A new immunotoxin built by linking a hemolytic toxin to a monoclonal antibody specific for immature T lymphocytes

Abstract

Hybrid molecules built by conjugation between monoclonal antibodies (MAbs) and toxins are currently being experimentally tested as potential new anti-cancer agents. These immunotoxins have mainly used the plant toxin ricin as the toxic component, which inhibits protein synthesis at the ribosome level. We present an alternative for toxic components using a hemolytic toxin acting at the membrane level, due to its phospholipase activity. The hemolytic toxin (HT), isolated from the sea anemone Stoichactis helianthus, has been conjugated to an antibody towards an antigen expressed on immature T lymphocytes (IOR-T6), by means of an artificial disulphide bridge. The hybrid IOR-T6-HT exhibits no hemolytic activity unless it is reduced. It is toxic for cells (CEM) expressing the IOR-T6 antigen and non-toxic for cells (K562) not bearing the antigen. An excess of unconjugated antibody reverses the toxicity. Immunotoxins based on membrane-active, hemolytic toxins can be a useful alternative when directed towards antigens which do not mediate internalization, as is the case for most carcinoma antigens.

The anticancer effects of actinoporin RTX-A from the sea anemone Heteractis crispa (=Radianthus macrodactylus)

“…The results shown in Table 1 indicated that RTX-A demonstrated potent cytotoxic activity (IC 50 = 1–5 nM) against human caner cell lines, including HL-60, MDA-MB-231, HeLa, THP-1, and SNU-C4. The active concentrations of RTX-A, 10 −9 M, agree with the values of the cytotoxicity obtained earlier for the α-PFT of the actinoporin family, ranging from 10 −10 to 10 −7 M, according to reviewed data (Alvarez et al, 2009; Avila et al, 1988; Avila et al, 1989; Tejuca et al, 2004). …”

Evolution of the Cytolytic Pore-Forming Proteins (Actinoporins) in Sea Anemones

“…Actinoporins have been used to elucidate cell membrane dynamics and to investigate pharmaceutically relevant biomedical applications [21,22,23,24]. Several residues have been manipulated to identify functionally important regions within the protein [25], revealing an aromatic-rich region that forms the phosphocholine (POC) binding site, with a single amino acid residue (W112 in Equinatoxin II (EqII)) taking on a key role in initiating sphingomyelin recognition and pore formation [11,26,27].…”

Construction of an immunotoxin with the pore forming protein StI and ior C5, a monoclonal antibody against a colon cancer cell line

“…Although other membrane-acting toxins have been investigated for their use in immunoconjugates [7] the first efforts involved a hemolytic toxin from the sea anemone Stichodactyla (formerly Stoichactis) helianthus, which was linked to an antibody that recognizes specific Ag expressed on immature T lymphocytes [8] or to a mAb directed against a carcinoembryonic antigen [9]. More recently, another sea anemone cytolysin, equinatoxin II (EqtII) from Actinia equina, was conjugated with promising results to transferrin, which is a major regulator of cellular growth, and a potent mitogen for a variety of tumors [10].…”