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

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.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). Rapid internalization, however, is not always synonymous with fast intoxication rates of the target cells as a result of cell mechanisms leading to inactivation of the internalized IT molecules (e.g. recycling, degradation, slow routing to subcellular compartments competent for toxin translocation) (1). The ricin toxin A chain (RTA) is a potent ribosome-inactivating enzyme used in the synthesis of highly selective IT. However, RTA-based IT exert their effect at relatively high concentrations due to poor translocation of RTA to the cell cytosol from the endocytic compartments where the IT are internalized (1).Viruses utilize specialized envelope structures that allow them to enter the cytosol of the infected cells. We reasoned that it might be possible to modify a cytotoxic enzyme (i.e. RTA) by fusing it to a protein structure derived from viral envelopes, thus conferring to the cytotoxic enzyme the cytosol targeting properties of the virus. A peptide representing the primary sequence of the 25 N-terminal amino acids of protein G of the vesicular stomatitis virus envelope (KFT25) was found to have pH-dependent membrane destabilizing properties (2, 3). In particular, at low pH, KFT25 was shown to be hemolytic, to mediate hemagglutination, to be cytotoxic for mammalian cells, and to effect gross changes in cell permeability (2, 3). Such a virus-derived structure might be endowed with the ability to facilitate the translocation of heterologous proteins across cell membranes when they are routed to acidic intracellular compartments.The transferrin receptor is a cell-surface structure known to de...

Genetic grafting of membrane-acting peptides to the cytotoxin dianthin augments its ability to de-stabilize lipid bilayers and enhances its cytotoxic potential as the component of transferrin-toxin conjugates

“…To clone the 3 chimeric dianthins (KFT25DIA, pHA2DIA and pJVEDIA), the 5Ј end of the gene was modified by PCR to insert a SoI restriction site just downstream of NcoI. The SoI site and the upstream site NcoI were used to ligate in 5Ј with respect to the toxin gene one of 3 double stranded oligonucleotides coding for: (i) the peptide pHA2 (sequence GLFEAIAGFIENGWEGMID, from the N terminus of the influenza virus hemagglutinin) (Wagner et al, 1992), (ii) the peptide pJVE (sequence GLFEALLELLE-SLWELLLEA, designed upon known properties of membraneacting peptides) (Pécheur et al, 1999), or (iii) the peptide KFT25 (sequence KFTIVFPHNQKGNWKNVPSNYHYCP, from the Nterminus of vesicular stomatitis virus protein G) (Chignola et al, 1995). The 3 new plasmids were designated pET11d-pHA2DIA, pET11d-pJVEDIA and pET11d-KFT25DIA respectively.…”

“…Moreover, ITs are essentially hydrophilic, therefore cell entry is a somewhat low-efficiency process in general, and especially in the case of ITs made with RTA or RIP-I toxins. We have shown (Chignola et al, 1995) that genetic grafting of a "membraneacting" peptide (KFT25) (Schlegel and Wade, 1984) onto RTA increased the cytotoxicity of transferrin-bound RTA, while reducing the intrinsic toxicity of unbound chimeric toxin for cells in vitro. We have here genetically linked 3 different membraneacting peptides to wild-type dianthin.…”

“…We have here genetically linked 3 different membraneacting peptides to wild-type dianthin. The 3 peptides utilised are the KFT25 peptide from the N terminus of VSV G protein (Chignola et al, 1995;Schlegel and Wade, 1984), a 19 aa peptide from the N-terminal portion of HA2 hemagglutinin of influenza virus (pHA2) (Wagner et al, 1992) and a 19 aa peptide (pJVE) based on known properties of membrane-acting peptides (Pécheur et al, 1999). To investigate whether the addition of membraneacting peptides conferred new biological properties to the chimeric dianthins, we evaluated their ability to interact with artificial membranes and to intoxicate cells.…”

Enhancement of immunotoxin activity using chemical and biological reagents

“…Thus HA-2 peptide can be used to enhance the cytotoxicity of ITs by facilitating their entry into target cells. Chignola et al (1995) modified RTA by fusing it to a protein structure derived from viral envelope, thus conferring the cytosoltargeting properties of the virus onto the cytotoxic enzyme-modified RTA. A peptide representing the primary sequence of the 25 N-terminal amino acid of protein G of the vesicular stomatitis virus envelope (KFT25) was found to have pH-dependent membrane-destabilizing properties.…”

Diving through Membranes: Molecular Cunning to Enforce the Endosomal Escape of Antibody-Targeted Anti-Tumor Toxins

“…However, immunospecificity was seriously reduced for the peptide modified targeted toxins, which were equally cytotoxic for target antigen negative cell lines (Flavell, unpublished data). Similarly, Chignola and coworkers [69] genetically fused a 19 amino acid peptide derived from the N-terminus of HA2 to ricin A chain (RTA) coupled to transferrin, but this failed to increase the cytotoxicity of this chimeric targeted toxin molecule for human transferrin receptor expressing cell lines. In contrast the same workers showed that KFT25, a 25 amino acid peptide derived from the N-terminal end of the fusogenic G protein from vesicular stomatitis virus when fused to RTA in turn conjugated to transferrin showed an almost four-fold increase in toxicity for the same cell lines over the unmodified targeted toxin transferrin-RTA.…”