Báo cáo khoa học: A guide to taming a toxin – recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer ppt

REVIEW ARTICLE
A guide to taming a toxin – recombinant immunotoxins
constructed from Pseudomonas exotoxin A for the
treatment of cancer
John E. Weldon and Ira Pastan
Laboratory of Molecular Biology, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA
Introduction
The natural world abounds with an enormous variety
of toxins, comprising poisonous substances that are
naturally produced by living organisms [1]. Typically,
only small quantities of toxins are necessary to damage
cells, although the specific target and the toxic dose
may vary extensively. Microorganisms secrete toxins as
virulence factors during pathogenic infection, and as
secondary metabolites that can contaminate local envi-
ronments. Well known examples include diphtheria
toxin and ergot alkaloids. Higher organisms use toxins
as components in complex venoms and accumulate
them as defense factors to deter predators. Overall,
toxins can take many forms, appear in sizes ranging
from small molecules to large proteins, and have
diverse mechanisms of action, although they normally
serve similar functions related to predation and ⁄ or
defense.
Although frequently hazardous and occasionally
lethal, many toxins have the potential for therapeutic
application by removing the molecule from its natural
Keywords
antibody conjugates; cancer therapy;
intracellular trafficking; moxetumomab
pasudotox; Pseudomonas exotoxin A;

recombinant immunotoxins
Correspondence
I. Pastan, Laboratory of Molecular Biology,
National Cancer Institute, 37 Convent Drive,
Room 5106, Bethesda, MD 20892-4264,
USA
Fax: +1 301 402 1344
Tel: +1 301 496 4797
E-mail:
(Received 6 April 2011, accepted 16 May
2011)
doi:10.1111/j.1742-4658.2011.08182.x
Pseudomonas exotoxin A (PE) is a highly toxic protein secreted by the
opportunistic pathogen Pseudomonas aeruginosa. The modular structure
and corresponding mechanism of action of PE make it amenable to exten-
sive modifications that can redirect its potent cytotoxicity from disease to a
therapeutic function. In combination with a variety of artificial targeting
elements, such as receptor ligands and antibody fragments, PE becomes a
selective agent for the elimination of specific cell populations. This review
summarizes our current understanding of PE, its intoxication pathway, and
the ongoing efforts to convert this toxin into a treatment for cancer.
Abbreviations
aEF2, archaeal translation elongation factor 2; ALL, acute lymphoblastic leukemia; CE, cholera exotoxin; CT, cholera toxin; dsFv, disulfide-
stabilized variable fragment; DT, diphtheria toxin; eEF2, eukaryotic translation elongation factor 2; ER, endoplasmic reticulum; ERAD,
ER-associated degradation; Fv, variable fragment; HCL, hairy cell leukemia; IL, interleukin; KDEL-R, KDEL receptor; LRP, low density
lipoprotein receptor-related protein; PDI, protein disulfide-isomerase; PE, Pseudomonas exotoxin A; RIT, recombinant immunotoxin;
scFv, single-chain Fv.
FEBS Journal 278 (2011) 4683–4700 Journal compilation ª 2011 FEBS. No claim to original US government works 4683
context. Strategies such as altering the route of deliv-
ery, changing the dose, eliminating supporting or syn-

ergizing molecules (e.g. from a complex mixture such
as venom) or even modifying the structure of the mole-
cule may convert a dangerous toxin into a valuable
therapeutic resource. One recent example comprises
the botulinum toxins, which are potent paralytic neu-
rotoxins produced by the microbes of the Clostridium
genus, most notably Clostridium botulinum. Botulinum
toxin type A has been approved as the drug onabotuli-
numtoxinA (Botox
Ò
and Botox Cosmetic
Ò
; Allergan,
Inc., Irvine, CA, USA) for both therapeutic and cos-
metic purposes. Although the toxin has an estimated
human LD
50
of approximately 1 ngÆkg
)1
body weight
[2], the extremely low dose employed clinically and its
delivery via a site-specific injection make the agent safe
for widespread use.
Other toxins must be more heavily modified for
therapeutic purposes. Diphtheria toxin (DT) is an
extremely potent cytotoxic protein that is the primary
virulence factor secreted by the bacterium Corynebacte-
rium diphtheriae, which is the pathogen that causes the
disease diphtheria [3]. The LD
50

of diphtheria toxin in
humans has been reported as £ 100 ng kg
)1
body
weight [2], yet the toxin was converted into the first
recombinant toxin to be approved by the Food and
Drug Administration for the intravenous therapy of
cutaneous T-cell lymphoma. Denileukin diftitox (On-
tak
Ò
; Eisai Inc., Woodcliff Lake, NJ, USA) is a
recombinant form of DT that has been engineered by
replacing the native receptor-binding domain of DT
with interleukin (IL)-2. This substitution alters the tar-
get of the toxin from the membrane-associated hepa-
rin-binding epidermal-growth-factor-like growth factor
[4] to the IL-2 receptor, redirecting its potent cytotoxi-
city toward a therapeutic purpose [5,6].
A comparable strategy to alter the target of an intra-
cellular toxin has been employed for Pseudomonas exo-
toxin A (PE), a protein toxin with many similarities to
DT. PE and DT are only distantly related, although
they both belong to a class of cytotoxic proteins (i.e.
the A-B toxins) that require cellular uptake through
receptor-mediated endocytosis for activity. The overall
structure of these proteins consists of a receptor-binding
domain (B subunit) linked to a domain with cytotoxic
activity (A subunit) that is delivered to the cytosol.
Although their B subunits have very different targets,
the A subunit of both PE and DT is a NAD

+
-diphtha-
mide ADP-ribosyltransferase (EC 2.4.2.36), which tar-
gets and inactivates eukaryotic translation elongation
factor 2 (eEF2). This halts protein synthesis and eventu-
ally leads to cell death. A recently identified third mem-
ber of the NAD
+
-diphthamide ADP-ribosyltransferase
toxin subfamily, cholera exotoxin (CE, also known as
cholix toxin) from Vibrio cholerae, has extensive
sequence (36% identity, 50% similarity) and structural
(2.04 A
˚
C
a
rmsd) resemblance to PE and presumably
utilizes a similar intoxication pathway [7,8]. PE, CE,
DT and other toxins that utilize receptor-meditated
endocytosis can potentially be redirected for therapeutic
purposes by replacing their native receptor-binding
domains with other targeting elements. This review dis-
cusses our current understanding of PE intoxication
and efforts to convert PE into a viable therapeutic
agent.
PE
Pseudomonas aeruginosa is a ubiquitous, Gram-nega-
tive, aerobic bacillus that is often encountered as an
opportunistic human pathogen, although infections in
healthy individuals are rare. Approximately 10% of

hospital-acquired infections are caused by P. aerugin-
osa, and certain patient populations, such as individu-
als with cystic fibrosis or burn wounds, are especially
prone to this infection [9]. The bacterium is known to
possess a number of virulence determinants, the most
toxic of which is the protein PE [10]. Studies in mice
have identified the median lethal dose of PE as being
approximately 200 ng, and evidence suggests that PE
may play a major role in the virulence of P. aeruginosa.
Strains of P. aeruginosa deficient in PE production
are less virulent than strains producing PE, and
patients who survive infection from PE-producing
strains typically have high antibody titers against PE
[3,11].
PE (GenBank accession number AAB59097) is syn-
thesized as a single 638 residues (69 kDa) polypeptide
that is processed by the removal of a 25 residues
N-terminal sequence before secretion as the 613 resi-
dues (66 kDa) native toxin (all sequence numbering in
this review is based on the 613 residues native toxin).
The initial X-ray crystallographic structure of native
PE revealed three major structural domains [12]. The
N-terminal domain I is divided into nonsequential but
structurally adjacent domains Ia (residues 1–252) and
Ib (365–404). The residues between domains Ia and Ib
comprise domain II (253–364) and the remaining
C-terminal residues make up domain III (405–613).
Native PE contains eight cysteines that form four disul-
fide bonds in sequential order: two lie in domain Ia
(C11-C15 & C197-C214), one lies in domain II (C265-

C287) and one lies in domain Ib (C372-C379). Figure 1
illustrates the domain structure of native PE.
Functionally, domain I of PE is the receptor-binding
domain, and is the major component of the B subunit.
Cancer therapy based on Pseudomonas exotoxin A J. E. Weldon and I. Pastan
4684 FEBS Journal 278 (2011) 4683–4700 Journal compilation ª 2011 FEBS. No claim to original US government works
It targets the low density lipoprotein receptor-related
protein (LRP)1 (also known as CD91 or the a
2
-macro-
globulin receptor) or the closely-related variant LRP1B
for subsequent cellular internalization by receptor-
mediated endocytosis [14,15]. Domain III is the cata-
lytically active domain, and is the primary constituent
of the A subunit. It catalyzes the inactivation of eEF2
by transferring an ADP-ribosyl group from NAD
+
to
the diphthamide residue, a highly conserved, post-
translationally modified histidine that is unique to
eEF2. Although domain III is structurally defined by
residues 405–613 of the native toxin, full catalytic
activity requires a portion of domain Ib [16,17]. We
have defined the catalytically functional domain III as
consisting of residues 395–613 [18]. Domain II was
proposed to be involved in toxin translocation and
intracellular trafficking, although supporting evidence
for this function is not consistent.
PE-based therapeutics
PE can be converted into an agent that selectively

eliminates cells by changing its target to a different cell
surface receptor. The new target is typically specified
by attaching either an anti-receptor antibody or a
receptor ligand to PE through chemical conjugation or
recombinant protein engineering. Our laboratory has
focused efforts over many years on the generation of
PE-based recombinant immunotoxins (RITs), which
are recombinant proteins that combine antibodies with
protein toxins. Initial studies in which full-length PE
was chemically conjugated to whole mAbs or receptor
ligands [19,20] gradually gave way to the more efficient
production of recombinant molecules in which domain Ia
of PE was replaced by a ligand [21] or the variable
fragment (Fv) of a mAb [22]. Single-chain Fv (scFv)
molecules, which utilize the heavy chain (V
H
) and light
chain (V
L
) fragments of the Fv covalently connected
with a flexible polypeptide linker sequence [23,24],
were recombinantly inserted at the N-terminus of a
cytotoxic fragment of PE. To enhance the stability of
Native Pseudomonas exotoxin A (PE)
dsFv-PE38 RIT
PE38
PE[LR]
dsFv-PE[LR] RIT
Ia II IIIIb
1 613252/253

364/365
404/405
(1-250)
(365-380)
(1-273) (285-394)
PE38
F
V
PE[LR]
F
V
Fig. 1. PE and PE-based RITs. Native PE consists of three struc-
tural domains organized from a single polypeptide sequence.
Domain I is separated into the structurally adjacent but discontinu-
ous domain Ia (blue; residues 1–252) and domain Ib (green; 365–
404) by domain II (yellow; 253–364). Domain III (red; 405–613) lies
at the C-terminus. A cartoon model, created using
VMD [13], based
on the X-ray crystal structure of PE (Protein Data Bank code: 1IKQ)
is shown, excluding those residues absent from the electron den-
sity map (607–613). RITs based on PE are chimeric molecules that
fuse antibodies to fragments of PE, most frequently a 38 kDa
truncation known as PE38 that contains extensive deletions in
domain Ia (D1–250) and domain Ib (D365–380). Recently, a smaller
fragment, PE[LR] (D1–273 and D285–394), has been developed for
use in RITs. Structural models of RITs using a dsFv joined to PE38
or PE[LR] are presented. The Fv is shown in purple. Models are
hypothetical only and do not represent actual structural determina-
tions. The dsFv-PE38 RIT contains a gap in the structure that corre-
sponds to the deletion of residues 365–380 in domain Ib. Disulfide

bonds in PE and the Fv are shown in orange. The site of furin
cleavage is indicated with a black arrow.
J. E. Weldon and I. Pastan Cancer therapy based on Pseudomonas exotoxin A
FEBS Journal 278 (2011) 4683–4700 Journal compilation ª 2011 FEBS. No claim to original US government works 4685
recombinant immunotoxins, disulfide-stabilized Fv
(dsFv) molecules were subsequently developed. The
dsFv divides the V
H
and V
L
into separate polypeptides
that are covalently connected through a disulfide bond
engineered into the framework region of the Fv
[25–27]. A cytotoxic fragment of PE can be inserted at
the C-terminus of one of the two Fv polypeptide
chains (Fig. 1). The generation and production of
PE-based RITs has been described previously [28].
The most commonly employed cytotoxic fragment
of PE in RITs is a 38 kDa version known as PE38 [29]
(Fig. 1). PE38 contains a deletion of the majority of
domain Ia (D1–250) and a portion of domain Ib
(D365–380) from native PE. Several RITs incorporat-
ing a 38 kDa fragment of PE are in preclinical evalua-
tion or have already reached clinical trials (Table 1).
PE38 RITs undergoing preclinical testing include an
antiglycoprotein NMB (scFv) for the treatment of
malignant gliomas and melanomas [30], an anti-HIV-1
gp120 (scFv) for the treatment of HIV [31,32] and a
RIT targeted to osteosarcomas using a dsFv from the
TP-3 mAb [33,34].

RITs that have progressed to clinical trials include the
anti-CD22 RIT RFB4(dsFv)PE38, also known as BL22
or CAT-3888, for the treatment of B-cell malignancies
[35–37]. The RFB4 Fv was subsequently affinity-opti-
mized by phage display selection to create the second-
generation molecule RFB4[GTHW](dsFv)-PE38 [38],
known variously as HA22 or CAT-8015, and now called
moxetumomab pasudotox. Moxetumomab pasudotox is
currently undergoing extensive clinical testing for the
treatment of hematologic malignancies [39,40] (ongoing
studies also can be found under ClinicalTrial.gov identi-
fiers: NCT00462189, NCT00457860, NCT00515892,
NCT01086644, NCT00659425 and NCT00586924).
Other RITs from our laboratory in clinical trials include
the anti-mesothelin SS1(dsFv)PE38, called SS1P, for the
treatment of lung cancer and mesothelioma [41,42]
(ongoing studies also can be found under ClinicalTri-
al.gov identifiers: NCT01041118, NCT00575770 and
NCT01051934) and the anti-TAC(scFv)PE38, called
LMB-2, which targets the IL-2 receptor for the treat-
ment of hematologic malignancies [43] (ongoing studies
also can be found under ClinicalTrial.gov identifiers:
NCT00924170, NCT00077922, NCT00080535 and
NCT00321555). Extensive lists of PE-based therapeutics
at both the preclinical and clinical stages have been pub-
lished [44,45] and additional agents continue to be devel-
oped. We have recently generated a new variant of PE,
PE[LR] (Fig. 1), which shows decreased immunogenic-
ity and nonspecific toxicity in mice at the same time as
retaining cytotoxicity against malignant cells [46].

The strategy of re-routing A-B toxins, such as DT
and PE, through a different cellular target works well
for several reasons. The cytotoxic A domain is stable
and fully active independent of the receptor-binding B
domain, which can be replaced by a component that
confers alternate specificity, such as a ligand or an anti-
body. Additionally, the available tools for recombinant
DNA manipulation and protein expression allow us to
easily generate these chimeric molecules, and protein
engineering techniques provide powerful methods for
developing and selecting improved variants. Further-
more, we can differentiate between normal and malig-
nant cells using tumor-associated cell-surface receptors
as markers. By specifically targeting these receptors
with PE, we can eliminate cancers at the same time as
avoiding toxicities to normal tissue that are frequently
associated with general chemotherapeutic strategies.
Lastly, these proteins are extremely potent toxins that
Table 1. Several PE-based recombinant toxins currently in development for the treatment of cancers.
Agent Alternative names Target Stage of development Cancer
BL22 RFB4(dsFv)-PE38
CAT-3888
CD22 Clinical trials completed;
superseded by
moxetumomab pasudotox
B cell malignancies
Moxetumomab
pasudotox
RFB4[GTHW](dsFv)-PE38
HA22

CAT-8015
CD22 Clinical trials B cell malignancies
LMB-2 anti-TAC(scFv)-PE38 CD25 (IL-2R a chain) Clinical trials T and B cell malignancies
SS1P SS1(dsFv)-PE38 Mesothelin Clinical trials Mesothelioma, lung cancer
MR1-1 MR1-1KDEL
MR1(scFv)-PE38KDEL
Epidermal growth
factor receptor vIII
Clinical trials Brain tumors
Cervene TP-38
TGFa-PE38
Epidermal growth
factor receptor
Clinical trials Brain and central nervous
system tumors
Cintredekin besudotox IL13-PE38QQR Interleukin-13 receptor Clinical trials Glioblastoma multiforme
G49[F6V](scFv)-PE38 – Glycoprotein NMB Preclinical Glioblastoma multiforme
Cancer therapy based on Pseudomonas exotoxin A J. E. Weldon and I. Pastan
4686 FEBS Journal 278 (2011) 4683–4700 Journal compilation ª 2011 FEBS. No claim to original US government works
have been naturally selected for their ability to kill
eukaryotic cells. Their activities typically require no
major enhancement to function at a therapeutic level.
The PE intoxication pathway
A basic outline of the PE intoxication pathway is well
understood. The secreted toxin binds to an LRP1 or
LRP1B cell surface receptor, is internalized by recep-
tor-mediated endocytosis, and undergoes intracellular
trafficking to reach the cytosol. In the cytosol, PE
encounters eEF2 and transfers an ADP-ribosyl group
from NAD

+
to the diphthamide residue. This irrevers-
ibly inactivates eEF2, halts protein synthesis and, ulti-
mately, leads to cell death. A general description of
the pathway is deceptively simple, and many of the
specifics are not clear. Figure 2 attempts to presents a
comprehensive description of PE intoxication, the
details of which are discussed below. The pathway
described in Fig. 2 is not necessarily complete,
although it represents our current understanding of PE
intoxication.
PE in the endocytic pathway
Similar to DT, native PE is a secreted as a proenzyme
that must be activated before it displays catalytic
activity [47]. Full activation can be accomplished
under reducing and denaturing conditions and proteol-
ysis, and appears to involve structural rearrangements
that reveal the previously obscured NAD
+
binding
cleft in domain III [48]. RITs using versions of PE
without domain Ia do not require a structural arrange-
ment to expose the NAD
+
binding site. This differ-
ence is unlikely to affect PE intoxication in RITs,
although it does eliminate the requirement for catalytic
activation.
After endocytosis, PE undergoes an essential proteo-
lytic processing step at a cleavage site between residues

R279 and G280 of domain II [49,50]. Using
SDS ⁄ PAGE, two bands corresponding to the A and B
subunits of PE were initially observed: a 28 kDa N-ter-
minal fragment (B subunit) and a cytotoxic 37 kDa
C-terminal fragment (A subunit), which was enriched
in the cytosolic fraction of treated cells. PE that had
been mutated so that it did not undergo this processing
step failed to kill cells. Subsequent research implicated
the intracellular protease furin (EC 3.4.21.75) in this
process [51–53] and supporting evidence has accumu-
lated [54–59]. PE that is treated with furin before intox-
ication is more active than untreated PE. In addition,
PE is less active on cell lines that are furin deficient or
on cells treated with furin inhibitors.
Furin is a ubiquitous, Ca
2+
-dependent, transmem-
brane serine endoprotease that is a member of the sub-
tilisin-like family of proprotein convertases [60]. It
plays an active role in the maturation of many cellular
proteins, and its prevalence is frequently exploited by
bacterial toxins and viruses during intoxication and
infection. Furin contains a luminal catalytic domain
and a cytoplasmic domain that controls its cycling
between the trans-Golgi network and the plasma mem-
brane. PE could potentially encounter furin at either
of these sites or in the endosomal network during
intracellular trafficking between them.
In addition to furin cleavage of the PE polypeptide
backbone, separation of the A and B fragments must

be preceded by the reduction of a disulfide bond
between residues C265 and C287, which provides a
second covalent linkage. Thus, both a reduction and a
proteolysis step are necessary for PE intoxication [61].
The C265-C287 disulfide bond is buried in the crystal
structure of native PE [12] and must be exposed by
unfolding before it can be reduced [61]. This observa-
tion suggests that furin cleavage precedes reduction,
although the order of events in vivo has not been
established experimentally.
The subcellular location of the reduction event is dif-
ficult to pinpoint. The general redox state of the extra-
cellular environment is normally more oxidizing,
whereas the intracellular environment is more reducing
[62], although numerous factors can influence the redox
balance and different subcellular compartments can
have very different redox potentials. One suggestion
has been that the reduction of PE is accomplished by
protein disulfide-isomerases (PDIs; EC 5.3.4.1) because
in vitro experimental evidence suggests that PE can be
reduced by PDIs [61]. PDIs are a family of enzymes
that catalyze the formation and breakage of disulfide
bonds in proteins [63]. They are abundant not only in
the endoplasmic reticulum (ER) and Golgi, but also in
other intracellular locations and on the cell surface
[64,65]. PE could potentially encounter PDIs at every
stage of the intoxication pathway. The relative abun-
dance of PDIs in the ER, however, suggests that PE
would be more likely to encounter PDIs there.
Indirect support for the involvement of PDIs in PE

intoxication comes from the pathways of other protein
toxins. The protein toxins ricin and cholera toxin (CT)
both follow routes through the ER and into the cyto-
sol after receptor-mediated endocytosis. Evidence
obtained both in vivo and in vitro supports the involve-
ment of PDIs in a reductive separation event essential
to ricin and CT [66–70]. The PDI family of proteins
has additionally been associated with retrograde trans-
port of polypeptides from the ER in the process of
J. E. Weldon and I. Pastan Cancer therapy based on Pseudomonas exotoxin A
FEBS Journal 278 (2011) 4683–4700 Journal compilation ª 2011 FEBS. No claim to original US government works 4687
ER-associated degradation (ERAD), a mechanism
that may be exploited by PE to reach the cytosol, as
discussed below.
The precise role played by intracellular processing of
PE in its intoxication pathway is not entirely clear.
Separation of the A and B subunits serves to activate
the PE proenzyme, although RITs that do not require
activation for catalytic activity still need a cleavable
furin site for full activity (J. E. Weldon, unpublished
results). Separation of the catalytic and binding
domains may therefore serve an additional function,
perhaps by exposing sequences in domain II necessary
for intracellular trafficking. PE38 RITs retain all of
domain II, including the furin cleavage site and C265-
C287 disulfide bond (Fig. 1). Unlike native PE, how-
ever, separation of the catalytic and binding fragments
is not always essential for cytotoxicity. The RIT HA22
(anti-CD22 ⁄ PE38) remains active on CD22-positive
cells even with an R279G mutation that prevents furin

cleavage, although it is three-fold less active than wild-
type HA22 (J. E. Weldon, unpublished results). The
same R279G mutation in the RIT SS1-LR ⁄ GGS (anti-
mesothelin ⁄ PE[LR]) is completely inactive on mesoth-
elin-positive cells. Current research is exploring these
Nucleus
PE
Endoplasmic
reticulum
B
Carboxypeptidase
AB
REDLK
Furin
LRP-1/B
Sec61
A
REDL
REDL
A
B
AB
REDL
AB
REDL
Lysosome
NAD
+
eEF2
ADP-Ribose

eEF2
Extracellular
Intracellular
Early
endosome
A
REDL
Protein synthesis
Apoptosis
AB
REDL
A
B
REDL
PDI
Late
endosome
1
11
10
9
5b
7
6
5a
8
2
3
4
Clathrin-coated

Pit
Tumor-associated receptor
(e.g. CD22)
I
III
Nicotinamide
Golgi
KDEL receptor
REDL
A
B
(dsFv)-PE38 RIT
A
REDLK
II
Fig. 2. PE intoxication pathway. Native PE can be divided into two fragments with functions of receptor binding (B) and catalytic activity (A).
After secretion into the extracellular environment, PE is cleaved by a carboxypeptidase (1) to remove the C-terminal lysine residue and
expose the ER localization signal (REDL). The B fragment subsequently recognizes its cell-surface receptor, LRP1 or LRP1B (2), and is inter-
nalized via receptor-mediated endocytosis in clathrin-coated pits (3). Within the endocytic pathway, PE encounters the endoprotease furin,
which cleaves at a site in domain II and separates the polypeptide backbone between the A and B fragments (4). A disulfide bond preserves
a covalent linkage between the two fragments. When in the endocytic pathway, PE can either follow a productive trafficking route to the
Golgi (5b) or continue to the lysosome for terminal degradation (5a). In the Golgi, PE encounters KDEL receptors that recognize the REDL C-
terminal signal and transport PE to the ER in a retrograde manner (6). At an undetermined point in the pathway, possibly by PDI in the ER,
the disulfide bond connecting the A and B fragments is reduced and the two fragments separate (7). The A fragment is subsequently trans-
ported into the cytosol (8), possibly by exploiting the ERAD pathway through the Sec61 translocon. In the cytosol, PE transfers an ADP-ribo-
syl (ADPr) group from NAD
+
to the diphthamide residue of eEF2 (9). This halts protein synthesis (10) and ultimately leads to apoptotic cell
death (11). RITs based on PE (I) target tumor-associated cell surface receptors for internalization (II), and are generally considered to undergo
an intoxication pathway similar to that of PE (III).

Cancer therapy based on Pseudomonas exotoxin A J. E. Weldon and I. Pastan
4688 FEBS Journal 278 (2011) 4683–4700 Journal compilation ª 2011 FEBS. No claim to original US government works
differences. Both the cell line and the target receptor
appear to play major roles in determining the outcome
of intoxication.
PE in the endoplasmic reticulum
The intoxication pathways of DT and PE are remark-
ably similar in several respects [71]. Both are secreted
as proenzymes, internalized by receptor-mediated
endocytosis, processed by furin, and reduced to sepa-
rate the catalytic (A) from the binding (B) fragments.
Subsequent to these steps, however, their respective
pathways diverge dramatically. Although DT pursues
a route directly from acidified endocytic vesicles into
the cytosol [72], PE follows a path through the ER.
The evidence for an ER-dependent PE intoxication
pathway is extensive. It was initially observed that the
R
609
EDL
612
sequence immediately adjacent to the
C-terminal residue of PE was essential for cytotoxicity
[73]. Deletions in the REDL sequence of PE eliminate
its cytotoxicity, although replacement with a similar
sequence, KDEL, restores activity. The KDEL
sequence is a well defined ER retention and retrieval
signal in mammalian cells [74] that is recognized by
integral membrane proteins known as KDEL receptors
(KDEL-R) [75,76]. The subcellular localization of

KDEL-R appears to be a dynamic cycle between the
Golgi and the ER [77,78]. This is consistent with
the proposed function of KDEL-Rs in returning to the
ER proteins that have escaped into the Golgi.
The REDL C-terminal sequence of PE, which also
occurs on several ER-resident proteins, is a variant of
the canonical KDEL sequence and is recognized and
retained in the ER by KDEL-R [79]. As anticipated,
the overexpression of KDEL-R1 (hERD2) sensitizes
cells to PE. Conversely, cells become resistant to PE
when KDEL transport is restricted by microinjected
antibodies to KDEL-R1 or by expression of lysozyme-
KDEL, which competes for binding to free receptor
[80]. Before KDEL-R can recognize PE, however, the
C-terminal residue, K613, must be removed to expose
the REDL signal sequence. Binding to KDEL-R is
seriously impaired if the terminal lysine residue is not
removed [81]. The removal of K613 appears to occur
early in the intoxication process, possibly by plasma
carboxypeptidase(s) in the bloodstream [82].
Analysis of KDEL-R binding to oligopeptides end-
ing with various sequences showed that the REDL
native sequence of PE had an almost 100-fold weaker
affinity than the canonical KDEL sequence [81]. This
result suggests that replacing the native REDL
sequence with KDEL might enhance the cytotoxicity
of PE-based RITs by increasing the efficiency of Golgi
to ER transport, and multiple studies have supported
this hypothesis [81,83]. Unfortunately, the therapeutic
benefit of enhanced cytotoxicity is offset by an accom-

panying increase in nonspecific toxicity in laboratory
animals (R. J. Kreitman, J. E. Weldon and I. Pastan,
unpublished results).
On the basis of the perturbation of different traffick-
ing pathways, it has been suggested that PE can
exploit routes to the ER other than through KDEL-R
[84]. Although alternative pathways to the ER cer-
tainly exist and are used by other toxins, most notably
a KDEL-R-independent lipid transport route used by
Shiga toxin [85,86], the evidence indicates that the vast
majority of PE reaches the ER through KDEL-R.
Deletion of the ER localization signal at the C-termi-
nus of PE reduces its activity by 1000-fold or more
[73]. Our experience with PE-based RITs has shown
that the C-terminal ER localization sequence of PE is
essential for cytotoxicity (J. E. Weldon & I. Pastan,
unpublished observations). An additional mechanism
has been suggested in which PE can translocate
directly from acidified endocytic vesicles into the cyto-
sol, using an approach similar to DT [87]. This
proposal also conflicts with the observation that the
C-terminal ER localization signal of PE is essential. It is
possible that differences between cell lines may account
for the conflicting experimental observations, and more
work needs to be carried out to clarify the matter.
An exit pathway from the ER to the cytosol is sug-
gested by the evidence for an association between PE
and the Sec61p ER translocation pore [88,89]. This
suggests that PE may be exported from the ER into
the cytosol through the Sec61p membrane channel in a

manner similar to the retrotranslocation (also know as
dislocation) of polypeptides destined for proteasomal
degradation by luminal ER-associated degradation
[90]. Presumably, this would entail a chaperone-
assisted unfolding step in the ER followed by translo-
cation and refolding in the cytosol. It is possible that
processed PE and other protein toxins such as CT and
Shiga toxin mimic the presence of a misfolded protein
in the ER to exploit the ERAD system for transport
across the ER membrane to the cytosol [91,92]. To
date, we are unaware of direct evidence for transport
of PE through the Sec61p translocon.
Additional support for the hypothesis that PE
exploits the ERAD system is the amino acid bias
against lysine residues in its catalytic fragment [93].
Sequence analyses of the catalytic (A) fragments of PE
and other protein toxins show that arginine residues
are much more highly preferred over lysine when
examining the occurrence of basic amino acids. Inter-
estingly, this paradigm does not hold true for the B
J. E. Weldon and I. Pastan Cancer therapy based on Pseudomonas exotoxin A
FEBS Journal 278 (2011) 4683–4700 Journal compilation ª 2011 FEBS. No claim to original US government works 4689
fragments, in which lysine residues occur with normal
frequency. In total, there are 15 lysine residues in
native PE but only three lysines in its A fragment (res-
idues 280–613): K590, K606 and K613. All three of
these residues are located near the C-terminus of PE,
and K613 must be removed to expose the C-terminal
REDL ER localization signal. This suggests a selective
pressure against the inclusion of lysine residues in the

protein sequence of the A fragment but not the B
fragment of PE. Because only the A fragment must
traffic to the cytosol for activity, the lack of lysine res-
idues may protect it from the ubiquitin ⁄ proteasome
system, comprising the terminal step of ERAD in
which proteins are targeted for degradation by poly-
ubiquitination of lysine e-amino groups [94]. Both
ricin and abrin toxins engineered to contain additional
lysine residues have shown enhanced ubiquitin-medi-
ated proteasomal degradation [95]. PE may similarly
lack lysine residues to avoid degradation in the cytosol
at the same time as exploiting an ERAD transport
pathway.
PE in the cytosol
Once PE reaches the cytosol, it exerts its catalytic
activity on EF2. The translation factor EF2 [96] is an
essential component of protein synthesis, during which
it catalyzes the coordinated movement of the growing
polypeptide chain along the ribosome. In eukaryotes
(eEF2) and archaea (aEF2), but not bacteria (EF2,
formerly EF-G), the protein contains a unique and rig-
idly conserved post-translationally modified histidine,
known as a diphthamide residue. The purpose of the
diphthamide residue is unclear, although it is strictly
conserved among eukaryotes and archaea. Gene
knockout studies in mice have shown that enzymes in
the diphthamide biosynthesis pathway are essential for
normal development [97,98], although it is not clear if
the diphthamide residue itself is essential. The lack of
a diphthamide did not have a significant impact on the

activity of aEF2 in vitro [99]. In addition, mammalian
and yeast cultured cells lacking the diphthamide modi-
fication on EF2 are viable and resistant to NAD
+
diphthamide ADP-ribosyltransferases, although they
may show effects such as temperature sensitivity and a
decreased growth rate [100–107]. Several hypotheses
for the necessity of the diphthamide have been
proposed, including its involvement in protection from
ribosome-inactivating proteins such as icin [108] or
preservation of translational fidelity [109], although no
consensus has been reached. The existence of bacterial
NAD
+
-diphthamide ADP-ribosyltransferases (PE, DT
and CE), however, demonstrates that bacteria have
found the diphthamide residue an appealing target to
differentiate themselves from archaea and eukaryotes.
Because the initial determination that PE halts pro-
tein synthesis in a manner identical to DT [110], the
catalytic mechanism of PE has been extensively studied
[111–117]. Several residues in domain III of PE have
been identified as playing important roles in catalysis,
including Glu553, His440, Tyr481 and Tyr470. Studies
of the reaction itself indicate that an ADP-ribosyl
group derived from NAD
+
is transferred to the N3
atom of the diphthamide imidazole using a random
third-order S

N
1 mechanism. NAD
+
is cleaved to pro-
duce nicotinamide, which is released, and an ADP-ri-
bosyl oxacarbenium ion intermediate, which contains a
positively charged ribosyl group that reacts with the
diphthamide imidazole N3 atom. The molecular mech-
anism by which the ADP-ribosylation of eEF2 halts
protein synthesis remains unclear, although it is possi-
ble that the ADP-ribose moiety interferes with an
interaction between eEF2 and RNA at the diphtha-
mide site [118].
We also do not know precisely how ADP-ribosyla-
tion of eEF2 leads to cell death, although halting
translation almost certainly leads to growth inhibition
and arrest. Studies that have examined cell death after
treatment with PE or PE-based RITs have reported
results consistent with apoptotic cell death [119–122],
although little is known about the intermediate steps
after ADP-ribosylation of eEF2 and before caspase
activation. Recently, it was reported that apoptosis
induced in mouse embryonic fibroblasts by PE or other
protein synthesis inhibitors was dependent on the
degradation of Mcl-1 and release of Bak [123]. The
anti-apoptotic protein Mcl-1 is rapidly turned over in
the cell, and inhibition of its synthesis may shift the bal-
ance of apoptotic signals towards cell death [124]. It is
possible that this mechanism could be common among
different cell types and protein synthesis inhibitors.

Unanswered questions
At this point, it should be clear that our understanding
of PE intoxication is incomplete. One important miss-
ing element is an understanding of the role of domain
II in PE intoxication. It has been suggested that
domain II assists in the translocation of the toxin into
the cytosol [16,87] and that it plays a role in proper
folding, stability and secretion by P. aeruginosa [125–
127], although there is no consensus. Domains Ia and
III have independent, experimentally verified functions
that can be directly assessed, although speculation con-
cerning the function of domain II has been made pri-
marily by inference. Domain Ib also has no
Cancer therapy based on Pseudomonas exotoxin A J. E. Weldon and I. Pastan
4690 FEBS Journal 278 (2011) 4683–4700 Journal compilation ª 2011 FEBS. No claim to original US government works
independent function but is structurally contiguous
with domain Ia, and a portion of domain Ib is func-
tionally essential to the catalytic activity domain III.
At least a portion of domain II is devoted to maintain-
ing the covalent attachments between the A and B
toxin fragments; it contains the furin protease cleavage
site and flanking cysteines (Cys265-Cys287) that form
a disulfide bond. It is unlikely, however, that the
entirety of domain II exists simply to provide a site for
the separation of the A and B fragments.
Work on PE-based RITs has shown that the major-
ity of domain II is not essential for activity, although
it can have a large influence on cytotoxicity [46].
Depending on the cells examined and the receptor
targeted, mutations that eliminate all of domain II

except for the furin cleavage site can enhance, reduce
or have no impact on cytotoxicity. Eliminating the fu-
rin cleavage site by deletion or preventing cleavage
with a point mutation in the site has either reduced
the cytotoxicity of the RIT or completely abolished it.
An explanation for these differing effects is unknown
and currently under study, although it raises the issue
that our understanding of the PE intoxication path-
way can be complicated by the use of recombinant
immunotoxins. Much of the information accumulated
over years of study concerns PE-based RITs rather
than native PE. Not only is the protein heavily modi-
fied from its native form, but also the target receptor
is changed. This could potentially influence cytotoxic-
ity in a variety of ways, from changing the number of
receptor sites per cell to altering the rate of internali-
zation of the receptor or influencing the intracellular
trafficking. The proteome of the target cell also influ-
ences the pathway. We have observed large differences
in the cytotoxicity of PE and PE-based RITs on dif-
ferent cell lines. The assumption that the route of
trafficking is conserved after internalization in differ-
ent cell lines and through different receptors is not
necessarily accurate, although our understanding of
PE trafficking is currently insufficient to make such
distinctions.
Another unanswered question concerns the fraction
of the internalized PE that productively traffics to
the cytosol. On the basis of studies on DT [128]
and unpublished data from our laboratory using PE

(I. Pastan, unpublished results), it has been proposed
that as few as one molecule of PE in the cytosol may
be sufficient to kill a cell. Typically, cells in culture
require treatment with concentrations of PE greater
than 1000 molecules per cell (approximately
10
)16
gÆcell
)1
) to ensure cell death. This number is close
to an estimate of the toxin load ⁄ cell in a mouse xeno-
graft tumor model. Tumor-bearing mice treated with a
PE-based RIT required 400–750 molecules per cell to
ensure tumor remission [129]. Taken together, these
studies suggest that less than 1% of the internalized
toxin may successfully traffic into the cytosol. The
remainder appears to follow an unproductive path into
lysosomes. This estimate agrees with observations of
cells treated with labeled PE [130,131] (J. E. Weldon,
unpublished observations). The stability of the A frag-
ment of PE in the cytosol has also not been examined,
although its relative lack of lysine residues may hamper
ubiquitination-dependent proteasomal degradation and
enhance cytosolic stability.
Clinical trials of PE-based RITs
Although no PE-based therapies have been approved
by the Food and Drug Administration, several have
reached the point of advanced clinical trials in their
development (Table 1). The examples provided in this
review do not constitute an exhaustive list. At the time

of this review, a search for ‘immunotoxin’ in the NIH
clinical trials database ()
revealed at least 16 active studies involving PE that
has been redirected to selectively eliminate cells. The
majority of these trials involve PE-based RITs devel-
oped in our laboratory, and they are discussed below.
The RIT BL22 (anti-CD22 ⁄ PE38) has undergone
several early-phase clinical trials for the treatment of B
cell malignancies [35–37]. These trials have validated
the use of CD22 as a target and highlighted several
potential problems with this treatment. BL22 was most
effective in patients with drug-resistant hairy cell leuke-
mia (HCL), whose response rates were 81% (25 ⁄ 31) in
a phase I trial [35] and 69% (25 ⁄ 36) in a phase II trial
[36]. Dose-limiting toxicity was related to a completely
reversible hemolytic uremic syndrome resulting from
the destruction of red blood cells. High levels of neu-
tralizing antibodies developed in 24% (11 ⁄ 46) of
patients in the phase I trial and 11% (4 ⁄ 36) of patients
in the phase II trial.
Clinical trials of BL22 have been superseded by
moxetumomab pasudotox, a modified RIT whose Fv
has undergone selection for enhanced CD22 affinity by
phage display [38]. As previously discussed, there are
at least six active clinical trials of moxetumomab pa-
sudotox. Preliminary results from a phase I study in
patients with relapsed or refractory HCL (trial identi-
fier NCT00462189) show a response rate of 81%
(26 ⁄ 32), even though neutralizing antibodies eventually
developed in 44% (14 ⁄ 32) of patients [132]. There is a

notable lack of dose-limiting toxicity as a result of
hemolytic uremic syndrome with moxetumomab
pasudotox, and a maximum tolerated dose has not yet
J. E. Weldon and I. Pastan Cancer therapy based on Pseudomonas exotoxin A
FEBS Journal 278 (2011) 4683–4700 Journal compilation ª 2011 FEBS. No claim to original US government works 4691
been established. An additional phase I clinical trial in
pediatric patients with acute lymphoblastic leukemia
(ALL) or non-Hodgkin’s lymphoma (trial identifier
NCT00659425) shows activity in patients with ALL
[133]. Of the ALL patients evaluated, 25% (3 ⁄ 12) had
complete responses, 50% (6 ⁄ 12) had partial responses
(hematologic activity), 17% (2 ⁄ 12) had stable disease
and 8% (1 ⁄ 12) had progressive disease. Two patients
eventually developed high levels of neutralizing anti-
bodies, and two patients developed a dose-limiting
capillary leak syndrome.
In addition to CD22, CD25 (IL-2 receptor a chain)
has been targeted for the treatment of various leuke-
mias and lymphomas. The anti-CD25 RIT LMB-2 has
undergone a phase I clinical trial [43] showing an over-
all response rate of 23% (8 ⁄ 35), and there are at least
four active clinical trials of LMB-2 (listed above).
Immunogenicity and nonspecific toxicities continue to
be problematic. Of the patients evaluated in the phase I
study, 29% (10 ⁄ 34) showed high levels of neutrali-
zing antibodies to PE38. Toxicities were reversible and
most commonly low level transaminase elevations and
mild fever. LMB-2 has also been used clinically in a par-
tially successful effort to deplete patients of CD25+
regulatory T lymphocytes and thereby enhance the

immune response to vaccination with tumor-specific
antigens [134].
Another PE-based RIT that has reached clinical trials
is the anti-mesothelin SS1P. Two phase I trials treating
patients with mesothelioma, pancreatic cancer or ovar-
ian cancer have been completed [41,42], and at least
two studies are currently active. Patient responses to
SS1P were modest, with a few minor responses. Toxici-
ties associated with treatment were typically mild.
Immunogenicity appears to constitute the major obsta-
cle to SS1P treatment. In the two studies, 88% (30 ⁄ 34)
and 75% (18 ⁄ 24) of patients developed high levels of
neutralizing antibodies to SS1P after a single cycle of
treatment. These rates were significantly higher than the
immunogenicity observed when treating hematologic
malignancies, possibly because patients with blood can-
cers have an immune system that is compromised as a
result of disease and ⁄ or previous chemotherapy. Pri-
marily as a result of the immunogenicity, very few
patients qualified to receive more than a single cycle of
treatment, which might account for the low efficacy of
SS1P. Preliminary results from a phase I clinical trial
combining SS1P with chemotherapy to treat patients
newly diagnosed with advanced-stage pleural mesotheli-
oma (trial identifier NCT00575770) show good results
[135]. SS1P is well tolerated when combined with
pemetrexed and cisplatin, and 50% (7 ⁄ 14) of patients
showed a partial response to treatment.
The future of PE-based RITs
Many obstacles have been overcome in the develop-

ment of RITs for the treatment of cancer, and striking
responses have been observed in many patients with
HCL, although several properties of RITs still need
improvement. One of the most significant problems we
have encountered in the clinical trials is immune
response leading to the generation of neutralizing anti-
bodies. Immunogenicity can be a major difficulty for
protein therapeutics, particularly those derived from
nonhuman sources [136]. For PE-based RITs, neutral-
izing antibodies are a common occurrence and com-
prise a major limitation in patients with solid tumors
who have an intact immune system. Antibody forma-
tion is much less of a barrier to treating patients with
hematologic malignancies, whose immune systems are
typically suppressed, and multiple treatment cycles can
usually be given. Mouse studies show that PE38 RITs
are no more immunogenic than most foreign proteins.
Antibody responses typically do not occur until several
weeks after the initial treatment [137–139]. Neverthe-
less, it is clear that lower immunogenicity would bene-
fit PE-based RITs. This is especially apparent with
SS1P; in approximately 80% of patients, only a single
cycle (three doses) can be administered before the
development of neutralizing antibodies.
Several strategies have been attempted to overcome
the issue of immunogenicity in PE-based RITs.
Poly(ethylene glycol)ylation is a common strategy to
reduce the immunogenicity and alter the pharmacoki-
netics of proteins [140]. We have poly(ethylene gly-
col)ylated various PE RITs [141–143] and found that

their efficacy was greatly diminished. An alternate
strategy is to treat patients with general immunosup-
pressive drugs concurrent with RIT therapy to prevent,
delay or otherwise limit the production of neutralizing
antibodies. This strategy is currently being assessed
clinically using LMB-2 in conjunction with fludarabine
and cyclophosphamide [40] (ClinicalTrials.gov study
identifier: NCT00924170), although previous attempts
to reduce immunogenicity in this manner have been
unsuccessful. Clinical trials using cyclophosphamide
[144] or cyclosporine A [145] in combination with a
ricin-based immunotoxin failed to decrease the anti-
body response. An attempt to treat patients with ritux-
imab (anti-CD20 mAb) before treatment with a PE-
based RIT also failed to suppress the antibody
response [146].
A third strategy is the elimination of immunogenic
epitopes in PE by mutation. The targeted removal of
B cell (antibody) epitopes [147,148] in PE38 has pro-
gressed the furthest [137–139,149]. This strategy has
Cancer therapy based on Pseudomonas exotoxin A J. E. Weldon and I. Pastan
4692 FEBS Journal 278 (2011) 4683–4700 Journal compilation ª 2011 FEBS. No claim to original US government works
been effective at dramatically decreasing immunogenic-
ity in mice, although much work remains to be carried
out to ensure low immunogenicity in humans. The
identification and elimination of T cell epitopes is a
complementary approach [150]. We propose that the
strategy of epitope elimination offers the best promise
for the deimmunization of PE-based RITs.
Other potential strategies to avoid immunogenicity

have a more uncertain forecast. One option is to utilize
human proteins that can act as cytotoxins because
fully human proteins should theoretically not elicit an
immune response in patients. Examples include pro-
apoptotic proteins and RNase. Although these
‘humanized’ RITs [151] are generally beyond the pur-
view of this review, the potential existence of an
endogenous eEF2 ADP-ribosyltransferase in mammals
[152–155] suggests that a human equivalent to PE may
exist. A RIT engineered out of such a protein might be
non-immunogenic, although it must first be identified
and characterized.
Strategies to enhance the efficacy of RITs might also
be practical. If only a single cycle of treatment is
required to achieve a complete tumor remission, the
appearance of neutralizing antibodies after that cycle of
treatment might be irrelevant to the outcome. One
strategy is to combine PE with other chemotherapeutic
drugs to achieve a synergistic effect without nonspecific
toxicity. Combination therapy with standard
chemotherapy drugs has shown promise in preclinical
studies of SS1P [156–158] and clinical trials are cur-
rently underway to test SS1P within a combination
therapy regimen (ClinicalTrials.gov study identifiers:
NCT01041118 and NCT00575770). Research into the
observed synergy suggests that it may be a result of
both chemotherapy-induced tumor cell depletion and
lower levels of free mesothelin shed into the extracellu-
lar space [158]. Free mesothelin competes with cell sur-
face mesothelin for SS1P, acting as an unproductive

sink for the immunotoxin [159]. Other potential combi-
nation drugs include that could act at specific stages in
the PE intoxication pathway to enhance productive
intoxication or those that can enhance the initiation of
apoptotic cell death in PE-treated cells. For example, a
pro-apoptotic BH3 domain mimetic has recently been
shown to synergistically enhance the cytotoxicity of PE-
based recombinant immunotoxins [160].
Further engineering of PE to enhance productive
intoxication might also be possible. As previously
described, the initial development of immunotoxins
utilized full-length native PE chemically conjugated to
mAbs. Improved protein engineering techniques per-
mitted the combination of PE and mAb fragments
(PE38 and Fv) into RITs, which were more efficient to
produce and highly active, with fewer side effects. Var-
iant RITs have subsequently been developed to
enhance ER trafficking by replacing the C-terminal
residues with a KDEL sequence [161] and to limit
endolysosomal proteolytic degradation by deleting pro-
tease-sensitive regions [46]. Other potential engineering
targets include the furin cleavage site of PE, which is
remarkably inefficiently cleaved by furin [162] and may
benefit from enhanced cleavage efficiency.
Additional obstacles exist outside of PE and its
intoxication pathway. Target selection and the target-
ing element are at least as important as the toxin por-
tion of RITs. The ability of a RIT to discriminate
between normal and malignant cells is fundamental to
its success, making the identification and validation of

a target the most important stage in their early devel-
opment. In addition to selectivity, factors such as
receptor site density, internalization rate and internali-
zation route can influence RIT efficacy. For example,
both CD19 and CD22 represent excellent targets for
RITs in the selective elimination of mature B cells
and associated malignancies. Although CD19 is more
heavily expressed than CD22, CD22 internalizes much
more rapidly and is a much better target for PE-based
RITs [163]. The stability of the targeting element is
also essential. The dsFv immunotoxin variants are
typically much more stable than the scFv molecules
originally developed for RITs, and are thus more clin-
ically useful [25]. A detailed discussion of receptor tar-
geting is beyond the scope of this review, although it
plays an essential role in the therapeutic efficacy of
RITs.
Concluding remarks
Substantial progress has been made in the development
of PE-based therapeutics over the past 30 years. Initial
tentative steps to transform a potent bacterial toxin
into a selective agent for the elimination of cells have
become purposeful strides to generate the immunotox-
ins of today and, we anticipate, the medicines of
tomorrow. Advances in our understanding of PE and
its intoxication pathway have fueled the translation of
basic research into clinical therapies that have the
opportunity to make a large positive impact on human
health. High expectations should be tempered by the
realization that obstacles remain to be overcome for

these RITs to achieve their maximum potential. Many
of the details of the PE intoxication process remain
uncertain and must be addressed before we can claim
success. Future advances in PE therapeutics will be
dependent on a clear and comprehensive grasp of PE
and its mechanism.
J. E. Weldon and I. Pastan Cancer therapy based on Pseudomonas exotoxin A
FEBS Journal 278 (2011) 4683–4700 Journal compilation ª 2011 FEBS. No claim to original US government works 4693
Acknowledgements
The authors would like to thank David Fitzgerald and
Dawn Walker for their helpful comments during the
preparation of this manuscript. This work is supported
in part by the Intramural Research Program of the
National Institutes of Health, National Cancer Insti-
tute, Center for Cancer Research. Work on BL22 and
moxetumomab pasudotox is supported in part by
MedImmune, LLC under a Cooperative Research and
Development Agreement.
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