Stefan Wirths

Vaccination of patients with cancer using dendritic cells (DCs) was shown to be effective for B-cell lymphoma and malignant melanoma. Here we provide evidence that patients with advanced breast and ovarian cancer can be efficiently vaccinated with autologous DCs pulsed with HER-2/neu- or MUC1-derived peptides. Ten patients were included in this pilot study. The DC vaccinations were well tolerated with no side effects. In 5 of 10 patients, peptide-specific cytotoxic T lymphocytes (CTLs) could be detected in the peripheral blood using both intracellular IFN-gamma staining and (51)Cr-release assays. The major CTL response in vivo was induced with the HER-2/neu-derived E75 and the MUC1-derived M1.2 peptide, which lasted for more than 6 months, suggesting that these peptides might be immunodominant. In addition, in one patient vaccinated with the MUC1-derived peptides, CEA- and MAGE-3 peptide-specific T-cell responses were detected after several vaccinations. In a second patient immunized with the HER-2/neu peptides, MUC1-specific T lymphocytes were induced after 7 immunizations, suggesting that antigen spreading in vivo might occur after successful immunization with a single tumor antigen. Our results show that vaccination of DCs pulsed with a single tumor antigen may induce immunologic responses in patients with breast and ovarian cancer. This study may be relevant to the design of future clinical trials of other peptide-based vaccines.

A phase I trial was conducted to evaluate the feasibility, safety, and efficacy of a dendritic cell-based vaccination in patients with metastatic renal cell carcinoma (RCC). Autologous mature dendritic cells derived from peripheral blood monocytes were pulsed with the HLA-A2-binding MUC1 peptides (M1.1 and M1.2). For the activation of CD4(+) T-helper lymphocytes, dendritic cells were further incubated with the PAN-DR-binding peptide PADRE. Dendritic cell vaccinations were done s.c. every 2 weeks for four times and repeated monthly until tumor progression. After five dendritic cell injections, patients additionally received three injections weekly of low-dose interleukin-2 (1 million IE/m(2)). The induction of vaccine-induced T-cell responses was monitored using enzyme-linked immunospot and Cr release assays. Twenty patients were included. The treatment was well tolerated with no severe side effects. In six patients, regression of the metastatic sites was induced after vaccinations with three patients achieving an objective response (one complete response, two partial responses, two mixed responses, and one stable disease). Additional four patients were stable during the treatment for up to 14 months. MUC1 peptide-specific T-cell responses in vivo were detected in the peripheral blood mononuclear cells of the six patients with objective responses. Interestingly, in patients responding to the treatment, T-cell responses to antigens not used for vaccinations, such as adipophilin, telomerase, or oncofetal antigen, could be detected, indicating that epitope spreading might occur. This study shows that MUC1 peptide-pulsed dendritic cells can induce clinical and immunologic responses in patients with metastatic RCC.

The epithelial mucin MUC1 is overexpressed on the cell surface of many epithelial malignancies as well as on some B-cell lymphomas and multiple myelomas. Recently, we identified two HLA-A2-restricted T-cell epitopes derived from the MUC1 protein. To further extend the potential application of these peptides, we analyzed the expression of MUC1 on blast cells from patients with acute myelogenous leukemia (AML; n = 43) and several other hematological malignancies including acute lymphoblastic leukemia (n = 24), chronic lymphocytic leukemia (n = 36), hairy cell leukemia (n = 9), follicular lymphoma (n = 7), and multiple myeloma (n = 12). Using reverse transcription-PCR and MUC1-specific monoclonal antibodies, MUC1 expression was found in 67% of AML samples and 92% of myeloma samples. To analyze the presentation of MUC1 peptides by primary AML blasts, we induced MUC1-specific CTLs in vitro using peptide-pulsed dendritic cells from HLA-A2+ healthy donors as antigen-presenting cells. These CTLs efficiently lysed in an antigen-specific and HLA-A2-restricted manner not only target cells pulsed with the antigenic peptide but also tumor cell lines including multiple myeloma cells and primary AML blasts that constitutively expressed both MUC1 and HLA-A2. The specificity of the CTLs was confirmed in a cold target inhibition assay. Our data demonstrate that MUC1-derived peptides are tumor antigens in AML and several other hematological malignancies that could potentially be used for immunotherapeutic approaches.

The exact identification of B cell subsets is instrumental to understand their dynamics under physiological and pathological conditions. Human memory B cells are currently identified according to the expression of CD27, which is absent on naive B cells. We report here that the ATP-binding cassette (ABC)B1 transporter is exclusively present on mature CD27- naive B cells, while it is absent in CD27+ memory B cells and in a heterogeneous subset of CD27- cells that comprise both switch memory and transitional B cells. Thus, ABCB1 activity precisely discriminates naive from transitional and all memory B cells. Using this improved method to discriminate human B cell subsets, and Ki67 staining to identify recently divided cells, we show that in both cord blood and adult peripheral blood, mature naive B cells are quiescent while transitional B cells and memory B cells have a high in vivo turnover.