ANTI-T CELL IMMUNOTOXINS - A LOOK AT POST-ENDOCYTOTIC RECEPTOR-MEDIATED ROUTING

Many types of targeted drug delivery systems utilize cell surface receptor binding to achieve targeting specificity. Receptor-mediated endocytosis also provides internalization of the targeted drug. What has not been previously appreciated is the fact that cell surface receptors play an important post-endocytotic role. After endocytosis the receptor ligand interaction determines how the ligand-drug complex will be routed. In many cases this routing uniquely determines the overall efficacy of the drug delivery system. In this paper we will show that a unique anti-T cell immunotoxin, anti-CD3+-CRM9, constructed with a diphtheria toxin binding site mutant, CRM9, is a highly effective reagent for inducing in vivo T cell ablation. The high degree of efficacy and the wide therapeutic margin of this reagent is a consequence of the obligatory intracellular routing pathway required for diphtheria toxin (DT) intoxication. The DT receptor (DTR) typically fulfills this function in diphtheria toxin intoxication. In the case of the anti-CD3+ toxin conjugate the routing function is performed by CD3. This epitope routes parallel with DTR. This fact accounts for the high toxicity of anti-CD3 conjugates made with diphtheria toxin binding site mutants such as CRM9. CRM9 a one is incapable of entering the optimal routing pathway and hence has very low systemic toxicity. The anti-CD3 antibody doubly complements the CRM9 defect: It facilitates ( 1 ) the entrance of CRM9 into the targeted cell endosomes and (2 directs the entrance of CRM9 into the DT intoxication pathway. It is likely that these concepts will have relevance to other cell type-specific immunotoxins and other cell type-specific targeted delivery systems which depend on the membrane translocation of bio-active macromolecules into the cytosol or nuclear compartments.