Self-potentiation of Ligand-Toxin Conjugates Containing Ricin A Chain Fused with Viral Structures (∗)

• Published in: The Journal of biological chemistry Vol. 270; no. 40; pp. 23345 - 23351
• Main Authors: Chignola, Roberto, Anselmi, Cristina, Serra, Mauro Dalla, Franceschi, Antonia, Fracasso, Giulio, Pasti, Marcella, Chiesa, Elena, Lord, J. Michael, Tridente, Giuseppe, Colombatti, Marco
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 06.10.1995; American Society for Biochemistry and Molecular Biology
• Subjects: Amino Acid Sequence; Biological Transport, Active; Cell Death - drug effects; Cell Line; Cell Membrane - metabolism; Cytosol - metabolism; Humans; Immunotoxins - pharmacokinetics; Immunotoxins - toxicity; In Vitro Techniques; Leukocytes - drug effects; Ligands; Molecular Sequence Data; Receptors, Transferrin - metabolism; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - pharmacokinetics; Recombinant Fusion Proteins - toxicity; Ricin - pharmacokinetics; Ricin - toxicity; Transferrin - genetics; Transferrin - metabolism; Vesicular stomatitis Indiana virus - genetics; vesicular stomatitis virus; Viral Proteins - genetics; Viral Proteins - pharmacokinetics; Viral Proteins - toxicity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.270.40.23345

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.