Development of a Novel Fusion Toxin Reagent for Targeting Antigen Presenting Cells In Vivo: DT390-CTLA-4

Abstract

Targeted cell therapies are possible through the generation of chimeric recombinant proteins that combine a toxin, such as Diptheria toxin (DT), with an antibody or other molecule that confers specificity. Upon binding of the chimeric protein to the cell of interest, the DT is internalized which results in protein synthesis inhibition and subsequent cell death. The Cytotoxic T lymphocyte antigen-4 (CTLA-4), also known as CD152, is expressed on T helper cells and functions to down-regulate T cells via binding to CD80/CD86 (B7 complex) on antigen presenting cells (APC). Blockade of the interactions between CD28/CTLA-4 and their ligands, CD80/CD86, has potential for induction of peripheral tolerance in autoimmune disease and organ transplantation. Currently, CTLA-4-Ig has proven to be an effective reagent for blocking these interactions in vivo. In our lab, we have a unique large animal model, the MGH MHC-defined miniature swine, for studying immune regulation and transplantation tolerance. We hypothesized that linking DT to CTLA-4 may provide a novel approach for in vivo depletion of APCs. We have expressed and purified recombinant soluble porcine CTLA-4 both with and without N-glycosylation in Yeast Pichia Pastoris for in vivo use in our large animal model. The glycosylated and non-N-glycosylated versions of this recombinant protein competitively inhibit the binding of an anti-CD80 antibody to a CD80 expressing porcine B cell lymphoma cell line (13271) with equal affinity (KD = 13nM). When linked to a truncated diphtheria toxin the non-N-glycosylated version of this recombinant protein proved to efficiently inhibit protein synthesis in target cells after 18 hours and significant cell death was observed after 52 hours in vitro. We are currently analyzing the effects of this DT-linked non-N-glycosylated version of recombinant CTLA-4 in vivo.