Activity of a recombinant fusion protein between transforming growth factor type alpha and Pseudomonas toxin.

Abstract

Pseudomonas exotoxin has been crystallized, and x-ray diffraction analysis of the crystal structure of PE shows that the toxin is composed of three distinct domains (7). Using the three-dimensional structure as a guide, deletion mutants of the PE structural gene were constructed and the proteins were encoded by the different domains of PE expressed in Escherichia coli (8). These studies have shown that domain I of PE is responsible for cell recognition, domain II is responsible for translocation of the toxin across membranes, and domain III is responsible for ADP-ribosylation of elongation factor 2, the step actually responsible for cell death (Fig. 1) (3.7 kb) was ligated to the 155-bp fragment. Restriction analysis using BamHI and Sph I was used to identify the plasmid with TGF-a gene in the proper orientation (pVC33).

Targeted inhibition of tumour cell growth by a bispecific single-chain toxin containing an antibody domain and TGF α

“…Although insertion at certain result in reduced cytotoxic activity, Pseudoi A has proved to be surprisingly flexible possible integration sites for small heterologous binding domains. ETA fusion proteins containing TGFa, either Nterminal of the ETA translocation domain II (Siegall et al, 1989) similar to TGFcx-ETA in our study, or at the Cterminus of the enzymatic domain III (Chaudhary et al, 1987), have been derived and were biologically active. The activity of proteins with a heterologous binding domain inserted between domains II and III of ETA seems to be dependent upon the size and/or nature of the ligand.…”

“…Several recombinant Pseudomonas exotoxin A fusion proteins binding to EGFR or ErbB-2 have been described (Chaudhary et al, 1987;Wels et al, 1992Wels et al, , 1995Batra et al, 1992). Since many tumour cells express both ErbB-2 and EGFR, therapeutic reagents binding to both receptor proteins might offer advantages over monospecific molecules by inducing the formation of receptor heterodimers which in turn might lead to more rapid uptake of toxin-receptor complexes.…”

EGFR‐targeted Chimeras of Pseudomonas ToxA released into the extracellular milieu by attenuated Salmonella selectively kill tumor cells

“…Multiple forms of ToxA chimeras have previously been generated that are capable of specifically killing cancer cells overexpressing EGFR. One of the early constructs had the binding domain Ia (DIa; amino acids 1–252 of the mature peptide as numbered following cleavage of the signal sequence) deleted and substituted with the epidermal growth factor receptor (EGFR) ligand TGFα fused to the remaining domains II, Ib, and III consisting of amino acids 253–613 and was designated TGFα-PE40 (Chaudhary et al, 1987; Siegall et al, 1989b). A reduction of a portion of PE40 by removal of amino acids 365–380 from domain Ib (DIb) was used to generate PE38 (Siegall et al, 1989a), which exhibited increased cytotoxicity to cancer cells.…”

A novel vascular endothelial growth factor-directed therapy that selectively activates cytotoxic prodrugs

“…Prime examples are the fusions of cellbinding ligands with the catalytic subunit of Pseudomonas exotoxin A, which lacks cell-binding activity. The first example was fusion with transforming growth factor-type alpha (Chaudhary et al, 1987). Since then, a wide range of fusion partners have been considered, including interleukins (Chaudhary et al, 1989;Batra et al, 1991Batra et al, , 1992Kreitman et al, 1992;Gawlak et al, 1993;Kunwar et al, 1993;Francisco et al, 1995;Siegall et al, 1995), other growth factors (Prior et al, 1991;Gawlak et al, 1993;Kunwar et al, 1993;Mesri et al, 1993;Siegall et al, 1995), single chain antibody fragments (Chaudhary et al, 1989;Batra et al, 1991Batra et al, , 1992Kreitman et al, 1992;Francisco et al, 1995) and hormones (Ben Yehudah et al, 1999).…”