Prospective, randomized, single-blinded, multi-center phase II trial of two HER2 peptide vaccines, GP2 and AE37, in breast cancer patients to prevent recurrence.

PURPOSE: AE37 and GP2 are HER2 derived peptide vaccines. AE37 primarily elicits a CD4+ response while GP2 elicits a CD8+ response against the HER2 antigen. These peptides were tested in a large randomized trial to assess their ability to prevent recurrence in HER2 expressing breast cancer patients. The primary analyses found no difference in 5-year overall disease-free survival (DFS) but possible benefit in subgroups. Here, we present the final landmark analysis. METHODS: In this 4-arm, prospective, randomized, single-blinded, multi-center phase II trial, disease-free node positive and high-risk node negative breast cancer patients enrolled after standard of care therapy. Six monthly inoculations of vaccine (VG) vs. control (CG) were given as the primary vaccine series with 4 boosters at 6-month intervals. Demographic, safety, immunologic, and DFS data were evaluated. RESULTS: 456 patients were enrolled; 154 patients in the VG and 147 in CG for AE37, 89 patients in the VG and 91 in CG for GP2. The AE37 arm had no difference in DFS as compared to CG, but pre-specified exploratory subgroup analyses showed a trend towards benefit in advanced stage (p = 0.132, HR 0.573 CI 0.275-1.193), HER2 under-expression (p = 0.181, HR 0.756 CI 0.499-1.145), and triple-negative breast cancer (p = 0.266, HR 0.443 CI 0.114-1.717). In patients with both HER2 under-expression and advanced stage, there was significant benefit in the VG (p = 0.039, HR 0.375 CI 0.142-0.988) as compared to CG. The GP2 arm had no significant difference in DFS as compared to CG, but on subgroup analysis, HER2 positive patients had no recurrences with a trend toward improved DFS (p = 0.052) in VG as compared to CG. CONCLUSIONS: This phase II trial reveals that AE37 and GP2 are safe and possibly associated with improved clinical outcomes of DFS in certain subgroups of breast cancer patients. With these findings, further evaluations are warranted of AE37 and GP2 vaccines given in combination and/or separately for specific subsets of breast cancer patients based on their disease biology.

Effects of HLA status and HER2 status on outcomes in breast cancer patients at risk for recurrence - Implications for vaccine trial design.

Immunotherapy, using peptide-based cancer vaccines is being studied to assess its potential in breast cancer. Trials of HLA-restricted peptide vaccines have been difficult to enroll given HLA subtype restrictions. It is necessary to determine the prognostic significance of HLA-status in breast cancer if patients who are ineligible to receive a vaccine due to their HLA-status are used as controls. The impact of targeted tumor associated antigen expression, when it effects eligibility is also important. We examined control patients from two randomized phase II trials that tested HER2-peptide vaccines to determine the effect of HLA-A2 status and HER2 expression on disease-free survival. The analysis showed that HLA-A2-status does not affect disease-free survival, regardless of HER2 expression suggesting that HLA-A2 negative patients can be used as control patients. Additionally, HER2 over-expression was associated with a better disease-free survival in this population, underscoring the need for additional therapies in HER2 low-expressing breast cancer. ClinicalTrials.gov Identifier: NCT00524277.

Prostate cancer vaccines: the long road to clinical application.

Cancer vaccines as a modality of immune-based cancer treatment offer the promise of a non-toxic and efficacious therapeutic alternative for patients. Emerging data suggest that response to vaccination largely depends on the magnitude of the type I immune response generated, epitope spreading and immunogenic modulation of the tumor. Moreover, accumulating evidence suggests that cancer vaccines will likely induce better results in patients with low tumor burden and less aggressive disease. To induce long-lasting clinical responses, vaccines will need to be combined with immunoregulatory agents to overcome tumor-related immune suppression. Immunotherapy, as a treatment modality for prostate cancer, has received significant attention in the past few years. The most intriguing characteristics that make prostate cancer a preferred target for immune-based treatments are (1) its relative indolence which allows sufficient time for the immune system to develop meaningful antitumor responses; (2) prostate tumor-associated antigens are mainly tissue-lineage antigens, and thus, antitumor responses will preferentially target prostate cancer cells. But, also in the event of eradication of normal prostate epithelium as a result of immune attack, this will have no clinical consequences because the prostate gland is not a vital organ; (3) the use of prostate-specific antigen for early detection of recurrent disease allows for the initiation of vaccine immunotherapy while tumor burden is still minimal. Finally, for improving clinical outcome further to increasing vaccine potency, it is imperative to recognize prognostic and predictive biomarkers of clinical benefit that may guide to select the therapeutic strategies for patients most likely to gain benefit.

A pilot study in prostate cancer patients treated with the AE37 Ii-key-HER-2/neu polypeptide vaccine suggests that HLA-A*24 and HLA-DRB1*11 alleles may be prognostic and predictive biomarkers for clinical benefit.

Recently, several types of immunotherapies have been shown to induce encouraging clinical results, though in a restricted number of patients. Consequently, there is a need to identify immune biomarkers to select patients who will benefit from such therapies. Such predictive biomarkers may be also used as surrogates for overall survival (OS). We have recently found correlations between immunologic parameters and clinical outcome in prostate cancer patients who had been vaccinated with a HER-2/neu hybrid polypeptide vaccine (AE37) and received one booster 6 months post-primary vaccinations. Herein, we aimed to expand these retrospective analyses by studying the predictive impact of HLA-A*24 and HLA-DRB1*11 alleles, which are expressed at high frequencies among responders in our vaccinated patients, for clinical and immunological responses to AE37 vaccination. Our data show an increased OS of patients expressing the HLA-DRB1*11 or HLA-A*24 alleles, or both. Vaccine-induced immunological responses, measured as interferon γ (IFN-γ) responses in vitro or delayed-type hypersensitivity reactions in vivo, were also higher in these patients and inversely correlated with suppressor elements. Preexisting (i.e., before vaccinations with AE37) levels of vaccine-specific IFN-γ immunity and plasma TGF-ß, among the HLA-A*24 and/or HLA-DRB1*11 positive patients, were strong indicators for immunological responses to AE37 treatment. These data suggest that HLA-DRB1*11 and HLA-A*24 are likely to be predictive factors for immunological and clinical responses to vaccination with AE37, though prospective validation in larger cohorts is needed.

AE37 peptide vaccination in prostate cancer: identification of biomarkers in the context of prognosis and prediction.

A fundamental challenge in administering immunotherapies for cancer is the establishment of biomarkers that can predict patients' responsiveness to treatment. In this study, our aim was to predict the immunologic and clinical responses of vaccination therapy with an Ii-key-modified HER-2/neu peptide (Ii-key/HER-2(776-790) or AE37), applied in our recent phase I study in patients with prostate cancer. To this end, we retrospectively analyzed our data derived from immunologic determinations before, during and after primary series of vaccinations with AE37, as well as after one AE37 booster injection. Using the obtained data, we then observed the relationship between the immunologic parameters and clinical outcome of patients. We found that preexisting levels of transforming growth factor beta (TGF-ß) had an inverse correlation with in vivo and in vitro immunologic responses to the AE37 vaccine which were measured as delayed-type hypersensitivity (DTH) and interferon gamma (IFN-γ) production in response to the native HER-2(776-790) (or AE36) peptide, respectively. Patients with preexistent IFN-γ immunity to AE36 developed positive DTH reactions after primary vaccinations and booster. Moreover, we could detect a direct correlation between IFN-γ production and DTH reactions in response to AE36 challenge in our vaccinated patients. DTH reactions were a stronger indicator for patients' overall survival (OS) than preexistent or vaccine-induced IFN-γ immunity. In contrast, we found that preexisting TGF-ß levels were correlated with shorter patients' OS. These retrospective analyses suggest that the above biomarkers at the time-points measured offer promise for evaluating immunologic and clinical responses to AE37-based vaccinations.

NK cell adoptive transfer combined with Ontak-mediated regulatory T cell elimination induces effective adaptive antitumor immune responses.

Previous work from our laboratory showed that hydrocortisone (HC) combined with IL-15 induces expansion of activated human NK cells. We set up an experimental tumor model to evaluate the use of adoptively transferred, HC plus IL-15 (HC/IL-15)-activated and -expanded murine NK cells in the treatment of syngeneic mice carrying established lung metastases of the CT26 transplantable tumor. We also examined the effect of denileukin diftitox (Ontak) on the depletion of regulatory T cells to enhance the in vivo antitumor immunity induced by the adoptively transferred NK cells. Our results clearly demonstrate that murine DX5(+) NK cells are largely expanded in the presence of IL-15 plus HC while retaining intact their functional status. Moreover, when intravenously infused, they mediated significant antitumor responses against CT26 lung tumors in syngeneic BALB/c animals that were further enhanced upon pretreatment of the tumor-bearing animals with Ontak. Total splenocytes and isolated splenic T cells from NK-treated mice responded in vitro against CT26 tumor cells as evidenced by IFN-γ-based ELISPOT, proliferation, and cytotoxicity assays. Importantly, animals treated with Ontak plus adoptive transfer of HC/IL-15-expanded NK cells significantly retarded CT26 tumor growth after a rechallenge with the same tumor s.c. in their flanks. Taken altogether, our data suggest that NK cell adoptive transfer can trigger adaptive antitumor T cell responses, and regulatory T cell depletion by Ontak is mandatory for enabling HC/IL-15-activated NK cells to promote long-lasting adaptive antitumor immunity.

Effect of the simultaneous administration of glucocorticoids and IL-15 on human NK cell phenotype, proliferation and function.

We have previously reported a synergistic effect between hydrocortisone (HC) and IL-15 on promoting natural killer (NK) cell expansion and function. In the present study, we extend our findings to methylprednisolone (MeP) and dexamethasone (Dex), thus ascribing to glucocorticoids (GCs) a general feature as positive regulators of IL-15-mediated effects on NK cells. We demonstrate that each GC when combined with IL-15 in cultures of peripheral blood (PB)-derived CD56(+) cells induces increased expansion of CD56(+)CD3(-) cells displaying high cytolytic activity, IFN-γ production potential and activating receptor expression, including NKp30, NKp44, NKp46, 2B4, NKG2D and DNAM-1. Furthermore, GCs protected NK cells from IL-15-induced cell death. The combination of IL-15 with GCs favored the expansion of a relatively more immature CD16(low/neg) NK cell population, with high expression of NKG2A and CD94, and significantly lower expression of KIR (CD158a and CD158b) and CD57, compared to IL-15 alone. IL-15-expanded NK cells, in the presence or absence of GCs, did not express CD62L, CXCR1 or CCR7. However, the presence of GCs significantly increased the density of CXCR3 and induced strong CXCR4 expression on the surface of NK cells. Our data indicate that IL-15/GC-expanded NK cells, apart from their increased proliferation rate, retain their functional integrity and exhibit a migratory potential rendering them useful for adoptive transfer in NK cell-based cancer immunotherapy.

Identification and characterization of a HER-2/neu epitope as a potential target for cancer immunotherapy.

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.

A phase I trial of adoptive transfer of allogeneic natural killer cells in patients with advanced non-small cell lung cancer.

HLA-mismatched natural killer (NK) cells have shown efficacy in acute myeloid leukemia, and their adoptive transfer in patients with other malignancies has been proven safe. This phase I clinical trial was designed to evaluate safety (primary endpoint) and possible clinical efficacy (secondary endpoint) of repetitive administrations of allogeneic, in vitro activated and expanded NK cells along with chemotherapy in patients with advanced non-small cell lung cancer (NSCLC). Patients with unresectable, locally advanced/metastatic NSCLC receiving 1st/2nd line chemotherapy were eligible to receive 2-4 doses of activated NK cells from two relative donors. Donor's CD56(+) cells were cultured for 20-23 days with interleukin-15 (IL-15) and hydrocortisone (HC) and administered intravenously between chemotherapy cycles. Premedication with corticosteroids and/or H1 inhibitors was allowed. Sixteen patients (performance status 0-1) with adenocarcinoma (n = 13) or squamous cell carcinoma (n = 3) at stage IIIb (n = 5) or IV (n = 11) receiving 1st (n = 13) or 2nd (n = 3) line treatment were enrolled. Fifteen patients received 2-4 doses of allogeneic activated NK cells (0.2-29 × 10(6)/kg/dose, median 4.15 × 10(6)/kg/dose). No side effects (local or systemic) were observed. At a median 22-month follow-up (range, 16.5-26 months) 2 patients with partial response and 6 patients with disease stabilization were recorded. Median progression free survival and overall survival were 5.5 and 15 months, respectively. A 56% 1-year survival and a 19% 2-year survival were recorded. In conclusion, repetitive infusions of allogeneic, in vitro activated and expanded with IL-15/HC NK cells, in combination with chemotherapy are safe and potentially clinically effective.

Cancer immunotherapy.

Recent scientific advances have expanded our understanding of the immune system and its response to malignant cells. The clinical goal of tumour immunotherapy is to provide either passive or active immunity against malignancies by harnessing the immune system to target tumours. Monoclonal antibodies, cytokines, cellular immunotherapy, and vaccines have increasingly become successful therapeutic agents for the treatment of solid and haematological cancers in preclinical models, clinical trials, and practice. In this article, we review recent advances in the immunotherapy of cancer, focusing on new strategies and future perspectives as well as on clinical trials attempting to enhance the efficacy of immunotherapeutic modalities and translate this knowledge into effective cancer therapies.

Combinatorial treatments including vaccines, chemotherapy and monoclonal antibodies for cancer therapy.

Accumulating evidence suggests that despite the potency of cytotoxic anticancer agents, and the great specificity that can be achieved with immunotherapy, neither of these two types of treatment by itself has been sufficient to eradicate the disease. Still, the combination of these two different modalities holds enormous potential for eliciting therapeutic results. Indeed, certain chemotherapeutic agents have shown immunomodulatory activities, and several combined approaches have already been attempted. For instance, chemotherapy has been proven to enhance the efficacy of tumor cell vaccines, and to favor the activity of adoptively transferred tumor-specific T cells. A number of mechanisms have been proposed for the chemotherapy-triggered enhancement of immunotherapy response. Thus, chemotherapy may favor tumor cell death, and by that enhance tumor-antigen cross-presentation in vivo. Drug-induced myelosuppression may induce the production of cytokines favoring homeostatic proliferation, and/or ablate immunosuppression mechanisms. Furthermore, the recently reported synergy between monoclonal antibodies and chemotherapy or peptide vaccination is based upon the induction of endogenous humoral and cellular immune responses. This would suggest that monoclonal antibodies may not only provide passive immunotherapy but can also promote tumor-specific active immunity. This article will review several strategies in which immunotherapy can be exploited in preclinical and clinical studies in combination with other agents and therapeutic modalities that are quite unique when compared with "conventional" combination therapies (ie, treatments with chemotherapeutic drugs or chemotherapy and radiotherapy based protocols). The results from these studies may have significant implications for the development of new protocols based on combinatorial treatments including vaccines, chemotherapy and monoclonal antibodies, suggesting an exciting potential for therapeutic synergy with general applicability to various cancer types. Given the complicity of immune-based therapies and cancer pharmacology, it will be necessary to bring together cancer immunologists and clinicians, so as to provide a robust stimulus for realizing the successful management of cancer in the near future.

CD4+CD25+ regulatory T-cell frequency in HER-2/neu (HER)-positive and HER-negative advanced-stage breast cancer patients.

PURPOSE: CD4(+)CD25(bright) regulatory T cells (Tregs) are increased in patients with several malignancies and correlate with disease stage and prognosis. Breast cancer patients represent a heterogeneous population with unpredictable disease progression even at advanced stages. Circulating Tregs in correlation with HER-2/neu (HER) status and treatment with chemotherapy, either alone or in combination with trastuzumab therapy, were monitored in advanced-stage breast cancer patients. EXPERIMENTAL DESIGN: Circulating Treg frequency and absolute counts of 46 HER(+) and 28 HER(-), stage III and IV, breast cancer patients before therapy and during trastuzumab therapy and/or chemotherapy have been compared with 24 healthy donors and correlated with plasma HER extracellular domain concentration and clinical outcome. RESULTS: Treg frequency in HER(+) patients was significantly increased compared with both HER(-) patients and healthy donors. Trastuzumab therapy, with or without combined chemotherapy, resulted in a progressive decrease of circulating Tregs. Percentage change in Tregs statistically correlated with percentage change in plasma HER extracellular domain. Furthermore, decrease in Tregs correlated with either objective clinical response or stable disease, whereas increased Treg frequency during trastuzumab therapy coincided with disease progression. No statistically significant change in Treg frequency following chemotherapy was observed in HER(-) patients. CONCLUSIONS: Treg cell frequency does not directly correlate with clinical stage in breast cancer, as stage III and IV HER(+) and HER(-) patients exhibit significantly different Treg profiles. Trastuzumab therapy, either alone or combined with chemotherapy, results in decreased Treg frequency in HER(+) advanced patients with an objective clinical response.

Vaccination with human HER-2/neu (435-443) CTL peptide induces effective antitumor immunity against HER-2/neu-expressing tumor cells in vivo.

HER-2/neu is a self-antigen expressed by tumors and nonmalignant epithelial tissues. The possibility of self-tolerance to HER-2/neu-derived epitopes has raised questions concerning their utility in antitumor immunotherapy. Altered HER-2/neu peptide ligands capable of eliciting enhanced immunity to tumor-associated HER-2/neu epitopes may circumvent this problem. The human CTL peptide HER-2/neu (435-443) [hHER-2(9(435))] represents a xenogeneic altered peptide ligand of its mouse homologue, differing by one amino acid residue at position 4. In contrast to mHER-2(9(435)), vaccination of HLA-A*0201 transgenic (HHD) mice with hHER-2(9(435)) significantly increased the frequency of mHER-2(9(435))-specific CTL and also induced strong protective and therapeutic immunity against the transplantable ALC tumor cell line transfected to coexpress HLA-A*0201 and hHER-2/neu or rHER-2/neu. Similar results were also obtained with wild-type C57BL/6 mice inoculated with HER-2/neu transfectants of ALC. Adoptive transfer of CD8(+) CTL from mice immunized with hHER-2(9(435)) efficiently protected naive syngeneic mice inoculated with ALC tumors. In conclusion, our results show that HER-2(9(435)) serves as a tumor rejection molecule. They also propose a novel approach for generating enhanced immunity against a self-HER-2/neu CTL epitope by vaccinating with xenogeneic altered peptide ligands and provide useful insights for the design of improved peptide-based vaccines for the treatment of patients with HER-2/neu-overexpressing tumors.

A novel quantitative flow cytometric method for measuring glucocorticoid receptor (GR) in cell lines: correlation with the biochemical determination of GR.

Currently, a time consuming biochemical method is used for GR quantification. Here we compare the biochemical approach with a newly developed flow cytometric method of measuring GR in cell lines, which is less time consuming and does not requires the use of radioactive materials. The biochemical assay is easy to apply but the cells need to be grown in media free of endogenous glucocorticoids, in order to prevent them from interfering with radiolabelled hormone binding to the receptor. The presence of endogenous GR ligands is known to reduce receptor levels and to often produce false negative results. The immunofluorescent method is free of such limitations, as it depends entirely on detecting the receptor using a highly specific monoclonal antibody. Additionally, the biochemical assay cannot measure heterogeneity in individual cells, in contrast the flow cytometric one enables the enumeration of the receptor on a per cell basis, allowing exact description of differences in receptor levels amongst intact cells. Our results demonstrate that the flow cytometric method is of similar accuracy but of higher precision compared to the biochemical one. Also, the data we obtained using the immunofluorescent method correlated well with the biochemical one (R(2)=0.9712). In conclusion, flow cytometric method requires small cell numbers, is more accurate and lesser time consuming than the biochemical one. Thus, it could be useful for the quantification of GR in lymphocyte subpopulations, in lymphoproliferative disorders and in tumor cells from cancer patients, in order to design more efficient clinical treatment protocols.

Immune properties of mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells isolated by various relatively easily accessible tissues, such as bone marrow and cord blood. MSCs gained attention because of their ease for in vitro expansion together with their multilineage potential. More recently, in vitro and in vivo immunosuppressive properties have been ascribed to them, as they are able to modulate the function of all major immune cell populations, thus impeding immune responses. The underlying mechanisms of their differentiation and function are not thoroughly understood, but still they represent important candidates for tissue regeneration and manipulation of the immune response in graft rejection, graft versus host disease, and autoimmune disorders. Characteristics and immunogenic profile of MSCs, their interface with immune system and their potential use as immunosuppressive elements in cellular therapeutic protocols are reviewed in this chapter.

Clinical grade expansion of human bone marrow mesenchymal stem cells.

Mesenchymal stem cells or marrow stromal cells (MSCs) represent a multipotent adult cellular population with immunomodulatory functions. In the adult human body, they are present in various niches, but their main source is bone marrow (BM). The regeneration capability of MSCs, their ease to undergo gene modification, as well as their immunosuppressive capacity render them as popular candidates for tissue engineering, gene therapy, and immunotherapy. They exhibit a unique in vitro expansion capacity, which however does not always compensate for the large number of cells required for cellular therapeutic applications. Unfortunately, to date, a uniform worldwide approach to MSC-culture is not available. Thus, in this chapter, we try to describe the optimal conditions for the successful isolation and ex vivo expansion of human MSCs from BM, to be used in all types of cellular therapeutic approaches. Moreover, we describe the methods for identification of their quality in terms of both multilineage potentiality and immunosuppressive ability. Detailed protocols for fetal calf serum selection and other culture parameters that affect the final outcome are described.

Increased frequency of CD4+ cells expressing CD161 in cancer patients.

PURPOSE: Although the function of natural killer receptors on T cells infiltrating tumors and their potential effect on antitumor immunity has been investigated, little is known about T cells expressing NKR-P1A (CD161) in cancer patients. In the present study, we examined T cells expressing CD161 in the peripheral blood, the tumor tissue and in malignant effusions of patients with several types of malignancies. EXPERIMENTAL DESIGN: Expression of CD161 in CD4(+) or CD8(+) (lacking CD56) T cells isolated from peripheral blood (n = 61), tumor specimens (n = 8), and malignant effusions (n = 37) of cancer patients was examined using four-color flow cytometry. Proliferative capacity and cytokine production of purified CD4(+)CD161(+)CD56(-) cells were studied after weak or strong stimulation, with or without costimulation, in the presence or absence of interleukin 2. The possible regulatory function of activated CD4(+)CD161(+)CD56(-) cells on T-cell alloresponses was also investigated. RESULTS: CD4(+) cells expressing CD161 were increased in cancer patients, compared with healthy individuals. This increase in the peripheral blood of cancer patients positively correlated with disease stage and was augmented at the tumor site. Phenotypic analysis revealed that CD4(+)CD161(+) cells are memory T cells, with low expression of activation markers. CD4(+)CD161(+) cells play an immunoregulatory role through cytokine production, because upon receiving costimulatory signals via CD28, they exert suppressive activity on autologous peripheral blood mononuclear cell alloresponses. CONCLUSIONS: CD4(+)CD161(+)CD56(-) cells represent a distinct memory T-cell population significantly increased in cancer patients. Depending on the type of signals provided by the tumor microenvironment, CD4(+)CD161(+) cells may regulate the immune response.

A HER-2/neu peptide admixed with PLA microspheres induces a Th1-biased immune response in mice.

The elimination of cancer cells requires strong cellular immune responses, and these responses are induced by the activation of Th1 lymphocytes. In this work, the possibility of inducing a Th1 type of immune response in vivo by mixing a HER-2/neu synthetic CTL (cytotoxic T lymphocyte) peptide [HER-2/neu (789-797)], with poly-lactide (PLA) microspheres was investigated. Various formulations of the peptide were administered to HLA-A2.1 transgenic (HHD) mice. Cellular experiments, assessing proliferation and cytokine determination in splenocyte culture supernatants, were carried out in order to evaluate the type of immune response to the antigen. The in vivo administration of the peptide antigen admixed with the PLA microspheres induced a potent immune response which was comparable to that induced by the combination of the antigen in complete Freund's adjuvant (CFA). Furthermore, the cytokine profile produced by the T lymphocytes of the immunized animals indicated that the combination of the peptide antigen with the PLA microspheres induced a strong Th1 biased immune response to the antigen. The time of peptide incubation with the microspheres prior to administration did not affect the immune response, which further simplifies the preparation of this type of vaccine. The results justify further investigation of the possibility of inducing effective cellular immune responses against cancer cells overexpressing HER-2/neu molecules by simply mixing appropriate HER-2/neu peptide antigens with PLA microspheres.

Characterization of a novel cell penetrating peptide derived from Bag-1 protein.

A highly cationic peptide (BagP), located within the normally expressed human protein Bag-1, was tested for its capacity to act as a cell penetrating peptide. BagP was found to translocate and transport high molecular weight cargos in several cell types, in varying degrees with a preference for adherent cells. The penetration phenomenon was not found to be subject to saturation for the highest amount of peptide tested (100 microM), whereas the time needed for maximum translocation to be achieved, was cell type-dependent. Finally, BagP internalization depends on its charge, cellular metabolism and cell-surface heparan sulfate proteoglycans.