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HER-2/ neu is the human epidermal growth factor receptor 2, which is associated with the progression of prostate cancer (PCa). HER-2/ neu -specific T cell immunity has been shown to predict immunologic and clinical responses in PCa patients treated with HER-2/ neu peptide vaccines. However, its prognostic role in PCa patients receiving conventional treatment is unknown, and this was addressed in this study. The densities of CD8+ T cells specific for the HER-2/ neu (780-788) peptide in the peripheral blood of PCa patients under standard treatments were correlated with TGF-β/IL-8 levels and clinical outcomes. We demonstrated that PCa patients with high frequencies of HER-2/ neu (780-788) -specific CD8+ T lymphocytes had better progression-free survival (PFS) as compared with PCa patients with low frequencies. Increased frequencies of HER-2/ neu (780-788) -specific CD8+ T lymphocytes were also associated with lower levels of TGF-β and IL-8. Our data provide the first evidence of the predictive role of HER-2/ neu -specific T cell immunity in PCa.

The use of synthetic peptides as vaccines aimed at the induction of therapeutic CD8-positive T-cell responses against tumor cells initially experienced great enthusiasm, mostly because of advances in vaccine technology, including design, synthesis, and delivery. However, despite impressive results in animal models, the application of such vaccines in humans has met with only limited success. The therapeutic activity of vaccine-stimulated, tumor-specific, CD8-positive T cells can be hampered through the physical burden of the tumor, tolerance mechanisms, and local factors within the tumor microenvironment. Recently, accumulating evidence has suggested that combining a peptide-based therapeutic vaccination with conventional chemotherapy can uncover the full potential of the antitumor immune response, increasing the success of immunotherapy. In addition, therapeutic vaccination in the preventive setting has been extremely effective in eliciting antitumor responses in preclinical tumor models and has demonstrated good promise clinically in patients with minimal residual disease. The rationale behind preventive vaccination is that patients with minimal tumor burden still have a fully competent immune system capable of developing robust antitumor responses. Finally, therapeutic CD8-positive T-cell peptide vaccines have been improved by coimmunization with T-helper epitopes expressed on long peptides., ((c) 2010 American Cancer Society.)

Our aim is to develop peptide vaccines that stimulate tumor antigen-specific T-lymphocyte responses against frequently detected cancers. We describe herein a novel HLA-A*0201-restricted epitope, encompassing amino acids 828-836 (residues QIAKGMSYL), which is naturally presented by various HER-2/neu (+) tumor cell lines. HER-2/neu(828-836), [HER-2(9(828))], possesses two anchor residues and stabilized HLA-A*0201 on T2 cells in a concentration-dependent Class I binding assay. This peptide was stable for 3.5 h in an off-kinetic assay. HER-2(9(828)) was found to be immunogenic in HLA-A*0201 transgenic (HHD) mice inducing peptide-specific and functionally potent CTL and long-lasting anti-tumor immunity. Most important, using HLA-A*0201 pentamer analysis we could detect increased ex vivo frequencies of CD8(+) T-lymphocytes specifically recognizing HER-2(9(828)) in 8 out of 20 HLA-A*0201(+) HER-2/neu (+) breast cancer patients. Moreover, HER-2(9(828))-specific human CTL recognized the tumor cell line SKOV3.A2 as well as the primary RS.A2.1.DR1 tumor cell line both expressing HER-2/neu and HLA-A*0201. Finally, therapeutic vaccination with HER-2(9(828)) in HHD mice was proven effective against established transplantable ALC.A2.1.HER tumors, inducing complete tumor regression in 50% of mice. Our data encourage further exploitation of HER-2(9(828)) as a promising candidate for peptide-based cancer vaccines.

BALB/c mice transgenic (Tg) for the transforming rat neu oncogene (BALB-neuT) are genetically predestined to develop mammary carcinogenesis in a process similar to that in humans. We crossed HLA-A2.1/HLA-DR1 (A2.1/DR1) Tg mice with BALB-neuT mice to generate A2.1/DR1 x BALB-neuT triple Tg (A2.1/DR1 x neuT(+)) mice, which represent an improvement over BALB-neuT mice for evaluating vaccination regimens to overcome tolerance against HER-2/neu. A vaccine formulation strategy, consisting of synthetic peptides from the rat HER-2/neu oncogene combined with granulocyte macrophage colony-stimulating factor, was highly effective in preventing the growth of established transplantable tumors in male A2.1/DR1 x neuT(+) mice. Vaccination with HER-2(435-443) (p435) CTL peptide alone induced weak antitumor responses, which were characterized by increased numbers of regulatory T cells (Treg) and low numbers of vaccine-specific CD8(+) CTL and helper T cells (Th). The administration of p435 plus HER-2(776-790) (p776; helper peptide) reversed this situation, inducing functionally active, peptide-specific CTL and Th. There was a striking change in the intratumoral balance of Tregs (decrease) and vaccine-specific Th (increase) that directly correlated with tumor rejection. Intratumoral administration of anti-FasL antibody promoted tumor growth. The decrease in Tregs (Fas(+)) was due to apoptosis induced by cell contact with Fas ligand(+) (L)(+) Th. Mice vaccinated with p435 plus p776 exhibited long-lasting antitumor immunity. Our vaccine regimen also significantly delayed the outgrowth of mammary carcinomas in female A2.1/DR1 x neuT(+) animals. We provide a mechanism to overcome tolerance against HER-2/neu, which proposes a combined vaccination with two (Th and CTL) HER-2 peptides against HER-2/neu-expressing tumors.

A novel modality toward the treatment of HER-2/neu-positive malignancies, mostly including breast and, more recently prostate carcinomas, has been the use of vaccines targeting HER-2/neu extracellular and intracellular domains. HER-2/neu-specific vaccines have been demonstrated to generate durable T-cell anti-HER-2/neu immunity when tested in Phase I and II clinical trials with no significant toxicity or autoimmunity directed against normal tissues. Targeting of HER-2/neu in active immunotherapy may involve peptide and DNA vaccines. Moreover, active anti-HER-2/neu immunization could facilitate the ex vivo expansion of HER-2/neu-specific T cells for use in adoptive immunotherapy for the treatment of established metastatic disease. In addition, early data from trials examining the potential use of HER-2/neu-based vaccines in the adjuvant setting to prevent the relapse of breast cancer in high-risk patients have shown promising results. Future approaches include multiepitope preventive vaccines and combinatorial treatments for generating the most efficient protective anti-tumor immunity.

The HER-2/neu oncoprotein is a promising cancer vaccine target. We describe herein a novel HLA-A2.1-restricted epitope, encompassing amino acids 657-665 (AVVGILLVV), which is naturally presented by human breast and ovarian cell lines. HER-2/neu(657-665), [HER-2(9(657))], binds with high affinity to HLA-A2.1 molecules as revealed by a prediction algorithm (SYFPEITHI) and in functional assays. This peptide was found to be immunogenic in HLA-A2.1 transgenic (HHD) mice inducing peptide-specific CTL, which responded with increased IFNgamma production, degranulation, and in vitro as well as in vivo cytotoxicity. Most important, HER-2(9(657)) functioned as a therapeutic vaccine by enabling HHD mice to reject established transplantable tumors. Cured mice resisted tumor growth when re-challenged with the same tumor, demonstrating the capacity of HER-2(9(657)) to generate tumor-specific memory immune response. Finally, this peptide was also found to be immunogenic in PBMCs from HLA-A2.1(+) patients with HER-2/neu(+) breast cancer. Our data encourage further exploitation of HER-2(9(657)) as a promising candidate for peptide-based cancer vaccines.

We have demonstrated that coupling an immunoregulatory segment of the MHC class II-associated invariant chain (Ii), the Ii-Key peptide, to a promiscuous MHC class II epitope significantly enhances its presentation to CD4+ T cells. Here, a series of homologous Ii-Key/HER-2/neu(776-790) hybrid peptides, varying systematically in the length of the epitope(s)-containing segment, are significantly more potent than the native peptide in assays using T cells from patients with various types of tumors overexpressing HER-2/neu. In particular, priming normal donor and patient PBMCs with Ii-Key hybrid peptides enhances recognition of the native peptide either pulsed onto autologous dendritic cells (DCs) or naturally presented by IFN-gamma-treated autologous tumor cells. Moreover, patient-derived CD4+ T cells primed with the hybrid peptides provide a significantly stronger helper effect to autologous CD8+ T cells specific for the HER-2/neu(435-443) CTL epitope, as illustrated by either IFN-gamma ELISPOT assays or specific autologous tumor cell lysis. Hybrid peptide-specific CD4+ T cells strongly enhanced the antitumor efficacy of HER-2/neu(435-443) peptide-specific CTL in the therapy of xenografted SCID mice inoculated with HER-2/neu overexpressing human tumor cell lines. Our data indicate that the promiscuously presented vaccine peptide HER-2/neu(776-790) is amenable to Ii-Key-enhancing effects and supports the therapeutic potential of vaccinating patients with HER-2/neu+ tumors with such Ii-Key/HER-2/neu(776-790) hybrid peptides.

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.