Selective Silencing of Disease-Associated B Lymphocytes from Hashimoto's Thyroiditis Patients by Chimeric Protein Molecules.

Hashimoto's thyroiditis is one of the most common endocrine disorders, affecting up to 20% of the adult population. No treatment or prevention exists except hormonal substitution for hypothyroidism. We hypothesize that it may be possible to selectively suppress anti-thyroglobulin (Tg) IgG antibody-producing B lymphocytes from HT patients by a chimeric protein molecule containing a monoclonal antibody specific for the human inhibitory receptor CR1, coupled to peptide epitopes derived from Tg protein. We expect that this treatment will down-regulate B-cell autoreactivity by delivering a strong inhibitory signal. Three peptides-two epitope-predicted ones derived from Tg and another irrelevant peptide-were synthesized and then coupled with monoclonal anti-human CR1 antibody to construct three chimeric molecules. The binding to CD35 on human B cells and the effects of the chimeric constructs on PBMC and TMC from patients with HT were tested using flow cytometry, ELISpot assay, and immunoenzyme methods. We found that after the chemical conjugation, all chimeras retained their receptor-binding capacity, and the Tg epitopes could be recognized by anti-Tg autoantibodies in the patients' sera. This treatment downregulated B-cell autoreactivity and cell proliferation, inhibited Tg-specific B-cell differentiation to plasmablasts and promoted apoptosis to the targeted cells. The treatment of PBMCs from HT patients with Tg-epitope-carrying chimeric molecules affects the activity of Tg-specific autoreactive B lymphocytes, delivering to them a strong suppressive signal.

Suppression of Disease-Associated B Lymphocytes by GAD65 Epitope-Carrying Protein-Engineered Molecules in a Streptozotocin-Induced Mouse Model of Diabetes.

Type 1 diabetes mellitus is an autoimmune syndrome defined by the presence of autoreactive T and B cells, which results in destruction of insulin-producing beta cells. Autoantibodies against GAD65 (glutamic acid decarboxylase 65)-a membrane-bound enzyme on pancreatic beta cells, contribute to beta cells' destruction and the loss of pancreatic functions. Mouse FcγRIIb on B lymphocytes possesses an inhibitory effect on the activity of these cells. We hypothesized that it may be possible to suppress GAD65-specific B cells in mice with diabetes using chimeric molecules, containing an anti-FcγRIIb antibody, coupled to peptide B/T epitopes derived from the GAD65 protein. With these engineered chimeras, we expect to selectively co-cross-link the anti-GAD65-specific B cell receptor (BCR) and FcγRIIb, thus delivering a suppressive signal to the targeted B cells. An anti-FcγRIIb monoclonal antibody and two synthetic peptide epitopes derived from the GAD65 molecule were used for chimeras' construction. The suppressive activity of the engineered molecules was tested in vivo in mice with streptozotocin (STZ)-induced type 1 diabetes. These chimeric molecules exclusively bind disease-associated B cells by recognizing their GAD65-specific BCR and selectively deliver a strong inhibitory signal through their surface FcγRIIb receptors. A reduction in the number of anti-GAD65 IgG antibody-secreting plasmocytes and an increased percentage of apoptotic B lymphocytes were observed after treatment with protein-engineered antibodies of mice with STZ-induced type 1 diabetes.