CD7-Targeted Cytotoxic Potency of Diphtheria Toxin- and Ricin-Based Immunotoxins in Targeted Therapy for T-Cell Acute Lymphoblastic Leukemia.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive and heterogeneous hematologic malignancy, underscoring the urgent need for innovative treatments such as immunotoxins, which combine specific antigen targeting with potent cytotoxic activity. In this study, we developed two distinct immunotoxins (aCD7Nb/DTA and RTA/aCD7Nb) by combining the CD7-binding nanobody (aCD7Nb) with recombinant diphtheria toxin (DTA) and ricin (RTA), utilizing SpyCatcher/SpyTag (SC/ST) post translational protein ligation system for targeted therapy in T-cell acute lymphoblastic leukemia (T-ALL). Both aCD7Nb/DTA and RTA/aCD7Nb specifically bind to three CD7-expressing T-ALL cell lines, CCRF-CEM, Jurkat, and MOLT-4 cells, based on CD7 expression levels, but not to the CD7-negative Raji B-ALL cells. Both aCD7Nb/DTA and RTA/aCD7Nb demonstrated high cytotoxic against T-ALL cells, with IC₅₀ values inversely correlated to CD7 expression. Notably, RTA/aCD7Nb exhibited approximately 2-fold higher anticancer efficacy compared to aCD7Nb/DTA in both CCRF-CEM and Jurkat cells. In an orthotopic model of CCRF-CEM T-ALL-engrafted NSG mice, systemic administration of RTA/aCD7Nb effectively inhibited T-ALL progression and extended survival, without any adverse effects. These findings underscore the potential of combining a CD7-binding ligand with an appropriate active toxin moiety to significantly enhance the efficacy of immunotoxins against T-ALL.