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DNA vaccines against cancer come of age
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DNA vaccines against cancer come of age Freda K Stevenson, Christian H Ottensmeier and Jason Rice Genetic technology allows construction of DNA vaccines encoding selected tumor antigens together with molecules to direct and amplify the desired effector pathways. Their enormous promise has been marred by a problem of scaling up to human subjects. This is now largely overcome by electroporation, which increases both antigen expression and the inflammatory milieu. While the principles of vaccine design can be developed in mouse models, the real operative test is in the clinic, using patients in temporary remission. Monitoring of induced immunity, although commonly limited to blood, is providing objective qualitative and quantitative data on T-cell and antibody responses. Prolongation of remission is the goal and an activated immune system should achieve this. Address Cancer Sciences Division, University of Southampton School of Medicine, Southampton General Hospital, Southampton SO16 6YD, UK Corresponding author: Stevenson, Freda K ([email protected]), Ottensmeier, Christian H ([email protected]) and Rice, Jason ([email protected])
Current Opinion in Immunology 2010, 22:264–270 This review comes from a themed issue on Tumour Immunology Edited by Freda Stevenson and Anna Karolina Palucka Available online 19th February 2010 0952-7915/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2010.01.019
pathway [1]. Global control of immune responses, however, lies with CD4+ T cells, involved in both activation and suppression of immunity. Strategies to remove regulatory CD4+ T cells are now popular [2], but we take the view that vaccines should reverse tolerance, especially if CD4+ T-cell help can be acquired from an undamaged immune repertoire [1]. DNA vaccines produce a small amount of antigen, apparently sufficient for effective priming. However, such vaccines often induce strong CD8+ T-cell responses which can remove antigen-presenting cells at the time of boosting. This has led to ‘prime/boost’ strategies which amplify antigen levels [3]. Although viral vectors are effective, they have the major disadvantage of inducing anti-vector immunity, making further boosts, likely to be needed for cancer control, less effective [4]. Electroporation is an attractive alternative, increasing both antigen levels and inflammatory activity [5]. The vexed question of scaling up from preclinical models to clinical application may be solved by this technology and clinical trials are now in progress. In spite of sophisticated mouse models, there are many unknowns in our understanding of human immunity and certainly in our knowledge of its ability to suppress cancer. Delivery of specific antigens via DNA vaccines at least allows objective measurement of immune responses induced against the specific target, as well as analyzing what really matters, the effect on cancer cells in patients. This review will focus on the development of novel DNA vaccine designs and their evaluation in the clinic.
Introduction
Activating innate immunity
Mobilizing the powerful immune system to attack cancer cells is appealing but attention must be paid to both vaccine design and clinical context. DNA vaccines not only have an inbuilt ability to activate multiple pathways of innate immunity, but also offer a unique opportunity to guide defined antigens, accompanied by specific activator molecules, through a patient’s compromised immune system. Any therapeutic vaccination may fail if tumor load is high, so there is obvious reliance on initial treatments to bring patients into remission, a setting becoming increasingly available. However, some immunodeficiency or tolerance may persist, requiring powerful but specific vaccines. There is now a range of potential target antigens including cell surface molecules, susceptible to antibody attack, and a multitude of intracellular antigens, requiring cytotoxic T cells. Vaccine design can modify the molecular format of antigen to select the desired effector
The innate immune system detects microbial molecular arrays, including plasmid DNA, using PRRs (pattern recognition receptors). The first receptor identified for DNA was TLR9 (Toll-like receptor 9) [6], highly expressed by B cells and DCs, and able to recognize dsDNA delivered into the cell by endocytosis or autophagy (Figure 1). The classical TLR9 ligand is unmethylated CpG dinucleotide, with certain flanking motifs, with binding leading to a cascade of activation, proliferation and differentiation of immune cell subsets [7]. However, although these sequence features are found in plasmid DNA, deductions on the role of TLR9 have been complicated by the use of synthetic phosphorothioate oligonucleotides, which differ in binding specificity from natural phosphodiester DNA [8]. The importance of TLR9 for activation by plasmid DNA has also been diminished by the finding that TLR9/ mice are capable
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Figure 1
Innate immune sensors of dsDNA. DNA is a potent activator of innate immunity and this attribute is thought to contribute to the efficacy of DNA vaccines in activating antigen-specific immune responses. The mechanisms for sensing DNA appear to be multi-layered and incorporate redundancy. HMGB (high-mobility group box) proteins act as universal sensors of nucleic acids and are required for subsequent activation of innate immunity by the more discriminative PRRs (pattern recognition receptors). PRR activation leads to a final common pathway of type I interferon production except for AIM2 engagement which triggers the maturation of pro-inflammatory cytokines with no interferon response. Clearly plasmid DNA engages multiple routes to activate innate immunity. Abbreviations: dsDNA, double-stranded DNA; ppp-dsRNA, 50 -triphosphate double-stranded RNA; IRF, interferonregulatory factors; NF-kB, nuclear factor-kappaB.
of responding to DNA vaccines [9]. Clearly there are additional pathways, and these are now being described. Several cytosolic DNA sensors have been identified, the first being DAI (DNA-dependent activator of interferonregulatory factors) [10] (Figure 1). DNA binding to DAI appears to be sequence independent but lengthrestricted [11]. However, proteomic–genomic screening and gene knock-down studies revealed another potential sensor, AIM2 (absent in melanoma 2) [12,13]. This recruits the AIM2 inflammasome, a multiprotein complex that activates caspase 1, leading to secretion of IL-1b and IL-18 as well as pyroptosis [13]. A novel DNA-sensing pathway involving RNA Pol-III (polymerase III) and RIG-I (retinoic acid-inducible gene-I) has also been described: Pol-III converts AT-rich cytosolic dsDNA into dsRNA with a 50 -triphosphate moiety. This serves as a ligand for RIG-I activation, leading to the final common pathway of type I interferon production [14,15]. Our understanding of the pathways leading to the interferon response is also increasing, with the recent description of STING (stimulator of interferon genes) which acts as a key regulator of innate immune signalling in response to intracellular DNA [16]. It has also been discovered that the discriminative sensing afforded by these different PRR may be preceded by, and reliant on, a more prowww.sciencedirect.com
miscuous sensing mechanism in which HMGB (highmobility group box) proteins serve as universal sentinels for nucleic acids [17]. Clearly plasmid DNA not only delivers antigen, but also engages multiple routes to activate innate immunity (Figure 1).
Activating adaptive immunity against cancer antigens Activating the innate immune system is the first step for induction of immunity against weak tumor antigens but, to activate adaptive immunity, we need to harness the knowledge and tools of modern immunology. CD4+ T cells are pivotal both in providing help and in regulating responses. A successful DNA vaccine should circumvent regulation, and, to achieve this, we have engaged T-cell help from an undamaged anti-microbial repertoire (Figure 2) [1]. Others have used alternative molecules [18–20]. For the induction of antibody, this mimics conjugate vaccines [21], and we have used selected sequences from a favored conjugate vaccine component, tetanus toxin, with dramatic effects in preclinical models [1] and encouraging results in a clinical trial (see below) (Figure 2). Conjugation is also effective for inducing effector T cells [1,22] and additive sequences can easily be genetically Current Opinion in Immunology 2010, 22:264–270
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Figure 2
Engagement of T-cell help for the induction of anti-tumor immunity. A DNA vaccine encoding a tumor antigen fused to the FrC (Fragment C) of tetanus toxin is injected into muscle. The fusion protein is cross-presented to DCs (dendritic cells) [1] which then express MHC Class II-associated immunogenic peptides derived from FrC. Responding CD4+ T cells interact and ‘license’ DCs for the induction of T-cell responses against weakly immunogenic tumor antigen-derived peptides.
combined with tumor antigen for induction of CD4+ and/ or CD8+ T cells [1]. The mode of action is likely through ‘licensing’ of APCs (antigen-presenting cells) via interaction with non-suppressed CD4+ T cells specific for tetanus toxin-derived peptides within the vaccine (Figure 2) [1]. Multiple interacting ligand/receptor pairs should protect APCs against apoptosis and OX40/OX40L interaction is known to reverse regulatory T cells [23]. These immune mechanisms are also likely involved in xenogeneic DNA vaccine strategies against melanoma [24]. The approach uses xenogeneic counterparts of autologous tumor antigens and has shown efficacy in dogs where a vaccine encoding human tryrosinase gained conditional FDA approval in 2007 [24]. The rationale is that target MHC Class I-binding epitopes are presented in a more immunogenic context, with CD4+ T-cell help probably provided via the xenogeneic sequences, although this has not been proved. Induction of cytolytic CD8+ T cells is a clear goal for targeting cancer cells. Establishing long-term memory requires T-cell help [25], and therefore, in our case, microbial sequences, but immunological principles have to be heeded. The phenomenon of immunodominance requires focusing of CD8+ T-cell responses onto tumorCurrent Opinion in Immunology 2010, 22:264–270
derived peptides, and must avoid competition from microbe-derived peptides [26]. We have achieved this by minimizing the microbial sequence and optimizing presentation of tumor-derived epitopes (reviewed in [1]). This design is in clinical trial for patients with prostate cancer (see below). No doubt there are many ways to harness immunological principles, but this strategy can break tolerance and activate high levels of epitope-specific CD8+ T cells able to suppress tumor in numerous models [1]. It demonstrates superior performance compared to peptide vaccination [27]. Additional epitopes from the same or other target antigens can be placed in separate vaccines and delivered in different sites thereby avoiding competition [28] and this approach will soon be tested in a trial of chronic myeloid leukaemia (see below). A remaining unknown is whether there is a need to include tumor-derived sequences to induce CD4+ responses, or indeed whether such sequences are best avoided in case regulatory T cells are stimulated. Flexibility of DNA construction is allowing investigation of these questions.
DNA vaccine delivery DNA vaccines can induce strong, protective immunity in a wide range of mouse cancer models, but initial clinical www.sciencedirect.com
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testing proved disappointing. In retrospect, for intramuscular routes, this was predictable, given the dependence of efficacy on the volume injected [29]. High hydrostatic pressure apparently increases plasmid uptake and the resulting tissue injury can attract APCs to the injection site [30]. Scaling up to human subjects would require an unacceptable injection volume, and other developments were required. The problem may be avoidable using the skin route, where gene gun or other delivery methods have shown some success, for example in prophylactic influenza vaccination [31]. Another popular approach for increasing efficacy has been to follow priming with DNA with boosting using viral vectors, and this appears useful for infectious diseases [3]. The amplification of response by viral vectors is likely to be via the attraction and infection of antigen-presenting cells. This will overcome the tendency of primed cytotoxic T cells to remove transfected muscle cells and/or APCs which could curtail the response. However, viral vector delivery is not readily applicable to cancer where preexisting or induced antivector immunity would undermine repeated boosting efficacy [4]. Alternative physical strategies include formulations with nanoparticles, microparticles, and liposomes, or delivery with electroporation, particle bombardment, jet injection, and tattooing [31–34]. Although some have reached clinical testing [31,33–35], electroporation has emerged as a preferred and very effective strategy [34,36]. EP (electroporation) involves electrical stimulation across the muscle (or skin) site coincident or immediately following DNA vaccine injection. The improved transfection efficiency increases antigen expression (10–100-fold) and entry of inflammatory cells [5,37,38]. These factors will likely increase cross-presentation of DNA vaccineencoded antigen [39] resulting in improved immune responses [40], although transfected muscle cells may also contribute directly [41]. EP overcomes the failure of low-volume injection to induce immunity in mice [29], and the combination of DNA priming with DNA/EP at boosting is particularly effective [29]. Also, in large animals [42] and human subjects [34], EP significantly increases DNA vaccine-specific immune responses with rhesus macaques showing improved vaccine-specific Tcell responses [42] and a reduction of viremia [43]. Initial concern that improved transfection efficiency may increase integration levels, appear unfounded [42,44]. Thus, EP is a safe delivery system that appears to overcome the translational block to effective DNA vaccination in the clinic.
Clinical evaluation DNA vaccines against infectious diseases
DNA vaccines are now licensed for the prevention of infection in horses (West Nile virus) and salmon (infecwww.sciencedirect.com
tious hemorrhagic necrosis virus) [1]. In human subjects, reports of prophylactic DNA vaccines able to induce antibody and CD4+ T-cell responses, with potential efficacy against several dangerous viruses, are accumulating [45,46]. Prime/boost approaches with viral vectors have been widely tested, especially for vaccines against malaria, where the variable influence of the boosting vector on outcome has been noted [47]. In the more challenging therapeutic setting, where parallels can be drawn with cancer, DNA vaccination can induce immune responses, with, in the case of persistent hepatitis C infection, apparent clinical benefit [48]. Certainly the speed of construction of DNA vaccines presents a real advantage for both prophylaxis and therapy of new pathogens, including influenza [49]. Beyond confirming that DNA vaccines can be effective clinically, testing of these vaccines in human subjects is revealing how human immune responses differ from those of mice and, in conjunction with careful studies of immune responses to conventional vaccines [50], will be critical for the optimal design of clinical trials against cancer. DNA vaccines against cancer
While there are parallels with persistent infection, the clinical settings for cancer patients differ. Variable factors include the nature of the cancer, any potentially preexisting immunity and the effects of previous treatment. Ideally small trials of defined groups, with rapid assessment of efficacy of vaccine design and delivery are required. However, in common with other vaccines early evaluation of DNA vaccination followed classical drug development pathways in recruiting patients who had exhausted standard treatment options (reviewed in [51,1]; also see [52]). Clearly, successful vaccination requires an intact immune system [53] and studies are therefore now moving toward patients with minimal disease. In terms of evaluating immune responses, this is straightforward for antibody, but more difficult for T cells. Specific responses to encoded antigens can be measured but there is argument about connecting responses to clinical outcome [54]. The major problem is effector cells may be located in tumor tissues or in vaccination sites, and analysis of blood samples may therefore underestimate significant responses. Alternatively, if effector cells are unable to enter the tumor site, measurements in blood may overestimate efficacy. While tumor biopsies are rarely available, the increasing use of skin challenge is extending knowledge of migratory potential [55], although the fate of effector cells in tumor sites remains less well understood. We undertook an early study in patients with follicular lymphoma targeting idiotypic Ig. We used our fusion gene design consisting of idiotypic determinants assembled as single chain Fv, linked to the FrC (Fragment C) portion of Current Opinion in Immunology 2010, 22:264–270
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tetanus toxin [1]. Individual naked DNA scFv-FrC vaccines were delivered by intramuscular injection, and we detected a boost of anti-FrC responses, in 75% of patients, with anti-idiotypic immunity induced in 38% [1]. Although encouraging, responses were relatively weak, as found in a similar trial using DNA-delivered chimeric Ig [56], although the use of individual vaccines makes any comparisons difficult. Clearly, since the goal is to induce anti-idiotypic antibody or CD4+ T cells, electroporation would be an ideal addition to any new trial. Our next trial tested a single vaccine aimed at induction of immunity against an antigen expressed in prostate cancer, PSMA (prostate-specific membrane antigen), already being tested in different vaccine trials [57]. Several questions were asked: first, if the modified design using a DOM (domain) of Fragment C of tetanus toxin fused to an epitope sequence from PSMA would induce anti-tumor CD8+ T cells; second, if EP would enhance responses. We used escalating doses (0.8–3.2 mg) EP. The patient group had minimal (radiologically undetectable) tumor load at biochemical failure of disease control [34]. Electroporation was safe and well tolerated, as reported for DNA cytokine delivery [58]. We had the opportunity to measure antibody and CD4+ T-cell responses against the tetanus toxin-derived DOM sequence, which, in addition to indicating vaccine performance, provided some indication of non-tolerized immune capacity. The majority of patients (21/30) showed clear responses with EP improving anti-DOM antibody responses (17) [34], although the effect on CD4+ T cells was less marked. +
The main goal was to induce CD8 T-cell responses against the PSMA peptide. Here we need to consider the kinetics of the response, since it would be expected that effector cells would increase following vaccination, migrate to antigen-expressing sites, with some then converting to memory T cells. Unless the blood samples ‘catch’ the peak of the induction, it is unlikely that ex vivo functional assays will be useful, although tetramer staining, when available, will be. Instead, central memory T cells should be sought and these require stimulation in vitro. While this complicates quantitation, most clinical trials follow this procedure [59,60]. It is vital that expertise is shared with inter-laboratory comparisons, and multinational collaborations, such as CIMT and CVC [59,61] are critical not only to standardize assay systems but also to identify the limits of reproducible detection of immune responses. In our prostate cancer trial, we are detecting IFNgproducing CD8+ T-cell responses against the target PSMA peptide in 60% of cases [60]. Preliminary evidence indicates that electroporation stimulates CD8+ Tcell responses more quickly and also that the overall level appears to be higher [60]. Using an xenogeneic approach, Current Opinion in Immunology 2010, 22:264–270
a phase I trial in 19 patients with high risk but completely resected melanoma was recently reported [52]. DNA vaccines encoding human and mouse GP100 were injected sequentially with evidence of expansion of tetramer+ CD8+ T cells in 5/18 patients [52].
Conclusions The data so far indicate that DNA vaccines can induce selected immune responses against tumor antigens in patients. We are learning that the human immune response shows similarities to mouse models but also differences, particularly in kinetics. While new principles will continue to emerge from mice, the challenge is to increase our understanding of human immunity, which, in spite of the success of prophylactic vaccination against infection, remains enigmatic. For cancer vaccines, measurements in blood provide only a limited snapshot of CD8+ T-cell responses, and skin challenges could be helpful. Correlation of immunity with clinical outcome is awaited and those tumors with biomarkers will allow early insights. Combinations with a judicious use of chemotherapy, which could have differential effects on suppressor mechanisms, are an emerging new approach. The ultimate goal of changing tumor behavior for patients is in sight but flexible small trials are still needed to guide the way.
Acknowledgements We thank David Anstee, Paul Lloyd-Evans and Iacob Mathiesen for ongoing support for our portfolio of DNA vaccine clinical trials. This research was supported by Leukaemia Research (grants 0306 and 08025), Cancer Research UK (project grant 7576, programme grant C491/A4363), Inovio and the Alan Willett Foundation.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Rice J, Ottensmeier CH, Stevenson FK: DNA vaccines: precision tools for activating effective immunity against cancer. Nat Rev Cancer 2008, 8:108-120. This review outlines the way that immunological principles can be harnessed for the induction of effective immunity against different molecular targets. It focuses on strategies for inducing T-cell help to activate immunity and to break tolerance against cancer antigens. 2.
Rech AJ, Vonderheide RH: Clinical use of anti-CD25 antibody daclizumab to enhance immune responses to tumor antigen vaccination by targeting regulatory T cells. Ann N Y Acad Sci 2009, 1174:99-106.
3.
Gilbert SC, Moorthy VS, Andrews L, Pathan AA, McConkey SJ, Vuola JM, Keating SM, Berthoud T, Webster D, McShane H et al.: Synergistic DNA–MVA prime-boost vaccination regimes for malaria and tuberculosis. Vaccine 2006, 24:4554-4561.
4.
Smith CL, Mirza F, Pasquetto V, Tscharke DC, Palmowski MJ, Dunbar PR, Sette A, Harris AL, Cerundolo V: Immunodominance of poxviral-specific CTL in a human trial of recombinantmodified vaccinia Ankara. J Immunol 2005, 175:8431-8437.
5.
Ahlen G, Soderholm J, Tjelle T, Kjeken R, Frelin L, Hoglund U, Blomberg P, Fons M, Mathiesen I, Sallberg M: In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake, protein expression, inflammation, and infiltration of CD3+ T cells. J Immunol 2007, 179:4741-4753. www.sciencedirect.com
DNA Vaccines Against Cancer Stevenson, Ottensmeier and Rice 269
6.
Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K et al.: A Toll-like receptor recognizes bacterial DNA. Nature 2000, 408:740-745.
7.
Krieg AM: CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002, 20:709-760.
8.
Haas T, Metzger J, Schmitz F, Heit A, Muller T, Latz E, Wagner H: The DNA sugar backbone 20 deoxyribose determines toll-like receptor 9 activation. Immunity 2008, 28:315-323.
9.
Spies B, Hochrein H, Vabulas M, Huster K, Busch DH, Schmitz F, Heit A, Wagner H: Vaccination with plasmid DNA activates dendritic cells via Toll-like receptor 9 (TLR9) but functions in TLR9-deficient mice. J Immunol 2003, 171:5908-5912.
10. Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T, Lu Y, Miyagishi M, Kodama T, Honda K et al.: DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 2007, 448:501-505. 11. Wang Z, Choi MK, Ban T, Yanai H, Negishi H, Lu Y, Tamura T, Takaoka A, Nishikura K, Taniguchi T: Regulation of innate immune responses by DAI (DLM-1/ZBP1) and other DNA-sensing molecules. Proc Natl Acad Sci U S A 2008, 105:5477-5482. 12. Burckstummer T, Baumann C, Bluml S, Dixit E, Durnberger G, Jahn H, Planyavsky M, Bilban M, Colinge J, Bennett KL et al.: An orthogonal proteomic–genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat Immunol 2009, 10:266-272. 13. Fernandes-Alnemri T, Yu JW, Datta P, Wu J, Alnemri ES: AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 2009, 458:509-513. 14. Ablasser A, Bauernfeind F, Hartmann G, Latz E, Fitzgerald KA, Hornung V: RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat Immunol 2009, 10:1065-1072. 15. Chiu YH, Macmillan JB, Chen ZJ: RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 2009, 138:576-591. 16. Ishikawa H, Ma Z, Barber GN: STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 2009, 461:788-792. 17. Yanai H, Ban T, Wang Z, Choi MK, Kawamura T, Negishi H, Nakasato M, Lu Y, Hangai S, Koshiba R et al.: HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses. Nature 2009, 462:99-103. The response of the innate immune system to nucleic acids, mediated by transmembrane TLRs and cytosolic receptors, is a critical prelude to the induction of immunity via DNA vaccination. These authors reveal that universal sentinels, the high-mobility group box proteins, bind nucleic acids, integrating a hierarchy of selective stimulatory pathways of the innate immune response. 18. Hung CF, Cheng WF, Hsu KF, Chai CY, He L, Ling M, Wu TC: Cancer immunotherapy using a DNA vaccine encoding the translocation domain of a bacterial toxin linked to a tumor antigen. Cancer Res 2001, 61:3698-3703. 19. Savelyeva N, Munday R, Spellerberg MB, Lomonossoff GP, Stevenson FK: Plant viral genes in DNA idiotypic vaccines activate linked CD4+ T-cell mediated immunity against B-cell malignancies. Nat Biotechnol 2001, 19:760-764. 20. Wolkers MC, Toebes M, Okabe M, Haanen JB, Schumacher TN: Optimizing the efficacy of epitope-directed DNA vaccination. J Immunol 2002, 168:4998-5004. 21. Peltola H: Worldwide Haemophilus influenzae type b disease at the beginning of the 21st century: global analysis of the disease burden 25 years after the use of the polysaccharide vaccine and a decade after the advent of conjugates. Clin Microbiol Rev 2000, 13:302-317. 22. Zanetti M: T for two: when helpers need help. Autoimmun Rev 2005, 4:571-578. 23. Piconese S, Valzasina B, Colombo MP: OX40 triggering blocks suppression by regulatory T cells and facilitates tumor rejection. J Exp Med 2008, 205:825-839. www.sciencedirect.com
24. Liao JC, Gregor P, Wolchok JD, Orlandi F, Craft D, Leung C, Houghton AN, Bergman PJ: Vaccination with human tyrosinase DNA induces antibody responses in dogs with advanced melanoma. Cancer Immun 2006, 6:8. 25. Harty JT, Badovinac VP: Shaping and reshaping CD8+ T-cell memory. Nat Rev Immunol 2008, 8:107-119. 26. Yewdell JW, Bennink JR: Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu Rev Immunol 1999, 17:51-88. 27. Chaise C, Buchan SL, Rice J, Marquet J, Rouard H, Kuentz M, Vittes GE, Molinier-Frenkel V, Farcet JP, Stauss HJ et al.: DNA vaccination induces WT1-specific T-cell responses with potential clinical relevance. Blood 2008, 112:2956-2964. 28. Liu J, Ewald BA, Lynch DM, Nanda A, Sumida SM, Barouch DH: Modulation of DNA vaccine-elicited CD8+ T-lymphocyte epitope immunodominance hierarchies. J Virol 2006, 80:11991-11997. 29. Buchan S, Gronevik E, Mathiesen I, King CA, Stevenson FK, Rice J: Electroporation as a ‘‘prime/boost’’ strategy for naked DNA vaccination against a tumor antigen. J Immunol 2005, 174:6292-6298. 30. Babiuk S, Babiuk LA, van Drunen Littel-van den Hurk S: DNA vaccination: a simple concept with challenges regarding implementation. Int Rev Immunol 2006, 25:51-81. 31. Yager EJ, Dean HJ, Fuller DH: Prospects for developing an effective particle-mediated DNA vaccine against influenza. Expert Rev Vaccines 2009, 8:1205-1220. 32. van den Berg JH, Nuijen B, Schumacher TN, Haanen JB, Storm G, Beijnen JH, Hennink WE: Synthetic vehicles for DNA vaccination. J Drug Target 2010, 18:1-14. 33. van den Berg JH, Nujien B, Beijnen JH, Vincent A, van Tinteren H, Kluge J, Woerdeman LA, Hennink WE, Storm G, Schumacher TN et al.: Optimization of intradermal vaccination by DNA tattooing in human skin. Hum Gene Ther 2009, 20:181-189. 34. Low L, Mander A, McCann K, Dearnaley D, Tjelle T, Mathiesen I, Stevenson F, Ottensmeier CH: DNA vaccination with electroporation induces increased antibody responses in patients with prostate cancer. Hum Gene Ther 2009, 20:1269-1278. 35. Gribben JG, Ryan DP, Boyajian R, Urban RG, Hedley ML, Beach K, Nealon P, Matulonis U, Campos S, Gilligan TD et al.: Unexpected association between induction of immunity to the universal tumor antigen CYP1B1 and response to next therapy. Clin Cancer Res 2005, 11:4430-4436. 36. Best SR, Peng S, Juang CM, Hung CF, Hannaman D, Saunders JR, Wu TC, Pai SI: Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery. Vaccine 2009, 27:5450-5459. 37. Roos AK, Eriksson F, Timmons JA, Gerhardt J, Nyman U, Gudmundsdotter L, Brave A, Wahren B, Pisa P: Skin electroporation: effects on transgene expression. DNA persistence and local tissue environment. PLoS One 2009, 4:e7226. 38. Liu J, Kjeken R, Mathiesen I, Barouch DH: Recruitment of antigen-presenting cells to the site of inoculation and augmentation of human immunodeficiency virus type 1 DNA vaccine immunogenicity by in vivo electroporation. J Virol 2008, 82:5643-5649. The authors have analyzed the cellular infiltrates which occur following injection of an HIV-specific DNA vaccine into mouse muscle. They show that electroporation augmented the numbers of T cells, B cells, macrophages and dendritic cells leading to amplification of immunity. In that setting, addition of DNA-delivered chemokine adjuvants did not result in any further effects. 39. Radcliffe JN, Roddick JS, Friedmann PS, Stevenson FK, Thirdborough SM: Prime-boost with alternating DNA vaccines designed to engage different antigen presentation pathways generates high frequencies of peptide-specific CD8+ T cells. J Immunol 2006, 177:6626-6633. Current Opinion in Immunology 2010, 22:264–270
270 Tumour Immunology
40. Heath WR, Belz GT, Behrens GM, Smith CM, Forehan SP, Parish IA, Davey GM, Wilson NS, Carbone FR, Villadangos JA: Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens. Immunol Rev 2004, 199:9-26. 41. Shirota H, Petrenko L, Hong C, Klinman DM: Potential of transfected muscle cells to contribute to DNA vaccine immunogenicity. J Immunol 2007, 179:329-336. 42. Luckay A, Sidhu MK, Kjeken R, Megati S, Chong SY, Roopchand V, Garcia-Hand D, Abdullah R, Braun R, Montefiori DC et al.: Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques. J Virol 2007, 81:5257-5269. 43. Rosati M, Bergamaschi C, Valentin A, Kulkarni V, Jalah R, Alicea C, Patel V, von Gegerfelt AS, Montefiori DC, Venzon DJ et al.: DNA vaccination in rhesus macaques induces potent immune responses and decreases acute and chronic viremia after SIVmac251 challenge. Proc Natl Acad Sci U S A 2009, 106:15831-15836. Electroporation appears to be a key requirement for translating DNA vaccination to large animals. In this study, using an HIV gag-encoding plasmid, rhesus macaques showed dramatically enhanced cellular responses following electroporation, leading to control of viremia. Interestingly, DNA was able to strongly boost responses in SIV-infected animals treated with anti-retroviral drugs, paving the way for a possible vaccination strategy against HIV. 44. Ledwith BJ, Manam S, Troilo PJ, Barnum AB, Pauley CJ, Griffiths TG 2nd, Harper LB, Beare CM, Bagdon WJ, Nichols WW: Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice. Intervirology 2000, 43:258-272. 45. Jin X, Newman MJ, De-Rosa S, Cooper C, Thomas E, Keefer M, Fuchs J, Blattner W, Livingston BD, McKinney DM et al.: A novel HIV T helper epitope-based vaccine elicits cytokine-secreting HIV-specific CD4+ T cells in a Phase I clinical trial in HIVuninfected adults. Vaccine 2009, 27:7080-7086. 46. Martin JE, Sullivan NJ, Enama ME, Gordon IJ, Roederer M, Koup RA, Bailer RT, Chakrabarti BK, Bailey MA, Gomez PL et al.: A DNA vaccine for Ebola virus is safe and immunogenic in a phase I clinical trial. Clin Vaccine Immunol 2006, 13:1267-1277. 47. Li S, Locke E, Bruder J, Clarke D, Doolan DL, Havenga MJ, Hill AV, Liljestrom P, Monath TP, Naim HY et al.: Viral vectors for malaria vaccine development. Vaccine 2007, 25:2567-2574. 48. Alvarez-Lajonchere L, Shoukry NH, Gra B, Amador-Canizares Y, Helle F, Bedard N, Guerra I, Drouin C, Dubuisson J, GonzalezHorta EE et al.: Immunogenicity of CIGB-230, a therapeutic DNA vaccine preparation, in HCV-chronically infected individuals in a Phase I clinical trial. J Viral Hepat 2009, 16:156167. Most cancer vaccines will need to induce immunity against persistent antigen. Knowledge of responses against persistent viruses may therefore be useful. These authors have successfully used a DNA vaccine against hepatitis C structural antigens, delivered with a core protein, to treat chronically infected patients. Clinical results appear promising. 49. Jones S, Evans K, McElwaine-Johnn H, Sharpe M, Oxford J, Lambkin-Williams R, Mant T, Nolan A, Zambon M, Ellis J et al.: DNA vaccination protects against an influenza challenge in a double-blind randomised placebo-controlled phase 1b clinical trial. Vaccine 2009, 27:2506-2512.
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50. Wrammert J, Miller J, Akondy R, Ahmed R: Human immune memory to yellow fever and smallpox vaccination. J Clin Immunol 2009, 29:151-157. As we seek to vaccinate patients against cancer, there is a need to understand normal human cellular immunity. Dissection of the response to the successful conventional vaccines against yellow fever and smallpox has given these authors insights into the induction and maintenance of immune memory against acute viral infections which will have relevance for vaccine strategies. 51. Lowe DB, Shearer MH, Jumper CA, Kennedy RC: Towards progress on DNA vaccines for cancer. Cell Mol Life Sci 2007, 64:2391-2403. 52. Yuan J, Ku GY, Gallardo HF, Orlandi F, Manukian G, Rasalan TS, Xu Y, Li H, Vyas S, Mu Z et al.: Safety and immunogenicity of a human and mouse gp100 DNA vaccine in a phase I trial of patients with melanoma. Cancer Immun 2009, 9:5. 53. Lotem M, Machlenkin A, Hamburger T, Nissan A, Kadouri L, Frankenburg S, Gimmon Z, Elias O, David IB, Kuznetz A et al.: Autologous melanoma vaccine induces antitumor and selfreactive immune responses that affect patient survival and depend on MHC class II expression on vaccine cells. Clin Cancer Res 2009, 15:4968-4977. 54. Rosenberg SA: Overcoming obstacles to the effective immunotherapy of human cancer. Proc Natl Acad Sci U S A 2008, 105:12643-12644. 55. Vukmanovic-Stejic M, Agius E, Booth N, Dunne PJ, Lacy KE, Reed JR, Sobande TO, Kissane S, Salmon M, Rustin MH et al.: The kinetics of CD4+Foxp3+ T cell accumulation during a human cutaneous antigen-specific memory response in vivo. J Clin Invest 2008, 118:3639-3650. 56. Houot R, Levy R: Vaccines for lymphomas: idiotype vaccines and beyond. Blood Rev 2009, 23:137-142. 57. Fishman M: A changing world for DCvax: a PSMA loaded autologous dendritic cell vaccine for prostate cancer. Expert Opin Biol Ther 2009, 9:1565-1575. 58. Daud AI, DeConti RC, Andrews S, Urbas P, Riker AI, Sondak VK, Munster PN, Sullivan DM, Ugen KE, Messina JL et al.: Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Clin Oncol 2008, 26:5896-5903. 59. Janetzki S, Britten CM, Kalos M, Levitsky HI, Maecker HT, Melief CJ, Old LJ, Romero P, Hoos A, Davis MM: ‘‘MIATA’’minimal information about T cell assays. Immunity 2009, 31:527-528. 60. Ottensmeier C, Low L, Mander A, Williams A, Tjelle T, CamposPerez J, Heath C, Dearnaley D, Mathiesen I, Stevenson F: DNA fusion gene vaccination, delivered with or without in vivo electroporation — a potent and safe strategy for inducing anti-tumor immune responses in prostate cancer. American Association for Cancer Research Annual Meeting 2008:300. abstract. 61. Britten CM, Gouttefangeas C, Welters MJ, Pawelec G, Koch S, Ottensmeier C, Mander A, Walter S, Paschen A, Muller-Berghaus J et al.: The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays. Cancer Immunol Immunother 2008, 57:289-302.
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DNA vaccines against cancer come of age Freda K Stevenson, Christian H Ottensmeier and Jason Rice Genetic technology allows construction of DNA vaccines encoding selected tumor antigens together with molecules to direct and amplify the desired effector pathways. Their enormous promise has been marred by a problem of scaling up to human subjects. This is now largely overcome by electroporation, which increases both antigen expression and the inflammatory milieu. While the principles of vaccine design can be developed in mouse models, the real operative test is in the clinic, using patients in temporary remission. Monitoring of induced immunity, although commonly limited to blood, is providing objective qualitative and quantitative data on T-cell and antibody responses. Prolongation of remission is the goal and an activated immune system should achieve this. Address Cancer Sciences Division, University of Southampton School of Medicine, Southampton General Hospital, Southampton SO16 6YD, UK Corresponding author: Stevenson, Freda K ([email protected]), Ottensmeier, Christian H ([email protected]) and Rice, Jason ([email protected])
Current Opinion in Immunology 2010, 22:264–270 This review comes from a themed issue on Tumour Immunology Edited by Freda Stevenson and Anna Karolina Palucka Available online 19th February 2010 0952-7915/$ – see front matter # 2010 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2010.01.019
pathway [1]. Global control of immune responses, however, lies with CD4+ T cells, involved in both activation and suppression of immunity. Strategies to remove regulatory CD4+ T cells are now popular [2], but we take the view that vaccines should reverse tolerance, especially if CD4+ T-cell help can be acquired from an undamaged immune repertoire [1]. DNA vaccines produce a small amount of antigen, apparently sufficient for effective priming. However, such vaccines often induce strong CD8+ T-cell responses which can remove antigen-presenting cells at the time of boosting. This has led to ‘prime/boost’ strategies which amplify antigen levels [3]. Although viral vectors are effective, they have the major disadvantage of inducing anti-vector immunity, making further boosts, likely to be needed for cancer control, less effective [4]. Electroporation is an attractive alternative, increasing both antigen levels and inflammatory activity [5]. The vexed question of scaling up from preclinical models to clinical application may be solved by this technology and clinical trials are now in progress. In spite of sophisticated mouse models, there are many unknowns in our understanding of human immunity and certainly in our knowledge of its ability to suppress cancer. Delivery of specific antigens via DNA vaccines at least allows objective measurement of immune responses induced against the specific target, as well as analyzing what really matters, the effect on cancer cells in patients. This review will focus on the development of novel DNA vaccine designs and their evaluation in the clinic.
Introduction
Activating innate immunity
Mobilizing the powerful immune system to attack cancer cells is appealing but attention must be paid to both vaccine design and clinical context. DNA vaccines not only have an inbuilt ability to activate multiple pathways of innate immunity, but also offer a unique opportunity to guide defined antigens, accompanied by specific activator molecules, through a patient’s compromised immune system. Any therapeutic vaccination may fail if tumor load is high, so there is obvious reliance on initial treatments to bring patients into remission, a setting becoming increasingly available. However, some immunodeficiency or tolerance may persist, requiring powerful but specific vaccines. There is now a range of potential target antigens including cell surface molecules, susceptible to antibody attack, and a multitude of intracellular antigens, requiring cytotoxic T cells. Vaccine design can modify the molecular format of antigen to select the desired effector
The innate immune system detects microbial molecular arrays, including plasmid DNA, using PRRs (pattern recognition receptors). The first receptor identified for DNA was TLR9 (Toll-like receptor 9) [6], highly expressed by B cells and DCs, and able to recognize dsDNA delivered into the cell by endocytosis or autophagy (Figure 1). The classical TLR9 ligand is unmethylated CpG dinucleotide, with certain flanking motifs, with binding leading to a cascade of activation, proliferation and differentiation of immune cell subsets [7]. However, although these sequence features are found in plasmid DNA, deductions on the role of TLR9 have been complicated by the use of synthetic phosphorothioate oligonucleotides, which differ in binding specificity from natural phosphodiester DNA [8]. The importance of TLR9 for activation by plasmid DNA has also been diminished by the finding that TLR9/ mice are capable
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Figure 1
Innate immune sensors of dsDNA. DNA is a potent activator of innate immunity and this attribute is thought to contribute to the efficacy of DNA vaccines in activating antigen-specific immune responses. The mechanisms for sensing DNA appear to be multi-layered and incorporate redundancy. HMGB (high-mobility group box) proteins act as universal sensors of nucleic acids and are required for subsequent activation of innate immunity by the more discriminative PRRs (pattern recognition receptors). PRR activation leads to a final common pathway of type I interferon production except for AIM2 engagement which triggers the maturation of pro-inflammatory cytokines with no interferon response. Clearly plasmid DNA engages multiple routes to activate innate immunity. Abbreviations: dsDNA, double-stranded DNA; ppp-dsRNA, 50 -triphosphate double-stranded RNA; IRF, interferonregulatory factors; NF-kB, nuclear factor-kappaB.
of responding to DNA vaccines [9]. Clearly there are additional pathways, and these are now being described. Several cytosolic DNA sensors have been identified, the first being DAI (DNA-dependent activator of interferonregulatory factors) [10] (Figure 1). DNA binding to DAI appears to be sequence independent but lengthrestricted [11]. However, proteomic–genomic screening and gene knock-down studies revealed another potential sensor, AIM2 (absent in melanoma 2) [12,13]. This recruits the AIM2 inflammasome, a multiprotein complex that activates caspase 1, leading to secretion of IL-1b and IL-18 as well as pyroptosis [13]. A novel DNA-sensing pathway involving RNA Pol-III (polymerase III) and RIG-I (retinoic acid-inducible gene-I) has also been described: Pol-III converts AT-rich cytosolic dsDNA into dsRNA with a 50 -triphosphate moiety. This serves as a ligand for RIG-I activation, leading to the final common pathway of type I interferon production [14,15]. Our understanding of the pathways leading to the interferon response is also increasing, with the recent description of STING (stimulator of interferon genes) which acts as a key regulator of innate immune signalling in response to intracellular DNA [16]. It has also been discovered that the discriminative sensing afforded by these different PRR may be preceded by, and reliant on, a more prowww.sciencedirect.com
miscuous sensing mechanism in which HMGB (highmobility group box) proteins serve as universal sentinels for nucleic acids [17]. Clearly plasmid DNA not only delivers antigen, but also engages multiple routes to activate innate immunity (Figure 1).
Activating adaptive immunity against cancer antigens Activating the innate immune system is the first step for induction of immunity against weak tumor antigens but, to activate adaptive immunity, we need to harness the knowledge and tools of modern immunology. CD4+ T cells are pivotal both in providing help and in regulating responses. A successful DNA vaccine should circumvent regulation, and, to achieve this, we have engaged T-cell help from an undamaged anti-microbial repertoire (Figure 2) [1]. Others have used alternative molecules [18–20]. For the induction of antibody, this mimics conjugate vaccines [21], and we have used selected sequences from a favored conjugate vaccine component, tetanus toxin, with dramatic effects in preclinical models [1] and encouraging results in a clinical trial (see below) (Figure 2). Conjugation is also effective for inducing effector T cells [1,22] and additive sequences can easily be genetically Current Opinion in Immunology 2010, 22:264–270
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Figure 2
Engagement of T-cell help for the induction of anti-tumor immunity. A DNA vaccine encoding a tumor antigen fused to the FrC (Fragment C) of tetanus toxin is injected into muscle. The fusion protein is cross-presented to DCs (dendritic cells) [1] which then express MHC Class II-associated immunogenic peptides derived from FrC. Responding CD4+ T cells interact and ‘license’ DCs for the induction of T-cell responses against weakly immunogenic tumor antigen-derived peptides.
combined with tumor antigen for induction of CD4+ and/ or CD8+ T cells [1]. The mode of action is likely through ‘licensing’ of APCs (antigen-presenting cells) via interaction with non-suppressed CD4+ T cells specific for tetanus toxin-derived peptides within the vaccine (Figure 2) [1]. Multiple interacting ligand/receptor pairs should protect APCs against apoptosis and OX40/OX40L interaction is known to reverse regulatory T cells [23]. These immune mechanisms are also likely involved in xenogeneic DNA vaccine strategies against melanoma [24]. The approach uses xenogeneic counterparts of autologous tumor antigens and has shown efficacy in dogs where a vaccine encoding human tryrosinase gained conditional FDA approval in 2007 [24]. The rationale is that target MHC Class I-binding epitopes are presented in a more immunogenic context, with CD4+ T-cell help probably provided via the xenogeneic sequences, although this has not been proved. Induction of cytolytic CD8+ T cells is a clear goal for targeting cancer cells. Establishing long-term memory requires T-cell help [25], and therefore, in our case, microbial sequences, but immunological principles have to be heeded. The phenomenon of immunodominance requires focusing of CD8+ T-cell responses onto tumorCurrent Opinion in Immunology 2010, 22:264–270
derived peptides, and must avoid competition from microbe-derived peptides [26]. We have achieved this by minimizing the microbial sequence and optimizing presentation of tumor-derived epitopes (reviewed in [1]). This design is in clinical trial for patients with prostate cancer (see below). No doubt there are many ways to harness immunological principles, but this strategy can break tolerance and activate high levels of epitope-specific CD8+ T cells able to suppress tumor in numerous models [1]. It demonstrates superior performance compared to peptide vaccination [27]. Additional epitopes from the same or other target antigens can be placed in separate vaccines and delivered in different sites thereby avoiding competition [28] and this approach will soon be tested in a trial of chronic myeloid leukaemia (see below). A remaining unknown is whether there is a need to include tumor-derived sequences to induce CD4+ responses, or indeed whether such sequences are best avoided in case regulatory T cells are stimulated. Flexibility of DNA construction is allowing investigation of these questions.
DNA vaccine delivery DNA vaccines can induce strong, protective immunity in a wide range of mouse cancer models, but initial clinical www.sciencedirect.com
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testing proved disappointing. In retrospect, for intramuscular routes, this was predictable, given the dependence of efficacy on the volume injected [29]. High hydrostatic pressure apparently increases plasmid uptake and the resulting tissue injury can attract APCs to the injection site [30]. Scaling up to human subjects would require an unacceptable injection volume, and other developments were required. The problem may be avoidable using the skin route, where gene gun or other delivery methods have shown some success, for example in prophylactic influenza vaccination [31]. Another popular approach for increasing efficacy has been to follow priming with DNA with boosting using viral vectors, and this appears useful for infectious diseases [3]. The amplification of response by viral vectors is likely to be via the attraction and infection of antigen-presenting cells. This will overcome the tendency of primed cytotoxic T cells to remove transfected muscle cells and/or APCs which could curtail the response. However, viral vector delivery is not readily applicable to cancer where preexisting or induced antivector immunity would undermine repeated boosting efficacy [4]. Alternative physical strategies include formulations with nanoparticles, microparticles, and liposomes, or delivery with electroporation, particle bombardment, jet injection, and tattooing [31–34]. Although some have reached clinical testing [31,33–35], electroporation has emerged as a preferred and very effective strategy [34,36]. EP (electroporation) involves electrical stimulation across the muscle (or skin) site coincident or immediately following DNA vaccine injection. The improved transfection efficiency increases antigen expression (10–100-fold) and entry of inflammatory cells [5,37,38]. These factors will likely increase cross-presentation of DNA vaccineencoded antigen [39] resulting in improved immune responses [40], although transfected muscle cells may also contribute directly [41]. EP overcomes the failure of low-volume injection to induce immunity in mice [29], and the combination of DNA priming with DNA/EP at boosting is particularly effective [29]. Also, in large animals [42] and human subjects [34], EP significantly increases DNA vaccine-specific immune responses with rhesus macaques showing improved vaccine-specific Tcell responses [42] and a reduction of viremia [43]. Initial concern that improved transfection efficiency may increase integration levels, appear unfounded [42,44]. Thus, EP is a safe delivery system that appears to overcome the translational block to effective DNA vaccination in the clinic.
Clinical evaluation DNA vaccines against infectious diseases
DNA vaccines are now licensed for the prevention of infection in horses (West Nile virus) and salmon (infecwww.sciencedirect.com
tious hemorrhagic necrosis virus) [1]. In human subjects, reports of prophylactic DNA vaccines able to induce antibody and CD4+ T-cell responses, with potential efficacy against several dangerous viruses, are accumulating [45,46]. Prime/boost approaches with viral vectors have been widely tested, especially for vaccines against malaria, where the variable influence of the boosting vector on outcome has been noted [47]. In the more challenging therapeutic setting, where parallels can be drawn with cancer, DNA vaccination can induce immune responses, with, in the case of persistent hepatitis C infection, apparent clinical benefit [48]. Certainly the speed of construction of DNA vaccines presents a real advantage for both prophylaxis and therapy of new pathogens, including influenza [49]. Beyond confirming that DNA vaccines can be effective clinically, testing of these vaccines in human subjects is revealing how human immune responses differ from those of mice and, in conjunction with careful studies of immune responses to conventional vaccines [50], will be critical for the optimal design of clinical trials against cancer. DNA vaccines against cancer
While there are parallels with persistent infection, the clinical settings for cancer patients differ. Variable factors include the nature of the cancer, any potentially preexisting immunity and the effects of previous treatment. Ideally small trials of defined groups, with rapid assessment of efficacy of vaccine design and delivery are required. However, in common with other vaccines early evaluation of DNA vaccination followed classical drug development pathways in recruiting patients who had exhausted standard treatment options (reviewed in [51,1]; also see [52]). Clearly, successful vaccination requires an intact immune system [53] and studies are therefore now moving toward patients with minimal disease. In terms of evaluating immune responses, this is straightforward for antibody, but more difficult for T cells. Specific responses to encoded antigens can be measured but there is argument about connecting responses to clinical outcome [54]. The major problem is effector cells may be located in tumor tissues or in vaccination sites, and analysis of blood samples may therefore underestimate significant responses. Alternatively, if effector cells are unable to enter the tumor site, measurements in blood may overestimate efficacy. While tumor biopsies are rarely available, the increasing use of skin challenge is extending knowledge of migratory potential [55], although the fate of effector cells in tumor sites remains less well understood. We undertook an early study in patients with follicular lymphoma targeting idiotypic Ig. We used our fusion gene design consisting of idiotypic determinants assembled as single chain Fv, linked to the FrC (Fragment C) portion of Current Opinion in Immunology 2010, 22:264–270
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tetanus toxin [1]. Individual naked DNA scFv-FrC vaccines were delivered by intramuscular injection, and we detected a boost of anti-FrC responses, in 75% of patients, with anti-idiotypic immunity induced in 38% [1]. Although encouraging, responses were relatively weak, as found in a similar trial using DNA-delivered chimeric Ig [56], although the use of individual vaccines makes any comparisons difficult. Clearly, since the goal is to induce anti-idiotypic antibody or CD4+ T cells, electroporation would be an ideal addition to any new trial. Our next trial tested a single vaccine aimed at induction of immunity against an antigen expressed in prostate cancer, PSMA (prostate-specific membrane antigen), already being tested in different vaccine trials [57]. Several questions were asked: first, if the modified design using a DOM (domain) of Fragment C of tetanus toxin fused to an epitope sequence from PSMA would induce anti-tumor CD8+ T cells; second, if EP would enhance responses. We used escalating doses (0.8–3.2 mg) EP. The patient group had minimal (radiologically undetectable) tumor load at biochemical failure of disease control [34]. Electroporation was safe and well tolerated, as reported for DNA cytokine delivery [58]. We had the opportunity to measure antibody and CD4+ T-cell responses against the tetanus toxin-derived DOM sequence, which, in addition to indicating vaccine performance, provided some indication of non-tolerized immune capacity. The majority of patients (21/30) showed clear responses with EP improving anti-DOM antibody responses (17) [34], although the effect on CD4+ T cells was less marked. +
The main goal was to induce CD8 T-cell responses against the PSMA peptide. Here we need to consider the kinetics of the response, since it would be expected that effector cells would increase following vaccination, migrate to antigen-expressing sites, with some then converting to memory T cells. Unless the blood samples ‘catch’ the peak of the induction, it is unlikely that ex vivo functional assays will be useful, although tetramer staining, when available, will be. Instead, central memory T cells should be sought and these require stimulation in vitro. While this complicates quantitation, most clinical trials follow this procedure [59,60]. It is vital that expertise is shared with inter-laboratory comparisons, and multinational collaborations, such as CIMT and CVC [59,61] are critical not only to standardize assay systems but also to identify the limits of reproducible detection of immune responses. In our prostate cancer trial, we are detecting IFNgproducing CD8+ T-cell responses against the target PSMA peptide in 60% of cases [60]. Preliminary evidence indicates that electroporation stimulates CD8+ Tcell responses more quickly and also that the overall level appears to be higher [60]. Using an xenogeneic approach, Current Opinion in Immunology 2010, 22:264–270
a phase I trial in 19 patients with high risk but completely resected melanoma was recently reported [52]. DNA vaccines encoding human and mouse GP100 were injected sequentially with evidence of expansion of tetramer+ CD8+ T cells in 5/18 patients [52].
Conclusions The data so far indicate that DNA vaccines can induce selected immune responses against tumor antigens in patients. We are learning that the human immune response shows similarities to mouse models but also differences, particularly in kinetics. While new principles will continue to emerge from mice, the challenge is to increase our understanding of human immunity, which, in spite of the success of prophylactic vaccination against infection, remains enigmatic. For cancer vaccines, measurements in blood provide only a limited snapshot of CD8+ T-cell responses, and skin challenges could be helpful. Correlation of immunity with clinical outcome is awaited and those tumors with biomarkers will allow early insights. Combinations with a judicious use of chemotherapy, which could have differential effects on suppressor mechanisms, are an emerging new approach. The ultimate goal of changing tumor behavior for patients is in sight but flexible small trials are still needed to guide the way.
Acknowledgements We thank David Anstee, Paul Lloyd-Evans and Iacob Mathiesen for ongoing support for our portfolio of DNA vaccine clinical trials. This research was supported by Leukaemia Research (grants 0306 and 08025), Cancer Research UK (project grant 7576, programme grant C491/A4363), Inovio and the Alan Willett Foundation.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Rice J, Ottensmeier CH, Stevenson FK: DNA vaccines: precision tools for activating effective immunity against cancer. Nat Rev Cancer 2008, 8:108-120. This review outlines the way that immunological principles can be harnessed for the induction of effective immunity against different molecular targets. It focuses on strategies for inducing T-cell help to activate immunity and to break tolerance against cancer antigens. 2.
Rech AJ, Vonderheide RH: Clinical use of anti-CD25 antibody daclizumab to enhance immune responses to tumor antigen vaccination by targeting regulatory T cells. Ann N Y Acad Sci 2009, 1174:99-106.
3.
Gilbert SC, Moorthy VS, Andrews L, Pathan AA, McConkey SJ, Vuola JM, Keating SM, Berthoud T, Webster D, McShane H et al.: Synergistic DNA–MVA prime-boost vaccination regimes for malaria and tuberculosis. Vaccine 2006, 24:4554-4561.
4.
Smith CL, Mirza F, Pasquetto V, Tscharke DC, Palmowski MJ, Dunbar PR, Sette A, Harris AL, Cerundolo V: Immunodominance of poxviral-specific CTL in a human trial of recombinantmodified vaccinia Ankara. J Immunol 2005, 175:8431-8437.
5.
Ahlen G, Soderholm J, Tjelle T, Kjeken R, Frelin L, Hoglund U, Blomberg P, Fons M, Mathiesen I, Sallberg M: In vivo electroporation enhances the immunogenicity of hepatitis C virus nonstructural 3/4A DNA by increased local DNA uptake, protein expression, inflammation, and infiltration of CD3+ T cells. J Immunol 2007, 179:4741-4753. www.sciencedirect.com
DNA Vaccines Against Cancer Stevenson, Ottensmeier and Rice 269
6.
Hemmi H, Takeuchi O, Kawai T, Kaisho T, Sato S, Sanjo H, Matsumoto M, Hoshino K, Wagner H, Takeda K et al.: A Toll-like receptor recognizes bacterial DNA. Nature 2000, 408:740-745.
7.
Krieg AM: CpG motifs in bacterial DNA and their immune effects. Annu Rev Immunol 2002, 20:709-760.
8.
Haas T, Metzger J, Schmitz F, Heit A, Muller T, Latz E, Wagner H: The DNA sugar backbone 20 deoxyribose determines toll-like receptor 9 activation. Immunity 2008, 28:315-323.
9.
Spies B, Hochrein H, Vabulas M, Huster K, Busch DH, Schmitz F, Heit A, Wagner H: Vaccination with plasmid DNA activates dendritic cells via Toll-like receptor 9 (TLR9) but functions in TLR9-deficient mice. J Immunol 2003, 171:5908-5912.
10. Takaoka A, Wang Z, Choi MK, Yanai H, Negishi H, Ban T, Lu Y, Miyagishi M, Kodama T, Honda K et al.: DAI (DLM-1/ZBP1) is a cytosolic DNA sensor and an activator of innate immune response. Nature 2007, 448:501-505. 11. Wang Z, Choi MK, Ban T, Yanai H, Negishi H, Lu Y, Tamura T, Takaoka A, Nishikura K, Taniguchi T: Regulation of innate immune responses by DAI (DLM-1/ZBP1) and other DNA-sensing molecules. Proc Natl Acad Sci U S A 2008, 105:5477-5482. 12. Burckstummer T, Baumann C, Bluml S, Dixit E, Durnberger G, Jahn H, Planyavsky M, Bilban M, Colinge J, Bennett KL et al.: An orthogonal proteomic–genomic screen identifies AIM2 as a cytoplasmic DNA sensor for the inflammasome. Nat Immunol 2009, 10:266-272. 13. Fernandes-Alnemri T, Yu JW, Datta P, Wu J, Alnemri ES: AIM2 activates the inflammasome and cell death in response to cytoplasmic DNA. Nature 2009, 458:509-513. 14. Ablasser A, Bauernfeind F, Hartmann G, Latz E, Fitzgerald KA, Hornung V: RIG-I-dependent sensing of poly(dA:dT) through the induction of an RNA polymerase III-transcribed RNA intermediate. Nat Immunol 2009, 10:1065-1072. 15. Chiu YH, Macmillan JB, Chen ZJ: RNA polymerase III detects cytosolic DNA and induces type I interferons through the RIG-I pathway. Cell 2009, 138:576-591. 16. Ishikawa H, Ma Z, Barber GN: STING regulates intracellular DNA-mediated, type I interferon-dependent innate immunity. Nature 2009, 461:788-792. 17. Yanai H, Ban T, Wang Z, Choi MK, Kawamura T, Negishi H, Nakasato M, Lu Y, Hangai S, Koshiba R et al.: HMGB proteins function as universal sentinels for nucleic-acid-mediated innate immune responses. Nature 2009, 462:99-103. The response of the innate immune system to nucleic acids, mediated by transmembrane TLRs and cytosolic receptors, is a critical prelude to the induction of immunity via DNA vaccination. These authors reveal that universal sentinels, the high-mobility group box proteins, bind nucleic acids, integrating a hierarchy of selective stimulatory pathways of the innate immune response. 18. Hung CF, Cheng WF, Hsu KF, Chai CY, He L, Ling M, Wu TC: Cancer immunotherapy using a DNA vaccine encoding the translocation domain of a bacterial toxin linked to a tumor antigen. Cancer Res 2001, 61:3698-3703. 19. Savelyeva N, Munday R, Spellerberg MB, Lomonossoff GP, Stevenson FK: Plant viral genes in DNA idiotypic vaccines activate linked CD4+ T-cell mediated immunity against B-cell malignancies. Nat Biotechnol 2001, 19:760-764. 20. Wolkers MC, Toebes M, Okabe M, Haanen JB, Schumacher TN: Optimizing the efficacy of epitope-directed DNA vaccination. J Immunol 2002, 168:4998-5004. 21. Peltola H: Worldwide Haemophilus influenzae type b disease at the beginning of the 21st century: global analysis of the disease burden 25 years after the use of the polysaccharide vaccine and a decade after the advent of conjugates. Clin Microbiol Rev 2000, 13:302-317. 22. Zanetti M: T for two: when helpers need help. Autoimmun Rev 2005, 4:571-578. 23. Piconese S, Valzasina B, Colombo MP: OX40 triggering blocks suppression by regulatory T cells and facilitates tumor rejection. J Exp Med 2008, 205:825-839. www.sciencedirect.com
24. Liao JC, Gregor P, Wolchok JD, Orlandi F, Craft D, Leung C, Houghton AN, Bergman PJ: Vaccination with human tyrosinase DNA induces antibody responses in dogs with advanced melanoma. Cancer Immun 2006, 6:8. 25. Harty JT, Badovinac VP: Shaping and reshaping CD8+ T-cell memory. Nat Rev Immunol 2008, 8:107-119. 26. Yewdell JW, Bennink JR: Immunodominance in major histocompatibility complex class I-restricted T lymphocyte responses. Annu Rev Immunol 1999, 17:51-88. 27. Chaise C, Buchan SL, Rice J, Marquet J, Rouard H, Kuentz M, Vittes GE, Molinier-Frenkel V, Farcet JP, Stauss HJ et al.: DNA vaccination induces WT1-specific T-cell responses with potential clinical relevance. Blood 2008, 112:2956-2964. 28. Liu J, Ewald BA, Lynch DM, Nanda A, Sumida SM, Barouch DH: Modulation of DNA vaccine-elicited CD8+ T-lymphocyte epitope immunodominance hierarchies. J Virol 2006, 80:11991-11997. 29. Buchan S, Gronevik E, Mathiesen I, King CA, Stevenson FK, Rice J: Electroporation as a ‘‘prime/boost’’ strategy for naked DNA vaccination against a tumor antigen. J Immunol 2005, 174:6292-6298. 30. Babiuk S, Babiuk LA, van Drunen Littel-van den Hurk S: DNA vaccination: a simple concept with challenges regarding implementation. Int Rev Immunol 2006, 25:51-81. 31. Yager EJ, Dean HJ, Fuller DH: Prospects for developing an effective particle-mediated DNA vaccine against influenza. Expert Rev Vaccines 2009, 8:1205-1220. 32. van den Berg JH, Nuijen B, Schumacher TN, Haanen JB, Storm G, Beijnen JH, Hennink WE: Synthetic vehicles for DNA vaccination. J Drug Target 2010, 18:1-14. 33. van den Berg JH, Nujien B, Beijnen JH, Vincent A, van Tinteren H, Kluge J, Woerdeman LA, Hennink WE, Storm G, Schumacher TN et al.: Optimization of intradermal vaccination by DNA tattooing in human skin. Hum Gene Ther 2009, 20:181-189. 34. Low L, Mander A, McCann K, Dearnaley D, Tjelle T, Mathiesen I, Stevenson F, Ottensmeier CH: DNA vaccination with electroporation induces increased antibody responses in patients with prostate cancer. Hum Gene Ther 2009, 20:1269-1278. 35. Gribben JG, Ryan DP, Boyajian R, Urban RG, Hedley ML, Beach K, Nealon P, Matulonis U, Campos S, Gilligan TD et al.: Unexpected association between induction of immunity to the universal tumor antigen CYP1B1 and response to next therapy. Clin Cancer Res 2005, 11:4430-4436. 36. Best SR, Peng S, Juang CM, Hung CF, Hannaman D, Saunders JR, Wu TC, Pai SI: Administration of HPV DNA vaccine via electroporation elicits the strongest CD8+ T cell immune responses compared to intramuscular injection and intradermal gene gun delivery. Vaccine 2009, 27:5450-5459. 37. Roos AK, Eriksson F, Timmons JA, Gerhardt J, Nyman U, Gudmundsdotter L, Brave A, Wahren B, Pisa P: Skin electroporation: effects on transgene expression. DNA persistence and local tissue environment. PLoS One 2009, 4:e7226. 38. Liu J, Kjeken R, Mathiesen I, Barouch DH: Recruitment of antigen-presenting cells to the site of inoculation and augmentation of human immunodeficiency virus type 1 DNA vaccine immunogenicity by in vivo electroporation. J Virol 2008, 82:5643-5649. The authors have analyzed the cellular infiltrates which occur following injection of an HIV-specific DNA vaccine into mouse muscle. They show that electroporation augmented the numbers of T cells, B cells, macrophages and dendritic cells leading to amplification of immunity. In that setting, addition of DNA-delivered chemokine adjuvants did not result in any further effects. 39. Radcliffe JN, Roddick JS, Friedmann PS, Stevenson FK, Thirdborough SM: Prime-boost with alternating DNA vaccines designed to engage different antigen presentation pathways generates high frequencies of peptide-specific CD8+ T cells. J Immunol 2006, 177:6626-6633. Current Opinion in Immunology 2010, 22:264–270
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40. Heath WR, Belz GT, Behrens GM, Smith CM, Forehan SP, Parish IA, Davey GM, Wilson NS, Carbone FR, Villadangos JA: Cross-presentation, dendritic cell subsets, and the generation of immunity to cellular antigens. Immunol Rev 2004, 199:9-26. 41. Shirota H, Petrenko L, Hong C, Klinman DM: Potential of transfected muscle cells to contribute to DNA vaccine immunogenicity. J Immunol 2007, 179:329-336. 42. Luckay A, Sidhu MK, Kjeken R, Megati S, Chong SY, Roopchand V, Garcia-Hand D, Abdullah R, Braun R, Montefiori DC et al.: Effect of plasmid DNA vaccine design and in vivo electroporation on the resulting vaccine-specific immune responses in rhesus macaques. J Virol 2007, 81:5257-5269. 43. Rosati M, Bergamaschi C, Valentin A, Kulkarni V, Jalah R, Alicea C, Patel V, von Gegerfelt AS, Montefiori DC, Venzon DJ et al.: DNA vaccination in rhesus macaques induces potent immune responses and decreases acute and chronic viremia after SIVmac251 challenge. Proc Natl Acad Sci U S A 2009, 106:15831-15836. Electroporation appears to be a key requirement for translating DNA vaccination to large animals. In this study, using an HIV gag-encoding plasmid, rhesus macaques showed dramatically enhanced cellular responses following electroporation, leading to control of viremia. Interestingly, DNA was able to strongly boost responses in SIV-infected animals treated with anti-retroviral drugs, paving the way for a possible vaccination strategy against HIV. 44. Ledwith BJ, Manam S, Troilo PJ, Barnum AB, Pauley CJ, Griffiths TG 2nd, Harper LB, Beare CM, Bagdon WJ, Nichols WW: Plasmid DNA vaccines: investigation of integration into host cellular DNA following intramuscular injection in mice. Intervirology 2000, 43:258-272. 45. Jin X, Newman MJ, De-Rosa S, Cooper C, Thomas E, Keefer M, Fuchs J, Blattner W, Livingston BD, McKinney DM et al.: A novel HIV T helper epitope-based vaccine elicits cytokine-secreting HIV-specific CD4+ T cells in a Phase I clinical trial in HIVuninfected adults. Vaccine 2009, 27:7080-7086. 46. Martin JE, Sullivan NJ, Enama ME, Gordon IJ, Roederer M, Koup RA, Bailer RT, Chakrabarti BK, Bailey MA, Gomez PL et al.: A DNA vaccine for Ebola virus is safe and immunogenic in a phase I clinical trial. Clin Vaccine Immunol 2006, 13:1267-1277. 47. Li S, Locke E, Bruder J, Clarke D, Doolan DL, Havenga MJ, Hill AV, Liljestrom P, Monath TP, Naim HY et al.: Viral vectors for malaria vaccine development. Vaccine 2007, 25:2567-2574. 48. Alvarez-Lajonchere L, Shoukry NH, Gra B, Amador-Canizares Y, Helle F, Bedard N, Guerra I, Drouin C, Dubuisson J, GonzalezHorta EE et al.: Immunogenicity of CIGB-230, a therapeutic DNA vaccine preparation, in HCV-chronically infected individuals in a Phase I clinical trial. J Viral Hepat 2009, 16:156167. Most cancer vaccines will need to induce immunity against persistent antigen. Knowledge of responses against persistent viruses may therefore be useful. These authors have successfully used a DNA vaccine against hepatitis C structural antigens, delivered with a core protein, to treat chronically infected patients. Clinical results appear promising. 49. Jones S, Evans K, McElwaine-Johnn H, Sharpe M, Oxford J, Lambkin-Williams R, Mant T, Nolan A, Zambon M, Ellis J et al.: DNA vaccination protects against an influenza challenge in a double-blind randomised placebo-controlled phase 1b clinical trial. Vaccine 2009, 27:2506-2512.
Current Opinion in Immunology 2010, 22:264–270
50. Wrammert J, Miller J, Akondy R, Ahmed R: Human immune memory to yellow fever and smallpox vaccination. J Clin Immunol 2009, 29:151-157. As we seek to vaccinate patients against cancer, there is a need to understand normal human cellular immunity. Dissection of the response to the successful conventional vaccines against yellow fever and smallpox has given these authors insights into the induction and maintenance of immune memory against acute viral infections which will have relevance for vaccine strategies. 51. Lowe DB, Shearer MH, Jumper CA, Kennedy RC: Towards progress on DNA vaccines for cancer. Cell Mol Life Sci 2007, 64:2391-2403. 52. Yuan J, Ku GY, Gallardo HF, Orlandi F, Manukian G, Rasalan TS, Xu Y, Li H, Vyas S, Mu Z et al.: Safety and immunogenicity of a human and mouse gp100 DNA vaccine in a phase I trial of patients with melanoma. Cancer Immun 2009, 9:5. 53. Lotem M, Machlenkin A, Hamburger T, Nissan A, Kadouri L, Frankenburg S, Gimmon Z, Elias O, David IB, Kuznetz A et al.: Autologous melanoma vaccine induces antitumor and selfreactive immune responses that affect patient survival and depend on MHC class II expression on vaccine cells. Clin Cancer Res 2009, 15:4968-4977. 54. Rosenberg SA: Overcoming obstacles to the effective immunotherapy of human cancer. Proc Natl Acad Sci U S A 2008, 105:12643-12644. 55. Vukmanovic-Stejic M, Agius E, Booth N, Dunne PJ, Lacy KE, Reed JR, Sobande TO, Kissane S, Salmon M, Rustin MH et al.: The kinetics of CD4+Foxp3+ T cell accumulation during a human cutaneous antigen-specific memory response in vivo. J Clin Invest 2008, 118:3639-3650. 56. Houot R, Levy R: Vaccines for lymphomas: idiotype vaccines and beyond. Blood Rev 2009, 23:137-142. 57. Fishman M: A changing world for DCvax: a PSMA loaded autologous dendritic cell vaccine for prostate cancer. Expert Opin Biol Ther 2009, 9:1565-1575. 58. Daud AI, DeConti RC, Andrews S, Urbas P, Riker AI, Sondak VK, Munster PN, Sullivan DM, Ugen KE, Messina JL et al.: Phase I trial of interleukin-12 plasmid electroporation in patients with metastatic melanoma. J Clin Oncol 2008, 26:5896-5903. 59. Janetzki S, Britten CM, Kalos M, Levitsky HI, Maecker HT, Melief CJ, Old LJ, Romero P, Hoos A, Davis MM: ‘‘MIATA’’minimal information about T cell assays. Immunity 2009, 31:527-528. 60. Ottensmeier C, Low L, Mander A, Williams A, Tjelle T, CamposPerez J, Heath C, Dearnaley D, Mathiesen I, Stevenson F: DNA fusion gene vaccination, delivered with or without in vivo electroporation — a potent and safe strategy for inducing anti-tumor immune responses in prostate cancer. American Association for Cancer Research Annual Meeting 2008:300. abstract. 61. Britten CM, Gouttefangeas C, Welters MJ, Pawelec G, Koch S, Ottensmeier C, Mander A, Walter S, Paschen A, Muller-Berghaus J et al.: The CIMT-monitoring panel: a two-step approach to harmonize the enumeration of antigen-specific CD8+ T lymphocytes by structural and functional assays. Cancer Immunol Immunother 2008, 57:289-302.
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