Our purpose is to continue our basic and clinical research determining the usefulness of immunotoxins (IT) in bone marrow transplantation (BMT). It will be utilized by the U. of Minnesota BMT Program to treat a variety of otherwise lethal clinical disorders including acute and chronic leukemia. In vivo use of IT - We will determine the usefulness of IT for systemic therapy. Because hemitoxins do not bind eukaryotic cells, we will link them to monoclonal antibodies and test their potential for systemic treatment of GVHD, for promotion of engraftment in allogeneic BMT, and for more effective conditioning for BMT for leukemia. We will also study IT synthesized using recombinant toxins which may have the added in vivo advantages of homogeneity, absence of carbohydrate residues that may effect homing, and the capacity for genetic alteration. Animal experiments will also be used to study homing of IT to target cells, biodistribution, pharmacokinetics, LD50, and damage to nontarget tissue. Ex vivo use of IT - We will continue using IT to purge GVHD-causing cells from normal donor marrow prior to allogeneic BMT. We will optimize our current approach using intact ricin IT by investigating various IT, e.g. T cell subpopulation directed IT, and pan-T IT sparing NK cells. We will emphasize the use of various in vitro functional assays as models of in vivo GVHD. Efficacy of IT will be determined at the clonal level using a variety of phenotypically and functionally different human T cell clones. We will continue our ongoing clinical trials to determine which IT will be the most efficacious for GVHD prophylaxis without concomitant engraftment problems. We will continue the clinical use of IT to purge residual leukemia cells from BM prior to autologous BMT. Choice of IT for purging T or B-lineage leukemia will be based on IT activity against fresh leukemic progenitor cells measured in novel colony assays and IT potency measured using clonogenic assays by limiting dilution. We will study ways of producing better IT. We will determine the role of differences in antibody subclass, FC portion, epitope recognition, and affinity for the potency of IT. Each IT will be evaluated in numerous biological and biochemical assays for purity, specificity, and potency. Our major goals are 1) to develop protocols for the in vivo use of IT in the treatment of GVHD, leukemia, and graft promotion; 2) to optimize our in vitro IT protocols that are currently undergoing clinical evaluation.
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