Ribociclib plus letrozole in patients with hormone receptor-positive, HER2-negative advanced breast cancer with no prior endocrine therapy: subgroup safety analysis from the phase 3b CompLEEment-1 trial.

BACKGROUND: The CDK4/6 inhibitor, ribociclib in combination with endocrine therapy significantly improved progression-free survival in the first line setting in post-menopausal patients with HR+/HER2- advanced breast cancer (ABC) in a pivotal phase 3, placebo-controlled trial (MONALEESA-2) and demonstrated superior overall survival in premenopausal patients with HR+/HER2- ABC (MONALEESA-7). The multinational, phase 3b, CompLEEment-1 trial, which assessed the safety and efficacy of ribociclib plus letrozole in a broader population of patients who have not received prior endocrine therapy for advanced disease, is the largest phase 3 clinical trial to date to evaluate the safety and efficacy of a CDK4/6 inhibitor. We report a subanalysis of data from patients (N = 339) enrolled in the central and south European countries of the SERCE (Southern Europe, RUC, Central Europe) cluster of CompLEEment-1. PATIENTS AND METHODS: Men and women of any menopausal status with HR+/HER2- ABC received once-daily oral ribociclib 600 mg (3-weeks on/1-week-off), plus letrozole 2.5 mg continuously. Men/premenopausal women also received a GnRH-agonist. The primary outcome was the number of patients with adverse events (AEs) over a timeframe of approximately 36 months. Time-to-progression, overall response rate, and clinical benefit rate were also measured. RESULTS: Safety results in the SERCE subgroup were consistent with those in the pivotal clinical trials of ribociclib in combination with endocrine therapy. Treatment-related AEs leading to dose adjustments/interruption occurred in 63.1% of patients but led to treatment discontinuation in only 10.6%. The most common treatment-related AEs of grade ≥ 3 were neutropenia and transaminase elevations. There were no fatal treatment-related events. CONCLUSIONS: These findings from the SERCE subgroup support the safety and manageable tolerability of ribociclib in a broad range of patients with HR+/HER2- ABC more representative of patients in real-world clinical practice.

Harnessing the Immunomodulatory Properties of Bacterial Ghosts to Boost the Anti-mycobacterial Protective Immunity.

Tuberculosis (TB) pathogenesis is characterized by inadequate immune cell activation and delayed T cell response in the host. Recent immunotherapeutic efforts have been directed at stimulating innate immunity and enhancing interactions between antigen presenting cells and T cells subsets to improve the protective immunity against TB. In this study, we investigated the immunostimulatory properties of bacterial ghosts (BG) as a novel approach to potentiate the host immunity against mycobacterial infection. BG are intact cytoplasm-free Escherichia coli envelopes and have been developed as bacterial vaccines and adjuvant/delivery system in cancer immunotherapy. However, BG have yet to be exploited as immunopotentiators in the context of infectious diseases. Here, we showed that BG are potent inducers of dendritic cells (DC), which led to enhanced T cell proliferation and differentiation into effector cells. BG also induced macrophage activation, which was associated with enhanced nitric oxide production, a key anti-mycobacterial weapon. We further demonstrated that the immunostimulatory capability of BG far exceeds that of LPS and involves both TLR4-dependent and independent pathways. Consistently, BG treatment, but not LPS treatment, reduced the bacterial burden in infected mice, which correlated with increased influx of innate and adaptive effector immune cells and increased production of key cytokines in the lungs. Finally and importantly, enhanced bacilli killing was seen in mice co-administered with BG and second-line TB drugs bedaquiline and delamanid. Overall, this work paves the way for BG as potent immunostimulators that may be harnessed to improve mycobacteria killing at the site of infection.

Bacterial ghosts as adjuvant to oxaliplatin chemotherapy in colorectal carcinomatosis.

Colorectal cancer (CRC) is one of the most commonly diagnosed cancers and a major cause of cancer mortality worldwide. At late stage of the disease CRC often shows (multiple) metastatic lesions in the peritoneal cavity which cannot be efficiently targeted by systemic chemotherapy. This is one major factor contributing to poor prognosis. Oxaliplatin is one of the most commonly used systemic treatment options for advanced CRC. However, drug resistance - often due to insufficient drug delivery - is still hampering successful treatment. The anticancer activity of oxaliplatin includes besides DNA damage also a strong immunogenic component. Consequently, the aim of this study was to investigate the effect of bacterial ghosts (BGs) as adjuvant immunostimulant on oxaliplatin efficacy. BGs are empty envelopes of gram-negative bacteria with a distinct immune-stimulatory potential. Indeed, we were able to show that the combination of BGs with oxaliplatin treatment had strong synergistic anticancer activity against the CT26 allograft, resulting in prolonged survival and even a complete remission in this murine model of CRC carcinomatosis. This synergistic effect was based on an enhanced induction of immunogenic cell death and activation of an efficient T-cell response leading to long-term anti-tumor memory effects. Taken together, co-application of BGs strengthens the immunogenic component of the oxaliplatin anticancer response and thus represents a promising natural immune-adjuvant to chemotherapy in advanced CRC.

Oncolysate-loaded Escherichia coli bacterial ghosts enhance the stimulatory capacity of human dendritic cells.

The natural adjuvant properties of bacterial ghosts (BGs) lie within the presence of intact pathogen-associated molecular patterns on their surface. BGs can improve the direct delivery, natural processing and presentation of target antigens within dendritic cells (DCs). Moreover, sensitization of human DCs by cancer cell lysate (oncolysate)-loaded BGs in the presence of IFN-α and GM-CSF enhanced DC maturation as indicated by an increased expression of maturation markers and co-stimulatory molecules, higher production of IL-12p70 and stimulation of significantly increased proliferation of both autologous CD4+ and CD8+ T cells compared to DCs matured in the presence of purified lipopolysaccharide. The induced T cells efficiently recognized oncolysate-derived tumor-associated antigens expressed by cancer cells used for the production of oncolysate. Our optimized one-step simultaneous antigen delivery and DC maturation-inducing method emerges as a promising tool for the development and implementation of next-generation cellular cancer immunotherapies.

Bacterial ghosts as adjuvants in syngeneic tumour cell lysate-based anticancer vaccination in a murine lung carcinoma model.

Instead of relying on external anticancer factors for treatment, immunotherapy utilizes the host's own immune system and directs it against given tumour antigens. This study demonstrated that it is possible to overcome the documented immunosuppressive properties of tumour cell lysate by supplementing it with appropriate adjuvant. Lewis lung carcinoma (LLC)­challenged C57BL/6 mice were treated with LLC cryo­lysate mixed with either bacterial ghosts (BGs) generated from E. coli Nissle 1917 or B. subtilis 70 kDa protein as adjuvants. Median and overall survival, the size of metastatic foci in lung tissue and levels of circulating CD8a+ T cells were evaluated and compared to the untreated control mice or mice treated with LLC lysate alone. After primary tumour removal, a course of three subcutaneous vaccinations with LLC lysate supplemented with BGs led to a significant increase in overall survival (80% after 84 days of follow­up vs. 40% in untreated control mice), a significant increase in circulating CD8a+ T cells (16.57 vs. 12.6% in untreated control mice) and a significant decrease in metastasis foci area and incidence. LLC lysate supplemented with B. subtilis protein also improved the inspected parameters in the treated mice, when compared against the untreated control mice, but not to a significant degree. Therefore, whole cell lysate supplemented with BGs emerges as an immunostimulatory construct with potential clinical applications in cancer treatment.

Modulation of bacterial ghosts--induced nitric oxide production in macrophages by bacterial ghost-delivered resveratrol.

The present study aimed to investigate the capacity of resveratrol (RV) delivered into macrophages by bacterial ghosts (BGs), representing intact empty nonliving envelopes of Gram-negative bacteria, to modulate nitric oxide (NO) production related to the presence of the pathogen-associated molecular patterns on the surface of BGs. Incubation of the murine macrophage cell line RAW 264.7 with BGs leads to a dose-dependent activation of inducible NO synthase. To modify BG-induced NO formation in RAW 264.7 cells by RV; BGs were loaded with RV (RV-BGs) and incubated with murine macrophages in a dose-dependent manner. RV-BGs delivering RV to the target macrophages significantly reduced BG-induced NO production with concentration of RV more than one order of magnitude lower than the amount of RV capable of reducing NO formation when applied directly. Moreover, no cytotoxic impact of BGs on the viability of RAW 264.7 cells added to macrophages alone or loaded with RV was detected after a mutual 24 h incubation, whereas cell viability slightly decreased (~ 10%) when RV concentrations of 30 µm alone were applied. The results obtained in the present study clearly indicate that the intracellular delivery of RV by BGs significantly enhances the total RV effect.

Rectal single dose immunization of mice with Escherichia coli O157:H7 bacterial ghosts induces efficient humoral and cellular immune responses and protects against the lethal heterologous challenge.

Bacterial ghosts (BGs) have been applied through oral, aerogenic, intraocular or intranasal routes for mucosal immunization using a wide range of experimental animals. All these applications required a booster after primary immunization to achieve protective immunity against the lethal challenge. Here we report for the first time that a single rectal dose of BGs produced from enterohaemorrhagic Escherichia coli (EHEC) O157:H7 fully protects mice against a 50% lethal challenge with a heterologous EHEC strain given at day 55. BGs from EHEC O157:H7 were prepared by a combination of protein E-mediated cell lysis and expression of staphylococcal nuclease A guaranteeing the complete degradation of pathogen residual DNA. The lack of genetic material in the EHEC BGs vaccine abolished any potential hazard for horizontal gene transfer of plasmid encoded antibiotic resistance genes or pathogenic islands to the recipient's gut flora. Single rectal immunization using EHEC O157:H7 BGs without any addition of adjuvant significantly stimulated efficient humoral and cellular immune responses, and was equally protective as two immunizations, which indicates the possibility to develop a novel efficacious single dose mucosal EHEC O157:H7 BGs vaccine using a simplified immunization regimen.

Bacterial ghosts as carriers of protein subunit and DNA-encoded antigens for vaccine applications.

Bacterial ghosts (BGs) represent vaccine delivery systems gifted with outstanding natural adjuvant properties. BGs are empty cell envelopes of Gram-negative bacteria lacking cytoplasmic content yet retaining all unaltered morphological and structural features of their living counterparts. The intact surface make-up of BGs is easily recognized by professional APCs through pattern-recognition receptors, making them ideal for mucosal administration through oral, ocular, intranasal or aerogenic routes, which represent the most desirable methods of application in advanced vaccine use. BGs have been designed to be used as carriers of active substances and foreign antigens (protein and/or DNA) for vaccine development. This review highlights the salient features of the BGs' versatile multipurpose vaccine platform for application in a wide range of human and veterinary medicines.

Bacterial Ghosts as antigen and drug delivery system for ocular surface diseases: Effective internalization of Bacterial Ghosts by human conjunctival epithelial cells.

The purpose of the presented investigation was to examine the efficiency of the novel carrier system Bacterial Ghosts (BGs), which are empty bacterial cell envelopes of Gram-negative bacteria to target human conjunctival epithelial cells, as well as to test the endocytic capacity of conjunctival cells after co-incubation with BGs generated from different bacterial species, and to foreclose potential cytotoxic effects caused by BGs. The efficiency of conjunctival cells to internalize BGs was investigated using the Chang conjunctival epithelial cell line and primary human conjunctiva-derived epithelial cells (HCDECs) as in vitro model. A high capacity of HCDECs to functionally internalize BGs was detected with the level of internalization depending on the type of species used for BGs generation. Detailed analysis showed no cytotoxic effect of BGs on HCDECs independently of the used bacterial species. Moreover, co-incubation with BGs did not enhance expression of both MHC class I and class II molecules by HCDECs, but increased expression of ICAM-1. The high rates of BG's internalization by HCDECs with no BG-mediated cytotoxic impact designate this carrier system to be a promising candidate for an ocular surface drug delivery system. BGs could be useful for future therapeutic ocular surface applications and eye-specific disease vaccine development including DNA transfer.

Epitope hierarchy of spontaneous CD4+ T cell responses to LAGE-1.

NY-ESO-1 and LAGE-1 represent highly homologous cancer-germline Ags frequently coexpressed by many human cancers, but not by normal tissues, except testis. In contrast to NY-ESO-1, little is known about spontaneous immune responses to LAGE-1. In the current study, we report on spontaneous LAGE-1-specific CD4(+) T cells isolated from PBLs of patients with advanced LAGE-1(+)/NY-ESO-1(+) melanoma and directed against three promiscuous and immunodominant epitopes. Strikingly, although the three LAGE-1-derived epitopes are highly homologous to NY-ESO-1-derived epitopes, LAGE-1-specific CD4(+) T cells did not cross-react with NY-ESO-1. LAGE-1-specific CD4(+) T cells produced Th1-type and/or Th2-type cytokines and did not exert inhibitory effects on allogenic T cells. We observed that most patients with spontaneous NY-ESO-1-specific responses exhibited spontaneous CD4(+) T cell responses to at least one of the three immunodominant LAGE-1 epitopes. Additionally, nearly half of the patients with spontaneous LAGE-1-specific CD4(+) T cell responses had circulating LAGE-1-specific Abs that recognized epitopes located in the C-terminal portion of LAGE-1, which is distinct from NY-ESO-1. Collectively, our findings define the hierarchy of immunodominance of spontaneous LAGE-1-specific CD4(+) T cell responses in patients with advanced melanoma. These findings demonstrate the capability of LAGE-1 to stimulate integrated cellular and humoral immune responses that do not cross-react with NY-ESO-1. Therefore, they provide a strong rationale for the inclusion of LAGE-1 peptides or protein in vaccine trials for patients with NY-ESO-1(+)/LAGE-1(+) tumors.

Escherichia coli ghosts promote innate immune responses in human keratinocytes.

Bacterial ghosts (BGs) as non-living bacterial envelopes devoid of cytoplasmic content with preserved and intact inner and outer membrane structures of their living counterparts have been used to study the ability of their surface components for the induction of antimicrobial peptides and pro-inflammatory cytokines in human primary keratinocytes (KCs). Quantitative real-time PCR analysis revealed that incubation of KCs with BGs generated from wild-type Escherichia coli induced the mRNA expression of antimicrobial psoriasin (S100A7c) in a BGs particle concentration-dependent manner. Using immunoblot analysis we showed that BGs generated from the flagellin-deficient (ΔFliC) E. coli strain NK9375 were as effective as its isogenic wild-type (wt) E. coli strain NK9373 to induce psoriasin expression when normalized to BG particles being taken up by KCs. However, results obtained from endocytic activity of KCs reflect that internalization of BGs is greatly dependent on the presence of flagellin on the surface of BGs. Moreover, BGs derived from wt E. coli NK9373 strongly induced the release of the pro-inflammatory cytokines IL-6 and IL-8, compared to ΔFliC E. coli NK9375 BGs. Taken together, obtained data demonstrate that non-living BGs possessing all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat have the capacity to induce the expression of innate immune modulators and that these responses are partially dependent on the presence of flagellin.

Bacterial ghosts (BGs)--advanced antigen and drug delivery system.

Bacterial ghosts (BGs) are empty bacterial envelopes of Gram-negative bacteria produced by controlled expression of cloned gene E, forming a lysis tunnel structure within the envelope of the living bacteria. BGs are devoid of cytoplasmic content and possess all bacterial bio-adhesive surface properties in their original state while not posing any infectious threat. BGs are ideally suited as an advanced drug delivery system (ADDS) for toxic substances in tumor therapy. The inner space of BGs can be loaded with either single components or combinations of peptides, drugs or DNA which provides an opportunity to design new types of (polyvalent) drug delivery vehicles. Uptake of BGs loaded with Doxorubicin (Dox) by CaCo2 cells led to effective Dox release from endo-lysosomal compartments and accumulation in the nucleus. Viability and proliferative capacity of the cells were significantly decreased (2-3 orders of magnitude) after internalization of Dox loaded BGs as compared to cells incubated with free Dox. The same effect was observed with leukemia cells. Melanoma cells also revealed a high capability to internalize BGs. These results indicate that BGs are able to target a range of types of cancer. BGs have also been investigated as DNA delivery vectors. Studies show DNA loaded BGs are efficiently phagocytosed and internalized by both professional APCs and tumor cells with up to 82% of cells expressing the plasmid-encoded reporter gene. Our studies with BGs as an ADDS system contribute (i) to optimize drug delivery for the treatment of cancer; (ii) define specific conditions for selection and preparation of BG formulations; (iii) and provide a background for the clinical application of BGs in cancer therapy.

Human tumor antigen-specific helper and regulatory T cells share common epitope specificity but exhibit distinct T cell repertoire.

CD4(+) regulatory T cells (Tregs) accumulate at tumor sites and play a critical role in the suppression of immune responses against tumor cells. In this study, we show that two immunodominant epitopes derived from the tumor Ags (TAs) NY-ESO-1 and TRAG-3 stimulate both CD4+ Th cells and Tregs. TA-specific Tregs inhibit the proliferation of allogenic T cells, act in a cell-to-cell contact dependent fashion and require activation to suppress IL-2 secretion by T cells. TRAG-3 and NY-ESO-1-specific Tregs exhibit either a Th1-, a Th2-, or a Th0-type cytokine profile and dot not produce IL-10 or TGF-beta. The Foxp3 levels vary from one Treg clone to another and are significantly lower than those of CD4+CD25high Tregs. In contrast to NY-ESO-1-specific Th cells, the NY-ESO-1-specific and TRAG-3-specific Treg clonotypes share a common TCR CDR3 Vbeta usage with Foxp3+CD4+CD25high and CD4+CD25- T cells and were not detectable in PBLs of other melanoma patients and of healthy donors, suggesting that their recruitment occurs through the peripheral conversion of CD4+CD25- T cells upon chronic Ag exposure. Collectively, our findings demonstrate that the same epitopes spontaneously stimulate both Th cells and Tregs in patients with advanced melanoma. They also suggest that TA-specific Treg expansion may be better impaired by therapies aimed at depleting CD4+CD25high Tregs and preventing the peripheral conversion of CD4+CD25- T cells.

The Bacterial Ghost platform system: production and applications.

The Bacterial Ghost (BG) platform technology is an innovative system for vaccine, drug or active substance delivery and for technical applications in white biotechnology. BGs are cell envelopes derived from Gram-negative bacteria. BGs are devoid of all cytoplasmic content but have a preserved cellular morphology including all cell surface structures. Using BGs as delivery vehicles for subunit or DNA-vaccines the particle structure and surface properties of BGs are targeting the carrier itself to primary antigen-presenting cells. Furthermore, BGs exhibit intrinsic adjuvant properties and trigger an enhanced humoral and cellular immune response to the target antigen. Multiple antigens of the native BG envelope and recombinant protein or DNA antigens can be combined in a single type of BG. Antigens can be presented on the inner or outer membrane of the BG as well as in the periplasm that is sealed during BG formation. Drugs or supplements can also be loaded to the internal lumen or periplasmic space of the carrier. BGs are produced by batch fermentation with subsequent product recovery and purification via tangential flow filtration. For safety reasons all residual bacterial DNA is inactivated during the BG production process by the use of staphylococcal nuclease A and/or the treatment with ß-propiolactone. After purification BGs can be stored long-term at ambient room temperature as lyophilized product. The production cycle from the inoculation of the pre-culture to the purified BG concentrate ready for lyophilization does not take longer than a day and thus meets modern criteria of rapid vaccine production rather than keeping large stocks of vaccines. The broad spectrum of possible applications in combination with the comparably low production costs make the BG platform technology a safe and sophisticated product for the targeted delivery of vaccines and active agents as well as carrier of immobilized enzymes for applications in white biotechnology.

PD-1 is a regulator of NY-ESO-1-specific CD8+ T cell expansion in melanoma patients.

The programmed death 1 (PD-1) receptor is a negative regulator of activated T cells and is up-regulated on exhausted virus-specific CD8(+) T cells in chronically infected mice and humans. Programmed death ligand 1 (PD-L1) is expressed by multiple tumors, and its interaction with PD-1 resulted in tumor escape in experimental models. To investigate the role of PD-1 in impairing spontaneous tumor Ag-specific CD8(+) T cells in melanoma patients, we have examined the effect of PD-1 expression on ex vivo detectable CD8(+) T cells specific to the tumor Ag NY-ESO-1. In contrast to EBV, influenza, or Melan-A/MART-1-specific CD8(+) T cells, NY-ESO-1-specific CD8(+) T cells up-regulated PD-1 expression. PD-1 up-regulation on spontaneous NY-ESO-1-specific CD8(+) T cells occurs along with T cell activation and is not directly associated with an inability to produce cytokines. Importantly, blockade of the PD-1/PD-L1 pathway in combination with prolonged Ag stimulation with PD-L1(+) APCs or melanoma cells augmented the number of cytokine-producing, proliferating, and total NY-ESO-1-specific CD8(+) T cells. Collectively, our findings support the role of PD-1 as a regulator of NY-ESO-1-specific CD8(+) T cell expansion in the context of chronic Ag stimulation. They further support the use of PD-1/PD-L1 pathway blockade in cancer patients to partially restore NY-ESO-1-specific CD8(+) T cell numbers and functions, increasing the likelihood of tumor regression.

Immunization with analog peptide in combination with CpG and montanide expands tumor antigen-specific CD8+ T cells in melanoma patients.

Analog peptides represent a promising tool to further optimize peptide-based vaccines in promoting the expansion of tumor antigen-specific cytotoxic T lymphocytes. Here, we report the results of a pilot trial designed to study the immunogenicity of the analog peptide NY-ESO-1 157-165V in combination with CpG 7909/PF3512676 and Montanide ISA 720 in patients with stage III/IV NY-ESO-1-expressing melanoma. Eight patients were immunized either with Montanide and CpG (arm 1, 3 patients); Montanide and peptide NY-ESO-1 157-165V (arm 2, 2 patients); or with Montanide, CpG, and peptide NY-ESO-1 157-165V (arm 3, 3 patients). Only the 3 patients immunized with Montanide, CpG, and peptide NY-ESO-1 157-165V in arm 3 developed a rapid increase of effector-memory NY-ESO-1-specific CD8+ T cells, detectable ex vivo. The majority of these cells exhibited an intermediate/late-stage differentiated phenotype (CD28-). Our study further demonstrated that our vaccine approach stimulated spontaneous tumor-reactive NY-ESO-1-specific CD8+ T cells in 2 patients with advanced disease, but failed to prime tumor-reactive NY-ESO-1-specific T cells in 1 patient with no spontaneously tumor-induced CD8+ T-cell responses to NY-ESO-1. Collectively, our data support the capability of the analog peptide NY-ESO-1 157-165V in combination with CpG and Montanide to promote the expansion of NY-ESO-1-specific CD8+ T cells in patients with advanced cancer. They also suggest that the presence of tumor-induced NY-ESO-1-specific T cells of well-defined clonotypes is critical for the expansion of tumor-reactive NY-ESO-1-specific CD8+ T cells after peptide-based vaccine strategies.

Effective gene transfer to melanoma cells using bacterial ghosts.

Bacterial ghosts (BG) are cell envelopes preparations of Gram-negative bacteria devoid of cytoplasmic content produced by controlled expression of PhiX174 plasmid-encoded lysis gene E. Eight melanoma cell lines were investigated for their capacity to bind and phagocyte BG derived from Escherichia coli NM522 and Mannheimia haemolytica A23. High capability to bind BG was observed in almost all of the analyzed cell lines, furthermore cells were able to take up BG independently of the used bacterial species. Further, transfection efficiency of BG loaded with DNA in vitro was measured. The Bowes cells exhibited a high expression level of GFP and the incubation of cells with plasmid loaded BG led up to 82% transfection efficiency.

Cross-reactive CD4+ T cells against one immunodominant tumor-derived epitope in melanoma patients.

TCRs exhibit a high degree of specificity but may also recognize multiple and distinct peptide-MHC complexes, illustrating the so-called cross-reactivity of TCR-peptide-MHC recognition. In this study, we report the first evidence of CD4(+) T cells recognizing the same tumor peptide-epitope from NY-ESO-1, in the context of multiple HLA-DR and HLA-DP molecules. These cross-reactive CD4(+) T cells recognized not only autologous but also allogenic dendritic cells previously loaded with the relevant protein (i.e., the normally processed and presented epitope). Using clonotypic real-time RT-PCR, we have detected low frequencies of CD4(+) T cells expressing one cross-reactive TCR from circulating CD4(+) T cells of patients with stage IV melanoma either spontaneously or after immunization but not in normal donors. The maintenance of cross-reactive tumor Ag-specific CD4(+) T cells in PBLs of cancer patients required the presence of tumor Ag/epitope in the context of the MHC molecule used to prime the Ag-specific CD4(+) T cells. Our findings have significant implications for the optimization of TCR gene transfer immunotherapies widely applicable to cancer patients.

Spontaneous CD4+ T cell responses against TRAG-3 in patients with melanoma and breast cancers.

The taxol resistance gene TRAG-3 was initially isolated from cancer cell lines that became resistant to taxol in vitro. TRAG-3 is a cancer germline Ag expressed by tumors of different histological types including the majority of melanoma, breast, and lung cancers. In the present study, we report that patients with stage IV melanoma and breast cancers developed spontaneous IFN-gamma-producing CD4+ T cell responses against a single immunodominant and promiscuous peptide epitope from TRAG-3 presented in the context of multiple HLA-DR molecules. The TRAG-3-specific CD4+ T cells and clones were expanded in vitro and recognized not only peptide pulsed APCs but also autologous dendritic cells (DCs) loaded with the TRAG-3 protein. All stage IV melanoma patients with TRAG-3-expressing tumors developed spontaneous CD4+ T cell responses against TRAG-3, demonstrating its strong immunogenicity. None of these patients had detectable IgG Ab responses against TRAG-3. TCRbeta gene usage studies of TRAG-3-specific CD4+ T cell clones from a melanoma patient and a normal donor suggested a restricted TCR repertoire in patients with TRAG-3-expressing tumors. Altogether, our data define a novel profile of spontaneous immune responses to cancer germline Ag-expressing tumors, showing that spontaneous TRAG-3-specific CD4+ T cells are directed against a single immunodominant epitope and exist independently of Ab responses. Because of its immunodominance, peptide TRAG-3(34-48) is of particular interest for the monitoring of spontaneous immune responses in patients with TRAG-3-expressing tumors and for the development of cancer vaccines.

Bacterial ghosts as a novel advanced targeting system for drug and DNA delivery.

Although there are powerful drugs against infectious diseases and cancer on the market, delivery systems are needed to decrease serious toxic and noncurative side effects. In order to enhance compliance, several delivery systems such as polymeric micro- and nanoparticles, liposomal systems and erythrocyte ghosts have been developed. Bacterial ghosts representing novel advanced delivery and targeting vehicles suitable for the delivery of hydrophobic or water-soluble drugs, are the main focus of this review. They are useful nonliving carriers, as they can carry different active substances in more than one cellular location separately and simultaneously. Bacterial ghosts combine excellent natural or engineered adhesion properties with versatile carrier functions for drugs, proteins and DNA plasmids or DNA minicircles. The simplicity of both bacterial ghost production and packaging of drugs and/or DNA makes them particularly suitable for the use as a delivery system. Further advantages of bacterial ghost delivery vehicles include high bioavailability and a long shelf life without the need of cold-chain storage due to the possibility to freeze-dry the material.