Expert Opinion on Biological Therapy

Editorial Strategies for preventing influenza: future perspectives in influenza vaccine technology 1. Commentary Gian...

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Editorial

Strategies for preventing influenza: future perspectives in influenza vaccine technology 1.

Commentary

Gian Vincenzo Zuccotti† & Valentina Fabiano

Expert Opin. Biol. Ther. Downloaded from informahealthcare.com by HINARI on 12/14/10 For personal use only.

Department of Pediatrics, Luigi Sacco Hospital, Universita` degli Studi di Milano, Italy

Prevention of influenza transmission and containment of epidemics and pandemics require effective strategies that can be efficiently and easily addressed to the whole population. Annual vaccination is undoubtedly the most effective way to provide protection against influenza infection. Numbers of vaccines are actually available for yearly immunisation. However, the continuous increasing demand for rapidly available vaccine doses for immunisation of a larger proportion of population represents the stimulus for study and development of more efficient vaccine production technologies, which can guarantee reduction of vaccine manufacture times and better compliance by availability of easier routes of administration. New perspectives in influenza vaccination technology are making their way in the future panorama of influenza prevention strategies. Keywords: administration route, influenza, vaccine technology Expert Opin. Biol. Ther. (2011) 11(1):1-4

1.

Commentary

Effective prevention of infectious diseases transmission has always represented a challenge for scientists and public health authorities. Recently, the worldwide spread of the novel swine influenza H1N1 pandemic in 2009 highlighted the urgent need for rapid development of infectious diseases containment strategies for protecting population and preventing increasing morbidity and mortality. Influenza is a highly infectious respiratory disease for which the World Health Organisation (WHO) has reported annually from three to five million cases [1]. Although generally benign, influenza is considered responsible for a number of complications in patients with chronic underlying diseases which result in increasing direct and indirect costs for the affected subjects and, globally, for the whole society. Strategies for mitigation and containment of influenza epidemics can rely on well-known effective non-pharmaceutical and pharmaceutical interventions, such as individual hygiene, wearing face masks, quarantine, social distancing and administration of antiviral drugs and vaccines. Influenza vaccination represents actually the most effective strategy for preventing the disease and its eventually associated complications. For the 2010 -- 2011 influenza season, the centers for Disease Control (CDC) suggested universal vaccination for all healthy and at risk subjects from 6 months of age onwards. Traditional trivalent inactivated influenza vaccines are largely available and, recently, a live-attenuated intranasally administered influenza vaccine has been licensed in the USA for healthy non-pregnant subjects from 2 to 49 years of age. Recent studies demonstrated a better immunogenicity of this vaccine particularly in children; however, safety data in the paediatric population are already contrasting [2]. Numbers of new influenza vaccines are being studied and developed by exploring either new production technologies or new, easier routes of administration.

10.1517/14712598.2010.539047 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

1


Strategies for preventing influenza

The first challenge is the one associated with circumventing the problems of egg-based vaccine manufacture, characterised by poor flexibility and long-lasting production times, which cannot effectively respond to the eventually increasing demand for rapidly available vaccine doses in case of a new pandemic. Encouraging results are being observed with studies on cellbased vaccines. The use of mammalian or insect cell-culture lines has numerous advantages: shortening of production times, easier production of avian strain influenza vaccines which generally poorly grow in egg substrates, absence of sterility issues and improved immunogenicity because of better antigen presentation. Madin Darbin Canine Kidney (MDCK) cells, VERO cells, from African green monkey kidney cells and PER C.6 from fetal retinal human cells are mammalian cell lines used in new influenza vaccination technologies. The MDCK cell line has been extensively studied and a numbers of experimental MDCK-grown influenza inactivated vaccines have been reported to have a good immunogenicity, at least comparable to the traditional vaccines, and a reassuring safety profile, with similar tolerability to the egg-grown vaccines [3,4]. A MDCK live cold-adapted attenuated influenza vaccine has been recently evaluated, too [5]. VERO-cell-based influenza vaccines were demonstrated to be highly immunogenic, with good humoral and cellular immune responses, with the latter being even more efficient than that of traditional inactivated vaccines [6]. As regards the PER C.6 cell-line, a Phase III study on adult subjects is being performed by Crucell and an application for license of the new vaccine is expected in 2014 [7]. Another promising alternative egg-independent vaccine production strategy is the one of viral vectors. Viral vectors (adenovirus, poxvirus, alphavirus) have been extensively studied for being used in influenza antigen presentation; however, they have not been initially considered for vaccine production because of safety concerns. Subsequent preclinical and clinical trials have later demonstrated that genetically modified viral vectors cannot multiply but continue presenting antigens effectively, so that they can be safely used for vaccine production. A preclinical study on animal models showed that an adenoviral-vector-based, adjuvant- and egg-independent pandemic influenza vaccine (HAd) is able to efficiently present influenza virus antigens and elicit good and long-lasting humoral and cellular responses against H5N1 strains [8]. Moreover, the adenovirus-vectored strategy, as intranasal or epicutaneous administered vaccine, has been evaluated in 24 healthy adult subjects, proving to be both immunogenic and safe [9]. Non-replicating virus-like particles (VLPs), resulting from a self-assembly process in a viral life cycle, are other candidates for new influenza vaccines. VLPs can be produced both from enveloped and non-enveloped viruses. Because of their non-infectious nature, VLP-based vaccines can be safely administered even in high-risk populations. A good 2

immunogenicity of this vaccine candidate has been observed in animal models; in humans, preliminary results from a running clinical trial showed that the vaccine could elicit a good immune response against H5N1 strains without safety concerns [10]. DNA technology was first used in influenza vaccine manufacture in 1993. A number of subsequent influenza vaccine preclinical trials in animal models suggested the possibility of eliciting both humoral and T cell-based immune responses. However, despite these encouraging preliminary results, the first human DNA-based intramuscularly administered influenza vaccine study showed no protective immune response. Subsequent human studies demonstrated positive results and a very recent Phase I clinical trial in 103 healthy adult subjects immunised with an adjuvanted DNA-based H5N1 intradermally administered influenza vaccine showed a good immunogenicity and safety [11]. Antigenic drift of viral membrane proteins haemoaggluitinin (HA) and neuroaminidase (NA) is the reason for the annual update of influenza vaccine formulations. Highly conserved viral proteins, such as membrane protein M2e and nucleoprotein (NP), represent new molecular targets for influenza vaccine production which have been evaluated both in preclinical and clinical trials with promising initial results. A good immunogenicity against highly pathogenic viruses has been observed with the combination of the two viral proteins [12]. Several studies of new vaccine administration routes have been recently published. Making vaccine administration easier is an important goal in a successful influenza prevention strategy. Other administration routes apart from intramuscular and deep-subcutaneous ones are intranasal, pulmonary, epidermal and oral. New anatomical administration sites offer in fact some important advantages if compared with the traditional ones: easier conservation and distribution of vaccines, reduction of adverse effect rates, in particular those associated with needle use, a better mucosal immune response through a local IgA-mediated stimulus, increasing patient compliance and cost savings. A liquid intranasally administered influenza vaccine has been already licensed in the USA however a new drypowder intranasal formulation is being evaluated. Powder formulation vaccines show better stability and sterility and can be stored at room or even somewhat higher temperatures, without necessity for cold chain facilities and so much easier conservation. Moreover, the stability of drypowder formulations results in an increase of vaccine shelf-life, facilitating stockpiling of readily available vaccine doses in case of new pandemics. All these features make the new powder intranasally administered vaccine one of the best candidates for mass influenza vaccination. A preclinical study on rats demonstrated the generation of strong nasal mucosal and systemic immune response by intranasal delivery of a dry-powder-formulated influenza vaccine [13].

Expert Opin. Biol. Ther. (2011) 11(1)


Zuccotti & Fabiano

Pulmonary delivery of powder-formulated influenza vaccines can represent an alternative to intranasal administration. Lungs have a much larger highly vascularized absorptive surface area, contain mucosa-associated lymphoid tissue and an abundance of macrophages and dendritic cells which act as local antigen-presenting cells. Dry-powder formulations for pulmonary delivery are designed as micrometer size particles which can reach the lower airways, smaller bronchioles and alveoli, and induce both a local and systemic immunity. A pulmonary-delivered spray-freeze-dried influenza subunit vaccine has been administered in mice demonstrating a good mucosal, humoral and cell-mediated immune response which proved to be superior both to conventional intramuscular and liquid aerosolised administration routes [14]. The epidermis represent another promising anatomical target for influenza vaccine delivery. A great portion of the immune system has its localisation in the cutis: Langerhans cells, local antigen-presenting cells, can effectively stimulate a CD4 and CD8 T-cell-mediated immune response and favour antigen presentation by production of local stimulating cytokines. A number of epidermal delivery methods are being evaluated. Jet injectors for powder formulations have been developed to obtain high speed acceleration of 20 -- 70 µm particles which can penetrate the stratum corneum and effectively reach the epidermis. In a Phase I clinical trial, epidermal powder influenza immunisation in humans using a jet injector device resulted in humoral immune response with a good safety profile [15]. The ultimate nanotechnologies have been recently applied to manufacture of microscopic and minimally invasive devices for epidermal delivery of influenza vaccines. Microneedle devices are being developed for effective and pain-free administration of vaccines across the skin barrier layer.

Microneedles allowed delivery of vaccines in the underlying skin compartments inducing local and, through cutaneous circulation, systemic effects. Recent studies demonstrated that the delivery of vaccine by microneedle provides immunological responses at least equal if not superior to the intramuscular injection [16,17]. Functional miniaturisation of vaccine delivery devices has been recently achieved with development of a densely packed dissolving microprojection array, Nanopatch. This consists of highly dense silicon projections which are coated with vaccines in dry form and applied to the skin: stratum corneum is crossed and vaccine is directly delivered to the immunologically sensitive cells in the skin [18]. Oral administration is another stimulating possibility for influenza immunisation. It is a simple, safe, pain-free, noninvasive and cheap administration route. Oral vaccination can elicit an IgA-mediated mucosal immune response in the respiratory tract which protects the subject at the port of entry of infection and might offer broader protection against antigenically drifted influenza strains, too [19]. Unfortunately oral immunisation resulted in scarce IgG responses and studies are needed to evaluate if an IgA immune response could alone provide adequate protection against influenza infection. Lastly, eye mucosa represent a new anatomical target and a fascinating alternative vaccine administration route. A first study in animal models demonstrated effective virus-specific humoral mucosal and systemic responses after eyedrop influenza vaccination [20].

Declaration of interest The authors state no conflict of interest and have received no payment in preparation of this manuscript.


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Strategies for preventing influenza

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Affiliation

Gian Vincenzo Zuccotti† MD & Valentina Fabiano MD † Author for correspondence Department of Pediatrics, Luigi Sacco Hospital, Universita` degli Studi di Milano, Via GB Grassi, 74, 20157, Milan Italy Tel: +39 0239042253; Fax: +39 0239042254 E-mail: [email protected]

Review

siRNA therapy for cancer and non-lethal diseases such as arthritis and osteoporosis 1.

Introduction

2.

siRNA gene therapy strategies

3.

siRNA therapy applied to


arthritis and other musculoskeletal disorders 4.

Conclusion

5.

Expert opinion

Qin Shi, Xiao-Ling Zhang, Ke-Rong Dai, Mohamed Benderdour & Julio C Fernandes† †

University of Montreal, Sacre´-Coeur Hospital, Research Center, Orthopaedics Research Laboratory, Montreal, Quebec, Canada

Importance of the field: Gene silencing mediated by siRNA has been widely investigated as a potential therapeutic approach. The success of these therapies depends on effective systems capable of selectively and efficiently conveying siRNA to targeted cells/organs with minimal toxicity. Areas covered in this review: This review discusses current experimental approaches to siRNA delivery strategies available for arthritis treatment and the management of other musculoskeletal disorders. The review covers literature on the subject from 2000 to 2010. What the reader will gain: In the last decade, extensive improvements have been made to optimize siRNA-based gene therapy and have been tested on several arthritis and orthopedic conditions. However, except for Phase I -- II DNA-based gene therapy trials on arthritis, no clinical studies have reported siRNA application in these domains. Take home message: Most musculoskeletal disorders, such as rheumatoid arthritis, osteoarthritis, fracture, aseptic loosening, cartilage and intervertebral disc degeneration are non-fatal and age-related chronic inflammatory conditions, but represent significant morbidity and a socio-economic burden. siRNA-based gene therapy offers treatment opportunities that are less invasive, more effective and less expensive than existing modalities. Future directions for siRNA therapy include the development of safe and more efficient delivery systems and the selection of optimal gene targets for disease control. Keywords: arthritis, gene therapy, musculoskeletal diseases, siRNA Expert Opin. Biol. Ther. (2011) 11(1):5-16

1.

Introduction

Musculoskeletal disorders, such as rheumatoid arthritis (RA), osteoarthritis (OA), cartilage and intervertebral disc degeneration and osteoporotic bone fracture, are leading worldwide causes of long-term morbidity and work disability and associated with socio-economic burdens. They are also non-fatal conditions, but age-related and chronic inflammation conditions that do not respond well to conventional treatments. However, new approaches, namely, biological therapy and gene therapy, have shown initial promise. For example, biological therapy of RA represents specially-engineered medications that block the actions of pro-inflammatory factors, such as TNF-a and IL-1b [1]. Current biological treatments of fracture include the local application of osteo-inductive bone morphogenetic proteins (e.g., BMP-2) [2], but they are costly, inconvenient and only partially effective in patients.


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siRNA therapy for cancer and non-lethal diseases such as arthritis and osteoporosis

Article highlights. . .

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siRNA therapy has been widely investigated as a potential therapeutic approach. An effective delivery system (viral or non-viral system) would enclose siRNA in carriers for protection and transport to the cytoplasm of targeted cells. Gene transfer associated with the siRNA approach is reasonably being targeted to manage the inflammation of arthritis. Other potential applications of siRNA-based gene therapy include the management of other musculoskeletal disorders. Over 10 RNA interference therapeutic clinical trials have been evaluated in diverse diseases, such as age-related muscular degeneration and cancer. However, no clinical trials of siRNA therapy for arthritis and other musculoskeletal disorders have been performed.

This box summarizes key points contained in the article.

Human diseases can be treated by the transfer of therapeutic genes (transgene) into specific cells or tissues of patients to correct or supplement defective, causative genes. Gene therapy offers a solution to controlled and specific delivery of genetic materials (DNA and RNA) to targeted cells. The main challenge is to develop a method that delivers the transgene to selected cells in which a proper gene expression can be achieved. The 2006 Nobel Prize in Medicine was awarded to Andrew Z Fire and Craig C Mello, pioneers in the field of RNA interference (RNAi), who again drew the attention of research scientists to gene therapy. The discovery of siRNAs has given renewed vision to the treatment of incurable diseases and genetically-associated disorders. Short double-stranded RNA of 21 -- 23 bp were cleaved by the RNAse III-like protein Dicer and incorporated into RNA-induced silencing complexes (RISCs) [3]. Chemically-synthesized siRNAs and short hairpin RNA (shRNA) expression plasmids, which are sequence-specific for mRNA targeting, are methods commonly used to mimic Dicer cleavage [4]. However, siRNAs are susceptible to nuclease destruction and cannot penetrate the cell membrane because of their highly-charged backbone. An effective delivery system would enclose siRNA in carriers for protection and transport to the cytoplasm of targeted cells but should have no detrimental effects such as specific and nonspecific off-targeted effects. Off-target effects can be divided into two categories, specific and nonspecific offtargeted effects. Off-targeted effects may cause inflammation including IFN response, cell toxicity and unintended gene knock-down. Gene transfer can occur through two delivery systems, viral-mediated or non-viral vectors. Viral gene therapy consists of using viral vectors which, given their structure and mechanisms of action, are good candidates or models to carry therapeutic genes efficiently, leading to long-term 6

expression. Viruses are obvious first choices as gene transport. They have the natural ability to enter cells and express their own proteins. Nowadays, most viral vectors used are retroviruses, herpes virus, adenoviruses and lentiviruses. Non-viral gene transfer systems offer several potential advantages over virus vectors. They are non-infectious, relatively non-immunogenic, have low acute toxicity, can accommodate large DNA plasmids or RNA, and may be produced on a large scale [5,6]. Non-viral gene therapy has been explored by physical approaches (transfer by gene gun, electroporation, ultrasound-facilitated and hydrodynamic delivery) as well as chemical approaches (cationic lipid-mediated gene delivery and cationic polymer-mediated gene transfer). Synthetic and natural cationic polymers (positively-charged) have been widely used to carry DNA or siRNA (negatively-charged) and condense it into small particles, facilitating cellular internalization via endocytosis through charge--charge interactions with anionic sites on cell surfaces. The main drawback to nonviral vectors as gene carriers is their typically low transfection efficiency (e.g., chitosan) [7]. Some of the polymers also suffered from the disadvantage of high cell toxicity. (e.g., polyethylenimine, PEI) [8]. Current gene therapeutic approaches to diseases of the musculoskeletal system have made use of gene transfer technology to improve delivery systems for emerging proteins and other biological agents whose activities may have salutary value. An increasing amount of evidence indicates that gene transfer can help in inflammation and pain management of arthritis, bone-healing and repair of articular cartilage, menisci, intervertebral discs, ligaments and tendons. These developments have the potential to transform many areas of musculoskeletal care, leading to treatments that are less invasive, more effective and less expensive than existing modalities. Phase I -- II clinical trials of plasmid DNA-based gene therapy of RA have already been completed [9]. siRNA therapy has been shown to have tremendous potential in preclinical studies of arthritis and other musculoskeletal disorders. To date, no siRNA therapy-based clinical trials for arthritis and other musculoskeletal disorders have been undertaken. We review the scientific basis and remarkable progress of siRNA therapy with arthritis and musculoskeletal applications and provide an overview of preclinical investigations that have been performed to date, with a discussion and elaboration of future directions. 2.

siRNA gene therapy strategies

Barriers to siRNA delivery Turning siRNA into drugs is a three-step process. The design and in vitro screening of target siRNAs are followed by incorporating stabilizing chemical modifications in lead siRNAs, as required, and end in the selection as well as in vivo evaluation of delivery technologies that are appropriate for the target cell type/organ and disease setting [10]. After nearly 10 years of 2.1



Shi, Zhang, Dai, Benderdour & Fernandes

study and development, many problems have been resolved, such as improving the stability of siRNAs and avoiding two types of off-target effects. A recent anti-influenza study showed that the antiviral activity of siRNA was found due to active siRNA. However, a different non-targeting control siRNA also had significant antiviral activity [11]. siRNA targeting VEGF for patients with age-related macular degeneration are currently in clinical trials. But further study showed that the inhibition is a siRNA classic effect, which is sequenceand target-independent [11]. The off-target effect can be minimized by optimizing the rules and algorithms for siRNA design [10]. However, several other factors limit the utility of siRNAs as therapeutic agents, such as competition with endogenous RNA, induction of immune responses and degradation in lysosomes after endocytosis [12,13]. Unprotected, naked siRNAs are relatively unstable in blood and serum and have short half-lifes in vivo [14]. Naked siRNAs do not freely cross cellular membranes because of their large molecular mass (~ 13 kDa) and strong anionic charge. They are rapidly degraded by nuclease. It is important to have a clear overview of obstacles to the in vivo treatment with siRNAs. Barriers to siRNA delivery depend on the targeted organ and routes of administration. Different in vivo administration routes will encounter different physiological barriers and complications may be furthered by different cells in organs and tissues. For example, intravenous (i.v.) administration is the most commonly used technique. The endothelial wall in the vasculature presents the primary delivery barrier to siRNAs. The endothelial barrier is often altered by inflammatory processes (e.g., RA, infection) [15]. siRNAs leave a blood vessel to enter tissue. After reaching target cells, they undergo internalization via endocytosis, escape from endosomes, release into the cytosol and, finally, load onto RISC. At the same time, siRNAs undergo elimination. The mononuclear phagocyte system is responsible for removing circulating foreign particles from the bloodstream by the phagocytosis of resident macrophages [15]. A variety of serum proteins, such as IgG and IgM as well as complement components (C3, C4, C5), are known as opsonins. Opsonization is the process whereby a foreign particle is tagged by opsonins and becomes visible to macrophages. Phagocytosis involves three distinct steps: first, the foreign particle must be recognized by opsonization in the bloodstream, then opsonized particles adhere to macrophages or other related cells and, ultimately, the particles are managed or controlled [16]. Opsonization occurs before phagocytosis itself; together with phagocytosis, it constitutes the main blood clearance mechanism for particles, resulting in the accumulation of foreign particles in organs, mostly in the liver, spleen and bone marrow. Thus, opsonization is recognized as a critical step that should be blocked to avoid phagocytic clearance from the bloodstream, ensuring its long-term circulation. Increased circulation times in blood permit the particles to reach their target.

Physiological barriers hinder siRNAs from reaching their targets, thereby, reducing their therapeutic efficacy. Moreover, siRNA molecules have unfavorable physicochemical properties (negative charge, large molecular mass and instability). Therefore, they need delivery systems to overcome physiological obstacles and prolong vascular circulation by reducing renal clearance, protecting them from serum nucleases, improving their effective biodistribution as well as targeted cellular uptake with endosomal escape and, finally, promoting trafficking to the cytoplasm and loading onto RISC. Therefore, delivery systems are required to facilitate siRNA access to intracellular sites of action. Viral vectors Numerous gene transfer vectors, including viral and nonviral-based systems, have been evaluated as gene therapies for DNA delivery. siRNA-mediated gene silencing is transient and lasts for only several days compared to plasmid DNAs, but siRNAs have advantages over plasmid DNAs. For delivery, siRNAs do not require transport into the nucleus and act in the cytosol. Because DNA and siRNA have similar physicochemical properties, vectors developed for DNAs have also been applied to siRNAs [6]. So far, siRNA gene therapy research has focused on several types of viral vectors: adeno-associated viruses (AAV), adenoviruses, retroviruses, lentiviruses and herpes simplex viruses. siRNA therapeutics has been assessed in numerous diseases, including genetic and viral diseases, cancer, as well as non-lethal disorders, such as arthritis and osteoporosis. Among these viral vectors, lentiviruses have progressed to clinical trials on metastatic melanoma and HIV infection [17,18]. Viruses are obvious first choices as gene delivery vehicles because they transfer their own genetic material and therapeutic genes very efficiently to the cells they infect. Unfortunately, certain viral vectors (e.g., adenoviruses) can elicit a robust cellular immune response against viral and some transgenic proteins, and so their application has been limited to studies in immunecompromised animals [19]. AAV, which have been considered safe, appear to be immunogenic in several experimental settings [20] and clinical trials [21]. 2.2

Non-viral vectors For practical and safety reasons, non-viral vectors would be preferable in treating chronic, non-lethal rheumatic diseases and other orthopedic disorders. They are noninfectious, relatively non-immunogenic, have low acute toxicity, can accommodate large siRNA/DNA plasmids and may be produced on a large scale [22]. Numerous non-viral gene transfer systems have been proposed, including chemical modification of siRNAs, cationic liposomes and polymers, cell-penetrating peptides and targeted delivery [5,23-25]. Chitosan and PEI are the most widely investigated polymers for transporting DNA/siRNA in vitro and in vivo. Chitosan and its derivatives, as favorable non-viral vectors involved in plasmid DNA 2.3


7



delivery, have attracted attention in the field of siRNA delivery in vitro and in vivo [26,27]. Chitosan was once believed to be less effective than most other nonviral vectors because of its low stability and buffering capacity. However, recent technological advances in the chemical modification of chitosan have instituted improvements of its transfection efficiency without disturbing its biocompatibility and biodegradability. The transfection effectiveness of non-viral vectors may depend on several factors, such as the chemical structure of polycations, the size and composition of complexes, interactions between cells and complexes and cell type. Small size complexes have the advantage of entering cells through endocytosis or pinocytosis and crossing nuclear pore complexes, thereby, increasing transfection rates [28]. PEIs are a series of synthetic polymers, one of the most prominent examples of cationic polymers demonstrating high transfection efficiency in vitro and in vivo [29]. PEIs exist as both branch and linear structures. Their transfection efficiency and toxicity depend on molecular mass (600 -- 70,000 kDa). High molecular mass PEI shows high transfection activity and cytotoxicity. The optimal molecular mass for PEI polyplex formation is typically between 5 and 25 kDa. Other non-viral siRNA vectors involve cationic cell-penetrating peptides [30], cationic polymers and dendrimers, cationic lipids and targeted delivery systems. Cyclodextrin-containing cationic polymers and dendrimes have been also studied as gene carriers in the last decade [5,24,25]. Other polymers are poly-L-lysine, poly-D,L-lactide-co-glycolide, poly (alkylcyanoacrylate) and gelatine [12]. siRNAs are also conjugated with small molecules (e.g., cholesterol, bile acids, and lipids) to improve their stability and cellular internalization [12]. Numerous cell-targeting ligands including small molecules (e.g., galactose, lactose, folic acid), peptides (e.g., RGD peptide), protein (e.g., transferrin) and antibodies (e.g., anti-b-7 integrin, anti-CD19) have been incorporated for cell-specific, active delivery of nucleic acids [31]. The selection of siRNA non-viral delivery systems depends on the properties of siRNAs, the targeted cell type and delivery routes in vivo. The main drawback of non-viral vectors as gene carriers is their typically low transfection efficiency [23]. Cytotoxic effects and inflammatory responses have been reported in vitro in three murine pulmonary target cell lines, alveolar epithelial (LA4), alveolar macrophage (MH-S) and macrophage-monocyte-like (RAW 264.7) cells after stimulation with PEI--siRNA complexes [32]. Furthermore, the in vivo delivery of non-viral liposome--siRNA complexes triggers an immune response [33]. siRNA-based gene therapy has already been tested in clinical trials dealing with the treatment of age-related macular degeneration, viral infection, skin disorders and cancer [6,12]. Clinical trials on gene therapy of musculoskeletal disorders are limited to RA management with plasmid DNAs delivered by viral vectors. siRNA-based therapy remains in the experimental stage. 8

Systemic and local siRNA delivery Numerous in vivo siRNA delivery approaches have been published. The route of administration varies from local, direct transport to systemic conveyance. Systemic delivery involves a generalized approach in which exogenous genes are delivered to cells in a certain tissue or tissues and secreted gene products are released into the circulatory system where they could modulate disease processes throughout the body. Systemic non-viral siRNA delivery has become an attractive alternative to viral vectors because of safety, versatility and ease of preparation [34]. siRNAs can be transported systemically (intramuscularly, intravenously, subcutaneously or, in animals, intraperitoneally). Intravenous administration is the most commonly adopted technique. Hydrodynamic-based gene delivery through systemic siRNA/DNA injection offers a convenient, efficient and powerful means of high-level gene expression in animals [35]. This method is expected to be evaluated in patients soon [36]. Naked siRNAs, lipidformulated siRNAs, plasmid expression shRNAs and cationic polymer-formulated siRNAs have been administered systemically in mice [37]. Naked and formulated siRNAs have also been injected directly into xenograft tumors in mice [38]. Several studies have demonstrated that systemic siRNA delivery in vivo may have side effects due to systemic downregulation of the targeted gene. To date, local siRNA delivery involves the local transport of naked siRNA in saline-based formulations or formulated siRNAs (siRNA conjugates, liposomes/lipoplexes, polymers/ polyplexes) to tissues, such as the eyes, lungs and CNS. Local siRNA delivery approaches, including ocular, respiratory, CNS, skin and vaginal sites, have been investigated most actively [10]. As joints are housed in a closed environment surrounded by an articular capsule, local administration (intraarticular injection) is thus helpful in terms of safety for OA patients with a single afflicted joint. Local gene delivery involves local conveyance of exogenous genes to afflicted joints, where cells populating articular tissues could be modified genetically to express the protein or biological agent of choice. With local gene therapy of RA and OA, the idea is to modify cells genetically within specific joints so that the activity of the affected cells can be re-directed towards inhibiting disease pathogenesis. The target tissue for genetic modification has primarily has been the synovium. With the exception of cartilage, synovial tissue covers all internal surfaces of joint capsules. In vivo electroporation has been developed to efficiently and safely deliver siRNA to articular synovial tissues [39]. They have a large surface area, readily capture particles injected intra-articularly and have no membrane separating synovial cells from the joint space. Local delivery is simple and avoids the substantial risk associated with systemic delivery [40]. Indeed, local intra-articular gene delivery is wellsuited for the treatment of OA, a disease that affects local bearing joints lacking important systemic components. Ex vivo siRNA delivery strategies are compatible with cell-based gene therapy. The ex vivo method of genetically 2.4



modifying cells in vitro and then introducing the altered cells back into the host may be the most advantageous for studying the efficacy of gene therapy in clinical settings. Ex vivo siRNA delivery to primary mouse bone marrow-derived dendritic cells for potential application as a cancer vaccine has been reported [41].

siRNA therapy applied to arthritis and other musculoskeletal disorders

3.

RA RA, a common, chronic, autoimmune inflammatory disease, is characterized by synovial inflammation associated with progressive cartilage and bone destruction. Despite significant progress in RA treatment in recent years, the disease is incurable with currently available therapeutic options. Arthritis was the first orthopedic condition to be targeted by plasmid DNA gene therapy [42]. Several excellent reviews on orthopedic gene therapy have been published with in-depth discussions on arthritis gene therapy trials [9,42]. Although the exact etiology of RA has not been fully elucidated, some evidence supports a role of TNF-a and IL-1 in cartilage and bone erosion. Blocking TNF-a or IL-1 with anti-TNF-a or anti-IL-1 mAbs significantly reduces cartilage and bone destruction. The use of mAb therapy for rheumatic diseases has increased in recent years [1]. Indeed, several TNF-a-blocking agents were approved in 2009 for RA treatment [1]. A natural antagonist, IL-1 receptor antagonist (IL-1Ra), has been shown to slow OA and RA progression in several in vivo models [42]. The FDA approved recombinant IL-1Ra therapy for RA management in November 2001. Unfortunately, high-yield, daily, subcutaneous injections of recombinant proteins (100 mg/day) are needed to maintain therapeutic serum levels in humans. In human trials, IL-1Ra biological therapy against RA and other rheumatic diseases such as ankylosing spondylitis, psoriatic arthritis, systemic lupus erythematosus and OA showed modest or no improvement results. Gene therapy with IL-1Ra, effective in reducing joint destruction in experimental RA [42], is an alternative strategy in arthritis. Transfer of IL-1Ra cDNA to human arthritic joints has already been accomplished in two clinical trials of retroviral vectors [42]. However, the clinical trials using retrovirus-IL-1Ra are closed and there is no ongoing project concerning IL-1Ra gene therapy in arthritis treatments (www.clinicaltrials.org). Several non-viral vectors are being tested in animal models of RA and OA [42]. Other genes reported to be efficient against RA are those of the anti-inflammatory cytokines, or receptors of inflammatory cytokines, such as IL-4, IL-1 and TNFsoluble receptors, TGF-b and IL-13 [43,44]. Silencing diseaserelated gene expression with siRNAs has therapeutic potential against RA. AAV type 5-mediated intra-articular administration of TNF--siRNA improves collagen-induced arthritis in mice [45]. Lentiviral vector-mediated TNF superfamily member B cell-activating factor gene silencing suppresses TH17-cell generation and ameliorates autoimmune arthritis


3.1

in murine macrophage 113.35 cells and mice [46]. siRNAs against Fc gRIII (CD16), complexed with PEI and injected into the superior joint space of the arthritic temporomandibular joint, decreases the number of Fc gRIII-positive cells responsible for binding with IgG, cell activation and cytokine release [47]. TNF-a knock-down by chitosan/siRNA nanoparticles diminishes both local and systemic inflammation in collagen-induced arthritic mice [27]. Intravenous delivery of a cationic lipoplex containing TNF siRNA markedly inhibits TNF production in knee joints, demonstrating a complete cure of arthritis in a mouse model of RA [48]. The expression of folate receptors (FRs) - in synovial mononuclear cells and CD14+ cells from RA patients - was described in 1999 [49]. Folate-mediated transfection has been shown to facilitate DNA internalization into cells via membrane receptors both in vitro and in vivo [50]. Folic acid was conjugated with PEI to increase transfection efficiency for targeting purposes in view of its low immunogenicity, low toxicity and high affinity to FRs. Folate-PEG-PEI has been determined to be a potentially safe and efficient gene vector, particularly for FR-positive cell gene delivery [51]. siRNA transfer ex vivo has been investigated in arthritis models. Ex vivo gene manipulation in dendritic cells by siRNA silencing CD40, CD80 and CD86 suppresses the onset of collagen-induced arthritis [52]. Aouadi et al. reported the engineering of b1,3-D-glucan-capsulated siRNA particles as efficient oral delivery vehicles that potently silenced genes in mouse macrophages in vitro and in vivo [53]. Silencing MAPK kinase kinase kinase 4 (Map4k4) in macrophages in vivo protected mice from lipopolysaccharide (LPS)-induced lethality by inhibiting TNF-a and IL-1b production [53]. This technology defines a new strategy for the oral delivery of siRNAs to attenuate systemic inflammatory responses in diseases such as RA, atherosclerosis and inflammatory bowel disease [53]. Local intra-articular delivery of TNF-a or IL-1b-specific siRNA effectively ameliorates collagen-induced arthritis in rats at the gross morphological, radiographic and histological levels [54]. The biological agent anti-IL-6 is also helpful in RA treatment at arthritis initiation and early progression [55]. However, silencing IL-6 and receptor activator of NF-kB ligand (RANKL) expression in vivo does not exert therapeutic effects on progression of inflammation and bone destruction [54]. Some studies have reported that combined blockade or silencing of TNF-a and RANKL has more significant therapeutic outcomes than blockade of RANKL alone [54,56,57]. The different delivery vectors help siRNA to increase its circulation time following systemic administration and aid in the passage of siRNA to across the cellular membrane. However, the relatively large molecular mass size of nanoparticles dramatically reduces the diffusion of these particles through spaces of tissues. For RA, the biodistribution of siRNA nanoparticles is also influenced by the presence of activated immune cells (e.g., T-regulatory cells and activated macrophage) in both circulation and tissues. It is unclear


9


how siRNA nanoparticles can selectively inhibit autoreactive response while leaving other immune functions intact. OA OA is a disease characterized by the progressive degradation of articular cartilage accompanied by secondary inflammation of the synovial membranes and abnormal subchondral trabecular bone remodeling. Current OA therapies with NSAIDs are largely aimed at symptomatic relief of pain and inflammation, but they may have limited effects on the underlying cascade of joint degradation. New strategies have been developed, including anti-cytokine therapy, gene therapy, delivery of growth factors and stem cell therapy. TNF-a and IL-1b are also considered as principal inflammatory cytokines. IL-1 inhibition has proven to result in the amelioration of OA-like pathology in animal models. Gene therapy with IL-1Ra, effective in reducing joint destruction, as reported in our earlier publication, transferred the IL-1Ra gene in vivo to treat rabbit knee OA [58]. Another promising target is the anabolic growth factor TGF-b which may constitute an alternative approach to promoting cartilage integrity and repair. Two Phase I trials have been conducted for gene therapy of OA, with TGF-b1 cDNA transduced retrovirally to an established line of human primary chondrocytes [59]. A variety of transcription factors, such as NF-kB, play major roles in inflammatory responses. They regulate IL-1b and TNF-a expression and are also activated by these two cytokines. siRNA therapy with ad-siRNA (NF-kB p65) suppresses the progression of early experimental OA produced surgically in rats [60]. Cathepsin K gene and protein expression is upregulated in the synovium in a rabbit model of anterior cruciate ligament transection-induced OA. Downregulation of cathepsin K by an electroporation-assisted siRNA transduction in the synovium leads to OA progression in rabbits, indicating that cathepsin K is involved in cartilage degeneration [61]. So far, siRNA therapy of OA (e.g., ad-siRNA (NF-kB p65) or electrotransfer of siRNA against cathepsin K) is in various preclinical development stages [60,61] and no clinical trials have been reported.


3.2

Osteoporosis and other bone-related diseases Osteoporosis is a disease in which bone density and quality are reduced with increasing risk of fracture. According to the WHO (www.who.int), several million peoples around the world suffer from osteoporosis. In 1990, there were 1.7 million osteoporosis-induced hip fractures alone worldwide; with changes in population demographics, this figure is expected to rise to 6 million by 2050 (WHO Technical Report Series 921). Currently available treatment agents include bisphosphonates, calcitonin, estrogens, selective estrogen receptor modulators, calcium, vitamin D and parathyroid hormone (PTH). The most common fractures associated with osteoporosis occur at the hip, spine and wrist. Osteoporotic bone-healing remains a challenge for the orthopedic community. Much effort has been expended in improving surgical approaches for the 3.3

10

management of osteoporotic fractures. Biological treatments, which enhance the healing potential of osteoporotic fractures, have been considered as an adjunct to surgery, particularly the local application of growth factors to influence bone formation. Another potential approach could be bone tissue-engineering with scaffolding material and mesenchymal stem cells (MSCs) [62]. Finally, gene therapy has been investigated as a possible solution. The delivery of osteogenic genes to precise anatomical locations has shown great potential in bone regeneration and fracture healing. Additional approaches include siRNAs to knock-down inhibitors of BMPs, such as noggin [63] and chordin [64]. Although the mechanisms of bone destruction in osteoporosis have not been fully elucidated, it is believed that osteoclasts are the main cells responsible for bone resorption. Recently, treatment options for primary osteoporosis, for example, bisphosphonates, calcitonin, estrogens, selective estrogen receptor modulators, calcium and vitamin D supplements, have been proposed for their beneficial effects and most of them target osteoclasts. The RANKL, a key differentiation and activation factor for osteoclasts, is released by osteoblasts, activated T cells, synovial fibroblasts and bone marrow stromal cells. RANKL subsequently binds to its specific membrane receptor activator of NF-kB (RANK) and promotes osteoclast differentiation, activation and survival. siRNA delivery targeting RANK to both RAW264.7 and primary bone marrow cell cultures produces short-term repression of RANK expression without off-target effects and significantly inhibits both osteoclast formation and bone resorption [65]. siRNA-mediated knock-down of nuclear factor of active T cells (NFATc1), a transcription factor involved in cytokine production and osteoclast formation, can inhibit LPS-induced osteoclast generation in murine monocyte RAW264.7 cells [66]. Degradation of bone extracellular matrix (e.g., native collagen) is initiated by the combined actions of several proteases, such as MMPs. MMP-9 [67], found in both human osteoblasts and osteoclasts, is likely to be crucial in pathophysiological bone processes. Silencing of NFATc1 also significantly decreases RANKL-induced MMP-9 activity during osteoclast differentiation [68]. Cathepsin K of the superfamily of cysteine proteases is characterized as the key enzyme in bone resorption by osteoclasts. Targeted knockdown of cathepsin K by siRNA transfected with lipid cation into mature human primary osteoclasts inhibits cathepsin K-mediated bone resorption [69]. RANKL can stimulate cathepsin K mRNA and protein expression as well as the phosphorylation of JNK and JunB (both involved in the activator protein-1 signaling pathway), and siRNAs targeted against c-jun or JunB suppress RANKL-induced cathepsin K expression [70]. Less attention has been paid to the role of osteoblasts in bone destruction. Bone loss could also be caused by a defect in osteoblasts under certain conditions, such as aging [71]. Osteoblasts are not only central to bone formation by




synthesizing multiple bone matrix proteins, but they also influence osteoclastic recruitment, differentiation and functions by releasing pro-inflammatory factors [72,73]. PTH peptide is the only known approach to stimulating bone formation and osteoblast activity [74]. Other therapeutic strategies in force or under development, including cathepsin K inhibitors, H+-ATPase inhibitor and p38 inhibitors, also act by blocking osteoclast activity [75]. Liposome-based transient siRNA suppression of PPAR-g can increase alcohol-induced osteogenesis on human bone marrow-derived mesenchymal cells [76]. Knock-down of PPAR-g or adiponectin receptor 1 in osteoblastic cells by liposome-based siRNA transfection prevents the downregulation of mRNA expression of runxrelated transcription factor 2 (Runx2), an important transcription factor for osteoblast differentiation [77]. Simultaneous suppression of MMP-1 and -13 by siRNA transfection in vitro results in significant increases of all osteoblastic markers, such as Runx2, osteonectin, osteopontin, bone sialoprotein and osteocalcin [78]. Aseptic loosening Implant loosening caused by peri-prosthetic osteolysis is a major clinical problem affecting total hip arthroplasty survivorship. Wear debris from articulating joint surfaces have been recognized as a key factor in osteolysis. Phagocytosis of wear particles is thought to be the initiating event in the inflammatory response leading to osteolysis [79]. In response to phagocytosis, osteoblasts and macrophages produce cytokines and inflammatory mediators. This affects, in an autocrine/paracrine manner, macrophages and the recruitment of osteoclasts, resulting in a net shift of bone homeostasis toward bone resorption. Local delivery of lentivirus-mediated TNF-a siRNA into the modified murine air pouch, stimulated by poly(methyl methacrylate) particles, culminates in significant downregulation of TNF-a, IL-1 and IL-6 for up to 4 weeks [80]. The transcription factor NF-kB is essential for osteoclastogenesis and is considered to be an immunemodulator of RA and inflammatory osteolysis. Intraarticular injection of NF-kB siRNA could also inhibit wear particle-induced inflammatory osteolysis [81]. Activation of NF-kB subunits is regulated by upstream IkB kinase (IKK) complexes containing IKKa, IKKb and IKKg. IKKg is also known to be a NF-kB essential modulator (NEMO) [82]. NEMO siRNA knock-down can inhibit NF-kB activation, impede osteoclastogenesis and arrest inflammatory osteolysis [83]. 3.4

Orthopedic tumors Primary malignant bone tumors, such as Ewing’s sarcoma, are rare but are the second most common malignancy in children and young adults [84]. Ewing’s sarcoma is characterized by specific t (11;22) chromosomal translocations leading to the formation of EWS-FLI1 fusion protein that is indispensable for sustained Ewing’s sarcoma growth. EWS-FLI1 fusion protein is considered to be an ideal therapeutic target in this disease. 3.5

siRNAs may hold great promise as fusion gene-specific agents. RNAi-mediated suppression of EWS-FLI1 is likely to result in an altered tumor cell phenotype, including changes in chemo-sensitivity and restored differentiation potential [84]. Systemic delivery of siRNA against the EWS-FLI1 gene product by a targeted non-viral system dramatically inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma. The non-viral system harnesses cyclodextrin-containing polycation to bind and protect siRNA and transferrin as a targeting ligand for delivery to transferrin receptor-expressing tumor cells [85]. Human Ewing’s sarcoma cells overexpress soluble VEGF 165 and vector-mediated siRNAs targeting VEGF inhibit Ewing’s sarcoma growth in a xenograft mouse model [86]. Oncogenic potential of the hedgehog pathway is mediated by increasing the activity of the GLI family of transcription factors. Recent evidence indicates that EWS-FLI1 enhances GLI1 expression via an unknown mechanism. GLI1 gene expression is decreased in an Ewing’s sarcoma family of tumors cell line where EWS-FLI1 is knocked down by RNAi [87,88]. BMPs induce the differentiation of osteoblast precursor cells to osteoblasts whose overexpression in osteosarcoma results in resistance to differentiation. This resistance to differentiation by BMPs may be overcome by siRNA silencing the expression of transcription factors related to osteoblast differentiation, such as Runx2 [89]. Chondrosarcomas account for about 25% of osteosarcomas, the most common primary malignancy of the skeleton. Recent advances in understanding the progression or development of chondrosarcoma suggest several molecular targets for the future configuration of new therapies, such as siRNAs, to knock down Bcl-2 [90]. Tumor invasion and metastasis are critical steps in determining the aggressive phenotype of human cancers, such as those of the breast, colon, prostate and ovaries. These tumors have a remarkable affinity to metastasis in bone. Once they metastasize to bone, they are a major cause of morbidity and mortality as they evoke significant skeletal remodeling, fractures, pain and anemia. Several discrete steps are discernible in the biological cascade of metastasis: loss of cellular adhesion, increased motility and invasiveness, entry and survival in the circulation, exit into new tissue and eventual colonization of distant sites [91]. Interactions between cancer cells and osteoblasts or osteoclasts provoke the activation and elevated expression of certain paracrine signaling factors, such as TGF-b, PTH-related peptide, MMP and COX-2, which lead to either osteolytic or bone metastasis [92]. Much research into siRNA therapy of bone metastasis has been undertaken. These approaches include siRNAs to block osteoclast-mediated bone resorption [92]. Tissue engineering Other orthopedic siRNA therapies embrace damaged musculoskeletal tissue (cartilage, ligaments, tendons and menisci) repair and intervertebral disc regeneration [93]. Tissue repair can be achieved by direct in vivo gene delivery or by ex vivo 3.6


11



cell-based gene therapy combined with tissue engineering. Such combinations have been termed gene therapy-based tissue engineering, in which cells are removed from the body, expanded in culture, genetically modified, seeded onto a scaffold, re-cultured and re-implanted [94,95]. Numerous growth factors and osteo-inductive cytokines have been reported to enhance healing processes in orthopedic disorders. The most prominent of these factors are the BMPs [2]. siRNAs for reducing the expression of a target gene (e.g., transcription factor) can elicit the expression of one or more tissueinductive factors, direct the differentiation of stem or progenitor cells, or remove factor(s) that inhibit(s) regeneration, which have been useful in fundamental studies of tissue formation or in applications to promote in vivo regeneration. Core-binding factor-a1 (Cbfa1), also known as Runx2 or Osf2, is a transcriptional regulator of both osteoblast differentiation and function. Cbfa1 controls the expression of osteocalcin, the last gene to be expressed during osteoblast differentiation. Cbfa1 activity is regulated by the a-chain of heterotrimeric G protein (Gsa) and is transcribed by the gene guanine nucleotide-binding protein (G protein) a-stimulating activity polypeptide 1 (GNAS1). Downregulation of the GNAS1 gene via siRNAs may lead to heightened Cbfa1 expression that could stimulate the production of bone-differentiating genes and result in the regulated osteogenic differentiation of MSCs [93]. Chondrocyte-based tissue engineering has emerged as a promising approach to repair injured cartilage tissues. Lentivirus-mediated RNAi has been used to target both aggrecanase-1 and -2 in primary rat chondrocytes, and transduced cells have been seeded into chitosan-gelatin 3D scaffolds. siRNA-mediated inhibition of aggrecanases increases chondrocyte proliferation and differentiation in chitosan-gelatin 3D scaffolds and facilitates in vitro cartilage formation [96]. The etiology of degenerative disc disease is unknown. Several investigators have reported the presence of proteolytic enzymes, such as MMP and ADAMTS5 (a disintegrin and metalloproteases with thrombospondin-like motifs 5) families, in degenerated human discs. A single injection of ADAMTS5 siRNA inhibits the production of ADAMTS5 and suppresses intervertebral disc degeneration in the rabbit anular needle-puncture model [97]. 4.

Conclusion

Gene therapy is being applied to various health problems, such as cancer, AIDS and cardiovascular diseases. Although most arthritis and musculoskeletal disorders are non-fatal, they represent a significant source of morbidity and a heavy socio-economic burden. The main challenge for gene therapy is to develop a method that delivers the transgene to selected cells, in which a proper gene expression can be achieved. Viral vectors (e.g., adenovirus) are very effective in term of transfection efficiency, but they have limitations in vivo, particularly by their safety concern and non-tissue-specific transfection. 12

Non-viral delivery systems for gene therapy have been increasingly proposed as safer alternatives to viral vectors because they evoke a minimal host immune response, stable in storage, easy to produce in large quantities and are low cost. But non-viral gene transfer systems are limited by their lower gene transfer efficiency, low tissue specificity and transient gene expression. DNA-based gene therapy has shown tremendous potential, and RA has been the subject of clinical Phase II efficacy protocols. siRNA therapies have also attracted considerable attention. Gene transfer associated with the siRNA approach is reasonably being targeted to manage the inflammation of arthritis and treat osteoporosis, aseptic osteolysis, genetic disease and tumors. Other potential applications of siRNA-based gene therapy include the repair and regeneration of bones, cartilage and intervertebral discs. To date, however, no clinical trials of siRNA therapy for arthritis and other musculoskeletal disorders have been performed. 5.

Expert opinion

Gene therapy offers new possibilities for the clinical management of orthopedic conditions that are difficult to treat by traditional surgical or medical means. To date (July 2010), > 10 RNAi therapeutic clinical trials are ongoing [10]. siRNA therapeutics has been evaluated in diverse diseases, such as age-related muscular degeneration, diabetes, hepatitis, respiratory disease, HIV infection and cancer. One of first siRNA therapies (siRNA to VEGF) for the treatment of wet neovascular age-related macular degeneration is now currently in Phase III clinical trial. For more information on siRNA clinical trials, see [10]. In the last decade, extensive improvements have been made to optimize gene therapy and tested on several orthopedic conditions. RA has been the subject of clinical Phase II efficacy protocols, and OA gene therapy efficacy has been investigated in various animal models. Gene transfer associated with the tissue engineering approach is reasonably being targeted to aid the repair and regeneration of bones, cartilage, ligaments, tendon, meniscus and intervertebral disks; other potential applications of gene therapy in orthopedic disorders include osteoporosis, aseptic osteolysis, genetic disease and tumors. Given the promising results and successes demonstrated in preclinical or experimental studies of siRNA therapy in orthopedic applications, no clinical investigations or translational trials in this field have been conducted [9]. It does not seem too surprising given the ongoing concerns about long-term safety and clinical applications of gene therapy with viral vector. The key concern is safety (e.g., with viral vectors). Safety has always emerged as a dominant issue in gene therapy after the development of leukemia in children receiving cells transduced with recombinant retrovirus in 2000 and one death of an arthritis patient from administration of AAV vector in July 2007 (the cause of death is unclear) [98]. Safety concerns about viral vectors may be overcome by the use of non-viral vectors for gene delivery,




but although non-viral gene transfer may be safer, further exploration is needed to justify such decisions. Another constraint of gene therapy for several orthopedic applications is that it is time-consuming and expensive. In particular, ex vivo strategies of bone-healing need multiple stages, such as cell harvest, culture expansion, transfection and re-implantation in vivo. Nevertheless, siRNA-based gene therapy is not only a promising treatment innovation in cancer, but also offers the opportunity to provide prolonged systemic or local drug delivery in non-lethal conditions, such as various orthopedic problems and musculoskeletal disorders. Future directions for siRNA therapy in musculoskeletal diseases include identification of the safest and most effective strategies for siRNA delivery (non-viral vectors versus viral vectors) and optimal cell targeting for disease control.

Acknowledgements The authors thank OM Da Silva (International research & trade communications, Inc., Montreal, Quebec, Canada) for editing this manuscript.

Declaration of interest This work was supported by the grants from National Natural Science Foundation of China (No. 30811120440 and 30911120261), Shanghai International Collaboration Foundation (No. 08410701800) and Canadian Institutes of Health Research (CCI-92212, CCL-99636 and CCM 104888) and the Fonds de la Recherche du Sante du Quebec (FRSQ).

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Affiliation Qin Shi1, Xiao-Ling Zhang2,3, Ke-Rong Dai2,3, Mohamed Benderdour1 & Julio C Fernandes†1 † Author for correspondence 1 University of Montreal, Sacre´-Coeur Hospital, Research Center, Orthopaedics Research Laboratory, 5400 West Gouin Blvd., Montreal, Quebec H4J 1C5, Canada Tel: +514 338 2222 ext. 3043; Fax: +514 338 2694; E-mail: [email protected] 2 Shanghai Jiao Tong University, Institute of Health Sciences, School of Medicine and Shanghai Institutes for the Biological Sciences, Chinese Academy of Sciences, 225 South Chongqing Road, Shanghai 200025, China 3 Shanghai Jiao Tong University, Ninth People’s Hospital, School of Medicine, Department of Orthopaedics, 639 Zhizaoju Road, Shanghai 200011, China

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Review

Analysis of the roles of HIV-derived microRNAs Aarthi Narayanan, Kylene Kehn-Hall, Charles Bailey & Fatah Kashanchi†


†

1.

miRNAs

2.

Viral miRNAs

3.

Conclusion

4.

Expert opinion

George Mason University, National Center for Biodefense and Infectious Diseases, Discovery Hall, Manassas, VA, USA

Importance of the field: HIV-1 is a retrovirus that has infected millions in recent decades. The level of life cycle complexity and host control exerted by this small virus with only nine proteins is astonishing. An interesting direction that has emerged in recent years is the role of small non-coding RNAs in viral gene expression. Areas covered in this review: We focus on HIV-1 produced microRNAs (miRNAs), namely, TAR, Nef and miR-H1, and their roles in HIV-1 biogenesis. The article provides insights into TAR miRNA-mediated downregulation of viral and host gene expression by recruitment of chromatin remodeling components (HDAC1). What the reader will gain: We address the influence of TAR miRNA on host cell cycle progression and apoptosis, and the role of Nef miRNA in the regulation of viral and host gene expression. The review also highlights an intriguing connection between miR-H1 and HIV-1-associated neurological pathogenesis, and the influence of the miRNA machinery in the establishment of latency. In the Expert Opinion section, we analyze the issue of hostbased therapeutics against HIV-1 and how transcription inhibitors are influenced by viral miRNA production. Take home message: HIV-derived miRNAs are of significance not only to understand host-virus interactions, but also for the design of effective therapeutics. Keywords: cdk inhibitors, HIV-1, latency, microRNA, miR-H1, Nef, TAR Expert Opin. Biol. Ther. (2011) 11(1):17-29

1.

miRNAs

MicroRNAs (miRNAs) are small non-coding RNA molecules that are encoded by life forms ranging from plants to higher order mammals [1]. Lin-4 [2] and let-7 [3] miRNAs were the first identified miRNAs and they were demonstrated to negatively regulate gene expression in Caenorhabditis elegans. Since then, miRNAs have been discovered in plants and many metazoans, where they play crucial roles in regulation of gene expression [4]. miRNAs exert their regulatory influence by destruction of target mRNA molecules (transcriptional repression) or inhibition of translation of target mRNAs (translational repression). Extensive research has been conducted and multiple manuscripts have been published on cellular miRNAs and their roles in the regulation of gene expression pertinent to cellular processes such as development, differentiation, stress response and apoptosis [5-9]. The focus of this review, however, is on the newly discovered HIV-1-derived miRNAs. Viral origin of miRNAs is an accepted phenomenon and many DNA viruses produce miRNAs. Retroviral miRNAs, specifically HIV-1-derived miRNAs, are an exciting novel discovery that have enormous implications in understanding viral regulation of susceptible host cells, cellular tropism, establishment of a latent state and,


17

Analysis of the roles of HIV-derived microRNAs

Article highlights. .

.

. .


.

MicroRNAs (miRNAs) are of cellular and viral origin. Retroviruses including HIV-1 are known to produce miRNAs in the infected host cells. The TAR miRNA of HIV is an abundant viral miRNA that plays important roles in regulating viral and host gene expression. The Nef miRNA and miR-H1 of HIV have been demonstrated to be involved in disease progression. Viruses also encode suppressor of RNA silencing function to counter the effect of the host RNA silencing machinery. Availability of a complete miRNA machinery could have significant implications for the establishment of viral latency in susceptible cells. Additionally, the cellular miRNA machinery may be an important component in influencing viral inhibition by antivirals.


ultimately, in their application towards the design of novel and effective inhibitors of viral multiplication. The miRNA pathway The biogenesis of miRNAs has been very well characterized and described in multiple articles [10-15]. Figure 1 illustrates the miRNA pathway. miRNAs are usually transcribed by RNA polymerase II. A primary RNA transcript (primiRNA) [16] that is ~ 80 nucleotides long is processed in the nucleus by the enzyme Drosha and its cofactor DGCR8 [17,18]. Drosha cleaves the miRNA to generate an ~ 60 nucleotide long pre-miRNA. The pre-miRNA is exported out of the nucleus by the exportin-5--RanGTP complex [19,20]. In the cytoplasm, the pre-miRNA is bound by Dicer. Dicer cleaves the hairpin and the stem portion is carried on in the pathway as a miRNA duplex. One strand of this duplex (the ‘passenger strand’) is degraded while the other (the ‘guide strand’) is incorporated into RISC (RNA-induced silencing complex) and the HIV-1 TAR RNA binding protein (TRBP) is thought to assist in the loading process [21-23]. The strand that is less stably base-paired at the 5¢ end of the duplex is usually the guide strand [24]. The catalytic components of RISC are the Argonaute proteins (Ago 1 -- 4). Ago 2 is shown to have endonuclease activity and can cleave target mRNAs that show complementarity to the guide strand. The RISC complex and the associated miRNA usually bind to the 3¢ UTR region of the target mRNAs. Nucleotides 2 -- 7 of the miRNA, called ‘the seed sequence’, play an important role in the positioning of the RISC complex and the associated miRNA on the target mRNA [25,26]. In case of perfect complementarity between the target and the miRNA, the mRNA is degraded. However, when the complementarity is incomplete, the mRNA is translationally repressed. The mRNA--RISC complex is transported to P bodies that are cytoplasmic structures containing RNA remodeling 1.1

18

components [27]. While the above mentioned processes occur at the post transcriptional level, RNA-mediated silencing can operate at the chromatin level as well to regulate gene expression. miRNAs can associate with the RNA-induced initiation of transcriptional silencing (RITS) complex and be guided to complementary regions in the chromosomal DNA [28,29]. Following association with such genomic regions, the RITS complex recruits factors such as histone modifying enzymes, which alter the chromatin structure and induce transcriptional silencing [30]. 2.

Viral miRNAs

Viral encoded miRNAs could negatively regulate the virus. However, viral miRNAs could also confer significant advantages to the virus. For example, given that the requirements of miRNA-mediated regulation of gene expression are flexible in terms of target recognition, even a single mutation in the miRNA could sharply alter the target range thus increasing adaptability of the virus during viral evolution. In fact, it is estimated that the cellular pool of miRNAs could regulate the expression of a third of the entire cellular genome because of the flexibility [31]. Timely suppression of viral multiplication in the appropriate cell type is a crucial deciding factor in the establishment of latency. Indeed, viruses have also evolved means to suppress the cellular RNA interference (RNAi) machinery so that the repression can be relieved when no longer needed by the virus such as a switch from latency to lytic phase. DNA viruses and miRNAs The discovery of viral encoded miRNAs was initiated by Pfeffer et al. when they reported in 2004 that they were able to clone five Epstein--Barr virus (EBV) miRNAs from human B cells that were latently infected with EBV [32]. Since then, > 140 virally encoded miRNAs have been discovered in herpesviruses. A total of 25 EBV encoded miRNAs have been identified [33-35]. In the case of Kaposi’s sarcomaassociated herpesvirus (KSHV), a total of 12 pre-miRNAs that resulted in 17 mature miRNAs have been reported [36]. In the case of human cytomegalovirus (HCMV), 11 miRNA precursors were identified that lead to the expression of 14 mature RNA species [37]. In the case of a herpesviruses, HSV1 and 2, MDV1 and 2 have been demonstrated to encode for miRNAs [38]. Functionally, in a herpesviruses, similar miRNAs have been found in the latency-associated transcript regions of all a herpesviruses suggesting that an important function of the only RNA species produced during latency is to express miRNAs. These miRNAs may contribute to the establishment and maintenance of latency [38]. Both HCMV and EBV miRNAs have been shown to target host and viral genes and regulate their expression. In the case of KSHV, although several host genes have been reported to be regulated by the viral miRNAs, there does not appear to be any report on viral genes being regulated. A general theme 2.1


Narayanan, Kehn-Hall, Bailey & Kashanchi

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Figure 1. miRNA biogenesis. A primary miRNA (Pri-miRNA) with a 5’ cap and a 3’poly A tail is processed by Drosha to yield PremiRNAs. They are transported to the cytoplasm by exportin-5 and the cytoplasmic pool of pre-miRNAs is processed by Dicer to mature miRNAs. The mature miRNAs participate in transcriptional and translational silencing to regulate gene expression. RISC: RNA-induced silencing complex; RITS: RNA-induced initiation of transcriptional silencing; TRBP: TAR RNA binding protein.

for viral miRNAs in herpesviruses appears to be latency/ lytic switch, immune evasion, cell survival and proliferation. Retroviruses and miRNAs While there have been numerous reports on miRNAs derived from DNA viruses, RNA virus-derived miRNAs appear to be somewhat controversial. It was first reported in 2005 by Pfeffer et al. that cDNA cloning of small RNAs from HIV-1 infected HeLa cells did not yield any viral encoded miRNAs [39]. A second report was published in 2007 by Lin and Cullen [40] that small RNAs (18 -- 24 nt) cloned from HTLV-1 and HIV-1 infected cells 2.2

(MT-2 and ACH-2) cells did not yield any viral encoded miRNAs. They report 1098 clones from ACH2 cells and state that the majority of clones corresponded to cellular miRNAs and a minor population of clones consisted of breakdown products of cellular mRNAs, tRNAs and rRNAs. The authors indicate that it may be possible for viral miRNAs to be present at very low levels (< 0.5% of the total cellular miRNAs). Another report [41] also explained that the reason why no viral miRNAs were identified in these experiments could be very low levels of viral miRNA expression. Such low levels of miRNAs may render their isolation by methods such as cloning difficult, more so


19



in the presence of abundant species such as ribosomal and transfer RNAs. Also, expression of viral miRNAs may vary based on cell type and inability to detect viral miRNAs in one cell type does not mean they are absent in all. Hence, the successful capture of viral miRNAs by small RNA cloning methods is governed by the specific stage of infection, robustness of the infection and the infected cell type [42] which could directly and indirectly affect total viral miRNA abundance. More sensitive methods such as RNase Protection Assays (RPA) and next-generation sequencing [43] could be advantageous in identifying low abundance small non-coding RNA species. Yeung et al. report that they identified multiple small non-coding RNAs in HIV-1 infected cells [43]. They sequenced a total of 47,773 clones of which 60% were miRNAs. Within this population, they identified 125 non-coding RNAs that were HIV-1 specific. They also report that the TAR non-coding RNAs were the most abundant followed by the RRE and NEF non-coding RNAs. These sensitive methods provide convincing evidence that HIV-1 encoded miRNAs do in fact exist and can be effectively identified in infected cells. Additionally, recent evidence points to small viral RNAs (svRNAs) encoded by other RNA viruses such as influenza virus. The svRNAs were suggested to trigger the viral switch from transcription to replication through their interaction with the viral polymerase machinery [44]. The TAR miRNA The HIV-1 TAR element is a hairpin structure measuring about 50 nucleotides in length that is found at the 5¢ end of the viral mRNA (Figure 2) [45,46]. The TAR element is encoded by the 5¢ and the 3¢ ends of HIV-1 RNA [47]. Structural features and RNA--protein interactions provide tantalizing clues to the potential of the TAR element to yield miRNAs. First, the hairpin structure of this element is in accordance with the structure requirements of Dicer substrates. Second, computer modeling has predicted the TAR element as one of five potential Dicer substrates in HIV-1 [48]. Third, TAR being bound by two proteins that are integral to the miRNA machinery, namely, TRBP and Dicer argues convincingly for the involvement of TAR in miRNA-mediated regulation of gene expression. Interestingly, a 124-mer TAR RNA domain present at the 5¢ end of HIV-2 is considered to possibly encode pre-miRNAs and may target a wide variety of cellular targets [49]. 2.2.1

TRBP TRBP is the human homologue of the Drosophila Loquacious protein. Loquacious and TRBP bind Dicer and are required for the efficient loading of the miRNA into the RISC complex. The importance of TRBP in RNAi was realized when intracellular depletion of TRBP resulted in a loss in cellular RNA silencing function [21]. It is of interest that TRBP was discovered by its association with the TAR element [50] and was demonstrated to be involved in the transactivation and 2.2.1.1

20

inhibition of IFN-induced protein kinase R. It was demonstrated that HIV-1 may use TAR to target TRBP and sequester it. This would result in reduced availability of TRBP to Dicer and hence affect miRNA processing. Dicer and TAR miRNA biogenesis The TAR element is bound by endogenous Dicer protein, as demonstrated by biotin pull down assays [51]. Biotin labeled TAR was utilized to pull down ~ 0.3 -- 2.8% of the total cellular Dicer protein from infected and uninfected cell lines. Competition experiments with unlabeled TAR revealed that the TAR--Dicer association was specific to the presence of the TAR element and not to any generic RNA. A mutant version of TAR called TAR-D (a shortened structure incapable of Dicer binding) and a generic poly-U RNA molecule were also used to demonstrate the specificity of the TAR--Dicer interaction. It has also been demonstrated by ethidium bromide staining and radioactive labeling that the TAR--Dicer association results in cleavage of the TAR RNA [51]. Both methods revealed the presence of an approximate 21 nucleotide fragment consistent with the cleavage of the TAR substrate. The appearance of this 21 nucleotide fragment was contingent on the availability of enzymatically active Dicer as using heat inactivated Dicer in in vitro studies did not yield the same smaller processed RNA fragment. Knock down of cellular Dicer expression using siRNAs showed that Dicer is indeed required for the processing of the TAR element. TAR mutants with alterations in structural elements (basepair inversions, absence of a pyrimidine bulge and mutations in the terminal loop element) while maintaining the overall integrity of the stem loop structure were also processed by Dicer comparably emphasizing the structural requirement for this enzymatic processing reaction. In addition to demonstrating that the TAR element could be bound and cleaved by Dicer in vitro, TARderived miRNA could also be detected in chronically infected CD4+ T cells by RPA [51]. The RPA results revealed the presence of a miRNA derived from the 5¢ end of the TAR element. TAR-derived miRNA was detectable in other cell lines including latent cell lines (OM10.1 and HLM-1 cells). Recently, TAR miRNA has also been observed in HAART treated patient samples (F. Romerio, IHV Institute, unpublished data). The 5¢ and the 3¢ arms of the TAR-derived miRNA have been cloned [50]. Comparison of the cloned sequences to the predicted sequences in the Sanger miRNA database revealed these clones to be different from previous predictions. Four clones of the 5¢ stem (miR-TAR-5p) and about fourteen clones of the 3¢ stem (miR-TAR-3p) of TAR were obtained [52] suggesting that the 3¢ stem of TAR may be more abundant in infected cells. miR-TAR-3p exhibited superior inhibition of gene expression comparatively and it is probably due to the preferential release and accumulation of the 3¢ element from the HIV-1 TAR RNA in vivo [53]. 2.2.1.2



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C C U C C A U U GUA UGGGU UCUCUGGUUAG CCAGA GAGC G GGUCU CUCG CGU ACCCA AGGGAUCAAUC U C A G

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UG A A C A GA A AG U UGG CC UG GA GAAGUGUUAG GUGG GU U GCC GG GC CU CUUUACGAUC CGCC CA A C U A A CA G

Figure 2. HIV-1-derived miRNAs. Positions of the TAR, HIV-miR-H1, nef and RRE-derived miRNAs in the context of the viral genome are indicated [45,52,53].

2.2.1.3 Regulation of viral and host gene expression by TAR miRNA

Downregulation of viral gene expression by TARderived miRNA has been demonstrated [51]. When utilizing a HIV-1 LTR controlled luciferase reporter, the TAR RNA was able to suppress luciferase gene expression in a concentration-dependent manner [51]. Additionally, it was shown that the TAR-derived miRNA can downregulate gene expression by recruiting chromatin remodeling components to the viral LTR. Using chromatin immunoprecipitation experiments, the recruitment of HDAC-1 to the viral LTR of both transfected reporter plasmids and an integrated HIV-1 LTR was observed [49]. This recruitment of HDAC-1 to the HIV-1 LTR by the TAR element suggested that the TAR element is capable of transcriptional silencing via the RITS mechanism. Additionally, it was found that the TAR miRNA affected host cell cycle [52]. 293T cells were transfected with wildtype and mutant TAR RNA molecules and effects on cell cycle progression were determined by flow cytometry. These experiments showed that under conditions of stress, TAR minus cells were arrested in G1 phase and subsequently proceeded to apoptosis. In contrast, TAR miRNA-containing cells progressed through S and G2 phases, displayed a block at G1/S and reduced apoptosis suggesting that the TAR miRNA protects the cells from stress-induced cell death [52]. The same phenotype was also observed in chronically infected cell lines (HLM-1 and ACH-2) suggesting that the antiapoptotic effect of the TAR miRNA was independent of the source of the miRNA. Using an antagomir with specific sequence complementarity to the mature TAR miRNA, this anti-apoptotic effect was blocked thus underscoring the specificity of TAR miRNA-mediated regulation of apoptosis of the host cell. The fact that the TAR miRNA is expressed in both latent and actively infected cells suggests that the miRNA may regulate a wide range of target genes including those

related to immune surveillance and evasion, cell survival or increased viral production [52]. Microarray analysis of RNA isolated from TAR transfected cells using an Affymetrix human focus array identified 18 human genes that were downregulated and 14 genes that were upregulated. The downregulated genes were connected to several targets related to replication, receptor signaling, DNA repair, apoptosis and mitochondrial function. Further validation of four of these genes showed possible links to apoptosis and cell survival including ERCC1 (excision repair cross complementing-group 1), PIASg (protein inhibitor of activated STATg), GIT2 (GPCR interacting protein) and IER3 (intermediate early response 3). Interestingly, in infected monocytes (which lack Dicer), the levels of these proteins were upregulated. This suggests that the viral infection may be upregulating the expression of these proteins while the viral miRNA was countering this effect. This serves as additional support for the notion that the viral miRNA downregulated ERCC1, GIT2 and IER3 expression (PIASg expression remained unaltered in infected cells). As further evidence, it was demonstrated that depletion of ERCC1 in 293T cells using siRNAs made the cells more resistant to apoptosis. Interestingly, other analysis also revealed that the resistance to apoptosis by downregulation of ERCC1 involved activation of p53. Sequence analysis of the ERCC1 mRNA using the search algorithm miRanda revealed that there are six potential target sites and that the TAR 5¢ miRNA was likely to be the effector RNA strand. Cloning of these target sites into luciferase reporter constructs and analysis of reporter gene expression in the presence of TAR miRNA revealed a suppression of luciferase expression thus confirming that TAR miRNA directed downregulation of the ERCC1 gene. Additional studies revealed that the repression of ERCC1 and IER3 expression involved alteration of protein expression without any effect on the respective mRNAs (translational repression of the target). This


21


downregulation of the host machinery by viral miRNAs is a vital component to prolonging the life span of an infected cell so as to allow ample time for viral multiplication or increased life span in latently infected cells [52]. Nef miRNA Nef is a HIV-1 accessory gene located at the 3¢ end of the viral genome, partially overlapping the 3¢ LTR (Figure 2) [48,54,55]. It is important for viral replication in vivo and is conserved in HIV-1, HIV-2 and SIV. Nef is expressed during infection and is said to account for up to 80% of HIV-specific RNA transcripts during early stages of viral replication. Introduction of defective variants of nef dsRNA [56] or cis expression of nef [57] inhibits HIV-1 replication suggesting that nef may function as a cis-regulatory element of HIV-1 replication. In fact, nef-derived miRNAs have been shown in HIV-1 infected cells [56]. Total RNA from MT-4 T cells persistently infected with HIV-1 IIIB strain was analyzed by northern blot with probes directed to the nef region. The analysis revealed small RNA molecules about 25 nucleotides in length. These molecules were resistant to RNase A and T1 suggesting that they are likely to be double stranded. These miRNA species have been cloned and sequenced [56]. Interestingly, nef miRNAs have been detected in HIV-1 infected long time non-progressors that display low viremia. Using EGFP and luciferase reporter constructs downstream of the nef target, nef miRNAs have been shown to downregulate nef expression in Jurkat T cells. Impressively, the nef miRNA (miR-N367) was effectively able to downregulate nef expression in the subcapsular regions of the spleen in mice, as determined by immunofluorescence and RT-PCR assays [58]. One of the downstream targets of nef is PPAR-g and nef suppresses PPAR-g expression. In mice expressing nef miRNA, significant suppression of PPAR-g expression was seen in the intestinal adipose tissue. Loss of nef expression also appeared to afford protection to the mice by preventing fatigue and drastic weight loss. This opens the door to the possibility of utilizing HIV-1-derived miRNAs as therapeutics to control viral multiplication in vivo.


2.2.2

HIV miR-H1 A third HIV-1-derived miRNA that has been reported is miR-H1. This is an 81 nucleotide stem-loop structure that is located downstream of the two NF-kB sites in the LTR (Figure 2) [48]. MiR-H1 has been demonstrated to degrade the apoptosis antagonizing transcription factor (AATF) gene product [41]. Downregulation of AATF is accompanied by lowered cell viability and lowered Bcl-2, c-myc, Par-4 and Dicer levels. In this aspect, miR-H1 seems to be antagonistic to the anti-apoptotic effect afforded by TAR miRNA. It was also noted further that HIV miR-H1 downregulated expression of the cellular miRNA miR149, which is considered to target the Vpr gene encoded by HIV-1 [41]. Interestingly, the effects of miR-H1 have been examined in the context of blood mononuclear cells. Monocytes not being able to sustain 2.2.3

22

a latent infection and miR-H1 negatively influencing Dicer levels (hence, influencing HIV-1 pathogenesis) are discussed later in the review. Strikingly, a study on miR-H1 variability in AIDS patients published earlier this year revealed that there is a strong relationship between the miRNA sequence and the development of HIV-1-associated dementia (HAD) [59]. All patients with HAD shared a comparable miR-H1 sequence that was similar to the published sequence. It is hypothesized that miRNAs that specifically target the AATF pathway could be involved in HAD development. The study also revealed that another disease state, AIDS-related lymphoma, may be associated with a less stable version of miR-H1 [59]. The authors hypothesize that the less stable versions may not be able to negatively regulate AATF; this may provide alternate means for activated macrophages to avoid apoptosis and produce factors that could stimulate lymphoma growth. Therefore, the connection between distinct miRNA species and HIV-1-related pathology in patients with AIDS opens the possibility for therapeutics. Suppressor of RNA silencing The presence of RNAi-based antiviral mechanisms in modern vertebrates, in additional to the protein-based immunity, contributes to a strong selection force that drives evolution of viral genomes. The HIV-1 genome clearly has multiple regions that produce interfering RNAs that could elicit the cellular RNAi-based defense mechanism. However, viruses are also equipped with suppressor of RNA silencing (SRS). Jeang’s lab has published evidence for the existence of one such SRS [60]. While studying the ability of short hairpin RNA (shRNA) constructs to suppress reporter gene expression when controlled by a Tat-independent promoter, it was found that whenever Tat was present in the experiment, the shRNAs were ineffective in suppressing the reporter gene. This lead to the conclusion that Tat was a suppressor of RNAi-induced silencing. Because Tat is a protein that binds to the TAR element, one could hypothesize that Tat-mediated suppression of the RNAi phenotype may be the result of Tat coating the RNA and making it unavailable to the RNAi machinery. However, studies using non-HIV-1-derived siRNAs revealed that Tat is in fact a generic suppressor of RNAi. Using Tat point mutants, it was shown that the two functions of Tat were distinct. Use of Tat deletion mutants revealed that amino acids 38 -- 72 in Tat contained its SRS function where this region is transcriptionally incompetent. It was also demonstrated that the SRS function of Tat was necessary for the virus to multiply in human cells. Importantly, while Tat was able to suppress silencing mediated by shRNAs, it was ineffective against siRNAs. This suggests that the target of Tat is upstream of the siRNA--RISC complex, possibly at the level of Dicer. Tat was seen to affect the functionality of Dicer without influencing the total Dicer RNA or protein levels. The connection of Tat with Dicer, Tat with the robust multiplication of the virus, lack of Dicer in monocytes and inability of monocytes to effectively sustain 2.3




a latent state provide important clues about miRNAmediated regulation of HIV-1 latency in Dicer containing target CD4+ T cells. However, there is some controversy surrounding the ability of Tat to function as a global RNAi suppressor. Lin and Cullen report that HIV-1 Tat and HTLV-Tax fail to inhibit RNAi in human cells. Their studies demonstrated that stable expression of Tat did not inhibit global miRNA production in infected human cells [40]. SRS function has been observed in other viruses as well [61]. The viral 7a accessory protein of severe acute respiratory syndrome coronavirus was found to suppress both transgene- and virus-induced gene silencing. The SRS function was attributed to the middle region (amino acids 32 -- 89) of 7a [62] Interestingly, in case of adenovirus, viral-associated RNAs VA RNAI and VA RNAII function as suppressors of RNAi by interfering with Dicer function [63]. The ebolavirus VP35 protein has also been demonstrated to possess a SRS function that is equivalent to the HIV-1 Tat protein [64]. In case of influenza virus, the NS1 protein of the strain A/WSN/33 (H1N1) was potent in suppressing RNAimediated silencing, although this function varied among influenza strains and was suggested to contribute to alterations in viral replication and pathogenicity [65]. It has also been demonstrated that the NS3 protein encoded by rice hoja blanca virus (plant virus) can complement the SRS function of HIV-1 Tat [66]. Availability of the miRNA machinery and implications for latency

2.4

Suppression of viral replication and/or alteration of cellular gene expression (e.g., delay of apoptosis) may be important mechanisms by which HIV-1 establishes latency. It is easy to conceive a mechanism by which HIV-1 miRNAs may be more abundant in latent cells. As transcription is restricted in the latent state, there is a low level of viral proteins produced including the viral transactivator Tat. This might result in latent cells producing abortive short transcripts that are between 50 and 100 nucleotides in length and containing the TAR stem-loop structure. Such short transcripts have been shown to be more abundant in non-viremic patients. As these TAR containing transcripts are the only HIV-1 RNA species produced in appreciable quantities in latent cells, it is possible that the miRNAs produced by the TAR element contribute to the maintenance of the latent state. If the TAR miRNA is crucial to the maintenance of latency, it would follow that only cells that are competent to generate miRNAs will be able to maintain the virus in the latent state. Dicer is present at strikingly low levels in monocytic cell lines and primary cells when compared to CD4+ T cells [67]. Analysis of CD4+ cell lines (Hut78, Molt4, H9, Jurkat, CEM) and HIV-1 infected cell lines (J1.1, ACH2, BE5) revealed that Dicer expression is similar in these multiple cells and remains unaffected during HIV infection. Studies of cells of the myeloid lineage including monocytes (THP-1), pro-monocytes (U937) and HIV-1 infected pro-monocytes (U1) revealed

that there was no detectable level of Dicer in these cells. Such analyses have been carried out in PBMCs obtained from healthy donors as well. Monocytes and lymphocytes from these donor cells and primary cells of the myeloid lineage were also found to be sub-optimal for Dicer protein. The levels of detectable Dicer in these monocytic cells were the same irrespective of the method of preparation of cell lysates. Interestingly, RT-PCR analysis revealed that 40 -- 60% less Dicer mRNA is present in U937 and U1 cells than CEM and ACH2 cells. However, Dicer protein becomes detectable in monocytes when they are differentiated to macrophages. When U937 cells were treated with increasing amounts of phorbol-myristate acetate (PMA) for 48 h and then probed for Dicer expression, Dicer protein was detected, although two to four times lower than what is seen in T cells. The absence of a complete, welldefined miRNA machinery in monocytes coincides with the inability of monocytes to sustain a latent state while the CD4+ T cells are targets of latency. This striking difference in Dicer levels observed between monocytes and macrophages raises questions about other miRNA components in monocytes. Drosha, Ago 1 and 2 were detectable in monocytic cells, but at lower levels than what were present in macrophages. When these cells were treated with PMA or M-CSF, Drosha, Ago 1 and 2 could be observed readily. Extensive analysis with multiple cell types revealed a clear distinction between cells of the lymphoid and myeloid lineage with respect to the miRNA machinery. Specifically, cells of the myeloid lineage were deficient for proteins relevant to miRNA biogenesis until induced to terminally differentiate. The absence of Dicer expression in monocytes brought up the question whether there was a transcriptional or a translational block. RT-PCR analysis of monocyte extracts revealed that Dicer mRNA was present at all times and did not show any particular increase following PMA treatment. This indicates a post-transcriptional block which could be the result of a specific miRNA--mRNA interaction. Interestingly, the cellular miRNA miR106a was involved in the suppression of Dicer expression in monocytes. While monocyte-derived macrophages (MDMs) produced detectable Dicer protein, HIV-1 infection of MDMs suppressed Dicer expression. If monocytes are deficient for Dicer protein, do the cells produce any host cellular miRNAs? Microarray analysis of RNA isolated from monocytes and MDMs using an Agilent V2 Human miRNA chip capable of detecting 722 unique human miRNAs revealed that miRNAs are in fact produced in monocytes and both PMA treatment and HIV-1 infection altered the miRNAs [67]. Consistent with HIV-1-mediated suppression of Dicer in MDMs, the array studies revealed that cells transfected with an HIV-1 clone produced fewer mature miRNAs when compared with control untransfected cells. Additionally, using mutant HIV-1 constructs, it was determined that the viral protein Vpr contributes to lowered Dicer levels in MDMs. Tat, as mentioned earlier, was the first discovered viral suppressor of Dicer activity. Vpr and nef have been demonstrated to be additional HIV-1 proteins that can


23



suppress the miRNA machinery by suppressing Dicer protein production [67]. The presence of multiple host cell miRNAs in a cellular environment that is defective for Dicer expression raises questions about the origin of these miRNAs. In addition to siRNAs and miRNAs, another group of small RNA molecules that are detected in multiple cell types are the piRNAs. These are generated by members of the PIWI family in a Dicer-independent manner. PIWI does not function in an enzymatic capacity. Instead, it protects double-stranded RNAs from ribonuclease degradation. Contrary to initial reports that PIWI expression is restricted to reproductive tissues, PIWI expression has been reported in somatic cells as well. Corroborating this, PIWIL4 expression has been observed in many cells of the lymphocyte and myeloid lineage and HIV-1 infection had no effect on endogenous PIWIL4 levels [67]. Mature miRNAs ranging in size between 26 and 33 base-pairs were produced in U937 cells, which were processed by PIWIL4. Collectively, analysis of the RNAi machinery in lymphoid and myeloid cells revealed that HIV-1 suppresses the miRNA machinery in both cell types, but by alternate means. 3.

Conclusion

An array of literature in the recent 5 -- 7 years has demonstrated the existence of HIV-1-derived miRNAs. These miRNAs appear to be abundantly expressed and derived from coding and non-coding regions of the viral genome. The miRNAs regulate both viral and host gene expression to alter the host milieu to support viral multiplication and establishment of latency. HIV-1, in addition to coding for miRNAs, also encodes a suppressor of the RNAi machinery. Finally, the availability of a functional, miRNA machinery is an important factor in the establishment of latency. 4.

Expert opinion

The influence of endogenous miRNA machinery on effectiveness of anti-HIV inhibitors

4.1

HIV-1 continues to be the subject of much scrutiny when it comes to therapeutics. Anti-HIV compounds directed against viral proteins eventually contribute to the evolution of resistance. While HAART treatment does save many lives and suppresses evolution of resistance, there is continuing concern on this matter [68,69]. A portion of the viral genome that appears to be highly conserved even in the presence of antivirals is the TAR element. The TAR element is the target for the viral transactivator protein Tat and both Tat and TAR are essential for HIV-1 replication [70,71]. Comparison of human and simian immunodeficiency viruses reveals that there is significant structural conservation (by co-variations of base-pairs) in spite of sequence divergence in the viral repeat (R) region that codes for the TAR RNA motif [72]. It was demonstrated that mutant viruses harboring base substitution mutations in the lower portion 24

of the TAR RNA stem were ‘dead viruses’ and that the base-pairing at the lower portion of the stem was critical for viral multiplication [73]. The critical role played by the TAR element in viral multiplication may be the reason for the observed resistance to mutation and this may make the TAR element more suited for the development of long term RNAi-based therapeutics. There has been mixed reports of success in utilizing the TAR region as a target. For example, in the study by Das et al., only one out of eight target regions (including the 5¢ and the 3¢ UTRs) was capable of inhibiting HIV-1 and the successful siRNA was directed against the Nef region [74]. While Leonard et al. suggested that HIV-1 could potentially evade the TAR directed siRNA inhibition by developing compensatory mechanisms based on the SP1 sites [75], Berkhout had suggested in an evaluation of the report that the HIV-1 strain used in their experiments modulates SP1 sites even when there is no RNAi-based inhibitory pressure [76]. Cellular miRNAs have been shown to function in an antiviral capacity. There is a large body of literature that focuses on the interactions between the host miRNA machinery and HIV-1 [77,78]. The cellular miRNAs display distinct antiviral roles against HIV-1 and there are multiple references in the literature relevant to this subject [79-82]. However, in this article, we restrict the focus to virally derived miRNAs and their influence on HIV-1. The cellular RNAi machinery has also been utilized in an anti-HIV-1 manner by introducing antiviral RNA molecules. For example, Son et al. have demonstrated that HIV-1 can be effectively suppressed by co-targeting multiple conserved sequence elements in the HIV-1 genome. When co-expressed as an artificial miRNA molecule from a miR-155-based vector, sequences directed against Tat and Vif genes mediated effective and sustained inhibition of HIV-1 [83]. In another such approach to target multiple HIV-1 regions and to attack escape-prone viruses, Liu et al. tested a multiplex miRNA expression system by inserting multiple anti-HIV-1 siRNA sequences into the miRNA polycistron mir-17 -- 92 and demonstrated that there is efficient inhibition of HIV-1 replication [84]. A similar study is described by Zhang and Rossi where they express multiple miRNA effector molecules as a single Pol II driven polycistronic transcript [85]. An alternate way to overcome the problem of drug resistance is to develop antivirals by targeting host components that are essential for viral replication. Important host candidate proteins that have been the subject of extensive research as anti-HIV-1 targets are the chemokine receptors CCR5 and CXCR4 (co-receptors for HIV-1 entry). Among the CCR5 inhibitors developed so far, maraviroc is the first drug that has been approved by the FDA. Many CXCR4 inhibitors have been studied, but have not reached the stage of FDA approval and research efforts in this arena continue [86]. Chable-Bessia et al. have demonstrated that downregulation of miRNA effector RCK/p54 protein in the infected cells also affects HIV-1 multiplication [87].



Cdk Inhibitors P

Abortive transcription (noncoding RNAs)

Pol II +1

Noncoding RNA (TAR)

(TAR)

RITS complex recruitment

RITS Chp1


HDAC

?

Chromatin Remodeling Factor recruitment

DNMT

HP1 Me

Me

Me

RITS Repressive Histone methylation ? Me

Possible LTR DNA methylation (heterochromatin)

Me

Me

Me

Me

Me

Me

Non coding RNA (either bound to DNA or nascent RNA)

Figure 3. Cdk inhibitors and transcriptional repression. Abortive transcription due to cdk inhibitors results in increased production of TAR miRNAs. The TAR miRNAs are incorporated into the RITS complex, which leads to recruitment of repressive chromatin. This ultimately culminates in repressive histone methylation, which could potentially lead to DNA methylation and formation of heterochromatin. DNMT: DNA methyltransferase; HDAC: Histone deacetylase; RITS: RNA-induced initiation of transcriptional silencing.

Another example of such targets is cyclin-dependent kinases (cdks). HIV-1 is influenced by both cdk2/cyclin E complex and cdk9/cyclin T1 [88-91]. Thus, cdk/cyclin inhibitors are ideal candidates for host-based antivirals against HIV-1 [92,93]. Roscovitine and flavopiridol are two cdk inhibitors that are most studied in relation to HIV-1 [94-99]. Cyc202 (R-roscovitine) targets the cdk2--cyclin E complex and causes apoptosis in HIV infected cells. CR8 is a derivative of Cyc202 with an alteration in the purine ring. CR8#13 is a third generation derivative that is a potent inhibitor of HIV-1 transcription without affecting host gene expression or cell viability [100]. The cellular miRNA machinery was necessary for effective viral inhibition by Cyc202 and CR8#13 as the drugs were

more effective in T cells than in monocytes. Specifically, the 5¢ TAR miRNA was required for CR8#13-mediated inhibition. CR8#13 was shown to decrease RNA polymerase II phosphorylation, which in turn inhibits transcription elongation. This leads to increased TAR production which results in the recruitment of the miRNA machinery and chromatin remodeling complexes to the viral LTR. The outcome of recruitment of remodeling complexes to the LTR is transcriptional repression mediated by the cdk inhibitors. This model has been illustrated in Figure 3. This dependence of antivirals on the endogenous miRNA machinery for effective inhibition of HIV-1 demonstrates that inhibitors that are effective in suppressing HIV-1 in lymphoid cells may not work in myeloid cells. Understanding viral pathology in multiple


25


target cells is hence an important prerequisite to designing effective anti-HIV-1 therapeutics. An important avenue for future research is to understand if the TAR miRNA affects the translational machinery in any manner. As mentioned above, while the role played by TAR in regulating transcription by controlling heterochromatin formation is being addressed, the potential role of TAR in modulating the host translational machinery to influence both viral and host translation requires attention. Also, it would be interesting to determine what happens to the host miRNA machinery (including Dicer) during later stages of an infectious process. It is not known if Dicer undergoes a

suppression of function as the viral infection progresses. Tackling these questions will be crucial for developing a wellrounded and coherent picture of miRNA-mediated regulation of the retroviral life cycle.

Declaration of interest This paper was supported by NIH grants AI 078859, AI 074410 and Department of Energy grant number DE-SC0001599 awarded to F. Kashanchi. The other authors declare no conflict of interest and have received no payment in preparation of this manuscript.


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Affiliation Aarthi Narayanan1 PhD, Kylene Kehn-Hall2 PhD, Charles Bailey3 PhD & Fatah Kashanchi†4 PhD † Author for correspondence 1 Research Assistant Professor, George Mason University, National Center for Biodefense and Infectious Diseases, Discovery Hall, Room 306, 10900 University Blvd. MS 1H8, Manassas, VA 20110, USA 2 Assistant Professor, George Mason University, National Center for Biodefense and Infectious Diseases, Discovery Hall, Room 306, 10900 University Blvd. MS 1H8, Manassas, VA 20110, USA 3 Center Director, George Mason University, National Center for Biodefense and Infectious Diseases, Discovery Hall, Room 306, 10900 University Blvd. MS 1H8, Manassas, VA 20110, USA 4 Director of Research, Professor of Microbiology, George Mason University, National Center for Biodefense and Infectious Diseases, Discovery Hall, Room 306, 10900 University Blvd. MS 1H8, Manassas, VA 20110, USA Tel: +1 703 993 9160; Fax: +1 703 993 7022; E-mail: [email protected]


29

Review


Stem cell transplantation as a biological therapy for peripheral T-cell lymphomas 1.

Introduction

2.

Autologous HSCT for PTCL

3.

Allogeneic transplant

4.

Conclusion

5.

Expert opinion

Abeer N Abouyabis, Pareen J Shenoy, Mary Jo Lechowicz & Christopher R Flowers† †

Emory University, School of Medicine, Winship Cancer Institute, Atlanta, GA, USA.

Importance of the field: Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of non-Hodgkin’s lymphomas in which conventional chemotherapy has been characterized by poor outcomes when compared with most aggressive B-cell lymphomas. Autologous, and to a lesser extent allogeneic, hematopoietic stem-cell transplant (HSCT) have been advocated as potential means to improve and consolidate remissions in PTCL. Areas covered in this review: Given the absence of large-scale randomized clinical controlled trials of transplantation for PTCL, we review existing data addressing the role and timing of HSCT in PTCL. What the reader will gain: A detailed overview of the available data of overall and progression-free survival associated with HSCT in PTCL with discussion of existing studies, data on determinants of HSCT outcome in PTCL, and future directions for research. Take home message: The optimal roles and timing of HSCT in PTCL remain unclear. Accordingly, clinicians are encouraged to register their patients in PTCL registry studies and enroll them in clinical trials investigating the role of HSCT. Risk-adapted treatment strategies utilizing identified prognostic parameters may provide future means for identifying the optimal use of HSCT for patients with PTCL, but additional studies are needed before such approaches can be routinely applied. Keywords: hematopoietic stem cell transplantation, peripheral T-cell lymphoma Expert Opin. Biol. Ther. (2011) 11(1):31-40

1.

Introduction

Peripheral T-cell lymphoma (PTCL) constitutes a small subgroup of non-Hodgkins lymphoma (NHL) with a worldwide prevalence that varies between 1.5 and 20% of NHL depending on the geographic location [1-10]. Historically, treatment of PTCLs with conventional chemotherapy has been characterized by poor outcome [3,4,11-13]. While combination chemotherapy regimens are associated with high response rates, the major difficulty is sustaining complete remissions (CR) [3,7,14-16]. Hematopoietic stem cell transplantation (HSCT) is a potential means of consolidating remissions and prolonging survival among PTCL patients. Autologous stem cell transplantation (ASCT) has been more widely studied in this setting than allogeneic HSCT (AlloSCT), but whether ASCT should be performed in the frontline or relapsed setting remains a debatable issue. The inability to reach a consensus on the optimal timing of ASCT in PTCL is due to the fact that most studies are small, PTCLs are heterogeneous group of NHL (Table 1) and most studies included patients with anaplastic lymphoma kinase- (ALK) positive anaplastic large cell lymphoma (ALCL), a subgroup with much more favorable outcomes. With respect to AlloSCT, its use in PTCL has been limited by its higher treatment-related mortality


31

Stem cell transplantation as a biological therapy for peripheral T-cell lymphomas

Autologous hematopoietic stem-cell transplantation at relapse

2.1


.

.


.

Conventional chemotherapy for peripheral T-cell lymphomas is characterized by poor treatment outcome. Autologous stem-cell transplantation in first remission may confer better overall survival to a subset of peripheral T-cell lymphoma patients, but additional studies are needed. Allogenic stem-cell transplantation should be considered for patients with poor response to therapy and those with relapsed/refractory disease. Large prospective randomized trials are required to clarify the role of hematopoietic stem-cell transplantation and define specific PTCL groups that benefit most.


(TRM), compared with ASCT [17-27]. Thus, AlloSCT has been reserved for heavily pre-treated, relapsed or chemorefractory patients. However, recent studies suggest more favorable outcomes for AlloSCT with reduced intensity conditioning (RIC), deriving benefit primarily from the graft-versuslymphoma effect in T-cell lymphoma patients [23,24,28-31] To examine the role of HSCT in the treatment of PTCL, we reviewed available prospective and retrospective studies and extracted data on outcomes for PTCL subtypes removing the more favorable ALK-positive ALCL subset. Studies were identified by searching Medline and Google Scholar databases through 2010. Additionally, we electronically and hand-searched the conference proceedings of American Society of Hematology and American Society of Clinical Oncology for the years 2003 -- 2009. Each search used combinations of the terms ‘Peripheral T-Cell Lymphoma’, ‘T-cell Lymphoma’, ‘Non Hodgkin Lymphoma’, ‘NK/T-cell lymphoma’, ‘Angioimmunoblastic lymphoma’, ‘Anaplastic large cell lymphoma’, ‘Enteropathy-type T-cell lymphoma’, ‘ALK-negative’, ‘Non-ALK positive’, and ‘hematopoietic stem cell transplant’. Citations of all studies were reviewed for additional references. Two reviewers (Abeer N AbouYabis and Pareen J Shenoy) performed study selection, quality assessment, and data extraction independently using standardized forms. Any disagreement was resolved by a third reviewer (Christopher R Flowers or Mary Jo Lechowicz). 2.

Autologous HSCT for PTCL

There have been conflicting data regarding the optimal timing of HSCT. In one study, the outcome did not differ significantly in patients who received ASCT during the first or subsequent remissions [17], while others reported a statistically significant longer survival in patients that received transplants in first CR compared to those receiving them in the second or later CR or partial remission (PR) [32-34]. 32

Overall, the role of high dose chemotherapy (HDCT) and ASCT in PTCL and other lymphomas is better established in the salvage than the frontline setting (Table 2). A retrospective review of 36 heavily pretreated PTCL (including ALCL) patients who underwent ASCT revealed a 3-year overall survival (OS) and progression-free survival (PFS) of 39 and 32%, respectively [18]. When compared with B-cell NHL, one study demonstrated that the CR rate for ASCT in patients with recurrent T-cell NHL was not significantly different (59 versus 42%; p-value is not specified in the paper) [35]. The 2-year OS and disease-free survival (DFS) were 35 and 28%, respectively, in PTCL, compared with 30 and 17%, respectively, in B-NHL [35]. Analysis of the Spanish Group of Lymphoma and Autologous Transplantation (GEL-TAMO) experience with HDCT and ASCT as salvage therapy in PTCL patients revealed a 5-year OS of 45%, which also compared favorably with the results in patients with B-cell histology [33,36] In another retrospective analysis, the 3-year OS and event-free survival (EFS) in PTCL patients who underwent ASCT were not statistically different from those for diffuse large B-cell lymphoma (DLBCL) (3-year OS 48 versus 53%, p = 0.41and 3-year EFS 37 versus 42% respectively, p = 0·29) [37]. Although one could argue that these favorable comparisons with B-cell NHLs may be due to the inclusion of ALK-positive ALCL patients, a similar finding was also reported in non-ALCL PTCL patients. One retrospective review of ASCT outcome among patients with PTCL (excluding ALK-positive ALCL) showed no statistically significant difference in the 5-year PFS or OS between PTCL and DLBCL patients [38]. These results, however, are unlikely to hold true when each PTCL subtype is evaluated separately. For example, in the same studies, PTCL not otherwise specified (PTCL-NOS) was characterized by an inferior EFS when compared with DLBCL (23 versus 42%, p = 0·028) while ALCL had a superior OS after HSCT in comparison with other subtypes (85 versus 35%, p = 0.007) [27,39] The retrospective nature of most studies makes it hard to draw meaningful conclusions, however, it appears that there is marked heterogeneity across PTCL subtypes in their response to ASCT at relapse. In particular, for patients with PTCL-NOS the outcomes with ASCT at relapse suggest that this approach may be of limited value for this patient group. Prospective PTCL-subtypespecific clinical trials examining the benefits of ASCT for patients with chemosensitive relapse are needed. Autologous hematopoietic stem-cell transplantation in first CR 2.2.1 Is transplant better than consolidative chemotherapy in PTCL? 2.2

Conclusive prospective, randomized data from trials focused on PTCL are not available to address this question. However,


Abouyabis, Shenoy, Lechowicz & Flowers

Table 1. WHO classification of mature T-cell and NK-cell neoplasms.


T-cell and NK-cell neoplasm subtypes T-cell prolymphocytic leukemia T-cell large granular lymphocytic leukemia Chronic lymphoproliferative disorder of NK-cells* Aggressive NK cell leukemia Systemic EBV+ T-cell lymphoproliferative disease of childhood (associated with chronic active EBV infection) Hydroa vacciniforme-like lymphoma Adult T-cell leukemia/lymphoma Extranodal NK/T cell lymphoma, nasal type Enteropathy-associated T-cell lymphoma Hepatosplenic T-cell lymphoma Subcutaneous panniculitis-like T-cell lymphoma Mycosis fungoides Se´zary syndrome Primary cutaneous CD30+ T-cell lymphoproliferative disorder Lymphomatoid papulosis Primary cutaneous anaplastic large-cell lymphoma Primary cutaneous aggressive epidermotropic CD8+ cytotoxic T-cell lymphoma* Primary cutaneous gamma-delta T-cell lymphoma Primary cutaneous small/medium CD4+ T-cell lymphoma* Peripheral T-cell lymphoma, not otherwise specified Angioimmunoblastic T-cell lymphoma Anaplastic large cell lymphoma (ALCL), ALK+ Anaplastic large cell lymphoma (ALCL), ALK--* *These represent provisional entities or provisional subtypes of other neoplasms. Diseases shown in italics are newly included in the 2008 WHO classification [82].

some information can be gained from available trials. In the randomized trial LNH87-2 which compared consolidative sequential chemotherapy (ifosfamide plus etoposide, asparaginase and cytarabine) to HDCT using cyclophosphamide, carmustine, and etoposide followed by ASCT in patients with aggressive NHL in CR1, there was no difference between HDCT and sequential chemotherapy in terms of 5-year OS (63.6% and 66.7%, respectively) and DFS (54.5 and 55.6%, respectively) [40]. In a matched-control analysis by pooling the data from the Groupe d’Etude des Lymphomes de l’Adulte trials LNH-87 and LNH-93, for non-ALCL PTCL patients after controlling for the age-adjusted International Prognostic Index (IPI) score, ASCT versus chemotherapy had no significant effects on OS (relative risk 0.93; p = 0.83) or DFS (relative risk 0.86; p = 0.61) [41]. In another prospective Phase II study of high-dose cyclophosphamide, hydroxydaunorubicin (doxorubicin), Oncovin (vincristine), and prednisone/ prednisolone (CHOP) alternating with Etoposide, methylprednisolone, high-dose cytarabine, and cisplatin (ESHAP) as first-line treatment for ALK-negative ALCL patients, followed by ASCT in responders, the 4-year OS was of 39% [42]. There was no significant difference in the 4-year EFS or OS among transplant patients versus non-transplant patients (4-year OS 57 versus 71%, and 4-year EFS 59 versus 29%;

both p > 0.1) suggesting a lack of benefit for ASCT even among responders [43]. On the other hand, in another prospective multicenter study the estimated 3-year OS rate was 71% for patients who underwent ASCT compared with only 11% for patients who did not undergo ASCT [44]. 2.2.2

Does disease status at transplant matter? Retrospective studies

2.2.2.1

One retrospective analysis showed that the 2-year OS and DFS were both 83% for patients receiving transplants in CR1 compared with 45% (p = 0.03) and 35% (p = 0.006), respectively, for those receiving transplants beyond first remission [45]. Univariate models showed the risk of death and/ or relapse was significantly less among the patients receiving transplants in CR1. In another study, angioimmunoblastic T-cell lymphoma (AITL) patients who underwent HSCT as a part of first-line therapy had a 5-year OS and EFS of 60 and 37%, respectively, versus 44 and 39%, in those who received transplants as second- or third-line therapy [19]. While many studies suggest that patients who do not achieve remission with chemotherapy do not benefit from ASCT [46,47], the distinction between outcome of transplant in CR1 versus PR1 is less clear. The GELTAMO experience with ASCT for AITL showed patients who underwent transplants in CR1 or PR1 had similar outcomes with a 5-year OS of 60% [48]. In another report, similar 5-year DFS were shown in patients receiving transplants in CR1 or PR1 (79 and 61% respectively; p = 0.3) but a significantly longer survival was seen in patients receiving transplants in CR1 compared with PR or second or greater CR pre-transplant [33]. In another retrospective multicenter study by the European blood and marrow transplantation (EBMT) lymphoma working group examining146 patients with AITL who received ASCT, the estimated PFS rates for patients who received their transplants during CR were 70 and 56% at 24 and 48 months, respectively, 42 and 30% for patients with chemotherapysensitive disease, and 23% at both time points for patients with chemotherapy-refractory disease [49]. Prospective studies Corradini et al. have shown that patients attaining CR before ASCT had a statistically significant benefit in terms of OS and EFS compared with those who did not (10-year OS 48 versus 22%, 10-year EFS 47 versus 11%, both p < 0.0001) [50]. On the other hand, Reimer et al. recently reported a nonsignificant trend for longer OS in PTCL patients who underwent transplantation in CR versus PR (p = 0.22; Specific OS rates are not mentioned in the paper [44]. 2.2.2.2

Are there factors other than disease status at transplant that affect ASCT outcome?

2.2.3

Some studies have incorporated factors such as components of the international prognostic index (IPI; age, Lactate dehydrogenase (LDH) level, performance status, Ann Arbor stage and extranodal disease) and the prognostic index for T-cell


33


Table 2. Characteristics and outcomes of studies on autologous stem cell transplantation for peripheral T-cell lymphoma (PTCL). Study, Year (Ref.)

Disease type

Disease status

n

5-yr OS

DFS/EFS/PFS

Rodriguez, 2001 [18] Schetelig, 2003 [19]

PTCL (including ALCL) AITL

CR/PR/SD/PD

29 29 13 11 52 23 29 37 18 19 14 14 28

3-yr 39% 44% 60% 44%

3-yr 5-yr 5-yr 5-yr

PFS EFS EFS EFS

91% 54% 54% 63% 45% 85% 35% 3-yr 42%

5-yr 5-yr 5-yr 5-yr 5-yr 5-yr 5-yr 5-yr

DFS 77% DFS 45% PFS 44% PFS 64% PFS 28% PFS 62% PFS 34% EFS 24%

34 62

4-yr 38% 57%

43

52%

4-yr DFS 63% 5-yr DFS 61%, EFS 42% 5-yr DFS 30%, EFS 32%

19 12 24 32 58 15 32 11 64 31 19 14 30 20 74 51 23 123 44 20 44 139 54 58

79% 20% 33% 34% 49% 76% 41% 36% 3-yr 53% 2-yr 62% 2-yr 49% 2-yr 34% 3-yr 36% 3-yr 60% 68% 61% 84% 45% 50% 33% 57% 42% 65% 34%

15 41

33% 4-yr 39%

Mounier, 2004 [41]

Jantunen, 2004 [39]

T-NHL (including ALCL) ALCL ALK-neg PTCL PTCL (including ALCL)

CR/PR1 > CR/PR1 CR/PR1


CR/PR1 > CR/PR1

Bang, 2005 [83]

ALCL PTCL, NOS PTCL (including ALCL)

Lopez-Guillermo, 2005 [84] Corradini, 2006 [50]

ALK-neg PTCL PTCL (including ALCL)

initially poor risk/Chemosensitive Rel/Chemoresistant Rel CR/PR1 CR/PR1

ALK-neg PTCL

Jantunen, 2006 [85] Kewalramani, 2006 [38] Smith, 2006 [86] Chen, 2007 [87]

Feyler, 2007 [30]

ALCL PTCL (including ALK-neg PTCL PTCL (including PTCL (including PTCL (including

ALCL) ALCL) ALCL) ALCL)

CR/PR1/Ref/Rel > CR/PR1 CR/PR1/Ref/Rel CR/PR1 > CR/PR1 Ref

PTCL (including ALCL) CR1 > CR/PR2/SD Ref

Rodriguez, 2007 [52]

Rodriguez, 2007 [21]

PTCL, NOS ALCL PTCL (including ALCL) ALK-neg PTCL ALCL PTCL (including ALCL)

CR1

PR1 > PR1 > CR1 Yang, 2007 [34]

Mercadal, 2008 [42]

PTCL (including ALCL)

ALK-neg PTCL

CR/PR1 Chemosensitive Rel CR/PR Ref CR/PR CR/PR1

32% 37% 37% 39%

5-yr 5-yr 5-yr 5-yr

RFS 18% PFS 24% PFS 51% PFS 17%

3-yr 2-yr 2-yr 2-yr 3-yr

PFS PFS PFS PFS PFS

50% 59% 49% 37% 36%

5-yr 5-yr 5-yr 5-yr 5-yr

PFS PFS PFS PFS PFS

63% 55% 80% 34% 47%

5-yr PFS 42% 5-yr PFS 61% 5-yr PFS 33%

4-yr EFS 59%

AITL: Angioimmunoblastic T-cell lymphoma; ALCL: Anaplastic large cell lymphoma; ALK: Anaplastic lymphoma kinase; CR: Complete remission; DFS: Diseasefree survival; EFS: Event-free survival; n: Number of patients; NHL: Non-Hodgkin’s lymphoma; NOS: Not otherwise specified; OS: Overall survival; PD: Progressive disease; PFS: Progression-free survival; PR: Partial remission; Ref: Refractory; Rel: Relapse; SD: Stable disease.

lymphoma (PIT; age, performance status, LDH level, and bone marrow involvement) score [51], or other factors such as b-2-microglobulin in their analyses of ASCT outcomes [20,21,42,44,52]. For instance, in one retrospective study with prolonged follow-up of patients who presented with unfavorable prognostic factors at diagnosis, consolidation with ASCT in CR substantially increased the OS and PFS 34

when compared with conventional chemotherapy [52]. In this study, the PIT risk system identified 14% of patients without benefit from ASCT consolidation [52]. In the GELTAMO experience, a prognostic model consisting of b2-microglobulin and age-adjusted IPI differentiated three distinct prognostic groups. Group I consisted of patients with no pretransplant adverse factors. These patients had an



OS and PFS at 5 years of 60 and 43%, respectively. Group II was patients who displayed one adverse factor. These patients’ OS was 28% and PFS 24%. Group III was patients with two adverse factors pre-transplant; all of whom died of their disease [21]. Notably, those latter patients all received transplants as a first line therapy (six cases of first PR and two cases of first progressive disease (PD)), making those other prognostic factors at least as important as disease status at transplantation [21]. 3.

Allogeneic transplant

Is there a role for AlloSCT in PTCL? The role of AlloSCT in PTCL is not yet clearly defined, since most published studies have been limited to heavily pre-treated, relapsed or chemorefractory patients (Table 3). In this setting, the 3-year OS rates have ranged between 35 and 40% and the 3-year PFS rates between 30 and 35% [22,23,28].


3.1

What are the limitations for the use of AlloSCT earlier in PTCL management?

3.2

Toxicity associated with AlloSCT has been a major obstacle in expanding the utilization of this important treatment modality in PTCL. TRM was observed in 15 -- 40% of PTCL patients undergoing AlloSCT, compared with less than 8% with ASCT [17,18,24,29,44,50,52]. In one retrospective analysis of outcome of full intensity AlloSCT in PTCL, including ALCL, collected from the national transplant registry databases of the British Society of Bone Marrow Transplantation and the Australasian Bone Marrow Transplant Recipient Registry, the 3-year OS and PFS were 39 and 33%, respectively [30], however, TRM was 39%. Similar TRM was observed in a retrospective analysis of 77 pretreated PTCL patients who underwent AlloSCT conducted on behalf of the Socie´te´ Française de Greffe de Moe¨lle et de The´rapie Cellulaire, despite the relatively favorable 5-year OS and EFS rates (57 and 53%, respectively) [23]. Attempts to improve outcome of RIC--AlloSCT in PTCL have led to the investigation of the role of antibodybased immunotherapy in this setting. The addition of the anti-CD52 antibody, alemtuzumab, may help with disease control and provide immunosuppression which may result in improved outcomes [31,53] Additional studies are needed to pursue these preliminary findings and monitor outcomes for possible prolonged immunosuppression with its use in the transplant setting. Are there any prognostic predictors of outcome in AlloSCT?

3.3

In one retrospective analysis of 77 PTCL patients treated with AlloSCT conducted by the Socie´te´ Française de Greffe de Moe¨lle et de The´rapie Cellulairethe, the 5-year OS and EFS rates were 80 and 80% for AITL patients, 63 and 58% for PTCL patients, 55 and 48% for ALCL patients,

respectively [23]. However, these differences in outcome across PTCL subtypes did not achieve statistical significance. Similarly, IPI score and ALK-status did not predict for differences in survival. However, both grade 3 -- 4 acute graft-versus-host disease and chemoresistant disease at time of AlloSCT had a negative prognostic effect on OS while only disease status affected EFS. 4.

Conclusion

PTCL is a group of NHL subtypes that is characterized by poor treatment outcome. Despite reports of initial success of commonly used chemotherapy regimens in inducing responses in PTCL patients, sustaining those responses remains a major challenge. ASCT in first remission seems to confer better OS to a subset of PTCL patients. There appears to be differences in the benefit of ASCT in CR1 according to prognostic risk group and PTCL subtype, however additional prospective studies are needed to define specific groups that benefit most. PTCL patients with poor prognostic factors and those with relapsed/refractory disease also should be considered for AlloSCT given the described benefits of graft-versus-lymphoma effects. A number of novel approaches have the potential to improve transplant outcomes, including the incorporation of immunotherapy prior to transplant or in the conditioning regimens, the introduction of new investigational agents and/or pralatrexate prior to ACT and the use of early response assessment by sensitive imaging modalities to tailor treatment. Significant improvements in PTCL treatment in general, and HSCT outcomes in particular, may be possible in the future with these and other approaches. 5.

Expert opinion

Although the 30 -- 70% CR rates obtained with CHOP chemotherapy in PTCL patients seems similar to the 44 -- 63% CR rates obtained with DLBCL, [4,7,8,14-16,25-27,54-57]. PTCL patients have higher relapse rates (43 versus 29%, p < 0.001) and significantly shorter freedom-from-relapse survival (median: 34 months versus not reached for p = 0.002) than their B-NHL counterparts [4]. In addition event-free survival (EFS; 33 versus 42%, p < 0.0001) and 5-year OS rate (41 versus 53%; p = 0.0004) for patients with PTCL are significantly shortened when compared with B-NHL [3,4,11-13,26,58]. These data suggest that the current conventional chemotherapy regimens are not sufficient to induce long-term disease control in PTCL. Hence, HSCT has been investigated as a reasonable way of consolidating remissions and prolonging survival among PTCL patients. Nevertheless, the role and timing of HSCT remains controversial. This is mainly due to the lack of large scale, prospective, randomized trials in PTCL. The main factors impeding such large scale research can be summarized as follows. First, PTCL is an uncommon disease compromising


35



Table 3. Characteristics and outcomes of studies on allogenic stem cell transplantation for peripheral T-cell lymphoma. Study, Year [Ref.]

Disease type

Rodriguez, 2001 [18] Corradini, 2004 [88] Tanosaki, 2006 [28] Feyler, 2007 [30] Hamadani, 2008 [81] Le Gouill, 2008 [23]

PTCL(including PTCL(including PTCL(including PTCL(including PTCL(including PTCL(including AITL PTCL, NOS ALCL

ALCL) ALCL) ALCL) ALCL) ALCL) ALCL)

n

OS

7 17 15 18 14 77 11 27 27

5-yr 3-yr 3-yr 3-yr 3-yr 5-yr 5-yr 5-yr 5-yr

EFS/PFS 29% 81% 81% 39% 35% 57% 80% 63% 55%

3-yr 3-yr 3-yr 3-yr 3-yr 5-yr 5-yr 5-yr 5-yr

PFS PFS PFS PFS PFS EFS EFS EFS EFS

14% 64% 81% 33% 31% 53% 80% 58% 48%

AITL: Angioimmunoblastic T-cell lymphoma; ALCL: Anaplastic large cell lymphoma; EFS; Event-free survival; n: Number of patients; NOS: Not otherwise specified; OS: Overall survival; PFS: Progression-free survival.

only 1.5 -- 20% of all NHL [2-8]. Second, PTCL is a heterogeneous group of NHL (Table 1). Although the variability across PTCL-histological subtype in the outcomes of treatment in general, and ASCT. In particular, in terms of PFS and OS has been widely demonstrated, [30,39,41,59] most studies, report results on all PTCL patients as a homogeneous group of patients. Third, there is inconsistency in reporting outcomes across studies, as some studies considered first remission as CR1 and PR1 status while others differentiated between CR1 and PR1. Given the limitations of prospective trials, it is very important to encourage oncologists to register their patients in a PTCL registry, such as the Comprehensive Oncology Measures for Peripheral T-cell Lymphoma Treatment study, so that a larger number of patients becomes available to assess the longitudinal benefits of existing treatment approaches. PTCL subtype as well as baseline prognostic factors should be incorporated in the assessment of the efficacy of newer treatment modalities. Moreover, consistent definition of disease status prior to transplantation will clarify many of the current controversial outcome questions. Yet, some broad guidelines for incorporating HSCT in general and ASCT in particular, in the treatment of PTCL can be drawn from available data. First, PTCL therapy should be individualized given the differences in sensitivity to chemotherapy across PTCL subtypes.[4,7,8,14-16,25-27,56-58]. Second, given the unsatisfactory relapse rates among PTCL patients (especially for non-ALCL PTCL) with conventional chemotherapy [4,7,8,14-16,25-27,56,57,60,61] we recommend that newly diagnosed PTCL patients should be considered for investigational therapy whenever possible. The addition of etoposide, bortezomib and/or purine nucleoside analogs to CHOP-like chemo-regimens as well as the introduction of immunotherapy, such as alemtuzumab and denileukin diftitox alongside conventional initial chemotherapy may improve the durability of remissions [62-75]. Third, available prognostic factors, such as IPI and PIT scores, b2-microglobulin, and Ann Arbor stage should be validated to determine their usefulness in predicting which 36

patients should undergo transplantation in CR1/PR1. Variations in these factors might explain the lack of beneficial effect of ASCT over consolidative chemotherapy in chemosensitive PTCL patients observed in some studies [40-43,76]. Data from one study suggest that for PTCL patients who present with a PIT score of 1 or 2 and achieve a CR with chemotherapy it is reasonable to consider upfront consolidative ASCT [52]. The same study also suggests that for PTCL patients with a PIT score greater than 2 at presentation who achieve a CR with initial chemotherapy consolidative ASCT does not seem to offer any advantage over conventional treatment. Such patients may be candidates for RIC-AlloSCT, especially if a matched-related donor is available [52]. This approach needs to be further evaluated in a larger series. In relapsed/refractory PTCL patients, two factors: the age adjusted-IPI [77] and b2-microglobulin may play a role in directing further therapy. For example, one study showed that patients with none of these pretransplant adverse factors may benefit from intensified therapy followed by ASCT, with OS and PFS at 5 years of 60 and 43%, respectively [21]. Given that, patients with one or more adverse factors had poorer outcomes they may be reasonably considered for experimental agent and/or additional intensified chemotherapy followed by RIC-AlloSCT if a matched donor becomes available. Moreover, since disease status at transplant plays an important role in determining outcome after ASCT [19,20,45,48-50], a response-adapted treatment strategy may be beneficial in improving ASCT outcome [78]. Aggressive or investigational chemotherapy may overcome pretransplant residual disease and improve transplant outcome [47]. Last, but not least, less toxic but more effective investigational treatment regimens, rather than the traditional CHOP-like chemotherapy, are needed as upfront therapy prior to ASCT, especially in highrisk patients. Toxicity from chemotherapy along with disease progression prior to ASCT rank among the top reasons why 20 -- 40% of PTCL patients do not proceed to their planned HSCT [29,43,50,59] Manipulation of pretransplant chemotherapy such as introducing newer agents or incorporating



immunotherapy, as well as considering newer less toxic, therapeutic modalities for patients unable to achieve adequate responses may improve transplant outcome [60,67,68,73]. For instance, the Pralatrexate in Patients With Relapsed or Refractory Peripheral T-Cell Lymphoma (PROPEL) trial demonstrated that patients with relapsed or refractory PTCL can proceed to subsequent HSCT after achieving a response with pralatrexate, permitting these patients a transplant option with the possibility of improved DFS [79]. Given its high TRM compared with ASCT, AlloSCT has been historically reserved for relapsed/refractory, heavily pretreated PTCL patients [17,18,23,24,28-30,32,44,50,52,80].

However, recent studies have demonstrated an earlier role for AlloSCT in cases where initial chemotherapy fails to produce at least a PR, or if a patient has adverse prognostic factors and only achieves a PR with initial chemotherapy, or in cases of relapse post-ASCT. In such instances the role of graft-versus-lymphoma effect associated with RIC-AlloSCT can be exploited to improve outcomes [30,81].

Declaration of interest The authors declare no conflict of interest and have received no payment in preparation of this manuscript.


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Affiliation Abeer N Abouyabis1 MD, Pareen J Shenoy2 MBBS MPH, Mary Jo Lechowicz2 MD & Christopher R Flowers†,2 MD MS † Author for correspondence 1 Mercer University, Hematology Oncology Care of Central Georgia, Department of Internal Medicine, 770 Pine Street, Suite 520, Macon, Georgia 31201, USA 2 Emory University, School of Medicine, Winship Cancer Institute, 1365 Clifton Road, N.E. Building B, Suite 4300, Atlanta, GA 30322, USA Tel: +1 404 778 5554; Fax: +1 404 778 5520; E-mail: [email protected]

Review

Stem cell-based strategies for the treatment of type 1 diabetes mellitus Introduction

2.

Pluripotent stem cells: ESC and iPSC


Yujie Wen, Bo Chen & Suzanne T Ildstad†

1.

3.

Organ-specific facultative stem/progenitor cells

4.

Hematopoietic stem cells

5.

Multipotent mesenchymal stromal/stem cells

6.

Expert opinion

†

University of Louisville, Institute for Cellular Therapeutics, Louisville, KY, USA

Importance of the field: b-Cell regeneration and b-cell preservation are two promising therapeutic approaches for the management of patients with type 1 diabetes (T1D). Stem cell-based strategies to address the problems of shortage in b cells, autoimmune and alloimmune responses have become an area of intense study. Areas covered in this review: This review focuses on the progress that has been made in obtaining functional, insulin-producing cells from various types of stem/progenitor cells, including the current knowledge on the immunomodulatory roles of hematopoietic stem cell and multipotent stromal cell in the therapies for T1D. What the reader will gain: A broad overview of recent advancements in this field is provided. The hurdles that remain in the path of using stem cellbased strategies for the treatment of T1D and possible approaches to overcome these challenges are discussed. Take home message: Stem cell-based strategies hold great promise for the treatment of T1D. In spite of the progress that has been made over the last decade, a number of obstacles and concerns need to be cleared before widespread clinical application is possible. In particular, the mechanism of ESC and iPSC-derived b-cell maturation in vivo is poorly understood. Keywords: regenerative medicine, stem cells therapy, transplantation, type 1 diabetes Expert Opin. Biol. Ther. (2011) 11(1):41-53

1.

Introduction

Type 1 diabetes (T1D), also known as insulin-dependent diabetes, is an autoimmune disease that targets insulin-secreting pancreatic b cells for destruction. T1D remains a major cause of long-term morbidity and mortality, affecting > 1% of the population worldwide. All patients with T1D and a third of the patients with type 2 diabetes require insulin treatment to prevent death from hyperglycemia resulting from the loss of pancreatic islets. T1D has various complications including renal failure, proliferative retinopathy leading to blindness, peripheral neuropathy and vascular disease. The discovery of insulin has prevented death from acute diabetes. However, even tight glucose control does not prevent the systemic complications [1]. Transplantation of whole pancreas or purified insulin-producing islets is the preferred approach to achieve glucose homeostasis, especially for a specific population of T1D patients who do not respond to conventional therapy [2]. Even though promising results including reduction or freedom from insulin, better glucose stability and less risk of complications have been achieved, the major obstacle to widespread use of pancreatic or islet transplantation is the shortage of suitable donor tissues [2,3] and the inferior long-term results, including graft rejection and nephrotoxicity from the immunosuppressive agents [4]. An attractive strategy to address this problem would be to generate islets from other types of cells, such as stem cells. This review focuses on the progress made in obtaining functional, 10.1517/14712598.2011.540235 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

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Stem cell-based strategies for the treatment of type 1 diabetes mellitus


.

.


.

.

.

Several embryonic stem cell (ESC) differentiation protocols recapitulating pancreatic development in vivo have been used to guide differentiation of ESC toward definitive endoderm, pancreatic progenitors, followed by endocrine progenitors in vitro. The in vivo studies confirm the potential of using ESC-derived b cells for the treatment of diabetes. Type 1 diabetes (T1D)-specific induced pluripotent stem cells could potentially provide treatment for diabetes in the future. Potential plasticity in adult pancreatic cells was reported previously and recent studies reveal the previously unrecognized function of a cells as b-cell progenitors. Independence from exogenous insulin can be achieved in most new-onset T1D patients following immunoablation and reconstitution of the immune system with autologous hematopoietic stem cell transplantation. As non-myeloablative conditioning approaches become refined and less toxic, the establishment of mixed chimerism may not only induce tolerance to islet allografts, but also halt the progression of the autoimmune process. Multipotent mesenchymal stromal/stem cells (MSC) offer an attractive source of stem cells for generation of surrogate b cells. MSC also play an important role in immunomodulation.


insulin-producing cells from various types of stem/ progenitor cells, emphasizes the current knowledge on the immunomodulatory role of hematopoietic stem cells and multipotent stromal cells in the therapies for T1D (Figure 1) and discuses the continuing obstacles that must be addressed. 2.

Pluripotent stem cells: ESC and iPSC

Embryonic stem cells (ESCs) are pluripotent cells derived from the inner cell mass of a blastocyst and have the potential to form derivatives from all three germ cell layers (mesoderm, ectoderm and endoderm) [5]. Embryology has offered important insights into key developmental pathways regulating ESC differentiation, resulting in advances in modeling gastrulation in culture and in the efficient induction of endoderm, mesoderm and ectoderm and many of their downstream derivatives [6]. The key signaling pathways regulating differentiation of ESC to pancreatic hormone-expressing endocrine cells include the TGF-b signaling pathway, the Wnt/bcatenin signaling pathway, the Hedgehog signaling pathway, Notch signaling pathway and PI3K signaling pathway [7]. The studies of embryonic pancreas development have been used as the basis for the directed, step-wise differentiation of mouse and human ESCs into pancreatic endocrine cells [8]. By applying modulators to reproduce the aforementioned developmentally active or inactive signaling pathways, several ESC differentiation protocols recapitulating pancreatic 42

development in vivo have been used to guide differentiation of ESC toward definitive endoderm, pancreatic progenitors, followed by endocrine progenitors in vitro. Lumelsky et al. first described a five-step protocol for in vitro generation of insulin-expressing cells and other pancreatic endocrine hormones from mouse embryonic stem cells (mES) [9]. Although later studies did not confirm insulin production by the differentiated ES cells [10,11], but rather demonstrated uptake of insulin present in the media supplements in which the cells were cultured [12,13], the initial study generated interest in the development of a future differentiation protocol. D’Amour et al. [14,15] further developed a differentiation process that converts human embryonic stem (hES) cells to endocrine cells capable of synthesizing the pancreatic hormones: insulin, glucagon, somatostatin, pancreatic polypeptide and ghrelin. This process mimics in vivo pancreatic organogenesis by directing cells through stages resembling definitive endoderm, gut-tube endoderm, pancreatic endoderm and endocrine precursors. Moreover, the hES cell-derived insulin-expressing cells have an insulin content approaching that of adult islets. More recently, Bouwens and co-workers modified the protocol of D’Amour et al. by shifting developmental pathways from hepatic to pancreatic cell differentiation at the definitive endoderm stage to favor pancreatic over hepatic differentiation, and reported improved and more efficient differentiation of pancreatic progenitors from multiple human ESC lines [16,17]. These proof-of-concept studies demonstrate that in vitro recapitulating signals controlling the development of the endocrine pancreas in vivo offer a promising strategy for b-cell generation [7]. The obvious prerequisite for embryonic stem (ES) cellbased b-cell replacement therapies is that ES cell-derived b cells must be capable of secreting fully processed insulin in response to glucose in a physiologic manner. However, functional characterization of endocrine cell populations differentiated from hES cells by the protocol D’Amour et al. showed that those cells did not respond to glucose stimulation to secrete insulin in vitro [15]. To overcome this limitation, researchers attempted to optimize the protocol and tested the survival and function of ES cell-derived b-like cells in vivo. Shi et al. [18] developed a novel three-stage shortterm induction method to induce mES cells into insulinproducing cells, which are able to lower blood glucose in mice rendered diabetic by treatment with the b-cell cytotoxic agent streptozotocin (STZ). By combining activin A and all-trans retinoic acid in a chemically defined medium, and other maturation factors such as basic fibroblast growth factor (bFGF) and nicotinamide in DMEM/F12, this group further demonstrated an optimized approach in serum-free culture medium without a feeder layer of stromal cells to induce hES cells to differentiate into functional insulin-producing cells [19]. The secretion of insulin by these cells was responsive to variations in glucose levels and the percentage of C-peptide positive cells exceeded 15%. However, in in vivo functional tests after transplantation of the differentiated cells into the


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Beta cell regeneration

Pluripotent stem cells

iPSC

T1D-specific iPSC

ESC Organ-specific facultative stem/progenitor cells Increase beta-cell mass

Pancreas-derived Liver-derived Spleen-derived

Beta cell preservation

HSC

HSCT

Endocrine Exocrine

Pre-existing beta cells Alfa cells Acinar cells Duct cells

auto-HSCT allo-HSCT

ESC-derived HSC MSC

Immunomodulatory properties


Tropic effects ESC-derived MSC

Figure 1. A schematic that represents the elements of stem cell-based strategies for the treatment of type 1 diabetes mellitus. ESC: Embryonic stem cell; HSC: Hematopoietic stem cell; HSCT: Hematopoietic stem cell transplantation; iPSC: Induced pluripotent stem cell; MSC: Mesenchymal stem cell/Multipotent stromal cell; T1D: Type 1 diabetes.

renal capsules of STZ-induced diabetic mice, only 30% of animals showed an obvious rescue of their hyperglycemic phenotype. Another group applied a similar serum-free protocol for differentiating hES cells into insulin-producing islet-like clusters (ILC) [20]. The cells that expressed pancreatic endocrine markers within ILC are likely to represent an immature phenotype as they contained significantly less insulin than adult b cells; however, they responded to high glucose challenge in vivo and extended the survival of graft recipients when transplanted them into STZ-induced diabetic mice [21]. Recent research from Baetge and colleagues [22] described pancreatic endoderm derived from hES cells by a modified five-stage protocol efficiently generates glucoseresponsive endocrine cells after implantation into mice. The cells are morphologically and functionally similar to normal pancreatic islets. And after a few months of maturation in vivo, implantation of hES cell-derived pancreatic endoderm protects against STZ-induced hyperglycemia in the majority (92%) of implanted mice expressing high levels of C-peptide before STZ treatment. Their results provide definitive evidence that hES cell-derived pancreatic endoderm can differentiate further in vivo and hES cells are competent to generate glucose-responsive, insulin-secreting cells. Their findings also suggest the need for in vivo differentiation to derive functionally mature b cells from ESC [23]. Two subsequent studies from other groups also support that the presence of in vivo factors is important for the final stages of maturation [24,25]. A recent report demonstrates that final maturation to isletspecific cells and a high yield of pancreatic islet cells are achieved by co-culturing the ESC-derived pancreatic endocrine cells with endothelial cells [26], which suggests that the signals received from interactions of pancreatic endocrine cells with endothelial cells and extracellular matrix are necessary for ESC-derived b-cell maturation. Collectively, these in vivo

studies confirm the potential of using ES cell-derived b cells for the treatment of diabetes. To bring ES cell-based b-cell replacement therapies for diabetes closer to reality, one of the hurdles that must be overcome is to generate mature and functional b cells in sufficient quantity. Current protocols for direct differentiation of ES cells into b cells use recombinant protein and growth factors and are not cost-effective to generate therapeutic quality b cells for clinic transplantation purposes [27]. Therefore, the focus has shifted to the possible use of small-molecules as imitators of the cellular signaling events for large-scale, reproducible, directed ES cell differentiation under good manufacturing practice conditions [28]. Implementation of high-throughput and high-content screening assays of chemical libraries to ESC research has led to the identification of several small-molecules that promote short-term hES maintenance and direct early lineage choice during differentiation [29,30]. Zhu et al. identified a small-molecule named stauprimide that increases the efficiency of the directed differentiation of mouse and human ES cells in synergy with defined extracellular signaling cues [31]. Affinity-based methods revealed that stauprimide interacts with NME2 and inhibits its nuclear localization and downregulates expression of c-Myc. In a screen of 4000 compounds, Melton and colleagues [32] also identified two cell-permeable small-molecules that direct differentiation of ESC into the endodermal lineage through induction of Smad2 phosphorylation. These compounds induce nearly 80% of ESC to form definitive endoderm, a significantly higher efficiency than that achieved by Activin A or Nodal. Moreover, the chemically-induced endoderm expresses multiple endodermal markers, can participate in normal development when injected into developing embryos and can form pancreatic progenitors. For promoting differentiation from definitive endoderm to pancreatic


43



progenitors, the same group identified indolactam V, an agonist of PKC, which induces differentiation of a substantial number of Pdx1-expressing cells from human ES cells. In addition, the Pdx1-expressing cells express other pancreatic markers and some of these Pdx1-expressing cells form insulin-expressing cells after transplantation into the kidney capsule of nude mice. Taken together with the discovery of small-molecules that specifically promote endoderm and pancreatic differentiation, the ES cell field is entering a new phase in which the possibility of using stem cells for human therapies edges closer to feasibility [27]. A landmark breakthrough of pluripotent stem cell research was made in 2006 when Takahashi and Yamanaka developed strategies to induce adult somatic cells to become pluripotent stem cells, also known as iPSC (induced pluripotent stem cells), by introducing a few key transcription factors or pluripotency genes including Oct3/4, Sox2, c-Myc and Klf4 [33]. iPSC derived from mouse or human adult fibroblasts showed similar capacities to ESC in terms of genetic, epigenetic and developmental criteria. iPSC are attractive cell therapy candidates derived from autologous cells because they avoid the inferior outcomes of b-cell replacement therapy in diabetes for transplanted allogeneic pancreas, isolated islets and engineered insulin-producing cells that are destroyed by the recipient immune system [34]. Recently, disease-specific iPSC have been generated from a wide variety of adult somatic cell types from donors with various disease conditions, including T1D, to fulfill therapeutic applications [35-42]. Maehr et al. reported that patient-specific iPSC can be generated from patients with T1D by reprogramming their adult fibroblasts with the transcription factors Oct4, Sox2 and Klf4. T1D-specific iPSC derived from patients with T1D, termed DiPS cells, have hallmarks of pluripotency and can be spontaneously differentiated into insulin-producing cells, as well as offering a few significant advantages including containing the genotype responsible for the human disease, providing an immunologically matched autologous cell population and making possible patient-specific disease modeling wherein the initiation and progression of T1D can be studied [37]. Using a serumfree protocol, Tateishi et al. confirmed that human iPSC can be derived from human skin cells by retroviral expression of Oct4, Sox2, c-Myc and Klf4. Moreover, they successfully generated ILC from the iPSC under feeder-free conditions. ILC not only contain C-peptide-positive and glucagonpositive cells but also release C-peptide on glucose stimulation [43]. More recently, Alipio et al. demonstrated that iPS derived from skin fibroblast could be differentiated into insulin-secreting b-like cells, which respond to glucose stimulation under physiological or pathological conditions. These b-like cells stably engrafted and corrected the hyperglycemia in two mouse models of types 1 and 2 diabetes [44]. These data raise the possibility that patient-specific iPSC could potentially provide a treatment for diabetes in the future. For improved induction of human iPSC to achieve high reprogramming efficiency, a chemical screening platform 44

was configured to determine the small-molecules that have direct impact on the signaling pathways of reprogramming process [45,46]. More recently, Amit et al. developed a suspension culture system for undifferentiated hES cells and iPSC using medium supplemented with IL6RIL6 chimeras (IL-6 receptor fused to IL-6) and bFGF. They demonstrate that the IL6RIL6 chimera supports the self-renewal and expansion of undifferentiated ESC and iPSC in suspension, and thus present another efficient system for large-scale propagation of undifferentiated pluripotent cells for clinical and translational applications [47]. The formation of teratomas or other tumors known to arise from ESC and iPSC is a major safety concern [22,48]. Cell sorting-based approaches may prove suitable for selection of residual undifferentiated ESC and iPSC ex vivo prior to transplantation [49], and building reporter constructs engineered to encode a suicide gene expression is a method for tracking and ablating any transformed cells that appear in vivo in the patient [50-52]. A recent study identified a new signaling pathway that the undifferentiated cells use for propagation, which depends on the phosphorylation of the transcription factor Nanog [53]. By inhibiting this pathway with small-molecule compounds, there was a stark reduction in resulting teratomas. Their study suggests that teratomas can be eliminated. The use of iPSC to produce differentiated b cells induced with retroviral vectors to deliver oncogenes as reprogramming factors may cause intentional mutagenesis and malignant diseases. Zhang et al. [54] developed a novel chemicaldefined culture system to induce insulin-producing cells from patient-specific human fibroblasts. A panel of human iPSC was generated by transducing the lentivirus containing Oct4, Sox2 and Klf4; cMyc was omitted in order to decrease the risk of tumorigenicity. The generated iPSC expressed pluripotency marker genes, such as Sea4, Nanog, TRA-1 -- 60 and TRA-1 -- 81. These cells were also coexpressed b-cell markers Pdx1, Mafa, Glut2 and insulin. In addition, the possibility that virus-free and transgenefree iPSC might be generated using only valproic acid and recombinant proteins or a non-viral minicircle vector is currently being explored in the murine system [55,56].

Organ-specific facultative stem/progenitor cells

3.

Endocrine stem/progenitor cells in the pancreas The pancreas is comprised of two main compartments, endocrine and exocrine. The exocrine cell compartment consists of acinar and duct cells. As the most abundant cell type in pancreas, the acinar compartment has been regarded as the possible site where endocrine progenitor cells reside in the pancreas. Several studies have suggested that functional insulin producing cells can be generated by in vitro transdifferentiation from rat exocrine tissue by adding agonists of the JAK2/STAT3 signaling pathway (EGF and leukemia inhibitory factor) to the medium [57]. The acinar cells that 3.1



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undergo exocrine to endocrine transdifferentiation first need to re-express a lineage-defining transcription factor, Neurogenin 3 (Ngn3), and then escape inhibition by Notch signaling. The insulin-expressing cells that are generated with this approach are glucose-regulated and can normalize hyperglycemia after transplantation into immunoincompetent diabetic mice [58]. Analysis by both non-genetic and genetic lineage tracing systems indicated these newly made insulin-producing cells originate from acinar cells [59,60]. Okuno et al. demonstrated that insulin-secreting cells can be generated by transdifferentiation from pancreatic acinar cells of diabetic rodents and further suggest that pancreatic acinar cells represent a potential source of autologous transplantable insulin-secreting cells for treatment of T1D [61]. Substantial evidence has also suggested that the duct compartment contains facultative endocrine stem/progenitor cells that can differentiate into b cells [62,63]. Yatoh et al. first showed that affinity-purified duct cells from adult human pancreas can differentiate to insulin-producing cells [64]. Hao et al. marked non-endocrine epithelial cells derived from human pancreas and showed that non-endocrine epithelial cells can differentiate into endocrine cells in response to inductive factors present in the fetal pancreas [65]. The potential of duct cells to undergo endocrine differentiation was further confirmed by in vivo experiments. Xu et al. found Ngn3-positive endocrine progenitors can be activated in injured adult mouse pancreas following partial duct ligation (PDL) and that the Ngn3-positive cells emerge from the pancreatic ducts. Differentiation of the adult progenitors is Ngn3-dependent and gives rise to all islet cell types, including glucose-responsive b cells that subsequently proliferate, both in situ and when cultured in embryonic pancreas explants [66]. Recently, Zhou et al. reprogrammed differentiated pancreatic exocrine cells of adult mice into cells that closely resemble b cells by re-expressing key developmental regulators (a combination of three transcription factors Ngn3, Pdx1 and Mafa) in vivo [67]. These in vivo experiments showed that mature pancreatic duct cells do contribute to pancreatic regeneration after partial pancreatectomy, and mature ducts can regress and recapitulate the embryonic differentiation program to form all differentiated pancreatic cell types [68]. However, the ductal origin of endocrine cell formation in adult injured pancreas remains controversial. By genetically marking ductal cells using carbonic anhydrase II (CAII) as a duct cell-specific promoter to drive Cre recombinase in the Cre/loxP-based direct cell lineage tracing system, the Bonner-Weir group showed that CAII-positive pancreatic cells are progenitors for both endocrine and exocrine pancreas after birth or PDL in adult mice [69]. The Ferrer group used a similar system with the Hnf1-b promoter to label ductal epithelium and found pancreatic exocrine duct cells give rise to insulin-producing b cells during embryogenesis but not after birth [70]. By genetically marking duct and acinar cells according to their expressing the mucin gene Muc1, another recent study also came to the same conclusion [71]. The contradictory

results may be explained by the hypothesis that there is functional heterogeneity of the morphologically defined ductal cells and duct cells are heterogeneous in their potential to act as pancreatic progenitors [72]. A recent report in the American Diabetes Association’s 70th Scientific Sessions by the Bonner-Weir group supported this hypothesis [73]. They found heterogeneity of protein expression for Sox9 and Hnf1-b in duct cells within the ductal tree of adult mice and humans and provided evidence for ductal subpopulations serving as postnatal pancreatic progenitors. b-Cell regeneration might be achieved from other endocrine cells. Collombat et al. demonstrated that ectopic and conditional expression of the transcription factor Pax4 in mouse pancreas using different cell-specific promoters converts progenitor cells into a and subsequently b cells. Notably, ectopic expression of Pax4 in a cells promotes the reconstitution of the insulin-expressing cell mass and cures diabetes in animals that have been chemically depleted of b cells [74]. Chung et al. recently developed a new mouse model of b-cell regeneration by combining PDL with elimination of pre-existing b cells with alloxan. In this model, they found that virtually all b cells observed are neogenic and large numbers of b cells were generated within 2 weeks. Most importantly, the neogenic b cells arose primarily from a cells [75]. In a transgenic mouse model of diphtheria toxininduced acute selective near-total b-cell ablation, under the condition of insulin treatment and in the absence of autoimmunity, mature glucagon-producing a cells are identified as the origin of b cells regeneration by genetic lineage tracing [76]. Whether the observed b-cell regeneration through spontaneous conversion of a cells also occurs in human pancreas of T1D patients with pre-existing autoimmunity to b cells is not known, but the approaches to modulate immune system of T1D patients might enhance b-cell preservation by promoting spontaneous conversion of a cells to b cells. In summary, the results of these studies confirms the potential plasticity in adult pancreatic cells reported previously and reveals the unrecognized function of a cells as b-cell progenitors. Liver cells Liver and pancreas arise from the same region of ventral foregut endoderm in response to the same signals during development. They share some specific differentiation pathways and expression of many specialized genes such as transcription factors, glucose transporter type 2 and glucokinase [77-79]. It is hypothesized that because liver cells share many transcription factors with b cells, they may retain the potential to produce endocrine pancreatic hormones. These characteristics make liver cells an attractive source for transdifferentiation into b-like cells. A variety of liver cells including hepatocytes, intra/extra hepatic biliary epithelial cells, and gall bladder epithelium have been demonstrated to show the phenotype of pancreatic lineages after viral introduction of different pancreatic transcription factors such as Pdx1 [80-82]. Recently, 3.2


45


Yechoor et al. reported that transfer of a single lineagedefining transcription factor Ngn3 is sufficient to induce a transdetermination from a hepatic progenitor cells to an islet lineage, which resulted in a rapid reversal of hyperglycemia as well as the significantly reduced blood glucose [83]. Spleen cells Whether spleen-derived stem cells can be used to generate b cells to treat T1D is still controversial. In 2003, Kodama et al. reported the existence of stem cells in the spleen of mice that could increase the speed of pancreas regeneration if the underlying autoimmune disease was controlled by administration of complete Freund’s adjuvant, donor spleen cells and temporary islet transplantation. More than 90% of non-obese diabetic (NOD) mice maintained euglycemia after the transplanted islets had been removed [84]. These results were very attractive as the spleen is not as essential to humans compared to islets, and it would dramatically increase the donor supply. In addition, spleen cells also share a very close developmental relationship with the pancreas, as the splenic mesenchyme bud off from the pancreatic mesenchyme early in development [85]. Some research teams also identified that splenic stem cells contribute to the regeneration of many tissues including bone, heart and salivary glands in animals [86,87]. Faustman and co-workers recently reported that the spleen of a variety of adult species (including humans) possesses a reservoir of multilineage adult CD45 negative stem cells that express the developmental transcription factor Hox11/Tlx1, which is essential for development of the spleen, pancreas and portions of the nervous system [88]. However, other groups failed to reproduce their findings using a similar treatment protocol. The results achieved by three independent efforts were remarkably consistent to Kodama’s conclusion: no splenocyte contribution to the islets was observed, even though diabetes could be reversed in this mouse model. Therefore, the recovered host b cells, rather than spleen cells, were believed be responsible for this reversal [89-91]. Additional studies are underway to confirm and enhance the role of splenocytes in islet neogenesis.


3.3

VSEL cells The infusion of bone marrow (BM) cells has been reported to improve damaged pancreas function in STZ-treated mice, and restored blood glucose and serum insulin levels to almost normal within a relatively short period [92,93]. Recent studies suggest that hematopoietic stem cells (HSC) may not be the main candidate for b-cell differentiation [94,95] but that another type of stem cells, CD45-/Sca-1+/Lin- very small embryonic-like (VSEL) stem cells, may contribute to tissue repair and regeneration [96]. Using a low-dose STZ-induced moderate pancreatic damage and hyperglycemia model, we recently reported that VSEL stem cells are mobilized in response to injured pancreatic tissue and contribute to b-cell regeneration, which were significantly enriched for developmental pancreatic transcription factors including Pdx1, Nkx6.1 and Ptf1. However, normoglycemia was not sustained long term, suggesting that additional stimuli are required for a more durable and robust effect [97]. 3.4

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4.

Hematopoietic stem cells

The best-known and best-studied example of stem cells is HSC. HSC are located in the BM niche and are characterized by the expression of cell-surface markers, which allows for isolation by cell-sorting-based approaches [98]. HSC can be readily harvested from BM and umbilical cord blood. It also can be collected from peripheral blood after mobilization from the marrow with growth factor mobilization agents [99]. Hematopoietic stem cell transplantation (HSCT) has been used for the treatment of autoimmune disease [100]. The transient lymphoablation by conditioning regimens before autologous HSCT allows immune regeneration and a resetting of immune self-tolerance from the multipotent HSC [101]. The Diabetes Control and Complications Trial demonstrated a reverse relationship between residual C-peptide production and development of chronic complications [102]. It suggests preservation of endogenous b-cell function is a potential target in the management of patients with T1D. The first autologous HSCT study in T1D patients was performed at a center in Brazil. After a mean follow-up of 29.8 months post autologous HSCT, C-peptide levels increased significantly and the majority of patients (20 of 23 newly diagnosed T1D patients) achieved insulin independence with good glycemic control, in which 12 of them stayed free of insulin for an average of 31 months and 8 patients had periods ranging from 6 to 47 months in which they were free from insulin [103,104]. More recently, a group in Poland reported the results of eight patients receiving autologous HSCT for treatment of T1D [105]. Patients became independent of exogenous insulin after transplantation. One patient resumed low-dose insulin 7 months after transplantation. Six out of eight patients were given acarbose for better glycemic control after transplantation. All patients exhibited good glycemic control: the average HbA1c concentrations were 12.3% at diagnosis, and 5.6 and 6.2% at 3 and 6 months after transplantation, respectively. Both studies suggest that independence from exogenous insulin can be achieved in most new-onset T1D patients following immunoablation and reconstitution of the immune system with autologous HSCT. The clinical experience of allogeneic HSCT in T1D patients is limited but shows promise. When patients with insulin-dependent T1D received BM transplants due to leukemia or other blood-borne diseases, there was no improvement of diabetes after the transplant [106]. In a T1D mouse model, allogeneic BM transplant reversed autoimmune process and prevented development of T1D if allogeneic HSCT was performed in pre-diabetic animals [107-109]. In overtly diabetic animals, allogeneic HSCT induced hematopoietic chimerism without causing graft-versus-host disease and reversed hyperglycemia in new-onset diabetic animals [110,111]. Notably, recovery of endogenous b-cell function occurs in both pre-diabetic and new-onset diabetic animals rendered chimeric [111,112], which may suggest that residual b cells play a significant role in determining the outcome of



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allogeneic HSCT. On the other hand, islet transplantation is currently limited by the toxicity of immunosuppressive agents and failure to control the autoimmune response. Induction of tolerance to alloreactive and auto-reactive response by allogeneic HSCT has been achieved in T1D animal models [113-116]. However, combined islet and HSC allotransplantation under an ‘Edmonton-like’ immunosuppression, without ablative conditioning, did not lead to stable chimerism and graft tolerance in a clinical pilot trial [117]. Taken together, these results suggest that durable mixed chimerism may prove to be an important approach to halt the autoimmune process and induce tolerance in T1D. As non-myeloablative conditioning approaches become refined and less toxic, the establishment of mixed chimerism may not only induce tolerance to islet allografts, but also halt the progression of the autoimmune process.

Multipotent mesenchymal stromal/ stem cells

5.

Multipotent mesenchymal stromal/stem cells (MSCs) are a heterogeneous population of adult stem cells originally isolated from BM. They have also been identified in cord blood, the perivascular niche and adipose tissue [118-122]. MSC have been recognized as a promising source for regenerative medicine due to their ability to undergo multilineage differentiation to various cell types of mesenchymal origin, and their ability to stimulate proliferation and differentiation of resident progenitor cells, as well as secrete multiple cytokines and chemokines [123]. MSC can be isolated from the BM and be easily separated from the HSC by their adherence to plastic [124]. However, there are no uniform phenotypic markers to characterize MSC, and investigators are using diverse methods to isolate and expand. The International Society for Cellular Therapy recommended that their name be changed to ‘multipotent mesenchymal stromal cells’ because the majority of MSC lack complete ‘stemness’ property. They also proposed minimal criteria for standardization of preparations [125], in which MSC must express CD105, CD73 and CD90, and lack expression of CD45, CD34, CD14 or CD11b, CD79-a or CD19 and histocompatibility complex antigen (HLA)-DR surface molecules. Recently, evidence suggests that MSC also play an important role in immunomodulation mainly through the direct inhibition of cell differentiation or proliferation, the alteration of cytokine secretion profiles of T cells, B cells, dendritic cells (DC) and NK cells, and the induction of T-regulatory cells (Treg) [126-130]. These immunomodulatory properties make MSC a promising source for therapeutic applications to treat numerous autoimmune diseases and induce allograft transplant tolerance [129,131]. Madec et al. recently reported that MSC could prevent autoimmune b-cell destruction and subsequent diabetes by inducing IL-10-secreting CD4+ FoxP3+ Treg [132]. Using a monkey model of islet/BM transplantation, Berman et al. demonstrated that MSC may provide an

important approach for enhancement of islet engraftment and function, and the mechanism was associated with increased Treg numbers in peripheral blood [133]. MSC offer an attractive source of stem cells for generation of surrogate b cells [122,134]. Expression of Pdx1 gene has been found in BM-derived MSC from human donors, followed by the activation of expression of all four islet hormones in vitro. Transplantation of these cells into STZ-diabetic immunodeficient mice resulted in further differentiation to insulin producing cells, including induction of neurogenic differentiation 1, and reduction of hyperglycemia [134]. Neshati et al. also demonstrated that BM-derived MSC expressed characteristics of insulin-producing cells including spherical, grape-like morphology, secretion of insulin and being positive for dithizone in an in vitro environment with high glucose concentration, nicotinamide and b-mercaptoethanol [135]. MSC treatment of experimental T1D resulted in long-term reversal of hyperglycemia, and therapy was shown to alter diabetogenic cytokine profile, to diminish T-cell effector frequency in the pancreatic lymph nodes, to alter antigen-presenting cell frequencies and to augment the frequency of the plasmacytoid subset of DC [135,136]. MSC from human umbilical cord blood and murine adipose tissue were also studied and showed the capacity to differentiate into the pancreatic lineage with insulin secretion functions [137,138]. However, this cell lineage transition has not been confirmed. Lee et al. found that the infusion of human BM-derived MSC to diabetic NOD/SCID mice resulted in significantly increased mouse insulin levels accompanied by improved pancreatic islets, even though rare human cells were found within the islets. Therefore, they proposed the possibility that MSC may be useful in enhancing insulin secretion instead of differentiation to insulin producing cells [139,140]. Sordi et al. indicate that MSC express negligible levels of islet-specific genes both in basal and stimulated conditions and might not be optimal candidates for generation of physiologically competent b cells [141]. MSC could exert an indirect role of ‘helper’ cells and provide trophic support to the injured tissues in tissue repair process. Park et al. reported that co-culture of islets and MSC improved the ATP:ADP ratio and insulin secretory function in vitro [142]. Ding et al. demonstrated that MSC could prevent islet allograft rejection leading to stable and long-term normoglycemia. These function mainly through the MMP secreted by MSC, in particular MMP-2 and -9, to reduce surface expression of CD25 on responding T cells [143]. Other mechanisms underlying MSC’s protective role in experimental autoimmune T1D include the T-cell cytokine secretion pattern shift toward IL-10/-13 production and the induction of Treg [144], as well as the promotion of pancreatic islet graft vascularization [145,146]. 6.

Expert opinion

Generation of insulin-secreting cells from ESC and iPSC holds great promise for the treatment of T1D. Despite recent progress in research on b-cell regeneration from ESC and iPSC, a number of scientific issues and medical


47



limitations need to be addressed and be overcome before ESC or iPSC-derived b cells can be considered safe for clinical applications. The first hurdle is the tumorigenic potential of ESC and iPSC. One strategy for dealing with this problem is to select and develop pure populations of more committed cells for transplantation. Therefore, future development of an ESC and iPSC cell-based therapy will require the purification of properly specified endodermal populations and the elimination of inappropriate cell types. Further research to study mechanisms of b-cell maturation in vivo and signals that drive differentiation to endocrine progenitors and/or mature b cells is required to realize the full potential of this therapy. The second hurdle is the fact that both generation of iPSC and differentiation of ESC or iPSC to mature b cells still suffer from low efficiency and high cost. Small-molecule library screening identifies simple imitators of the cellular signaling events that normally guide formation of the pancreas and its insulin-secreting b cells, enabling cost-effective differentiation methods for largescale generation of transplantation quality b cell from ESC or iPSC [147]. Marked differences in differentiation propensity among hES cell lines and iPSC generated from various tissues have been reported [148,149]. Therefore, screening and deriving lines is important to achieve efficient lineagespecific differentiation. The third hurdle is prevention of alloimmune rejection and recurrent autoimmune destruction. Transplantation of hES cell-derived b cells into patients is limited by potential HLA incompatibility. Strategies to reduce immune rejection include: i) establishing a large ‘bank’ of ESC for the purpose of matching its HLA phenotype to recipient transplant patients [150]; ii) microencapsulation techniques to protect grafted tissues from the immune system [28] and iii) using ESC generated from parthenogenetic embryos as a source of histocompatible cells and tissues for cell therapy [151]. Although generating autologous patient-specific b cells from iPSC solves the allorecognition issue, and bypasses some of the ethnical problems of ESC, both iPSC- and ESC-derived b cells will likely be the targets of persistent autoimmune response in T1D patients. In this regard, possible approaches to suppress pre-existing autoimmunity will be developed and integrated for induction of long-term tolerance to autoantigens. Recent studies suggest that hES cell-derived CD34+ cells not only have a potential for long-term in vivo endocrine cellular activity without teratomas [152], but also prevent diabetes in a T1D mouse model by induction of hematopoietic mixed chimerism [153]. Thus, combined ESC-derived b-cells islet and ESC-derived HSC transplantation may become an approach to suppress pre-existing autoimmunity. Most recently, MSC have been derived from hESC through several different methodologies [154]. Human MSC derived from hESC have been shown to possess characteristics very similar to BM-derived MSC. Thus, immunomodulatory properties of hESC-derived MSC are likely to prove valuable for inducing immune tolerance toward other cells or tissues 48

derived from the same hESC lines [119]. The fourth hurdle is ethical and policy issue that arises by hES cells research. Although in 2009 the Obama administration overturned the restriction access and use of hES cells by revoking statements and orders made during the former President Bush’s administration, a federal judge recently issued an injunction barring the Obama’s stem cell policy from funding research involving hES cells [155,156], which leads to the limitation of grants by NIH for the stem cell research. At the same time, the guidelines are recommended by the major stem cell organizations to enforce the regulation of stem cell therapies [157,158]. Experimental data suggest that the adult human pancreas contains facultative endocrine stem/progenitor cells that can differentiate to b cells under specific circumstances [59,68,76]. Induction of facultative endocrine stem/progenitor cells in pancreas and other organs to differentiation into b cells could become a viable therapeutic approach. However, any effort to replace b cells in diabetes, in particular by endogenous progenitors, will require an approach for dealing with recurrent autoimmunity in patients with T1D [62]. Thus, the amelioration of autoimmune stress together with stimulus for regeneration of endogenous b cells could be a feasible approach to improve endogenous insulin production in a substantial number of patients with T1D. A recent clinical study suggests residual functional b cells remain after as long as 50 years in the majority of patients with T1D [159]. In T1D patients, autologous HSCT may allow the residual b cells to recover and may induce facultative endocrine stem/progenitor cells in pancreas to transdifferentiate into b cells [58,66,76]. Induction of tolerance to alloreactive and autoreactive response by allogeneic HSCT has been achieved in experimental models of T1D. Future studies need to address the possible immunomodulatory effects of donor HSC and to optimize reduced-intensity non-myeloablative conditioning protocols. MSC have the capacity to differentiate into various cell types of mesenchymal origin, and also are well recognized for their immunomodulatory roles through direct inhibiting cell differentiation or proliferation and the altering of cytokine secretion profiles of many immune cell types. These unique characteristics make them an attractive source for cellular therapy of T1D based on the combined benefits not only from insulin-producing cells regeneration but also the negative regulation of autoimmunity. Although an emerging body of evidence shows the infusion of MSC resulted in increased insulin levels accompanied by improved pancreatic islets, some unaddressed questions may limit their clinical application. A standard protocol must be established to produce MSC and the optimal passage and doses of cells should be defined as these may have significant influence on MSC’s immunomodulatory properties. Clinically applicable assays should also be considered for monitoring the genetic and phenotypic stability of transplanted cells and the safety of recipients.


Wen, Chen & Ildstad

In conclusion, stem cell-based therapies continue to show great promise for the treatment of T1D, and significant progress has brought these therapies closer to a clinical reality through research efforts.

Acknowledgements The authors thank Y Huang and E Yolcu for reading the manuscript and C DeLautre for manuscript preparation. We apologize to those colleagues whose work we could not cite here because of space limitation.

Declaration of interest This work was supported in part by the following: The American Diabetes Association-Takeda Pharmaceuticals Beta Cell postdoctoral fellowship (07-09-BETA-03), NIH R01 DK069766, and the W. M. Keck Foundation. ST Ildstad and B Chen are sponsored by the Department of Defense, Office of Army Research, National Institute of Health. ST Ildstad is Chief Scientific Officer of Regenerex, LLC.


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from human embryonic stem cells. Exp Hematol 2010;38(6):516-25.e4 153. Verda L, Kim DA, Ikehara S, et al. Hematopoietic mixed chimerism derived from allogeneic embryonic stem cells prevents autoimmune diabetes mellitus in NOD mice. Stem Cells 2008;26(2):381-6 154. Karlsson C, Emanuelsson K, Wessberg F, et al. Human embryonic stem cell-derived mesenchymal progenitors-Potential in regenerative medicine. Stem Cell Res 2009;3(1):39-50 155. Cash G. Embryonic stem cells: court decision a threat to science itself. Nature 2010;467(7313):271 156. Wadman M. Stem-cell work thrown into limbo. Nature 2010;467(7311):12-3 157. Hyun I, Lindvall O, Ahrlund-Richter L, et al. New ISSCR guidelines underscore major principles for responsible translational stem cell research. Cell Stem Cell 2008;3(6):607-9 158. Dolgin E. Survey details stem cell clinics ahead of regulatory approval. Nat Med 2010;16(5):495 159. Keenan HA, Sun JK, Levine J, et al. Residual insulin production and pancreatic beta cell turnover after 50 years of diabetes: Joslin Medalist Study. Diabetes 2010;58(11):2846-53

Affiliation Yujie Wen1, Bo Chen1 & Suzanne T Ildstad†2 MD † Author for correspondence 1 University of Louisville, Institute for Cellular Therapeutics, 570 South Preston Street, Suite 404, Louisville, KY 40202-1760, USA 2 Director, Institute for Cellular Therapeutics, The Jewish Hospital Distinguished Professor of Transplantation, Distinguished University Scholar, Professor of Surgery, Microbiology and Immunology, Physiology, University of Louisville, 570 South Preston Street, Suite 404, Louisville, KY 40202-1760, USA Tel: +1 502 852 2080; Fax: +1 502 852 2079; E-mail: [email protected]

53

Review

1.

Immune monitoring of pancreatic islet graft: towards a better understanding, detection and treatment of harmful events

Introduction: why is there a need for an immune

Ste´phanie Lacotte, Thierry Berney, AM James Shapiro & Christian Toso†

monitoring? 2.

†

University of Geneva, Department of Surgery, Geneva, Switzerland

Early post-transplantation monitoring of the innate immunity


3.

Pre- and post-transplant monitoring of the adaptive immunity

4.

Pre- and post-transplant monitoring with non antigen-specific assays

5.

Expert opinion

Importance of the field: Long-term clinical outcomes of islet transplantation are hampered by rejection and recurrence of autoimmunity, which lead to a gradual decrease in islet function usually taking place over the first five years after transplantation. An accurate monitoring strategy could allow for the detection and treatment of harmful immune events, potentially resulting in higher rates of insulin-independence. Areas covered in this review: This article provides a critical review of the various assays currently available for the assessment of allo- and autoimmunity both prior to and after islet transplantation. The accuracy in predicting clinical outcome is specifically addressed. What the reader will gain: Most current tests based on the assessment of alloand auto-immune antibody are of minimal help in clinical practice. Cellbased tests (including the assessment of cytotoxic T lymphocyte precursors, proliferation tests, enzyme-linked immunospot) have the potential to allow earlier and more accurate detection of harmful events. Take home message: A specific and accurate immune monitoring has the potential to significantly improve islet transplant outcomes. The development and use of such tests (favouring cell-based tests) should be promoted. Keywords: alloimmunity, autoimmunity, immune monitoring, immunosuppression, islet transplantation Expert Opin. Biol. Ther. (2011) 11(1):55-66

Introduction: why is there a need for an immune monitoring?

1.

After the publication of the Edmonton protocol for islet transplantation in 2000, some believed that all patients with type 1 diabetes undergoing the procedure would be cured of diabetes [1]. Unfortunately, with more follow-up, it became apparent that insulin-independence was only transient in most recipients, although an improved glucose homeostasis with the absence of hypoglycaemia was maintained in the majority, which outcome should not be underestimated [2,3]. This decrease in function occurs in a two-step fashion. Early losses are primarily non specifically immunological, linked to low rates of engraftment, thought to be the result of damage sustained during the isolation procedure or in the graft microenvironment within the liver, secondary to ischemia--reperfusion-like injury and to non-specific inflammatory phenomena such as the instant blood mediated inflammatory reaction (IBMIR) [4]. Early non-immune islet injury may lead to a secondary immunological exposure to inflamed islet cells from exposed antigens. Subsequent islet losses are usually more progressive and involve allogeneic rejection, recurrence of autoimmunity or immunosuppressive drug toxicity [1,5-7]. The presence of allo-immune factors has been strongly suggested by the poorer 10.1517/14712598.2011.536530 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

55

Immune monitoring of pancreatic islet graft

Early post-transplantation monitoring of the innate immunity

2.


.

.


.

.

Long-term clinical outcome after islet transplantation is hampered by rejection and recurrence of autoimmunity, which lead to a decrease in islet function. Current tests based on the assessment of allo- and auto-immune antibodies are of minimal help for immune monitoring in clinical practice (with the exception of pre-transplant allogeneic antibody). Cell-based tests (allowing alloimmunity and autoimmunity assessments) have the potential to allow earlier and more accurate detection of harmful events. To determine the whole immune system activation level, non-antigen-specific assays are promising and need validation in the setting of islet transplantation. An ideal immune monitoring technique would allow tailoring immunosuppression according to the risk of immune events.


clinical outcomes in case of prior human leukocyte antigen (HLA) sensitisation, and conversely, by the more favorable results achieved with more potent immunosuppression induction [8-12]. The presence of autoimmune factors has also been demonstrated by the post-transplant increased frequency of b-cell-reactive T cells detected utilizing HLAA2-GAD65114--122 pentamers and HLA-DR-GAD65555--567 tetramers, and by the association of anti-glutamate decarboxylase 65 (GAD65), anti-insulinoma-associated protein 2 (IA2) and anti-insulin auto-antibodies with shorter graft survivals [13-15]. On the one hand, newly implemented, more potent induction protocols appear to be required to prevent posttransplant immunological events, but also expose the recipient to higher rates of potentially severe side effects from profound immunological depletion [16]. An accurate immune monitoring strategy therefore appears necessary. It would allow for the use of more tailored (and milder) immunosuppression combinations and prompt intervention and treatment of acute immunological events. In addition, such a monitoring may provide a better understanding and characterization of the various mechanisms of destruction involved in the loss of islet grafts and would allow for a more refined assessment of new therapies, specifically developed to prevent harmful events. Immunological tests require the implementation of advanced assays ideally according to common technical standards. They should be easy to perform and provide results within a clinically acceptable time. The present review explores the various monitoring strategies tested both in animal models and in clinical studies of islet transplantation. It also urges the community for a more accurate immune monitoring after clinical islet transplantation, which is likely to become a key step towards improved long-term results. 56

Several animal and clinical studies have suggested that about half of the transplanted islets are lost during the first minutes to hours after transplantation [17,18]. This loss is to a large extent driven by pre-transplant factors. To illustrate, donor characteristics, including age, body mass index (BMI) and the level of liver function tests, alter the quality of the transplanted islets [19]. Similarly, isolated human islet function is decreased by an elevated pancreas core temperature during harvesting or a long harvesting time [19-21]. Pancreas preservation solutions effect on islet yield and function, and the current techniques of human islet isolation, requiring tissue digestion at ‘high’ temperature add another level of injury to the islets [22,23]. In addition, tissue digestion leads to a disruption of pancreatic extracellular matrix and a loss of adhesion molecules, which affect islets survival and function (islet cell functional mass may not be 100% viable/functional at the time of transplant) [24,25]. Beyond the alteration of isolation outcomes, many of these factors increase the level of metabolic stress of the islets, which leads to the release of mediators activating the coagulation cascade and the innate immune system [26,27]. All these events are part of the IBMIR reaction, a key component of the early post-transplant loss of islets [26,28-30]. Notably, the liver implantation site, with early and direct contact between islet grafts and blood may be especially prone to non-specific inflammatory (IBMIR) reactions. Alternative sites may be more appropriate and should be explored and validated [31]. Several promoters and markers of these events have been identified, and could be potential targets of a peri-transplant monitoring. One of the most studied is tissue factor (TF), which is produced by most endocrine cells within the islets [26,28,30,32]. Its secretion can be prevented and islet graft outcome improved with the use of nicotinamide or low-molecular-weight dextran sulphate, an inhibitor of complement and coagulation activation [28,30,33,34]. Monocyte chemoattractant protein-1 (MCP-1) also correlates with the level of isolated human islet injury and post-transplant outcomes [30,33,35]. Along the same line, several other proinflammatory mediators have been shown to be expressed and/or secreted by isolated human islets, including macrophage migration inhibitory factor (MIF), VEGF, TNF, IL-1b, IL-8 and IFN-g [26,34,36-38]. Several cell subsets of innate immunity also take part in the early post-transplant loss of islets. NKT cells, granulocytes, dendritic cells and to a larger extent macrophages can migrate toward chemoattractant molecules released by injured islets and take part in the destruction of the graft [36,39-42]. Notably, injured islets also undergo shedding of donor antigens and probably contribute to the very first steps of the activation of the adaptive immunity. This said early islet loss cannot be attributed (or only to a very minor extent) to allogeneic immune events, nor to the recurrence of autoimmune


Lacotte, Berney, Shapiro & Toso

diabetes, as shown by a similar rate of occurrence after syngeneic and allogeneic islet transplantation [39,40]. Altogether the immune monitoring of peri-islet transplant events could assess the level of soluble mediators, including TF and MCP-1, or could measure the frequency or the level of activity of innate immune cells. Such strategies would still need to be further explored and validated in clinical practice. They would primarily be helpful in the development of treatment strategies aiming at decreasing early islet loss and would probably not be relevant for long term monitoring.

Pre- and post-transplant monitoring of the adaptive immunity


3.

While many islets are lost early at the time of transplantation, the remainder engraft in the liver and are exposed to allo- and autoimmune injury. In animal models without systemic immunosuppression, such immune events are usually very acute and destroy all remaining islets within a few days [43]. In clinical practice, however, rejection and recurrence of autoimmunity are slower and more insidious in the presence of background potent immunosuppression. As a consequence they may be less sharp and more difficult to detect in comparison to baseline condition. Conversely, the more indolent kinetics of the immune events in humans allows more time for their detection and treatment. Islet transplant monitoring could target either the humoral or cellular aspects of the adaptive immunity (Figure 1, Table 1). Monitoring humoral immunity Pathogenic antibodies can be either pre-formed during the pre-transplant period or acquired after exposure to donor antigens. While the former have been extensively studied in the setting of solid organ transplantation, the nature and the long term risks associated with the latter still remain to be explored. In the setting of islet transplantation, a cellular transplantation supposedly devoid of donor endothelial cells, the effects of antibodies have often been thought to be secondary. The detection of pre-formed antibodies, both allo- and autoimmune, has however entered clinical pre-transplant routine. We explore this practice and whether it should also be conducted after transplantation. Patients receiving islet transplants in the setting of type 1 diabetes represent transplantation in the presence of a pre-primed potent immune response, and elimination of memory responses to autoantigens has been a clinical challenge. 3.1

Alloimmune antibody Panel-reactive antibody assays (PRA) are routinely performed prior to most solid organ transplantation and new flow cytometry PRA assays are highly sensitive [44]. They allow for the detection of recipient antibodies thanks to panels of lymphocytes or beads expressing a wide spectrum of HLA class I and class II antigens. Prior to transplant, patients 3.1.1

with positive PRA are considered to have developed HLA sensitization, usually due to a previous exposure to antigens, via a previous transplant, blood transfusion or pregnancy. The specificity of anti-HLA antibodies can be determined using beads coated with identified recombinant HLA determinants in a single antigen (SA) assay. Donor-specific antibodies (DSA) are established with the help of donor HLA typing. Cross-match assay may also allow detection of DSA with the use of donor cells as alloantigens. This type of assay allows for a general assessment of the recipient antibody/donor antigen interaction, but does not provide information about one specific antigen. Although an islet is not a ‘classic’ vascularized organ, pretransplant HLA sensitization with PRA > 15% and donor-specific antibodies, is associated with an accelerated post-transplant loss of islet function [8,45]. This suggests that current immunosuppression protocols do not fully control the allo-immune response in the presensitized population. Such individuals may require more aggressive induction and maintenance immunosuppressive treatment, and donors against whom recipient antibodies were previously developed should be avoided. The development of de novo post-transplant antibody against HLA class I and II antigens has been observed after islet transplantation [45-50]. While some claim that the presence of such antibody is not acceptable after islet transplantation, the detected rates of immunization (mean PRA level: 5 -- 34%) were similar to those after kidney transplantation alone, and multiple islet infusions do not represent a specific risk for the development of anti-HLA antibodies [46-48]. In addition, it remains unclear whether the detected antiHLA antibodies are the cause or the consequence of the loss of islets. One the one hand, several patients have developed antibodies after immunosuppression withdrawal, following the occurrence of side-effects or complete islet graft failure [46-48]. This supports the need for some degree of continuous immunosuppression after islet transplantation, in order to avoid de novo anti-HLA antibodies, which would hamper the chance of a subsequent transplantation. In contrast, some patients have shown a direct decline in islet graft function in relation to newly produced antibody (anti-class I or anti-class II) [51,52]. In one case, islet function recovered after treatment with anti-CD20 antibody and intravenous immunoglobin [52]. Altogether, the monitoring of anti-HLA antibodies is especially important prior to islet transplantation. The monitoring of de novo antibodies is less clear as their correlation with the clinical outcome has not been well documented. They could however help in tuning the level of post-transplant immunosuppression. Autoimmune antibody While autoimmune antibodies, such as anti-insulin, antiIA2 and anti-GAD65 are routinely used at the time of diagnosis of type 1 diabetes, their prognostic and pathogenic 3.1.2


57


Detection of soluble factors in serum: cytokines, sCD30, tissue factor Antigen-presenting cells sCD30 Helper T lymphocyte B cells and plasma cells


IL2 Inflammatory soluble factors

Uptake and processing

Antibodies

β cell

β cell

β cell

β cell

Cytotoxic T lymphocyte

IFN-γ granzyme B, perforin

Insulin

Alloantibodies: panel reactive and donors-specific Autoantibodies: anti-GAD65, anti-IA2, anti-insulin

Cell-based tests for the assessment of allo-and auto-immunity - CTLp determination - IFN-γ or granzyme B ELISpot - Tertramers - Gene analysis - Proliferation - ATP production - Assessment of cytokine profile

Figure 1. Immune events altering islet graft function and survival, with potential monitoring tests.

roles are less clear prior to and after islet transplantation [53,54]. The accuracy of pre-formed pre-transplant autoimmune antibodies is weak in the prediction of post-transplant outcomes [2,55-59]. The assessment of these antibodies cannot be used to predict clinical outcome and to exclude patients from a transplantation waiting list but provides a general idea of the level of autoimmunity. It is likely however that some anti-self humoral events may alter the fate of islet grafts, as suggested by worse outcomes in case of high pre-transplant B lymphocyte counts or by a liver biopsy report showing B cell accumulation around islet cells [55]. In addition, the emergence of newly formed autoantibodies can be detected during the first months after islet transplantation and correlate, at least in one study, with the risk of graft failure [13,14]. 58

This said it is clear that the loss of islets can happen without any increase in the level of auto-antibody. Such events could involve other mechanisms than a recurrence of autoimmunity [60]. One could also speculate that the antibodies are sequestrated in the graft environment upon reaction with their epitopes and that they cannot be detected in the peripheral sera. Conversely, some patients with high auto-antibody titers do well off insulin [61]. Increases in auto-antibody titers may only be markers of an immunological status and may not harm the islet graft. They would be the consequence of a cellular response which could, itself, be pathogenic. Overall, auto-antibodies are considered at best as a marker of autoimmune activity but not as an indicator of autoimmune graft damage; their assessment is secondary both prior to and after islet



Number of cytotoxic cells through target cell (donor cell) lysis measure Pattern of cytokine release following Incubation with donor cells Number of IFN-g and/or IL-2 secreting cells following incubation with donor cells ELISA with autoantigen peptides. Proliferation assessment following incubation with autoantigens

Quantification of antigen-specific cells with multimers of peptide-MHC molecules linked by fluorochrome. sDC30 determination in serum Genes of interest analysis on PBMC (Granzyme B. perforin and FasL)

CTLp

Cytokine secretion

Peptide-MHC multimers

Gene expression

sDC30

Proliferation

Antibodies

ELISpot

PRA detection or anti-donor antibody determination

Antibodies

Technique description

Allows determination of multiple genes with only few cells

Easy to perform

Quick Reproducible

Easy to perform and to calibrate Can be associated with phenotypic markers Measures function

Easy to perform Routinely used in clinical settings Reproducible Well validated Measures function and frequency Allow determination of cytokine pattern with multiple cytokine detection Short incubation time Measures function and frequency

Advantages

Requires optimization and validation

Requires validation

Long incubation period (5 days) Low sensibility Requires optimization and validation Restricted to HLA.A2 patients No analysis of function Requires validation

Long incubation period (5 days) Requires optimization and validation Requires validation

Long incubation period (5 days) Need for radioactivity

Disadvantages

*Correlation with clinical outcome: +: Low; ++: Intermediate; +++: High level of evidence; NA: No data available. CTLp: Cytotoxic T lymphocyte precursors; ELISpot: Enzyme-linked immunospot; HLA: Human leukocyte antigen; PRA: Panel reactive antibodies.

Assessment of general level of immunity

Assessment of autoimmunity

Assessment of alloimmunity

Assay

Table 1. Comparison of assays for immunological monitoring.

NA

NA

++

+++

+

++

+

++

+++

++

++

++

++

+

NA

++

+++

+

Post transplant

Correlation with clinical outcome* Pre transplant


[71]

[79,80]

[6,15,67]

[2,13,14, 53-55,59-61] [5,6,55,61,64]

[45]

[62]

[5,60-64]

[8,45-52]

Ref.


59


transplantation, as they currently cannot be used to guide clinical intervention. Monitoring cellular immunity Similar to the assessment of antibodies, the detection of a T-cell response can be used to quantify the degree of autoand/or alloimmunity. Cell-based assays are without doubt more difficult to implement than antibody assays, but they provide a probably better and earlier view of the pathogenic course of a disease. 3.2

Alloreactive cells T-cell alloreactivity was originally determined as the specific number of cytotoxic lymphocytes against target cells (cytotoxic T lymphocyte precursors, CTLp). Roep et al. were the first investigators to implement this test in the setting of islet transplantation and demonstrated that an increased antidonor specific CTLp frequency is associated with decreasing C-peptide levels [5]. Since then, most reports (except two, based on the same group of patients [61,62]) have confirmed the correlation between pre-transplant or de novo CTLp and clinical outcome [5,63,64]. These types of alloreactive cell monitoring are important and should be performed more widely in the setting of islet transplantation. The detection of alloreactive cells should promote the maintenance of higher immunosuppression levels. It can also guide a subsequent listing for retransplantation, as the risk of failure increases with the presence of alloreactive cells [60]. In addition, the CTLp method has shown that the level of alloreactivity against HLA mismatches present both on the newly transplanted islets and a previous kidney or islet graft is lower than the one against novel HLA mismatches [60,63]. This observation is probably associated to the previous use of anti-thymoglobulin (ATG) treatment and is helpful in the selection of new donors [5]. Cell-based tests are extremely useful, and their use should be promoted especially prior to transplantation. However, they are complicated, time consuming and only available for class I antigens. In addition, the level and the type of immunosuppression can inhibit or/and suppress the ex vivo results [65]. As a result, further improvements are still required.


3.2.1

Autoreactive cells The presence of pre-transplant autoreactive cells can be determined as the number of proliferating T cells after incubation with islet-specific auto-antigens like IA2 and GAD. It correlates very clearly with the achievement of post-transplant insulin-independence and should be used in the pre-transplant assessment of patients [55,61,64]. The effect of post-transplant auto-reactivity is less clear. The appearance of such a reactivity has been associated with the loss of islets in only some studies [5,61]. These more ambiguous post-transplant results may be related to the background immunosuppression treatment and/or to the migration of donor-specific CTLp and autoreactive T cells to the graft, 3.2.2

60

which may not be detectable in the blood stream anymore [6]. As a consequence, post-transplant tests should be refined, potentially with the design of faster (current tests require a five-day incubation) and more sensitive tests, with or without the study of more specific cell types. Flow cytometry analysis with tetramers can also allow for the quantification of antigen-specific T cells. Tetramers consist of four biotinylated MHC molecules covalently linked by a fluorescent-labeled streptavidin. The increase in avidity caused by multimerisation allows consistent binding of autoantigenderived peptides to T cells receptors. The presence of in vivo insulin B10 -- 18-specific CD8+ T cells has been identified in recipients with recurrence of autoimmunity after islet transplantation and subsequent graft failure, but not in patients with persistent b-cell function or without autoimmunity recurrence [15]. Similarly, proliferation of autoreactive T cells has been highlighted with the help of HLA-A2-GAD65114--123 pentamers in islet-transplant recipients [6]. As a further development, a kit has been developed to detect changes in the frequency of autoreactive CD8+ T cells. It allows for a simultaneous monitoring of several known epitopes like GAD65114--123, IA-2797--805 and IGRP265--273 with the help of HLA-A2 multimers and quantum-dot-labeled streptavidin [66]. This assay is able to efficiently highlight multiple islet-specific CD8+ T-cell responses and has the potential to enter routine monitoring, pending further development and validation. Due to the current nature of the tetramers, their use is currently however restricted to HLA-A2 patients, including only about 60% of the western diabetic population. Of note, IFNgsecreting GAD555-567-specific CD4+ T cells have recently demonstrated their deleterious effect in pancreas transplant patients [67]. Thus, class II-tetramers are designed with HLA-DR specificity matched to patients’ HLA-DR type. Ex vivo cytokine profile assessment HLA class II-restricted CD4+ T cells from islet transplant recipients have been recently shown to proliferate upon incubation with HLA-DR mismatched donor cells in mixed lymphocyte culture (MLC) [62]. This proliferation, combined with the assessment of a specific cytokine profile, appears as a valuable biomarker of islet loss. Allograft-specific cytokine profiles are skewed toward a TH2 or regulatory phenotype after transplantation in insulin-independent patients. As a result, decreased ex vivo IFNg:IL10 ratio and MLC proliferation rates after transplantation are favorable and increase the chance of achieving insulin-independence [62]. 3.2.3

ELISpot Enzyme-linked immunospot (ELISpot) is performed by culturing recipient cells with specific stimulators (e.g., donor cells). The secreted cytokine of interest is further assessed as spots around the cells using a sandwich ELISA technique. Because of the short incubation time (< 24 h) each detected spot represents a cell that has been previously primed in vivo 3.2.4




(effector or memory T cells). IFN-g is the cytokine most often assessed, but other molecules like granzyme B and perforin could be more sensitive and specific (they still need further assessment). While ELISpot has been used in kidney transplantation, only one study reports data after islet transplantation [45]. A high number of pre-transplant IFNg and IL2-producing cells after stimulation with irradiated donor PBMCs correlated with the loss of islets. This reaction was specifically directed towards donor cells and not third party cells [45]. Due to the limited number of studied patients included in the described study, further validation of this technique is required both prior and after transplantation. ELISpot is easier to perform than previously described tests, with a shorter incubation time [5,63,64].

Pre- and post-transplant monitoring with non antigen-specific assays

4.

Measurement of ATP production The assessment of ATP production after overnight incubation of whole blood with a mitogen (phytohemagglutinin (PHA)) reflects the level of T cell activity. ATP level helps identify patients at risk of rejection or infection (over-immunosuppression), and allows for a better tuning of the immunosuppression, as demonstrated after solid organ transplantation [68-70]. This assay has been accepted by the FDA for detection of cell-mediated immunity in an immunosuppressed population. Notably, this test has several limitations. ATP can be produced by any cell type within the blood and the measured levels do not always reflect the activity of pathogenic cells. Results are not specific for donor antigens, and the test only provides a sense of the overall immune activity without predicting punctual donor-specific adverse events like rejection. The test also needs validation in the setting of islet transplantation. 4.1

4.2

Gene expression analysis

Gene PCR analysis on PBMC is also an interesting and attractive way of monitoring transplant recipients. The detection of granzyme B, perforin and/or FasL has been associated with the occurrence of harmful events after islet transplantation [71]. The main limitation of the test is its lack of specificity for rejection, as any non-related immune event (infection or inflammation) can also increase the marker. It has therefore been suggested to combine this test with the detection of insulin mRNA in the blood, in order to detect specific immune events directed towards islet grafts [72,73]. This approach still needs validation. The immune activity can also be assessed by the analysis of the T cell receptor (TCR) gene repertoire. Some Vb transcripts have been shown to be overexpressed in PBMC of patients undergoing acute kidney rejection. In addition, T cells with this specific pattern accumulate in the graft micro-environment [74].

Another approach targets a wider range of protein genes. The analysis of 33 genes in 75 renal-transplant patients correctly segregated tolerance and chronic rejection phenotypes with high specificity. Forkhead box p3 (Foxp3) and glucocorticoid-induced TNFR-related protein (gitr) were increased and costimulatory molecule genes were decreased in patients showing peripheral tolerance [75]. This test could highlight a tolerance pattern, but found no marker of rejection. However, another preliminary study detected rejection episodes with a sensitivity of 73% and a specificity of 91% [76]. These gene analysis techniques have mainly targeted solid organ recipients. Their use after islet transplantation still needs to be explored. Soluble CD30 CD30 is a member of the TNF-R superfamily expressed on a subset of activated and memory T lymphocytes. Soluble CD30 (sCD30) is proteolytically cleaved from the membrane and released into the serum after activation of CD30expressing cells. sCD30 detection is an accurate marker of acute rejection after kidney transplantation with 100% specificity and 88% sensitivity [77]. Moreover, serum sCD30 measured one-year post transplant provides predictive information regarding kidney outcomes [78]. In islet transplantation, sCD30 correlates with CD4-cells activation during rejection of mouse allograft [79]. In humans, sCD30 levels are higher in patients with failing islet allotransplantation [79]. A reduction in sCD30 post-transplant level correlated with a full graft function at one year and is associated with an ATG immunosuppressive protocol [80]. These preliminary data suggest that sCD30 is a potential monitoring tool. 4.3

5.

Expert opinion

Current routine immune monitoring of islet transplant recipients is based on the assessment of alloimmune and autoimmune antibody and is of minimal help in clinical practice (with the exception of preformed alloreactive antibodies for the selection of a suitable donor). Such a pre- and post transplant monitoring should be improved, validated and promoted in clinical studies. The aims are: 1) To allow tailored immunosuppression according to the risk of immune events. This risk varies a lot from one individual to another, as shown by in vitro PBMC proliferation experiments following PHA stimulation in presence of common immunosuppression drugs [81]. The sensitivity to a drug also changes over time according to the level of activation of the immune system, the presence of costimulatory molecules and cytokines and the degree of metabolic stress of the islet graft [82]. Finally, for the same recipient, the risk of immune


61


+ Treatment

Immune markers in the peripheral blood Non-conclusive

+ Treatment

Islet graft imaging: • MRI • PET

-

Non-conclusive + Treatment

Liver biopsy


Figure 2. Future post-transplant monitoring.

events is changing tremendously from one donor to the other. As a consequence, ideal monitoring should include repeated assessments of anti-donor/ autoimmune-specific responses, as well as their inhibition by immunosuppression drugs in order to determine the best dose-response. 2) To detect and treat harmful immune events in a timely fashion. When immunosuppression is not appropriately tailored, rejection or recurrence of autoimmunity can occur. These events should be detected early, when only a marginal number of islets has been damaged, in order to prompt treatment and salvage of the remainder of the graft. As such, one should have a better understanding of the rejection process and monitor the earliest steps of the cascade. Regarding this issue, the development of antibodies appears only late during a post-transplant rejection, and a cell monitoring would be more appropriate (with the problem of the interfering efffects of immunosuppression still to be solved). 3) To improve the rates of insulin independence and overall quality of life after islet transplantation [83,84]. This aim should be easily achieved, when one or both of the previous ones have been fulfilled. The clinical relevance of a monitoring test varies a lot from one technique another (Table 1). Pre-transplantation PRA and cross-matching are currently the only assays routinely used. We argue that cell-based tests would allow earlier and more accurate detection of rejection events, and that they should be favored. Among them, the detection of CTLp has been successfully used and validated. This test however is time-consuming, expensive and relatively difficult to implement, and alternative techniques, including ELISpot and the measurement of gene expression, may become more widely accepted in the coming years. The former can be rapidly performed, is reproducible and allows the assessment of cytokines including IFNg and granzyme B (other cytokines may also be tested). Notably, donor PBMCs used as stimulators can be 62

lacking after multiple testing, and the use of a cell panel from different donors can be an attractive alternative (similar to panels used for PRA) [85,86]. Similar to the monitoring of rejection, cell-based techniques appear most attractive to assess autoimmunity. Auto-reactive cells can be detected with the use of class I- or class IItetramers or of proliferation assays utilizing islet proteins (including GAD65 and IA2) as stimulators [5,15,55,61,64,66]. While pre-transplant autoimmunity monitoring probably correlates with the clinical outcome, the relevance of post-transplant monitoring remains controversial [5,15,55,61,64]. Overall, we believe that the pre-transplant islet graft monitoring of the future will be more developed, including cell-based tests. The post-transplant assessment will be multistep. Immune tests will be part of the first assessment, as they can be performed each time the patient comes to the clinic (Figure 2). If these first tests are positive, they would lead to a treatment and in case of doubt, the clinician will proceed with the next step of monitoring, involving more invasive tests, which could not be performed on a routine basis. MRI monitoring is currently the most promising option, but positron-emission tomography (PET) monitoring using new tracers more specific for pancreatic b-cells may also be of interest in the future [87-89]. The last step of monitoring, in case of further doubt would probably include the use of biopsy, but this technique would probably remain the last option as only about 30% of needle biopsies can sample islets [90]. Notably, several ongoing clinical trials assess the efficacy of subcutaneous and intraperitoneal human and porcine alginate-encapsulated islet transplantation [91-93]. Thanks to encapsulation, islet grafts have the potential to be protected against IBMR and other allo-, auto- or xeno-immune mechanisms, and immunosuppressive treatment can be reduced or suppressed. However, the protective efficiency of this type of encapsulation remains to be proven in the long term. In a similar manner, some studies support the idea of an acellular matrix to mimic the pancreatic environment [94,95]. While these prototypes are promising, their ability to prevent rejection remains to be demonstrated. Islet-specific immune monitoring is also important to assess humoral and cellular reactivity in view of a possible kidney transplantation. PRA sensitization subsequent to islet transplantation is well studied and is similar to the one after kidney transplantation alone [46]. Cellular pretransplant ‘sensitization’ has been shown to correlate with kidney allograft dysfunction and anti-islet donor cellular memory could represent a specific risk for subsequent organ transplantation [96]. While the detection of harmful immune events is currently still a challenge after islet transplantation, nothing is known about the treatment of such events. One can speculate however that steroid boluses routinely used for the treatment of solid organ graft rejection will not be applicable in the setting of islet transplantation because of their islet toxicity. Treatment may include a dose



increase of the maintenance drugs or the use of antibodies or other biologics. Overall, we strongly believe that specific and accurate immune monitoring has the potential to significantly improve islet transplant outcomes. The development and use of such tests, including cell-based tests, should be promoted.

Declaration of interest Christian Toso was supported by the Swiss National Science Foundation (SCORE grant 3232230-126233). The authors declare no conflict of interest and have received no payment in preparation of this manuscript.

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Affiliation Ste´phanie Lacotte1, Thierry Berney1, AM James Shapiro2 & Christian Toso†1 † Author for correspondence 1 University of Geneva, Department of Surgery, Rue Gabrielle-Perret-Gentil 4, 1211 Geneva 14, Switzerland 2 University of Alberta, Department of Surgery, Edmonton, Canada Tel: +41 22 3723311; Fax: +41 22 3727755; E-mail: [email protected]

66


Review


The therapeutic potential of engineered human neovessels for cell-based gene therapy 1.

Introduction

Luı´s A´lvarez-Vallina & Laura Sanz†

2.

Engineering neovessels in vivo

3.

Factory neovessels

Molecular Immunology Unit, Hospital Universitario Puerta de Hierro, 28222 Majadahonda, Madrid, Spain

4.

Expert opinion

Importance of the field: Several works have shown the feasibility of engineering functional blood vessels in vivo using human endothelial cells and mural cells. In this context, the genetic modification of endothelial cells would ensure the secretion of a therapeutic protein into the systemic circulation for a prolonged period of time. Areas covered in this review: We discuss the different strategies aimed at the formation of long-lasting neovessels in vivo, using human endothelial and mural cells. The main focus is the potential of these constructs in gene therapy strategies for the in vivo production of therapeutic proteins. What the reader will gain: The reader will have an outline of the different types of cells that have been used for microvessel engineering in vivo, as well as scaffolds employed to seed these cells. We provide a critical review of their advantages and drawbacks, along with examples of their potential in cell-based gene therapy strategies. Take home message: There is a real potential for neovessels derived from human endothelial and mural cells to be incorporated in clinical interventions, either as a cell-based gene therapy to produce a therapeutic protein or as a component of engineered tissue constructs in regenerative medicine. Keywords: angiogenesis, animal model, microvessel engineering Expert Opin. Biol. Ther. (2011) 11(1):67-76

1.

Introduction

Angiogenesis is a complex process implying a plethora of molecular and cellular interactions that is difficult to recapitulate in vitro. Classical in vivo models of angiogenesis relied on blood vessel formation by endothelial cells (ECs) provided by the host. Although these models have certainly been useful, they are based on the extrapolation of results obtained in nonhuman systems. In order to engineer a human model of angiogenesis in vivo, with stable and functional neovessels, several groups have use human ECs of different origins seeded in scaffolds and inoculated into immunodeficient mice (Figure 1, Table 1). Blood vessel engineering has an obvious application in regenerative medicine, where the formation of a functional vascular network is a major challenge. However, we intend here to focus on the potential of ECs as ‘cell factories’ for the in vivo production of therapeutic proteins in a variety of pathological conditions, including haemophilia and cancer. As an alternative to the administration of recombinant proteins, gene therapy may provide a novel means for in vivo systemic delivery of therapeutic proteins, resulting in effective and persistent plasma levels. The use of ex vivo genetically modified cells offers several advantages with respect to in vivo gene delivery with viral vectors: no safety concerns about the risk of unwanted virus dissemination; levels of expression by transduced cells can be measured in vitro and plasma levels can be predicted; the possibility of selection for high-expression clones 10.1517/14712598.2011.538378 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

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The therapeutic potential of engineered human neovessels for cell-based gene therapy


.

.

.

Human endothelial cells seeded in appropriate scaffolds are able to form functional vascular networks in immunodeficient mice. Co-implantation of mural cells along with endothelial cells improves the survival of endothelial cells and their incorporation into host vasculature. The use of human mesenchymal stem cells as a source of mural cells promotes the formation of long-lasting human neovessels. Endothelial cells genetically modified to express a therapeutic protein and organized into neovessels in vivo represent a new vehicle for cell-based gene therapy.



prior to administration and less likelihood of immune responses against the vehicle if autologous cells are used. ECs, because of their strategic contiguity with the bloodstream, are especially appealing as cell-based delivery vehicles. 2.

Engineering neovessels in vivo

Human endothelial cells form functional vascular networks in immunodeficient mice

2.1

Blood vessels are not only composed of ECs: mural cells (pericytes and vascular smooth muscle cells) envelop the surface of the vascular tube in mature vessels [1]. The basement membrane (BM), a specialized form of extracellular matrix (ECM), is also an important structural component of vascular networks and BM deposition is known to play a key role in the functional organization and maturation of newly formed vessels [2,3]. More than 20 years ago, Kubota et al. [4] plated ECs on a reconstituted ECM obtained from a murine sarcoma (Matrigel) and found that they rapidly attached, aligned, and formed capillary-like tubules. Matrigel is a commercially available, soluble BM extract [5] that contains laminins, collagens, heparan sulfate proteoglycans and nidogens/entactins, takes the form of a liquid at 4 C and gels rapidly at 22 -- 35 C. This was the origin of the most popular in vitro capillary morphogenesis assay, widely used for screening of pro-angiogenic and anti-angiogenic factors [6]. In the Matrigel plug assay in vivo [7,8], a liquid Matrigel solution is inoculated into the subcutaneous (s.c.) space of mice, where it reconstitutes into a plug at body temperature. Then, host ECs migrate into the plug and form vessellike structures in response to pro-angiogenic agents incorporated into the gel. Matrigel plugs are removed after one to three weeks and angiogenesis is quantified by histological analysis and immunohistochemistry. Another method for quantitative in vivo study of angiogenesis was based on s.c. implantation of biocompatible porous matrices (sponges) in the rat using surgical procedures, a method that was 68

subsequently applied to mice [7-10]. Neovascularization was also induced by implantation of gelatine sponges loaded with pro-angiogenic factors [11]. These assays have been widely used to evaluate the proangiogenic or antiangiogenic potential of drugs in vivo, but suffer from one main disadvantage: the vascular network obtained is provided by the host, and angiogenesis-modulating agents may be species-specific [12,13]. A critical step forward was the identification of conditions for in vivo engineering of human microvessels constituted by human dermal microvascular endothelial cells (HDMECs) or HUVECs, using naturally occurring or synthetic matrices as scaffolds. N€or et al. suspended HDMECs in Matrigel and seeded them in biodegradable polymer matrices before s.c. implantation into severe combined immunodeficiency (SCID) mice [14,15]. HDMECs organized in vivo into human microvessels that connected to the host vascular bed (inosculation), as assessed by the presence of mouse blood cells, recruited smooth-muscle a-actin (SMA)-positive host mural cells, and were stable for at least 21 days [15]. This novel bioassay, ‘the SCID mouse model of human angiogenesis’, was proposed for the study of physiological or pathological angiogenic processes and validation of strategies aimed to inhibit or promote angiogenesis. Alternatively, HUVECs were embedded in collagen/ fibronectin gels in vitro and allowed to organize into tubes for 20 h, before s.c. implantation. Schechner et al. demonstrated that such preformed networks incorporated into the mouse vascular bed and could be monitored for 60 days [16,17]. Survival of preformed microvessels is dependant on their ability to connect to the host systemic circulation, process that may take several days [18]. In some of these early works, HUVEC and HDMEC survival and tube formation in vivo were improved by transduction of EC with the anti-apoptotic B cell leukamia/lymphoma associated gene 2 (Bcl-2) [14,16,17]. Another means to prolong EC life span is to gene-modify them for the expression of human telomerase reverse transcriptase (hTERT). ‘Telomerized’ HDMECs formed functional microvessels when s.c. implanted in SCID mice, and vessel density remained stable for 6 weeks, whereas primary HDMEC-derived vessel numbers decreased with time [19]. Using a different strategy, Skovseth et al. [20] demonstrated that unmodified HUVEC could be mixed with Matrigel and inoculated as a suspension, giving rise to similar microvascular structures that were stable up to 100 days. This technique offered two advantages: injection of cells in liquid gel is less traumatic than surgical implantation of solidified gels or polymer matrices, and it eliminated the concerns about the effects of apoptosis suppression or telomerase-based life prolongation in ECs. Bagley et al. used endothelial precursor cells (EPCs) developed in vitro from CD133+, CD34+ bone marrow cells instead of HUVECs and demonstrated that after Matrigelembedding and implantation into mice, similar vascular networks were formed [21]. Moreover, EPCs exhibited greater proliferative and invasive capacities than HUVECs.


´ lvarez-Vallina & Sanz A

5

199

Human endothelial cells

Mural cells

HDMEC Nör et al. 1999 0

200

HUVEC Schechner et al. 2000 [17] Fibroblasts (b) Siemisnki et al. 2002 [22]


10T1/2 (m) Koike et al. 2004 [23] 5

200

ESC-EC Wang et al. 2007 [25] EPC Melero-Marín et al. 2007 [32] Au et al. 2008 [33] 0 1 0

MSC (h) Sanz et al. 2008 [35] ASC (h) Traktuev et al. 2009 [39]

2

In vivo long-lasting human blood vessels

Figure 1. Main landmarks in the engineering of in vivo long-lasting human blood vessels, in chronological order. ASC: Adipose stromal cells; b: Bovine; EPC: Endothelial progenitor cell; ESC-EC: Embryonic stem cell-derived endothelial cell; h: Human; HDMECs: Human dermal microvascular endothelial cells; m: Murine; MSC: Mesenchymal stem cells; Underlined: Stem/progenitor cells.

Interestingly, in most of these models, human ECs were shown to recruit SMA-positive, pericyte-like cells from the surrounding mouse tissue [14,16,17,20], giving rise to microvessels of mixed origen: human endothelium supported by murine mural cells. In fact, mural cell covering is a requisite of mature vessels, with an important role in EC homeostasis and vessel stability [1]. In all these pioneering works, the histological endpoint was still required. Providing human neovessels with human mural cells

2.2

In a seminal work, Sieminski et al. [22] demonstrated the ability of non-endothelial cells to improve the survival of implanted ECs and their incorporation into host vasculature. When mixed with fibroblasts and suspended in Matrigel, viable bovine aortic endothelial cells (BAECs) were present for at least 6 weeks at a higher density than in implants containing BAECs alone at 3 weeks. To our knowledge, this is the first report of the ability of coimplantated fibroblasts to

promote EC survival and function in vivo [22]. Subsequent studies demonstrated that mesenchymal precursor cells (the murine 10T1/2 cell line) co-implanted with human ECs act as a source of perivascular cells and generate a stable functional vasculature. HUVECs had been transduced for the expression of enhanced green fluorescent protein (EGFP), and engineered vessels were monitored by intravital multiphoton laser-scanning microscopy [23]. Four months after implantation, HUVEC-10T1/2 constructs were still stable and functional. Using immunohistochemistry, the authors demonstrated that these vessels were supported by SMApositive, 10T1/2-derived mural cells. In a previous work, Hirsch et al. had demonstrated that 10T1/2 cells could be incorporated in vivo into host-derived neovessels [24]. A similar strategy was used by Wang et al., with HUVECs being substituted by ECs derived from human embryonic stem (hES) cells and murine 10T1/2 cells also used as a source of mural cells [25]. Ferreira et al. employed hES as a source of both ECs and SMCs and showed that both cell types


69

70


ApoE GH (h)

GH (h) ApoE Bcl-2 Bcl-2 Flag, AP TERT (h) N/A GH (h), LacZ

N/A EGFP Bcl-2 Factor VIII (c)

EGFP N/A N/A Luciferase EGFP

Luciferase-EGFP EGFP N/A Luciferase N/A Diabody CEA-CD3/ Luciferase

MVEC (nd) YSC (m)

YSC (m) YSC (m) HDMEC HUVEC HDMEC HDMEC HUVEC BAEC

EPC (h) HUVEC HUVEC BOEC (c, m)

ESC-EC (h) EPC (h) ESC-EC (h) HDMEC EPC (h)

HUVEC HUVEC EPC (h) HUVEC EPC (h) HUVEC

MSC (h) MSC (h) MSC (h) N/A ASC (h) MSC (h)

10T1/2 (m) SMC (h) ESC-SMC (h) N/A 10T1/2 (m)

N/A 10T1/2 (m) N/A N/A

N/A N/A N/A N/A N/A N/A N/A Fibroblasts (b)

N/A N/A

Mural cell

Matrigel COL I/FN gel Matrigel Matrigel/fibrin gel COL I/FN gel Matrigel

COL I/FN gel Matrigel Matrigel PLLA COL I/FN gel

Matrigel COL I/FN gel COL I/FN gel Matrigel

Matrigel Matrigel PLLA sponges COL I/FN gel PLLA sponges Matrigel Matrigel Matrigel

COL I gel Matrigel

Scaffold

N/A ON N/A 24 h ON N/A

24 h N/A N/A 30 min 24 h

N/A 24 h 20 h N/A

N/A N/A N/A 20 h N/A N/A N/A 48 h

ON N/A

In vitro pre-formation

(m) (m) (m) (m) (m) (m)

(m) (m) (m) (m) (m)

(m) (m) (m) (m)

(m) (m) (m) (m) (m) (m) (m) (m)

Nude SCID Nude SCID SCID Nude

SCID Nude Nude SCID SCID

Nude SCID SCID SCID

C57BL/6 C57BL/6 SCID SCID SCID SCID Rag2-/Nude

(r) (nd) (m) Balb/c

Host

120 130 28 40 14 49

151 7 28 21 119

7 112 60 189

340 112 14 60 21 42 100 42

14 196

Follow-up (days)

ApoE plasma levels GH immunostaining and plasma levels GH plasma levels Cholesterol levels Immunostaining Immunostaining Immunostaining Immunostaining Immunostaining Plasma GH levels, X-gal staining Immunostaining Intravital ML-S microscopy Immunostaining Factor VIII immunostaining and plasma levels Intravital ML-S microscopy Immunostaining Immunostaining BLI, immunostaining Intravital ML-S microscopy, immunostaining BLI, immunostaining Intravital ML-S microscopy Immunostaining BLI, immunostaining Immunostaining BLI, diabody plasma levels

Tracking system

AP: Alkaline phosphatase; ASC: Adipose stromal cells; b: Bovine; BAEC: Bovine aortic endothelial cells; BLI: Bioluminescence imaging; BOEC: Blood outgrowth endothelial cells; c: Canine; COL I: Collagen type I; EC: Endothelial cell; EPC: Endothelial progenitor cell; ESC: Embryonic stem cell; FN: Fibronectin; h: Human; HDMECs: Human dermal microvascular endothelial cells; m: Murine; ML-S: Multiphoton laser scanning; MSC: Mesenchymal stem cells; nd: Not determined; N/A: Not applicable; ON: Overnight; pb: Peripheral blood; PGA: Polyglycolic acid; PLLA: Poly-L-lactic acid; SMC: Smooth muscle cells; YSCs: Yolk sac cells.

Transgene

Endothelial cell

Table 1. In vivo angiogenesis models.


[35] [37] [38] [42] [39] [51]

[25] [32] [26] [36] [33]

[21] [23] [16] [50]

[48] [49] [14] [17] [15] [19] [20] [22]

[46] [47]

Ref.




contribute to the formation of human microvasculature in vivo [26]. Interesting though it is, the use of ES cells can raise ethical concerns and murine mesenchymal cells are not applicable in a clinical setting. Levenberg et al. successfully produced skeletal muscle constructs in vitro by co-culturing myoblasts, fibroblasts and HUVECs in a porous scaffold [27]. The embryonic fibroblasts promoted increases in total area of ECs and the number and size of endothelial lumens compared with those of cultures without fibroblasts. In addition, fibroblast-containing cocultures markedly increased the number of SMA--positive cells around the engineered blood vessels at 4 weeks. This skeletal muscle construct was then successfully transplanted in vivo and the in vitro developed vasculature was maintained in vivo. The in vitro formation of microvessels within an engineered tissue was first described using a skin equivalent obtained by coculture of dermal fibroblasts, keratinocytes and HUVEC on a collagen biopolymer scaffold [28]. In this kind of tissue constructs with preformed vasculature, immature microvessels not only are less stable but also have a limited potential to anastomose to the host vasculature [29]. And rapid vascularization is critical for the survival and long-term maintenance of implanted constructs [30,31]. In this context, Melero-Martıń and colleagues demonstrated the vasculogenic potential of blood-derived EPCs (from human umbilical cord blood or adult peripheral blood) and human smooth muscle cells (SMCs) from saphenous vein as a source of peripheral cells, mixed in Matrigel and s.c. implanted [32]. Microvascular density was significantly superior to that generated by HDMECs but similar to that generated by HUVECs. Unfortunately, implants were evaluated by histological analysis and immunohistochemistry one week later so no data are provided about vessel stability. A related work demonstrated the superior stability in vivo of vessels constituted by umbilical cord derived EPCs and 10T1/2 cells when compared with adult peripheral blood EPCs in the same model [33]. In fact, a previous work had demonstrated in vitro that the proliferative rate and clonogenic potential of cord blood derived EPCs were considerably greater than those of adult peripheral blood EPCs [34]. Towards long-lasting human neovessels using human mesenchymal stem cells

2.3

Taking the best of previous work, a new model of in vivo human angiogenesis was proposed based on the following criteria: i) only human adult primary cells (both endothelial and mural cells) would be used, to avoid ethical concerns and risks implied by immortalized cell lines, and to achieve maximal clinical applicability; ii) the vascular network should be stable and long-lasting; iii) use of a technically simple preimplantation procedure, (no pre-establishment in vitro); iv) use of a minimally invasive method for implantation (cranial windows, for example, are highly invasive procedures that require surgical skills); v) use of a non-invasive, relatively easy-to-perform and affordable method for in vivo EC

tracking (sophisticated strategies such as intravital microscopy are technically demanding and time-consuming). This work by Sanz et al. was the first to explore the potential of bone marrow-derived, human mesenchymal stem cells (MSC) as a source of mural cells and demonstrated that a network of human mature blood vessels can be formed in immunodeficient mice by co-implantation of HUVECs expressing the firefly luciferase gene and MSC. The vessel walls engineered under these conditions were found to be substantially similar to native vessels at both molecular and cellular level. These vascular networks were stable and functional for more than three months in vivo and could be assessed noninvasively and quantitatively by in vivo bioluminescent imaging (BLI) [35]. This study demonstrated the feasibility of technically simple, rapid and non-invasive monitoring of the angiogenic process based on BLI, offering the opportunity to perform serial analysis of the molecular processes underlying angiogenesis and monitoring responses to anti-angiogenic agents in living animals. As a proof of principle, the authors demonstrated that systemic treatment of mice with the antiangiogenic agent SU-5416 decreased photon emission by their vascular implants significantly when compared with untreated animals. The use of BLI for quantification of human angiogenesis had been validated shortly before by Dong et al. [36]. In this work, HDMECs expressing luciferase were implanted in the absence of a source of mural cells, and bioluminescence began to decrease 21 days after implantation. One possible explanation for this decrease could be the failure of HDMECs in the recruitment of perivascular cells that would eventually stabilize the newly formed vessels. Later on, a related paper was published [37] where the authors evaluated human MSCs for use in vascular tissue engineering. They described that MSCs, co-administered with HUVECs in preformed collagen gels and implanted into a cranial window, efficiently stabilized nascent blood vessels in vivo by functioning as perivascular precursor cells, but did not spontaneously differentiate into endothelial tubes. The fate of EGFP-labeled HUVECs was tracked by intravital imaging with multiphoton laser scanning microscopy, with engineered vessels stable and functional for more than 130 days in vivo. Shortly after, Melero-Martin et al. [38] reported the formation of functional microvascular beds in immunodeficient mice by s.c. co-implantation of human EPCs and MSCs isolated from blood and bone marrow, respectively, and embedded in Matrigel. In previous works by the same group, the role of perivascular cells was assumed by SMC from saphenous vein, not a clinically relevant source due to risks associated with obtaining cells from this source. EPCs were infected with a lentiviral vector encoding luciferase and evaluated by BLI. The engineered vascular networks remained stable and functional for up to 4 weeks in vivo. Exploring a new and more accessible source of perivascular cells, Traktuev et al. used MSC obtained from adipose tissue (ASC) in neovessel engineering [39]. They had previously


71



demonstrated that ASC possessed pericyte-like properties and were able to stabilize newly formed vessels in vitro [40]. In mice, subcutaneous collagen/fibronectin implants containing EPC from umbilical cord and ASC gave rise to a functional vasculature, as assessed by immunostaining of implants retrieved at day 14. Interestingly, coimplantation of EPCs, ASCs and parenchymal cells produced ‘neoorgans’ vascularized by human vessels. In vitro, ASCs exhibited significantly higher potential to stimulate vascular network formation than smooth muscle cells and fibroblasts [41]. A technically more challenging approach is the implantation of ECs aggregated in vitro as ‘spheroids’, embedded in a Matrigel/fibrin matrix [42]. Spheroid cell culture systems had previously proven useful for the in vitro analysis of nontransformed cells, such as primary ECs [43]. Luciferaseexpressing HUVEC spheroids implanted in immunodeficient mice formed a vascular network constituted of human ECs and recruited host-derived mural cells, that was stable for at least 40 days as assessed by BLI. According to the authors, spheroidal delivery of ECs facilitates the generation of 3D vascular networks. 3.

Factory neovessels

If ECs can be genetically modified to express a reporter or an anti-apoptotic gene, they can also be transduced to produce a therapeutic protein, in a cell-based gene therapy approach (Figure 2). One obvious advantage of this approach is that such neovessels could provide direct secretion of protein into the bloodstream, given their capacity to anastomose with the host vascular bed. In fact, the possible use of genetically modified EC for gene therapy strategies, taking advantage of their strategic bloodstream location, was envisioned 20 years ago [44,45], long before the quest for an in vivo human angiogenesis model. In a seminal work, Zwiebel et al. used retroviral vectors to transduce rabbit aortic ECs, and demonstrated the expression in vitro of rat growth hormone (GH) for at least four weeks [45]. Squinto et al. implanted s.c. plugs with ApoE-producing microvascular ECs (MVECs) in rats and reported detectable ApoE serum levels for at least two weeks [46]. As a an alternative cellular source, Wei et al. used murine yolk sac cells (YSCs), that could differentiate into ECs within Matrigel implants in vivo [47,48]. YSC transfected to express human GH (hGH), suspended in Matrigel and s.c. injected into syngenic mice, supported physiologically significant levels of GH in their serum for beyond four months [47]. The same strategy was successfully applied to immunosupressed allogeneic mice, with hGH levels detectable for over 340 days [48]. In a related study, an EC line derived from yolk sac was stably transfected to express human Apo E. These ECs were evaluated for their potential use in gene therapy as a gene delivery platform in the murine apoE knockout atherosclerosis model. After transplantation of the apoE secreting ECs into apoE-deficient mice, levels of cholesterol in the animals were significantly lower compared 72

with controls having received non-secreting ECs for at least 3 months. Concomitant with cholesterol reduction, atherosclerotic aortic plaques were noticeably reduced in the treated animals [49]. In the work by Sieminski et al. [22], s.c. implantation of Matrigel-embedded BAECs secreting hGH in mice initially resulted in detectable plasma hGH levels, which declined after 2 weeks. When mixed with fibroblasts, implantation of hGH-secreting BAECs resulted in plasma hGH levels that decreased slightly over 2 weeks and then remained stable for at least 6 weeks, reinforcing the role of supporting non-endothelial cells in promoting EC survival and transgene expression. In another EC-based gene therapy strategy for hemophilia A, blood outgrowth endothelial cells (BOECs) were isolated from canine and murine blood and transduced with a lentiviral vector encoding the canine factor VIII (FVIII) transgene. To enhance safety, these cells were implanted s.c. in a Matrigel scaffold. Therapeutic levels of FVIII were extended when the cytomegalovirus promoter was replaced with the thrombomodulin regulatory element. Studies in immunocompetent hemophilic mice, pretreated with tolerizing doses of FVIII or with transient immunosuppression to avoid the formation of neutralizing anti-FVIII antibodies, showed therapeutic FVIII expression for 27 weeks before the eventual return to baseline levels [50]. Taking advantage of the previously developed model of human angiogenesis in vivo, Compte et al. explored the potential of ‘factory neovessels’ after gene-modifying the ECs for the secretion of a therapeutic protein, based on the permissiveness of HUVEC to be transduced by lentiviral vectors and the safety of their use. Given that these neovessels connect to the host vascular bed, the therapeutic protein would be directly released into the bloodstream and exert a systemic effect. As a proof of principle, HUVEC transduced with a lentiviral vector were used for the expression of a recombinant bispecific anti-carcinoebryonic antigen (CEA) anti-CD3 antibody. These ECs, along with MSC as a source of mural cells, were embedded in Matrigel and s.c. implanted in nude mice. High antibody levels were detected in plasma for one month. Furthermore, the antibody exerted a therapeutic effect in mice bearing distant CEApositive tumors after inoculation of human T cells [51]. Interestingly, plasma concentration of diabody in mice bearing factory neovessels dropped less than 30% with respect to the peak of expression. In contrast, plasma levels of the recombinant antibody in mice that received implants with the same number of MSC transduced to express the diabody in the same period dropped more than 80% with respect to the peak of expression [52]. MSCs are a popular vehicle for cell-based gene therapy, given that they are easy to isolate and transduce and exhibit high proliferation rates [53-55]. However, MSCs administered s.c in ‘factory plugs’ do not organize or differentiate into recognizable structures. Perhaps the difference in diabody level maintenance may be



Gene-modified EC

MSC Bioscaffold

Endogenous vessel

Y

Mural cell

Y

Y

Y

Y Therapeutic protein

Y

Bioactive factors BM Factory neovessel components

Y Y

Y

MSC-derived mural cell

Y

Y

Y Gene-modified EC

Cell expansion

Factory neovessel

Viral transduction Time

Ex vivo manipulation

Ex vivo manipulation


Y Y

Therapeutic protein

Endothelial cells (EC)

Mesenchymal stem cells (MSC)

Figure 2. Schematic diagram of ‘factory’ neovessel generation. Autologous endothelial cells ex vivo transduced for the expression of a therapeutic gene, mixed with autologous MSC and embedded in a biocompatible scaffold form neovessels in vivo, that connect with the patient’s vascular bed and allow the release into the bloodstream of the therapeutic protein. BM: Basement membrane.

attributable to problems with systemic release of the expressed protein in plugs constituted exclusively by MSC. 4.

Expert opinion

EC-based in vivo therapeutic protein production shares challenges common to any cell-based gene therapy strategy, such as obtaining optimal therapeutic levels (dependant on number of cells inoculated and their level of expression) and duration of transgene expression over time (dependant on the life-span of producer cells, potential immune responses against the cell or the gene product, or transcriptional repression in vivo). It is obvious that the use of autologous cells would reduce the risk of an immune

response against the vehicle. The challenge is to find clinically viable sources of adequate numbers of both EC and mural cells, preferably obtained by non-invasive methods that could be expanded in vitro to yield the large number of cells required for therapeutic applications. HUVECs have been widely used in this context, but the possibility of obtaining autologous cells would be very limited. HDMECs constitute an option but they are difficult to obtain in sufficient quantities from a small skin biopsy. As an alternative to adult differentiated ECs, the use of embryonic and adult stem and progenitor cells has been explored. ECs derived from embryonic stem cells (ESCs) have been used to form blood vessels in some in vivo models [25,26]. However, ethical issues surrounding their use must be


73



resolved prior to their implementation in therapeutic strategies. The recent generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of three transcription factors provides a means of circumventing this problem. To date, iPS cells appear to be indistinguishable from ES cells. Recently, murine iPS cells from tail-tip fibroblasts were differentiated to both EC and EPC expressing major EC markers and able to secrete FVIII in vivo [56]. Despite the therapeutic potential of reprogrammed somatic cells, major technical challenges must be overcome, especially those concerning viral transduction and risk of malignant transformation. The identification of EPCs in adult bone marrow and peripheral blood offered an unprecedented opportunity to non-invasively obtain ECs [57]. Bone-marrow derived EPCs form tubes on Matrigel and migrate in vitro similarly to HUVECs and HMVECs [21], and have a high proliferation rate when compared to mature ECs [58]. Peripheral-bloodderived EPCs have also the capacity to form functional vascular networks in vivo [32], and obtaining them is minimally invasive. Umbilical cord blood is a rich source of EPCs, with some added advantages: the yield is considerable higher with respect to the same volume of peripheral blood and their capacity of proliferation is greater [59], but their therapeutic administration would be essentially allogenic. In the future, cryopreserved autologous cord blood cells could be used as a source of EPCs [60]. Currently, we believe that adult peripheral blood is the most feasible source of autologus EPCs, that in turn will most probably be the first choice as the source of therapeutic ECs. We and other authors have shown that in order to obtain stable, long-lasting vascular networks, EC require coimplantation with mural cells. In the previous work [23,25,33], the mouse embryonic cell line 10T1/2 served as the perivascular component of the vascular networks. Interesting though as a proof of principle, the use of murine cells has no applicability in a clinical setting. On the other hand, isolation of adult mural cells, as SMCs from human saphenous veins, would imply serious morbidity in patients [38]. Fibroblasts are also an option [22], easier to isolate in principle, but as terminally differentiated cells they possess a short lifespan, and this implies an obvious limitation to their application in gene therapy strategies. For these reasons, we believe in the potential of MSC as a source of mural cells: they have a great expansion capacity and they are easy to transduce and ready available from a diversity of human tissues. We have used bone-marrow-derived MSCs in our model of human angiogenesis in vivo, but recently MSCs from adipose tissue have been used successfully in the same context as a source of pericytes [39].

74

Another relevant issue is the nature of the scaffold in which ECs and mural cells are embedded. Matrigel has been widely used, but its murine origin may be not compatible with clinical applications. The FDA approved, collagen-based matrix Contigen is bovine. Not only the nature, but also the concentration of specific components within the scaffold modulate in vivo blood vessel formation. Recently, it has been reported that increasing collagen concentration significantly decreased EPC-derived vessel density, but increased vessel size [61]. As an alternative to Matrigel and collagen we have tested different hydrogel formulations, commercially available, that offer several advantages: they are synthetic, of non-animal origin, potentially less immunogenic, and their composition is the same batch to batch. Sophisticated hydrogel formulations specifically designed to promote angiogenesis include integrin binding sites, growth factors and proteasesensitive substrates, in an attempt to mimic the natural ECM matrix [62]. New generations of synthetic biomaterials containing the necessary signals to recapitulate capillary morphogenesis are being developed. In summary, a clinical scenario in which human neovessels are incorporated in therapeutic strategies, either as cell-based gene delivery vehicles to produce a therapeutic protein or as a component of engineered tissue constructs in regenerative medicine, can be envisaged. Initial clinical attempts at cell-based gene therapy were hampered by the poor posttransplant survival of ex vivo modified cells and the suboptimal plasma levels of therapeutic protein obtained. However, recent years have witnessed remarkable progress in developing new cell vehicles (such as ECs) that are efficiently transduced for the production of higher levels of the therapeutic protein and exhibit prolonged lifespans after being seeded in scaffolds that support long-term engraftment.

Acknowledgements This study was supported by grants from the Ministerio de Ciencia e Innovacioń (BIO2008-03233 and PSE-010002009-11), the Comunidad de Madrid (S-BIO-0236-2006), and the European Union (SUDOE-FEDER. IMMUNONETSOE1/P1/E014) to L A´lvarez-Vallina; and from the Fondo de Investigacioń Sanitaria/Instituto de Salud Carlos III (PI08/90856 and PS09/00227) and Fundacioń Investigacioń Biome´dica Hospital Puerta de Hierro to L Sanz.

Declaration of interest The author’s declare no conflicts of interest. Support is from non-profit public agencies, with no connection to industry.




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Affiliation

Luı´s A´lvarez-Vallina & Laura Sanz† † Author for correspondence Molecular Immunology Unit, Hospital Universitario Puerta de Hierro, Joaquıń Rodrigo 2, 28222 Majadahonda, Madrid, Spain Tel: +34 91917764; Fax: +34 913160644; E-mail: [email protected]

Review

1.

Introduction

2.

Methods for detecting


and/or measuring AECA 3.

Prevalence and isotypes of AECA in vasculitis

4.

Pathological roles of AECA

5.

Reported targets of AECA

6.

What is the role of AECA in systemic vasculitis, cause or consequence?

7.

Our research on targets of AECA by proteomics

8.

Expert opinion

Anti-endothelial cell antibodies (AECA) in patients with systemic vasculitis: our research using proteomics Rie Karasawa†, Kazuo Yudoh, Shoichi Ozaki & Tomohiro Kato †

Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Japan

Importance of the field: Anti-endothelial cell antibodies (AECA) may cause damage to endothelial cell (EC) functions and therefore may be of a pathophysiological role in systemic vasculitis. The pathophysiological role of AECA, however, is still uncertain. Areas covered in this review: To clarify the detailed roles of AECA, various methods for identification of target proteins of AECA have been developed, such as expression libraries and proteomic approaches combining two-dimensional electrophoresis and immunoblotting. What the reader will gain: Advances, including our research, have been made in defining the target antigens of AECA, which we summarize in this review. Furthermore, we discuss the possible significance of AECA in the pathophysiology of vascular damage and the value of AECA in systemic vasculitis. Take home message: To identify target antigens of AECA and to establish a standardized method for measuring AECA would be helpful in the search for a possible pathophysiological role of AECA in systemic vasculitis. Keywords: anti-endothelial cell antibodies (AECA), proteomics, systemic vasculitis, target antigens Expert Opin. Biol. Ther. (2011) 11(1):77-87

1.

Introduction

Vasculitis is pathologically defined as inflammation of the blood vessel walls. The causes and mechanisms of the blood vessel injury in vasculitis are not fully understood. The pathological factors proposed to date include autoantibodies, immune complexes and pathogenic T cell responses [1,2]. The autoantibodies specifically produced in patients with vasculitis may cause vascular inflammation directly or through the formation of immune complexes [3]. The representative autoantibodies in vasculitis are anti-neutrophil cytoplasmic antibodies (ANCA) and antiendothelial cell antibodies (AECA) [2]. Recently, ANCA testing has played a critical role in the pathogenesis, diagnosis and classification of vasculitides, including Wegener’s granulomatosis (WG), microscopic polyangiitis (MPA) and allergic granulomatous angiitis (AGA). Two major autoantigens for ANCA have been identified as proteinase 3 (PR3) and myeloperoxidase (MPO) [4,5]. PR3-ANCA are specifically detected in WG, and therefore used as a disease marker for WG, whereas MPO-ANCA are frequently detected in MPA, AGA and some other autoimmune diseases [1]. Terrier et al. [6] recently suggested that anti-MPO antibodies are a useful marker of disease activity and a good predictor of relapse in anti-MPO-associated vasculitis [6]. It has been suggested that ANCA trigger the degranulation, activation and apoptosis of neutrophils, which then cause endothelial cell (EC) damage [4]. AECA are antibodies that recognize a variety of antigens on the surface of ECs. In contrast to ANCA, AECA are widely detected in various types of systemic vasculitis, 10.1517/14712598.2011.540234 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

77

AECA in patients with systemic vasculitis


. .

.


. .

.

The prevalence of anti-endothelial cell antibodies (AECA) differ depending on the Endothelial cell (EC) types. Therefore, it is necessary to use EC lines based on consideration of the injured vessel sizes and affected organs. It would be desirable to combine at least two different methods for AECA detection. The prevalence of AECA was highly influenced by the coexistence of vasculitis in patients with rheumatoid arthritis. Antibodies to heat shock protein 60 (hsp60) would have pathogenic roles in vasculitis-associated systemic autoimmune diseases via EC apoptosis. This experimental animal model of vasculitis provides the first direct proof for the pathogenicity of AECA. Anti-peroxiredoxin 2 autoantibodies would be a useful marker for systemic vasculitis and would be involved in the inflammatory processes of systemic vasculitis. The possible implications of the redox state in the onset and progression of autoimmune diseases in a gender perspective is discussed.


including WG [7], MPA [8] and Kawasaki disease (KD) [9]. In some diseases, it has been reported that the presence and titer of AECA are correlated with the activity of systemic vasculitis [7-10]. For example, it was reported that AECA were only detected in the active phase of KD [9] and that the titers of AECA decreased after treatment in WG [7] and MPA [8]. In addition, AECA were reported to be associated with particular clinical manifestations, including vascular lesions in systemic lupus erythematosus (SLE) [11,12] and acute thrombotic events in Behçet’s disease (BD) [13,14]. These observations suggest an important role for AECA in the pathophysiology of vasculitis, including EC damage and vascular injury. However, compared with ANCA, the clinical and pathological significance of AECA in vasculitis is not fully understood. One of the reasons for this situation is that the target antigens for AECA have been poorly identified. In this article, we describe the recent findings regarding AECA, including antibodies against peroxiredoxin 2 (Prx2), which we identified as an AECA target using a proteomics technique.

Methods for detecting and/or measuring AECA 2.

AECA were first described in 1971 by Lindqvist and Osterland [15] and in 1972 by Tan and Pearson [16]. They used frozen mouse kidney sections as a substrate to detect AECA with a standard indirect immunofluorescence (IIF) technique. AECA are detectable using several methods, including IIF, FACS [17,18], radioimmunoassay [19,20], western blotting (WB) [18,21], immunoprecipitation [22] and ELISA [17,18,23]. 78

Each of these techniques has merits and demerits regarding sensitivity, specificity or operation. Therefore, use of the different techniques can provide somewhat different results. FACS of unfixed ECs was used to detect AECAs. However, the expression of some membrane proteins is modified in EC suspensions because of the lack of matrix. In addition, the surface antigen on ECs is also predicted to be altered by use of trypsin to detach ECs from the culture plates or flasks. WB is the method normally used to detect AECAs due to its high sensitivity. In WB, the pattern of reactivity can be well defined by densitometric analysis. Thus, it is difficult to analyze when the specific bands are weak against the background staining. It may be also difficult to set optimal conditions for measurement and to show good reproducibility. In addition, only antibodies to linear epitopes can be identified by the use of reducing agent, including dithiothreitol. Now, the technique used most widely is a cell-based ELISA system with a substrate of HUVECs. In this system, the antibodies that bind to HUVECs are detected non-selectively. Although cell-based ELISA is a useful method for detecting AECA, it has at least three problems to be overcome. First, when ECs fixed with glutaraldehyde or paraformaldehyde are used, antibodies against not only surface antigens of ECs but also cytoplasmic antigens would be detected because the fixation induces hyperpermeability of the EC membranes. The differences between assays in which fixed and unfixed ECs are used for the detection of AECA remain to be elucidated. For example, Westphal et al. [17] reported that AECA were not specifically detected by an ELISA using fixed ECs. On the other hand, Wangel et al. [24] reported that both assays gave similar results. Second, HUVECs cannot represent the AECA antigens of all EC types produced in humans. In addition, the prevalences of AECA differ depending on the EC types used for the measurements. Holmen et al. [7] reported that a high proportion of WG patients had AECA that bound to human nasal ECs (61%) and human kidney ECs (71%) compared with HUVECs (7%) and human liver sinusoidal ECs (0%) [7]. Third, even within one type of cells, HUVECs, the presented antigen profiles will differ according to the origin, passage numbers and culture conditions. Therefore, cell-based ELISAs using HUVECs sometimes provide controversial results. Other methods have similar problems regarding the profile of expressed antigens when a certain cell type as a whole is used for antigens. In view of the great variability in the antigen patterns of ECs from one umbilical cord to another, it would be desirable to use EC lines based on consideration of the injured vessel sizes and affected organs. Thus, it is recommended to combine at least two different methods for AECA detection to obtain data with high precision.

Prevalence and isotypes of AECA in vasculitis

3.

Table 1 shows the prevalences of AECA in different

diseases

[17,23,25-30].


AECA have been detected in a wide

Karasawa, Yudoh, Ozaki & Kato

Table 1. Prevalence of AECA in systemic diseases.


Diseases Primary vasculitis Takayasu arteritis Wegener’s granulomatosis Microscopic polyangiitis Churg-Strauss syndrome Polyarteritis nodosa Kawasaki disease IgG IgM Secondary vasculitis Polymyositis Systemic sclerosis Rheumatoid arthritis Behçet’s disease Systemic lupus erythematosus Mixed connective tissue disease Relatives of vasculitis Anti-phospholipid syndrome Thrombotic thrombocytopenic purpura Hemolytic uremic syndrome

Positive rate (%)

Ref.

89 -- 95 8 -- 100

[25,26] [27]

2 -- 60

[25]

69

[25]

56

[25] [28,29]

5 -- 26 42 -- 73 44 15 -- 84 0 -- 87 18 -- 80 15 -- 85

[25] [23,25,30] [25] [25] [25]

45 -- 58

[17,25]

60 -- 63

[25]

13 -- 100

[25]

93

[25]

variety of diseases, such as autoimmune diseases, systemic inflammatory diseases and infectious diseases, including systemic vasculitides, but they can also be observed in healthy individuals. For example, AECA were reported to be detected in 100% of patients with autoimmune hypoparathyroidism, 76% of female patients with hyperprolactinemia and 23 -- 75% of patients with multiple sclerosis [31]. According to the type of ECs used for detection, the prevalence of AECA ranges from 0 to 71% of WG patients [7]. Furthermore, in a comparison of the results obtained using different methods, the prevalences of AECA in WG patients were 17, 13 and 57% by cell-based ELISA, FACS and WB, respectively [18]. In addition, even when the same method was used for the detection of AECA, cell-based ELISA using cultured HUVECs, the prevalence of AECA in patients with systemic sclerosis (SSc) ranged from 22 to 71% in different studies [32]. AECA represent a heterogenous group of antibodies directed against a great variety of EC surface proteins. The EC antigens can be either constitutively expressed, or constitutively expressed and modulated by cytokines such as IL-1 and TNF-a, and physical effects such as shear stress. It was reported that AECA titers in SLE sera are decreased by extensive washes of radiolabeled preparations with high-molarity buffers. The evidence indicates that some of these autoantibodies are directed to non-constitutive proteins [33]. The demonstration has been also

supported by the description of monoclonal and polyclonal anti-DNA antibodies binding to ECs through DNA and histones attached to the cell membrane [34]. Furthermore, another possibility is the binding of various components to the EC membrane. For example, proteinase 3 (PR-3) is a key target protein of ANCA in patients with WG. On the other hand, it is reported that PR-3 is detected on the surface of EC. Mayet et al. reported that PR-3 is an EC target antigen of pseudo-AECAs in WG and that anti-PR-3 antibodies represent an important missing link in ANCA--endothelial interactions [35]. Equally, there are ‘planted’ antigens such as myeloperoxidase (MPO) and b2 glycoprotein I (b2-GPI). MPO may bind to EC released by activated neutrophils and/or monocytes. Del Papa et al. reported that b2-GPI binds to ECs through a cluster of lysine residues, that is involved in binding to anionic phospholipids [36]. In detecting and/or measuring AECA, these results must be interpreted with caution. In secondary autoimmune vasculitis, it was reported that the prevalence of AECA was highly influenced by the coexistence of vasculitis. By cell-based ELISA, AECA activity was detected in 68% of patients with both rheumatoid arthritis (RA) and vasculitis, but only 16% of patients with RA alone. Likewise, by radioimmunoassay, AECA activity was detected in 75% of patients with both RA and vasculitis, but only 13% of patients with RA alone. Therefore, AECA may represent a useful tool for the diagnosis of vasculitis in patients with RA [20]. Furthermore, SLE patients with AECA were found to have an increased prevalence of vascular lesions, including arterial and venous thromboses and leukocytoclastic vasculitis [11]. IgG, IgM and IgA isotypes of AECA have been reported. Each isotype may have its own unique role. IgM AECA were detected in 42 -- 73% of KD patients, while the reactivity for IgG AECA was reported to be 5 -- 26% [28,29]. It was reported that IgM AECA mediated complement-dependent cytotoxicity (CDC) against ECs in KD patients [29]. Yang et al. [37] reported that IgA AECA were detected in 45% of patients with acute Henoch-Sch€onlein purpura, while IgG and IgM AECA were not detected. In addition, the values for IgA AECA were reported to be correlated with the disease activity [37]. IgM and IgG AECA were detected in 83.0 and 68.1% of patients with Takayasu’s arteritis (TA), respectively, whereas both isotypes were detected in 3.3% of healthy individuals [26]. Furthermore, IgM and IgG AECA were respectively detected in 92.3 and 69.2% of patients in the active disease group and 66.7 and 61.9% of patients in the stable disease group. In addition, the TA patients with paired blood samples (active and stable stages) showed similar findings. In conclusion, these findings showed that the IgM AECA titers were well correlated with disease activity in TA patients. 4.

Pathological roles of AECA

AECA are considered to play pathological roles in vasculitis. The proposed mechanisms are described below and shown schematically in Figure 1. The first is EC cytotoxicity, in which binding of AECA to ECs mediates antibody-dependent


79


Activation of complement

VCAM-1 E-selectin ICAM-1 etc., IL-1, IL-6, IL-8 MCP-1 etc.,

Complement Upregulate the expression of adhesion molecules

Induction of inflammatory cytokines

Membrane attack complex Complement-dependent cytotoxicity (CDC) Fc receptor

AECA

Damage


Induction of apoptosis

Effector cell

Endothelial cells

Induction of coagulation

Antibody-dependent cell cytotoxicity (ADCC)

Figure 1. The schema for endothelial damage by AECA. AECA: Anti-endothelial cell antibody; ICAM: Intracellular cell adhesion molecule; MCP: Monocyte chemotactic protein; VCAM: Vascular cell adhesion molecule.

cytotoxicity (ADCC) or CDC. The second is apoptosis of ECs triggered by AECA. The third is activation of ECs triggered by AECA, including upregulation of the expressions of adhesion molecules such as E-selectin, intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) on ECs and the secretion of cytokines and chemokines from ECs, which in turn cause leukocyte recruitment and adhesion to ECs. The fourth is the induction of coagulation, since AECA can exhibit procoagulation properties. Fujieda et al. [29] demonstrated that IgM AECA mediate CDC against HUVECs in KD patients, and that gammaglobulin may reduce the CDC of AECA against HUVECs. Furthermore, the cytotoxicity was significantly enhanced by pretreating HUVECs with TNF-a. In addition, the IgM AECA serum titers were positively correlated with cytotoxicity in KD patients [29]. Tripathy et al. [38] suggested that AECA in TA patients mediate CDC against HUVECs. In addition, treatment of HUVECs with IL-1b and TNF-a results in enhanced CDC of AECA-positive cytotoxic sera in TA patients [38]. Marks et al. [39] found that sera from SSc patients mediated ADCC against ECs, including microvascular ECs, and the aortic endothelium. Recently, it was reported that Hsp60 is an important target for AECA in patients with SLE, periarteritis nodosa (PAN), WG, MPA, Churg--Strauss syndrome and BD [40]. Schett et al. [41] found that anti-Hsp60 antibodies were able to lyse ECs in vitro via either CDC or ADCC. Furthermore, Jamin et al. [40] demonstrated that binding of antiHsp60 antibodies to Hsp60 on ECs stressed by exposure to heat did indeed induce apoptosis. In addition, ECs displayed a dose-dependent apoptotic response when they were incubated with various concentrations of an anti-Hsp60 monoclonal antibody. Furthermore, apoptosis of ECs was triggered by anti-Hsp60-containing AECA-positive sera and inhibited by 80

preincubation of the ECs with recombinant Hsp60. Bordron et al. [42] found that apoptosis-inducing AECA appeared to be present in patients with SSc, giant cell arteritis, PAN, TA, WG and SLE, especially in SSc patients. In addition, apoptosis-inducing AECA did not recognize the Fas (CD95) receptor [42]. On the other hand, Sgonc et al. [43] reported that EC apoptosis in SSc was induced by ADCC via the Fas pathway. Apoptosis induction was observed in human dermal microvascular ECs but not in HUVECs. Furthermore, immunofluorescence analyses of cryosections from SSc skin samples revealed Fas expression by ECs [43]. On the other hand, Williams et al. demonstrated that AECA from patients with active SLE did not induce apoptosis compared with normal IgG [44]. Though, they also showed that there was increased binding of AECA to apoptotic ECs. Upregulation of IL-8 and MCP-1 has been reported in BD, KD and WG [45-48]. The upregulation of IL-8 and MCP-1 suggests enhanced migration of neutrophils and monocytes. IL-8 is also a potent chemoattractant that may recruit and activate polymorphonuclear cells to induce further EC damage [49]. Previous studies in TA patients revealed AECA-induced production of IL-6 and IL-8 by HUVECs and human aortic ECs [50,51]. IL-6 is known to upregulate the production of fibrinogens and to stimulate immature megakaryocytes to produce platelets [52]. The serum IL-6 levels are positively correlated with the disease activity in TA patients [53]. Growth-related oncogene alpha (GROa) could be involved in atherogenesis and plaque destabilization [54] and collaboration between GM-CSF and plateletactivating factor synthesized by activated ECs strikingly potentiated neutrophil activation [55]. Monoclonal AECA from a TA patient induced the expressions of adhesion molecules such as VCAM-1, ICAM-1 and E-selectin in ECs and increased the adhesion of U937 monocytic cells to ECs [50].




Carvalho et al. [56] demonstrated that IgG from AECApositive SLE patients and AECA-positive systemic vasculitis patients upregulated adhesion molecule expressions and leukocyte adhesion to ECs. Pretreatment of HUVECs with AECA-positive sera from scleroderma patients or IgG purified from these sera led to a dose- and time-dependent increase in the ability of the cells to bind to human U937 monocytic cells through increased expressions of ICAM-1, VCAM-1 and E-selectin. In addition, IL-1 generated by HUVECs in response to AECA was involved in the upregulation of adhesion molecules and U937 cell adhesion [57]. ECs play roles in the initiation and regulation of both coagulation and fibrinolysis. The EC surface changes from an antithrombotic surface to a thrombotic surface under the influence of a wide variety of cytokines and factors. Derangement of coagulation and fibrinolysis was reported to be mediated by IL-6 and IL-8 [58]. The procoagulant activity of ECs was increased after incubation with recombinant IL-1b [59]. IL-1 is mainly expressed within the endothelium of atherosclerotic plaques and increased expression of GROa was confirmed within carotid plaques located with ECs [53]. Tannenbaum et al. [60] demonstrated that HUVECs produced a procoagulant tissue factor after exposure to AECA from SLE patients. To date, pathological roles of AECA supported by all studies are unknown; however, recently it was shown that antibodies to some target proteins of AECA induced apoptosis. Given that there are many reports in patients with vasculitis, apoptosis may be the most pathogenic effect of the antibodies. In addition, these experimental demonstrations may indicate that pathological roles of AECA vary among the individual target protein of AECA. 5.

Reported targets of AECA

As described above, the reported prevalences of AECA differ among studies. The main reasons for this situation are the different detection and measurement methods. Identification of autoantigens for AECA and establishment of antigen-specific measurement systems are needed to determine the pathological and diagnostic roles of AECA. To date, considerable efforts have been made for the identification of AECA autoantigens. As shown in Table 2, more than 15 candidate autoantigens for AECA have been reported [26,31,40,61-65], including Hsp60 [26,40], tropomyosin [63] and DNA topoisomerase I [64].

What is the role of AECA in systemic vasculitis, cause or consequence?

6.

AECA have been detected in not only patients with autoimmune, inflammatory and infectious diseases, but also in healthy individuals. Therefore, the presence of AECA is most likely to be a marker for endothelial damage, rather than to be involved in the pathoetiology of systemic vasculitis.

It is also reported that presence of AECA have been shown to be clinical signs of vasculitis in the patients with RA [20] and SLE [11,12]. Furthermore, it is relevant to note that titers of AECA has been correlated with disease activity in KD [9], WG [7] and MPA [8]. However, that is not to say that this is specific to almost all cases with systemic vasculitis. In our data, anti-Prx2 antibodies changed from negative to positive after disease onset in serum samples from a patient with TA. In addition, the anti-Prx2 antibodies changed from positive to negative after therapy [66]. These results suggested that the appearance of anti-Prx2 antibodies may be a consequence, rather than a cause, of the vasculitis damage in patients with TA. There is at least one observation to support the probability that AECA can trigger the pathogenesis of vasculitis. Damianovich et al. reported that idiotypic manipulation of naı¨ve mice with human AECA can induce the production of specific mouse AECA leading to vasculitis-like lesions in mouse lungs and kidneys [67]. In addition to lymphoid cell infiltration, the most prominent findings in mouse kidneys were deposition of Igs at the outer part of the blood vessel walls. It remains possible that the AECA bound to ECs induced a series of cellular modifications that were essential for lymphocyte activation and for induction and/or maintenance of the inflammatory process in vasculitis. However, it remains unclear what the appearance of AECA means, cause or consequence, in patients with systemic vasculitis.

Our research on targets of AECA by proteomics

7.

Recently, the techniques in proteomics studies have rapidly progressed, thereby facilitating the screening and identification of autoantigens by 2-dimensional electrophoresis (2-DE) and WB followed by mass spectrometry. To detect EC autoantigens for AECA, we separated proteins extracted from HUVECs and HeLa cells by 2-DE, transferred them onto membranes and analyzed them by WB using serum samples from patients with systemic vasculitis [66]. We selected autoantigenic spots that were detected in the HUVEC sample but not in the HeLa cell sample, which should be candidates for HUVEC autoantigens. Next, we identified the detected proteins by peptide mass fingerprinting and characterized their antigenicities by preparing recombinant autoantigens and raising antibodies against them. Finally, we compared the clinical parameters between the autoantibody-positive and -negative patients to assess their clinical importance (Figure 2). We detected approximately 150 candidate autoantigenic spots by the differential 2-DE and WB for AECA and have identified more than 50 protein spots so far. One of the identified proteins was Prx2, an antioxidative enzyme. Oxygen free radicals produced in the body cause various types of oxidative damage to cells and tissues. One of several antioxidative systems in the body is the redox regulatory mechanism involving Prx proteins (Figure 3).


81



Table 2. The target antigens of AECA in systemic diseases. Disease

AECA target antigens

Ref.

Systemic lupus erythematosus

Ribosomal P protein PO Elongation factor 1a Ribosomal protein L6 Adenyl cyclase-associated protein Profilin II Plasminogen activator inhibitor Fibronectin DNA or DNA-histone Heparin sulfate Hsp70 b-glycoprotein I

[31] [31] [31] [31]

Systemic lupus erythematosus Antiphospholipid syndrome Behçet’s disease Systemic lupus erythematosus Wegener’s granulomatosis Behçet’s disease Microscopic polyangiitis Periarteritis nodosa Takayasu’s arteritis Thrombotic thrombocytopenic purpura Kawasaki disease Diffuse cutaneous systemic sclerosis Limited cutaneous systemic sclerosis

[31] [31]

Glycoprotein CD36

[31] [31] [31] [61] [31] [31] [62] [40] [40] [40] [40] [40] [26] [31]

Tropomyosin T-plastin DNA topoisomerase I

[63] [63] [64]

CENP-B

[65]

a-enolase Hsp60

significantly increased the secretion of various inflammatory cytokines, including IL-6, IL-1b, IL-1ra, GROa, G-CSF, GM-CSF, IL-8 and monocyte chemotactic protein 1 (MCP-1). In particular, the IL-6 levels in anti-Prx2 antibody-stimulated culture supernatants were found to be more than six times higher than those in control IgG-stimulated culture supernatants. Based on the observations described above, ECs appear to be activated by binding of anti-Prx2 antibodies to Prx2 on their cell surface, thereby resulting in the productions of inflammatory cytokines and chemokines by ECs. In addition, intracellular Prx2 may be inactivated by excess oxidative stress, resulting in a promotional effect on endothelial damage [68]. Recently, it was reported that treatment with tocilizumab, a humanized anti-human IL-6 receptor (IL-6R) monoclonal antibody, improved the inflammatory manifestations and laboratory findings of a patient with TA [69]. In addition, the serum IL-6 levels were reported to be greatly elevated in patients with TA and positively correlated with the disease activity [48]. Interestingly, in our data, the autoantibodies against Prx2 tended to be rather specifically detected in TA patients and the disease activity was correlated with the anti-Prx2 antibody titers in all the TA patients examined. Furthermore, IL-6 was remarkably induced in HUVECs by the antibodies against Prx2. These findings indicate that tocilizumab, an IL-6R inhibitor, may be an effective therapeutic option for anti-Prx2-antibody-positive patients with TA. 8.

Expert opinion

Approachfor detecting the target antigens of AECA

In our data obtained by proteomics analyses, antibodies against Prx2, a target autoantigen of AECA, were detected in 60% of patients with systemic vasculitis, compared with only 7.3% of patients with collagen diseases with obvious vasculitis and 0% of healthy individuals. Furthermore, the frequency of anti-Px2 antibodies tended to be higher in largevessel vasculitis such as TA than in small-vessel vasculitis such as ANCA-associated vasculitis. Consequently, anti-Prx2 antibodies may be a good diagnostic marker for systemic vasculitis, especially large-vessel vasculitis, and may be a useful marker for early diagnosis of secondary vasculitis. In immunocytochemistry, antibodies against Prx2 were found to bind to the cell surface of unfixed HUVECs. Therefore, antiPrx2 antibodies would be able to access Prx2 on vascular ECs. Clinically, the D-dimer and thrombin--antithrombin complex titers were found to be significantly higher in antiPrx2 antibody-positive patients with vasculitis than in antiPrx2 antibody-negative patients with vasculitis. It is suggested that the anti-Prx2 antibodies would upregulate the coagulation activity in patients with systemic vasculitis. Furthermore, the anti-Prx2 antibody titers tended to change in parallel with the activity of the vasculitis. The anti-Prx2 antibodies also 82

Several groups have tried to identify the target antigens of AECA to date. However, the target antigens for clinical applications, including MPO and PR3 of ANCA, remain unknown. Recently, certain approaches such as expression libraries, 2-DE-based proteomics studies and N-terminal sequence analyses have been developed to identify the target antigens of AECA. Kaneko et al. [63] successfully applied serological analyses to a recombinant cDNA expression library in KD patients and identified some EC antigens as targets of AECA, including tropomyosin and T-plastin. Margutti et al. reported a new autoantigen of AECA using a human microvascular endothelial cell cDNA library with sera from patients with BD [70]. The identified autoantigen is the C-terminus of Ral binding protein1 (RLIP76) that catalyzes the ATP-dependent transport of glutathione (GSH) conjugates including GSH-4-hydroxy-t-2,3-nonenal (4-HNE), in the serum of a significant percentage of patients with various diseases characterized by immune-mediated endothelial dysfunction, including BD and carotid atherosclerosis. Futhermore, these autoantibodies are involved in oxidative stress-mediated EC apoptosis because they increased intracellular levels of 4-HNE, decreased levels of GSH and



HUVEC

HeLa

Protein extracts 2-Dimensional electrophoresis (2DE) Western blotting (WB) Detection of HUVEC-specific autoantigens Mass fingerprinting Identification of the proteins


Further analysis by preparing recombinant autoantigens (Prevalence of AECA in vasculitis, clinical importance, role of target antigens etc.,)

Figure 2. Basic strategy for identification of AECA autoantigens in our research using proteomics.

SH SH

Oxidized TRX S NADPH

Protein Reduced protein

S

S

TRX

S

Protein Oxidized protein Reduced Prx

H2O2 R-O-O-H

TRX reductase Peroxiredoxin (Prx) SH SH NADP

TRX Reduced TRX

Oxidized Prx

H2O R-O-H

Figure 3. The role of peroxiredoxin (Prx) in the redox system. TRX: Thioredoxin.

activated JNK signaling. In detail, 4-HNE is an end product of lipid peroxidation that induces oxidative stress, causes apoptosis and activates several signaling pathways. Thus, antiRLIP76 autoantibodies have an important role in the redox balance, leading to increased intracellular levels of 4-HNE that result in oxidative stress and apoptosis of ECs [71]. In addition, our research concerned with redox system also implies that it is important role in pathophysiology of systemic vasculitis. In the case of Hsp60, proteins extracted from the human EC line EA.hy 926 were surveyed by 2-DE plus WB and ELISA using recombinant Hsp60. In addition, anti-Hsp60 antibodies were detected in about 40% of patients with SLE, PAN or WG. Lee et al. [62] applied a similar method

of 2-DE and WB to extracted proteins from human dermal microvascular ECs and identified alpha-enolase as a target antigen of IgM AECA in BD patients. Specific positive signals against recombinant human alpha-enolase were detected by IgM ELISA in 37.5% of BD patients. Furthermore, using a combination of Edman’s degradation and N-terminal sequencing, Garcıá de la Penã-Lefebvre et al. [64] and Servettaz et al. [65] identified two EC antigens, topoisomerase I and centromeric protein B (CENP-B), respectively, as targets of AECA in SSc patients. However, the expressions of the reported antigens, including Hsp60 and alpha-enolase, are not specific for or dominant in ECs. In addition, clear evidence for roles of these target proteins in systemic vasculitis has been lacking thus far. If proteins


83


dominantly expressed on ECs are targeted by AECA, such proteins would be more suitable candidates for considering the EC-specific pathological mechanisms of AECA. A considerable number of reports have indicated that AECA would be involved in the pathology and/or pathogenesis of systemic vasculitis. However, the exact roles of AECA remain unclear. To understand the roles of AECA in more detail, it will be necessary to identify target antigens of AECA and to measure AECA by a standardized method. Investigations on individual


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Affiliation

Rie Karasawa†1, Kazuo Yudoh1, Shoichi Ozaki2 & Tomohiro Kato3 † Author for correspondence 1 Institute of Medical Science, St. Marianna University School of Medicine, Kawasaki, Japan 2-16-1, Sugao, Miyamae, Kawasaki, Kanagawa, 216-8512, Japan Tel: +81 44 977 8111; Fax: +81 44 976 6401; E-mail: [email protected] 2 St. Marianna University School of Medicine, Department of Internal Medicine, Kawasaki, Japan 2-16-1, Sugao, Miyamae, Kawasaki, Kanagawa, 216-8512, Japan 3 St. Marianna University School of Medicine, Department of Biochemistry, Kawasaki, Japan 2-16-1, Sugao, Miyamae, Kawasaki, Kanagawa, 216-8512, Japan


87

Drug Evaluation

Influvac, a trivalent inactivated subunit influenza vaccine Gian Vincenzo Zuccotti† & Valentina Fabiano Universita` degli Studi di Milano, Luigi Sacco Hospital, Department of Pediatrics, Italy 1.

Introduction

2.

Overview of seasonal influenza vaccines market

3.

The trivalent inactivated subunit formulation: composition and mechanisms


of immune response 4.

Influvac: efficacy and safety studies

5.

Conclusions

6.

Expert opinion

Importance of the field: Influenza represents a major sanitary and socioeconomic burden and vaccination is universally considered the most effective strategy for preventing the disease and its complications. Traditional influenza vaccines have been on the market since the late 1940s, with million of doses administered annually worldwide, and demonstrated a substantial efficacy and safety. Areas covered in this review: The trivalent inactivated subunit vaccine has been available for more than 25 years and has been studied in healthy children, adults and the elderly and in people affected by underlying chronic medical conditions. We describe vaccine technology focusing on subunit vaccine production procedures and mode of action and provide updated information on efficacy and safety available data. What the reader will gain: A review of efficacy and safety data in healthy subjects and in high risk populations from major sponsor- and investigator-driven studies. Take home message: The vaccine showed a good immunogenicity and a favorable safety profile in all target groups. In the panorama of actually available influenza vaccines, trivalent inactivated subunit vaccine represents a well-established tool for preventing flu and the associated complications. Keywords: children, influenza vaccine efficacy, influenza vaccine safety, Influvac, trivalent subunit influenza vaccine Expert Opin. Biol. Ther. (2011) 11(1):89-98

1.

Introduction

Influenza is an acute respiratory highly infectious disease and an important cause of morbidity and mortality especially in the elderly, in children aged < 2 years and in people with chronic underlying medical conditions, such as chronic respiratory, cerebro-cardiovascular and metabolic diseases, and primary or acquired immunodeficiencies, which represent major risk factors for potentially severe flu complications [1,2]. The World Health Organization (WHO) estimated annually from three to five million influenza cases of severe illness with 250,000 -- 500,000 deaths worldwide [3]. Influenza is also a relevant public health matter with important implications for healthcare systems, due to increased number of hospitalizations and outpatient visits [2-4], and a socio-economic burden, secondary to impaired quality of life and loss of productivity, all resulting in increased direct and indirect healthcare costs [5]. The most effective strategy for preventing the disease and its complications is annual vaccination [1]. Influenza vaccines are trivalent preparations, containing surface antigens from two influenza A virus strains and one B strain. Because of antigenic drift of influenza virus, the composition of influenza vaccines has to be updated yearly as recommended by the WHO on the basis of worldwide surveillance [6] and mathematical modeling [7]. After the 2009 influenza A (H1N1) pandemic, health authorities recommendations for the 2010 -- 2011 influenza season have been updated and vaccination is recommended for all persons aged ‡ 6 months [1]. This decision is based on three major reasons. Firstly the observation that morbidity and mortality even in previously healthy young adults 10.1517/14712598.2011.541436 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

89

Influvac

Box 1. Drug summary. Drug name Phase Indication Pharmacology description Route of administration Pivotal trial(s)

Influvac III Influenza Immunostimulant Parenteral [81,83,84,87]


Pharmaprojects - copyright to Citeline Drug Intelligence (an Informa business). Readers are referred to Pipeline (http://informa-pipeline. citeline.com) and Citeline (http://informa.citeline.com).

occurs in every influenza season; secondly the unawareness of being at risk of influenza complications; and lastly a higher risk of influenza complications among adults < 50 years because of the predicted pandemic 2009 A (H1N1)-like virus circulation in the 2010 -- 2011 influenza season [8] together with the evidence that a great proportion of young adults do not yet have natural immunity against the virus [9]. Strong recommendation is however provided for children and adolescents aged 6 months -- 18 years, patients with underlying chronic diseases, all persons aged ‡ 50 years, pregnant and breastfeeding women, household and close contacts with high risk people and healthcare personnel [1]. Annual vaccination is a cost-effective strategy, in the elderly [10,11], in healthy adolescents and adults [12,13] and in working settings [14]. Efficacy and cost-effectiveness of influenza vaccination in healthy children is a matter of current discussion. Although influenza in children is a benigncourse disease, it has been considered responsible either for a significant disease burden even in the absence of highrisk conditions [4] or for a social and economic effect [5]. Some studies suggested that annual influenza vaccination of healthy children would result in health benefits and be costsaving for the whole community [15-17], in contrast, other studies raised doubts [18] or stated that modest or even no effects on mortality, serious complications and community transmission of the disease would result from influenza vaccination of healthy children [19].

Overview of seasonal influenza vaccines market 2.

Inactivated influenza vaccines have been available since the 1940s [20] and their production underwent a constant increase so that in the 2004 -- 2007 period an estimated 408 million doses had been distributed worldwide [21]. There are currently three available inactivated influenza vaccine types: subunit inactivated vaccines, made up only of surface haemagglutinin (HA) and neuraminidase (NA) antigens; split-virion inactivated vaccines, which contain both surface and internal antigens; and whole-virion inactivated vaccines. The seasonal version of whole-virion inactivated vaccine is manufactured by Baxter and will be available in Austria and the Czech 90

Republic in the 2010 -- 2011 influenza season. Both subunit and split-virion are egg-derived inactivated vaccines. The production process starts with the amplification of influenza viruses in embryonated chicken eggs and is followed by a fragmentation of the virus lipid membrane through the use of detergents, obtaining split influenza vaccines, or by the separation of the surface HA and NA antigens through a purification process, obtaining the subunit vaccines. Although initially considered less immunogenic than the whole-virion vaccines [22,23], a subsequent review by Beyer et al. showed equal immunogenicity of the three vaccine types [24]. Currently available trivalent subunit and split-virion influenza vaccines demonstrated a substantial efficacy, offering protection against the circulating strains in 70 -- 90% of healthy adult < 65 years of age [25,26], in 60 -- 90% and in 50 -- 60% of healthy children and elderly adults, respectively [27]. Trivalent inactivated influenza vaccines (TIV) provide significant health benefits in people with underlying medical conditions, such as the elderly with chronic heart diseases [28], patients with chronic obstructive pulmonary disease [29] or HIVinfected individuals [30]. Since the immune response to TIV is suboptimal in certain people such as older adults [31], new approaches to enhance influenza vaccine immunogenicity are currently being evaluated, including the use of adjuvants. Two adjuvanted seasonal influenza vaccines were firstly available in several countries since late 1990s: an MF59-adjuvanted subunit vaccine (Fluad, Novartis Vaccines, Siena, Italy) and a virosomal-adjuvanted subunit vaccine (Inflexal V, Berna Biotech Ltd, Berne, Switzerland). MF-59, a submicron squalene oil-in-water emulsion capable of activating antigen-presenting cells (APCs) [32], has been used in influenza vaccine formulations, showing a good immunogenic effect even in target groups such as the elderly [33] and people at risk of influenza complications [34,35]. Enhanced immunogenicity of MF59-adjuvanted influenza vaccines has been observed in healthy infants and young children, too [36,37]. Moreover, recent studies suggested that MF59-adjuvanted subunit influenza vaccines could offer cross-protection against antigenically drifted influenza virus strains, too [38,39]. MF59-adjuvanted vaccines show a good safety profile [40], with only a higher rate of local side effects, due to the local pro-inflammatory response induced by the squalene emulsion when injected in the muscle [32]. Another strategy is the novel virosomal antigen presentation [41] through which the viral surface glycoproteins are presented in a manner similar to that of the intact virus, by mimicking and maintaining the cell-entry and membranefusion properties and acting as antigen delivery and presenting system [42], thus resulting in good vaccine immune response [43,44] even in high-risk populations, such as the elderly [45] or asthmatic [46], diabetic [47] and HIV-infected children and adolescents [48,49]. In 2003 the FDA approved the use of Flumist (Medimmune, Gaitherburg, MD, USA) [50], a trivalent live-attenuated cold-adapted temperature-sensitive influenza



Zuccotti & Fabiano

vaccine (CAIV-T), administered intranasally, in which the live virus can only multiply in the lower temperatures of nasal passages stimulating both mucosal antibody and cellular responses with potential efficacy against variant circulating strains [51]. This vaccine is available for healthy children and non-pregnant adults between 2 and 49 years of age. Liveattenuated influenza vaccines seem to be less efficacious than conventional trivalent inactivated vaccines in healthy adults [52], in contrast, they are capable of eliciting a good immune response in children (aged 6 -- 59 months) [53], even if safety data about hospitalization rates for any cause, including wheezing, are contrasting [51,54]. Intradermally administered influenza vaccines are available, too. The skin shows unique immunological properties. In the skin, two types of dendritic cells (DCs), which act as potent antigen-presenting cells, are present: the Langerhans cells (LCs) in the epidermis and dermal DCs (dDCs) in the dermis. Intradermal vaccination targets directly APCs, which, by migrating to the draining lymph nodes, are able to trigger T- and B-cell activation [55]. Moreover, intradermal vaccination could favour lymphatic drainage of free antigens and their subsequent capture by DCs in the lymph nodes [56]. Several major studies evaluated immunogenicity and safety of intradermally administered influenza vaccines, which demonstrated its similar immunogenicity and safety to the intarmuscolar ones in adult subjects [57-59]. Few studies evaluated intradermal vaccine administration in children; however, available data suggested a good immunogenicity. Local reactions were more common in intradermally immunized children, but all adverse events were mild and transient [60,61]. A recent new horizon of influenza vaccine manufacturing is the one of cell-culture-grown vaccines. Despite the undeniable success of egg-derived vaccine production, the increased demand for seasonal influenza vaccination, the reduced and/or variable vaccine supply, and the threat of avian H1N5 pandemic influenza and the subsequent need for millions of rapidly available vaccine doses are reasons for which manufacturers are pursuing alternative approaches for influenza vaccine production. The use of tissue cell-culture lines could eliminate the long lead times and the limited flexibility of egg-based production, overcome potential sterility problems and enable production of avian strains influenza vaccines, which generally poorly grow in eggs. Mammalian cell lines, PER.C6, Madin Darbin Canine Kidney (MDCK), Vero cells and insect cell lines are being in use in Europe and the USA for seasonal and pandemic influenza vaccines manufacture [62,63]. In 1993 Ulmer et al. reported for the first time that a DNA vaccine can prevent influenza infection in mice [64] and subsequent preclinical studies demonstrated that DNA-technologybased influenza vaccination is successful in enhancing both a humoral and a cellular immune response against influenza antigens in various animal models [65,66]. In humans, after some disappointing initial results, a Phase I study by Drape and colleagues has demonstrated the induction of protective

antibody titres after DNA influenza vaccination [67]. A new ‘universal’ influenza vaccine strategy targeting highly conserved viral proteins NP, M1 and M2 is under development [68,69].

The trivalent inactivated subunit formulation: composition and mechanisms of immune response

3.

Influvac (Abbott, Chicago, USA) has been on the market since the early 1980s and is a trivalent subunit inactivated influenza vaccine made up of surface antigens haemagglutinin and neuraminidase of the A/H1N1, A/H3N2 and B virus strains according to annual WHO recommendations for the upcoming influenza season. Influvac is one of the leading influenza vaccines worldwide with more than 250 million doses manufactured so far (Box 1). Every year, in February for the northern hemisphere, in August for the southern hemisphere, each of the primary seed virus strains is separately propagated in chicken eggs to produce secondary seed viruses. These are inoculated in millions of 10 -- 11-day old embryonated eggs and, after a three-day incubation period, virus particles are extracted from the eggs. Viral RNA is inactivated and viral particles are ultracentrifuged to obtain a highly purified virus suspension. These viruses are dead and cannot replicate anymore. NA and HA antigens, the subunits, are removed from viral membrane using acetyltrimethylammonium bromide detergent. A second ultracentifugation is than necessary for separating the subunits from the viral membrane and viral core. Finally, the components are combined in the correct proportion in 0,5 ml pre-filled syringes for intramuscular or deep-subcutaneous injection containing 15 µg HA and 7.5 µg NA per strain. From 2004 onwards, Influvac has not contained thimerosal as preservative. Protection against influenza infection is achieved through the production, by B-lymphocytes, of specific antibodies, serum haemagglutination inhibition antibodies and neutralizing antibodies [70]. Specific antibody response starts to appear as early as 2 -- 6 days after immunization [71,72], and by 2 weeks postvaccination, 90% of subjects have protective antibody titres [71-73]. The majority of healthy adults and children have high antibody titres after vaccination [74,75], typically defined as the achievement of a 1:40 haemagglutination titre threshold. Serum immune response is characterized principally by influenza-specific IgG antibodies (mainly IgG1) with a lower concentration of IgA and IgM [61]. Although postvaccination antibody response is type-specific [73], cross-reaction towards newer viral strains is possible. After trivalent inactivated influenza vaccination, a local immune response is induced in the tonsils and saliva. In the tonsillar tissue and in the peripheral blood, IgG and IgA influenza-specific antibody secreting cells (ASCs) can be predominantly detected, in the oral fluid, the immune response is dominated by serum IgA1 (SIgA1) and, to a less extent, SIgA2 [76]. In young, influenza-infection-naı¨ve


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children immune response is mainly characterized by IgM, with lower IgA titres and no or very little salivary SigA [77]. Specific immunity persists usually from 6 to 12 months after vaccination, even if some studies suggest that vaccination-induced immunity could last even longer [78,79].


4.

Influvac: efficacy and safety studies

Since Influvac has been available on the market from 1982 to 2006, 76 clinical efficacy and safety studies in healthy adults and in the elderly, including the annual update studies required by European Union regulations [80], have been performed giving a sample of more than 6400 vaccinated subjects [81]. Influvac has been studied in children, too. Immunogenicity in healthy adults, in the elderly and in at-risk populations

4.1

Target population of the majority of the above mentioned studies were healthy adults and older subjects; 23 were randomised double- or single-blind studies, 2 studies were performed in subjects at risk of influenza complications and 2 in nursing home residents. All studies were performed in accordance with the Good Clinical Practice Guidelines of the International Conference on Harmonization standards and with the Declaration of Helsinki. Study design was more or less identical. Subjects were immunized with the intramuscularly administered vaccine Influvac. Blood samples were collected at baseline, before receiving vaccination and 2 -- 4 weeks afterwards. According to the criteria established by the Committee on Proprietary Medicinal Products (CPMP) [80], immunogenicity of Influvac was assessed considering the seroprotection rate, defined as the proportion of subjects with an haemoagglutination inhibition (HI) antibody titre ‡ 40; the seroconversion/ fourfold increase rate, defined as the proportion of subjects with a pre-vaccination HI titre < 10 and a post-vaccination titre ‡ 40, or at least a fourfold post-vaccination HI antibody relative increase; and the mean fold increase, defined as the geometric mean of the intra-individual fold increases in HI titre (GMT). For healthy adults aged 18 -- 60 years, seroprotection rate must exceed 70%, seroconversion rate 40% and the mean fold increase 2.5, 60%, 30% and 2.0 are the respective targets for the over 60 years old subjects. Based on these criteria, vaccination with Influvac was immunogenic both in healthy adults and in the elderly, with seroprotection rates of 80 -- 90% for all the three virus strains in the ones and of more than 70% in the latter. Seroconversion rate targets were also largely achieved, both in healthy adults and in the elderly. Mean fold increase was more than 10.0 for all three virus strains in adults and more than 6.0 in the older subjects. Immunogenicity of Influvac was confirmed in nursing home residents and, even if post-vaccination antibody titre in the at-risk population was lower, all the requested criteria were met in this subject group, too [81,82]. 92

Comparative studies of Influvac and other influenza vaccines have been published. In a randomized, observerblind, three-arms, parallel group, multi-centre trial, immunogenicity and safety of Influvac, a virosomal subunit vaccine (Invivac, Solvay Pharmaceuticals, Brussels, Belgium), and a MF59-adjuvanted subunit vaccine (Fluad) were studied in nearly 400 elderly subjects in four countries [83]. Immunogenicity was similar, with 84.1 -- 100% seroprotection rates, indicating a good humoral immune response for all three vaccines. In a randomized, controlled study conducted in China, immunogenicity and safety of Influvac and of another inactivated subunit influenza vaccine (Agrippal, Novartis Vaccines, Siena, Italy) were compared in healthy adults and the elderly [84]. Both Influvac and Agrippal proved to be highly immunogenic, with seroprotection rates > 85% and seroconversion rate > 70% for both vaccines in both age groups. In this study, post-vaccination GMTs were higher in the Influvac-vaccinated subjects than in those who received Agrippal, with an average of nearly 50% higher titres in the first group, in all age groups and for all the three virus strains. A comparative study has been conducted in elderly people in Russia. Immunogenicity was assessed for Influvac and four other inactivated influenza vaccines licensed in Russia, three split-virion vaccines, Begrivac (Novartis Vaccines and Diagnostics GmbH & Co., Marburg, Germany), Vaxigrip (Sanofi Pasteur SA, Lyon, France) and Fluarix (Glaxosmithkline Spa, Rixensart, Belgium); and an inactivated subunit vaccine Grippol (Petrovax, Moscow, Russia and Solvay Pharmaceuticals, Brussels, Belgium). All influenza vaccines demonstrated similar high immunogenicity in the elderly. The presence of chronic underlying medical conditions did not affect immunogenicity in all vaccines [85]. Effectiveness in healthy and at-risk children Influvac has been studied in both healthy and highrisk children, resulting in a substantial efficacy and safety. In a recent field study [86], a mass immunization programme was carried out in Russia, in the Moscow area, in kindergarten children aged 3 -- 6 years and in school children and adolescents from 7 to 17 years. A total of 28,309 children of both age groups were vaccinated with Influvac before the onset of the 2001 -- 2002 influenza season. Compared with a nearly 61,000 aged-matched subjects of the control group, efficacy of children influenza mass-vaccination with Influvac, evaluated as reduction of influenza-like illnesses (ILI), was 60.9% in kindergartner children and 68.8% in school children and adolescents. Moreover, mass childhood influenza immunization with Influvac resulted in a significant reduction of ILI and influenza-complications in unvaccinated non-institutionalized elderly showing a favourable Influvac effect on public health. Zhu and colleagues [84] evaluated immunogenicity and safety of Influvac compared with Agrippal in healthy children aged 3 -- 12 years. In this study two age subgroups were 4.2



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considered: 56 children aged 3 -- 5 years and 217 children from 6 to 12 years of age. Children of both subgroups were randomized to receive Influvac or Agrippal. In both younger and older children, Influvac and Agrippal resulted in good immunogenicity. GMTs were higher for Influvac especially in the 6 -- 12 years old children subgroup, in which GMT ratios for Influvac:Agrippal were between 1.44 and 1.70. Influenza vaccination is always recommended in children at risk of complications, the ones with chronic underlying medical conditions such as respiratory or cardiac diseases. Immunogenicity and tolerability of Influvac have been studied in high-risk children between 6 months and 4 years of age, with chronic lung disease or congenital heart disease [87]. In this open study, 52 high-risk children (48% with chronic lung disease, 46% with congenital heart disease and 6% with both conditions) were vaccinated with two 0.25 ml doses of Influvac, administered 4 weeks apart. Immunogenicity was assessed by dosage, three weeks after administration of the second vaccine dose, of HI-antibody titres. Influvac proved to be strongly immunogenic against all three virus strains in this population with an efficacy similar to that in healthy adults. Vaccination in asthmatic children is recommended [1]; however, influenza vaccination coverage in this high-risk group, although variable, is generally lower than in asthmatic adults [88]. Influenza vaccination with Influvac in asthmatic children has been found to have a moderate beneficial effect on quality of life [89], but scarce effects on reduction of the number, severity and duration of asthma exacerbations related to influenza infection [90]. However, in a randomized, doubleblind, placebo-controlled study, influenza vaccination did not prove to be associated with asthma exacerbations [91]. A comparative study of Influvac and Inflexal V was conducted in children and adolescents with cystic fibrosis (CF). In these patients, both vaccines were efficiently immunogenic [92]. Influvac and Inflexal V have been also compared in a study in diabetic children, adolescents and young adults [47]. Both vaccines resulted immunogenic but a better long-lasting immune response was assessed in patients vaccinated with Inflexal V, especially those without pre-existing antibodies to influenza strains. Efficacy of Influvac has been assessed in severely ill children, such as those affected by acute lymphoblastic leukemia. In these children Influvac proved to be efficiently immunogenic, both in previously vaccinated and in unprimed individuals [93]. Safety and tolerability In clinical trials and annual update studies in adults and the elderly, safety and tolerability of Influvac were assessed by subjects recording of local and systemic reactions and general inconvenience on a standard questionnaire for three or more days after vaccination. Local vaccine reactions are the ones that typically occur at the vaccination site, namely redness, induration, pain, bruises, warmth, swelling and arm 4.3

movement impairment. Systemic reactions are instead caused by immunological reaction to the vaccine, represented generally by headache, malaise, fever, insomnia, increase sweating and coughing. Both local and systemic reactions were scored as present or not present. If present, general inconvenience was scored as slight, moderate or severe. Influvac showed a good safety profile, with the majority of reported reactions being local and mostly represented by pain at the vaccination site. Other more frequently reported local reactions were redness, warmth and induration at the vaccination site. The most frequently reported systemic reaction was headache, followed by malaise and increased sweating. For both local and systemic reactions, there was an agedependent trend with fewer events reported by the elderly. The population at risk of influenza complications showed a little higher incidence of systemic reactions, the most frequent of which were always headache and malaise. The degree of inconvenience was reported as absent or mild by the majority of the subjects, both adults and elderly. Serious adverse events were collected only in the more recent studies and the ones possibly related to vaccination were three cases of dizziness, nausea and pneumonia respectively [82]. These data were confirmed by post-marketing surveillance. As regard nervous system disorders apart from headache, the most commonly reported events were acute polyneuropathy, paraesthesia or dysaesthesia, paralysis and dizziness. In more than 25 years experience with Influvac, only 46 cases of Guillain-Barre´ Syndrome have been reported [81,82]. Comparative studies confirmed safety and tolerability of Influvac. In study by De Bruijn and colleagues Influvac, Invivac and Fluad were all safe and substantially well-tolerated and a higher incidence of local and systemic reactions in the MF59-adjuvanted group was reported (46 and 32% respectively for the adjuvanted vaccine versus approximately 20% for the other two vaccine types) [83]. Comparison with Agrippal demonstrated that both vaccines presented a good safety profile in all age groups [84]. Low reactogenicity in the elderly was observed in the comparative Russian study, too [85]. In children, Influvac showed a good safety profile in all studies. Comparison with Agrippal resulted in good tolerabilty in both vaccines. The most frequently reported local reactions were pain and itching/pruritus at site of injection (nine children in the Influvac group). Systemic reactions were mainly mild and represented above all by headache. Fever after vaccination was observed in nine children in the Influvac group and four children in the Agrippal group. Systemic temperature increase was mild in all subjects and lasted no longer than 1 day. No serious adverse reaction was observed in large population mass vaccination [84]. In children with underlying chronic respiratory or cardiac diseases, most frequently reported local reactions were redness at the injection site (12% out of a total local reactions rate of 23%) whereas fever and increased irritability were the most common systemic ones (27 and 25% of reported systemic reactions respectively). In all cases, systemic reactions were


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described as mild and resolution was observed within a few days [87]. In asthmatic children and adolescents the most frequently reported local reactions were redness and pain at injection site, whereas sneezing and runny and/or stuffed nose were the most frequent systemic ones (74 and 47% respectively). Both local and systemic reactions lasted no more than 2 -- 3 days, and no serious adverse event, including asthma exacerbations, was observed [91]. In children affected by cystic fibrosis, local reactions were more frequently reported by Influvac-vaccinated patients (57%) than Inflexal V-vaccinated ones (44%). In this study, incidence of reported systemic reactions was higher than that reported in the literature, with 15 (71%) of children vaccinated with Influvac reporting one or more systemic adverse event. In this case however, incidence of systemic reactions in Influvac-vaccinated children was lower than in Inflexal V-vaccinated ones (36 vaccinees, 84%). Most of the events were represented by headache, fatigue, cough and coryza and classified as mild or moderate; there was no case of fever. These data were in contrast with the general tolerance of vaccination, judged as ‘very good’ or ‘good’ 4 weeks after immunization, and can be explained by difficulty in distinguishing between vaccine reactions and underlying CF symptomatology [92]. Influvac has been well tolerated in diabetic children and adolescents, without any significant difference of the severity of any local or systemic reaction compared with Inflexal V [47]. 5.

Conclusions

The analysis of data from annual update studies, clinical trials and post-marketing surveillance demonstrates the substantial immunogenicity and the favorable safety profile of the inactivated subunit influenza vaccine Influvac [81,94,95]. These observations are in line with literature data on efficacy and safety of influenza vaccines in children [96-98], in healthy, working adults [25-27,99], and in the elderly [27,100,101]. Seroprotection rates in healthy adults vaccinated with Influvac consistently met the CPMP serological criteria for immunogenicity and were between 80 and 90% in all studies. These data are in accordance with literature and surveillance reports, in which inactivated influenza vaccines are estimated to prevent the disease in 70 -- 90% of healthy adults [27,102]. Since serological HI antibody titre is considered a well-accepted correlate of protection [103], Influvac proved to be substantially efficacious in all studies. Seroresponses to vaccination with Influvac in the elderly were somewhat lower than in younger adults. This has been largely reported in the literature: ageing of the immune system is responsible for lower responsiveness to vaccination in general. However, Influvac seroprotection rate in the older subjects was over 70% in the majority of the studies, proving to be even better than the estimated 30 -- 70% efficacy in this subject group [91]. Immunogenicity of Influvac is in accordance with literature data about comparisons of 94

inactivated subunit vaccines with other inactivated vaccine types [24] or with cold-adapted live influenza vaccines [104] even if the latter have been found being able to elicit a longlasting, broader immune humoral and cellular response in healthy adults and children but not in the elderly [105]. In all studies, vaccination with Influvac was found to be safe and well-tolerated, with an extremely low prevalence of serious adverse events. The favorable safety profile of Influvac confirms what is reported for other trivalent inactivated influenza vaccines [98]. In children from 6 to 23 months of age, a large population-based study reported safety of trivalent inactivated vaccines. In a cohort of more than 45,000 vaccinated children, very few medically attended events significantly associated to the vaccine have been reported. None of these was serious [95]. Moreover, efficacy and safety of Influvac has been confirmed in high-risk children, too. 6.

Expert opinion

Influenza vaccination is overall considered the best way to prevent the infection and related complications [1]. As a result, recommendations for immunization have been regularly updated to meet the constantly increasing need for influenza protection, both from an individual and a societal point of view. With their long-lasting presence on the market and their worldwide distribution, traditional trivalent inactivated influenza vaccines represent actually the mainstay of influenza prevention. More recently, adjuvanted influenza vaccines, virosomal and MF59-adjuvanted vaccines have demonstrated to be particularly immunogenic in elderly people [34,35]. Recent head-to-head comparative studies with conventional subunit, virosomal-adjuvanted, and MF59-adjuvanted influenza vaccines have shown however a similar immunogenicity of the three vaccine types [83,106]. MF59 vaccine resulted in a greater reactogenicity than inactivated and virosome-adjuvanted ones. Seasonal influenza vaccines are safe and sufficiently immunogenic in children, virosome-adjuvanted vaccines show a greater immunogenicity than inactivated ones, especially in unprimed children [107]. Live-attenuated intranasally administered influenza vaccine showed a higher immunogenicity in children than conventional inactivated vaccines not only for wellmatched strains but also for antigenically drifted ones. However, data on safety are actually already contrasting. Influenza immunization is not licensed in infants aged less than 6 months, because of scarce immunogenicity, typical of this age. However, recent studies evaluated immunogenicity and safety of conventional trivalent inactivated influenza vaccines in infants less than 6 months old. TIV proved to be safe and well tolerated and showed a moderate immunogenicity particularly in infants without pre-existing maternally acquired antibodies [108,109]. Considering all available literature data together with the long-lasting post-marketing experience, TIV continue to represent a valid and safe choice for influenza immunization in children, adults and the elderly even in the presence of underlying chronic medical conditions. Influvac has been on


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the market for more than 25 years and millions of doses have been administered worldwide. Its efficacy and safety in all target populations have been demonstrated by several studies and post-marketing surveillance.

Declaration of interest The authors state no conflict of interest and have received no payment for the preparation of this manuscript.

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El-Madhun AS, Cox RJ, Haaheim LR. The effect of age and natural priming on the IgG and IgA subclass response after parenteral influenza vaccination. J Infect Dis 1999;180:1356-60 Foy HM, Cooney MK, McMahan R. A Hong Kong influenza immunity three years after immunization. JAMA 1973;226:758-61 Couch RB, Keitel WA, Cate TR. Prevention of influenza virus infection by current inactivated influenza vaccines. In: Brown LE, Hampson AW, Webster RG, editors. Options for the control of influenza III. Elsevier, Amsterdam, Netherlands; 1996. p. 97-106 Committee for Proprietary Medicinal Products. Note for Guidance on Harmonization of Requirements for Influenza Vaccines. CPMP/BWP/214/96, European Medicines Agency, London, UK; 1997. Available from: http://www. ema.europa.eu/docs/en_GB/ document_library/Scientific_guideline/ 2009/09/WC500003945.pdf [Last accessed 20 November 2010] Giezeman KM, Nauta J, de Bruijn IA, et al. Trivalent inactivated subunit influenza vaccine Influvac: 25-Year experience of safety and immunogenicity. Vaccine 2009;27:2414-17 Palache AM, Nauta J, de Regt V, et al. Twenty years’ experience with safety and immunogenicity of an inactivated subunit influenza vaccine. J Drug Assess 2005;8:1-15

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Brydak LB, Rokicka-Milewska R, Machala M, et al. Immunogenicity of subunit trivalent influenza vaccine in children with acute lymphoblastic leukaemia. Pediatr Infect Dis J 1998;17:125-9

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La Montagne JR, Noble GR, Quinnan GV, et al. Summary of clinical trials of inactivated influenza vaccine, 1978. Rev Infect Dis 1983;5:723-36 Brokstad KA, Cox RJ, Major D, et al. Cross-reaction but no avidity change of the serum antibody response after

children on the morbidity of the unvaccinated elderly. Epidemiol Infect 2006;134:71-8

influenza vaccination. Vaccine 1995;13:1522-8

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100. Corsetti E, Arniani S, Bordoni F, et al. Effectiveness of influenza vaccination in the elderly in a community in Italy. Eur J Epidemiol 2001;17:163-8 101. Vu T, Farish S, Jenkins M, et al. A meta-analysis of effectiveness of influenza vaccine in persons aged 65 years and over living in the community. Vaccine 2002;20:1831-6 102.

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1999-2000 seasons. Morbidity and Mortality Weekly Report 2002;51:1-10 103. de Jong JC, Palanche AM, Beyer WE, et al. Haemoagglutination-inhibiting antibody to influenza virus. Develop Biol 2003;115:63-73 104. Beyer WEP, Palanche AM, Osterhaus ADME, et al. Comparison of serology and reactogenicity between influenza subunit vaccines and whole virus or split vaccines. Clin Drug Invest 1998;15:1-12 105. Cox RJ, Brokstad KA, Ograt P. Influenza virus: immunity and vaccination strategies. Comparison of the immune response to inactivated and live, attenuated influenza vaccines. Scand J Immunol 2004;59:1-15 106. Ruf BR, Colberg K, Frick M, et al. Open, randomized study to compare the immunogenicity and reactogenicity of an influenza split vaccine with an MF59-adjuvanted subunit vaccine and a virosome-based subunit vaccine in elderly. Infection 2004;32:191-8 107. Kanra G, Marchisio P, Feiterna-Sperling C, et al. Comparison of immunogenicity


and tolerability of a virosome adjuvanted and a split influenza vaccine in children. Pediatr Infect Dis J 2004;23:300-6 108. Emmanuel BW, Englund JA, Blatter M, et al. Trivalent inactivated influenza virus vaccine given to two-months-old children. Pediatr Infect Dis J 2009;28:1099-104 109. Halasa NB, Gerber MA, Chen Q, et al. Safety and immunogenicity of trivalent inactivated influenza vaccine in infants. J Infect Dis 2008;197:1448-54

Affiliation

Gian Vincenzo Zuccotti† MD & Valentina Fabiano MD † Author for correspondence Universita` degli Studi di Milano, Luigi Sacco Hospital, Department of Pediatrics, Via GB Grassi, 74, 20157, Milan, Italy Tel: +390239042253; Fax: +390239042254; E-mail: [email protected]

Drug Evaluation


Immunotherapy for metastatic prostate cancer: where are we at with sipuleucel-T? 1.

Introduction

2.

Overview of the market

3.

Immunology

4.

Clinical efficacy

5.

Safety and tolerability

6.

Regulatory affairs

7.

Conclusions

8.

Expert opinion

Chen Lu¨, Andrew K Williams, Venu Chalasani, Carlos H Martıńez & Joseph Chin† University of Western Ontariot, Divisions of Urology and Surgical Oncology, Departments of Surgery and Oncology, London, Ontario, Canada

Importance of the field: Prostate cancer is the leading malignancy in North American men and despite improvements in treatments 20 -- 30% of patients will relapse. Immunotherapy using activated mononuclear cells is a way to harness the body’s adaptive immune response to fight metastatic prostate cancer. Areas covered in this review: In 2005, at least 10 therapeutic cancer vaccines, designed to confer active, specific immunotherapy against tumorassociated antigens, were in clinical trials. These covered potential fields of immunological strategy to overcome castration-resistant prostate cancer. What the reader will gain: A literature review was performed using the search terms sipuleucel-T, Provenge and APC8015 or APC-8015, and restricted to English language articles from 2000 to 2010. The immunological design and development of sipuleucel-T are summarized. The efficacy and safety of sipuleucel-T are discussed based on current data from clinical trials. Ongoing clinical trials involving sipuleucel-T are summarized. Take home message: Efficacy and safety with sipuleucel-T has been demonstrated in Phase I/II trials. The latest data from a Phase III trial shows that sipuleucel-T has met the primary endpoint of survival benefit. Further work is needed to understand the mechanisms behind cancer vaccine failure and elucidate the population for whom this vaccine will be suitable. Keywords: APC8015, cancer vaccine, immunotherapy, prostate cancer, Provenge, Sipuleucel-T Expert Opin. Biol. Ther. (2011) 11(1):99-108

1.

Introduction

Prostate cancer is the leading malignancy in North American men, with an estimated 192,280 new cases and 27,360 deaths in the USA for 2009 [1]. In the UK prostate cancer was the second most common cause of cancer death in men (10,239 deaths), accounting for 13% of all male deaths from cancer in 2007 [2]. In Canada, prostate cancer will continue to be the most commonly diagnosed cancer in men and the third leading cause of male cancer death [3]. Despite improvements in local treatment, 20 -- 30% of men with apparently localized disease will develop metastatic disease. A significant proportion (about 60%) of these patients progress to castration-resistant prostate cancer (CRPC) within a median of 18 to 24 months [4]. CRPC remains an incurable disease, with a median survival rate of 18 -- 20 months when treated with prednisone and docetaxel-based chemotherapy, currently the only FDA-approved chemotherapeutic agent for the treatment of CRPC [5,6]. This regimen has been reported to have a survival benefit of 2.4 months in men with CRPC, but with some serious adverse effects (e.g., grade 3 or 4 neutropenia, infection, anemia and neuropathy) [5,6].


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Sipuleucel-T

Box 1. Drug summary. Drug name Phase Indication


Pharmacology description/ mechanism of action Route of administration Pivotal trials

2.

Sipuleucel-T Phase III Metastatic, asymptomatic or minimally symptomatic castration-resistant prostate cancer Immunostimulant Intravenous infusion D9901, a randomized, double blind, placebo-controlled Phase III trial, enrolled 127 patients, to evaluate the safety and efficacy of sipuleucel-T D9902A, a randomized, double blind, placebo-controlled Phase III trial to evaluate the safety and efficacy of sipuleucel-T. Enrollment of D9902A was stopped at 98 patients based on initial disease progression results in D9901 D9002B, A randomized, double blind, placebo-controlled Phase III trial, also known as IMPACT. In D9902B, 512 patients were enrolled and overall survival is the primary endpoint


In the hope of a more effective therapy for CRPC, alternative treatment strategies including immunotherapy have been investigated. In 2005, at least 10 therapeutic cancer vaccines, designed to confer active, specific immune responses against tumor-associated antigens, were in clinical trials. These vaccines covered potential fields of immunological strategy to overcome CRPC, including whole tumor cell, dendritic cell (DC), protein and peptide, viral vectors and DNA vaccines [7]. However, nearly 5 years later, all but one of them have failed being approved by the US FDA or European Medicines Agency (EMEA). Sipuleucel-T, also known as Provenge (Dendreon Corporation) represents a novel cellular immunotherapy for the treatment of metastatic CRPC (Box 1). At present Sipuleucel-T is priced a $31,000 per injection resulting in a cost of $93,000 per patient. 3.

Immunology

Each dose of sipuleucel-T is made from autologous peripheral blood mononuclear cells collected from a 1.5 -- 2 l blood volume by leukapheresis. Erythrocytes, granulocytes, platelets and low-density monocytes are removed, resulting in a product consisting mainly of antigen presenting cells (APC) (e.g., dendritic cells (DC) [CD54+], T cells [CD3+], monocytes [CD14+], B cells [CD19+], and NK cells [CD56+]) [8,9]. CD54+ APC [9], which is responsible for antigen uptake, presentation, and stimulation of T-cell activity [8] has been shown to be the active component of sipuleucel-T. APC’s express co-stimulatory molecules and secrete immuneactivating cytokines to elicit a cellular immune response initiating primary, secondary and tertiary immune signaling [10]. APC’s include activated B lymphocytes, monocytes, and macrophages, but DC’s are considered the most potent APCs, capable of initiating anti-tumor immune responses from both naı¨ve and memory T-cells. DC’s internalize, process and display foreign and self antigens to B and 100

T lymphocytes and are critical for priming cytotoxic T lymphocytes (CTL). For sipuleucel-T, the tissue-specific protein target antigen for immunization is prostatic acid phosphatase (PAP), which is expressed in about 95% of prostate tumors, and has fairly specific expression for prostatic tissue. To enhance its antigenicity and DC-stimulating properties, PAP was linked to GM-CSF, using recombinant DNA techniques. The resultant fusion protein, termed PA2024, consists of human PAP fused through its carboxy terminus to the amino terminus of human GM-CSF via a Gly--Ser linker, which is expressed in a Baculovirus system and concentrated at > 90% purity [9]. GM-CSF was originally identified and cloned because of its ability to stimulate white blood cell production in lymphopenic patients. It was subsequently shown to be one of the most potent stimulators of APC, enhancing their differentiation, survival and antigen processing ability [11]. GM-CSF most profoundly affects DC development [12] compared with other factors, such as interferon-a, IL-12 and CD40 ligand [13]. The final vaccine product is produced by isolating an individual patient’s CD54+ white cells via leukapheresis, exposing the isolated cells ex vivo to PA2024, incorporating the stimulated cells into Dendreon’s antigen delivery cassette, and infusing the vaccine back into the patient intravenously. Sipuleucel-T therefore consists essentially of an individual patient’s APC primed by exposure to PA2024, but also is a mixed cell suspension containing also monocytes, macrophages, B and T cells, exposed to activated APCs [14]. The logistics of production of sipuleucel-T needs to be addressed. Processing the patient’s CD54+ white cells isolated via leukapheresis, exposing the isolated cells ex vivo to PA2024 and incorporating the stimulated cells into Dendreon’s antigen delivery cassette, is critically time-sensitive. Reliable and prompt transport of the isolated autologous peripheral blood mononuclear cells to the processing facility is imperative. In situations where significant delay is a possibility (e.g., being subjected to cross-border customs inspection), the patients


Lu¨, Williams, Chalasani, Martıńez & Chin

may have to be sent to the processing center for leukapheresis. Infusion of the vaccine is carried out at the patient’s local treatment facility. The number of isolated CD54+ cells is variable from patient to patient but is a key determinant of the potency of each doses and the quantity of sipuleucel-T. 4.

Clinical efficacy

Phase I trials The first Phase I clinical trial of sipuleucel-T in patients with CRPC was conducted at the Mayo Clinic between 1997 and 1998. This study enrolled 13 patients with metastatic CRPC, who were treated with two infusions of sipuleucel-T one month apart, followed by 3 monthly subcutaneous injections of PA2024 alone at one of three dosage levels (0.3, 0.6 or 1.0 mg/injection). One patient was not evaluated because of disease progression after the first dose of sipuleucel-T. After infusion of sipuleucel-T, patients experienced mild (grade 1 -- 2) toxicities consisting of short-lived fever, chills, myalgia, pain and fatigue. Development of antigen-specific T-cells was observed in all patients evaluated for immune response. Three patients had a drop in their serum prostate specific antigen (PSA) level by 50% or more [9]. Antibody response to sipuleucel-T was also measured in this trial. Compared with the findings of this study, a higher rate of antibody responses to PAP and higher titers of anti-PAP antibodies were reported in a study of sipuleucel-T alone, suggesting that the follow up injections of soluble antigen PA2024 in this trial actually suppressed antibody generation [9]. The administration of soluble antigen without adjuvant immune cells can suppress B-cell immune responses [15]. The next Phase I clinical trial of sipuleucel-T on CRPC was conducted at the University of California San Francisco Comprehensive Cancer Center (2000). DC precursors were harvested by leukapheresis at weeks 0, 4 and 8, loaded ex vivo with antigen and then infused intravenously. A fourth dose was administered at Week 24 to patients whose disease was stable or improving. Twelve patients were treated with sipuleucelT in a dose escalation fashion (0.2 109, 0.6 109, 1.2 109 or 2.0 109 nucleated cells/m2), and six patients were treated at the maximum dose of sipuleucel-T. Of the 12 patients 11 (92%) had received a second-line hormonal manipulation, such as ketoconazole, and 8 patients (66%) had also received chemotherapy, suramin or some other investigational agent. All patients developed evidence of an immunological response to the fusion protein PA2024. Maximum T cell proliferation responses to PA2024 were observed after either two or three infusions of sipuleucel-T, but the dose of Provenge did not correlate with the magnitude of the T-cell proliferation response. The median time to disease progression for twelve patients was 12 weeks [8].


4.1

Phase II trials Based on their first Phase I clinical trial, Small et al. enrolled 19 more patients with CRPC onto the Phase II clinical trial 4.2

(2000) [8]. All patients were treated with sipuleucel-T at the maximum manufacturable dose. Twelve (63.2%) had received a second-line hormone, and one had received prior therapy with hydrazine sulfate. The median time to progression for the Phase II patients was 29 weeks. Seven of the 19 Phase II patients had not progressed by the end of the planned 1-year follow-up period. Data of all 31 patients from both Phase I and II clinical trials were then combined for analysis. T-cell proliferation responses after infusion of sipuleucel-T developed in all patients, but different from T-cell or B-cell immune response to native PAP. The median time to disease progression was 34 weeks for patients who developed an immune response (n = 20) compared with 13 weeks for patients who did not (n = 11) (p < 0.027). Patients tolerated treatment well. Fifteen infusions (14.7%) were associated with febrile reactions that developed within 2 h [8]. A second Phase II clinical trial of sipuleucel-T on AIPC was conducted by Burch et al. [14]. A total of 21 patients with CRPC were enrolled. Sipuleucel-T was administered intravenously to eligible patients twice, at week 0 and week 2. Three subcutaneous injections of 1.0 mg of PA2024 were administered subsequently at weeks 4, 8 and 12. Of the 15 patients evaluated for immune response 13 developed antibodies specific for PA2024 with the highest titers measured between week 4 and week 8. The median time to disease progression was 118 days, and two patients exhibited a 25 -- 50% transient decrease in serum PSA. The treatment was again generally well tolerated in this Phase II study, with the most common adverse effects being mild, grade 1 -- 2 chills and fatigue. Two Phase II clinical trials treating men with biochemically relapsed androgen-dependent prostate cancer (ADPC) were designed to assess the PSA--modulating effects of sipuleucel-T. Beinart et al. enrolled 19 patients with nonmetastatic recurrent disease as manifested by increasing PSA levels (0.4 -- 6.0 ng/ml) and who had undergone previous definitive surgery or radiation therapy [16]. All patients received three scheduled infusions of sipuleucel-T at weeks 0, 2 and 4 and each infusion consisted of approximately 1.2 109 nucleated cells/m2. Treatment was well tolerated with grade 1 toxicity in all but one case. Out of 18 patients 13 demonstrated an increase in PSA doubling time (PSADT), with a median increase of 62%, though no decrease in PSA ‡ 50% from baseline levels was reported. Based on the hypothesis that inhibition of VEGF could augment the immuno-stimulatory and anti-tumor effects of sipuleucel-T, a Phase II trial of combination immunotherapy with sipuleucel-T plus bevacizumab was initiated by Rini et al. in patients with serologic progression of ADPC [11]. Bevacizumab is a recombinant antibody against VEGF, given at 10 mg/kg i.v. at weeks 0, 2 and 4 and every 2 weeks thereafter until significant toxicity resulted or disease progression. Sipuleucel-T was given intravenously at weeks 0, 2, and 4 at the dose of 1.2 109 nucleated cells/m2. A total of 22 patients were enrolled. All patients demonstrated induction of an


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Sipuleucel-T

immune response against PA2024. Nine patients exhibited some decrease in PSA from baseline, ranging from 6 to 72%, with the PSA of three patients decreasing at least 25%. The median pretreatment PSA doubling time was 6.9 months and the median post-treatment PSA doubling time was 12.7 months (p = 0.01). The majority of toxicities were Grade 1 or 2 including fatigue and infusion-related chills, myalgia and fever. A total of six Grade 3 toxicities, but no Grade 4, were observed leading to the discontinuation of therapy in four patients (congestive heart failure, gastrointestinal hemorrhage, cerebrovascular ischemia and proteinuria). Phase III trials The first Phase III clinical trial of sipuleucel-T, designated D9901, was initiated in January 2000 [17]. A total of 127 patients with asymptomatic metastatic CRPC from 19 centers were randomly assigned in a 2:1 ratio to receive three infusions of sipuleucel-T (n = 82) or placebo (n = 45). For generation of placebo, one-third of the cells collected at leukapheresis were processed in a manner similar to sipuleucel-T, but without recombinant fusion protein activation. All patients were followed for survival for 36 months. The trial was completed in 2004. This placebo-controlled study failed to show significant delay in the median time to disease progression (TTP) (11.7 weeks for sipuleucel-T versus 10.0 weeks for placebo, p = 0.052), but demonstrated a significant delay in TTP in a subset of patients with Gleason score of £ 7. Treatment remained a strong independent predictor of overall survival after adjusting for prognostic factors using a Cox multivariable regression analysis (p = 0.002). A median survival benefit of 4.5 months (25.9 months for sipuleucelT versus 21.4 months for placebo, p = 0.01) and a significant increase in estimated survival rate at 36 months (34% for sipuleucel-T versus 11% for placebo, p = 0.005) was observed. Therapy was generally well tolerated, displaying Grade 1 and at the most Grade 2 toxicity for each treatment group (70.7% for sipuleucel-T and 68.9% for placebo). Another identically designed double-blind, placebocontrolled Phase III randomized clinical trial (D9902) in asymptomatic, metastatic CRPC patients started in May 2000 [18]. Enrollment in D9902 was stopped at 98 patients (sipuleucel-T, n = 65; placebo, n = 33) based on initial disease progression results in D9901 and before the availability of survival results. The first part of the study was then designated as D9902A, and the study was amended to become D9902B, a study in which overall survival was the primary endpoint and in which 512 patients were enrolled. In D9902A, the median TTP was not significantly different between treatment groups (10.9 weeks for sipuleucel-T versus 9.9 weeks for placebo, p = 0.719). There was a 21% reduction in the risk of death for sipuleucel-T relative to placebo, which was not statistically significant (p = 0.331). To adjust for potential imbalances in baseline prognostic factors, a Cox multiple regression model developed from the D9901 data was applied to the D9902A data. After adjusting in this model for baseline


4.3

102

lactate dehydrogenase (LDH), PSA, localization of disease, number of bone metastases and weight, the magnitude of the survival effect for patients treated with sipuleucel-T increased (p = 0.023). To provide an additional estimate of the survival effect in this patient population, an exploratory analysis of the larger integrated dataset from D9901 and D9902A was performed [18]. Patients randomized to sipuleucel-T had a 21% reduction in the risk of disease progression (p = 0.111), and a 33% reduction in the risk of death (p = 0.011) compared with patients randomized to placebo. The median survival was 23.2 months for sipuleucel-T and 18.9 months for placebo, and the estimated survival rate at 36 months was 33 and 15%, respectively (log-rank test, p = 0.01). The overall incidence of adverse events was similar between patients treated with sipuleucel-T (98.6%) and patients treated with placebo (96.1%), including chills, pyrexia, headache, asthenia, dyspnea, vomiting and tremor. These adverse reactions were primarily Grade 1 -- 2, lasting 1 to 2 days. In 2009, the data from the Phase III trial the immunotherapy prostate adenoaarcinoma treatment (IMPACT), also known as D9902B [19] showed that sipuleucel-T has met the primary end points. The Phase III IMPACT trial enrolled 512 patients with asymptomatic or minimally symptomatic metastatic CRPC. The use of sipuleucel-T prolonged overall survival among men with metastatic CRPC, but no effect on the time to disease progression was observed. For inclusion within the trial, patients had to have a life expectancy of ‡ 6 months, serum PSA levels of ‡ 5.0 ng/ml, and testosterone levels of < 50 ng/dl following medical or surgical castration. Overall survival was chosen as the primary endpoint, and time to radiographic disease progression as the secondary endpoint. Treatment with sipuleucel-T extended median overall survival by 4.1 months compared with placebo, and it reduced the risk of death from any cause by 22% (hazard ratio [HR], 0.78; p = 0.03). The median overall survival was 25.8 months in the sipuleucel-T group, compared with 21.7 months in the placebo group. The survival benefit of sipuleucel-T was consistent among all patient subtypes, including those defined by bisphosphonate use, primary Gleason grade (‡ 4 versus £ 3), number of bone metastases (> 10 versus £ 10), disease localization (bone or soft tissue versus bone and soft tissue), or Eastern Cooperative Oncology Group (ECOG) performance status (1 versus 0). Time to objective disease progression, as assessed by independent radiological review, was similar in the sipuleucel-T and placebo groups (3.7 months and 3.6 months, HR, 0.95; p = 0.63). In the integrated analysis of findings from IMPACT, D9901 and D9902A, sipuleucel-T reduced the risk of death from any cause by 26.5% (HR: 0.735; p < 0.001) and extended median overall survival by 3.9 months compared with placebo. The 36-month overall survival was 33% in the sipuleucel-T group and 20% in the placebo group. Several adverse events were reported at a




significantly higher rate (p £ 0.05) in the sipuleucel-T group than in the placebo group, including chills (54.1 versus 12.5%), pyrexia (29.3 versus 13.7%), headache (16.0 versus 4.8%), influenza-like illness (9.8 versus 3.6%), hypertension (7.4 versus 3.0%) and hyperhidrosis (5.3 versus 0.6%). The rate of serious adverse events was similar in the sipuleucel-T (24.0%) and placebo (23.8%) treatment groups. 1.2% of patients in the sipuleucel-T group were unable to receive all three courses of sipuleucel-T therapy because of adverse events [19,20]. The transition from promising Phase I/II trials to decisive Phase III trials confronted the developers of sipuleucel-T with a challenge, particularly with regard to efficacy. In Phase I and II trials, sipuleucel-T reduced PSA levels more than 50% in approximately 10% of patients with CRPC [8,9,14]. Compared with patients who did not develop an immune response to PAP, TTP was prolonged 21 weeks in patients with CRPC who demonstrated an anti-PAP immune response [8]. In Phase III trials, the primary end point, TTP, failed to meet statistical criteria, but there was a notable TTP trend in D9901 with a hazard ratio of 1.45 (p = 0.052) [18]. The sipuleucel-T overall survival effect in D9901 was characterized by an HR estimate of 1.71 (p = 0.010). In D9902A, the treatment effect for overall survival (HR = 1.27) was consistent with that of D9901, despite imbalances in baseline prognostic factors which favored the placebo arm. Importantly, in D9901, despite the lack of statistical significance in the TTP analysis, there was a separation of curves 3 months post-randomization. This finding was consistent with the survival benefit demonstrated in the overall survival analysis, in which the arms started to separate and remained separated after 10 months [18]. In the IMPACT trial the overall survival curves for the sipuleucel-T and placebo arms separated at 6 months and did not converge again until 5 years later. Kaplan-Meier survival estimates consistently favored sipuleucel-T rather than placebo at all pre-specified timepoints, including 24 months (52.1 versus 41.2%), 36 months (31.7 versus 23.0%) and 48 months (20.5 versus 16.0%). Survival rates in the sipuleucel-T arm exceed the survival rates typically observed in other trials of patients with metastatic CRPC [19,20]. To date, the completed clinical trials of sipuleucel-T were all carried out in patients with asymptomatic or minimally symptomatic CRPC or ADPC. On March 29, 2007, the FDA Office of Cellular, Tissue and Gene Therapy’s advisory committee suggested that there was substantial evidence of efficacy for sipuleucel-T for asymptomatic metastatic CRPC and recommended FDA approval [21]. On April 29, 2010, sipuleucel-T was approved for this indication. [22].

4.4.1

Ongoing Phase II/III trials This history of sipuleucel-T has illustrated a number of questions to be answered, and more Phase II/III trials have been designed to establish efficacy and support market authorization.

5.

4.4

OpenACT (Open-Label Active Cellular ImmunoTherapy, NCT00901342)

This was a multicenter, open label, Phase II Study of sipuleucelT in men with metastatic castrate resistant prostate cancer (CRPC), to provide sipuleucel-T to men with metastatic CRPC while marketing approval was being pursued, to obtain safety data, evaluate the magnitude of immune responses to treatment with sipuleucel-T, and to further characterize the cellular components of sipuleucel-T [23]. This trial is closed to accrual. ProACT (treatment of PROstate cancer with Active Cellular ImmunoTherapy, NCT00715078)

4.4.2

This is a randomized, multicenter, single blind, Phase II trial of 120 patients with CRPC. The purpose of this study is to compare the changes in CD54 upregulation between each of the three groups of subjects treated with different c concentrations of PA2024 antigen. The study will also evaluate the levels of immune response, the duration of survival, the role of circulating tumor cell levels in the blood, and changes in quality of life. All subjects will be followed for this study for the remainder of their lives [24]. NeoACT (NEOadjuvant Active Cellular ImmunoTherapy, NCT00715104)

4.4.3

This is an open label, Phase II trial of immunotherapy with sipuleucel-T as neoadjuvant treatment. Patients (n = 40) with localized prostate cancer from two centers will be treated with three infusions of sipuleucel-T prior to a scheduled radical prostatectomy (RP) surgery. To assess the immune response following treatment with sipuleucel-T, tissue from the prostatectomy specimen will be compared with tissue from the core biopsy specimen obtained prior to treatment with sipuleucel-T. Following RP, patients will be randomized to receive either a booster infusion of sipuleucel-T or no further treatment with sipuleucel-T [25]. PROTECT (Provenge for the Treatment of Hormone Sensitive Prostate Cancer, NCT00779402)

4.4.4

The PROTECT (Provenge Treatment and Early Cancer Treatment) trial is a prospective, double-blind, controlled, randomized Phase III trial for patients with hormone-sensitive prostate cancer. The study is being conducted at over fifteen centers, and plans to enroll patients with rising PSA after radical prostatectomy and no evidence of metastasis. The purpose of the study is to determine if Provenge is effective for treatment of early-stage, non-metastatic prostate cancer. The primary efficacy endpoint is serologic (PSA) progression, and the secondary endpoint is time-to progression. Data from this study will be combined with earlier data to provide greater statistical power to detect a difference in the time to progression [26].

Safety and tolerability

In Phase III trial D9901, sipuleucel-T treatment was well tolerated at the maximum dose of 2.0 109 nucleated


103

104

Clinical trial

Phase I, CRPC

Phase I, CRPC

Phase II, CRPC

Phase I/II, CRPC (Integrated Data Analysis)

Phase II, CRPC

Phase II, ADPC

Phase II, ADPC, plus Bevacizumab

Phase III, CRPC (D9001)

Phase III, CRPC (D9002A)

Phase III, CRPC (D9001/D9002A)

Phase III, CRPC (IMPACT)

Author [Ref.]

Burch et al. 2000

Small et al. 2000 [8]



Burch et al. 2004 [14]

Beinart et al. 2005

Rini B.I. 2006 [11]


Higano et al. 2009

Higano et al. 2009 [18]

Schellhammer et al. 2009 [19]

Table 1. Clinical Trials of Sipuleucel-T.


Placebo, 45 98 Sipuleucel-T, 65 Placebo, 33 225 Sipuleucel-T, 147 Placebo, 78 512 Sipuleucel-T, 341 Placebo, 171

127 Sipuleucel-T, 82

22

19

21

31

19

12

13

No. of patients

-- 91) -- 89)

-- 85) -- 87)

-- 84) -- 87)

(3.5 -- 3,621) (7.9 -- 2,799)

(8.0 -- 936.5) (8.2 -- 1342.5)

(7.9 -- 2799) 47.9 N/A 61.3 44.0 N/A 50.7 45.8 N/A 51.7 47.2

-- 86)

71 (50 N/A 70 (51 71 (57 N/A 72 (47 71 (50 N/A 72 (49 70 (40

N/A 46.0 (3.5 -- 3621)

2.3 (0.5 -- 5.1)

70 (55 -- 79)

N/A 73 (47 -- 85)

1.6 (0.5 -- 3.1)

221 (21 -- 1147)

41.3 (3.4 -- 1007)

14.5 (3.4 -- 216)

209 (26.3 -- 1007)

323 (19.8 -- 920.9)

Baseline PSA (median, range)-ng/ml

67 (55 -- 78)

72 (57 -- 83)

69 (48 -- 83)

72 (50 -- 83)

67.5 (48 -- 79)

67.5 (59 -- 84)

Age (median, range)-yr

Three infusions of sipuleucel-T at weeks 0, 2, and 4 Three infusions of placebo at weeks 0, 2 and 4

Three infusions of sipuleucel-T at weeks 0, 2 and 4 Three infusions of placebo at weeks 0, 2 and 4

Three infusions of sipuleucel-T at weeks 0, 2 and 4 Three infusions of placebo at weeks 0, 2 and 4

Three infusions of sipuleucel-T (median, 3.65 109 nucleated cells/infusion) at weeks 0, 2 and 4 Three infusions of placebo at weeks 0, 2 and 4

Two infusions of sipuleucel-T at week 0 and 4; three subsequent subcutaneous injections of PA2024 (0.3, 0.6 or 1.0 mg/injection) at weeks 8, 12 and 16 Three infusions of sipuleucel-T (0.2 109, 0.6 109, 1.2 109 or 2.0 109 nucleated cells/m2) at weeks 0, 4, 8 and one infusion of sipuleucel-T at week 24 if disease was stable or improving Three infusions of sipuleucel-T (0.2 109, 0.6 109, 1.2 109 or 2.0 109 nucleated cells/m2) at weeks 0, 4, 8 and one infusion of sipuleucel-T at week 24 if disease was stable or improving Three infusions of sipuleucel-T (0.2 109, 0.6 109, 1.2 109 or 2.0 109 nucleated cells/m2) at weeks 0, 4, 8 and one infusion of sipuleucel-T at week 24 if disease was stable or improving Two infusions of sipuleucel-T at week 0 (median, 2.7 109 nucleated cells/infusion) and week 2 (median, 3.2 109 nucleated cells/infusion); three subsequent subcutaneous injections of PA2024 (1.0 mg/injection) at weeks 4, 8 and 12 Three infusions of sipuleucel-T (1.2 109 nucleated cells/m2) at weeks 0, 2 and 4 Three infusions of sipuleucel-T (1.2 109 nucleated cells/m2) at weeks 0, 2 and 4; Bevacizumab was given at a dose of 10 mg/kg i.v. on weeks 0, 2, 4, and every 2 weeks thereafter until toxicity or disease progression

Administration of Sipuleucel-T


Sipuleucel-T

Phase II, CRPC

Phase II, ADPC Phase II, ADPC, plus Bevacizumab Phase III, CRPC (D9001)

Phase III, CRPC (D9002A)

Phase III, CRPC (D9001/D9002A)

Phase III, CRPC (IMPACT)

Burch et al. 2004 [14]

Beinart et al. 2005 [16] Rini et al. 2006 [11]



Schellhammer et al. 2009 [19]


Phase Phase Phase Phase

Burch et al. 2000 [9] Small et al. 2000 [8] Small et al. 2000 [8] Small et al. 2000

I, CRPC I, CRPC II, CRPC I/II, CRPC

Clinical trial

Author [Ref.]

127 Sipuleucel-T, 82 Placebo, 45 98 Sipuleucel-T, 65 Placebo, 33 225 Sipuleucel-T, 147 Placebo, 78 512 Sipuleucel-T, 341 Placebo, 171

19 22

21

13 12 19 31

No. of patients

Table 1. Clinical Trials of Sipuleucel-T (continued).

N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A

25% (3/12) ‡ 50% N/A N/A 9.7% (3/31): ‡ 50% 9.7% (3/31): 25 -- 49% 10.5% (2/19): 25 -- 50% 5.3% (1/19): undetectable 38.9% (7/18): 6 -- 33% 42.9% (9/21): 6 -- 72%

PSA decline

N/A 11.7 weeks 9.1 weeks N/A 10.9 weeks 9.9 weeks N/A 11.1 weeks 9.7 weeks N/A 14.6 weeks 14.4 weeks

11.7 months 11.2 months

118 days

135 days 12 weeks 29 weeks N/A

Median time to disease progression

N/A 25.9 21.4 N/A 19.0 15.7 N/A 23.2 18.9 N/A 25.8 21.7

N/A N/A

N/A

N/A N/A N/A N/A


months months

months months

months months

months months

Median survival

Efficacy

N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 66.2% (100/151) 2.9% (2/70)

N/A 100%

86.7% (13/15)

100% 100% 100% 100%

Anti-PA2024 B cell response


N/A N/A N/A N/A N/A N/A N/A N/A N/A N/A 73.0% (46/63) 12.1% (4/33)

N/A 100%

N/A

100% 100% 100% 100%

Anti-PA2024 T cell response

N/A 70.8% 68.9% N/A 58.5% 67.7% N/A 65.3% 68.4% N/A 67.1% 61.3%

40.7% ‡ 73%

76.6%

50% N/A N/A 14.7%

Grade 1 -- 2

N/A 24.4% 24.4% N/A 44.6% 32.3% N/A 33.3% 27.6% N/A 31.7% 35.1%

1.9% 4/22

10.6%

0 N/A N/A 0

Grade 3 -- 4

Toxcity


105


Sipuleucel-T

cells/m2, with no dose-limiting toxicities observed [17], and in D9902A, a similar dose of 3.65 109 nucleated cells/ infusion was applied [18]. To date, the minimum and maximum cell threshold for each dose of sipuleucel-T has not been disclosed. Initial Phase I and II clinical trials of sipuleucel-T evaluated various administration frequencies and found that two infusions, without subsequent subcutaneous injections of PA2024, were adequate to stimulate cell-mediated immunity to the target antigen PA2024 [8,9]. Although monthly sipuleucel-T administration was studied in prior trials, biweekly administration was selected in subsequent Phase III trials. This modification was based on findings in which a similar degree of immune response was detected after infusion of sipuleucel-T at Weeks 0 and 4 and at Weeks 0 and 2 in two study cohorts [14]. As a cancer vaccine, sipuleucel-T typically has low toxicity. Immune response measurements can be confounded by the previous oncologic treatments prior to receiving sipuleucelT, causing compromised immune responses, rendering evocation or detection of an elicited immune response difficult. In Phase I and II trials, active immune response was observed, and was described in terms of anti-PAP response rate (38%), anti-PA2024 immune response rate (100%) and T-cell proliferation response rate (100%) (Table 1). In Phase III trials D9901 and D9902A, cumulative CD54 upregulation was introduced for monitoring the patient immune responses, and a strong relationship between cumulative CD54 upregulation and overall survival was demonstrated in a Cox regression model (p = 0.009). This association persisted after adjustment for baseline prognostic factors including bodyweight, PSA, lactate dehydrogenase, number of bone lesions and location of disease; p = 0.022) [18]. The adverse events associated with sipuleucel-T generally occurred soon after treatment, were low grade and transient, lasting one to two days. In the Phase III trials, a possible increased risk of cerebrovascular events was observed in the sipuleucel-T treatment arm [18]. The cerebrovascular events included hemorrhagic, ischemic and embolic strokes, transient ischemic attacks as well as bleeding from a dural metastatic lesion. The incidence of cerebrovascular events was 7.5% in the sipuleucel-T arm and 2.6% in the placebo arm. The cerebrovascular events were reported over a wide range of time (26 -- 859 days from the first infusion), and the majority of events were non-fatal. An ongoing study in ADPC has demonstrated an appropriate trend [27]. Additional studies are underway to better characterize the nature of these events. 6.

Regulatory affairs

7.

Conclusions

Sipuleucel-T has demonstrated efficacy and safety in Phase I/II trials. However in Phase III trials, results were seen with definite efficacy in some trials, and a trend towards efficacy in some other trials. The latest data from IMPACT showed that sipuleucel-T met the primary end points by extending median survival by 4.1 months compared with placebo and improving 3-year survival by 31.7% [20]. Further trials are ongoing to prove efficacy and support market authorization, as well as to improve understanding of the mechanisms behind cancer vaccine failure. Ongoing trials involving sipuleucelT will help clarify and probably expand the patient population for whom this vaccine will be most efficacious. 8.

Expert opinion

As one of the novel cancer vaccines for the treatment of metastatic, asymptomatic CRPC, sipuleucel-T has demonstrated efficacy, safety and tolerability in different clinical trials, as well as the personalized characteristic of active cancer immunotherapy. Sipuleucel-T is uniquely designed to use live human immune cells to engage the patient’s own immune system with the goal of eliciting a specific long-lasting response against prostate cancer. For the same reason, manufacturing a product that is customized to each individual patient is challenging. Hopefully, further work will further define true efficacy, support market acceptance and more clearly identify the patient population for whom this vaccine will be suitable.

Declaration of interest

On May 9, 2007 the US FDA decided not to approve sipuleucel-T, even though in March 2007 its own advisory committee reviewed the clinical safety and efficacy data and

106

voted in favor of both the safety and efficacy of sipuleucel-T. This decision was taken based on the fact that data from D9901 failed to meet its primary end points [17]. On November 2, 2009 the Dendreon Corp. announced it had completed the submission of the amended Biologics License Application (BLA) for Provenge to the FDA, seeking licensure for Provenge for men with metastatic CRPC. The amended BLA includes data from the IMPACT trial, which was conducted under a Special Protocol Assessment agreement with the FDA. On November 20, 2009, the FDA accepted Dendreon’s amended BLA as a complete response and set a Prescription Drug User Fee Act date of May 1, 2010 [28]. On April 29, 2010, the FDA approved sipuleucel-T for the treatment of asymptomatic or minimally symptomatic metastatic castrate resistant (hormone refractory) prostate cancer.

This paper has been sponsored by the Provincial Surgical Oncology Program for the Southwestern Ontario Region. The authors declare no conflicts of interest.



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Taussig MJ. Induction of hapten-specific B cell tolerance by low doses of hapten-carrier conjugate. Nature 1973;245:34-6 Beinart G, Rini BI, Weinberg V, Small EJ. Antigen-presenting cells 8015 (provenge) in patients with androgen-dependent, biochemically relapsed prostate cancer. Clin Prostate Cancer 2005;4:55-60 Small EJ, Schellhammer PF, Higano CS, et al. Placebo-controlled Phase III trial of immunologic therapy with sipuleucel-T (APC8015) in patients with metastatic, asymptomatic hormone refractory prostate cancer. J Clin Oncol 2006;24:3089-94 Higano CS, Schellhammer PF, Small EJ, et al. Integrated data from 2 randomized, double-blind, placebo-controlled, Phase 3 trials of active cellular immunotherapy with sipuleucel-T in advanced prostate cancer. Cancer 2009;115:3670-9 Schellhammer PF, Higano CS, Berger ER, et al. A randomized, double-blind, placebo-controlled,


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Affiliation Chen Lu¨, Andrew K Williams, Venu Chalasani, Carlos H Martıńez & Joseph Chin† † Author for correspondence University of Western Ontario, Divisions of Urology and Surgical Oncology, Departments of Surgery and Oncology, 800 Commissioners Road East, London, Ontario, N6A 4G5, Canada E-mail: [email protected]

108


Drug Evaluation

Adalimumab in ulcerative colitis: hypes and hopes


Gionata Fiorino, Laurent Peyrin-Biroulet, Alessandro Repici, Alberto Malesci & Silvio Danese† 1.

Introduction

2.

Methods

3.

Results

4.

Conclusions

5.

Expert opinion

†

IBD Unit, Gastroenterology, IRCCS Humanitas, Rozzano, Milan, Italy

Importance of the field: The advent of anti-TNF-a monoclonal antibodies has dramatically changed the management of inflammatory bowel diseases (IBD). Unlike Crohn’s disease (CD), only one anti-TNF-a agent, infliximab, is currently approved for active moderate-to-severe ulcerative colitis (UC). Adalimumab is a fully human anti-TNF-a antibody that is effective and safe for the treatment of luminal and fistulising CD. Areas covered in this review: This review of the literature summarizes available data on of efficacy and safety profile adalimumab in patients with UC. What the reader will gain: Adalimumab may be effective in inducing and maintaining clinical remission in patients with moderate-to-severe UC. It may also induce mucosal healing and reduce the need for colectomy in patients with severe disease. The safety profile of the drug in UC is consistent with previous experience with this drug in CD. Take home message: Adalimumab may be effective and well tolerated in UC. Its efficacy in maintaining clinical remission needs to be confirmed in a randomized controlled trial. Keywords: adalimumab, anti-TNF-a, inflammatory bowel disease, ulcerative colitis Expert Opin. Biol. Ther. (2011) 11(1):109-116

1.

Introduction

Tumour necrosis factor-a in inflammatory bowel disease Ulcerative colitis (UC) and Crohn’s disease (CD) are two inflammatory bowel diseases (IBD) characterized by chronic inflammation in the gut. Although their etiology remains unknown, they share similar pathogenetic mechanisms, especially the production of pro-inflammatory cytokines that trigger and maintain chronic inflammation. TNF-a is a key pro-inflammatory cytokine that is produced by macrophages, and activated T-cells in response to intestinal injury [1]. The production of TNF leads to further stimulation and recruitment of immune cells in the gut [2], and to the activation of fibroblasts and endothelial cells [3]. Increased TNF-a levels can be found in the gut mucosa of patients with IBD of the large bowel [4,5]. 1.1

Anti-TNF-a blockers in IBD The key-role of TNF-a in chronic immune-mediated diseases has led to the development of biological agents able to block TNF-a-mediated pathways [6]. Infliximab, adalimumab and certolizumab are three monoclonal antibodies that are currently approved by the FDA for the treatment of active moderate-to-severe CD [7]. Infliximab, a chimeric monoclonal IgG, is currently the only anti-TNF-a approved for the use in moderate-to-severe active UC. Two large studies, the acute ulcerative Colitis Treatment (ACT)-1 and ACT-2, have shown that it is effective and safe in UC and that it can significantly decrease the risk of colectomy at one year [8]. The chimeric structure of infliximab (Figure 1A) can lead to development of antibodies directed against the drug (ATI) that are associated with loss of response 1.2


109

Adalimumab

Box 1. Drug summary. Drug name Phase Indication Pharmacology description

Route of administration Pivotal trial(s)

Adalimumab Launched Ulcerative collitis Tumour necrosis factor receptor antagonist Immunosuppressant Parenteral [17]


Pharmaprojects - copyright to Citeline Drug Intelligence (an Informa business). Readers are referred to Pipeline (http://informa-pipeline. citeline.com) and Citeline (http://informa.citeline.com).

to the drug [9,10]. Adalimumab (Box 1) is a recently developed fully human monoclonal IgG directed against TNF-a. Because of the absence of any non-human protein in its structure (Figure 1B), it seems to be less immunogenic than infliximab [11]. Recently, four randomized trials have demonstrated that adalimumab is effective and safe for the induction and maintenance of clinical remission in patients with active moderate-to-severe CD [12-15]. However, all anti-TNF agents have the potential for immunogenicity. Consistent with this, CD patients under adalimumab can develop antiadalimumab antibodies, [16]. Recently, several studies have investigated the efficacy and safety of adalimumab in patients with UC [17-25]. Here, we review available data from uncontrolled and controlled trials that investigated adalimumab as first-line anti-TNF agent or in case of infliximab failure in UC. 2.

Methods

A review of the literature searching for the terms ‘TNF-a’, ‘anti-TNF’, ‘biological agent’ or ‘adalimumab’ matched with the terms ‘inflammatory bowel disease’ or ‘ulcerative colitis’ was performed, using PubMed, MEDLINE, EMBASE, Cochrane and Ovid databases. All relevant articles in English between January 2000 and August 2010 were reviewed. Relevant abstracts presented to United European Gastroenterology Week, Digestive Disease Week and European Crohn’s Colitis Organization Congress were also included in our review. 3.

Results

One randomized placebo-controlled double blinded trial (RCT) [17], five open-label studies [18-22], two retrospective studies [23,24], and one case report on two patients [25], were identified following literature search. In all studies, adalimumab was administered at 160/80 mg induction regimen and then 40 mg every other week, except in one patient who received 80/40 mg induction regimen [22], and in the only RCT, where the protocol was amended to include a 80/40 mg induction regimen group [17]. The interval between 110

adalimumab injections was shortened to 40 mg every week when necessary. Characteristics of the studies that evaluated efficacy of adalimumab in UC are summarized in Tables 1 and 2. 3.1

Efficacy of adalimumab in UC Randomized controlled trials

3.1.1

The only randomized controlled multicentre trial enrolled 576 patients with active UC [17]. Patients were all naı¨ve to anti-TNF-a. All patients were randomized 1:1 to receive adalimumab 160/80 mg subcutaneously or placebo and then 40 mg or placebo every 2 weeks. During the enrolment phase, the protocol was amended to include a third group receiving adalimumab at induction dose of 80/40 mg. All patients had Mayo Score equal or greater than 6 and endoscopic subscore greater than 1 at baseline. Primary endpoint was clinical remission, defined as a Mayo Score £ 2 at week 8. Secondary endpoints were clinical response, mucosal healing, and improvement in rectal bleeding, physician global assignment (PGA) and stool frequency at week 8. At that time point, remission rates of patients in the 160/80 group (19.2%) were significantly higher than those in the placebo group (9.2%, p = 0.021), while no significant differences were found between the 80/40 mg group (10% of patients, p = 0.833) and the placebo group. Regarding secondary endpoints, no differences were found in terms of clinical response and mucosal healing among the three arms, whereas induction with 160/80 mg was more effective than placebo for improvement of rectal bleeding, PGA and improvement in stool frequency. For all secondary endpoints, statistically significant differences were observed between 160/80 mg and placebo groups, whereas no differences were found between the 80/40 mg and placebo groups. Overall, despite of the lower remission rates in the 160/80 group compared with the results of the ACT 1 and 2 trials for infliximab [8], this study demonstrated that adalimumab (160/80 mg) is significantly more effective than placebo in inducing the remission of active UC, while efficacy of the 80/40 mg regimen is not different from that of placebo. Open-label trials Five open-label trials have evaluated so far the efficacy and safety profile of adalimumab in UC patients. Peyrin-Biroulet et al. [18] investigated the efficacy of 160/80 mg induction regimen in 10 patients with UC who were intolerant or lost response to infliximab. About 70% of patients had extensive disease (defined as extended proximal to the splenic flexure) and 60% of patients had severe disease at baseline; 60% of patients had lost response to infliximab while 40% were intolerant. In order to evaluate the clinical efficacy of adalimumab, the ulcerative colitis clinical activity index (CAI) was calculated at baseline and at week 4. Levels of C-reactive protein (CRP) were also measured. At week 4, one patient achieved clinical remission (10%), defined as CAI less than four, three patients had clinical improvement 3.1.2


Fiorino, Peyrin-Biroulet, Repici, Malesci & Danese


. . . .

Infliximab is more effective than placebo for inducing and maintaining clinical remission in patients with ulcerative colitis. Adalimumab may be effective and safe in inducing and maintaining steroid-free remission in ulcerative colitis. Adalimumab therapy may be associated with a lower need for colectomy in ulcerative colitis. Predictors of response to adalimumab as well as its efficacy to heal mucosa will require further investigation. Efficacy of adalimumab to maintain clinical remission needs to be confirmed in a randomized controlled trial.



A.

Infliximab

B.

Adalimumab

Murine Fv component

Human Fv component Human Fcγ1

Figure 1. Differences in structure between infliximab (A) and adalimumab (B).

(30%), defined as at least a four-point decrease of CAI, and six patients (60%) did not respond to the treatment. Two patients belonging to this group underwent total coloproctectomy after 2 weeks from the inclusion in the study. Patients with left-sided colitis responded better than patients with extensive colitis (50 versus 16.6%, respectively). Most patients with severe colitis (CAI > 12) did not respond to adalimumab. Half of the patients with prior intolerance to infliximab responded to treatment, and 33.3% of patients with prior loss of response gained improvement by adalimumab. Except for the two patients requiring colectomy, mean CRP rapidly decreased within 4 weeks. Subsequently, 13 patients, including those completing the above-mentioned trial, were evaluated for maintaining the long-term clinical remission (CAI < 4) under adalimumab (median follow-up 42 weeks) [19]. Eight patients lost response to infliximab, five patients were intolerant to infliximab. CAI and CRP levels were measured at each visit every 4 weeks. In this study, five patients were able to continue adalimumab treatment during follow-up, while eight patients discontinued

the drug because of drug intolerance (one patient), nonresponse (one patient), and need for colectomy (six patients). Out of 13 patients, 6 needed to shorten interval between injections from every other week to weekly. The cumulative probability of remaining colectomy-free was 92.3, 84.6, 69.2 and 49.5% respectively at month 1, 3, 6 and 23. Responders to adalimumab showed a decrease in CRP blood levels, while non-responders had higher CRP at last followup compared with baseline. No differences were found between patients that lost response to infliximab and patients intolerant to infliximab. Another open-label uncontrolled trial with 20 patients investigated the efficacy and safety of adalimumab in clinically and endoscopically active UC [20]. Of these patients, 13 were intolerant or had prior loss of response to infliximab and 7 patients were naı¨ve to biological therapy. Clinical response (a decrease of 30% of baseline Mayo Score with a decrease of rectal bleeding subscore ‡ 1 or equal to 0 or 1) was evaluated up to week 24. Of the 20 patients, 5 had clinical response at week 8 (3 patients previously exposed to infliximab and 2 naı¨ve), one patient who was naı¨ve to anti-TNF-a had clinical remission (defined as Mayo score £ 2 points with no individual score > 1 point) at the same time point. Ten patients had clinical response (50%), and in this subgroup five patients had clinical remission at week 24. Seven patients out of 12 (58%) were able to discontinue oral steroids at week 24. Six patients out of 20 (30%) showed mucosal healing at week 8 (defined as a decrease in the endoscopy subscore from 2 or 3 at baseline to 0 or 1), including four patients previously exposed to infliximab and two naı¨ve for biologics. A ‘real-life’ clinical practice single-centre prospective study has been conducted on a cohort of 53 patients evaluating the efficacy and safety of infliximab (first group, 28 patients) and adalimumab in UC (second group, 25 patients) [21]. All patients were naı¨ve to biologics and had active disease at inclusion in the study. The majority of patients (89.3% in the infliximab group and 96% in the adalimumab group) had pancolitis. All patients had high CRP levels at baseline. Definition of response was based on a PGA, which included normalization of bowel movements, absence of rectal bleeding and ability to discontinue steroids. This study showed that 80% of patients responded to adalimumab at week 14, without any significant difference compared with the infliximab group. A total of 20 patients were able to complete the induction phase of the study and were enrolled in the maintenance phase. At the end of the follow-up (mean period 54.5 weeks), 14 patients out of 20 (70%) were still responders while 6 patients discontinued the drug and underwent a new course of oral steroids (3 patients), a switch to infliximab (1 patient) or colectomy (2 patients). All patients under adalimumab were able to discontinue steroids during the maintenance phase. All results concerning the adalimumab group did not differ significantly from infliximab group. Recently, Taxonera et al. [22] treated 30 patients with prior exposure to infliximab (16 with loss of response, 2 primary


111

Adalimumab


Table 1. Characteristics of studies. Author (year) [ref.]

Type of study

Number of patients

Naı¨ve to anti-TNF

Clinical score

Induction regimen

Maintenance regimen

Reinisch et al. (2010) [17]

RCT

576

NA

Mayo

40 mg eow

Peyrin-Biroulet et al. (2007) [18] Oussalah et al. (2008) [19]

Open-label trial

10

None

CAI

160/80 80/40 Placebo 160/80

NA

Open-label trial

13

None

CAI

NA

40 mg eow

Afif et al. (2010) [20] Gies et al. (2010) [21] Taxonera et al. (2010) [22] Hudis et al. (2009) [23]

Open-label trial Open-label trial Open-label trial

20 25 30

7 25 None

Mayo Mayo Mayo

40 mg eow 40 mg eow 40 mg eow

Retrospective

9

1

Mayo

160/80 160/80 160/80 (n = 29) 80/40 (n = 1) 160/80

Garcıà-Bosch et al. (2010) [24] Barreiro de Acosta et al. (2010) [25]

Retrospective

48

10

Mayo

160/80

40 mg eow

Case report

2

2

Mayo

160/80 mg

40 mg eow

Notes

Six patients switched to 40 mg weekly

11 patients switched to 40 mg weekly 4 patients switched to 40 mg weekly

40 mg eow

CAI: Clinical activity index; eow: Every other week; NA: Not available; RCT: randomized controlled trial.

Table 2. Efficacy of adalimumab in ulcerative colitis. Author (year) [ref.]

Time point

Response rate

Remission rate

Mucosal healing

Colectomy rate

Notes


8 weeks

NA

160/80 80/40 Placebo

4 weeks

19.2%* 10.0% 9.2% 10%

46.9% 37.7% 41.5%

Peyrin-Biroulet et al. (2007) [18] Oussalah et al. (2008) [19] Afif et al. (2010) [20]

54.6% 51.5% 44.6% 40%

NA

20%

Gies et al. (2010) [21] Taxonera et al. (2010) [22] Hudis et al. (2009) [23]

Garcıà-Bosch et al. (2010) [24] Barreiro de Acosta et al. (2010) [25]

42 weeks (on average) 8 weeks 24 weeks Week 14 Week 54 Week 12 Week 40 NA

NE

38%

NA

49.5% at month 23

20% 50% 80% 70% 56.7% 50% NA

5% 25% 70%

30%

NE

NE

NE

26.7% 45.8% NA

NE

20%

NE

NE

Week Week Week Week

75.6% 47.1% 37.5% 100%

NA

NA

16%

100%

100%

0%

12 28 54 104

Mean decrease in Mayo Score of 4 points for all patients. 67% gained response after increasing the dose

CAI: Clinical activity index; eow: Every other week; NA: Not available; NE: Not evaluated; RCT: Randomized controlled trial; w: Weekly.

failures and 12 with intolerance to infliximab) with adalimumab 160/80 mg (29 patients) or 80/40 mg (1 patient). Clinical response was evaluated up to week 40. Remission rate, need for colectomy and predictors of response were also evaluated. Clinical response was defined as a 3-point decrease 112

in the Partial Mayo Score (PMS) or a final PMS £ 1, and clinical remission was defined as a PMS £ 1. After the induction phase, at week 12, 56.7% of patients achieved clinical response; 26.7% of patients achieved clinical remission at week 12. Half of the patients (n = 15) continued adalimumab



treatment up to 41 weeks on average. Eleven patients required dose intensification (40 mg weekly), and 6 patients underwent colectomy. The probability of remaining on adalimumab was 96.7, 90, 68, 60, and 45.8% at weeks 4, 12, 20, 30 and 40, respectively. The use of concomitant immunomodulators was the only independent predictor of clinical response to adalimumab. A total of 17 patients (56.7%) had clinical response at week 12 and were likely to maintain long-term remission (p = 0.03).

from mesalamine, one for a rash, one for urinary tract infection and worsening abdominal pain, one for intermittent heartburn and increase of INR, and one for gastroenteritis with dehydration. No relevant adverse events were reported in the other studies [21-25]. Data on safety of adalimumab in the only randomized controlled trial conducted so far in UC are currently not available [17]. Overall, its safety profile in UC is consistent with previous experience with this drug in CD. All available data are summarized in Table 3.

Retrospective studies Hudis et al. [23] have evaluated the efficacy of adalimumab in UC and the rate of steroid-free clinical remission in a retrospective study including nine patients. About half of the patients had pancolitis, 44% left-sided colitis, and eight patients (89%) had previous exposure to infliximab. Mean Mayo subscore decreased by four points on average during followup and all five patients on steroids at baseline were able to discontinue. Adalimumab was increased at 40 mg weekly in 43% of patients, which gained response in 67% of cases. Another retrospective study [24] of 48 patients evaluated predictors of response to adalimumab therapy and the need for colectomy in UC patients. Notably, 81.3% of patients had prior exposure to infliximab. Response rates were 75.6% at week 12, 47.1% at week 28, and 37.5% at week 54. About 16% of patients underwent colectomy after a median time of 29.4 weeks. Response to adalimumab at week 12 in those patients who previously achieved remission, response or nonprimary response to infliximab, was 89, 5, 53, 8 and 50% respectively. The only independent predictor of response to adalimumab was prior exposure to infliximab (p < 0.05).

4.


3.1.3

Case reports Two UC patients who had experienced infliximab failure were treated with adalimumab in combination thiopurines. Both patients were in clinical remission after week 8 and showed a complete mucosal healing after 2 years of treatment (Mayo endoscopic subscore equal to 0) [25]. 3.1.4

Safety No deaths or malignancies have been reported in UC patients treated with adalimumab (Table 3). In the study by PeyrinBiroulet et al. [18] only two mild adverse events were reported, none of them leading to discontinuation of the drug and easily recovered (one fungal dermatitis and one cutaneous rash). After long-term maintenance adalimumab therapy, five patients (38.5%) showed adverse efects: three patients had mild infections, one had arthralgia, and one had psoriasis de novo [19]. As mentioned above, only one patient enrolled in the study had to withdraw adalimumab due to exacerbation of psoriasis [19]. In the study by Afif et al. [20], serious adverse events were reported in 6 out of 20 patients, none of them were judged as probably related to the administration of adalimumab. Nine patients then required to discontinue adalimumab, four for worsening of UC, one for pneumonitis 3.2

Conclusions

There is an increasing evidence that adalimumab may be an effective and safe therapeutic option for patients with moderate-to-severe UC refractory to standard medications, such as steroids, thiopurines, calcineurine inhibitors and infliximab, avoiding colectomy in a significant number of individuals. It seems to be effective in both anti-TNF-naı¨ve patients and in those previously treated with infliximab. Further randomized controlled trials are needed to confirm the efficacy of adalimumab in maintaining clinical remission in UC patients and to identify predictors of response to this drug. The efficacy of adalimumab for inducing and maintaining mucosal healing in UC will also require further investigation. 5.

Expert opinion

In the last decade, biologics have brought a revolution in the management of IBD. Unfortunately, the main efforts in research have been primarily directed to CD, and all new treatments are generally investigated in CD patients first and then shifted to UC, thus considering UC as the ‘Cinderella’ of IBD. UC is actually different from CD and research should consider an independent pathway to follow for the ultimate goal, which is to cure definitively the colonic inflammation and avoid complications and surgery. Major endpoints in the clinical management of UC are mainly considered to be clinical steroid-free long-term remission, mucosal healing, reduction of colectomy rate and, hopefully, the reduction of risk of colorectal cancer. The role of anti-TNF-a in UC to achieve these endpoints is now well established, as infliximab has been shown to be effective and safe in cases of active moderate-to-severe UC, in terms of steroid-free remission and mucosal healing [8]. Infliximab is also the only drug able to reduce significantly the need for colectomy in patients with moderate-to-severe ulcerative colitis [26,27]. The latter can be considered a great success, because about 20% of all UC patients require colectomy during 10 years of disease, raising up to 40% colectomy rate in cases of long-lasting pancolitis [28]. Unfortunately, we currently do not have enough therapeutic choices in cases of UC patients with steroid-dependant or steroid-refractory disease. Compared with at least four biological agents that can be used in CD, infliximab is the only biological agent approved for UC, and if patients are primary


113

Adalimumab


Table 3. Safety of adalimumab in ulcerative colitis. Author (year) [ref.]

Adverse Serious adverse Malignancies events events


NA

4.0%

Two patients 0% (placebo group)

Peyrin-Biroulet et al. (2007) [18] Oussalah et al. (2008) [19] Afif et al. (2010) [20]

20% 38.5% 85%

40% NE 30%

0% 0% 0%

NA NA 0%

Gies et al. (2010) [21] Taxonera et al. (2010) [22] Hudis et al. (2009) [23]

4% 20% NA

0% 6% NA

0% 0% NA

0% 0% NA

NA 0%

NA 0%

NA 0%

NA Garcıà-Bosch et al. (2010) [24] Barreiro de Acosta et al. (2010) [25] 0%

Deaths Notes

SAE were respectively 3.8 and 7.6% in the 80/40 mg group and in the placebo group

All SAE were not related to the use of adalimumab

Mean decrease in Mayo Score of 4 points for all patients. 67% gained response after increasing the dose

NA: Not available; NE: Not evaluated.

non-responders or lose response or become intolerant, then there will be no other solution than colectomy. Evidence coming from large studies on CD [12-14] show that adalimumab is effective and safe and that it can certainly play a key role in the management of UC, although data coming from the only RCT show lower response and remission rates when compared with infliximab [8]. Unfortunately, it is not possible to compare both studies in terms of patient population, duration, severity and extent of the disease in order to find possible explanations for such difference. Data in the literature collected in our review show that major endpoints such as steroidfree remission, mucosal healing and the reduction in colectomy rate can be achieved with adalimumab. Anyway, studies are mostly open-label trials or retrospective studies and all data cannot be directly compared due to different patient populations, different extent and behaviour of the disease. Most of the patient populations in clinical studies include patients with long lasting disease that already failed anti-TNF-a therapy and then we cannot evaluate whether the use of anti-TNF-a in early disease, that is within 2 years from diagnosis, can positively affectthe achievement of major endpoints. Moreover, it seems clear that the 160/80 mg induction regimen is the only effective dosage in UC, as shown by the Reinisch et al. trial [17]. The use of adalimumab in early disease especially in patients who are naı¨ve for immunosuppressants or steroids may result in higher response and remission rates and maybe can dramatically improve the natural evolution of the disease. Recently, a ‘top down’ approach has been investigated in CD [29] showing that the use of early combined immunosuppression results in significantly higher remission rates than conventional ‘step-up’ strategy, with no increase in adverse events. A ‘top-down’ or ‘accelerated step-up’ approach have been never investigated in UC and it could probably lead to improvement of the clinical management of UC patients. Taxonera and colleagues [22] have found that concomitant immunosuppressant therapy with thiopurine is the only 114

predictive factor for response to adalimumab. Combination of infliximab and azathioprine has been recently investigated in CD [30]. This trial has shown that combination of infliximab and azathioprine results in significantly higher response rates at week 26 compared with monotherapy with infliximab or azathioprine. Recently, a large multicenter randomized trial has been completed investigating the efficacy of combination of infliximab and azathioprine versus monotherapy with infliximab or azathioprine [31]. Results have been not yet published, but they can probably add new evidence on which strategy is the most effective in active UC. Beside the urgent need for development of new therapeutic strategies and new drugs in UC, aetiology and pathogenesis in UC should be further investigated. Mechanisms triggering and maintaining chronic inflammation in the gut, the role of environmental factors and possible prognostic factors for disease evolution or response to therapy are needed to be elucidated in order to drive new therapeutics for specific pathogenetic targets. Concerning the use of anti-TNF-a agents, the identification of genetic, biological and clinical factors that can predict the successful response to these drugs may lead to tailoring of the therapy to a specific set of patients who would greatly benefit from it. In conclusion, studies on adalimumab in UC give the chance to increase the therapeutic armamentarium in the management of active moderate-to-severe disease. At the moment, data do not support the role of adalimumab as first-line choice in patients with UC requiring treatment with anti-TNF-a, therefore it should be used as rescue therapy after failure of steroids, immunosuppressants and infliximab. Further studies are needed to identify clear predictive factors of response and specific subpopulations that could successfully respond to the treatment. Research on antiTNF-a should also keep on finding new strategies to optimize UC management and dramatically affect the natural history of the disease.



Declaration of interest S Danese has served as a speaker, consultant and advisory board member for Schering-Plough, Abbott Laboratories, UCB, Ferring, Cellerix, Millenium Takeda, Actelion Bibliography Papers of special note have been highlighted as either of interest () or of considerable interest () to readers. 1.


2.

..

3.

.

4.

5.

6.

7.

..

8.

..

Watts TH. TNF/TNFR family members in costimulation of T cell responses. Annu Rev Immunol 2005;23:23-68 Tracey D, Klareskog L, Sasso EH, et al. Tumour necrosis factor antagonist mechanism of action: a comprehensive review. Pharmacol Ther 2008;117:244-79 This article gives a detailed overview of anti-TNF-a. Danese S, Fiocchi C. Etiopathogenesis of inflammatory bowel diseases. World J Gastroenterol 2006;12:4807-12 This paper focuses on the recent advances in the knowledge of aetiology and pathogenesis of IBD. Murch SH, Lamkin VA, Savage MO, et al. Serum concentrations of tumour necrosis factor alpha in childhood chronic inflammatory bowel disease. Gut 1991;32:913-17 Murch SH, Braegger CP, Walker-Smith JA, MacDonald. Location of tumour necrosis factor alpha by immunohistochemistry in chronic inflammatory bowel disease. Gut 1993;34;1705-9 Danese S. Mechanisms of action of infliximab in inflammatory bowel disease: an anti-inflammatory multitasker. Dig Liver Dis 2008;40(Suppl 2):S225-8 Peyrin-Biroulet L, Deltenre P, de Suray N, et al. Efficacy and safety of tumor necrosis factor antagonists in Crohn’s disease: meta-analysis of placebo-controlled trials. Clin Gastroenterol Hepatol 2008;6:644-53 A key paper that analyses efficacy and safety of anti-TNF-a in Crohn’s disease. Rutgeerts P, Sandborn WJ, Feagan B, et al. Infliximab for induction and maintenance therapy for ulcerative colitis. N Engl J Med 2005;233:2462-73 This is the first, to our knowledge, large randomized trial on infliximab in ulcerative colitis.

and Cosmo Pharmaceuticals. L Peyrin-Biroulet has received consulting fees and lecture fees from speaking at continuing medical education events from Abbott Laboratories. The other authors declare no conflict of interest.

9.

Gisbert JP, Panes J. Loss of response and requirement of infliximab dose intensification in Crohn’s disease: a review. Am J Gastroenterol 2009;104:760-7

10.

Baert F, Noman M, Vermeire S, et al. Influence of immunogenicity on the long-term efficacy of infliximab in Crohn’s disease. N Engl J Med 2003;341:601-8 This article investigates the role of immunogenicity in loss of response to anti-TNF-a.

.

11.

Breedveld FC. Therapeutic monoclonal antibodies. Lancet 2000;355(9205):735-40

12.

Hanauer SB, Sandborn WJ, Rutgeerts P, et al. Human anti-tumor necrosis factor monoclonal antibody (adalimumab) in Crohn’s disease: the CLASSIC-I trial. Gastroenterology 2006;130:323-33 This is the largest RCT on efficacy of adalimumab for the induction of remission in naı¨ve patients with active CD.

..

13.

..

14.

..

15.

..

Sandborn WJ, Hanauer SB, Rutgeerts P. Adalimumab for maintenance treatment of Crohn’s disease: results of the CLASSIC II trial. Gut 2007;56:1232-9 This is the largest RCT on efficacy of adalimumab for the maintenance of remission in naı¨ve patients with CD. Sandborn WJ, Rutgeerts P, Enns R, et al. Adalimumab induction therapy for Crohn disease previously treated with infliximab: a randomized trial. Ann Intern Med 2007;146:829-38 This is the most important RCT on the efficacy of adalimumab in gaining response for patients that failed infliximab. Colombel JF, Sandborn WJ, Rutgeerts P, et al. Adalimumab for maintenance of clinical response and remission in patients with Crohn’s disease: the CHARM trial. Gastroenterology 2007;132:52-65 This large trial investigated the maintenance dose of adalimumab in patients with CD.


16.

.

17.

..

18.

.

19.

.

Karmiris K, Paintaud G, Noman M, et al. Influence of trough serum levels and immunogenicity on long-term outcome of adalimumab therapy in Crohn’s disease. Gastroenterology 2009;137:1628-40 This study showed that immunogenicity can result in loss of response to adalimumab. Reinisch W, Sandborn WJ, Hommes DW, et al. Adalimumab for induction of clinical remission in moderately to severely active ulcerative colitis. J Crohn’s Colitis 2010;4(1):S5 This is the first RCT, to our knowledge, intended to investigate efficacy and safety of adalimumab in UC. Peyrin-Biroulet L, Laclotte C, Roblin X, Bigard MA. Adalimumab induction therapy for ulcerative colitis with intolerance or lost response to infliximab: an open-label study. World J Gastroenterol 2007;13:2328-32 This is the first study, to our knowledge, on adalimumab as induction therapy in active UC. Oussalah A, Laclotte C, Chevaux JB, et al. Long-term outcome of adalimumab therapy for ulcerative colitis with intolerance or lost response to infliximab: a single-centre experience. Aliment Pharmacol Ther 2008;28:966-72 This is the first study, to our knowledge, on adalimumab as maintenance therapy in UC.

20.

Afif W, Leighton JA, Hanauer SB, et al. Open-label study of adalimumab in patients with ulcerative colitis including those with prior loss of response or intolerance to infliximab. Inflamm Bowel Dis 2009;15:1302-7

21.

Gies N, Kroeker KI, Wong K, Fedorak RN. Treatment of ulcerative colitis with adalimumab or infliximab: long-term follow-up of a single-centre cohort. Aliment Pharmacol Ther 2010;32:522-8

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Taxonera C, Estelles J, Blanco I, et al. Adalimumab for ulcerative colitis patients previously treated with infliximab: outcomes at short and long term and predictors of response. Gastroenterology 2010;138(5):S61 Hudis N, Rajca B, Polyak S, et al. The outcome of active ulcerative colitis treated with adalimumab. Gastroenterology 2009;136(5):A661 Garcia-Bosch O, Aceituno M, Perez J, et al. Efficacy of adalimumab in the treatment of ulcerative colitis. J Crohn’s Colitis 2010;4(1):S55 Barreiro-de Acosta M, Lorenzo A, Dominguez-Munoz JE. Adalimumab in ulcerative colitis: two cases of mucosal healing and clinical response at two years. World J Gastroenterol 2009;15:3814-16 Sandborn WJ, Rutgeerts P, Feagan BG, et al. Colectomy rate comparison after treatment of ulcerative colitis with placebo or infliximab. Gastroenterology 2009;137:1250-60 This study shows that infliximab affects the reduction of colectomy rate in patients with active severe UC. Jarnerot G, Hertervig E, Friis-Liby I, et al. Infliximab as rescue therapy in severe to moderately severe ulcerative colitis: a randomized, placebo-controlled study. Gastroenterology 2005;128:1805-11

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30.

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31.

Langholz E, Munkholm P, Davidsen M, Binder V. Course of ulcerative colitis: analysis of changes in disease activity over years. Gastroenterology 1994;107:3-11 This is a milestone among epidemiological studies of IBD. D’Haens G, Baert F, van Assche G, et al. Early combined immunosuppression or conventional management in patients with newly diagnosed Crohn’s disease: an open randomised trial. Lancet 2008;371(9613):660-7 This is the first study, to our knowledge, comparing ‘top down’ and ‘step up’ approaches in IBD. Colombel JF, Sandborn WJ, Reinisch W, et al. Infliximab, azathioprine, or combination therapy for Crohn’s disease. N Engl J Med 2010;362:1383-95 This is a large study evaluating the efficacy of combined immunosuppression versus monotherapy in CD. Efficacy & Safety of Infliximab Monotherapy Vs Combination Therapy Vs AZA Monotherapy in Ulcerative Colitis (Part 1) Maintenance Vs Intermittent Therapy for Maintaining Remission (Part 2)(Study P04807AM3). Bethesda, MD: clinicaltrials.gov, 2007. Available from: http://clinicaltrials. gov/ct2/show/NCT00537316? term=NCT00537316.&rank=1 [Last accessed 20 November 2010]


Affiliation Gionata Fiorino1,2 MD, Laurent Peyrin-Biroulet3 MD PhD, Alessandro Repici4 MD PhD, Alberto Malesci5,6 & Silvio Danese†1 MD PhD † Author for correspondence 1 IBD Unit, Gastroenterology, IRCCS Humanitas, Via Manzoni 56, 20089 Rozzano, Milan, Italy Tel: +39 0282244771; Fax: +39 0282242591; E-mail: [email protected] 2 University of Messina, Messina, Italy 3 Inserm U954 and Department of Gastroenterology, Henri Poincare´ University Nancy 1, Nancy University Hospital, Vandoeuvre-le`s-Nancy, France 4 Gastroenterology and Digestive Endoscopy, IRCCS Humanitas, Rozzano, Milan, Italy 5 Professor, University of Milan, Department of Translational Medicine, Milan, Italy 6 Professor, Gastroenterology and Digestive Endoscopy, IRCCS Humanitas, Rozzano, Milan, Italy

Drug Evaluation

Sclerostin monoclonal antibody therapy with AMG 785: a potential treatment for osteoporosis 1.

Introduction

E Michael Lewiecki

2.


3.

Sclerostin

New Mexico Clinical Research & Osteoporosis Center, 300 Oak St. NE, Albuquerque, New Mexico 87106, USA

4.

AMG 785: chemistry, pharmacodynamics, pharmacokinetics and


metabolism 5.

Conclusions

6.

Expert opinion

Importance of the field: Osteoporosis is a common skeletal disease characterized by loss of bone strength that leads to increased risk of fractures. Fractures of the hip and spine are associated with disability, increased risk of death and high healthcare costs. Recent improvement in the understanding of the molecular regulators of bone metabolism has led to the investigation and development of new therapeutic agents with novel mechanisms of action that may offer advantages over currently available treatments for osteoporosis. Areas covered in this review: Sclerostin is a small protein secreted by osteocytes that downregulates osteoblast-mediated bone formation. This is a review of the rationale, mechanism of action, preclinical and clinical development of AMG 785 (CDP7851), an investigational humanized mAb that inhibits the activity of sclerostin, resulting in increased bone formation. What the reader will gain: The reader will gain an insight into the potential use of sclerostin mAb therapy for the treatment of osteoporosis. Take home message: Preclinical studies and an early report of a clinical study suggest that inhibition of sclerostin with AMG 785 may provide skeletal benefit for patients with osteoporosis. Keywords: anabolic, inhibition, osteoanabolic, osteoporosis, PTH, sclerostin Expert Opin. Biol. Ther. (2011) 11(1):117-127

1.

Introduction

Osteoporosis is a common skeletal disease characterized by low bone mineral density (BMD) and poor bone quality resulting in reduced bone strength and increased risk of fractures [1]. It is a major public health concern worldwide, with > 75 million people in the US, Europe and Japan affected, and > 8.9 million fractures annually [2]. In the US, an estimated 44 million people have osteoporosis or low bone mass (osteopenia) leading to an increased risk of fracture [3]. The lifetime risk of an osteoporotic fracture is about 50% for caucasian women [4], with 30% of women over the age of 50 having one or more prevalent vertebral fractures [5]. Fragility fractures of the spine and hip may result in chronic pain, deformity, depression, disability and death. About 50% of patients with hip fractures will never walk without assistance and 25% will require long-term care [6]. In the Americas and Europe, osteoporotic fractures are associated with 2.8 million disability-adjusted life-years annually, which is more than for hypertension and rheumatoid arthritis [2]. The mortality rate 5 years after a hip fracture or clinical vertebral fracture is about 20% greater than expected [7], with about 738,000 deaths attributed to osteoporotic fractures worldwide in 1990 [2]. The healthcare resources devoted to patients with osteoporotic fractures are great. In the US, osteoporotic fractures result in > 432,000 hospitalizations, almost 2.5 million medical office visits and 180,000 nursing home admissions a year [4]. Hip fractures account for 14% of incident fractures and 72% of fracture-related expenses, which were nearly 10.1517/14712598.2011.540565 © 2011 Informa UK, Ltd. ISSN 1471-2598 All rights reserved: reproduction in whole or in part not permitted

117

AMG 785, CDP-7851

Box 1. Drug summary.


Compound name Phase Indication Pharmacology Route of administration Chemical structure Pivotal trial

AMG 785, CDP-7851 I and II Treatment of osteoporosis Osteoanabolic Subcutaneous Humanized sclerostin mAb None

US$17 billion in the US for 2005 [8]. The cost of osteoporotic fractures in Europe is estimated to be > e36 million for 2000 [9]. With global increases in life expectancy, the number of elderly individuals at risk for fracture is increasing throughout the world. Worldwide, the number of men and women over the age of 65 is expected to rise from about 323 million currently to > 1.5 billion by 2050 [2]. The number of hip fractures and their costs are expected to double or triple by 2040 [4]. Osteoporosis is a disorder of bone remodeling, the physiological process by which adult bone is continually being broken down and reformed in small discrete packets on the surface of trabecular bone and in Haversian systems of cortical bone. At the cellular level, this occurs through the coordinated activity of osteoclasts (multinucleated bone resorbing cells originating from pluripotential hematopoietic cells) and osteoblasts (bone forming cells derived from mesenchymal stem cells in bone marrow). A third type of bone cells, osteocytes (former osteoblasts that have become buried in bone), are the most numerous of the bone cells. Osteocytes comprise > 90 -- 95% of all bone cells, compared with about 4 -- 6% osteoblasts and 1 -- 2% osteoclasts [10]. Osteocytes are embedded in the bone matrix throughout the skeleton and communicate with each other and with bone lining cells through extensive cytoplasmic connections. This network of cells appears to function as a ‘mechanostat’ that senses the magnitude of bone loading and produces a signal that modulates bone remodeling. The presence of such a mechanoregulatory process has been postulated since the early 1990s [11,12]. Sclerostin, a protein encoded by the SOST gene in osteocytes, has been shown to inhibit osteoblastic bone formation [13,14]. Compounds that reduce the production, increase the breakdown or inhibit the activity of sclerostin might be expected to increase osteoblastic bone formation and thereby have an osteoanabolic effect. One such compound is AMG 785 (CDP-7851; co-developed by Amgen, Thousand Oaks, CA, US, and UCB, Belgium), an investigational humanized sclerostin mAb that is now in Phase II clinical trials (Box 1). This is a review of the research that has led to the development of anti-sclerostin therapy and the data that are now available for AMG 785. 2.


Therapeutic agents for the treatment of postmenopausal osteoporosis act by modulating bone remodeling [15]. Antiresorptive (anti-catabolic) agents primarily work by 118

reducing bone resorption, while anabolic (bone-forming) drugs primarily work by increasing bone formation. Pharmacological therapy proven to reduce fracture risk includes the antiresorptive agents estrogen with or without medroxyprogesterone [16,17], alendronate [18-20], risedronate [21-23], ibandronate [24], zoledronate [25,26], denosumab [27], salmon calcitonin [28], raloxifene [29], bazedoxifene [30] and lasofoxifene [31]; osteoanabolic agents teriparatide [32] and recombinant human parathyroid hormone (PTH) (1-84) [33]; and one drug, strontium ranelate [34,35] that may have both antiresorptive and osteoanabolic properties. Oral bisphosphonates (e.g., alendronate, risedronate, ibandronate) are generally considered to be the firstline therapy for osteoporosis because of their proven efficacy in reducing fracture risk and good safety profile. Inexpensive generic alendronate is widely available. However, oral dosing of bisphosphonates is complex (pre-dose fasting, ingestion with plain water only, post-dose fasting in upright position) and may cause upper gastrointestinal (GI) distress in some patients [36]. Intermittent intravenous (i.v.) bisphosphonates (e.g., ibandronate, zoledronate) are not associated with GI intolerance, but must be given at a medical office or infusion center by office staff trained in their administration. Estrogen/medroxyprogesterone use has declined in recent years due to safety concerns, particularly regarding the risk of breast cancer, cardiovascular disease and venous thromboembolic events (VTEs). A selective estrogen receptor modulator (e.g., raloxifene) offers convenient daily dosing, but increases the risk of VTEs and leg cramps. Denosumab, a fully human mAb to receptor activator of NF-kB ligand, is administered by a healthcare professional at 6 month intervals; as a newly approved (mid-2010) therapeutic agent, its long-term utility in clinical practice has not yet been established. The osteoanabolic agents, teriparatide and PTH (1-84), are usually given by daily self-administered subcutaneous (s.c.) injection; their use is limited by factors that include high cost, concern about osteosarcoma in rats, and inconvenience of storage, handling and administration. The effectiveness of current drugs to reduce the burden of osteoporotic fractures has been adversely impacted by failure to identify patients who are likely to benefit from therapy [37-39], failure to initiate drug therapy in appropriate patients [40-42] and poor adherence to therapy when it is started [43-45]. Antiresorptive agents strengthen bone and reduce fracture risk by decreasing bone turnover. This reduces the bone remodeling space and stabilizes or increases BMD through prolongation of secondary mineralization, with preservation of bone microarchitecture, reduction in trabecular perforation and a decrease in cortical porosity. However, lost bone is not replaced and bone microarchitectural elements are not replaced. Osteoanabolic drugs strengthen bone and reduce fracture risk by increasing bone formation. They are associated with an increase in bone size and restoration or formation of new trabecular microarchitectural elements.


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The rebuilding of bone with this class of agents, alone or perhaps in combination with antiresorptive drugs, offers the potential of ‘curing’ osteoporosis [46] and enhancing healing of bone following fractures and orthopedic procedures. Therefore, a major effort is underway to identify and develop new therapeutic agents with osteoanabolic activity [47].


3.

Sclerostin

Sclerosteosis (cortical hyperostosis with syndactyly, OMIM: 269500) was first described in 1958 [48] and identified by that name (‘sklerosteose’ in German) in 1967 [49]. It is a rare autosomal recessive disorder characterized by progressive bone thickening and sclerosis leading to tall stature, syndactyly, enlargement of the skull, entrapment of cranial nerves resulting in facial palsy and increases in intracranial pressure that may cause sudden death due to impaction of the brainstem in the foramen magnum [50,51]. Affected patients appear normal at birth, except for the possible presence of syndactyly, with other manifestations commencing early in life. While sporadic cases have been reported in various parts of the world [52-55], it principally occurs in Afrikaners -- white settlers in South Africa -- who are mainly of Dutch origin. In this population, the carrier rate for the determinant SOST gene is thought to be about 1 in every 100 individuals [50]. Sclerosteosis is caused by loss of function mutations of SOST, a gene located on the chromosomal region 17q12 -- 21, resulting in decreased production of sclerostin by osteocytes. This leads to an increase in osteoblastic bone formation and the typical bone phenotype. Few, if any, patients with osteosclerosis have been known to have fractures [51]. Heterozygous carriers of this disorder are clinically normal, although some may develop age-related radiographic evidence of skull thickening [56]. In a study of 18 heterozygous carriers of sclerosteosis, BMD values were found to be consistently higher than normal subjects, without the complications encountered in homozygotes [50]. Van Buchem disease (OMIM: 23100) is a related rare autosomal recessive disorder, reported mostly in The Netherlands, with manifestations similar to sclerosteosis, without the finding of tall stature and syndactyly [57]. Van Buchem disease involves a 52-kb deletion in the same 17q12 -- 21 chromosomal region as SOST, resulting in downstream impairment of SOST function that leads to defective sclerostin production [58]. Interestingly, while patients with van Buchem disease have high BMD, heterozygote carriers may not have high BMD [57], at least as measured by radiogrammetry. Sclerostin is a SOST gene product that is a 190-residue secreted monomeric glycoprotein containing a cystine knotlike domain with homology to the Cerebrus/DAN family of bone morphogenetic protein (BMP) antagonists [59-61]. It is highly conserved across vertebrate species, with aminoacid sequences in the vervet (a monkey native to Africa), rat and mouse that are 98, 89 and 88% identical, respectively, to the human sequence [60]; thus, animal models may provide

a useful method of studying sclerostin that is applicable to humans. Sclerostin binds to low-density lipoprotein receptor-related proteins 5 and 6 (LRP6 and LRP6), thereby inhibiting Wnt/b-catenin signaling [62] and reducing bone formation by inhibiting osteoblast differentiation, proliferation and activity [63,64]. Although sclerostin/SOST appears to be predominately expressed in osteocytes [14], quantitative real time-PCR analyses have demonstrated a significant presence in cartilage, kidney and liver, with detectable lower levels in placenta and fetal skin [60]. The clinical significance of potential unintended off-target non-skeletal effects of anti-sclerostin therapy is unknown. Animal studies support the concept that sclerostin expression plays a role in bone mechanoregulation, the system by which bone loading is sensed and bone mass is adjusted accordingly. Male and female SOST knockout mice have a high bone mass phenotype, as with humans who have sclerosteosis and van Buchem disease [64]. In mice and rats subjected to ulnar loading, SOST transcription and sclerostin expression by osteocytes were dramatically decreased in the loaded bone, with histomorphometry showing an increase in bone formation rates [65]. Conversely, in mice with hindlimb unloading in tail suspension experiments (an animal model for disuse osteoporosis), a significant increase in SOST transcription was observed in the tibia [65], although no increase in sclerostin-positive osteocytes was seen, perhaps due to limitations in the sensitivity of the assay used. Mechanical unloading of wild-type mice is associated with upregulation of sclerostin and a decrease in Wnt/b-catenin signaling, and SOST knockout mice are resistant to mechanical unloadinginduced bone loss [66], suggesting that sclerostin-mediated inhibition of osteoblastic bone formation plays a role in the pathogenesis of disuse osteoporosis. Sclerostin expression is modulated by factors in addition to mechanical loading. For example, a single injection of PTH in mice is associated with a transient 50% decrease in expression of sclerostin within 2 h and continuous infusion of PTH results in sustained downregulation of sclerostin of 80 -- 90% in vertebral bone [67]. The pattern of transient decreases in sclerostin secretion after each PTH injection may result in bursts of BMP and/or Wnt signaling that could stimulate osteoblastogenesis and in part explain the osteoanabolic effect of therapy with teriparatide [68,69]. In a rat experiment, fibrous tissue of nonunion fractures was found to express significantly lower levels of BMPs as well as lower levels of sclerostin and other BMP antagonists compared with standard healing fractures [68]. These findings suggest that downregulation of osteogenic BMPs may in part account for nonunion fractures and that reduced expression of BMP antagonists, such as sclerostin, may be a response to reduced BMPs. The balance between BMPs and their endogenous antagonists may be important for optimal fracture healing [70]. Our current knowledge of the molecular basis of sclerosteosis and van Buchem disease, combined with the findings from many animal studies, suggest that interventions that


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downregulate sclerostin might increase BMD and potentially be used to treat osteoporosis and other skeletal diseases associated with low BMD and increased fracture risk. Sclerostin inhibition A range of anti-sclerostin mAbs have been raised using a modification of the selected lymphocyte antibody approach [61,71]. One of these antibodies (Scl-AbI) was found to block the protein inhibition of the Wnt/b-catenin signaling pathway in cell culture, and mice treated with this antibody showed a significant increase in BMD [61]. In a mouse model of chronic colitis, a condition associated with chronic inflammation and bone loss, treatment with Scl-AbI was found to halt bone loss and reverse the decline in intrinsic and extrinsic mechanical properties of the femur [72]. Another anti-sclerostin mAb (Scl-AbII) was evaluated in ovariectomized (OVX) rats [73], an animal model that is recommended by the FDA for preclinical evaluation of candidate compounds for the treatment of postmenopausal osteoporosis [74]. The effects of treatment with Scl-AbII 25 mg/kg subcutaneously two times per week for 5 weeks were assessed by BMD testing, bone histomorphometry and strength testing. Robust anabolic effects of Scl-AbII were reported. At 5 weeks, there were large increases in bone formation on trabecular, periosteal, endocortical and intracortical surfaces with Scl-AbII. There was not only a complete reversal of bone loss due to 1 year of estrogen deficiency at several skeletal sites, but additional increases in bone mass and bone strength compared with nonOVX control rats. The newly formed bone was normal in appearance, and despite the increase in osteoblast surface and mineralizing surface, there was a decrease in osteoclast surface, suggesting the possibility of at least partial uncoupling of osteoblastic bone formation and osteoclastic bone resorption. This is consistent with reports of uncoupling reported with SOST knockout mice [64] and histomorphometric data in a patient with sclerosteosis [54]. The proof-of-concept study with Scl-AbII established the role of sclerostin as a pivotal negative regulator of bone formation and stimulated further interest in inhibition of sclerostin as a promising novel approach for anabolic therapy of clinical disorders characterized by low BMD and skeletal fragility. It is notable, with clear clinical implications, that in OVX rodents concomitant treatment [75] and previous treatment with alendronate [76] did not blunt the anabolic effect of sclerostin mAb, and that the increase in bone mass was maintained when followed by administration of zoledronic acid [77]. This suggests that anti-sclerostin therapy may be effective in the common clinical scenario of previous treatment with an oral bisphosphonate (the usual first-line therapy for patients with osteoporosis), and that there may be benefit to following anti-sclerostin therapy with antiresorptive therapy, as is the case with teriparatide, another osteoanabolic agent [78]. The most appropriate animal model for evaluating skeletal effects of pharmacological agents is non-human primates, in which bone physiology (remodeling, changes following estrogen


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withdrawal) closely approximates that of humans. The effects of a humanized sclerostin mAb (Scl-AbIV) have been evaluated in cynomolgus monkeys. Healthy adolescent gonad-intact female cynomolgus monkeys (Macaca fascicularis) age 3 -- 5 years (mean = 3.9 years) received two once-monthly s.c. doses of Scl-AbIV 3 mg/kg (n = 2), 10 mg/kg (n = 3), 30 mg/kg (n = 3) or vehicle (n = 4) [79]. The aim of the study was to assess the effects of therapy on serum bone turnover markers (osteocalcin, intact N-terminal propeptide of type 1 procollagen (P1NP) and C-telopeptide (CTX)), bone density (by dual-energy X-ray absorptiometry (DXA) and peripheral quantitative CT (pQCT)), bone histomorphometry and bone strength by biomechanical testing. On day 61, the study was terminated and the bones harvested for analysis. There was a significant increase and subsequent decline in markers of bone formation (osteocalcin, P1NP) following each dose of Scl-AbIV, with no clear effect on the bone resorption marker CTX. Significant increases in bone mineral content (BMC) and/or BMD were observed at the femoral neck, radial metaphysic and tibial metaphysis, with marked dose-dependent increases in bone formation on trabecular, periosteal, endocortical and intracortical surfaces. There were significant increases in trabecular thickness and bone strength at the lumbar vertebrae in the highest-dose group. Systemic administration of anti-sclerostin therapy has been demonstrated to have beneficial effects on fracture healing and bone repair in rodent studies, with findings of increased callus density, increased bone strength at the fracture site and accelerated bone repair [80,81]. In 3 -- 5 year old male cynomolgus monkeys which underwent bilateral, transverse fibular osteotomies stabilized with an intramedullary pin, treatment with sclerostin mAb for 10 weeks improved fracture healing, as manifested by increased callus area, callus BMC and torsional stiffness compared with vehicle [82]. In addition, the same study showed for the first time that sclerostin inhibition increased bone formation, mass and strength in male nonhuman primates. Taken as a whole, these preclinical findings are consistent with a robust osteoanabolic effect of anti-sclerostin therapy, suggesting that this type of therapy might be beneficial for the treatment of osteoporosis and other skeletal disorders. At least three pharmaceutical companies (Amgen/UCB, Novartis, Eli Lilly) are developing mAbs to sclerostin and another (OsteoGeneX) is reportedly developing a small anti-sclerostin molecule [83]. Of these, the Amgen/UCB product, AMG 785, has advanced the furthest, and is now in Phase II clinical trials.

AMG 785: chemistry, pharmacodynamics, pharmacokinetics and metabolism

4.

AMG 785 is a humanized sclerostin mAb developed collaboratively by UCB and Amgen. The first in-human study of AMG 785 was a Phase I randomized, double-blind, placebo-controlled, ascending single-dose study in healthy men and postmenopausal women (ClinicalTrials.gov Identifier NCT01059435) conducted between December 2006 and



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July 2007 at a single investigative center in Miami, FL, USA [84]. The primary objectives of this study were to evaluate the safety and tolerability of AMG 785, with secondary objectives to assess the pharmacodynamics (PD), pharmacokinetics (PK), and the effects on bone turnover markers and BMD. The PD of AMG 785 was analyzed in all subjects who received investigational product and had both predose and postdose measurements; PK was analyzed in all treated subjects for whom the PK parameters could be estimated. Selected PD parameters between placebo and each AMG 785 dose group were tested in a mixed effects model using linear contrasts for change and percentage change from baseline. The study subjects were randomized to receive either AMG 785 or placebo in a 3:1 ratio. A total of 72 subjects received investigational product (54 AMG 785, 18 placebo), of whom 56 received it subcutaneously (42 AMG 785 with a dose of 0.1, 0.3, 1, 3, 5 or 10 mg/kg; 14 placebo) and 16 received it intravenously (12 AMG 785 with a dose of 1 or 10 mg/kg; 4 placebo). After review of data from the 10 mg/kg s.c. cohort, the dose for all subjects in the 10 mg/kg i.v. cohort was reduced to 5 mg/kg. All subjects who received investigational product completed the study. Most of the subjects were female (78%) and Hispanic (90%). The mean age for each cohort ranged from 49 to 55 years. Serum concentrations of AMG 785 were measured by a validated ELISA. After both s.c. and i.v. dosing, a greater than doseproportional increase in AMG 785 serum concentrations was observed, with clearance or apparent clearance decreasing as dose increased [84]. Nonlinear PK was observed, similar to what had been reported with other therapeutic mAbs [85], with this effect most pronounced between the 1 and 3 mg/kg s.c. dose groups. Peak AMG 785 serum concentrations were observed within the first week after s.c. administration. In the highest s.c. and i.v. dose groups, serum concentrations of AMG 785 decreased in a biphasic pattern with b half-lifes of 11 -- 18 days and g half-lifes of 6 -- 7 days. Exposures (AUC0-inf) in the 1 and 5 mg/kg s.c. dose groups were ~ 50 and 70% with respect to the corresponding i.v. doses. No in vitro or in vivo data have been published on the distribution, metabolism, excretion or drug interactions of AMG 785. It is likely that these features are similar to other therapeutic mAbs [85]. Antibody distribution is determined by the rate of extravasation in tissue, the rate of distribution within tissue, the rate and magnitude of antibody binding within tissue, and the rates of elimination from tissue. Renal elimination, the primary pathway for the clearance on small molecule drugs, is insignificant for mAbs, which are too large for glomerular filtration. Most antibody elimination is by intracellular catabolism following fluid-phase or receptor-mediated endocytosis [85]. Efficacy A Phase I randomized, double-blind, placebo-controlled, ascending single-dose study in healthy men and postmenopausal women included measurement of bone formation markers (serum P1NP, osteocalcin, bone-specific alkaline 4.1

phosphatase (BSAP)) and the bone resorption marker serum CTX. BMD was measured by DXA in all groups, except those receiving 0.1 and 0.3 mg/kg s.c., at baseline and on approximately days 29 and 57; for the 5 and 10 mg/kg groups, an additional BMD measurement was performed on approximately day 85. Following a single s.c. or i.v. dose of AMG 785, there was a dose-dependent increase in the serum levels of P1NP, BSAP and osteocalcin compared with baseline, calculated as the mean percentage change [84]. The maximum increases from baseline for P1NP, BSAP and osteocalcin were 184, 126 and 176% for the 10 mg/kg s.c. dose and 167, 125 and 143% for the 5 mg/kg i.v. dose, respectively (p < 0.01 compared with placebo). Serum CTX levels, calculated as mean percentage change from baseline, decreased in an approximately dose-dependent manner after a single s.c. or i.v. dose of AMG 785 [84]. The maximum significant decreases from baseline for CTX were 54% for the 10 mg/kg s.c. dose and 49% for the 5 mg/kg i.v. dose, respectively (p < 0.01 compared with placebo). In the placebo groups, a rapid but transient reduction in CTX levels, perhaps due to assay variability, was reported. The magnitude of the increase in bone formation markers at 1 month was comparable to what was reported after 6 months of teriparatide [86], suggesting a more rapid onset of osteoanabolic effect with AMG 785. Bone formation marker levels peaked about 2 -- 4 weeks after dosing with AMG 785, depending on the dose and route of administration, and then declined and returned to baseline, while the decrease in serum CTX was sustained over the period of observation. This may represent a prolongation of the ‘anabolic window’ in comparison with teriparatide. The pattern of an increase in bone formation markers and a decrease in bone resorption markers with sclerostin inhibition suggests uncoupling of osteoblast and osteoclast activity that is consistent with observations in preclinical studies in SOST knockout mice [64], OVX rats [87] and primates [79]. The mean baseline T-score for each cohort ranged from -1.09 to -0.17 at the lumbar spine and -0.57 to 0.27 at the total hip. Compared with placebo, a single s.c. dose of AMG 785 increased BMD at the lumbar spine and total hip in all cohorts that were measured at days 29, 57 and 85, with the exception of total hip BMD for the 5 mg/kg cohort at day 29 [84], in an approximately dose-dependent manner. The largest significant increase in lumbar spine (5.3%) and total hip (2.8%) BMD was observed on day 85 with an s.c. dose of 10 mg/kg (p < 0.01 compared with placebo). In the i.v. cohorts, the largest increase in lumbar spine (5.2%) and total hip (1.1%) BMD was also observed on day 85 with a dose of 5 mg/kg (p < 0.01 for lumbar spine; p > 0.05 for total hip). Safety and tolerability In the same Phase I trial, AMG 785 was generally well tolerated with all administered doses [84]. At least one adverse event (AE) 4.2


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was reported by 64 or 60% of the subjects who received s.c. placebo or AMG 785, respectively, and 50 or 25% of the subjects who received i.v. placebo or AMG 785, respectively. Most AEs were considered mild by the investigator, and none resulted in study discontinuation or death. The most commonly reported AEs with s.c. administration of either placebo or AMG 785 were injection site erythema, back pain, headache, constipation, injection site hemorrhage, arthralgia and dizziness, all of which were considered mild and not serious. One serious AE (SAE) was reported in a subject who received 10 mg/kg s.c. AMG 785: severe nonspecific hepatitis, with an elevated liver function test beginning 1 day after dosing and liver enzymes peaking at levels of 6 -- 13 times the upper limit of normal. Six to eight days after dosing, abdominal ultrasound tests and hepatitis panels were normal; resolution of the SAE occurred on day 26. No additional information was provided on confounding variables that might have contributed to the risk of hepatitis. In study subjects who received i.v. dosing of placebo or AMG 785, none reported more than one mild AE, and there were no SAEs. Mild, transient asymptomatic decreases in mean serum ionized calcium levels (about 4% below baseline) were reported after s.c. and i.v. dosing of AMG 785, with values returning to baseline during the course of the study or follow-up period. In association with the decreased serum calcium levels, there was a transient increase in serum intact PTH levels that returned to baseline levels by the end of the study. Some subjects who received higher doses of AMG 785 were reported to have increased total serum alkaline phosphatase levels, probably a consequence of osteoanabolic activity increasing the BSAP. In all, 6 (11%) of the 54 subjects receiving AMG 785 tested positive for binding anti-AMG 785 antibodies; of these who tested positive, 1 subject receiving the 10 mg/kg s.c. dose tested positive for AMG 785 neutralizing antibodies at study end and up to day 283, and 1 subject receiving 5 mg/kg i.v. tested positive for neutralizing antibodies during followup on day 132 and up to day 252. The finding of neutralizing antibodies was not associated with any AEs or abnormalities of other laboratory tests, vital signs or electrocardiogram. The effects of neutralizing antibody formation on the PK and PD of AMG 785 could not be definitively determined in this study due to the timing of antibody formation relative to when serum concentrations of AMG 785 and bone turnover markers began to return to baseline. Other clinical trials with sclerostin mAb Ongoing Phase I trials with AMG 785 include a fully enrolled single-dose study in 31 healthy postmenopausal Japanese women (NCT01101061) to evaluate drug safety and tolerability, with a completion date of January 2011; and a fully enrolled study to evaluate the effect of AMG 785 on bone quality of the forearm, assessed by pQCT, in 24 postmenopausal women with low bone mass (NCT00950950), with a completion date of December 2010. Ongoing Phase II trials with AMG 785 include a fully enrolled study in 419 postmenopausal 4.3

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women with low BMD (NCT00896532) to evaluate the change in lumbar spine BMD, with a completion date of August 2012; a study that is recruiting 330 subjects with intertrochanteric fractures of the proximal femur who are status post fixation with a sliding hip screw or intramedullary nail (NCT01081678), with a primary end point of functional healing measured by a timed up-and-go test; and a study that is recruiting 400 subjects with tibial diaphyseal fractures who are status post intramedullary nailing (NCT 00907296), with a primary end point of time to radiographic fracture healing. Another sclerostin mAb, AMG 167 (CDP1671 Amgen/ UCB), has been evaluated in clinical trials, including a Phase I first in-human study in 69 healthy men and postmenopausal women (NCT00902356), completed in November 2009, and a Phase I ascending multiple dose study recruiting 48 healthy men and postmenopausal women in low BMD (NCT20080389), with a planned completion date of August 2011. Regulatory affairs AMG 785 is an investigational sclerostin mAb with no regulatory approval for clinical use. 4.4

5.

Conclusions

Preclinical studies with a range of sclerostin mAbs have shown a robust osteoanabolic effect with increases in bone formation, bone mass and bone strength, as well as evidence for enhancement of fracture healing and bone repair. The first clinical trial of sclerostin inhibition with AMG 785, an investigational sclerostin mAb, showed a significant increase in bone formation markers, a significant decrease in bone resorption markers, and significant increases in BMD in healthy men and postmenopausal women. Treatment with AMG 785 was well tolerated with a generally favorable safety profile. It is not yet known whether it reduces the risk of osteoporotic fractures or improves fracture healing in humans. AMG 785 is a promising therapeutic agent for the treatment of osteoporosis and other skeletal conditions that might benefit from increased bone formation. 6.

Expert opinion

Pharmacological agents for the treatment of osteoporosis may be classified as antiresorptive or osteoanabolic, depending on whether the principal means of improving bone strength is by inhibition of osteoclastic bone resorption or stimulation of osteoblastic bone formation. The antiresorptive drugs (e.g., bisphosphonates, SERMs, salmon calcitonin, estrogen, denosumab) represent a wide range of antiresorptive potency, from very mild with salmon calcitonin to very great with denosumab. Due to physiological coupling of bone resorption and bone formation, as bone resorption decreases so does bone formation. With the approved antiresorptive agents, suppression of bone resorption occurs faster and is greater



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than suppression of bone formation, but nevertheless the linkage of the two components of bone remodeling is a limiting factor in improving BMD. These agents reduce fracture risk by reducing the remodeling space and increasing bone mineralization, but do not build new bone or restore the deteriorated bone microarchitecture. Osteoanabolic agents (e.g., teriparatide, recombinant human PTH 1-84) increase bone formation faster and greater than bone resorption, but the linkage of bone formation and resorption appears to limit the achievable response in terms of BMD increase. It has been suggested that some agents, such as strontium ranelate, may at least partly uncouple bone resorption and formation, thereby, having both antiresorptive and osteoanabolic features [88]. An investigative antiresorptive drug, odanacatib (Merck), is a cathepsin K inhibitor that may have the distinguishing feature of partially uncoupling bone resorption and formation, so that as bone resorption is decreased, bone formation is only minimally reduced [89]. Another approach to uncoupling bone resorption and formation might be to use an antiresorptive drug and osteoanabolic drug together, perhaps sequentially, cyclically or simultaneously. In a study of zoledronic acid, an antiresorptive agent, and teriparatide, an osteoanabolic agent, given as monotherapy or simultaneous combination therapy, it was found that teriparatide alone increased spine BMD more than zoledronic acid alone and zoledronic acid alone increased hip BMD more than teriparatide alone, but combination therapy provides the largest, most rapid increments when both spine and hip sites are considered [90]. It is not known whether monotherapy or combination therapy that uncouples bone resorption and formation reduces fracture risk more than an agent that acts primarily by reducing bone resorption or increasing bone formation alone. However, there are at least theoretical advantages to such an approach that might lead to additional therapeutic benefit [91,92]. How does sclerostin mAb therapy, and AMG 785 in particular, fit into this spectrum of therapies, and what advantages could it offer in comparison to currently available treatments for osteoporosis? It is clear that osteocyte expression of sclerostin inhibits osteoblastic bone formation; sclerostin mAb therapy increases osteoblastic bone formation and is, therefore, appropriately classified as an osteoanabolic agent. Teriparatide, the only osteoanabolic agent approved in the US, reduces the risk of vertebral and nonvertebral

fractures, but is limited in its use due to the inconvenience daily s.c. dosing, the requirement for refrigeration and high cost. Due to safety concerns (i.e., rat osteosarcoma) and limited efficacy data, it is restricted to no more than 24 months of lifetime exposure. The risk of osteosarcoma with AMG 785 is unknown; however, studies of human osteosarcoma cell lines showing activation of Wnt signaling by loss of Wnt inhibitory factor 1 [93] suggest the possibility that sclerostin inhibition could increase the risk of osteosarcoma and warrants diligence in assessing this important safety concern in future studies. The ‘anabolic window’ with teriparatide appears to be limited due to the transient nature of the increase in bone formation and the subsequent increase in bone resorption, as represented by the pattern of change observed with bone turnover markers [68,94]. The published clinical experience with AMG 785 is from a single Phase I trial, but nevertheless provides some insight into ways in which it might have a therapeutic profile that distinguishes it from teriparatide. The BMD response at the lumbar spine and hip 3 months after a single dose of AMG 785 10/mg/kg s.c. was similar to or greater than what was seen after 6 months of daily teriparatide [95]. The pattern of bone turnover marker changes with AMG 785 differs from teriparatide. AMG 785 appears to uncouple bone formation and resorption, with increased bone formation markers occurring at the same time that a bone resorption marker decreased. This suggests a large anabolic window that may allow for a greater BMD response and longer duration of treatment than with teriparatide. Much more study is needed to evaluate the longterm safety of AMG 785 and determine whether it reduces fracture risk, enhances the healing of fractures and improves repair of bone after surgical intervention. With what is known at this time, it is a promising investigational agent for the treatment of osteoporosis and other disorders that could benefit from an increase in bone formation.

Declaration of interest The author has received grant/research support from Amgen, Merck, Eli Lilly, Novartis, Warner Chilcott, and Genentech. He has served as a consultant, advisory board member, speakers’ bureau participant, or given presentations at sponsored speaking events for Amgen, Eli Lilly, Novartis, and Genentech.


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Affiliation


E Michael Lewiecki MD FACP FACE New Mexico Clinical Research & Osteoporosis Center, 300 Oak St. NE, Albuquerque, New Mexico 87106, USA Tel: +1 505 855 5525; Fax: +1 505 884 4006; E-mail: [email protected]


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