Genetic Construction and Characterization of an Anti-Monkey CD3 Single-Chain Immunotoxin with a Truncated Diphtheria Toxin

We have previously developed a chemically conjugated anti-rhesus monkey CD3 immunotoxin FN18-CRM9 that can deplete in vivo T cells and induce long term tolerance of mismatched renal allograft in rhesus monkeys. This immunotoxin is a monkey analogue of anti-human CD3 immunotoxin UCHT1-CRM9. In this study, we cloned the light and heavy chain variable regions of anti-monkey CD3 monoclonal antibody FN18 and constructed a single-chain Fv (sFv) by linking variable light and variable heavy regions with a (Gly4Ser)3 linker. The single-chain immunotoxin DT390-FN18sFv was constructed by ligating the sFv to the carboxyl terminus of DT390, a truncated form of diphtheria toxin. The DT390-FN18sFv fusion protein was expressed in Escherichia coli and purified with Ni-RTA affinity and anion exchange columns. Similar to the chemically conjugated immunotoxin FN18-CRM9, DT390-FN18sFv can also specifically inhibit protein synthesis in primary monkey T cells in a dose-dependent manner. DT390-FN18sFv at 10(-7) mol/L or FN18-CRM9 at 10(-8) mol/L is sufficient to reduce protein synthesis of monkey primary T cells to less than 5% of the control. The 50% inhibition dosage (IC50) of FN18-CRM9 is 1 x 10(-10) mol/L, while the IC50 of DT390-FN18sFv is 1 x 10(-8) mol/ L, reflecting the lowered affinity of monovalent Fab' FN18 to its parental divalent antibody. The availability of functional FN18sFv will provide the basis for the construction of divalent anti-CD3 immunotoxins for preclinical studies on the induction of tolerance in organ transplantation and experimental autoimmune diseases.