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: 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. 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 (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 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 [...]... 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 immunotoxins 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... 12 9–1 36 Siegall CB, Chaudhary VK, FitzGerald DJ & Pastan I (1989) Functional analysis of domains II, Ib, and III of Pseudomonas exotoxin J Biol Chem 264, 1425 6– 14261 Kihara A & Pastan I (1994) Analysis of sequences required for the cytotoxic action of a chimeric toxin composed of Pseudomonas exotoxin and transforming growth factor alpha Bioconjug Chem 5, 53 2–5 38 FitzGerald DJ, Padmanabhan R, Pastan I &... 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Merrill AR (2002) Insight into the catalytic mechanism of Pseudomonas aeruginosa exotoxin A Studies of toxin interaction with eukaryotic elongation factor-2 J Biol Chem 277, 4666 9–4 6675 Armstrong S & Merrill AR (2004) Toward the elucidation of the catalytic mechanism of the monoADP-ribosyltransferase activity of Pseudomonas aeruginosa exotoxin A Biochemistry 43, 18 3–1 94 Jørgensen R, Merrill AR, Yates... intoxication pathway Target selection and the targeting element are at least as important as the toxin portion 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 development In addition to selectivity, factors such as receptor site density, internalization... on Pseudomonas exotoxin A 148 Nagata S & Pastan I (2009) Removal of B cell epitopes as a practical approach for reducing the immunogenicity of foreign protein-based therapeutics Adv Drug Deliv Rev 61, 97 7–9 85 149 Onda M, Nagata S, FitzGerald DJ, Beers R, Fisher RJ, Vincent JJ, Lee B, Nakamura M, Hwang J, Kreitman RJ et al (2006) Characterization of the B cell epitopes associated with a truncated form... Shapira A & Benhar I (2010) Toxin- based therapeutic approaches Toxins 2, 251 9–2 583 Weldon JE, Xiang L, Chertov O, Margulies I, Kreitman RJ, Fitzgerald DJ & Pastan I (2009) A protease-resistant immunotoxin against CD22 with greatly increased activity against CLL and diminished animal toxicity Blood 113, 379 2–3 800 Leppla SH, Martin OC & Muehl LA (1978) The exotoxin P aeruginosa: a proenzyme having an... Cytotoxicity of the anti-CD22 immunotoxin HA22 (CAT8015) against paediatric acute lymphoblastic leukaemia Br J Haematol 150, 35 2–3 58 Du X, Youle RJ, FitzGerald DJ & Pastan I (2010) Pseudomonas exotoxin A- mediated apoptosis is Bak dependent and preceded by the degradation of Mcl-1 Mol Cell Biol 30, 344 4–3 452 Adams KW & Cooper GM (2007) Rapid turnover of mcl-1 couples translation to cell survival and apoptosis... display Clin Cancer Res 8, 99 5–1 002 Alderson RF, Kreitman RJ, Chen T, Yeung P, Herbst R, Fox JA & Pastan I (2009) CAT-8015: a second-generation Pseudomonas exotoxin A- based immunotherapy targeting CD22-expressing hematologic malignancies Clin Cancer Res 15, 83 2–8 39 Kreitman RJ (2009) Recombinant immunotoxins for the treatment of chemoresistant hematologic malignancies Curr Pharm Des 15, 265 2–2 664 Hassan... levels within tumors: an additional barrier to immunoconjugate therapy Clin Cancer Res 14, 798 1–7 986 Traini R, Ben-Josef G, Pastrana DV, Moskatel E, Sharma AK, Antignani A & Fitzgerald DJ (2010) ABT-737 overcomes resistance to immunotoxin-mediated apoptosis and enhances the delivery of Pseudomonas exotoxin- based proteins to the cell cytosol Mol Cancer Ther 9, 200 7–2 015 Kreitman RJ, Schneider WP, Queen . ARTICLE A guide to taming a toxin – recombinant immunotoxins constructed from Pseudomonas exotoxin A for the treatment of cancer John E. Weldon and Ira. this toxin into a treatment for cancer. Abbreviations aEF2, archaeal translation elongation factor 2; ALL, acute lymphoblastic leukemia; CE, cholera exotoxin;