Abstract
A chimeric protein was obtained by fusing together the ricin toxin A chain (RTA) gene and a DNA fragment encoding the N terminus of protein G of the vesicular stomatitis virus. Chimeric RTA (cRTA) retained full enzymic activity in a cell-free assay, but was 10-fold less toxic against human leukemic cells than either native RTA (nRTA) or unmodified recombinant RTA (rRTA). However, conjugates made with cRTA and human transferrin (Tfn) showed 10-20-fold greater cell killing efficacy than Tfn-nRTA or Tfn-rRTA conjugates despite equivalent binding of the three conjugates to target tumor cells. As a consequence, by fusion of the KFT25 peptide to the RTA sequence, the specificity factor (i.e. the ratio between nonspecific and specific cytotoxicity) of Tfn-cRTA was increased 90-240 times with respect to those of Tfn-nRTA and Tfn-rRTA. cRTA interacted with phospholipid vesicles with 15-fold faster kinetics than nRTA at acidic pH. Taken together, our results suggest that the ability of vesicular stomatitis virus protein G to interact with cell membranes can be transferred to RTA to facilitate its translocation to the cell cytosol. Our strategy may serve as a general approach for potentiating the cytotoxic efficacy of antitumor immunotoxins.
Highlights
Cell-surface structures mediating the efficient internalization of cell-bound molecules are frequently selected as targets of monoclonal antibody/ligand-toxin conjugates (immunotoxins (IT)1) [1]
We show that a KFT25-containing ricin toxin A chain (RTA) (chimeric RTA) exhibits a greater cytotoxic activity when delivered to tumor cells by Tfn than analogous conjugates containing either native RTA or unmodified recombinant RTA
Three forms of RTA were used in our experiments: native RTA (nRTA) (i.e. RTA prepared from native ricin that had been purified from the seeds of Ricinus communis), unmodified recombinant RTA (rRTA), and Chimeric RTA (cRTA)
Summary
Cell-surface structures mediating the efficient internalization of cell-bound molecules are frequently selected as targets of monoclonal antibody/ligand-toxin conjugates (immunotoxins (IT)1) [1]. Comparing Cytotoxicity of Free Toxins and of Their Tfn Conjugates—To compare the cytotoxic activity of nRTA, cRTA, and rRTA and their Tfn conjugates, the concentration of the cytotoxic agents inhibiting 50% of the protein synthesis of target tumor cells (IC50) was considered.
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