Autoimmune Diabetes Mellitus (ADM) is an autoimmune metabolic disorder characterized by chronic hyperglycemia, presence of autoreactive T and B cells and autoantibodies against self-antigens. A membrane-bound enzyme on the pancreatic beta-cells, GAD 65, is one of the main autoantigens in type 1 diabetes. Autoantibodies against GAD65 are potentially involved in beta-cells destruction and decline of pancreatic functions.
The human complement receptor type 1 (CR1) 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 ADM patients by using chimeric molecules, containing an anti-CR1 antibody, coupled to peptides resembling GAD65 B/T epitopes. These molecules are expected to bind selectively the anti-GAD65 specific B-cells by the co-crosslinking of the immunoglobulin receptor and CR1 and to deliver a suppressive signal.
Two synthetic peptide epitopes derived from GAD65 protein, and anti-CD35 monoclonal antibody were used for the construction of two chimeras. The immunomodulatory activity of the engineered antibodies was tested in vitro (Epitope prediction, Protein engineering, ELISA, FACS, ELISpot and Proliferation assay) and in vivo (NSG mice transfer) using PBMCs from 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 PBMCs from patients with ADM with engineered antibodies.
The constructed chimeric molecules are able to modulate selectively the activity of GAD65-specific B-lymphocytes and the production of anti-GAD65 IgG auto-antibodies by co-crosslinking of the inhibitory CR1 and the BCR.