Protein‐engineered molecules carrying GAD65 epitopes and targeting CD35 selectively down‐modulate disease‐associated human B lymphocytes

Summary Type 1 diabetes mellitus is an autoimmune metabolic disorder characterized by chronic hyperglycemia, the presence of autoreactive T and B cells and autoantibodies against self‐antigens. A membrane‐bound enzyme on the pancreatic beta‐cells, glutamic acid decarboxylase 65 (GAD65), is one of the main autoantigens in type 1 diabetes. Autoantibodies against GAD65 are potentially involved in beta‐cell destruction and decline of pancreatic functions. The human complement receptor type 1 (CD35) on B and T lymphocytes has a suppressive activity on these cells. We hypothesized that it may be possible to eliminate GAD65‐specific B cells from type 1 diabetes patients by using chimeric molecules, containing an anti‐CD35 antibody, coupled to peptides resembling GAD65 B/T epitopes. These molecules are expected to selectively bind the anti‐GAD65 specific B cells by the co‐cross‐linking of the immunoglobulin receptor and CD35 and to deliver a suppressive signal. Two synthetic peptides derived from GAD65 protein (GAD65 epitopes) and anti‐CD35 monoclonal antibody were used for the construction of two chimeras. The immunomodulatory activity of the engineered antibodies was tested in vitro using peripheral blood mononuclear cells (PBMCs) from type 1 diabetes patients. A reduction in the number of anti‐GAD65 IgG antibody‐secreting plasma cells and increased percentage of apoptotic B lymphocytes was observed after treatment of these PBMCs with the engineered antibodies. The constructed chimeric molecules are able to selectively modulate the activity of GAD65‐specific B lymphocytes and the production of anti‐GAD65 IgG autoantibodies by co‐cross‐linking of the inhibitory CD35 and the B cell antigen receptor (BCR). This treatment presents a possible way to alter the autoimmune nature of these cells.