVA IgE standard from Abeam (Cambridge, UK). Cytokine titers were determined in fresh BAL and lung tissue homogenate supernatant. Lung tissue was collected, homogenized at 100 mg/ml in HBSS (Gibco, Carlsbad, USA), centrifuged at 80Og for 10 min and the supernatant collected. The ELISA assays for cytokines were performed using the following kits: IL-10, IL- 13, IFN-γ, TNF-α (Peprotech, London, UK); IL-4, IL-5 (BD-Pharmingen); IL- 17 (R&D Systems, Minneapolis, MN, USA). All ELISA assays were performed according to the manufacturer's instructions.

Histology

Lungs were perfused with 4% formalin solution (Sigma), collected and sectioned. Staining was performed using hematoxilin/eosin, and mucus containing cells were revealed using a periodic acid-Schiff (PAS) stain. Photographs were taken using a Leica DM2500 microscope and a Leica DFC420 camera. Respiratory mechanics and methacholine responsiveness

Airway responsiveness was determined 24 hours after last intranasal OVA challenge. Changes in the respiratory input impedance (Zrs) were measured using a modification of the low frequency forced-oscillation technique (LFOT) in mice anesthetized with 10 μl/g of xylazine (2 mg/ml, Ronpum, Bayer, Germany) and ketamine (40 mg/ml, Merial, Lyon, France), tracheostomized and ventilated (FlexiVent, SciReq, Montreal, Canada). Mice were hyperventilated at 450 breaths/min and Zrs was measured during periods of apnea using a 16 s signal containing 19 prime sinusoidal frequencies as described elsewhere (21). Calculation of airway resistance (R_aw), tissue damping (G) and tissue elastance (H) is obtained from the Zrs spectrum using FlexiVent software (SciReq). AHR was measured by exposure to an aerosol containing increasing doses of methacholine (MCh, Sigma), following a baseline measurement after the delivery of a saline aerosol.

CFSE staining and adoptive cell transfers

Single cell suspensions from spleen and LN from DOl 1.10.Rag^"A mice were ressuspended at 5x10⁷ cells/ml and stained with 5 μM of Carboxyfluorescein succinimidyl ester (CFSE, Invitrogen, Carlsbad, USA). The cells were washed, ressuspended in saline and injected i.v., in the tail vein of BALB/c mice at a total of 3.5xlO⁷ cells per animal.

Flow cytometry

Cells were stained for flow cytometric analysis with CD25-AlexaFluor488 (PC61; produced and conjugated in house), CD25-PE-Cy7 (PC61.5; eBioscience), CD3-PE-Cy7 (145- 2Cl 1; eBioscience), CD8-APC-AlexaFluor750 (53-6.7; eBioscience), CD4-PerCp (RM4-5; BD Pharmigen), and the DO 11.10 TCR-specific KJ 1 -26 MAb conjugated with PE or APC (BD Pharmigen) in PBS containing 0,01% NaN₃, 2% fetal bovine serum (FBS, Gibco) and 10 μg/ml Fc receptor blocking 2.4G2 MAb (produced and purified in house). Cells were then washed and fixed using Foxp3 Staining Set (eBioscience). The cells were stained for intracellular cytokines and Foxp3 with Foxp3-APC (FJK- 16s; eBioscience), IFN-γ-FITC (XMG 1.2; eBioscience), IL-IO-PE (JES5-16E3; BD Pharmigen), IL- 13-PE (eBiol3A; eBioscience), IL-4-FITC (BVD6-24G2; eBioscience) and IL-5-APC (TRF K5; BD Pharmigen). Apoptotic cells were identified with Annexin V-biotin (BD Pharmigen) and Streptavidin-APC-Cy7 (eBioscience) labeled in Annexin V Binding Buffer (BD Pharmigen) according to manufacturer protocol. Propidium iodide solution was added right before the cells were analyzed. Four and six color analyses were performed using a FACSCalibur or a FACSCanto (BD Bioscience) with dual laser (488nm and 633nm) excitation. The analysis gate was set on the forward and side scatters to eliminate cell debris and dead cells.

Statistical analysis

Statistical significance was determined using the two-tailed non-parametric Student's t test (Mann- Whitney) and the Spearman test for correlation analysis. Statistical analysis was performed using Prism 4.0 (GraphPad, San Diego, USA) and P values <0.05 were deemed significant (*, /^><0.05; **, Z^O.Ol ; ^♦♦♦, P<0.001).

References

1. Agua-Doce A, Graca L. 2007. Induction of dominant tolerance using monoclonal antibodies. Methods MoI Biol 380: 405-29

2. Graca L, Le Moine A, Cobbold SP, Waldmann H. 2003. Antibody-induced transplantation tolerance: the role of dominant regulation. Immunol Res 28: 181-91

3. Holgate ST, Polosa R. 2008. Treatment strategies for allergy and asthma. Nat Rev Immunol 8: 218-30

4. Benjamin RJ, Waldmann H. 1986. Induction of tolerance by monoclonal antibody therapy. Nature 320: 449-51

5. Benjamin RJ, Cobbold SP, Clark MR, Waldmann H. 1986. Tolerance to rat monoclonal antibodies. Implications for serotherapy. J Exp Med 163: 1539-52

6. Winsor-Hines D, Merrill C, O'Mahony M, Rao PE, Cobbold SP, Waldmann H, Ringler DJ, Ponath PD. 2004. Induction of immunological tolerance/hyporesponsiveness in baboons with a nondepleting CD4 antibody. J Immunol 173: 4715-23

7. Reipert BM, Sasgary M, Ahmad RU, Auer W, Turecek PL, Schwarz HP. 2001. Blockade of CD40/CD40 ligand interactions prevents induction of factor VIII inhibitors in hemophilic mice but does not induce lasting immune tolerance. Thromb Haemost 86: 1345-52

8. Salooja N, Kemball-Cook G, Tuddenham EG, Dyson J. 2002. Use of a non-depleting anti-CD4 antibody to modulate the immune response to coagulation factors VIII and IX. BrJ Haematol 118: 839-42

9. Kool M, Soullie T, van Nimwegen M, Willart MA, Muskens F, Jung S, Hoogsteden HC, Hammad H, Lambrecht BN. 2008. Alum adjuvant boosts adaptive immunity by inducing uric acid and activating inflammatory dendritic cells. J Exp Med 205: 869-82 10. Kearley J, McMillan SJ, Lloyd CM. 2007. Th2-driven, allergen-induced airway inflammation is reduced after treatment with anti-Tim-3 antibody in vivo. J Exp Med 204: 1289-94

11. Krinzman SJ, De Sanctis GT, Ceraadas M, Mark D, Wang Y, Listman J, Kobzik L, Donovan C, Nassr K, Katona I, Christiani DC, Perkins DL, Finn PW. 1996. Inhibition of T cell costimulation abrogates airway hyperresponsiveness in a murine model. J Clin Invest 98: 2693- 9

12. Li L, Crowley M, Nguyen A, Lo D. 1999. Ability of a nondepleting anti-CD4 antibody to inhibit Th2 responses and allergic lung inflammation is independent of coreceptor function. J Immunol 163: 6557-66

13. Linhart B, Bigenzahn S, Haiti A, Lupinek C, Thaihamer J, Valenta R, Wekerle T. 2007. Costimulation blockade inhibits allergic sensitization but does not affect established allergy in a murine model of grass pollen allergy. J Immunol 178: 3924-31

14. Seshasayee D, Lee WP, Zhou M, Shu J, Suto E, Zhang J, Diehl L, Austin CD, Meng YG, Tan M, Bullens SL, Seeber S, Fuentes ME, Labrijn AF, Graus YM, Miller LA, Schelegle ES, Hyde DM, Wu LC, Hymowitz SG, Martin F. 2007. In vivo blockade of OX40 ligand inhibits thymic stromal lymphopoietin driven atopic inflammation. J Clin Invest 117: 3868-78

15. Mucida D, Kutchukhidze N, Erazo A, Russo M, Lafaille JJ, Curotto de Lafaille MA. 2005. Oral tolerance in the absence of naturally occurring Tregs. J Clin Invest 115: 1923-33

16. Waldmann H, Chen TC, Graca L, Adams E, Daley S, Cobbold S, Fairchild PJ. 2006. Regulatory T cells in transplantation. Semin Immunol 18: 111-9

17. Akkoc T, de Koning PJ, Ruckert B, Barlan I, Akdis M, Akdis CA. 2008. Increased activation-induced cell death of high IFN-gamma-producing T(H)I cells as a mechanism of T(H)2 predominance in atopic diseases. J Allergy Clin Immunol 121: 652-8 el

18. Graca L, Le Moine A, Lin CY, Fairchild PJ, Cobbold SP, Waldmann H. 2004. Donor- specific transplantation tolerance: the paradoxical behavior of CD4+CD25+ T cells. Proc Natl Acad Sci USA 101 : 10122-6

19. Qin SX, Wise M, Cobbold SP, Leong L, Kong YC, Parnes JR, Waldmann H. 1990. Induction of tolerance in peripheral T cells with monoclonal antibodies. Eur J Immunol 20: 2737-45

20. Watson CJ, Cobbold SP, Davies HS, Rebello PR, Thiru S, McNair R, Rasmussen A, Waldmann H, Calne RY, Metcalfe SM. 1994. Immunosuppression of canine renal allograft recipients by CD4 and CD8 monoclonal antibodies. Tissue Antigens 43: 155-62 21. Zosky GR, von Gamier C, Stumbles PA, Holt PG, Sly PD, Turner DJ. 2004. The pattern of methacholine responsiveness in mice is dependent on antigen challenge dose. Respir Res 5: 15

The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. The present invention is not to be limited in scope by examples provided, since the examples are intended as a single illustration of one or more aspects of the invention and other functionally equivalent embodiments are within the scope of the invention.

Various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. The advantages and objects of the invention are not necessarily encompassed by each embodiment of the invention.

Claims

Claims

1. A method for inducing immune tolerance in a subject, the method comprising: administering an isolated antigen, an adjuvant and a tolerogenic agent to a subject in need of increased tolerance to the antigen to induce antigen-specific immune tolerance in the subject.

2. The method of claim 1 , wherein the subject has an allergy and is sensitized to the isolated antigen .

3. The method according to any one of claims 1-2, wherein the antigen is an allergen from a mite, a venom, an insect, an animal, a fungi, smuts, a pollen, a food, dust or a drug.

4. The method according to any one of claims 1 -2, wherein the antigen is an allergen derived from a genus selected from the group consisting of:

Canine (Canis familiaris); Dermatophagoides (e.g. Dermatophagoides farinae); Felis (Felis domesticus); Ambrosia (Ambrosia artemiisfolia; Lolium (e.g. Lolium perenne or Lolium multiflorum); Cryptomeria (Cryptomeria japonica); Alternaria (Alternaria alternata); Aider; Alnus (Alnus gultinoasa); Betula (Betula verrucosa); Quercus (Quercus alba); Olea (Olea europa); Artemisia (Artemisia vulgaris); Plantago (e.g. Plantago lanceolata); Parietaria (e.g. Parietaria officinalis or Parietaria judaica); Blattella (e.g. Blattella germanica); Apis (e.g. Apis multiflorum); Cupressus (e.g. Cupressus sempervirens, Cupressus arizonica and Cupressus macrocarpa); Juniperus (e.g. Juniperus sabinoides, Juniperus virginiana, Juniperus communis and Juniperus ashei); Thuya (e.g. Thuya orientalis); Chamaecyparis (e.g. Chamaecyparis obtusa); Periplaneta (e.g. Periplaneta americana); Agropyron (e.g. Agropyron repens); Secale (e.g. Secale cereale); Triticum (e.g. Triticum aestivum); Dactylis (e.g. Dactylis glomerata); Festuca (e.g. Festuca elatior); Poa (e.g. Poa pratensis or Poa compressa); Avena (e.g. Avena sativa); Holcus (e.g. Holcus lanatus); Anthoxanthum (e.g. Anthoxanthum odoratum); Arrhenatherum (e.g. Arrhenatherum elatius); Agrostis (e.g. Agrostis alba); Phleum (e.g. Phleum pratense); Phalaris (e.g. Phalaris arundinacea); Paspalum (e.g. Paspalum notatum); Sorghum (e.g. Sorghum halepensis); and Bromus (e.g. Bromus inermis).

5. The method according to any one of claims 1-4, wherein the adjuvant is selected from the group consisting of: alum (aluminum hydroxide, aluminum phosphate); mineral oil, non-mineral oil, water- in-oii emulsions, oil-in-water emulsions, Seppic ISA series of Montanide adjuvants; MF-59, PROVAX; saponins (QS21); poly[di(carboxylatophenoxy)phosρhazene (PCPP), monophosphorlyl lipid (MPL), muramyl dipeptide (MDP) and threonyl muramyl dipeptide (tMDP); OM- 174; and Leishmania elongation factor; ISCOMS; SB-AS2; SB-AS4; CRL 1005; and Syntex Adjuvant Formulation.

6. The method according to any one of claims 1 -4, wherein the adjuvant is an immunostimulatory nucleic acid molecule.

7. The method according to any one of claims 1-6, wherein the tolerogenic agent is an agent that interferes with T-cell activation.

8. The method according to claim 7, wherein the tolerogenic agent is an agent that blocks a co-stimulation signal.

9. The method according to claim 7, wherein the tolerogenic agent binds to a cell surface molecule expressed on a T cell or on an antigen presenting cell.

10. The method according to claims 7, wherein the tolerogenic agent is selected from the group consisting of: anti-CD4, anti-CD3, anti-CD25, anti-CD28, anti-PDl, anti-BTLA, anti-B7 (anti-B7-l and anti-B7-2), anti-ICOS, anti-CTLA, anti-CD40, anti-CD40L, anti-CD99, anti-CD2, anti- LFA3, anti-CD27, anti-CD70, anti-DC8, anti-OX40, anti-OX40L, anti-LFA-1, anti-CDl la, anti-ICAMl, anti-CD26, anti-CD44, anti-CD137 (anti-4- IBBL), CTLA4-Ig, anti-MHC class II and anti-MHC class I.

13. The method according to any one of claims 1-and 5-10, wherein the subject has received or is going to receive an exogenous antigen administered as a therapy, and the exogenous antigen comprises the isolated antigen.

12. The method according to any one of claims 1-and 5-10, wherein the subject has received or is going to receive an exogenous peptide, and the exogenous peptide comprises the isolated antigen.

13. The method according to any one of claims I -and 5-10, wherein the subject has received or is going to receive gene therapy, and an expression product of the gene therapy comprises the isolated antigen.

14. The method according to any one of claims 1-and 5-10, wherein the subject has received or is going to receive gene therapy, and the gene therapy comprises the isolated antigen.

15.. The method according to any one of claims 11-14, wherein the subject has a peptide deficiency.

16. The method according to claim 11 , wherein the exogenous antigen is Factor VIII, Factor IX, Von Willebrand Factor (VWF), Factor XI, Factor VII, T4 endonuclease V, erythropoiesis stimulating protein, insulin, Protein C, neuregulin 1, Laminin-111, p53, Erythropoietin (EPO), alpha- 1 antitrypsin, Rod derived Cone Viability Factor (RdDVF), estrogen, estradiol, testosterone, Dehydroepiandrosterone, Phenylalanine hydroxylase, DNase I, Glucocerebrosidase, Growth hormone (GH), Alpha interferon, Gamma- Ib interferon, Interleukin-2 (IL-2), Interleukin-3 (IL-3), Interleukin-4 (IL-4), Tissue plasminogen activator tPA), monoclonal antibodies, adenosine deaminase, Nutropin (somatropin), Neupogen or Leukine (G-CSF, GM-CSF).

17. The method according to any one of claims 11-16, wherein the subject is previously sensitized to the peptide.

18. The method according to any one of claims 11-16, wherein the subject is not previously sensitized to the peptide.

19. The method according to any one of claims 11-18, wherein the subject has a disease or disorder selected from the group consisting of: hemophilia (A and B), Type I diabetes, DNA repair disorder, Xeroderma Prgmentosum (XP), Cockayne Syndrome (CS), Trichothiodystrophy (TTD), severe protein C deficiency, purpura fulminans (PF), disseminated intravascular coagulation (DIC), venous thromboembolism (VTE), chronic granulomatous disease, cystic fibrosis (CF), Gaucher 's disease, Duchenne muscular dystrophy (DMD), Alpha- 1 antitrypsin chronic deficiency, multiple sclerosis (MS), retinitis pigmentosa (RP), blood clots, pulmonary embolism, myocardial infarction, stroke, hormone deficiency, phenylketonuria, anemia, chronic renal disease, lysossomal storage diseases (e.g., Fabry Disease), Nanism hypophyseal, leukemia, Kaposi's sarcoma, hepatitis B, hepatitis C, Chronic granulomatous disease, cancer, Acute myocardial infarction, massive pulmonary embolism, rheumatoid arthritis, SLE, multiple sclerosis, inflammatory bowel disease and severe combined immune deficiency (SCID).

20. The method according to any one of claims 1-19, wherein the subject is human.