CLASSIC IMMUNOTOXINS WITH PLANT OR MICROBIAL TOXINS - Fusion Protein Technologies for Biopharmaceuticals: Applications and Challenges - page 263

TABLE 18.1 Toxins Used in Immunotoxin Preparation

Type Function Toxins

Cytosol-acting
toxins

Protein synthesis inhibition by
ADP-ribosylation of EF-2

Holotoxins: diphtheria toxin [1], Pseudomonas extotoxin [2]

Protein synthesis inhibition by
cleavage of ribosomal RNA

Holotoxins: ricin [3], abrin [4], viscumin [5], nigrin b [6]
Hemitoxins: gelonin [7], saporin [8], pokeweed antiviral protein [9], luffin [10],
bouganin [11], trichokirin [12], trichosanthin [13], barley ribosome inactivating
protein [14], ebulin [15], bryodin [16], momordin [17], momorchochin [18],
moschatin [19], dianthin 30 [20], ocymoidine [21], pyramidatine [21], colocin 1
[22], a -sarcin [23], mitogillin [24], clavin [25], restrictocin [26], barnase [27]
Modification of signaling pathways Holotoxins: Anthrax toxin [28]
Inhibition of exocytosis (secretion) Holotoxins: Botulinum neurotoxins [29]

Membrane-
acting
toxins

Disintegration of vital membrane
function

Hemitoxins: Clostridia perfringens phospholipase C [30], Pyrularia thionin [31]

Membrane pore formation Hemitoxins: proaerolysin [32], Cyt1Aa toxin [33]

induces anti-immunotoxin antibodies that neutralize biologi-
cal activities of immunotoxins. In particular, most individuals
have anti-diphtheria toxin antibody titers due to childhood
and adult vaccination programs of DPT (diphtheria, pertussis,
and tetanus). The preexisting anti-diphtheria antibodies may
reduce biological activities of diphtheria immunotoxins. In
this chapter, overview of toxins used in immunotoxin prepa-
ration, recent progress in immunotoxin design and synthesis,
clinical updates of immunotoxin trials, and challenges and
perspectives in the immunotoxin field are discussed.

immunotoxins, the receptor-binding domain is replaced with
tumor-selective ligands. Binding induces receptor-mediated
endocytosis followed by localization to the endosomes.
Because different receptors have different preferences for
intracellular routing some receptors achieve a limited pene-
tration to the late acidic endosome. These DT-ligand com-
binations exhibit suboptimal target toxicity [37]. In the
acidic environment of the endosomes, DT unfolds partially.
This exposes hydrophobic regions on the translocation
domain and triggers membrane insertion to form a channel
for translocation of the catalytic domain [38-40]. Unfolding
of the catalytic domain is also required for cytosolic entry
through the translocation channel [41,42]. Chaperonic
action of cytosolic Hsp90 (heat shock protein 90) and the
membrane-inserted translocation domain translocates
the unfolded catalytic domain to the cytosol [43,44]. For
the release of the catalytic domain into the cytosol, cytosolic
TrR-1 (thioredoxin reductase) reduces the disulfide bond
between the catalytic and translocation domains [44]. The
translocated catalytic domain refolds and catalytically inac-
tivates cellular protein synthesis by ADP-ribosylating the
diphthamide residue in domain IV of elongation factor-2
(EF-2) [45]. One single molecule of catalytic domain in the
cytosol is sufficient to block protein synthesis and lead to
death of the target tumor cell [46]. Three forms of DT have
been used in immunotoxin preparation (Figure 18.1).
CRM107 and CRM9 are full-length DT mutant forms
lacking binding function. DAB 486 ,DAB 389 ,DT 388 , and
DT390 are truncated forms of DT. The first 388 amino
acid residues of mature DT contain the catalytic domain
and translocation domain essential for cell intoxication.

18.2 TOXINS USED IN IMMUNOTOXIN
PREPARATION

Many toxins have been used in immunotoxin preparation
(Table 18.1). DT, PE, ricin, saporin, gelonin, and PAP
immunotoxins have been tested in clinical trials. DT and
PE immunotoxins currently in clinical trials are mostly
recombinant proteins because recombinant DT and PE
immunotoxins can be easily produced in Escherichia coli
and/or Pichia pastoris. DT and PE immunotoxins are more
potent against target cells and have less side effects in trials
as compared to plant toxins including ricin, saporin, gelonin,
and PAP. Ricin, saporin, gelonin, and PAP immunotoxins
tested in humans are chemically conjugated proteins.

18.2.1 Diptheria Toxin

DT is a single chain protein of 535 amino acid residues
produced by Corynebacterium diphtheriae. It consists of
two subunits, an A chain (catalytic domain) and a B chain,
connected by a furin-cleavable linker that is an arginine-rich
loop formed by one disulfide bond [34]. The cleavage of the
furin-cleavable linker is a prerequisite for DT intoxication of
cells [35,36]. The B chain contains a translocation domain
and a receptor-binding domain [34]. In recombinant DT

18.2.2 Pseudomonas Exotoxin

PE is a single chain protein of 613 amino acid residues
produced by Pseudomonas aeruginosa. It consists of three