Self-potentiation of ligand-toxin conjugates containing ricin A chain fused with viral structures.
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.
Chignola
R
R
Istituto di Immunologia e Malattie Infettive, Università di Verona, Italy.
Anselmi
C
C
Dalla Serra
M
M
Franceschi
A
A
Fracasso
G
G
Pasti
M
M
Chiesa
E
E
Lord
J M
JM
Tridente
G
G
Colombatti
M
M
D000595:Amino Acid Sequence; D001693:Biological Transport, Active; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006801:Humans; D000922:Immunotoxins / Q000493:pharmacokinetics / Q000633:toxicity*; D066298:In Vitro Techniques; D007962:Leukocytes / Q000187:drug effects; D008024:Ligands; D008969:Molecular Sequence Data; D011990:Receptors, Transferrin / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000493:pharmacokinetics / Q000633:toxicity*; D012276:Ricin / Q000493:pharmacokinetics / Q000633:toxicity*; D014168:Transferrin / Q000235:genetics / Q000378:metabolism; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000493:pharmacokinetics / Q000633:toxicity*
D000922:Immunotoxins; D008024:Ligands; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; D014168:Transferrin; D014764:Viral Proteins; D012276:Ricin