Immunogenic HER-2/neu peptides as tumor vaccines

Abstract

During the last decade, a large number of tumor-associated antigens (TAA) have been identified, which can be recognized by T cells. This has led to renewed interest in the use of active immunization as a modality for the treatment of cancer. HER-2/neu is a 185-KDa receptor-like glycoprotein that is overexpressed by a variety of tumors including breast, ovarian, lung, prostate and colorectal carcinomata. Several immunogenic HER-2/neu peptides recognized by cytotoxic T lymphocytes (CTL) or helper T lymphocytes (TH) have been identified thus far. Patients with HER-2/neu over-expressing cancers exhibit increased frequencies of peripheral blood T cells recognizing immunogenic HER-2/neu peptides. Various protocols for generating T cell-mediated immune responses specific for HER-2/neu peptides have been examined in pre-clinical models or in clinical trials. Vaccination studies in animals utilizing HER-2/neu peptides have been successful in eliminating tumor growth. In humans, however, although immunological responses have been detected against the peptides used for vaccination, no clinical responses have been described. Because HER-2/neu is a self-antigen, functional immune responses against it may be limited through tolerance mechanisms. Therefore, it would be interesting to determine whether abrogation of tolerance to HER-2/neu using appropriate adjuvants and/or peptide analogs may lead to the development of immune responses to HER-2/neu epitopes that can be of relevance to cancer immunotherapy. Vaccine preparations containing mixtures of HER-2/neu peptides and peptide from other tumor-related antigens might also enhance efficacy of therapeutic vaccination.

Introduction

Active immunization for cancer treatment is based on the existence of tumor-associated antigens (TAA), which are in principle both antigenic and at the same time immunogenic (i.e., they are recognized by and are capable of stimulating the immune system, respectively).

TAA have been classified into several categories including differentiation, tissue-specific, mutated and overexpressed antigens [103]. HER-2/neu, a member of the last category, is a transmembrane glycoprotein and member of the epidermal growth factor receptor family. Amplification and/or overexpression of HER-2/neu have been reported in 20–40% of primary breast cancers and also in ovarian (20–25%), colorectal and pancreatic adenocarcinomas (up to 80–85% for both). HER-2/neu has become an important target for cancer immunotherapy for several reasons. In particular, HER-2/neu is present in high proportions of tumor cells [19] and single tumor cells frequently show an intense expression of this molecule [66] suggesting that anti-HER-2/neu therapy would specifically target most tumor cells. In addition, CTL and IgG specific for HER-2/neu have been detected in 30–50% of patients with breast cancer [45] demonstrating that this molecule is capable of inducing both cellular and humoral immunologic responses in vivo. The fact that HER-2/neu-specific IgG levels are increased in patients with HER-2/neu⁺ breast cancer, suggests that TH cells can also be sensitized upon recognition of HER-2/neu epitopes. In this review, we shall focus on the identification and application of HER-2/neu peptides as tumor vaccines.

Identification of HLA-restricted cytotoxic T cell epitopes

The identification of HLA-restricted CTL peptides is essential for understanding the role of immunity in various circumstances, including its potential in the treatment of cancer. Specifically, it is instrumental in the development of defined vaccines that stimulate CTL responses as well as for the monitoring of immunotherapies of cancer. The peptide-binding based prediction of CTL epitopes in protein sequences has led to the identification of CTL epitopes on proteins from various sources, also including tumor antigens [44]. CTL epitopes are presented in the context of MHC class I alleles. The peptide-binding groove of HLA molecules contains highly polymorphic allele-specific pockets that accommodate side chains of the anchor residues of the bound peptide [8, 9, 88]. Peptides bound to MHC class I molecules are usually 8–10 amino acids in length, because the peptide-binding groove of the HLA class I molecules is closed at both sites [9] and thus accommodates only short peptides. Each allele has specific peptide-binding motifs, which were defined through the analysis of naturally presented peptides eluted from class I molecules [20, 22]. Each individual HLA class I molecule displays a preference for certain amino acids at the major peptide anchor positions (positions 2 and 9 for most class I alleles) that bind to the binding pockets. Amino acids at other positions in the peptide can significantly contribute to binding by their engagement in secondary pockets [50, 78, 87]. The knowledge of allele-specific peptide-binding motifs has led to the development of peptide-binding prediction algorithms by several groups [16, 67, 72]. Although peptides can bind MHC class I molecules, this does not necessarily mean that they are presented on the cell surface. Proteins are degraded intracellularly in the cytosol by the proteasome complex for presentation by MHC class I molecules. Not all of the putative MHC-binding peptides from a protein are generated in vivo and it is not an easy task to predict which peptides will be naturally processed. Various antigen-presenting cells, such as dendritic cells (DC) and tumor cells, can have different proteasome complexes that may, in turn, generate different peptides from the same protein. It has been shown that some TAA epitopes are generated by tumor cells but not by DC [57]. For these epitopes, peptide-based vaccines may be the only modality for vaccination.

Two approaches have been used to identify immunogenic CTL peptides for the HER-2/neu protein that are naturally processed and presented. First, tumor-specific CTL have been generated using tumor-infiltrating lymphocytes (TIL) and autologous tumor cells. Target cells pulsed with the peptides are then used to stimulate the same CTL and to identify peptide-specific T-cell reactivity [103]. The advantage of this method is the guarantee that peptides identified in this way are naturally processed. The disadvantage, however, is the difficulty in obtaining TIL and sufficient numbers of autologous tumor cells from many tumors.

The second approach to identifying HER-2/neu CTL peptides is “reverse immunology”. In this method, MHC class I-binding epitopes are identified using computer programs that take into account the presence of allele-specific major anchor residues, and specific secondary anchor residues [40, 98]. The corresponding synthetic peptides are then tested for their capacity to induce peptide- and tumor-specific CTL derived from healthy individuals or cancer patients [14, 40, 24, 54, 98].

Breaking tolerance to HER-2 self-protein through active immunization with HER-2/neu CTL peptides

The majority of TAA represent abnormally expressed or overexpressed normal gene products. Therefore, mechanisms responsible for self tolerance dampen immune responses against these antigens. Transgenic mouse model studies revealed that tolerance is capable of deleting reactive high-avidity T cells against the transgene, thereby leading to self-tolerance [30, 59, 60]. However, T cell elimination through tolerance is not absolute, since self-reactive T cells can be isolated from tolerant hosts [31, 39]. Moreover, it has been demonstrated that low-avidity T cells can be activated, expanded and involved in antitumor responses [57, 58]. Multiple immunizations with DC pulsed with two different HER-2/neu CTL peptides restarted tumor growth in HER-2/neu transgenic mice, despite the fact that CD8⁺ T cells from these mice were of low-avidity for the same peptides [56]. These results underscore the potential role of low-avidity T cells in antitumor immunity and offer an important component for vaccination immunotherapies. Similar results were also obtained in another syngeneic mouse/tumor model where injections of DC pulsed with tyrosinase- or gp100-derived CTL peptides significantly delayed melanoma outgrowth in HLA-A*0201-transgenic mice [61]. In this model, protection was enhanced by the use of peptide ligands containing conservative substitutions that were cross-reactive with the original antigens.

We have established a preclinical tumor model based on HLA-A*0201 transgenic, H-2k^b knockout (HHD) mice inoculated with syngeneic ALC tumor cells transfected to co-express human HER-2/neu and HLA-A*0201 (ALC.HER.A2.1). Immunization with murine HER-2/neu peptide spanning residues 435–443 plus GM-CSF only partially protected HHD mice from the growth of ALC.HER.A2.1 tumor cells. However, when mice were repeatedly vaccinated with human HER-2 (435–443), which differs in a single amino acid at position 4 from its murine homologue, protection was dramatically enhanced (manuscript in preparation). These results demonstrate that if immune responses from tolerant hosts are properly primed and effector function is maintained over time, the immune response will contribute to the destruction of the tumor. Further evaluation of the low-avidity T cell repertoire specific for self-HER-2/neu in tolerated hosts will ultimately lead to improvements of the methods for optimization of vaccination immunotherapies against HER-2/neu.

HER-2/neu CTL peptides

Peptide HER-2 (p369-377) was originally identified by Fisk et al. [24] as an immunodominant HLA-A2-binding epitope recognized by tumor-associated lymphocytes of ovarian cancer. The characteristic feature of HER-2 (p369-377) as an immunodominant peptide recognized by effector CTL in the context of HLA-A2 molecules on the surface of HER-2/neu overexpressing tumor cells was shown in our laboratory by two different means: first, we stimulated in vitro ascitic fluid T cells from HLA-A2.1⁺ patients suffering from HER-2/neu overexpressing ovarian cancer with autologous DC pulsed with membrane peptides (acid cell extracts; ACE) which were extracted from their tumors [5]. After two–three rounds of restimulation in the presence of cytokine combinations, we obtained ACE-specific CTL lines and, subsequently, T-cell clones which recognized synthetic HER-2 (p369-377) expressed on T2 cells. The same HER-2 (p369-377) specific CTL lines and clones also recognized the HER-2/neu overexpressing and HLA-A2.1⁺ SKOV3.A2 ovarian tumor cell line, demonstrating that this CTL epitope is naturally processed and presented. In the second approach [29], we immunized HHD mice with ACE extracted from HLA-A2.1⁺, HER-2/neu overexpressing ovarian and breast tumors; splenocytes from vaccinated HHD mice were then restimulated in vitro with syngeneic splenocytes pulsed with ACE and tested in cytotoxicity assays. HHD splenocytes specifically recognizing ACE from five patients with cancer (three patients with breast cancer and two patients with ovarian cancer) efficiently lysed T2 cells pulsed with HER-2 (p369-377) as well as the ovarian SKOV3.A2 and breast MCF-7 (both expressing HER-2/neu and HLA-A2.1) cancer cell lines. These HER-2 (p369-377)-specific HHD CTL, upon adoptive transfer, efficiently protected SCID mice from growth of SKOV3.A2 or MCF-7 induced tumors. Later on, p369-377 was also found to be expressed by several types of HLA-A2⁺ tumors, including renal cell carcinoma [12], breast carcinoma [48], and melanoma cells [77].

Rongcun et al. [77] were able to generate in vitro T-cell lines and clones from ascitic fluids of HLA-A2⁺ patients with epithelial ovarian cancer recognizing HER-2 peptides (p435-443), (p665-673), (p689-697), and (p952-960). These peptides are also expressed on a variety of tumor cell lines including ovarian, colon and breast carcinomas, and melanomas [45]. The HER-2 (p689-697) was also found to be recognized by gastric cancer specific CTLs [51]. More recently, we have found that besides classical CTL, p369-377, p435-443, p665-673, and p689-697 can elicit NKT cells specifically recognizing autologous HLA-A2⁺ HER-2/neu⁺ ovarian tumors [5]. HER-2 (p435-443) is also an immunodominant CTL epitope recognized by ACE-specific CTL effectors either loaded onto T2 cells or naturally presented in the context of HLA-A2.1⁺ and HER-2/neu⁺ tumor cell lines [5, 29]. Peptide HER-2 (p754-762) was shown to induce CTL from healthy donor-derived PBMC that were capable of killing the colon tumor cell line SW403 expressing HLA-A3 and HER-2/neu [42]. Additional MHC-binding studies with the most common HLA molecules belonging to the HLA-A3 superfamily (HLA-A*1101, HLA-A*3101, HLA-A*3301, and HLA-A*6801) demonstrated that p754-762 was able to bind these alleles [42]. Eberlein and co-workers [69] identified HER-2 peptide (p654-662) from the transmembrane region of this protein as a common epitope presented by various HLA-A27 tumor types, including breast, ovarian, pancreatic, and non-small lung cancer. HER-2 peptides p5-13, p48-56, and p1023-1032 were demonstrated to trigger CTL responses in both HLA-A2⁺ humans and HLA-A2 transgenic mice. Such CTL lysed HLA-A2⁺ HER-2/neu⁺ tumor cells of different origins (breast, colon, lung, and renal cancer) irrespective of the expression levels of HER-2/neu [81]. Shiku and collaborators [87] have identified two HER-2 peptides, p63-71 and p780-788, capable of inducing HLA-A24-restricted CTL responses against various targets, including HLA-A24⁺ HER-2/neu⁺ tumor cell lines. MHC class I-presented peptides from HER-2/neu are listed in Table 1.

Table 1.

MHC class I presented CTL HER-2/neu epitopes

Peptide	HLA-presenting allele	Reference
HER-2 (p5-13)	A2	[41]
HER-2 (p8-16)	A24	[41]
HER-2 (p48-56)	A2	[43]
HER-2 (p63-71)	A24	[87]
HER-2 (p106-114)	A2	[51]
HER-2 (p369-377)	A2, A3, A26	[24, 92]
HER-2 (p435-443)	A2	[77]
HER-2 (p654-662)	A2	[69]
HER-2 (p665-673)	A2	[77]
HER-2 (p689-697)	A2	[77]
HER-2 (p754-762)	A3, A11, A33	[42]
HER-2 (p773-782)	A2	[55]
HER-2 (p780-788)	A24	[87]
HER-2 (p785-794)	A2	[77]
HER-2 (p789-797)	A2	[24]
HER-2 (p799-807)	A2	[24]
HER-2 (p952-961)	A2	[77]
HER-2 (p1023-1032)	A2	[81]

HER-2/neu peptides presented in the context of MHC class II gene products

There is now increasing evidence that optimal cancer vaccines require the collaboration between CTL and TH cells [6]. In the murine system there are several reports demonstrating that both in vitro and in vivo generation of CTL, specific for a tumor protein-epitope, requires cross-priming of tumor antigens by antigen presenting cells and is strictly TH-cell dependent, since depletion of CD4⁺ T cells totally abrogated the generation of peptide-specific CTL responses [15, 75, 83, 108]. In humans, we were the first to show an absolute requirement of synergistic interactions between autologous tumor-specific CD4⁺ and CD8⁺ T cells and DC for optimal killing of autologous tumor cells in patients with various types of metastatic cancers [7]. To this end, we have also shown that an initial interaction of CD4⁺ T cells with tumor peptide-pulsed autologous DC is required for providing all necessary conditions to the latter to induce optimal CTL killing.

There are also several studies reporting the existence of MHC class II-restricted T-cell responses to HER-2/neu. CD4⁺ T helper cells from HER-2/neu⁺ breast and ovarian cancer patients can proliferate and produce lymphokines in response to stimulation with HER-2/neu recombinant protein or synthetic peptides corresponding to immunodominant regions of HER-2/neu such as HER-2 (396-406), HER-2 (776-788), and HER-2 (884-899) [4, 18, 26, 70, 91]. Some of these patients exhibited preexistent immunity to these peptides in that they responded moderately after a short-term stimulation period [26, 48]. We have recently reported [91], on the generation and characterization of CD4⁺ T cell clones specifically recognizing HER-2 (p776-788). Such clones yielded specific proliferative and cytokine responses in response to DC loaded with HER-2 (p776-788). By performing blocking experiments with monoclonal antibodies and by using DC from allogeneic donors sharing certain DR-alleles, we could show that HER-2 (p776-788) is a promiscuous peptide presented by at least three different alleles (i.e., DRB5*0101, DRB1*0701 and DRB1*0405). In another series of experiments [70], we successfully generated CD4⁺ T-cell clones recognizing HER-2 (p884-899) in an HLA-DR4-restricted fashion. These clones also recognized human HER-2/neu⁺ breast, colorectal and pancreatic adenocarcinomas constitutively expressed or induced to express HLA-DR4. The data from both studies provided novel information on the role of HER-2 (p776-788) and HER-2 (p884-899) as naturally processed epitopes expressed by various types of epithelial cancers and the capacity of the HER-2/neu protein to follow the endogenous class II processing pathways. Our data also suggested that these peptides might be attractive as applicable vaccines offering a broad population coverage. MHC class II-presented epitopes from HER-2/neu are listed in Table 2.

Table 2.

MHC class II presented TH HER-2/neu epitopes

Peptide	HLA-presenting allele	Reference
HER-2 (p62-76)	DR4/15, DR51, DR53, DQ6/7	[49]
HER-2 (p605-619)	DR4/15, DR51, DR53, DQ6/7	[49]
HER-2 (p765-783)	DR4/15, DR51, DR53, DQ6/7	[49]
HER-2 (p776-788)	DR51, DR7, DR4	[91]
HER-2 (p777-789)	DR4	[99]
HER-2 (p822-836)	DR1/11, DR51, DR52, DQ5/7	[49]
HER-2 (p883-889)	DR1/11, DR4, DR51, DR52, DR53, DQ6/7	[49]
HER-2 (p884-899)	DR4	[70]

Vaccination of cancer patients with HER-2/neu peptides

Most published vaccination trials in humans have used HER-2 (p369-377). Knutson et al. [47] showed that immunization of six patients with breast or ovarian cancer with HER-2 (p369-377) plus GM-CSF resulted in increased peptide-specific T-cell precursor frequencies (PF) in two of them, as measured in IFN-γ ELISPOT assays. The responses were short-lived and not detectable at 5 months after vaccination. In a phase I clinical study [109], patients with metastatic breast, ovarian, or colorectal cancer were immunized with HER-2 (p369-377) in IFA. Peptide-specific CTL were obtained from patients’ peripheral blood and upon restimulation in vitro were able to recognize and lyse target cells pulsed with the peptide but not tumor cells expressing HER-2/neu. In another phase I clinical study [62], HER-2 (p369-377) was administered with GM-CSF. Thirty percent of the patients developed peptide-specific T-cell proliferative responses and the vast majority of them gave peptide-specific DTH responses. Peptide-specific CTL precursors could be detected in only one of them (eight patients were enrolled). No clinical responses were observed.

Disis group [17, 48] vaccinated patients with HER-2/neu-overexpressing breast, ovarian or non-small-cell lung cancers with peptides derived from potential TH epitopes of the HER-2/neu protein admixed with GM-CSF. One of these peptides was HER-2 (p369-384), which also contained the p369-377 CTL epitope. Patients received mixtures of peptides, which included not only HER-2 (p369-384) but also HER-2 (p688-703) and HER-2 (p971-984). Ninety-two percent of patients developed T-cell immunity to HER-2/neu peptides, which was preferentially directed against HER-2 (p369-384). At 1-year follow-up, immunity to HER-2/neu protein persisted in 38% of patients. In a similar trial [47], nineteen HLA-A2 patients with HER-2/neu overexpressing breast and ovarian cancers received a vaccine preparation consisting of putative helper HER-2/neu peptides, which contained within their sequences HLA-A2-binding CTL peptides [also including HER-2 (p369-377)]. After vaccination, the mean peptide-specific T-cell PF to HER-2 (p369-377) was increased in the majority of patients. In addition, the peptide-specific T cells were able to lyse HER-2/neu⁺, HLA-A2⁺ tumors. The responses were long-lived and detectable for more than 1 year after the final vaccination. In a similar designed study, Disis et al. [18] demonstrated that FLt3 ligand could act as a vaccine adjuvant in association with HER-2/neu peptide-based vaccines. A summary of the clinical studies based on HER-2/neu peptide vaccines is summarized in Table 3.

Table 3.

HER-2/neu peptide-based vaccinations in patients with HER-2/neu⁺ cancers

Peptide-vaccine	Schedule	Immunological effects	Referencea
P369-377 in GM-CSF	500 μg/Month. Monthly×6	Increase of peptide-specific CTL precursors	[46]
Mixtures of p369-384 p686-703 p971-984 in GM-CSF	500 μg of each peptide/Month. Monthly×6	Increase of peptide-specific CTL precursors. Lysis of HER-2+ tumors	[17, 47]
Mixtures of p776-790 p927-941 p1166-1180	20 μg/kg FL/day × 14 days/Month. Monthly×6 500 μg of each peptide on day 7 of FL cycle alone or in GM-CSF	FL was effective in boosting the GM-CSF-induced IFN-γ secreting HER-2-specific TH cells	[18]
p369-377 in IFA	1 mg/3-4 Week. Weekly×4	Increased reactivity against targets pulsed with p369-377. No reactivity against HER-2+, HLA-A2+ tumors	[108]
p369-377 in GM-CSF	10 μg or 500 μg or 1000 μg/Week. Weekly×4 Monthly×6	Proliferative T-cell responses specific for p369-377 DTH responses	[62]

In none of these studies clinical activity was tested

^a In refs. [17, 47, 108] breast, ovarian, non-small-cell lung and colorectal cancer patients were enrolled. In refs. [46, 18, 62] breast and ovarian cancer patients were enrolled

In most of the patients examined, Disis group could detect increased PF to HER-2/neu CTL epitopes only after vaccination with the relevant peptide and only rarely non-immunized patients exhibited detectable CTL PF [17, 47, 48]. We were able to demonstrate increased CTL PF specific for HER-2 (p369-377) in non-vaccinated patients with HER-2/neu overexpressing tumors [92, 93]. A reason possibly accounting for this may lie in our ability to develop a more sensitive IFN-γ ELISPOT assay favouring the detection of preexisting CTL responses, eventually also in individuals who have developed physiological mechanisms of immunologic tolerance. We used DCs pulsed with HER-2 (p369-377) to stimulate, in the presence of IL-7 and IL-12, autologous PBMCs. We could thus detect IFN-γ producing CTL in 25% of HLA-A2⁺, 30% of HLA-A26⁺ and 60% of HLA-A3⁺ patients with breast, lung, colorectal and prostate cancers but not in healthy individuals [92]. PBMC from patients with increased CTP PF for HER-2 (p369-377) were capable of lysing autologous HER-2/neu⁺, HLA-A2⁺ tumors. By applying the same method, we could demonstrate increased PF of CTL specific for HER-2 (p435-443), HER-2 (p665-673), HER-2 (p689-697), HER-2 (p777-785) and HER-2 (p952-960) ranging from 6% to 31% in patients suffering from breast, ovarian, lung, colorectal and prostate cancers [93]. Autologous tumor-killing by patients’ PBMC with increased CTL PF was also observed. The detection of CTL PF for certain HER-2/neu epitopes in non-vaccinated cancer patients is of major importance because it is suggestive of the peptide or mixture of peptides to be included in vaccine formulations.

HER-2/neu CTL peptide analogs with enhanced immunogenicity

The immunogenicity of synthetic peptides which correspond to CTL epitopes in patients is usually limited. Two major reasons may account for this: first, peptide-specific CTL are actively tolerized, either through deletion or anergy induction among high avidity T cells [84, 85]; second, short synthetic peptides (i.e., octamers to decamers) are susceptible to rapid degradation by serum proteases, thus requiring repeated administration of relatively high doses [100, 52, 86, 25]. For these reasons there is a clear interest in designing alternative immunization regimens aiming at a more efficient induction of CTL responses towards tumor-associated antigens. These also include CTL peptide analogs carrying single or multiple amino acid substitutions that have improved immunogenicity compared to the native CTL peptides [79]. Enhancement of immunogenicity is determined by the ability of peptide analogs (or agonists) to induce higher levels of immune responses compared to those induced by the native CTL peptides. Such responses are illustrated by higher levels of cytokine secretion and cytotoxicity upon encounter with the native CTL peptide presented by DCs or the tumor itself. Strong agonistic peptides are designed by two general approaches: (1) modification of HLA anchor residues, resulting in higher HLA binding; and (2) modification of the residues involved in the TCR contact site, resulting in increased response by T cells. The first approach has been successfully used for normally low-affinity binding HLA-A2 CTL peptides from various tumor antigens such as NY-ESO-1 [11], gp100 [65] and Melan-A [100]. With respect to HER-2/neu peptides, Fisk et al. [25] investigated whether oligopeptide analogs can induce an improved anti-CTL response. To this end, PBMC were stimulated with HER-2/neu analogs and the CTL generated recognized the peptides used as immunogen as well as HLA-A2.1⁺, HER-2⁺ tumors. Peptide GP2 [HER-2 (9₆₅₄)] was found to bind poorly to HLA-A2.1 due to a lack of stabilizing contacts with the peptide-binding cleft [52]. In that case, substitutions at the peptide center had a potential impact in increasing peptide affinity. By substituting the amino acid at position 1 with a tyrosine (P1Y) Scardino et al. [81, 82] generated heteroclitic P1Y variants of CTL peptides, HER-2 (9₃₉₁), HER-2 (9₄₀₂), HER-2 (9₄₆₆) and HER-2 (9₆₅₀), all of which bound to HLA-A2.1 with much higher affinities than did the wild-type epitopes. Such heteroclitic variants were also immunogenic, generating in vivo strong antitumor responses.

However, improved immunogenicity of a given CTL peptide does not necessarily correlate with increased affinity for binding to MHC class I alleles. For instance, in a recently published report [21], HER-2 (10₄₂₀) restricted by H-2D^q was shown to generate CTL lines in vitro, which upon adoptive transfer, could highly protect non-transgenic mice from spontaneous mammary carcinomas. However, when alanine was substituted for glutamate at position 2, this heteroclitic peptide demonstrated markedly improved recognition by a T cell clone in a cytotoxicity assay as the wild-type peptide. Interestingly, the heteroclitic peptide was found to have a lower binding affinity than the wild-type peptide in a T2D^q stabilization assay, suggesting that its improved stimulatory capacity was instead due to the enhanced stability of the TCR/MHC/peptide complex. In a similar manner, Vertuani et al. [101] have most recently demonstrated that a HER-2 (9₃₆₉) analog generated by introduction of V at positions 2 and 9 proved to be a more potent immunogen compared to wild-type HER-2 (9₃₆₉) although the analog bound to HLA-A2.1 with the same affinity as the wild-type peptide. The authors demonstrated that the high biological activity of HER-2 (9₃₆₉) analog was associated with a slower dissociation kinetic profile, resulting in an epitope with greater HLA-A2.1 stability.

The second approach to designing epitopes from tumor antigens, which involves the replacement of residues responsible for TCR contact, has been mainly applied to melanoma differentiation antigens, and carcinoembryonic antigen [80, 76, 89]. Castilleja et al. [13] modeled the complex of HLA-A2.1 with HER-2 (9₃₆₉) in order to identify side chain orientation of the latter. They then modified HER-2 (9₃₆₉) at the central Ser (which is involved in the contact with the TCR) in different ways, producing three variants one of which was a weaker stimulator than native HER-2 (9₃₆₉) for induction of lytic function, indicating that this particular variant hindered TCR activation. However, CTL raised against this variant were less apoptotic and upon restimulation with the wild-type peptide became highly active, recognizing tumors endogeneously presenting HER-2 (9₃₆₉). Agonists produced by modifications at TCR contacts may circumvent apoptotic pathways and mechanisms of active tolerance and thus, may provide improved approaches to the rational design of cancer vaccines. The data from this study offer the prospect of sequential stimulation of PBMC with weak and strong gonists (produced by modifications at anchor residues 2 and 9 or at the central portion of the peptide, or a combination of both) for developing efficient and long-lasting antitumor responses.

Active immunization targeting HER-2/neu and other tumor-related antigenic molecules

Limited efficacy of tumor-specific CTL in cancer patients may be attributed to several factors including (a) active immunosuppression either via soluble factors released by the tumor cells [49] or via T regulatory cells [35], (b) down-regulation of MHC class I products and co-stimulatory molecules by the tumor cells [38, 49] and (c) inadequate TH function essential for optimal CTL activation [7, 75]. Early activation of type-1 innate effector cells such as DC, NK and NKT cells, which produce TH1-inducing cytokines, would be essential for supporting the generation and amplification of TH1-dependent tumor-specific CTL. This could be achieved by developing an efficient tumor peptide-vaccine protocol that can overcome strong immunosuppression in tumor-bearing hosts and at the same time promote the generation and activation of tumor-specific CTL and TH. Unmethylated cytosine-phosphorothioate-guanine (CpG) containing oligonucleotides are well-known to promote TH1-type immune response through binding to TLR-9 in DCs [1, 32, 36]. Internalization of CpG-ODN is a prerequisite for activation of TLR-9 and initiation of its signaling pathway [1] which suggests that encapsulation of CpG-ODN in liposomes might increase their uptake by DCs to produce higher levels of IL-12, which in turn may potentially induce higher amounts of IFN-γ producing NK and NKT cells. Such a pathway involving type-1 innate immune mediators through TLR engagement may trigger activation of bystander T cells [23, 26]. Accordingly, tumor-vaccines including CTL and TH peptides along with CpG-ODN would benefit from the generation of type-1 innate and adoptive immunity with the former exerting direct antitumor effects but also indirect via amplification of the latter.

Xenogeneic homologues of tumor-associated proteins have been shown upon immunization to induce strong in vivo antitumor responses in mice by overcoming tolerance to weakly immunogenic self-antigens [104]. DC-based xenovaccination for prostate cancer in humans has also produced strong TH1 immunity for self-prostate antigens, with no data available so far, however, on tumor response [27]. Chimeric fusion proteins resulting from translocations can be necessary for the persistence of the tumor [80, 107], thus critically marking the tumor cells as a target for immunotherapy. Peptides generated from proteolytic processing of fusion proteins would have a “non-self” sequence of amino acids, which, if displayed via MHC molecules, could result in potent T-cell-mediated immunity [106]. Cytogenetic studies have revealed a number of translocations in both breast cancer lines and primary tumors [28, 102]. There is also an association between translocations and HER-2/neu expression in tumor cells [97]. These findings suggest a potential utility of peptides from xenogeneic homologues of breast tumor antigens and for translocation-specific peptides combined with HER-2/neu peptides in novel active immunotherapies.

There is now evidence to suggest that tumor escape variants are likely to emerge after treatment with increasingly effective immunotherapies. Thus, it is important that tumor-associated antigens be part of molecules which are obligatory for the survival of tumor cells. Immunization against such tumor antigens would overcome tumor escape attributable to antigenic variation because losing the survival-related molecule may result in tumor cell death. One of the antigens that is needed by cancer cells to survive is the antiapoptotic protein, survivin, which is overexpressed in several types of cancer [105, 95] and correlates with a more aggressive disease and poor survival [90, 37]. So far, survivin CTL epitopes restricted by various HLA class I alleles have been identified and demonstrated to be naturally expressed on the surface of tumor cell lines [3, 34, 59, 74]. Telomerase is a ribonucleoprotein that mediates RNA-dependent synthesis of telomeric DNA, thus maintaining telomere length and chromosomal stability [10]. Normal tissues display little or no telomerase activity [46, 63]. Most of the human tumors express high telomerase activity [46, 63] and telomerase peptides have been recently demonstrated to induce strong CTL responses against tumor cell lines naturally expressing these epitopes [96, 68, 33, 2, 94]. Vaccines comprising of HER-2/neu CTL and TH peptides mixed with survivin and/or telomerase CTL peptides may more efficiently target tumor cells with improved clinical results.

Growth of tumor cells and metastasis are dependent on mechanisms inducing angiogenesis. Therapy against angiogenesis represents a new modality for cancer therapy. Vaccination protocols for active specific immunotherapy include endothelial cell vaccines, DNA vaccines, DC vaccines and peptide or protein vaccines [53, 64, 71, 73]. The rational for such vaccination strategies is to target antigens selectively expressed by tumor-associated endothelial cells, thus inducing tumor necrosis by blocking blood supply through angiogenesis inhibition. Combined vaccination with HER-2/neu peptides and peptides targeting angiogenesis-related molecules (e.g. vascular endothelial growth factor receptor [54]) may provide a new strategy for the rational design of cancer vaccines.

Conclusion

The HER-2/neu oncoprotein gene encompasses sequences that can activate, upon recognition in the context of certain alleles on DC, both CTL and TH cells. Such cells, with specificity for HER-2/neu peptides, have been isolated from in vitro cultures but also from patients’ peripheral blood. Moreover, HER-2/neu peptide-specific CTL have been shown to efficiently lyse only HER-2/neu⁺ tumor cells expressing the appropriate allele(s).

However, how such HER-2/neu specific T-cell responses can be translated into effective therapeutic responses remains so far unclear. Combination of active immunotherapy with other therapeutic modalities, such as surgery or chemotherapy, might be necessary for achieving improved clinical responses. Another topic of major importance is the definition of optimum vaccination protocols including vaccine dose, number of doses, and modus of administration. The identification and optimal use of new adjuvants which are necessary for breaking tolerance to self-proteins or peptides is also an important issue. A better understanding of host–tumor interactions will surely provide insights for the development of more effective vaccination strategies mostly based on mixtures of HER-2/neu peptides with other peptides targeting tumor cells directly or even indirectly. Moreover, in light of recent findings demonstrating the generation of negative regulatory mechanisms of anti-tumor immunity in the tumor-bearing host, approaches that overcome this immunosuppressive state should be developed. Potent adjuvants, administered along with peptide vaccines, that bridge type-1 innate and acquired immunity are essential for inducing tumor-specific CTL. Implementation of these modalities in the clinical setting will certainly help to more precisely define the role of HER-2/neu peptide vaccines as effective immunotherapy of HER-2/neu overexpressing cancers.