Generation and Characterization of an Anti-CD19 Single-Chain Fv Immunotoxin Composed of C-Terminal Disulfide-Linked dgRTA

Abstract

Our laboratory utilized two methods to produce the anti-CD19 immunotoxin 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 proteinase digestion site (CD), and RTA. FVS191CDRTA did not show CD19 antigen binding and cytotoxic activity. The second method generated a disulfide-linked FVS191cys-dgRTA from a FVS191cys, the FVS191 with an additional C-terminal cysteine, and a deglycosylated RTA (dgRTA). The formation of FVS191cys-dgRTA is efficient; up to 70% of the proteins participating in the reaction had formed FVS191cys-dgRTA when the molar ratio of FVS191cys to dgRTA was 1:1. A competitive ELISA assay indicated that FVS191cys-dgRTA and the parental monoclonal antibody B43 possessed comparable CD19 binding abilities. The protein synthesis inhibition assay revealed that FVS191cys-dgRTA was toxic to CD19 positive cell lines, but it was less potent than the intact antibody-conjugated B43-dgRTA, which had an IC50 = 2 x 10(-11) M. 125I-Labeled FVS191 and 125I-labeled B43 were internalized by Nalm-6 cells at 37 degrees C as demonstrated by internalization studies; this result indicates that cross-linking of CD19 antigen is not required for the endocytosis of CD19 and raises the possibility that the lower cytotoxity of FVS191cys-dgRTA is not due to the monovalent binding of CD19 by FVS191cys-dgRTA. Our study 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.