Björn Schöttker

Therapeutic vaccination against metastatic renal cell carcinoma by autologous dendritic cells: preclinical results and outcome of a first clinical phase I/II trial

Abstract. In this study we have presented in vitro data and results of a preliminary clinical trial using dendritic cells (DC) in patients with progressive metastatic renal cell carcinoma. DC precursor cells were obtained from peripheral blood mononuclear cells (PBMC). DC were pulsed with autologous tumor cell lysate if available. In total, 15 patients were treated with a median of 3.95×106 DC administered and ultrasound-guided into a lymph node or into adjacent tissue. Seven patients remained with progressive disease (PD), 7 patients showed stable disease (SD), and one patient displayed a partial response (PR). Most interestingly, the patient who was treated with the highest number of DC (14.4×106 DC/vaccine) displayed a PR. Delayed-type hypersensitivity (DTH) reaction using autologous tumor lysate was positive in 3 out of 13 patients, including the patient with PR. Two out of 3 patients receiving additional treatment with keyhole limpet hemocyanin (KLH) showed reactivity to KLH after vaccination. CD3+CD4+ and CD3+CD28+ cells as well as the proliferation rate of peripheral blood lymphocytes (PBL) increased significantly in the blood of patients during therapy. In conclusion, our observations confirm the capability of tumor-lysate pulsed autologous DC vaccines to stimulate an immune response in patients with metastatic renal cell carcinoma even in the presence of a large tumor burden. The lack of adverse effects together with immunologic effects support further investigation of this novel therapeutic approach. Further studies are necessary to demonstrate clinical effectiveness in cancer patients, in particular in patients with less advanced disease.

Interactions Between Dendritic Cells and Cytokine-induced Killer Cells Lead to an Activation of Both Populations

Dendritic cells (DCs) are major antigen-presenting cells. They are capable of capturing and processing tumor antigens, expressing lymphocyte costimulatory molecules, and secreting cytokines to initiate immune responses. Here, the authors tested the effect of cytokine-induced killer (CIK) cells, a population that includes CD3 + CD56 + cells (natural killer T cells), with regard to their capacity to immunomodulate DCs. Cytokine-induced killer cells were cocultured with autologous DCs generated from peripheral blood mononuclear cells. Expression of markers typical for both populations was measured using flow cytometry, and secretion of interleukin (IL)-12 was determined using enzyme-linked immunosorbent assays. Cytotoxicity assays were performed to investigate the role of IL-12 and the importance of cell–cell interactions. Considering this, receptors for IL-12 and CD40 were blocked and cocultures were performed with cell culture inserts. Coculture of CIK cells led to a significant increase of DC-specific, costimulatory, and antigen-presenting molecules in DC cultures. In addition, coculture resulted in a dramatically increase of IL-12 secretion by DCs and to a significant increase in cytotoxic activity of CIK cells toward carcinoma cells. Blockage of IL-12 uptake decreased the cytolytic activity of CIK cells. Cytokine secretion was shown to be important for activation of CIK cells, and also cellular interactions between DCs and effector cells caused a higher cytolytic capacity. Interactions between DCs and CIK cells caused changes in the surface molecule expression of both populations, led to an increase of IL-12 secretion, and rendered an improved cytotoxic activity. The natural killer T cell subpopulation seems to be responsible for this effect. Therefore, coculture of DCs with CIK cells may have a major impact on immunotherapeutic protocols for patients with cancer.

Novel non-viral method for transfection of primary leukemia cells and cell lines.

BackgroundTumor cells such as leukemia and lymphoma cells are possible targets for gene therapy. However, previously leukemia and lymphoma cells have been demonstrated to be resistant to most of non-viral gene transfer methods.MethodsThe aim of this study was to analyze various methods for transfection of primary leukemia cells and leukemia cell lines and to improve the efficiency of gene delivery. Here, we evaluated a novel electroporation based technique called nucleofection. This novel technique uses a combination of special electrical parameters and specific solutions to deliver the DNA directly to the cell nucleus under mild conditions.ResultsUsing this technique for gene transfer up to 75% of primary cells derived from three acute myeloid leukemia (AML) patients and K562 cells were transfected with the green flourescent protein (GFP) reporter gene with low cytotoxicity. In addition, 49(+/- 9.7%) of HL60 leukemia cells showed expression of GFP.ConclusionThe non-viral transfection method described here may have an impact on the use of primary leukemia cells and leukemia cell lines in cancer gene therapy.

Generation of activated and antigen-specific T cells with cytotoxic activity after co-culture with dendritic cells

Abstract. Co-culturing of immunological effector cells with antigen-pulsed DC leads to an increase of cytotoxic activity against antigen-expressing tumour cells. Using this approach, we could detect up to 2.8% antigen-specific CTLs after co-culture with antigen-pulsed DC. However, the required high effector cell numbers remain a major obstacle in immunotherapy. In this study, we show an approach for generating activated and antigen-specific effector cells that enables us to decrease effector to target cell ratios. We used an interferon-γ secretion assay to enrich activated effector cells after co-culture with antigen-pulsed dendritic cells (DC). Purified immunological effector cells lysed 58.3% of antigen-expressing tumour cells at an effector to target ratio of 1:1. Furthermore, using MHC-IgG complexes, we enriched effector cells expressing antigen-specific T-cell receptor after co-culture with DC. Performing ELISpot, flow cytometry and TCR analysis, we could show a significant increase of activated and specific TCR-expressing effector cells after co-culture with DC.

Increase of the immunostimulatory effect of dendritic cells by pulsing with CA 19-9 protein.

Previously, a relative resistance of solid tumor cells to immunologic effector cells was shown in vitro. This resistance could be one reason for the clinical phenomenon of resistance of patients with colon carcinoma or other solid tumors to immunologic therapeutic approaches. In this study, dendritic cells (DCs) pulsed with CA 19-9 protein were tested for their immunostimulatory capacity of immunologic effector cells against cells derived from colon and pancreatic carcinoma. Dendritic cell cultures coexpressed CMRF-44 and CD1a, markers typical of DCs, in 31.5% ± 5.3% after 13 days of culture. Coculture of NK-like T lymphocytes with DCs led to a significant increase in cytotoxic activity, as measured using a lactate dehydrogenase release assay. Cytotoxic activity could be further increased using DCs pulsed with CA 19-9 protein. The effect of CA 19-9 on increasing the cytotoxic effect of NK-like T lymphocytes was dose dependent. Similarly, cocultivation of DCs with NK-like T cells derived from patients with metastatic pancreatic cancer and elevated CA 19-9 serum levels led to a significant increase in cytotoxic activity. In conclusion, DCs pulsed with CA 19-9 protein can increase the cytotoxic activity of immunologic effector cells against colon carcinoma and pancreatic cancer cells. Dendritic cells pulsed with CA 19-9 protein may have an important effect on immunotherapeutic protocols for patients with cancer.

Effects of adenoviral wild-type p53 gene transfer in p53-mutated lymphoma cells.

The present study assessed the role of adenoviral vector–mediated wild-type p53 gene transfer in B lymphoma cells. Deficiency of p53-mediated cell death is common in human cancer contributing to both tumorigenesis and chemoresistance. Lymphoma cells are being considered as suitable targets for gene therapy protocols. Recently, we reported an adenoviral protocol leading to highly efficient gene transfer to B lymphoma cells. All lymphoma cell lines ( n =5) tested here showed mutations in the p53 gene locus. The aim of this work was to transduce lymphoma cells with the wild-type p53 gene. Using this protocol, 88% of Raji, 75% of Daudi, and 45% of OCI-Ly8-LAM53 cells were transfected with the reporter gene green fluorescent protein at a multiplicity of infection of 200. The expression of green fluorescent protein in CA46 and BL41 cells was 27% and 42%, respectively. At this multiplicity of infection, growth characteristics of lymphoma cell lines were not changed significantly. In contrast, cells transduced with wild-type p53 gene showed an inhibition of proliferation as well as an increase in apoptosis. Cell loss by apoptosis after p53 gene transfer was up to 40% as compared to transduction with an irrelevant vector. In addition, we determined the effects of DNA damage produced by the DNA topoisomerase II inhibitor etoposide on wild-type p53 transfected lymphoma cells. In Ad-p53–transfected Raji cells, treatment with the drug resulted in a marked increase of cell loss in comparison to Ad-β-Gal–transfected cells (45% vs. 77%). Interestingly, performing cytotoxicity studies, we could show an increased sensitivity of Raji and Daudi cells against immunological effector cells. In conclusion, transduction of wild-type p53 into lymphoma cells expressing mutated p53 was efficient and led to inhibition of proliferation and increase in apoptotic rate in some cell lines dependent on p53 mutation. This protocol should have an impact on the use of lymphoma cells in cancer gene therapy protocols. Cancer Gene Therapy (2001) 8, 430–439

Low Frequency of Enteric Infections by Salmonella, Shigella, Yersinia and Campylobacter in Patients with Acute Leukemia

AbstractBackground: Several authors found that isolation of Salmonella, Shigella, Yersinia and Campylobacter ssp. (SSYC) from stool cultures after the 3rd day of hospitalization is a rare event. The significance of enteric infections caused by these pathogens has not been systematically investigated in severely immunosuppressed patients with acute leukemia. Patients and Methods: We screened all patients treated on the leukemia ward of a university medical center. A total of 1,185 stool cultures from 371 episodes of diarrhea, mostly following myelosuppressive chemotherapy, were examined for the complete range of classic bacterial enteric pathogens (i. e. SSYC). Results: Only three (0.25%) cultures from one patient were positive for Salmonella enteritidis. This patient suffered from cholangitis. S. enteritidis could also be detected by liver biopsy. Other infections by classic enteric pathogens were not observed. Conclusion: Symptomatic infections by classical bacterial enteric pathogens in hospitalized patients with acute leukemia are very rare. Stool cultures for these pathogens cannot be recommended as a routine test in uncomplicated diarrhea occurring after the 3rd hospital day.

Increased Sensitivity of Myeloid Leukemia Cell Lines: Potential of Lovastatin as Bone-Marrow-Purging Agent

Lovastatin reduces the isoprenylation of p21ras via suppression of mevalonic acid generation. Lovastatin has been shown to reduce tumor cell proliferation in a dose-dependent manner. Here, the potential of lovastatin for purging leukemia cells from bone marrow was investigated using the myeloblastic cell lines K562 and KG-1 as a model system, derived from an erythroleukemia and an acute myelogenous leukemia, respectively. Optimal purging conditions were determined using an MTT proliferation and a leukemia colony assay. Elimination of leukemia cells was time- and dose-dependent. Depletion of K562 was 2.5 logs for 100 μM of lovastatin at 72 h of incubation. Compared to another purging agent, 100 μg/ml mafosfamide had an activity comparable to 100 mM lovastatin. Interestingly, KG-1 acute myelogenous leukemia cells were even more sensitive to lovastatin than K562 cells. In clonogenic assays, 100 μM of lovastatin resulted in a 3- to 4-log reduction of K562 colonies. Lovastatin had a progressive effect on normal hematopoietic progenitor cells. At a concentration of 100 μM of lovastatin, CFU-GM colonies were reduced by 1–2 logs. In conclusion, a differential effect on leukemia and normal progenitor cells could be detected in a clonogenic assay. These results suggest that lovastatin deserves further study as an agent for ex vivo marrow purging.