SBIR-STTR Award

Graves’ disease (GD), one of the most prevalent autoimmune diseases, is caused by stimulating autoantibodies (TSAb) to the Thyroid-Stimulating Hormone Receptor (TSHR), resulting in hyperthyroidism. Current methods for managing hyperthyroidism in GD target excess thyroid hormone secretion by ablation or removal of the thyroid or by blocking thyroid hormone synthesis. Each of these therapies is associated with adverse effects, most commonly hypothyroidism requiring life-long replacement therapy. Importantly, none of these approaches address the underlying anti-TSHR CD4 T cell response that contributes to the continued production of TSAb auto-antibodies. Autoimmunity is the result of an imbalance in Teffector:Tregulatory (Teff:Treg) immune-cell homeostasis, and a loss of central or peripheral immune tolerance. T regulatory epitopes (Tregitopes) found in IgG stimulate proliferation and activation of natural and induced Tregs, thereby downregulating a Teff response to self-antigens. These Tregitopes when administered with MHC Class II binding self-antigen peptides, will elicit Antigen- Specific Adaptive Tolerance Induction (ASATI). Hypothesis: Tregitopes, in combination with the target antigen —TSHR CD4 T cell effector peptides — will reduce CD4 T cell activation and T-dependent B cell production of TSAb. Due to the short half-life of peptides in vivo, a practical Tregitope BioTherapeutic (Treg-BT) will need to be administered in a suitable delivery system, that improves its PK/PD properties with minimal toxicity. This project will identify TSHR peptides that stimulate a CD4 effector immune response in PBMC from GD patients, and combine the optimal peptide(s) with Tregitopes in an effective delivery vehicle. In Specific Aim 1, TSHR peptides identified to stimulate a CD4 effector cell response in GD patients will be tested for inhibition by Tregitopes in a TSHR bystander suppression assay. Tregitope peptides with and without TSHR peptides will then be formulated into two Treg-BT delivery vehicles: a) chemically ligated to a recombinant FDA-approved human serum albumin (HSA) or b) formulated into poly(lactic-glycolic acid) (PLGA) microspheres. The Treg-BT will then be tested in the TSHR bystander suppression assay with PBMC from GD patients. In Specific Aim 2, the candidate Treg-BT (HSA- Tregitope + TSHR peptide or Tregitope + TSHR peptides in microspheres, with appropriate controls) will be tested in vivo in a mouse model of GD. HLA-DR3 mice will be genetically immunized with an expression plasmid coding for the extracellular domain of TSHR to generate TSAb. After TSAb response is established, the mice will be treated with the optimized Treg-BT and serum TSAb and CD4 T cell responses will be monitored. Overall Impact: These studies will produce candidate Treg-BT which, combined with TSHR peptides, stimulate a Treg-mediated down-regulation of TSAb autoantibodies, with potential fast-track applicability to the treatment of patients with clinical Graves’ disease.

Public Health Relevance Statement:
PROJECT NARRATIVE Graves' disease (GD) is an autoimmune condition that causes over-activity of the entire thyroid gland. Current therapies include removal of the gland using surgery or radioactive iodine, or chemical treatments, that don't address the underlying cause and can have side effects. Here, we propose to test a new, potentially curative, approach to the treatment of GD that engages natural regulatory immunity, in lab assays with human T cells and in animal models of Graves’ disease.

Project Terms:
Ablation; Address; Adverse effects; Animal Model; Antibodies; Antibody Response; Antigen Targeting; Antigens; Autoantibodies; Autoantigens; Autoimmune Diseases; Autoimmune Process; Autoimmunity; B-Lymphocytes; base; Binding; Biological Assay; Biological Response Modifier Therapy; Bystander Suppression; CD4 Antigens; CD4 Positive T Lymphocytes; Cells; Chemicals; Child; Clinical; clinical application; Clinical Treatment; Code; Disease; Down-Regulation; Effector Cell; Electroporation; Epitopes; Eragrostis; Evaluation; Excision; extracellular; Extracellular Domain; Eye diseases; FDA approved; Formulation; Future; Gland; Glycolates; Graves' Disease; Half-Life; HLA-DR3 Antigen; Homeostasis; Human; Hyperthyroidism; Hypothyroidism; Immune; Immune response; Immune Tolerance; Immunity; Immunize; Immunoglobulin G; immunoregulation; improved; in vivo; Life; Link; Lymphocyte; Mediating; Methods; MHC Class II Genes; Microspheres; Modeling; Monitor; mouse model; Mus; Muscle; novel; Operative Surgical Procedures; Pathogenesis; Patients; Peptide Hormones Receptors; Peptides; Peripheral; Peripheral Blood Mononuclear Cell; Phase; Plasmids; Pregnant Women; product development; Production; Property; Radioactive Iodine; receptor binding; Recombinants; Regulatory T-Lymphocyte; Replacement Therapy; Research; response; Serum; Serum Albumin; Splenocyte; System; T cell response; T-Lymphocyte; Testing; Tetanus Toxoid; Thyroid Gland; Thyroid Hormones; Thyroid stimulating immunoglobulins; Thyrotropin Receptor; Toxic effect

Significance: Graves' disease (GD), one of the most prevalent autoimmune diseases, is caused bystimulating autoantibodies (TSAb) to the Thyroid-Stimulating Hormone Receptor (TSHR), resulting inhyperthyroidism. Current methods for managing hyperthyroidism in GD target excess thyroid hormonesecretion by ablation or removal of the thyroid or by blocking thyroid hormone synthesis. Each of thesetherapies is associated with adverse effects, most commonly hypothyroidism requiring life-longreplacement therapy. Importantly, none of these approaches address the underlying anti-TSHR CD4 Tcell response that contributes to the continued production of TSAb auto-antibodies. Autoimmunity is theresult of an imbalance in Teffector:Tregulatory (Teff:Treg) immune-cell homeostasis, and a loss of centralor peripheral immune tolerance. T regulatory epitopes (Tregitopes) found in IgG stimulate proliferationand activation of natural and induced Tregs, thereby downregulating a Teff response to self-antigens.These Tregitopes when administered with MHC Class II binding self-antigen peptides, will elicit Antigen-Specific Adaptive Tolerance Induction (ASATI). Hypothesis: Tregitopes, in combination with the targetantigen —TSHR CD4 T cell effector peptides — will reduce CD4 T cell activation and T-dependent B cellproduction of TSAb. Due to the short half-life of peptides in vivo, a practical Tregitope BioTherapeutic(Treg-BT) will need to be administered in a suitable delivery system, that improves its PK/PD propertieswith minimal toxicity. This project will identify TSHR peptides that stimulate a CD4 effector immuneresponse in PBMC from GD patients, and combine the optimal peptide(s) with Tregitopes in an effectivedelivery vehicle. In Specific Aim 1, TSHR peptides identified to stimulate a CD4 effector cell response inGD patients will be tested for inhibition by Tregitopes in a TSHR bystander suppression assay. Tregitopepeptides with and without TSHR peptides will then be formulated into two Treg-BT delivery vehicles: a)chemically ligated to a recombinant FDA-approved human serum albumin (HSA) or b) formulated intopoly(lactic-glycolic acid) (PLGA) microspheres. The Treg-BT will then be tested in the TSHR bystandersuppression assay with PBMC from GD patients. In Specific Aim 2, the candidate Treg-BT (HSA-Tregitope + TSHR peptide or Tregitope + TSHR peptides in microspheres, with appropriate controls) willbe tested in vivo in a mouse model of GD. HLA-DR3 mice will be genetically immunized with anexpression plasmid coding for the extracellular domain of TSHR to generate TSAb. After TSAb responseis established, the mice will be treated with the optimized Treg-BT and serum TSAb and CD4 T cellresponses will be monitored. Overall Impact: These studies will produce candidate Treg-BT which,combined with TSHR peptides, stimulate a Treg-mediated down-regulation of TSAb autoantibodies, withpotential fast-track applicability to the treatment of patients with clinical Graves’ disease.

Public Health Relevance Statement:
PROJECT NARRATIVE Graves' disease (GD) is an autoimmune condition that causes over-activity of the entire thyroid gland. Current therapies include removal of the gland using surgery or radioactive iodine, or chemical treatments, that don't address the underlying cause and can have side effects. Here, we propose to test a new, potentially curative, approach to the treatment of GD that engages natural regulatory immunity, in lab assays with human T cells and in animal models of Graves’ disease.

Project Terms:
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