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MEETING REPORT Engineering toxins for 21st century therapies John A. Chaddock 1 and K. Ravi Acharya 2 1 Syntaxin Limited, Abingdon, Oxon, UK 2 Department of Biology and Biochemistry, University of Bath, UK Introduction It is a paradox of drug development that nature’s most powerful toxins can also be the active component of some of the most effective therapies for a range of conditions. For example, botulinum toxins produced by the genus Clostridia have the ability to cause botu- lism and tetanus following exposure to extremely small doses of protein. Conversely, botulinum neurotoxins (BoNTs, e.g. BOTOX Ò , Dysport Ò ; see Table 1, [1]) have become the first-line treatment for a variety of debilitating neuromuscular conditions; for example, various dystonia, spasmodic torticollis, blepharospasm and strabismus. Native-sourced botulinum products have also been approved for use in hyperhidrosis and, most recently, in chronic migraine. Similarly, up to 10% of diphtheria patients die (even if properly treated) and yet components of diphtheria toxin have been used to create successful new medicines (e.g. ONTAK Ò ;a CD25-directed cytotoxin) for the treatment of a range of cancers, such as persistent or recurrent cutaneous T-cell lymphoma. In September 2010, a small, focussed meeting was convened as part of the Royal Society (UK) Interna- tional Seminar series. The meeting was assembled to discuss the design, manufacture and regulatory consid- erations of developing novel therapies that utilize toxin domains, and to discuss the protein engineering Keywords biotechnology; botulinum neurotoxin; innovation; therapy; toxin Correspondence K. R. Acharya, Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK Fax: +44 1225 386779 Tel: +44 1225 386238 E-mail: bsskra@bath.ac.uk J. A. Chaddock, Syntaxin Limited, Units 4–10, Barton Lane, Abingdon, Oxon OX14 3YS, UK Fax: +44 1235 552200 Tel: +44 1235 552115 E-mail: john.chaddock@syntaxin.com (Received 5 November 2010, revised 21 December 2010, accepted 10 January 2011) doi:10.1111/j.1742-4658.2011.08013.x ‘Engineering Toxins for 21st Century Therapies’ (9–10 September 2010) was part of the Royal Society International Seminar series held at the Kavli International Centre, UK. Participants were assembled from a range of disciplines (academic, industry, regulatory, public health) to discuss the future potential of toxin-based therapies. The meeting explored how the current structural and mechanistic knowledge of toxins could be used to engineer future toxin-based therapies. To date, significant progress has been made in the design of novel recombinant biologics based on domains of natural toxins, engineered to exhibit advantageous properties. The meet- ing concluded, firstly that future product development vitally required the appropriate combination of creativity and innovation that can come from the academic, biotechnology and pharma sectors. Second, that continued investigation into understanding the basic science of the toxins and their targets was essential in order to develop new opportunities for the existing products and to create new products with enhanced properties. Finally, it was concluded that the clinical potential for development of novel biologics based on toxin domains was evident. Abbreviations BoNT, botulinum neurotoxin; LC, light chain; TSI, targeted secretion inhibitor. FEBS Journal 278 (2011) 899–904 ª 2011 The Authors Journal compilation ª 2011 FEBS 899 opportunities that exist for developing new medicines that harness components of some of nature’s most potent protein toxins. These objectives were high- lighted by K. Ravi Acharya (organizer of the meeting, University of Bath, UK) in his introduction to the meeting. The nature of the meeting was such that the majority of the discussion was related to clostridial neurotoxins (CNTs), specifically BoNT. This is primarily because BoNTs have emerged from a physician-led investiga- tional drug in the 1980s to become a multi-billion dollar product with a range of medical and cosmetic applications. Nevertheless, many of the concepts and conclusions that were described during the meeting have relevance to other similar toxins that may have therapeutic utility. The participants spent a consider- able amount of time evaluating how toxin structure– function information and understanding their unique mechanisms of action can provide new opportunities for the development of therapeutic interventions. Clostridial neurotoxins are multi-domain structures [2] of  150 kDa that consist of five major structural elements (Fig. 1): l An N-terminal 50 kDa light chain (LC) domain is a metalloprotease with specificity for SNARE pro- tein substrate. To the C-terminus of the LC is; l An H N domain ( 50 kDa) that forms a pore in intracellular membranes to effect translocation of the LC into the cytosol. The H N domain is cova- lently attached to the LC by; l A single disulfide bond that is reduced in the cyto- sol as part of the LC translocation event; l An H CN domain ( 25 kDa) that is C-terminal to the H N domain and is of unknown function. The H CN domain is a subdomain of the ‘binding domain’ formed with; l An H CC domain ( 25 kDa) that exhibits neuronal binding capability via two binding sites. Although the precise ‘shape’ of other bacterial pro- tein toxins, for example diphtheria toxin, can differ markedly from clostridial BoNTs [BoNTs with seven different serotypes (BoNT ⁄ A–BoNT ⁄ G)], many have been shown to be similar in concept: the delineation of a ‘binding’ domain, a ‘translocation’ domain and a ‘catalytic’ domain is common. This modular, domain- based structure has enabled drug development scien- tists to implement protein engineering approaches to create novel proteins that harness specific biology of the ‘parent’ toxins. The most advanced demonstration of this concept is exemplified in targeted secretion inhibitors (TSIs); a novel class of biotherapeutics for treating diseases where inappropriate cell secretion is a primary cause. Concepts such as the TSI platform and the similar advancements that have been made by sci- entific advancement in this ‘toxin’ field are described in the meeting highlights below. Meeting highlights This meeting brought together leaders in their respec- tive fields and major scientific and conceptual advance- ments are described in the following section. There are, however, a number of highlights that require specific mention: l Academic laboratories and small biotechnology com- panies are rich sources of creativity and entrepreneurial Fig. 1. Tertiary structure of BoNT ⁄ A (pdb code 3BTA [8]). Table 1. Summary of botulinum toxin products (taken from [1]). BOTOX â Dysport â Myobloc â ⁄ Neurobloc â NT201 ⁄ Xeomin â Company Allergan Inc. Ipsen Inc. Solstice Merz Pharmaceuticals Type Type A Type A Type B Type A Approvals In over 75 countries In over 65 countries Some EU + USA + CA Some EU, Mexico, Argentina Active substance Type A complex (900 kDa) Type A complex (900 kDa) Type B complex (700 kDa) Type A, free from complexing proteins (150 kDa) Mode of action SNAP-25 SNAP-25 VAMP SNAP-25 Engineering toxins for 21st century therapies J. A. Chaddock and K. R. Acharya 900 FEBS Journal 278 (2011) 899–904 ª 2011 The Authors Journal compilation ª 2011 FEBS drive; pharmaceutical industry has the infrastructure to innovate, i.e. apply the new ideas in the market place, and it is vital that these two approaches to drug dis- covery and development are brought together. Exam- ples of creativity in the design of toxin fragment-based therapeutics included next-generation Ontak, TSIs, BoNT hybrids and BoTIMs, TrapoX. l Investment in basic science research is an essential requirement for the development of new concepts and new opportunities. Throughout the meeting there were multiple examples of where opportunities for the devel- opment of new medicines could emerge from scientific exploration into fundamental biological mechanisms. l Small, focussed meetings of this type are an excel- lent forum for cross-discipline discussion and learning. Major achievements A number of advancements in scientific knowledge or conceptual thought emerged from the meeting, which have been collated into four themes: Understanding protein structure–function can lead to the development of new medicines In one of the most advanced examples of developing engineered toxins for the clinic, John Murphy (Boston University School of Medicine, USA) noted that ON- TAK Ò (a diphtheria toxin-interleukin-2 fusion protein [3]) had successfully completed studies in steroid-resis- tant graft versus host disease and refractory T-cell lym- phoma, but commented that the major adverse event is vascular leak syndrome. Murphy provided experimen- tal evidence for the identification and elimination of peptide motifs in diphtheria toxin that promote vascu- lar leak syndrome, which could be the basis of devel- oping a drug with similar efficacy but a much reduced adverse event profile, thereby widening the therapeutic opportunities for use of the diphtheria toxin cell ablation technology. The advances in structural knowledge in the BoNT field (as summarized by Subramanyam Swaminathan, Brookhaven National Laboratory, USA), has led to a large number of opportunities to manipulate those domains that nature has brought together as BoNT, for the creation of new therapeutic opportunities. In particular, Keith Foster (Syntaxin Ltd, UK) described the development of a platform of new biologicals based on the LC and H N domain of BoNT termed TSIs. TSIs do not possess the native BoNT binding domain within their structure and therefore can be retargeted to any cell of choice by incorporation of an appropriate ligand (peptide or protein) to a cell surface marker. Although the precise nature of the range of targeting ligands built into TSIs was not disclosed, the TSI approach takes SNARE cleavage beyond the neu- ronal target to alternative cell types. In this way, the SNARE cleavage activity of the LC can be redirected to cells that are secreting mediators that cause disease. Foster described the progress of TSIs into phase I clin- ical trials in pain and advanced preclinical studies in acromegaly. In contrast to the domain replacement path taken by Syntaxin Ltd, Andreas Rummel (Medizinische Ho- chschule, Hannover, Germany) described how the study of the mechanism of binding by the native neu- rotoxins had led to the development of TrapoX, a BoNT ⁄ A-based protein that incorporates the latest understanding of binding to increase the potency of BoNT ⁄ A. Rummel reported that TrapoX (which has a specific mutation of the H C binding site) has more than three-fold increased potency and could, therefore, lead to lower therapeutic dosages of BoNT. In a sec- ond example of engineering the native BoNT structure, Oliver Dolly (Dublin City University, Ireland) reported the construction of ‘BoTIMs’ (full-length BoNTs incorporating catalytic-inactive LC ⁄ A), which were recombinantly fused to LC ⁄ E domains to create a hybrid construct that utilized components within the LC ⁄ A element to extend the intracellular persistence of the LC ⁄ E and therefore the duration of action of LC ⁄ E-induced SNAP-25 cleavage. Dolly proposed that the LC ⁄ E-induced cleavage of SNAP-25 would be advantageous for specific conditions, for example pain. Finally, studies by Joseph Barbieri (Medical College of Wisconsin at Milwaukee, USA) have led to the identification of a new binding loop in BoNT ⁄ C and ⁄ D and the observation that TeNT, BoNT ⁄ C and BoNT ⁄ D enter cortical neurons via activity- independent endocytosis. Advances in the understanding of toxin fragment translocation A leader in the field of membrane protein transloca- tion, Mauricio Montal (University of California, San Diego, CA, USA), discussed the impact of the BoNT H C domain on the pH dependency of translocation. The established dogma states that low pH is essential for H N domain insertion into the endosomal mem- brane in order to form the pore for LC translocation. Using a precise membrane conductance assay he noted that no pH gradient was required for LHA (a frag- ment of BoNT ⁄ A comprising the LC and the H N domain) translocation, whereas BoNT ⁄ A required apHof 5 for efficient LC translocation. Montal J. A. Chaddock and K. R. Acharya Engineering toxins for 21st century therapies FEBS Journal 278 (2011) 899–904 ª 2011 The Authors Journal compilation ª 2011 FEBS 901 hypothesized that the H C domain acts as a chaperone for the LC; restricting membrane insertion until local- ized into an acidic endosome. Because this effect was seen in simple lipid bilayers, it is probably a structural effect rather than one based on binding. Montal also confirmed the importance of the disulfide bond to ensure LC translocation through the H N pore. In the field of diphtheria toxin, Murphy described data that indicated binding of diphtheria toxin to the COPI complex via KXKXX sequence motifs in the transmembrane domain of diphtheria toxin. He hypothesized that this binding event provides the drive for the translocation process of the diphtheria toxin C-domain across the intracellular vesicle membrane, and made the staggering observation that the same KXKXX signals for this process are present in anthrax lethal factor and elongation factor. The discussion on this topic addressed the question of whether there is a common motif within bacterial protein toxins that facilitates the translocation process by binding to host cell machinery. Murphy reported that a detailed under- standing of the amino acid requirements for the trans- location event had led to an experimentally testable opportunity to deliver nucleic acids into the cytosol for cell modulation. Murphy also noted the role of Grp78 and hypothesized that such a vesicle-located unfoldase may be involved in unfolding the catalytic domains of bacterial protein toxins prior to translocation. Although immunogenicity was not a key topic of the meeting, Jim Marks (University of California and San Francisco General Hospital, USA) noted that antibodies raised to the H N domain of BoNT were effective inhibitors of function and Montal noted that one specific antibody raised to a conserved epitope across serotypes did inhibit membrane insertion of the H N domain. Understanding the biology of toxin action can provide new ideas for medicine development Thierry Galli (INSERM, France) reported that BoNT ⁄ D inhibited fast endocytosis in a primary dorsal root ganglion neuronal culture system, thereby impli- cating VAMP2 to have a role in endocytosis. Also, expression of TeNT in epithelial cells (containing VAMP3) prevents the recycling of integrins. Hence, BoNTs have a role in the disruption of endocytosis and endocytic mechanisms, not just exocytosis. George Oyler (Synaptic Research LLC, USA) described the development of a designer VHH E3 ubiquitin ligase that is able to degrade LC ⁄ A. The VHH structure is the antigen binding fragment of cam- elid heavy chain antibodies. If suitably delivered to BoNT-intoxicated neurons, such a protein would have the potential to degrade the ordinarily persistent LC ⁄ A and thereby accelerate recovery from BoNT ⁄ A poison- ing. However, the potential for this approach is more widespread and could provide a mechanism for the delivery of ubiquitin ligases for the modification of cellular function per se. Giampietro Schiavo (Cancer Research UK London Research Institute, UK) described elegant studies into axonal transport of CNTs and proposed the use of labelled CNTs as diagnostic markers for neuronal dis- ease, e.g. motor neurone disease, utilizing live body imaging to assess the speed of axonal transport. Praveen Anand (Imperial College London, UK) described the wide range of toxins currently employed in providing medical benefit (e.g. capsaicin, resinifero- toxin, ziconotide, CNTs, chemotherapeutic agents). By understanding the expression of SV2A in neuronal samples from patients, Anand was able to identify potential and preferential sensory targets for BoNT therapy, including painful nerve injury, inflammatory bowel disease and irritable bowel syndrome. Development of new medicines requires a new mindset, a new model Melanie Lee (Syntaxin Ltd, UK) opened the meeting with a discussion of the rise and fall of the fully inte- grated pharmaceutical company model, pharma’s resis- tance to change and blindness to entrepreneurial opportunities. Acknowledging that the pharmaceutical industry faced many challenges to their pipeline and profitability (research and development, patent expiry, generics, high attrition rates), Lee commented that the pharmaceutical industry has evolved away from prod- uct innovation to focused process innovation and yet the pipelines required creativity, which in turn requires overcoming the major hurdles of new knowledge advancement. William Habig (former member of the Food and Drug Administration, USA) observed that there was generally a lower success rate for small com- panies ⁄ first applicants to the Food and Drug Adminis- tration and added that only 30% of approved pharmaceuticals recover the cost of their development. Murphy also noted the negative impact on product optimization when a small biotechnology company develops innovative medicines and funds are limited for extensive basic science investment. Emerging trends and future directions One of the clear outputs of the meeting was an acknowledgement that functional domains from toxins Engineering toxins for 21st century therapies J. A. Chaddock and K. R. Acharya 902 FEBS Journal 278 (2011) 899–904 ª 2011 The Authors Journal compilation ª 2011 FEBS can form the basis for new medicine development. Engineered toxins take advantage of the natural selec- tion process that has already created protein structures with specific functions that can modulate intracellular events. In the final session of the meeting, John Chad- dock (Syntaxin Ltd, UK) captured the immediate opinion of the participants regarding the major themes that had emerged from the meeting. These flipchart scribbles are reproduced in Table 2 and represent a snapshot of the opinions at the time. In preparing this meeting summary, many of the ideas and thoughts for the future have been crystal- lized into trends and future direction statements (Table 2). The general consensus of the meeting was that there is an opportunity for ‘science-driven evolu- tion’, i.e. using in-depth knowledge of the fine details of toxin structure–function in combination with a sig- nificantly enhanced understanding of the biology of the target cell. For example, in the botulinum toxin opportunities space, the participants appreciated the importance of understanding the diversity of the neu- ronal system and the detail of biological processes therein. For this to occur, there is an absolute require- ment for continued basic scientific research. There is also the need to combine the talents of the academic and entrepreneurial biotechnology sector with the resources of pharma that are necessary to innovate. Mechanistically, targeted toxins were known to have multiple medical applications based on their ability to affect: (a) secretion ⁄ exocytosis ⁄ endocytosis through SNARE cleavage, (b) cell viability through modifica- tion of essential cellular pathways, (c) general cellular modulation by facilitating delivery of protein ⁄ nucleic acid cargo. It was agreed that targeted toxins could have diagnostic potential. As noted above, naturally evolved toxins are a good framework for the design of cell modulation technologies, as they often target molecular mechanisms at the heart of disease condi- tions. A challenge to the community is to change the ‘reputation’ of clostridial (and other) toxins from ‘dan- gerous’ to be accepted as unique and effective medi- cines and diagnostic tools for the future (Fig. 2). The wider context Although not a topic that was specifically discussed at the meeting, it is useful to appreciate the wider con- text of the use of toxins and toxin fragments for the development of medical products, vaccines etc. In addition to the toxins described within the meeting, other researchers have, for a number of years, devel- oped novel molecule-based ribosome inactivating pro- teins (such as ricin, saporin and Shiga toxin) and ADP-ribosylating bacterial toxins such as Pseudomo- nas exotoxin. All of these approaches are linked by their desire to utilize warheads that lead to cell death. The most prevalent target for such novel molecules has been for the treatment of cancer and proliferative diseases. For example, the literature is rich with exam- ples of fragments of Pseudomonas exotoxin targeted to cancer cells via a range of targeting ligands and antibodies [4]. Such molecules have demonstrated some significant success in preclinical studies and have Table 2. Captured participant opinions on seminar themes. The LC protease in the cell is not well understood and could have a range of properties that are poorly understood and poorly predicted from simple in vitro experiments The role of the glycolipids and glycoproteins is greater than previously understood. Also, we need tools to better understand glyco-contributions It is difficult to translate ideas into products Targeting ubiquitin-like molecules is a therapeutic opportunity There is an ability to engineer proteins based on structural knowledge: science-driven evolution The neuronal system is highly diverse and there are many unknowns There is a need for new tools to enable better understanding of biological systems There is an appreciation of the increased number of applications for nonserotype A-based products Custom-designed molecules are a possibility, based on a better understanding of an individual’s specific needs and the opportunities to tailor the products. Opportunities for selected subpopulation treatment (at reduced cost) Interaction between the toxin and the target cell is an important understanding Fig. 2. Key elements of developing toxin-based therapies. J. A. Chaddock and K. R. Acharya Engineering toxins for 21st century therapies FEBS Journal 278 (2011) 899–904 ª 2011 The Authors Journal compilation ª 2011 FEBS 903 given much hope to the possibility of targeted cellular ablation technology being implemented within the clinical setting. However, despite over 20 years of investigative studies, no such molecules comprising Pseudomonas domains have yet transitioned through clinical trials to the clinic. Concerns have been raised over antibody formation, hepatic toxicity and nonspe- cific side-effects, such as vascular leak syndrome. Indeed, attempts to overcome potential immunological responses to targeted toxins have led to the develop- ment of agents based on human protein warheads, for example proapoptotic proteins or RNase [5]. Alterna- tively, protein engineering solutions have been used to create modified targeted toxins with reduced immuno- genicity [6]. Some of the pioneering work towards the implemen- tation of cell ablation technology in the clinic arose through the use of ricin and domain fragments of ricin targeted to specific cells by virtue of conjugation to an appropriate antibody. Ricin, and the ribosome-inacti- vating class, kills cells by preventing protein synthesis. Ricin-based approaches were promoted as exemplars of the ‘immunotoxin’ concept and, although very effec- tive in the preclinical setting, did not translate into widespread use in the clinic. The main issues faced included vascular leak syndrome (as described for the ONTAK strategy earlier) and hepatic toxicity. An alternative strategy for the use of cell ablation technol- ogy has been taken with the development of a conju- gate of substance P-saporin [7]. Using the substance P peptide to target NK1 receptors on the extracellular face of the cell membrane of pain-sensing neurons, such molecules are being developed to ablate specific neuronal populations. Such an approach has the potential to inhibit the symptoms of cancer pain, for example in patient populations that have become resis- tant to morphine and other opiates. After 30 years of research and development, where is the next clinical targeted-toxin product? Clearly, advancements have been made with the Pseudomonas exotoxin and diphtheria toxin platforms that give hope to successful completion of clinical studies and the expansion of the targeted toxin strategy. In the botu- linum field, TSIs have entered the clinic, bringing a new, nonablation strategy that will complement the various cell-kill strategies. Retargeting cell modifying proteins to target cells is entering an exciting phase of development. Acknowledgements KRA is supported by the Royal Society (UK) through an Industry Fellowship and is grateful to the Royal Society for sponsoring this international seminar. He also wishes to acknowledge Syntaxin Ltd (UK) for providing additional travel support for the partici- pants. References 1 Carruthers A & Carruthers J (2008) Botulinum toxin products overview. Skin Therapy Lett 13, 1–4. 2 Turton K, Chaddock JA & Acharya KR (2002) Botulinum and tetanus neurotoxins: structure, function and therapeutic utility. Trends Biochem Sci 27, 552–558. 3 Foss FM (2000) DAB(389)IL-2 (ONTAK): a novel fusion toxin therapy for lymphoma. Clin Lymphoma 1, 110–116. 4 Kreitman RJ (2006) Immunotoxins for targeted cancer therapy. AAPS J 8, E532–E551. 5 Mathew M & Verma RS (2009) Humanized immunotox- ins – a new generation of immunotoxins. Cancer Sci 100, 1359–1365. 6 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, 977–985. 7 Wiley RG & Lappi DA (2003) Targeted toxins in pain. Adv Drug Deliv Rev 55, 1043–1054. 8 Lacy DB, Tepp W, Cohen AC, DasGupta BR & Stevens RC (1998) Crystal structure of botulinum neurotoxin type A and implications for toxicity. Nat Struct Biol 5, 898–902. Engineering toxins for 21st century therapies J. A. Chaddock and K. R. Acharya 904 FEBS Journal 278 (2011) 899–904 ª 2011 The Authors Journal compilation ª 2011 FEBS . translocation, whereas BoNT ⁄ A required apHof 5 for efficient LC translocation. Montal J. A. Chaddock and K. R. Acharya Engineering toxins for 21st century therapies FEBS Journal 278 (2011) 899–904. structure of botulinum neurotoxin type A and implications for toxicity. Nat Struct Biol 5, 898–902. Engineering toxins for 21st century therapies J. A. Chaddock and K. R. Acharya 904 FEBS Journal. from complexing proteins (150 kDa) Mode of action SNAP-25 SNAP-25 VAMP SNAP-25 Engineering toxins for 21st century therapies J. A. Chaddock and K. R. Acharya 900 FEBS Journal 278 (2011) 899–904