Immunotoxins (IT) are a new class of pharmacologic agent consisting of monoclonal antibodies (mAb) linked to potent catalytic toxins such as ricin toxin A chain (RTA). While efficacy of IT has been observed, toxic side effects have limited the efficacy in established animal models and in clinical trials. Theoretically, IT should provide specific target cell elimination since the mAb portion is directed against a specific epitope and is required for the internalization of the toxin moiety which by itself can not efficiently internalize. Therefore, it is somewhat surprising that these reagents do not have a higher therapeutic index in vivo. The goal of this proposal is to perform a comprehensive, well-controlled study dealing with IT toxicity in order to understand the mechanisms involved with the impaired efficacy in vivo, and then, to design second and third generation IT which bypass these toxicity mechanisms. We have chosen to focus this proposal on anti-Ly1-RTA since anti-Ly1 recognizes the murine homologue of human CD5. Anti-human CD5-RTA is now in clinical trials for graft-versus- host-disease (GVHD) treatment. We will test the hypothesis that different regions of the IT molecule are responsible for different IT toxicities. We will focus on the most problematic IT toxicities including hepatic, vascular, and renal toxicities and we will determine the contribution of antibody activation to IT toxicity. Since the major interest of our group is the application of IT to the field of bone marrow transplantation, we will test the previously unexplored hypothesis that irradiation enhances in vivo anti-Ly1-RTA toxicity. We will analyze the relative contribution of the recipient's microenvironment as compared to lymphohematopoietic system by using non-transplanted or irradiated and syngeneically transplanted congeneic mice which differ in Ly1 alleleic determinants. Once we have established a relationship between certain regions of the IT molecule and toxicity, we will investigate modifications chosen on the basis of their ability to reduce toxicity related to the toxin or antibody moeity of IT. We will test the hypothesis that removal of sugars from RTA will reduce IT toxicity. We will test the hypothesis that toxicities can be further reduced by the removal of the Fc region of the mAb. If these modifications can reduce toxicity without sacrificing IT activity, we will test modified anti-Ly1-RTA for efficacy in our established GVHD model which has proven to be limited by the toxicity of unmodified anti-Ly1-RTA in past published studies. These studies should provide valuable insights into the clinical toxicities observed with current IT usage.
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