GENERATION AND CHARACTERIZATION OF AN ANTI-CD19 SINGLE-CHAIN FV IMMUNOTOXIN COMPOSED OF C-TERMINAL DISULFIDE-LINKED DGRTA

Our laboratory utilized two methods to produce the anti-CD19 immunotox in containing a single-chain Fv (scFv) FVS191 and a ricin A chain (RTA ). The first method produced the recombinant protein FVS191CDRTA from a fusing gene containing sequences encoding FVS191, catheptsin D prote inase digestion site (CD), and RTA. FVS191CDRTA did not show CD19 anti gen binding and cytotoxic activity. The second method generated a disu lfide-linked FVS191cys-dgRTA from a FVS191cys, the FVS191 with an addi tional C-terminal cysteine, and a deglycosylated RTA(dgRTA). The forma tion of FVS191cys-dgRTA is efficient; up to 70% of the proteins partic ipating in the reaction had formed FVS191cys-dgRTA when the molar rati o of FVS191cys to dgRTA was 1:1. A competitive ELISA assay indicated t hat FVS191cys-dgRTA and the parental monoclonal antibody B43 possessed comparable CD19 binding abilities. The protein synthesis inhibition a ssay revealed that FVS191cys-dgRTA was toxic to CD19 positive cell lin es, but it was less potent than the intact antibody-conjugated B43-dgR TA, which had an IC50 = 2 x 10(-11) M. I-125-Labeled FVS191 and I-125- labeled B43 were internalized by Nalm-6 cells at 37 degrees C as demon strated by internalization studies; this result indicates that cross-l inking of CD19 antigen is not required for the endocytosis of CD19 and raises the possibility that the lower cytotoxity of FVS191cys-dgRTA i s not due to the monovalent binding of CD19 by FVS191cys-dgRTA. Our st udy with anti-CD19 scFv immunotoxin indicates that the formation of a disulfide-linked scFv immunotoxin is an alternative to the recombinant method of producing scFv immunotoxin.