Cancer Immune Therapy Edited by G. Stuhler and P. Walden ...

(DAB486, DAB389 and DT388), but retain the translocation and ADP-ribosylation
activity of DT [53]. Recombinant antibody fusion proteins with such derivatives target
only cells that bind the antibody moiety of the immunotoxin.
17.3.1.3 Bacterial toxins: PE and PE derivatives
Two major research studies have enabled the use and genetic manipulation of PE for
the design of immunotoxins [1±3]: (1) the elucidation of the crystal structure of PE,
showing the toxin to be composed of three major structural domains, and (2) the
finding that these domains are different functional modules of the toxin.
PE is a single-chain 66-kDa molecule secreted by P. aeruginosa that, like DT, irreversibly
ADP-ribosylates the diphthamide residue of EF-2, using NAD + as cofactor
[54]. As a consequence, protein synthesis is inhibited and cell death ensues. PE is
composed of three major domains [55]. Different functions have been assigned to
each domain by mutational analysis [56]. The N-terminal domain la mediates binding
to the a2-macroglobulin receptor [57]. Domain lb is a small domain that lies between
domains II and III, and has no known function [58]. Domain II mediates
translocation of domain III, the C-terminal ADP-ribosylating domain, into the cytosol
of target cells [59](Fig. 17.2). Translocation occurs after internalization of the
Fig. 17.2 The biological activity of PE A. The Fv
portion of the immunotoxin targets domains II
and III of PE to a cell surface receptor or other
target molecule on the tumor cell (A). The immunotoxin
enters the cell by internalization and
is transferred into the endosome (B). Within the
endosome the molecule unfolds due to a fall in
pH. The conformational change exposes a proteolytic
site, and a proteolytic cleavage occurs in
the translocation domain between amino acids
279 and 280 (C). A disulfide bond is then broken,
thus creating two fragments: the Fv moiety
and a small part of domain II, and the rest of domain
II connected to domain III (D). The C-terminal
fragment containing the ADP-ribosylation
domain (domain III)and most of the translocation
domain (domain II)is carried into the endoplasmic
reticulum (E), and translocation occurs
from the endoplasmic reticulum into the cytosol
(F). The enzymatically active domain ADP-ribosylates
EF-2 at a diphtamide residue located at
His415, using NAD + as a cofactor. This modification
arrests protein synthesis and subsequently
leads to cell death by apoptosis. In DT,
the poteolytic processing occurs between residues
193 and 194. The catalytic A chain (amino
acids 1±193)then translocates to the cytosol
through the endosome with the help of translocation
domain residues 326±347 and 358±376
which form an ion channel.
17.3 The Development of Recombinant DNA-based Immunotoxins
353