Improved peptide vaccine strategies, creating synthetic artificial infections to maximize immune efficacy

Advanced Drug Delivery Reviews 58 (2006) 916 – 930 www.elsevier.com/locate/addr

Improved peptide vaccine strategies, creating synthetic artificial infections to maximize immune efficacy☆ Sjoerd H. van der Burg a,⁎, Martijn S. Bijker b , Marij J.P. Welters b , R. Offringa b , Cornelis J.M. Melief b a

Department of Clinical Oncology, Leiden University Medical Centre, Albinusdreef 2, 2333 ZA Leiden, The Netherlands b Department of Immunohematology and Blood Transfusion, Leiden University Medical Center, Albinusdreef 2, 2300 RC Leiden, The Netherlands Received 22 November 2005; accepted 10 July 2006 Available online 12 August 2006

Abstract Soon after it was realized that T-cells recognize their target antigens as small protein fragments or peptides presented by MHC molecules at the cell surface, these peptide epitopes have been tried as vaccines. Human testing of such vaccines, although protective in mouse models, has produced mixed results. Since these initial trials, there has been an tremendous increase in our understanding of how infectious organisms can induce potent immune responses. In this article we review the key changes in the design, formulation and delivery of synthetic peptide vaccines that are applied to improve peptide vaccine strategies. © 2006 Elsevier B.V. All rights reserved. Keywords: Peptides; Cell-mediated immunity; Adjuvants; Immunotherapy; Tumour antigens

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Enhancing the immunogenicity of minimal CTL peptide-epitope vaccines . . . . . . . . . . Inclusion of T-helper epitopes; improving the strength and quality of vaccines. . . . . . . . Increasing peptide length for top quality in vivo presentation; size matters! . . . . . . . . . HLA-type independent peptide vaccines aimed at multiple antigens; just a matter of mixing! Adding danger signals to improve the induction and polarization of T-cell immunity . . . .

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This review is part of the Advanced Drug Delivery Reviews theme issue on “Human Cancer Vaccines”, Vol. 58/8, 2006. ⁎ Corresponding author. Tel.: +31 71 526 1180, +31 71 526 3464 (secretary); fax: +31 71 526 6760. E-mail address: [email protected] (S.H. van der Burg).

0169-409X/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.addr.2005.11.003

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7. Synthetic peptides vaccines do their job. Change the immunotherapeutic strategy! . . . . . . . . . . . . . . . . 924 8. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 925 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 925

1. Introduction Synthetic peptide vaccines, aiming at the induction of T-cell immunity, hold great promise for prevention and therapy of infectious or malignant diseases. Synthetic peptides are easily produced, are chemically stable and are free of bacterial/viral contaminating substances as well as devoid of oncogenic potential. Importantly, the simplicity of producing clinical grade peptides allows swift changes in the design of peptide vaccines and, therefore, rapid translation of new immunological concepts into phase I/II trials in humans. In addition, the use of synthetic peptides offers an alternative for proteins, which are currently hard to produce by recombinant technology due to their inherent toxicity for the bacterial/viral expression system (e.g., Human Papilloma Virus type 16-E2, wild-type p53). Following the first two reports in 1991, in which it was clearly shown that vaccination with exact MHC class I binding CTL peptide-epitopes in incomplete Freund's Adjuvant (IFA) protected mice against a subsequent challenge with a live virus [1,2], questions surfaced with respect to the efficiency of this vaccine design. While in some of the model systems, vaccination with such a vaccine resulted in the protection, for instance, against the outgrowth of human papilloma virus type 16 (HPV16) positive tumour cells in mice [3] or metastases of established tumours [4], other reports indicated that this type of minimal CTL peptide-epitope vaccines elicited only low levels of CTL [5], caused the deletion of specific CTL [6,7], or enhanced the outgrowth of virus-transformed tumour cells in mice [7,8]. Notwithstanding these doubts concerning vaccine efficiency, numerous therapeutic vaccination trials were conducted, with the result that injection of unmodified minimal CTL peptide-epitopes in IFA elicited a T-cell response in only a few of over 200 vaccinated patients with solid tumours (reviewed in [9]). In hindsight, this is not a surprise, if only because we now know so much more about the requirements for the induction, maintenance and polarization of T-cell immunity, as well as about the

pharmacokinetics of peptide vaccines and adjuvant. We highlight here the key changes in the design, formulation and delivery of synthetic peptide vaccines in relation to their effects in clinical trials.

2. Enhancing the immunogenicity of minimal CTL peptide-epitope vaccines CD8+ cytotoxic T-cells (CTL) recognize their target antigens as small protein fragments presented by MHC class I molecules at the cell surface. Many of the recognized peptides presented on tumours originate from deregulated or mutated self-proteins. These selfpeptides can readily serve as molecular targets for fully activated CTL, provided that these CTL have escaped tolerance induction, but are generally poor immunogens unable to elicit a fully activated CTL response. One of the major reasons for this inherent lack of immunogenicity is that these peptides display a low MHC-binding affinity [10]. Alterations of the MHC-peptide binding affinity by conservative amino acid modifications at the MHC-binding residues of the peptides results in so-called heteroclitic (with higher biological potency) peptides able to induce more robust polyclonal CTL responses that are biologically active in vivo, as reflected by their capacity to recognize the wild-type epitope and to reject both newly implanted and established tumours [11]. This concept was validated in metastatic melanoma patients using an immunodominant peptide from the gp100 melanomaassociated antigen. Ten out of 11 patients receiving the modified gp100 peptide in IFA responded, while only 2 reactions were observed in the group of 8 patients vaccinated with the wild-type gp100 epitope in IFA [12]. The increased in vivo immunogenicity of the modified gp100 peptide was confirmed in larger clinical trials in which the number of responders increased to more than 75% in metastatic melanoma patients [13] and over 90% of patients with resected stage I to III melanoma [14,15]. Similarly, vaccination with a mix of wild-type and heteroclitic MART-1 peptide in IFA resulted in the expansion of specific CD8+ T-cells in 12

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out of 17 patients. The 2 strongest responders also showed clinical signs of regressions [16]. However, this concept of enhancing MHC peptide binding affinity was not applicable to all peptides since similar attempts to increase the CTL response to an epitope of the melanoma-associated tyrosinase antigen failed [13,14]. Furthermore, no difference in the magnitude of the response, the number of responders or clinical responsiveness was observed in patients receiving either a Wilms' tumour gene specific wild-type peptide (WT-1) or the heteroclitic WT-1 peptide [17]. This indicated that lack of appropriate MHC-peptide affinity was only part of the difficulties in getting synthetic peptide vaccines to work effectively.

3. Inclusion of T-helper epitopes; improving the strength and quality of vaccines A closer examination of the immune response inuced by the minimal MHC class I binding peptide vaccine used to induce a protective CTL against lymphocytic choriomeningitis virus (LCMV) [1] revealed that this small peptide epitope also contained a helper CD4+ Tcell epitope and induced a concomitant T-helper response [18]. In vivo elimination of CD4+ T-cells before immunization strongly reduced the virus-specific CTL response [18] demonstrating that the efficacy of this synthetic peptide vaccine depended on both CD4+ T-cells and CD8+ T-cells. Indeed, vaccination of mice with a mix of malaria-derived T-helper and CTL peptide epitopes in IFA resulted in stronger malaria-specific CTL responses than when mice were injected with CTL

peptide only (Fig. 1) [5]. In addition, vaccination of mice with a mix of murine leukemia virus (MuLV)-derived Thelper and CTL peptide-epitopes induced a protective anti-tumour response against MuLV-induced tumour cells, whereas vaccination with the CTL peptide epitope did not [19]. The results of a phase I clinical trial in patients at high risk for melanoma endorsed this principle. Patients were vaccinated with MHC class I restricted CTL epitopes of the melanoma-associated tyrosinase antigen and a non-specific helper protein KLH. Tyrosinase peptide-specific IFNγ-producing T cells were detected as early as 2 weeks after the second vaccination in 5 of the 9 patients vaccinated in combination with the T-helper protein but not in any of the patients vaccinated with tyrosinase peptides without KLH [20]. Although this difference was less pronounced after more vaccinations, the patient group vaccinated with tyrosinase peptide and KLH responded better than the other vaccine groups [20]. Physical linking of T-helper peptide and CTL peptide-epitope further increased the magnitude of the CTL response [21,22] suggesting that the presentation of both T-helper and CTL peptide epitopes on a single APC was more efficient than when the two epitopes are presented on different APC, which may occur when these epitopes are delivered as a mix (Fig. 1). These data fit well with recent studies indicating a pivotal role for antigenspecific CD4+ T-helper cells in the induction and expansion as well as in sustaining effective CD8+ T-cell responses [23–26]. CD4+ T-helper cells deliver help for CD8+ CTL by fully activating DC through the CD40/ CD40L signalling pathway [23,24,27], as well as by the secretion of IL-2. In addition, physical linking of T-

Fig. 1. Development of peptide vaccine in relation to their in vivo presentation. (a) Administered minimal CTL peptide epitopes can bind directly to MHC class I molecules present on T cells, B cells and dendritic cells (DC). Recognition of cognate antigen presented by either T cells and B cells will lead to an abortive proliferative response and dead of the responding CD8+ T cells. Presentation of the minimal CTL peptide epitope on immature DC may lead the expansion of antigen-specific CD8+ memory T cells, but in general, such T cells do not possess strong effector function. (b) Inclusion of a helper peptide in the vaccine improves the efficacy of the vaccine. The CTL peptide epitope can bind to cells as described in (a) and the helper peptide will be taken up and presented effectively in MHC class II of DC. In theory, this will lead to DC presenting either a CTL-peptide or a helper-peptide, or DC may present both peptides. When the latter occurs, DC will stimulate antigen-specific CD4+ Thelper cells and in return receive an activation signal that allows the DC to endow the CD8+ T cells with a licence-to-kill signal and to become full effector cells (indicated by granules in T cell). In addition, CD8+ T-cells receive direct help from the antigen-specific CD4+ T-helper cells. (c) Physically linking of helper-peptides and CTL-peptides ensures that any DC, which has taken up peptide antigen, will stimulate both CD4+ helper T cells and CD8+ T cells. When this vaccine is mixed with a DC-activating compound such as some of the well-known Toll-like receptor (TLR) agonists (e.g., CpG, LPS, Imiquimod), DCs acquire an improved capacity to stimulate both CD4+ and CD8+ T cells and this results in a stronger and more effective immune response. (d) By covalently linking the DC-activating agent to the peptide antigen, all DC that have taken up antigen will become strongly activated, precluding the theoretical situation presented in (c) where some DC presenting antigen are not activated and some DC become activated but do not present antigen. As a result, a strong and effective cellular immune response is induced, which can effectively cope with bacterial and viral infections as well as with recently established tumours.

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helper and CTL-epitopes increases the length of the peptide used and this positively influences the dynamics of peptide presentation (see below). However, not all the combinations of T-helper and CTL epitopes can be linked with impunity. In a Phase I trial in stage IV melanoma patients, a response to the melanoma-associated immunodominant gp100 CTL epitope was detected in patients injected with the minimal peptide but not in those patients injected with the CTL epitope that was C-terminally extended with a tetanus-derived T-

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helper peptide [28]. A potential explanation for this may be the change of context in which the C-terminus of the CTL epitope that now has to be liberated by proteasomal cleavage is presented to the proteasome. Important Cterminal cleavage sites can be destroyed by alterations in the surrounding amino acids [29], as is the case when two epitopes are just linked together. Alternatively, linkage of a completely unrelated T helper epitope may deliver help at the induction phase of tumour-specific CTL but still fails to induce effective help at the effector

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phase of the immune response. In the MuLV system described above, effective protection against tumours was only obtained when mice were vaccinated with a tumour-specific T-helper epitope, rather than an unrelated T-helper epitope [19], emphasizing the importance of activating both CTL and T-helper responses that are specific for the pathogen/tumour [19]. This effect is likely to occur because sustained activation of CTL in later phases of the immune response exists only through the help of T helper cells activated by APC that crosspresent pathogen/tumour-derived antigens taken up at the site of disease [30]. There are several examples of naturally linked, and usually overlapping, T-helper and CTL epitopes. In mice, the presence of a partially overlapping T-helper epitope significantly enhanced the magnitude of the human papilloma virus type 16 (HPV16) E7-specific CTL response in a prime-boost vaccination protocol [31]. In humans, the use of peptides with CD4+ and CD8+ T-cell epitopes embedded in the natural sequence resulted in the efficient generation of both NY-ESO-1-specific T helper cells and CTL in melanoma patients [32] and HER-2/Neu-specific CD4+ and CD8+ T-cell immunity was induced in 12 out of 15 breast or ovarian cancer patients [33]. In contrast to a previous trial, in which the use of a minimal HER-2/ Neu-specific CTL epitope resulted in low and shortlived CTL responses [34], the T-cell responses in this trial were shown to be long-lived (N1 year), and CTL were able to recognize and kill HER-2/Neu-positive tumour cells [33]. Furthermore, Lopez et al. showed that the injection of a 100-amino-acid-long peptide representing the C-terminal region of the circumsporozoite protein of Plasmodium falciparum resulted in the induction of robust IFNγ producing CD4+ and CD8+ T-cell immunity in 13 out of 15 healthy volunteers [35]. Thus, the simultaneous activation of CD4+ and CD8+ Tcell responses not only resulted in a higher response rate to the vaccine but also in sustained and stronger CD8+ T-cell responses compared to vaccines containing only minimal CTL peptide epitopes.

4. Increasing peptide length for top quality in vivo presentation; size matters! Although the inclusion of a T-helper component in synthetic peptide vaccines contributed strongly to the induction of effective CD8+ T-cell responses, this is not

the only reason why the longer peptides vaccines perform better than minimal MHC class I binding peptide-epitope vaccines. Under conditions that precluded T-cell help, a single vaccination with a long peptide vaccine induced significantly higher CD8+ T-cell responses in MHC class II knock-out mice and in CD40 knock-out mice than when a minimal peptide-epitope vaccine was used [31], indicating that also other mechanisms play a role in the enhanced immunogenicity of these longer peptides. One of these factors is the context in which peptides are presented to the immune system. Long synthetic peptides are not able to bind directly to MHC class I or II molecules and are, therefore, taken up, processed and presented by dendritic cells only. In contrast, minimal CTL epitopes are loaded exogenously in MHC class I molecules and can be presented by both professional APC (dendritic cells) and non-professional APC (T-cells and B-cells) in vivo (Fig. 1a) (Bijker et al., in preparation). The presentation of CTL peptide epitopes on Bcells is known to induce a transient CTL response and subsequent deletion of these CD8+ T-cells [36] and this may be one of the reasons why, for instance, Toes et al. observed an enhanced outgrowth of tumours after vaccination [7,8]. Loading of minimal MHC class I binding peptide-epitopes onto DC can convert a CTL tolerating peptide into a peptide that triggers the expansion of a tumour-protective CTL response [37]. Thus, presentation of peptides specifically by DC is essential for the proper activation of T-cells. The induction of strong CD8+ T-cell responses requires a sustained presentation of antigen in a stimulatory context [38]. Therefore, another aspect that may play a role in vivo is the biodegradation by extracellular proteases. Endo- and exo-peptidases present on the cell surface of DC and serum-derived proteases are able to trim minimal MHC class I binding peptides resulting in a decreased presentation of these CTL epitopes [39–41]. It is possible to produce non-natural derivatives of CTL epitopes that are fully resistant to such serum proteases and still retain the antigenicity and immunogenicity of the parental peptide [42]. Most often, however, these alterations result in a complete loss of immune reactivity (reviewed in [43]). Another method, to overcome the downside effects of proteolytic degradation, is to give a high number of repetitive injections with minimal CTL peptide epitopes within a week and for several courses. Vaccination of highrisk melanoma patients with heteroclitic gp100

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peptides, which bind with high affinity to HLA class I molecules, according to such a repetitive protocol resulted in a strong gp100-specific CD8+ CTL response, which persisted for at least 1 year [44]. Interestingly, other extracellular proteases are known to trim large peptides (e.g., CNBr-cleaved ovalbumin peptides) into minimal MHC class I binding peptides. As a result an increase in the presented immunodominant ovalbumin-derived CTL epitope can be detected [39]. Thus, while vaccines of small peptides can be rapidly biodegraded, larger peptides are relatively protected and may actually benefit from additional extracellular processing. Our recent experiments indicated that, depending on their MHC-binding affinity, minimal CTL peptide epitopes are presented in a stimulatory context for either a few days to almost 2 weeks after injection into mice (Bijker et al., in preparation). This shows that also the affinity of MHC class I binding peptides plays a role with respect to the capacity of vaccines to sustain T-cell responses. Possibly, small high-affinity peptides are protected for degradation by proteases through their ability to dock directly into either temporarily empty MHC class I molecules at the cell surface or by displacement of endogenous low-affinity binding peptides. This may also constitute a second explanation why heteroclitic CTL peptides display a higher capacity to prime Tcells in vivo. Interestingly, increasing the length of the lower affinity CTL peptides results in the longer duration of antigen presentation and an enhanced immunogenicity of these epitopes (Bijker et al., in preparation). This may be due to the requirement for processing of these long peptides resulting in the presentation of CTL peptides only in the highly stimulatory context of dendritic cells. Last but not least, vaccination of sarcoma patients with both the minimal 9-mer peptide (157–165) and a 11-mer peptide (157–167) comprising the CTL peptide-epitope of the NY-ESO-1 cancer testis antigen resulted in the induction of CD8+ T-cells of which a minor fraction recognized the appropriate processed and on tumours presented epitope 157–165. However, the majority of responding CD8+ T-cells recognized so-called cryptic epitopes, non-naturally processed Nterminally truncated forms of the 11-mer peptide, and as such, these CD8+ T-cells were not able to recognize tumour cells [45]. A similar result was obtained in metastatic melanoma patients receiving the heteroclitic

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157–165V peptide of NY-ESO-1 [46]. The induction of T-cells responding to cryptic epitopes in small peptides is not unique for CD8+ T-cells but has also been observed for CD4+ T-cells. Analysis of the immune response of mice injected with a minimal Thelper peptide epitope of myelin basic protein revealed the presence of an immunodominant non-naturally processed CD4+ T-cell epitope in the sequence of a subdominant epitope recognized by pathogenic T-cells [47]. In contrast to these minimal peptides, which may contain multiple epitopes liberated by proteases not involved in the natural processing pathway, the requirement for long peptides to be taken up by dendritic cells ensures the priming of T-cells reacting only to naturally processed epitopes.

5. HLA-type independent peptide vaccines aimed at multiple antigens; just a matter of mixing! To date, almost all peptide vaccine studies require the selection of patients eligible for vaccination based on their HLA type because these patients are generally injected with a low number of selected CTL epitopes [9]. A vaccine is likely to be versatile when it elicits T-cell responses against a background of many different HLA class I and II alleles [48]. Therefore, vaccines providing the immune system with complete proteins are favoured over single peptide-epitope vaccines, since the latter have to be identified per HLA type and may not contain all important epitopes. Currently, pools of overlapping peptides have been successfully used to detect specific CD4+ and CD8+ T-cell responses to a wide array of antigens in humans, independent of the HLA type present [49–55] as well as to isolate antigen-specific CD4+ and CD8+ T-cells [56]. In a comparison, the CD4 + tetanus-specific T-cell repertoire propagated by stimulation with either TTD (whole protein) or with pools of overlapping 20-mer peptides do not differ with respect to recognition of naturally processed epitopes [57]. This indicated that professional APC can handle pools of long peptides and are capable of properly excising multiple MHC class I and II peptide epitopes for presentation at the cell surface in each individual tested. It was, therefore, logical to assume that using similar sets of peptides for vaccination may solve the HLA-dependency problem. In a trial, Dutch belted outbred rabbits, which have considerable variability in their

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MHC genes, were persistently infected with cottontail rabbit papillomavirus (CRPV) and vaccinated with a set of 12 long overlapping CRPV E6- and E7-peptides formulated in Montanide ISA 51 after papillomas were established. Treatment resulted in the regression of established warts and a reduction of the viral load [58]. This concept is currently tested as a therapeutic vaccine for HPV16-induced intraepithelial neoplasia's in several phase II trials at our institute (Leiden University Medical Center, the Netherlands). While one can afford to make overlapping peptide vaccines of small proteins, such as HPV16 E6 (158 amino acids) and HPV16 E7 (98 amino acids), this will be much harder for larger proteins because of the number of peptides required to be synthesized. In these situations, one may make use of promiscuously presented peptides or overlapping peptides covering the regions that are highly immunogenic and comprise clustered T-cell epitopes. Several of these regions have been identified in viral proteins and tumour antigens [53,59–63]. In a phase II trial, patients with HER-2/Neu-overexpressing breast, ovarian or nonsmall lung cancers were injected with mixes of three different peptides comprising potential HER-2/Neuderived T-helper epitopes, independent of their HLA class II type. The great majority of patients responded to at least one of the peptides in this vaccine and this activity was associated with protein recognition and epitope spreading [64]. Once the immunogenic regions in a particular antigen have been identified, mixes of overlapping long peptides representing T-cell epitopes of different antigens (e.g., multiple melanoma antigens) can be used as vaccines. It is important to note that differences in the genetic HLA background of ethnically diverse populations may impact on the region displaying clustered T-cell epitopes in a particular antigen. Analyses of T-cell immunity against HIV-1 in a Chinese cohort and a Caucasian cohort showed that the two populations differed significantly with respect to the regions recognized [65]. Application of vaccines containing a limited set of overlapping peptides, therefore, may be geographically restricted. The ultimate long peptides are chemically synthesized proteins that consist of a number of chemically linked very long peptides. The advantage of such proteins over mixes of overlapping peptides is that the number of operations to produce a clinical grade vaccine is enormously reduced. Furthermore, expensive and time-consuming toxicity tests, inherent to the use

of recombinant technologies, can be avoided. We showed that a chemically synthesized GMP-compatible pure synthetic E7 protein (98 amino acids) of the human papillomavirus type 16 (HPV16-E7), which contains at least two different CD4+ T-helper epitopes and 1 CTL epitope, was able to induce strong HPV16specific T-cell reactivity and to protect mice against HPV16-positive tumours in both prophylactic and therapeutic vaccination regimens ([66] and unpublished observations). While the HPV16 E7 protein is relatively small, proteins of over 30 kDa have been chemically synthesized, indicating that also larger proteins (e.g., HPV16 E2) can be synthesized.

6. Adding danger signals to improve the induction and polarization of T-cell immunity The design of peptide vaccines, completely free of any endogenous adjuvant, provides us with the opportunity to test the true capacity of an adjuvant to deliver the appropriate danger signals to DC for enhancement of T-cell efficacy. Granulocyte-macrophage-colonystimulating factor (GM-CSF) has been used to recruit DC to the vaccination site in a number of trials, and at least in two studies the immune responses of patients upon injecting peptides in IFA has been compared to peptides plus GM-CSF. In stage II melanoma patients, the injection of two melanoma-associated heteroclitic CTL peptide epitopes in IFA with or without GM-CSF did not lead to significant differences in the vaccineinduced CTL response, albeit that overall, the responses in the GM-CSF group were always somewhat higher against the gp100-derived epitope but not the tyrosinase-derived epitope [67]. In stage III–IV melanoma patients, 4 out of 9 patients responded to the heteroclitic tyrosinase-epitope after injection with GMCSF, while none of the 9 patients vaccinated with this epitope in IFA mounted a response [68]. These data show that the use of GM-CSF is only a minor improvement for the induction of T-cell responses, which was to be expected since the biological role of GM-CSF is to support growth of DCs but not to fully mature and activate DC. The most devastating signal a T-cell can get is that of a DC presenting the cognate peptideepitope in the absence of expression of co-stimulatory molecules, since this may tolerate CTL precursors [69,70]. The main hallmark of good vaccines is,

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therefore, that they closely mimic the most successful natural triggers of dendritic cell (DC) activation, conducive not only to naïve CTL division, but also to CTL survival, expansion and migration as fully activated effector cells to tumour sites (reviewed in [71]). In most clinical trials, including our own [72], the strong DC-activation signals required to induce effective T-cell immunity have been missing; hence, only a short-lived suboptimal burst of CD8+ CTL effector expansion are observed. Effective DC-activation signals employ molecularly defined innate immunity receptors such as those belonging to the Toll-like receptor (TLR) family and/or adaptive immunity receptors such as CD40. Except for TLR9, which in humans is restricted to the plasmacytoid DC, the expression pattern of TLRs is similar in both humans and mice. TLR 2, 3, 4, 5 and 7 are broadly expressed on several DC types of both species [73–75]. An important aspect of these TLR agonists is that they are molecularly defined and most of them can be produced synthetically, which makes it possible to scrutinize the exact mechanism of function and efficacy of these immune stimulatory adjuvants. In several mouse models, agonists of TLR 3, 4, 7 and 9 significantly enhanced the magnitude and efficacy of virus and/or tumour-specific CTL responses when mixed with viral proteins, virus-like particles or peptide [31,76–81]. Notably, in each of the model systems used, particular agonist–antigen combinations failed to boost immunity. Administration of CD40 agonist antibodies to activate DC via CD40 in vivo considerably enhanced the efficacy of a peptide-based antitumour vaccine in mice [82,83]. Combinations of this CD40 agonist antibody with TLR agonists displayed a clear-cut synergism in DC activation [84] and CD8+ Tcell expansion [79]. However, these combinations did not result in a more effective tumour eradication because the activated and expanded T-cell effector populations accumulated predominantly in the vaccine-draining lymph nodes and hardly emigrated into the circulation (M. Schoenmaekers-Welters, M. Bijker, C. Melief, R. Offringa, S.H. van der Burg, submitted for publication). This demonstrates the importance of a rigorous preclinical test strategy in which all aspects of the desired immune response are examined. Despite the fact that vaccinations with peptide in IFA elicited tumour-specific IFNγ-secreting CD8+ T memory cells in some melanoma patients, these T-cells

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generally lacked features of effector cells such as the expression of perforin and granzyme B [85,86]. Adding a strong polarizing TLR agonist to the peptide in IFA vaccine solved this problem. Patients vaccinated with Melan-A peptide and CpG 7909 in IFA mounted CTL responses that were 1–3 orders of magnitude higher than in previous trials where this peptide was administered without CpG [85–87]. Importantly, the Melan-A-specific CD8+ T-cells did not only secrete IFNγ, but also expressed granzyme and perforin ex vivo [87]. Thus, inclusion of proper DC activating TLR agonists drastically enhances the magnitude of the Tcell response and endows them with full effector function as well. When applied in peptide vaccines, it will be possible to use only those minimal essential synthetic compounds needed to create an artificial infection, without having to care about potential unwanted bystander effects such as the induction of immunoregulatory and/or Th2 type cytokines, as was recently reported for recombinant vaccinia vector viruses [88]. The logical next step was to covalently link TLR agonists to model antigens. This resulted in the induction of rapid and long-lasting Th-immunity secreting high levels of IFNγ and a strong CTL response which exceeded the results of the mixed components [89]. In a model for antigen-induced murine airway eosinophilia, inhibition of disease was regulated a 100-fold more efficiently when CpG was conjugated to antigen rather than when administered as a mix [90]. Furthermore, Jackson et al. covalently linked a TLR 2 targeting lipid moiety (Pam2Cys) between a Th-peptide and a CTL peptide. In different models, they showed that this simple generic peptide-based vaccine concept could protect mice against a challenge with influenza virus, Listeria monocytogenes as well as B16-OVA tumour cells [91]. Similarly, Pam3CSS linked to an Her-2 CTL epitope and an influenza Th-epitope resulted in the protection of mice against HER-2-positive tumours. Importantly, a construct lacking the Th-epitope was less effective in this model [92] indicating that TLRmediated DC-activation is not able to substitute the impact of T-helper cells on CTL. The requirement for Thelper epitopes in covalently linked TLR-agonistpeptide vaccines to induce effective T-cell immunity was confirmed in another mouse model where mice were protected against a systemic challenge with live recombinant vaccinia-HIVpol virus after injection with a CpG-Th-CTL peptide conjugate [93]. The

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immunogenicity of a construct lacking this T-helper epitope was only minimal but was restored almost completely when mixed with a T-helper peptide [93]. Thus, the key to the induction of strong effective immune responses is the simultaneous presentation of both T-helper- and CTL-epitopes by DC as well as the activation of this particular antigen presenting DC (Fig. 1d). Covalently linking a TLR agonist to a Th-peptide and a CTL-peptide epitope not only meets the length requirement that ensures the presentation of these T-cell epitopes by DC only but also achieves that each DC, which takes up the antigen, is activated. In a way, such a vaccine mimics a normal inflammatory infection.

7. Synthetic peptides vaccines do their job. Change the immunotherapeutic strategy! It is clear that synthetic peptide vaccines aiming at the induction of virus- or tumour-specific T-cell immunity are fully functional, provided that peptides comprise both CD4+ and CD8+ T-cell epitopes and are codelivered with strong inflammatory signals. In rodents, peptide vaccines elicit robust CD4+ and CD8+ type 1 Tcell responses and this is associated with the protection against viral infections [1,2,58,94], the outgrowth of injected tumour cells [3] and the regression of tumours that were allowed to grow for a short period before animals were vaccinated [31]. It is important to realize that in all these cases the high clinical response rate is associated with a disease period that is probably too short to establish disease-mediated immune suppression and, therefore, does not mimic the situation in tumourbearing patients. Recently, in a mouse model of sporadic cancer, it was shown that immunogenic tumours induce T-cell tolerance during tumour development [95]. Similarly, human tumours develop slowly over time and establish themselves in the face of immunity. In some of the elder patients, vaccines may fail to induce immunity because of the collapse in CD4 T cell diversity which appears during the seventh and eight decades of life, suggesting that therapeutic measures to improve vaccine responses will have to include strategies for T cell replenishment [96]. In the other patients, synthetic peptide vaccines are still capable of triggering vigorous systemic type 1 CD4+ T-helper and CD8+ CTL responses. However, there are only few clinical responders. In this respect, the results of peptide vac-

cines do not differ from other vaccine strategies [9]. In two of our own vaccination studies, 13 patients with HPV16-induced pre-malignant lesions mounted a vaccine-induced type 1 T-cell response upon vaccination with a recombinant vaccinia virus expressing HPV16 E6 and E7, but only 7 patients showed a clinical response [97,98]. In addition, profoundly expanded numbers of vaccine-induced, gp100-specific T cells (up to 17% of all CD8+ T-cells) were detected in melanoma patients, but the detection of these tumour-specific T-cells per se could not serve as marker for clinical efficacy [99]. This clearly indicates that the mere presence of circulating, tumour-specific T-cells is not sufficient to induce a clinical response, but can we expect vaccines to do more than to launch the expansion and activation of tumourspecific T-cells? The induction of such T-cells is just one of several steps required for tumour clearance while more work needs to be done to get these T-cell to migrate to the tumour and to work within this microenvironment [86,100,101]. In some cases, this environment is conducive, as reflected by the infiltration of large numbers of immune cells into the lesion and HLA-DR expression by vascular endothelial cells on local small blood vessels, and such an immunologically active microenvironment may predispose patients to undergo clinical regression after subsequent vaccination [102– 104]. Forcing dendritic cells that are present in this microenvironment to become active by the intratumoural injection of DC-activating agonistic CD40specific antibodies [105–107] or inflammatory agents of innate immunity (CpG, LPS) [107] results in the awakening and expansion of more or less quiescent tumour-specific T-cell responses and subsequent eradication of tumours. Other mechanisms that add to this but are less well defined include the local innate immune response [108] plus associated inflammation as well as the activation and expansion of local resident T-cells. We recently showed a significant correlation between the presence of circulating HPV16-specific type 1 T-cells and the regression of HPV16-induced high-grade vulvar lesions topically treated with the inflammatory agent imiquimod [109]. Topical treatment, of course, is limited to superficial (pre-)malignant lesions such as non-melanoma skin cancer [110], melanoma [111] and HPVinduced lesions [112]. Alternatively, systemic IL-2 administration can be used to achieve the local activation of monocytes and NK cells as well as the production of chemoattractants [113]. Furthermore, the injection of IL-

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2 can lead to the direct activation of tumour-associated T-cells [86], enhancement of vaccine-induced T-cell expansion [114] and seems to induce the migration of vaccine-induced circulating T-cells to the tumour site [115,116]. Immunotherapeutic strategies aiming at improving the efficacy of vaccine-induced T-cell response should, therefore, start to focus on inducing local inflammation within the neoplastic lesion. Note, however, that some TLR agonists may enhance tumour-mediated suppression of T-cell proliferation and NK activity in vitro and in vivo resulting in tumour evasion [117].

[6]

[7]

[8]

[9]

8. Conclusion The development in the design of peptide vaccines has closely followed new emerging concepts in immunology. The current state-of-the-art peptide vaccine is a complete synthetic product able to artificially create an “infection” where DC take up MHC class I and II presented antigens and become fully activated. The result is a vigorous, sustained and effective T-helper and CTL response to acute and chronic bacterial and viral infections as well as transplantable tumours. Curative vaccination of established vascularized tumours requires a more robust therapeutic regimen, including the induction of inflammation at the site of disease.

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