Toxins 2013, 5, 1486-1502; doi:10.3390/toxins5081486
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`OPEN ACCESS
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`toxins
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`ISSN 2072-6651
`www.mdpi.com/journal/toxins
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`Review
`Immunotoxins: The Role of the Toxin †
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`Antonella Antignani * and David FitzGerald *
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`Biotherapy Section, Laboratory of Molecular Biology, Center for Cancer Research, National Cancer
`Institute, 37 Convent Dr, Bethesda, MD 20892, USA
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`† This review is dedicated to the memory of Phil Thorpe, an immunotoxin pioneer and esteemed
`colleague. He is sorely missed.
`
`* Authors to whom correspondence should be addressed; E-Mails: antignaa@mail.nih.gov (A.A.);
`fitzgerd@helix.nih.gov (D.F.); Tel.: +1-301-496-9457 (D.F.); Fax: +1-301-402-1344 (D.F.).
`
`Received: 15 July 2013; in revised form: 30 July 2013 / Accepted: 6 August 2013 /
`Published: 21 August 2013
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`Abstract: Immunotoxins are antibody-toxin bifunctional molecules
`that rely on
`intracellular toxin action to kill target cells. Target specificity is determined via the binding
`attributes of the chosen antibody. Mostly, but not exclusively, immunotoxins are
`purpose-built to kill cancer cells as part of novel treatment approaches. Other applications
`for immunotoxins include immune regulation and the treatment of viral or parasitic
`diseases. Here we discuss the utility of protein toxins, of both bacterial and plant origin,
`joined to antibodies for targeting cancer cells. Finally, while clinical goals are focused on
`the development of novel cancer treatments, much has been learned about toxin action and
`intracellular pathways. Thus toxins are considered both medicines for treating human
`disease and probes of cellular function.
`
`Keywords:
`toxin; cancer;
`immunotoxin; antibody;
`translocation; ricin; diphtheria; Pseudomonas
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`immunotherapy; apoptosis;
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`1. Introduction
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`In the late 1970s three seminal papers set the stage for future immunotoxin development. One by
`Yamaizumi et al. confirmed the potency of diphtheria toxin for mammalian cells [1] and coined the
`now famous phrase “one molecule of diphtheria toxin (DT) can kill a cell”. Thus the potency of DT
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`and similar protein toxins was established. Potency apparently resides in both the turnover rate and
`intracellular stability of the toxin’s enzyme domain. The second paper, by Thorpe et al., introduced the
`concept of using antibodies to redirect toxin killing activity in a purposeful way [2]. Specifically, the
`report described the use of anti-lymphocyte antibodies to kill lymphoblastoid tumor cells. The strategy
`involved the use of chemical linking agents to attach DT to these antibodies and so “early”
`immunotoxins were born. And, finally, the “antibody world” itself changed as monoclonal antibodies
`emerged onto the scene [3] allowing for the construction of bimolecular agents with toxins chemically
`attached to antibodies of a single defined specificity [4]. Then for a while “favorite” monoclonal
`antibodies were attached chemically to “favorite” toxins and new agents were produced on a regular
`basis, mostly for cancer therapy [5–9]. The next leap forward involved the application of molecular
`cloning techniques. This allowed for the production of fusion proteins composed of antibody
`fragments joined to enzymatically active toxin domains [10,11]. Mostly these fusion proteins were
`expressed in E. coli, which allowed for efficient production of a homogeneous product.
`Over 30 years of development, progress with immunotoxins as cancer treatment agents followed a
`predictable path: promising results in tissue culture systems led to experimentation in animal tumor
`models which progressed to large animal toxicology/pharmacology studies and then to the planning
`and implementation of clinical trials. Various immunotoxins derived either from the plant toxin ricin or
`the bacterial toxins DT or Pseudomonas exotoxin (PE) entered clinical trials. Many of these trials are
`now published with some describing very encouraging results [12–24]. However, also described are
`dose-limiting toxicities: including vascular leak syndrome, hemolytic uremic syndrome and
`pluritis [21,25,26]. Improved immunotoxin design should minimize these side effects. To date only
`one targeted toxin, DT-IL2 (termed denileukin diftitox—trade name Ontak), directed to the IL2
`receptor, has been approved for human use [27,28]. The approval of other immunotoxins awaits
`favorable results from Phase III trials. Despite having a reputation for potency, immunotoxins have
`been co-administered with “enhancing agents” even from the earliest days—in the hopes of making a
`good reagent even better [29–31]. Because cancer therapies usually require combination treatments
`this is not an unreasonable approach: and, in the future, successful immunotoxin development will
`likely depend on discovering the best agents for co-administration.
`While immunotoxins are most frequently studied as cancer therapy agents other uses have been
`suggested and evaluated—for a recent comprehensive immunotoxin review see Shapira and Benhar [32].
`These include modulating immune responses: such as preventing graft versus host disease [33,34],
`removing T-cells from grafts [35,36] or the elimination T-regulatory cells [37–40]. Some progress has
`been made also in producing immunotoxins with anti-viral [41–43] or anti-parasitic activity [44].
`Ex-vivo uses are also anticipated whereby unwanted cells are killed before infusing bone marrow or
`other stem cell like preparations [45,46].
`Immunotoxin experimentation with eukaryotic cells has led directly to the identification of novel
`toxin features and functional domains. Similarly, the concept of toxins-as-probes of eukaryotic biology
`has been exploited to uncover previously unknown pathways or properties of cells. A very early
`example of the latter stemmed from the observation in the 1960s by Kim and Groman that ammonium
`chloride protected cells from DT [47]. This led to the understanding that endocytic vesicles are
`maintained at acidic pH. And as we now know, acidic pH is required for DT transport to the
`cytosol [48,49].
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`2. Toxin Candidates
`
`Protein toxins first came to prominence as pathogenic factors released by bacteria or poisons
`ingested from toxic plants and were noteworthy because, as “single agents”, they caused such severe
`morbidity and mortality. Many decades later, it is intriguing to note that several of these toxins share a
`common biochemical mechanism i.e., they inhibit protein synthesis. DT and PE have similar
`mechanisms: they ADP-ribosylate elongation factor 2 and halt protein synthesis at the elongation
`step [50]. Ricin A chain is an N-glycosidase and is toxic because it depurinates a critical adenine in
`28S rRNA [51]. And it is from these toxins that investigators have turned most often to construct
`immunotoxins. Originally there were three toxin candidates: the plant toxin ricin (and similar toxins
`expressed from other plants [52,53]) and the bacterial toxins DT and PE. Because of toxin complexity
`and reagent loyalty, rarely did individual researchers use more than one toxin, so direct toxin-to-toxin
`comparisons were seldom undertaken. Even to this day, these three toxins remain among the top
`choices for immunotoxin development; although, other plant toxins and fungal toxins are also used to
`make immunotoxins [53,54]. So what were the features that characterize toxin utility?
`Toxin structure, orientation of domains, expression and purification yields, ease of cloning, sugar
`binding, immunogenicity, and non-specific toxicity have each contributed to researchers choosing to
`work with one toxin over another. Many of these issues have been discussed in a recent review [32]
`and won’t be discussed at length here but a few key points should be mentioned. Each toxin has an
`active enzyme domain that must reach the cell cytosol to kill cells. Each toxin also has a cell-binding
`domain that has to be eliminated or nullified before attachment to an antibody. Finally there is the
`“translocation” function, which may or may not be encompassed in a single functional domain. The
`“job” of the translocation domain is the transport of the toxin’s enzyme domain across an intracellular
`membrane into the cell cytosol. And, even today, understanding the mechanism or mechanisms of
`toxin translocation remains a challenge. In broad terms, DT translocates from acidic endosomes with
`the aid of its T-domain [55], while ricin and PE associate with the ER prior to translocation; although
`the case for the ER pathway is stronger for PE [56] (with a known KDEL-like sequence at the
`C-terminus) than it is for ricin [57,58]. For each toxin, translocation apparently involves unfolding
`prior to reaching the cytosol [59,60] and refolding once in the cytosol, leading the speculation that
`chaperones may be needed for the most efficient translocation [61]. DT and PE have distinct binding
`and enzymatic domains-at each termini and an alpha helical domain in the middle (Figure 1). The role
`of the helical domain is more clearly defined for DT than for PE but it is intriguing to note that
`multi-helical domains of protein toxins may be involved in membrane insertion and possible pore
`formation [62]. In fact, the membrane insertion of the T domain of DT has been used to model the
`molecular behavior of Bax and Bak, the proapoptosis Bcl2 proteins that cause pores in mitochondria,
`leading to the release of cytochrome C and the initiation of apoptosis [62]. In the case of ricin distinct
`binding (the B chain) and enzyme domains (the A chain) are also defined (Figure 1) while
`translocation activity is harder to locate precisely. However, it is noteworthy to point out the presence
`of a 5-helix structure in the middle of the A chain. Ricin A (RTA) is clearly able to translocate to the
`cytosol when coupled to some monoclonal antibodies [63]. And several trials are on-going evaluating the
`utility of this form of the toxin [19,63]. However, when RTA is coupled to other antibodies, there is
`poor cell killing and researchers are “forced” to include the B chain as well [22,64] suggesting that in
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`some instances the B chain is needed to direct the routing of the A chain. To nullify normal B chain
`binding to surface galactose residues, immunotoxins were developed using “blocked” ricin. Blocked
`ricin retains sugar-binding residues but their active sites are blocked via chemical modification.
`Retaining the entire B-chain, albeit with reduced binding activity, has also been reported for DT
`immunotoxins constructed with CRM9 [35,36].
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`Figure 1. Graphic representations of three toxins, diphtheria toxin (DT), Pseudomonas
`exotoxin (PE) and the plant toxin, ricin. Above each “domain” is a functional label. Below
`each domain is a common name that was used in early publications. DT has an N-terminus
`catalytic domain (C-domain) also known and the A fragment followed by a protease
`processing site,
`then a nine helix domain (commonly known as
`the “T” or
`translocation-domain) followed by a receptor binding domain (R-domain). The B-fragment
`includes both the T-domain and the R-domain. PE has an N-terminal receptor-binding
`domain followed by a processing domain. Then at the C-terminus there is a catalytic
`domain followed by a KDEL-like sequence. Ricin has a catalytic domain at the
`N-terminus, followed by a processing site and then a duplicated receptor-binding domain
`with a preference for binding galactose residues. Each toxin has a helical domain where
`several helices follow in close sequence. For DT there are nine helices while PE has 6; and
`these helices are arranged in what appears to be a separate domain between C and
`R-domains. Ricin also has a cluster of helices but these are located in the middle of its
`catalytic domain. A simple view of these helical domains is that they function in the
`translocation of each toxin’s C-domain. However, this has only been established for the
`T-domain of DT. The site of proteolytic processing is shown for each toxin.
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`Finally, toxins that interact with mammalian cells invariably need “processing” steps to convert a
`precursor molecule to an active one [65]. In addition, toxins act in the cell cytosol and must reach their
`destination via a collaboration between the toxin and the target cell [57]. By tracking the fate of toxin
`molecules one can learn about cellular functions and thus toxins are probes for the cells they attack.
`For DT and PE minimum processing includes a protease cleavage step [66] (Figure 1) and a reduction
`of a key disulfide bond [67]. Other features include a transient unfolding step-followed by
`refolding [61] For PE, there is a KDEL-like ER retention sequence at the C-terminus that is essential for cell
`killing activity [56]. So for PE and PE-derived immunotoxins there are four known steps prior to
`reaching the cytosol: (1) receptor binding, (2) furin cleavage, (3) disulfide reduction and (4) interaction
`with KDEL receptor 2. For DT in addition to protease “nicking” there is a cytosolic chaperone and
`reductase that have been identified as being important for toxin action [61]. Proteolytic processing of
`ricin occurs in the germinating castor bean, producing the A and B chains (Figure 1). Because ricin
`interacts with terminal galactose residues displayed on many different surface receptors, tracking its
`fate can be challenging [68,69]. Ricin also requires an intracellular reduction step. Recent studies using
`RNAi highlighted important genes in the ricin pathway and compared these with genes involved in PE
`intoxication [70,71]. These genetic screens along with chemical screens to identify anti-ricin
`compounds should provide new insights into ricin’s intracellular trafficking pathway [72].
`
`3. Early Immunotoxin Development
`
`Thorpe et al. set the stage for immunotoxin development by confirming that protein toxins could be
`redirected to kill selected cell types over bystander cells [2]. However, their result was achieved with a
`poorly defined antibody preparation. Using the same concept but with the benefit of Kohler and
`Milstein’s monoclonal antibody technology [3,73], well defined immunotoxins of a single specificity
`were produced. These included, ricin-, DT- and PE-derived immunotoxins. Besides antibody and toxin
`selection, other steps in the manufacture of immunotoxins included the use of different chemical
`“glues” (called cross linkers) to join the two molecules in a manner that kept both parts
`functional [74,75]. Early on it was appreciated that antibodies alone were rarely cytotoxic. This fueled
`research into making antibodies more potent by attaching protein toxins to them. Potency depended not
`only on internalization but also on the “correct” internal conditions within the cell. For instance, in the
`case of early immunotoxins to CD5 made with the T101 antibody, neutralization of acidic pH was
`deemed important for optimal killing [76]. In other immunotoxins, disulfide linkers allowed for
`cytotoxic activity while thioether linkers did not, confirming the need for the appropriate reducing
`environment to allow separation of toxin from antibody [75,77].
`For PE the first immunotoxins were made via thioether linkage from an intact monoclonal antibody
`to the native intact toxin (Figure 2B). When the functions of the toxin’s structural domain were
`discovered, it made sense to delete the receptor binding domain, producing a molecule termed
`PE40-based on its molecular weight. However, the deletion of the N-terminal domain (harboring many
`lysine residues for chemical conjugation) created a problem of how to attach PE40 to antibodies. This
`was solved by the introduction of a novel lysine residue near the terminus of PE40, producing
`Lys-PE40 (Figure 2C). Together, these chemical conjugates made up first and second generations of
`immunotoxins.
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`Figure 2. Immunotoxin construction-from oldest to newest. First generation immunotoxins
`were constructed by using chemical crosslinking agents to attach intact toxins to intact
`antibodies. Second generation
`immunotoxins used modified
`toxins
`lacking
`receptor-binding domains. Third generation molecules used cloned antibody fragments
`fused to modified toxin genes; allowing for the recombinant production of homogeneous
`protein. Further improvements of the third generation molecule might include the removal
`of immunogenic amino acids including (as shown) much of the multi-helical domain of PE.
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`4. Evolved Immunotoxins
`
`Molecular cloning techniques, producing gene fusions, together with prokaryotic expression
`systems revolutionized the development of immunotoxins and has produced a third generation molecule
`(Figure 2D). No longer was it necessary to purify large quantities of antibody and toxin and then
`combine the two using chemical cross linkers. The latter approach was not very efficient, produced
`heterogeneous products and risked interfering with antigen binding. The initial breakthrough for
`recombinant immunotoxins came with the expression of single chain Fv fragments in E. coli that
`retained antigen binding [78,79]. This led directly to the first recombinant antibody-toxin fusion
`protein [80]. For PE-based immunotoxins this placed the antibody fragment at the N-terminus leaving
`the KDEL-like sequence free at the C-terminus (Figure 2D). While this approach was very attractive
`for the design of recombinant cytotoxic molecules, several challenges remained for expression and
`purification of active monomeric species. Codon optimization (mostly avoiding rare Arg codons in the
`antibody clone), inclusion body production, refolding protocols that included redox-shuffling and a
`multi-step purification scheme solved most of the problems [81,82]. One problem that was not
`immediately obvious related to the propensity of single chain antibody fragments to aggregate. For
`unstable Fvs, this issue was overcome with the incorporation of a novel disulfide linker that replaced
`the flexible linker originally described to keep the heavy and light chains tethered to one
`another [83,84]. Once produced, immunotoxins directed to potential cancer targets were investigated
`for cell killing activity and for non-specific damage to non-target tissue. Of various candidate antigens,
`mesothelin, CD22 and CD25 remain options for clinical investigation [85–87]. Other antigens
`including HER2/neu, Lewis-Y, CD30 and CD19 were considered for pre-clinical development but
`were either abandoned because of systemic toxicity or never developed because of poor cytotoxic
`activity in tissue culture.
`More recent advances in the development of recombinant immunotoxins have focused on the
`production of smaller and less immunogenic versions of the original PE40/38 molecule [88–90]. By
`eliminating most of domain II of PE, a smaller molecule was produced that retained cytotoxic action
`with the added benefit of removal of one major and several minor immunogenic epitopes. The
`production of a smaller version of PE-derived immunotoxins was the by-product of an effort to
`stabilize the toxin intracellularly and prevent degradation in lysosomes. The deletion of domain II also
`removed many lysosomal cleavage sites and produced a molecule that was termed “LR” for lysosomal
`resistant [88]. Thus the LR version of PE-derived immunotoxins exhibits three new features: it is
`smaller, less immunogenic and more resistant to cleavage by lysosomal enzymes [89,90]. Generating
`molecules that lack domain II also produced a “puzzle”: these immunotoxins are more active against
`various cell types and less active against others [88]. A full explanation for this disparity remains to be
`uncovered as does a full appreciation of the role of domain II in PE-mediated killing. However, the
`removal of domain II allowed for the production of a “minimal” immunotoxin. This path of
`development was summarized in a recent review [91]. Data support the retention of an N-terminal
`antibody Fv, linked by a minimal furin site to domain III and the placement of a KDEL-like sequence
`at the C-terminus—see Figure 2E.
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`5. Gene Fusions with Cell-Binding Ligands
`
`Classically, immunotoxins describe an antibody-toxin molecule, either linked or fused together.
`However, receptor ligands can also be used to direct toxins to kill cells expressing specific target
`receptors. EGF was an early candidate in this class of ligand-toxin molecule followed by TGFalpha,
`IL-2, IL-4, IL-6, IL-13 and with diphtheria toxin, MSH, TF, and IL-2 [27,92–94]. Ligand-toxins can be
`efficient at killing cells displaying the corresponding receptor but potentially send a “mixed” message
`to cells. Many peptide ligands convey growth or survival signals via binding to surface receptors.
`These signals are often transmitted via phosphorylation cascades and happen quite rapidly. Thus the
`signal to grow or survive can be transmitted perhaps several hours before the toxin gains access to the
`cytosol and begins to shut down protein synthesis. Despite these concerns, it is noteworthy that the
`only “immunotoxin” approved for treatment to date is a DT-IL2 toxin termed, denileukin diftitox.
`
`6. Toxin Resistance
`
`Three kinds of toxin resistance are known: cellular, organismal and immunologic. Cellular
`resistance is further divided into inefficient delivery to the cytosol [95,96], failure to ADPr EF2 [97] or
`poor triggering of apoptosis [98]. At the level of the intact animal or person, delivery to every cell can
`be challenging and may require either repeated dosing or dosing together with agents that improve
`access to tumor cells [99,100]. Finally, immunologic resistance arises when patients with intact
`immune systems make neutralizing antibodies to the immunotoxin (usually the toxin portion of the
`immunotoxin) [26,101]. For each type of resistance listed above, it is possible to make improvements
`in design of the immunotoxin itself that should produce better anti-tumor results. That said, it will be
`additionally useful to design treatment options that include co-administration of “helper” agents that
`overcome the particular resistance of concern—see below.
`
`7. Immunotoxin-Drug Combinations
`
`Even from early days—combinations were sought to make immunotoxins more active. For
`example, ammonium chloride or monensin was added to cultured cells to make them more sensitive to
`ricin A immunotoxins [102,103]. Likewise calcium channel blockers enhanced the activity of
`PE-based immunotoxins [104]. Co-delivery with endosome-disrupting adenovirus also enhanced PE
`immunotoxins [105]. None of these approaches was ever likely to be useful in vivo because it would be
`difficult to achieve the necessary drug levels or overcome safety concerns. However, these results
`confirmed the utility of using combinations to increase the delivery of toxins to the cytosol and also
`highlighted a potential issue of inefficient trafficking of toxins within target cells. Expanding on this
`concept was a study by Youle et al. showing that retinoic acid disrupted Golgi structure and in the
`process enhanced the activity of a ricin A chain immunotoxin by 10,000-fold. Like earlier studies, the
`authors concluded that the enhanced killing was a result of increased delivery of toxin to the cytosol [106].
`A second reason for using enhancers has been proposed more recently and that is to decrease the
`influence of prosurvival factors that might otherwise prevent cells from succumbing to cytotoxic
`therapy [107–109]. A similar strategy employs targeting agents to “death” receptors has also been
`explored as a way to overcome resistance to apoptosis [110].
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`At the level of the intact animal, there are several reports of using combination treatments in vivo to
`enhance immunotoxin-mediated antitumor action. Agents that enhance immunotoxin access to tumor
`cells such as taxol, produce improved responses over either agent alone [100,111]. The report from
`2007 suggests that local concentrations of shed antigen were also reduced with taxol treatment,
`potentially allowing immunotoxin molecules less restricted access to tumor cells [100]. Similarly,
`agents that neutralize survival factors, such as Bcl-2 proteins, may enhance immunotoxin action. In
`several tumor systems this approach appears to have merit where the presence of the Bcl2/Bcl-xl
`inhibitor ABT-737 enhanced immunotoxin-mediated antitumor action [98,109].
`If we examine on-going clinical trials, we see these kinds of efforts persist. Combotox was
`developed to improve the antitumor action of ricin A chain-based immunotoxins: two immunotoxins
`are administered in a 1:1 ratio. By combining an anti-CD22 immunotoxin with an anti-CD19
`immunotoxin it is hoped to avoid resistance and to produce improved treatment outcomes compared to
`the use of either agent alone [20,21]. Phase 1 studies’ results with this combination approach produced
`encouraging results and a recent trial was opened using Combotox in conjunction with cytarabine for
`patients with B-cell ALL (ClinicalTrials.gov Identifier: NCT01408160). Likewise, in an effort to
`enhance patient responses to the SS1P immunotoxin targeting surface mesothelin, a current trial
`investigates the co-administration of the immunosuppressive agents cytoxan and pentastatin
`(ClinicalTrials.gov Identifier: NCT01362790).
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`8. Future Directions
`
`Combinations to improve killing kinetics of immunotoxins or those that prevent the expression of
`survival pathways are likely to be particularly useful. Likewise, agents that break up tumor masses and
`allow greater access of immunotoxin to individual malignant cells should improve outcomes. Finally,
`agents that suppress neutralizing antibodies and that can be administered without severe toxicity,
`should be explored.
`
`9. Conclusions
`
`Immunotoxins exhibit selectivity and potency for cancer cells and may be effective clinically as
`single agents only under exceptional circumstances (such as the targeting of circulating tumor cells
`expressing a high number of target antigens). Under more usual circumstances, immunotoxins may be
`beneficial as part of a combined treatment with other agents. The process of discovering agents that
`work best in combination with immunotoxins is currently ongoing. When combinations are needed,
`agents that increase toxin killing and reduce immunogenicity will likely be the most valuable ones.
`
`Acknowledgements
`
`This research was supported by funding from the Intramural Research Program, National Cancer
`Institute, Center for Cancer Research at the National Institutes of Health, Department of Health and
`Human Services. The authors also acknowledge the many contributions of their colleagues at the
`Laboratory of Molecular Biology.
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`Conflicts of Interest
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`The authors declare no conflict of interest.
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`References
`
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