Angela Märten

Allogeneic dendritic cells fused with tumor cells: preclinical results and outcome of a clinical phase I/II trial in patients with metastatic renal cell carcinoma.

Therapeutic vaccination with dendritic cells (DC) can lead to tumor regression in animal models and has shown promising results in the first clinical trials of metastatic renal cell carcinoma and malignant melanoma. In vitro data and results of a clinical phase I/II trial using DC tumor fusions in patients with progressive metastatic renal cell carcinoma are presented here. In addition to toxicity and feasibility, complex immune monitoring was a point of interest. DC precursor cells were obtained from the peripheral blood mononuclear cells (PBMCs) of healthy donors and were fused with either allogeneic (8 patients) or autologous (4 patients) renal tumor cells. In total, 12 patients with progressive metastatic renal cell carcinoma were treated with an average of 2.8 x 10(7) tumor cells fused with 1.8 x 10(7) DC each administered on days 0, 28, and 56 intradermally. Fusion efficacy for the tumor cells used was 14.3% +/- 7.8%. Cell viability was 59.8% +/- 6.8% after fusion and irradiation. We observed no adverse effects and no difference in clinical outcome between the allogeneic and the autologous treatment. Eight patients remained in a progressive disease state and four patients in a stable disease state. T-cell immunity was carefully monitored before, during, and after treatment. Delayed-type hypersensitivity (DTH) reaction using tumor cells was positive after treatment in 7 of 12 patients, 2 of whom were found to have stable disease. An increase in the reactivity against recall antigens was seen in most patients. Interestingly, cytotoxicity of peripheral blood lymphocytes (PBLs) against renal cell carcinoma cells increased during treatment as well as the percentage of interferon-gamma-secreting cells. This effect was significantly enhanced within the group that had stable disease. The lack of adverse effects together with positive immunologic signs justifies further investigation of this novel therapeutic approach. Further studies are necessary to test for clinical effectiveness in patients with tumors, especially those with less advanced disease.

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.

Soluble MIC is elevated in the serum of patients with pancreatic carcinoma diminishing γδ T cell cytotoxicity

Intestinal cells express MHC related molecules termed MICA/MICB, which are up‐regulated under stress and in many gastrointestinal tumors. These molecules can be recognized by the immunoreceptor NKG2D, which is present on NK and γδ T cells. Release of MIC molecules from the cell surface is thought to constitute an immune escape mechanism of tumor cells. The immediate effect of soluble MIC (sMIC) on cellular cytotoxicity of γδ T cells is yet not investigated. We determined sMIC levels in sera of patients with pancreatic carcinoma and the expression of MIC on the surface of tumor cells by FACS. The effect of sMIC content in patient serum on cellular cytotoxicity of γδ T and NK cells was investigated by cytotoxicity assays. Subsequently, the effect of IFN‐α treatment on MIC expression, release and cellular cytotoxicity was investigated. Pancreatic carcinoma cells express MIC, and patient sera contain elevated sMIC levels that correlate with tumor stage and differentiation. Furthermore, cellular cytotoxicity of γδ T cells and NK cells against pancreatic carcinoma is impaired by sMIC in patient sera which is prevented by sMIC neutralization. Incubation of pancreatic cancer cells with IFN‐α increases MIC expression without induction of sMIC resulting in enhanced lysis of tumor cells. Our results demonstrate that sMIC impairs NKG2D‐mediated immunity against pancreatic carcinoma by directly diminishing cytotoxicity of γδ T cells and NK cells. IFN‐α, which is used in adjuvant treatment of pancreatic carcinoma, might partly act via up‐regulation of MIC without induction of sMIC release.

Therapeutic Vaccination against Metastatic Carcinoma by Expression-Modulated and Immunomodified Autologous Tumor Cells: A First Clinical Phase I/II Trial

Therapeutic vaccination of tumor patients with cytokine gene-transfected tumor cells leads to tumor regression in animal models but has so far not resulted in significant clinical benefit. We and others demonstrated that tumor cells transfected to mediate overexpression of a cytokine gene activate immunologic effector cells for an improved proliferation rate and significantly higher antitumoral cytotoxic activity. Here, we performed a pilot study of therapeutic vaccination in patients with metastatic disease. Autologous tumor cells were simultaneously transfected with novel minimalistic, immunogenically defined, gene expression constructs (MIDGE) for overexpression of the two cytokines interleukin 7 (IL-7) and GM-CSF and newly designed double stem-loop immunomodulating oligodeoxyribonucleotides (d-SLIM) as a Th1-promoting and NK cell-stimulating adjuvant. Transfection was performed ex vivo by ballistomagnetic gene transfer. Patients received four subcutaneous injections of at least 1 x 10(6) of their expression-modulated and immunomodified autologous tumor cells. Ten patients have been enrolled in the study protocol. In all patients no adverse effects could be detected. IL-7 and interferon gamma levels were elevated in the serum of the patients after treatment. Interestingly, cytotoxicity of patient-derived PBLs increased significantly during treatment. All 10 patients had progressive disease when entering our protocol. One complete, one partial, and one mixed response with progression of abdominal metastases and regression of lung metastases were observed. Two patients showed a stable disease after treatment and five patients remained in progressive disease. Our observations confirm the capability of autologous expression-modified and immunomodulated tumor cell vaccines to stimulate a strong immune response in patients with metastatic cancer even in the presence of a large tumor burden.

Gemcitabine plus erlotinib followed by capecitabine versus capecitabine plus erlotinib followed by gemcitabine in advanced pancreatic cancer: final results of a randomised phase 3 trial of the ‘Arbeitsgemeinschaft Internistische Onkologie’ (AIO-PK0104)

Objective AIO-PK0104 investigated two treatment strategies in advanced pancreatic cancer (PC): a reference sequence of gemcitabine/erlotinib followed by 2nd-line capecitabine was compared with a reverse experimental sequence of capecitabine/erlotinib followed by gemcitabine. Methods 281 patients with PC were randomly assigned to 1st-line treatment with either gemcitabine plus erlotinib or capecitabine plus erlotinib. In case of treatment failure (eg, disease progression or toxicity), patients were allocated to 2nd-line treatment with the comparator cytostatic drug without erlotinib. The primary study endpoint was time to treatment failure (TTF) after 1st- and 2nd-line therapy (TTF2; non-inferiority design). KRAS exon 2 mutations were analysed in archival tumour tissue from 173 of the randomised patients. Results Of the 274 eligible patients, 43 had locally advanced and 231 had metastatic disease; 140 (51%) received 2nd-line chemotherapy. Median TTF2 was estimated with 4.2 months in both arms; median overall survival was 6.2 months with gemcitabine/erlotinib followed by capecitabine and 6.9 months with capecitabine/erlotinib followed by gemcitabine, respectively (HR 1.02, p=0.90). TTF for 1st-line therapy (TTF1) was significantly prolonged with gemcitabine/erlotinib compared to capecitabine/erlotinib (3.2 vs 2.2 months; HR 0.69, p=0.0034). Skin rash was associated with both TTF2 (rash grade 0/1/2–4:2.9/4.3/6.7 months, p<0.0001) and survival (3.4/7.0/9.6 months, p<0.0001). Each arm showed a safe and manageable toxicity profile during 1st- and 2nd-line therapy. A KRAS wild-type status (52/173 patients, 30%) was associated with an improved overall survival (HR 1.68, p=0.005). Conclusion Both treatment strategies are feasible and demonstrated comparable efficacy; KRAS may serve as biomarker in patients with advanced PC treated with erlotinib. Trial registration number This study was registered at ClinicalTrials.gov, number NCT00440167. Significance of this study What is already known on this subject? Gemcitabine-based chemotherapy remains an international standard of care for patients with non-resectable, advanced pancreatic cancer (PC). Anti-EGFR treatment with the tyrosine kinase inhibitor erlotinib, as well as chemotherapy intensification by application of the FOLFIRINOX regimen, both significantly improved overall survival in randomised phase 3 trials. The optimal (sequential) regimen for the use of gemcitabine, erlotinib and the oral fluoropyrimidine capecitabine remains unclear in advanced PC. Molecular predictors for the efficacy of anti-EGFR treatments in PC have not been defined up to now. What are the new findings? The sequential use of gemcitabine, erlotinib and capecitabine is safe and equally effective in PC; gemcitabine appears to be more effective in 1st- and 2nd-line therapy than capecitabine and therefore remains the preferred combination partner for erlotinib. Skin rash is strongly correlated with efficacy outcome measures in PC patients treated with erlotinib. KRAS wild-type status appears to be associated with improved overall survival in patients treated with erlotinib in this AIO study. Significance of this study How might it impact on clinical practice in the foreseeable future? The benefit of adding erlotinib to chemotherapy is restricted to patients that experience skin rash during treatment; non-rash patients are characterised by a very poor outcome and need to be offered novel treatment strategies. Second-line salvage chemotherapy is effective and safe in selected PC patients. KRAS could serve as the first biomarker for improved survival in erlotinib-treated patients; the predictive value of KRAS for erlotinib efficacy remains to be defined prospectively.

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.

PankoMab: a potent new generation anti-tumour MUC1 antibody

Recently, we described a new carbohydrate-induced conformational tumour-epitope on mucin-1 (MUC1) with the potential for improvement of immunotherapies [29, 30]. PankoMab is a novel antibody, which binds specifically to this epitope and was designed to show the highest glycosylation dependency and the strongest additive binding effect when compared to other MUC1 antibodies. This enables PankoMab to differentiate between tumour MUC1 and non-tumour MUC1 epitopes. It has a high-affinity towards tumour cells (e.g. KD [M] of 0.9 and 3×10−9 towards NM-D4 and ZR75-1, respectively) and detects a very large number of binding sites (e.g. 1.0 and 2.4×106 for NM-D4 and ZR75-1, respectively). PankoMab is rapidly internalised, and after toxin coupling is able to induce very effectively toxin-mediated antigen-specific tumour cell killing. PankoMab reveals a potent tumour-specific antibody-dependent cell cytotoxicity (ADCC). PankoMab is, therefore, distinguished by a combination of advantages compared to other MUC1 antibodies in clinical development, including higher tumour specificity, higher affinity, a higher number of binding sites, largely reduced binding to shed MUC1 from colon and pancreatic carcinoma patients, no binding to mononucleated cells from peripheral blood (except ~7% of activated T cells), stronger ADCC activity and rapid internalisation as required for toxin-mediated cell killing. This renders it a superior antibody for in vivo diagnostics and various immunotherapeutic approaches.

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.

Intratumoural injection of the toll-like receptor-2/6 agonist ‘macrophage-activating lipopeptide-2’ in patients with pancreatic carcinoma: a phase I/II trial

This phase I/II trial examined safety and efficacy of the toll-like receptor 2/6 agonist MALP-2 in combination with gemcitabine in patients with incompletely resectable pancreas carcinomas. MALP-2 is a toll-like receptor 2/6 agonist, acts as an immunological adjuvant, and has been described recently to prolong survival in a mouse model of an orthotopic, syngeneic pancreas tumour. Male and female patients with incompletely resectable pancreas carcinomas were eligible while those with R0 or R1 resections or with peritoneal carcinosis were excluded. Ten patients were injected intratumourally during surgery with 20–30 μg MALP-2 followed by postoperative chemotherapy. Samples were taken from peripheral blood and wound secretion, and assayed for cell content, cytokine and CRP levels, and NK activity. An MALP-2 dose of 20 μg was well tolerated. Clear signs of local MALP-2 effects were presented by the influx of lymphocytes and monocytes in wound secretions, and abolishment of inhibition of NK activity. The actual mean survival is 17.1±4.2 months; the median survival being 9.3 months. Two patients are still alive after 31 months. Up to 20 μg MALP-2 was well tolerated, and no systemic side effects were noted. The mean survival of 17.1 months is remarkably high.

Generation of dendritic cell-based vaccines for cancer therapy

Dendritic cells play a major role in the generation of immunity against tumour cells. They can be grown under various culture conditions, which influence the phenotypical and functional properties of dendritic cells and thereby the consecutive immune response mainly executed by T cells. Here we discuss various conditions, which are important during generation and administration of dendritic cells to elicit a tumouricidal T cell-based immune response.