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

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].. Our laboratory has focused efforts o

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: pastani@mail.nih.gov

(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.

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 and Botox Cosmetic; Allergan,

Inc., Irvine, CA, USA) for both therapeutic and

cos-metic purposes Although the toxin has an estimated

human LD50 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 LD50of 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; 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 exo-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˚ Ca 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

It targets the low density lipoprotein receptor-related

protein (LRP)1 (also known as CD91 or the a2

-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 (VH) and light chain (VL) 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

364/365 404/405

(1-250) (365-380)

(1-273) (285-394)

PE38

FV

PE[LR]

FV

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.

recombinant immunotoxins, disulfide-stabilized Fv

(dsFv) molecules were subsequently developed The

dsFv divides the VHand VLinto 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 toxicimmunogenic-ity 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.

CAT-3888

superseded by moxetumomab pasudotox

B cell malignancies

Moxetumomab

pasudotox

RFB4[GTHW](dsFv)-PE38 HA22

CAT-8015

LMB-2 anti-TAC(scFv)-PE38 CD25 (IL-2R a chain) Clinical trials T and B cell malignancies

MR1(scFv)-PE38KDEL

Epidermal growth factor receptor vIII

Clinical trials Brain tumors

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

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, Ca2+-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 reducobserva-tion, 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

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

A

Sec61

A

REDL

REDL A B

A

A B REDL

Lysosome

NAD +

eEF2

ADP-Ribose eEF2

Extracellular

Intracellular

Early endosome

A REDL

Protein synthesis Apoptosis

A B 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).

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

R609EDL612 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

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 SN1 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 understandmiss-ing 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

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 activmajor-ity, 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 differinflu-ences

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 differdiffer-ent 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)16gÆcell)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 (http://www.clinicaltrials.gov) 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

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 treatdevelop-ment 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