The long-term objective of this application is to develop novel recombinant immunotoxins for therapeutic use in human bone marrow transplantation and for the in vivo treatment of human leukemias and lymphomas. Recombinant immunotoxins comprise a new class of pharmacologic agents designed to be selectively cytotoxic. They are fusion proteins in which the antigen-binding (variable) regions of antibodies are linked to potent, catalytic toxins. Genetically engineered immunotoxins have several hypotheses concerning the efficacies of recombinant versus chemically-linked immunotoxins. Recombinant immunotoxins specific for the pan -T cell antigen CD7 will be used as a model immunotoxins may prove to be effective in the treatment of T -ALL. In the first specific aim, the variable region genes expressed by a CD7 will be used as a model system. CD7 is expressed on virtually all T cell acute lymphoblastic leukemias (T-ALL) and so these immunotoxins may prove to be effective in the treatment of T-ALL. In the first specific aim the variable region genes expressed by a CD7-specific hybridoma will be linked to the ricin toxin A chain (RTA) gene in different ways to determine which encodes the most efficacious immunotoxin. The potency of the these chemically conjugated to RTA. The in vivo comparisons will employ SCID mouse models for T-ALL. Internalization, pharmacokinetic, and biodistribution studies will examine what factors influence the in vivo efficacy of the recombinant and chemical immunoconjugates. In the second specific aim recombinant CD7-specific immunotoxins containing a variant of Pseudomonas exotoxin A (PE38KDEL) will be constructed and assayed. The purpose of these experiments is to determine if RTA and PE38KDEL differ in cytotoxicity when targeted to CD7. The third specific aim addresses a problem encountered in all clinical trials of RTA-immunotoxins: dose-limiting toxicity caused by endothelial cell damage. The hypothesis is that RTA alone can kill endothelial cells via receptor-mediated endocytosis. Mutants of RTA will be generated and screened for enzymatically active variants that no longer bind and kill endothelial cells. The identification of these mutants should allow for the construction of more therapeutically effective immunotoxins.
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