Biomedical subjects

Helga Bernhard

Publications and source records attributed to Helga Bernhard.

9 recordsLinked to original sources

PubMed2007

Ratio of trastuzumab levels in serum and cerebrospinal fluid is altered in HER2-positive breast cancer patients with brain metastases and impairment of blood-brain barrier.

Patients receiving trastuzumab for HER2-overexpressing metastatic breast cancer seem to suffer from an increased risk of brain metastases, even in cases with responsive disease. To evaluate whether trastuzumab is able to penetrate the blood-brain barrier, we measured trastuzumab levels in the serum and in cerebrospinal fluid of metastatic breast cancer patients with brain metastases receiving trastuzumab for HER2-overexpressing metastatic breast cancer. In a pilot study, metastatic breast cancer patients with brain metastases and HER2-overexpressing tumors (HercepTest; Dako, Copenhagen, Denmark) were included. At different time points, trastuzumab levels in the serum and cerebrospinal fluid were measured using a newly developed immunoenzymatic test for trastuzumab. Six out of eight patients were evaluable for determination of trastuzumab level in the serum and cerebrospinal fluid. Before radiotherapy, median trastuzumab level in the serum was 52 054 ng/ml compared with 124 ng/ml in cerebrospinal fluid (ratio 420 : 1). After completion of radiotherapy, median trastuzumab level was 20 185 ng/ml in the serum and 226 ng/ml in cerebrospinal fluid, respectively (ratio 76 : 1). With concomitant meningeal carcinomatosis, trastuzumab level in the serum after radiotherapy was 17 431 and 356 ng/ml in cerebrospinal fluid (ratio 49 : 1). For the first time, we present clinical evidence that trastuzumab levels in cerebrospinal fluid are increased under conditions of an impaired blood-brain barrier such as meningeal carcinomatosis or radiotherapy. This evidence supports the concept of continuing trastuzumab therapy in patients with brain metastases treated by radiotherapy. Monitoring of trastuzumab levels in the serum and cerebrospinal fluid may enable individualized therapy strategies in metastatic breast cancer patients with brain metastases, and lead to a better understanding of trastuzumab pharmacokinetics in the cerebrospinal fluid and serum.

Adult↗

PubMed2006

Infection of human dendritic cells with recombinant vaccinia virus MVA reveals general persistence of viral early transcription but distinct maturation-dependent cytopathogenicity.

Vector-infected dendritic cells (DC) are evaluated for antigen delivery in experimental therapy of cancer and infectious diseases. Here, we investigated infections of immature or mature, monocyte-derived human DC with recombinant vaccinia virus MVA producing human Her-2/neu, a candidate tumor-associated antigen. Assessment of the molecular virus life cycle in infected DC revealed a general arrest at the level of viral early gene expression. When monitoring the phenotype of MVA-infected DC, including expression of cell surface markers, we found immature cells readily undergoing apoptosis. Nevertheless, we detected significant populations of viable DC being characterized by high level Her-2/neu expression and unimpaired display of costimulatory molecules. While infected viable immature DC failed to undergo maturation despite cytokine treatment, both DC populations efficiently presented MVA-produced target antigen. These findings allow to better define the requirements for MVA-mediated antigen delivery to DC and help to derive optimized vectors for this advanced therapy option.

Apoptosis↗

PubMed2006

Designer T cells by T cell receptor replacement.

T cell receptor (TCR) gene transfer is a convenient method to produce antigen-specific T cells for adoptive therapy. However, the expression of two TCR in T cells could impair their function or cause unwanted effects by mixed TCR heterodimers. With five different TCR and four different T cells, either mouse or human, we show that some TCR are strong--in terms of cell surface expression--and replace weak TCR on the cell surface, resulting in exchange of antigen specificity. Two strong TCR are co-expressed. A mouse TCR replaces human TCR on human T cells. Even though it is still poorly understood why some TCRalpha/beta combinations are preferentially expressed on T cells, our data suggest that, in the future, designer T cells with exclusive tumor reactivity can be generated by T cell engineering.

Animals↗

PubMed2005

Isolation and expansion of tumor-reactive cytotoxic T-cell clones for adoptive immunotherapy.

Attempts to treat patients with tumor-reactive cytotoxic T-lymphocytes (CTL) have been limited. This is due to the difficulty of isolating and expanding functionally active T-cells, which are present at extremely low frequencies in the peripheral blood. Recently developed multimers of the HLA-peptide complex mimic the natural ligand of the T-cell receptor and, therefore, fluorochrome-labeled multimers allow visualization and isolation of rare T-cells with defined specificity. Multimer-guided T-cell sorting permits the in vitro culture of antigen-specific T-cells as lines or clones. Cytolytic T-cells capable of recognizing HLA-peptide complexes endogenously processed by tumor cells are selected for further expansion, since lysis of tumor cells in vitro is a prerequisite for effective tumor elimination in vivo. The expansion of tumor-reactive CD8+ T-cells yields cell numbers sufficient for adoptive transfer. Tumor-reactive T-cells retain the functional activity in terms of cytolysis after expansion, encouraging their use in the immunotherapy of cancer patients.

Antigens↗

PubMed2004

A phase I vaccination study with tyrosinase in patients with stage II melanoma using recombinant modified vaccinia virus Ankara (MVA-hTyr).

A significant percentage of patients with stage II melanomas suffer a relapse after surgery and therefore need the development of adjuvant therapies. In the study reported here, safety and immunological response were analyzed after vaccination in an adjuvant setting with recombinant modified vaccinia virus Ankara carrying the cDNA for human tyrosinase (MVA-hTyr). A total of 20 patients were included and vaccinated three times at 4-week intervals with 5x10(8) IU of MVA-hTyr each time. The responses to the viral vector, to known HLA class I-restricted tyrosinase peptides, and to dendritic cells transfected with tyrosinase mRNA, were investigated by ELISpot assay on both ex vivo T cells and on T cells stimulated in vitro prior to testing. The delivery of MVA-hTyr was safe and did not cause any side effects above grade 2. A strong response to the viral vector was achieved, indicated by an increase in the frequency of MVA-specific CD4+ and CD8+ T cells and an increase in virus-specific antibody titers. However, no tyrosinase-specific T-cell or antibody response was observed with MVA-hTyr in any of the vaccinated patients. Although MVA-hTyr provides a safe and effective antigen-delivery system, it does not elicit a measurable immune response to its transgene product in patients with stage II melanoma after repeated combined intradermal and subcutaneous vaccination. We presume that modification of the antigen and/or prime-boost vaccination applying different approaches to antigen delivery may be required to induce an effective tyrosinase-specific immune response.

Adult↗

PubMed2004

Melanoma-reactive class I-restricted cytotoxic T cell clones are stimulated by dendritic cells loaded with synthetic peptides, but fail to respond to dendritic cells pulsed with melanoma-derived heat shock proteins in vitro.

Immunization with heat shock proteins (hsp) isolated from cancer cells has been shown to induce a protective antitumor response. The mechanism of hsp-dependent cellular immunity has been attributed to a variety of immunological activities mediated by hsp. Hsp have been shown to bind antigenic peptides, trim the bound peptides by intrinsic enzymatic activity, improve endocytosis of the chaperoned peptides by APCs, and enhance the ability of APCs to stimulate peptide-specific T cells. We have investigated the potential capacity of hsp70 and gp96 to function as a mediator for Ag-specific CTL stimulation in an in vitro model for human melanoma. Repetitive stimulation of PBLs by autologous DCs loaded with melanoma-derived hsp did not increase the frequency of T cells directed against immunodominant peptides of melanoma-associated Ags Melan-A and tyrosinase. In contrast, repeated T cell stimulation with peptide-pulsed DCs enhanced the number of peptide-specific T cells, allowing HLA/peptide multimer-guided T cell cloning. We succeeded in demonstrating that the established HLA-A2-restricted CTL clones recognized HLA-A2(+) APCs exogenously loaded with the respective melanoma peptide as well as melanoma cells processing and presenting these peptides in the context of HLA-A2. We were not able to show that these melanoma-reactive CTL clones were stimulated by autologous dendritic cells pulsed with melanoma-derived hsp. These results are discussed with respect to various models for proving the role of hsp in T cell stimulation and to recent findings that part of the immunological antitumor activities reported for hsp are independent of the chaperoned peptides.

Antigen Presentation↗

PubMed2002

Identification of vaccinia virus epitope-specific HLA-A*0201-restricted T cells and comparative analysis of smallpox vaccines.

Despite worldwide eradication of naturally occurring variola virus, smallpox remains a potential threat to both civilian and military populations. New, safe smallpox vaccines are being developed, and there is an urgent need for methods to evaluate vaccine efficacy after immunization. Here we report the identification of an immunodominant HLA-A*0201-restricted epitope that is recognized by cytotoxic CD8(+) T cells and conserved among Orthopoxvirus species including variola virus. This finding has permitted analysis and monitoring of epitope-specific T cell responses after immunization and demonstration of the identified T cell specificity in an A*0201-positive human donor. Vaccination of transgenic mice allowed us to compare the immunogenicity of several vaccinia viruses including highly attenuated, replication-deficient modified vaccinia virus Ankara (MVA). MVA vaccines elicited levels of CD8(+) T cell responses that were comparable to those induced by the replication-competent vaccinia virus strains. Finally, we demonstrate that MVA vaccination is fully protective against a lethal respiratory challenge with virulent vaccinia virus strain Western Reserve. Our data provide a basis to rationally estimate immunogenicity of safe, second-generation poxvirus vaccines and suggest that MVA may be a suitable candidate.

Amino Acid Sequence↗

PubMed2002

Antihuman epidermal growth factor receptor 2 (HER2) monoclonal antibody trastuzumab enhances cytolytic activity of class I-restricted HER2-specific T lymphocytes against HER2-overexpressing tumor cells.

The monoclonal antibody trastuzumab (Herceptin) directed against the human epidermal growth factor receptor 2 (HER2) results in tumor regressions when administered to patients with HER2-overexpressing breast cancer. One of the underlying mechanisms of this antibody-induced tumor regression is based on the internalization and degradation of HER2 by tumor cells on interaction with trastuzumab, subsequently inhibiting signal transduction pathways. As antibody-induced degradation of HER2 is likely to be accompanied with increased numbers of HER2 peptides presented with MHC, we asked whether trastuzumab-treated tumor cells were more susceptible to CTL-mediated lysis. Here we show that the cytolytic activity of human, HER2-specific CD8(+) CTLs is augmented by anti-HER2 antibody trastuzumab. HER2-reactive CTL clones lyse class I-matched, HER2-overexpressing tumor cells more efficiently after treatment with trastuzumab. The potentially synergistic activity of HER2-specific antibody and CTL encourages the development of an HER2-targeted immunotherapy using a combination of inhibitory antibodies and CTLs for patients with HER2-overexpressing tumors.

Antibodies, Monoclonal↗

PubMed2002

Reversible MHC multimer staining for functional isolation of T-cell populations and effective adoptive transfer.

Recently developed major histocompatibility complex (MHC) multimer technologies allow visualization and isolation of antigen-specific T cells. However, functional analysis and in vivo transfer of MHC multimer-stained cells is hampered by the persistence of T-cell receptor (TCR) MHC interactions and subsequently induced signaling events. As MHC monomers do not stably bind to TCRs, we postulated that targeted disassembly of multimers into MHC monomers would result in dissociation of surface-bound TCR ligands. We generated a new type of MHC multimers, which can be monomerized in the presence of a competitor, resulting in rapid loss of the staining reagent. Following dissociation, the T cells are phenotypically and functionally indistinguishable from untreated cells. This 'reversible' T-cell staining procedure, which maintains the specificity and sensitivity of MHC multimer staining while preserving the functional status of T lymphocytes, may be of broad benefit for ex vivo investigation of T-cell functions and clinical applications.

Adoptive Transfer↗