Conjugated Killing Moieties for Targeted T cell Depletion in Cancer Immunology

Received January, 2009

Abstract
Immunotoxins are chimeric molecules designed to kill specific cells. They contain a targeting moeity that delivers a cytotoxic agent to cells of interest. These target cells are typically malignant cells, although non-transformed cells may also be chosen for elimination. The ultimate goal of this project is have the ability to selectively delete specific CD4+ T regulatory cells. Because T regulatory cells suppress anti-tumor immune responses, we reasoned that removing them should enhance the efficacy of cancer vaccines. The antigens recognized by CD4+ T cells are peptides presented by major histocompatability complex II (pMHCII). The avidity afforded by multimeric pMHCII complexes permits the detection of antigen specific CD4+ T cells, and by extension, immunotoxins containing multimeric pMHCII targeting moiety should kill antigen-specific cells. First we explored various approaches to the conjugation chemistries to disulfide link Ricin Toxin Alpha chain to an antibody that functions as a targeting moiety.
We then biotinylated this antibody to form a tetrameric immunotoxin with Streptavidin linked to Ricin Toxin Alpha chain. Both the monomeric and tetrameric forms of the immunotoxin were specifically cytotoxic. Additionally, we advanced proof of principle studies with conjugates containing appropriate chemical linkages for the delivery of nucleic acid based silencing and killing moieties. We are now poised to extend this technology to develop pMHCII tetramers to ablate antigen specific CD4+ T regulatory cells with surgical precision using both protein based toxins and nucleic acid based silencing and killing approaches.