B cell directed tolerogenic fusion proteins

We previously demonstrated that retroviral delivery of antigen-IgG fusion proteins in B cells ex vivo rendered these cells tolerogenic. Such B cells, upon adoptive transfer, could block or reverse the adverse immune responses in rodent models of multiple sclerosis, type 1 diabetes and hemophilia. In order to create a non-viral­ delivery system taking advantage of the tolerogenicity of IgG fusion proteins directed into B cells, we have prepared a fusion comprising anti-human CD20 single chain variable fragment and with target T-cell epitope on a human IgG4 scaffold. We term this fusion a B-cell-specific antigen IgG tolerogen (BAIT). As an example, the BAIT fusion containing a DR1 restricted T cell epitope FVIII 2191-2210 (BAIT-FVIII 2191 ) was prepared in a CHO cell expression system via stable transfection. Ex vivo analyses demonstrated that biotinylated BAIT-FVIII 2191 could be specifically taken up by CD20+ve B cells from human PBMC. In this report, we will evaluate the presentation of B-cell directed BAIT-FVIII 2191 to FVIII 2191-2210 specific human T cells, and then test in vivo using DR1-transgenic FVIII KO mice. The results obtained from these studies will facilitate the translation of our successful B-cell gene therapy approach into a novel protein therapeutic for adverse immune responses, such as anti-FVIII inhibitor development in hemophilia A.