Immunotoxin FN18-CRM9 induces stronger T cell signaling than unconjugated monoclonal antibody FN18

Background. The T-cell receptor (TCR)/CD3 complex is the target of therapeu tic strategies aimed at prolonging allograft survival. The immunotoxin FN18 -CRM9, composed of the anti-CD3 monoclonal antibody FN18 and the mutated di phtheria toxin CRM9, is useful for prolonging allograft survival in preclin ical models of transplantation. To explore the influence of conjugation of the mutated diphtheria toxin on functional activation of the TCR/CD3 comple x, we compared the effects of FN18-CRM9 and unconjugated FN18 on protein ty rosine phosphorylation and ligand/receptor internalization in purified monk ey peripheral blood T cells. Methods. Purified normal rhesus monkey T cells were incubated with unconjug ated FN18 or conjugated FN18-CRM9 and examined for differences in antibody binding, tyrosine phosphorylation, and CD3 internalization. Results. Binding cross-inhibition studies demonstrated that both compounds were able to inhibit fluorescein isothiocyanate-FN18 binding to CD3 with si milar efficacy and potency. However, FN18-CRM9 was more potent than FN18 in triggering the phosphorylation of several proteins on tyrosine residues an d in inducing CD3 internalization. The tyrosine kinase inhibitor genistein blocked FN18-CRM9-induced protein tyrosine phosphorylation and CD3 internal ization, suggesting that tyrosine phosphorylation is involved in the intern alization of the immunotoxin. Interestingly, in FN18-CRM9- but not FN18-tre ated cells, there was a gradual decrease in cellular CD3 protein levels wit hin 24 and 48 hr; such a decrease was not observed with the control protein Csk. Conclusions. Our findings suggest that the conjugation of the mutated dipht heria toxin CRM9 to FN18 modulates the monoclonal antibody-mediated crossli nking of the TCR/CD3 complex, leading to a stronger protein tyrosine phosph orylation and CD3 internalization. This may in turn contribute to the great er efficacy of the immunotoxin in prolonging allograft survival.