Heribert Bohlen

Vaccination of multiple myeloma patients with idiotype-pulsed dendritic cells: immunological and clinical aspects

Multiple myeloma (MM) is characterized by a clonal proliferation of malignant plasma cells in the bone marrow secreting a monoclonal immunoglobulin (paraprotein) with specific antigenic determinants, the idiotype (Id), which can be regarded as a tumour‐associated antigen (TAA). In order to analyse the impact of a dendritic cell (DC)‐based vaccine, 11 patients with advanced MM were treated with CD34 stem cell‐derived dendritic cells that were pulsed with Id peptides. Subsequently, the patients received three boost immunizations every other week with a combination of Id and granulocyte–macrophage colony‐stimulating factor (GM‐CSF) (nine patients) or with Id peptide‐pulsed dendritic cells again (two patients). The treatment was well tolerated with no side‐effects. The present clinical study was a proof of concept analysis of dendritic cell‐based vaccines in MM. The capacity of the dendritic cells to activate idiotype‐specific T cells was verified by in vitro stimulation experiments before the vaccination therapy. Immunological effects of the Id vaccination were analysed by monitoring changes in anti‐idiotype antibody titres and idiotype‐specific T‐cell activity. After vaccination, three out of 10 analysed patients showed increased anti‐idiotype antibody serum titres, indicating the induction of an idiotype‐specific humoral immune response. The idiotype‐specific T‐cell response analysed by ELISpot was increased in four out of 10 analysed patients after vaccination, and one patient had a decreased plasma cell infiltration in the bone marrow. In conclusion, five out of 11 patients showed a biological response after vaccination. Thus, our data indicate that immunotherapy with Id‐pulsed DCs in MM patients is feasible and safe. DC generated from CD34+ progenitor cells can serve as a natural adjuvant for the induction of clinically relevant humoral and cellular idiotype‐specific immune responses in patients suffering from advanced MM.

Cellular Cardiomyoplasty Improves Survival After Myocardial Injury

Background—Cellular cardiomyoplasty is discussed as an alternative therapeutic approach to heart failure. To date, however, the functional characteristics of the transplanted cells, their contribution to heart function, and most importantly, the potential therapeutic benefit of this treatment remain unclear. Methods and Results—Murine ventricular cardiomyocytes (E12.5–E15.5) labeled with enhanced green fluorescent protein (EGFP) were transplanted into the cryoinjured left ventricular walls of 2-month-old male mice. Ultrastructural analysis of the cryoinfarction showed a complete loss of cardiomyocytes within 2 days and fibrotic healing within 7 days after injury. Two weeks after operation, EGFP-positive cardiomyocytes were engrafted throughout the wall of the lesioned myocardium. Morphological studies showed differentiation and formation of intercellular contacts. Furthermore, electrophysiological experiments on isolated EGFP-positive cardiomyocytes showed time-dependent differentiation with postnatal ventricular action potentials and intact &bgr;-adrenergic modulation. These findings were corroborated by Western blotting, in which accelerated differentiation of the transplanted cells was detected on the basis of a switch in troponin I isoforms. When contractility was tested in muscle strips and heart function was assessed by use of echocardiography, a significant improvement of force generation and heart function was seen. These findings were supported by a clear improvement of survival of mice in the cardiomyoplasty group when a large group of animals was analyzed (n=153). Conclusions—Transplanted embryonic cardiomyocytes engraft and display accelerated differentiation and intact cellular excitability. The present study demonstrates, as a proof of principle, that cellular cardiomyoplasty improves heart function and increases survival on myocardial injury.

Whole-body positron emission tomography using 18F-fluorodeoxyglucose for initial staging of patients with Hodgkin's disease

Abstract. An accurate initial staging of patients with Hodgkins disease (HD) is important for the evaluation of clinical stage and risk factors, which are crucial for the choice of an appropriate treatment. 18F-fluorodeoxyglucose (FDG) positron emission tomography (PET) is useful for detecting active tumor tissue in patients with lymphoproliferative diseases and may contribute to conventional staging methods in patients with HD. Twenty-two patients who presented with newly diagnosed HD underwent conventional staging methods including computed tomography (CT) as well as FDG PET. Lesions apparent in FDG PET and CT were correlated to each other. Seventy-seven lesions were observed either in PET or CT or in both. In 48 (62%) lesions PET and CT were both positive. In 20 (26%) sites, PET was positive and CT negative. Of 22 patients (18%) 4 were upstaged due to these positive PET findings, and as a result one patient received a different therapeutic regimen. PET failed to detect nine (12%) CT-positive sites in six patients. Statistically, these data are reflected by a sensitivity for PET and CT of 88% and 74%, respectively. Specificity of both imaging modalities was 100%. PET can contribute valuable information as an additional staging examination and led to an upstaging in some patients with primary HD. However, PET should not be used as the only imaging modality as it failed to detect CT-positive, active tumor regions in some cases.

Identification and characterization of embryonic stem cell-derived pacemaker and atrial cardiomyocytes

The aim of this study was to identify and functionally characterize cardiac subtypes during early stages of development. For this purpose, transgenic embryonic stem cells were generated using the α‐myosin heavy chain promoter driving the expression of the enhanced green fluorescent protein (EGFP). EGFP‐positive clusters of cells were first observed as early as 7 days of development, thus, even before the initiation of the contractile activity. Flow cytometry and single‐cell fluorescence measurements evidenced large diversities of EGFP intensity. Patch‐clamp experiments showed EGFP expression exclusively in pacemaker and atrial but not ventricular cells. The highest fluorescence intensities were detected in pacemaker‐like cardiomyocytes. In accordance, multielectrode‐array recordings of whole embryoid bodies confirmed that the pacemaker center coincided with strongly EGFP‐positive areas. The cardiac subtypes displayed already at this early stage differential characteristics of electrical activity and ion channel expression. Thus, quantitation of the α‐myosin heavy chain driven reporter gene expression allows identification and functional characterization of early cardiac subtypes.

Poor clinical outcome of patients with Hodgkin's Disease and elevated interleukin-10 serum levels

Abstract Interleukin (IL)-10 is a pleiotropic cytokine with potent inhibitory effects towards TH-1 cells. IL-10 inhibits secretion of IL-2 and interferon (IFN)γ by T cells and downregulates major histocompatibility complex antigens. A variety of tumor cells secrete IL-10, which can inhibit growth of tumor-specific cytotoxic T cells. IL-10 expression has also been detected in B-cell lymphomas and Hodgkins disease (HD), and it has been suggested that the cytokine is involved in the pathogenesis of these tumors. We analyzed levels of IL-10 in pretreatment sera of 64 patients with HD and healthy controls using a sensitive enzyme-linked immunosorbent assay. Patients with biopsy-proven HD were enrolled in trials of the German Hodgkin Study Group (GHSG). Elevated IL-10 levels were detected in the sera of nine patients with HD (14.1%) (range 4.5–225.6 pg/ml with a mean of 61.5 pg/ml). IL-10 was not detectable in a control population of healthy volunteers (n=90). Multivariate analyses revealed a significant correlation between elevated IL-10 levels and higher age (over 45 years) but not with any other factors defined by the international prognostic factor score. Patients with elevated IL-10 levels had a significantly lower freedom from treatment failure rate as detected in univariate and multivariate tests. Thus, IL-10 may serve as an independent prognostic factor for HD patients.

Elevated Serum Levels of CC Thymus and Activation-Related Chemokine (TARC) in Primary Hodgkin's Disease: Potential for a Prognostic Factor

The CC thymus and activation-related chemokine (TARC) is a protein, which is highly expressed by Reed-Sternberg cells in Hodgkins disease and is found in the majority of Hodgkins disease patients. Within several trials conducted by the German Hodgkin study group, 62 Hodgkins disease patients were elected based on availability of serum samples post and prior therapy to assess TARC levels by ELISA. TARC levels from 33 patients with continuous complete response (CCR), 20 patients with relapse, and nine patients with progressive disease (PD) were correlated with freedom from treatment failure and survival. As defined in healthy donors (mean value +/- 2x SD), a TARC level of >500 pg/mL was considered as elevated. The median TARC levels of all patients at baseline and after completed primary treatment were 5,803 pg/mL (range, 116-73,074 pg/mL) and 663 pg/mL (50-24,709 pg/mL), respectively. TARC levels of patients with PD were higher than those of patients with CCR at baseline and after therapy. Baseline TARC correlated significantly with stage (P = 0.019), erythrocyte sedimentation rate (P = 0.004), leukocyte count (P < 0.001), and lymphocyte count (P = 0.026). A TARC level of >2,000 pg/mL after completed treatment was a significant risk factor for poorer survival (P = 0.02) but not for relapse. In conclusion, monitoring serum TARC levels in Hodgkins disease patients may add valuable information about therapy success in Hodgkins disease patients, especially those with PD and should therefore be prospectively evaluated in future trials.

Dynamic monitoring of beating periodicity of stem cell‐derived cardiomyocytes as a predictive tool for preclinical safety assessment

BACKGROUND AND PURPOSE Cardiac toxicity is a major concern in drug development and it is imperative that clinical candidates are thoroughly tested for adverse effects earlier in the drug discovery process. In this report, we investigate the utility of an impedance‐based microelectronic detection system in conjunction with mouse embryonic stem cell‐derived cardiomyocytes for assessment of compound risk in the drug discovery process.

Phase I/II combined chemoimmunotherapy with carcinoembryonic antigen-derived HLA-A2-restricted CAP-1 peptide and irinotecan, 5-fluorouracil, and leucovorin in patients with primary metastatic colorectal cancer

Purpose: We conducted a phase I/II randomized trial to evaluate the clinical and immunologic effect of chemotherapy combined with vaccination in primary metastatic colorectal cancer patients with a carcinoembryonic antigen–derived peptide in the setting of adjuvants granulocyte macrophage colony-stimulating factor, CpG-containing DNA molecules (dSLIM), and dendritic cells. Experimental Design: HLA-A2–positive patients with confirmed newly diagnosed metastatic colorectal cancer and elevated serum carcinoembryonic antigen (CEA) were randomized to receive three cycles of standard chemotherapy (irinotecan/high-dose 5-fluorouracil/leucovorin) and vaccinations with CEA-derived CAP-1 peptide admixed with different adjuvants [CAP-1/granulocyte macrophage colony-stimulating factor/interleukin-2 (IL-2), CAP-1/dSLIM/IL-2, and CAP-1/IL-2]. After completion of chemotherapy, patients received weekly vaccinations until progression of disease. Immune assessment was done at baseline and after three cycles of combined chemoimmunotherapy. HLA-A2 tetramers complexed with the peptides CAP-1, human T-cell lymphotrophic virus type I TAX, cytomegalovirus (CMV) pp65, and EBV BMLF-1 were used for phenotypic immune assessment. IFN-γ intracellular cytokine assays were done to evaluate CTL reactivity. Results: Seventeen metastatic patients were recruited, of whom 12 completed three cycles. Therapy resulted in five complete response, one partial response, five stable disease, and six progressive disease. Six grade 1 local skin reactions and one mild systemic reaction to vaccination treatment were observed. Overall survival after a median observation time of 29 months was 17 months with a survival rate of 35% (6 of 17) at that time. Eight patients (47%) showed elevation of CAP-1–specific CTLs. Neither of the adjuvants provided superiority in eliciting CAP-1–specific immune responses. During three cycles of chemotherapy, EBV/CMV recall antigen–specific CD8+ cells decreased by an average 14%. Conclusions: The presented chemoimmunotherapy is a feasible and safe combination therapy with clinical and immunologic efficacy. Despite concurrent chemotherapy, increases in CAP-1–specific T cells were observed in 47% of patients after vaccination.

Single-step purification of bispecific monoclonal antibodies for immunotherapeutic use by hydrophobic interaction chromatography

A method for large scale production and single-step purification of bispecific antibodies is described. Hybrid-hybridomas were grown in hollow-fibre bioreactors with an average yield of 8 to 12 g of immunoglobulin per month. Bispecific antibodies were purified from the bioreactor supernatant by hydrophobic interaction chromatography which resolves bispecific antibodies, monospecific immunoglobulins, and culture medium supplements in one single chromatographic step. Proteins were analyzed by ELISA, SDS-PAGE, isoelectric focussing, indirect fluorescence staining, CTL-stimulation and T-cell proliferation assays. Finally, antibody preparations were checked for the presence of endotoxin and mouse DNA. Our results suggest that functional bispecific antibodies for use in therapeutic applications can be batch purified from bioreactor harvest by hydrophobic interaction chromatography in a single step. Compared to other methods such as affinity chromatography (protein A/G), ion-exchange or hydroxyapatite chromatography, our protocol offers a substantial reduction in labor time, cost, protein loss, and risk of contamination.

Expression and a role of functionally coupled P2Y receptors in human dendritic cells

We investigated the physiology and function of P2Y receptors expressed in human dendritic cells (DCs) differentiated in vitro from CD14+ cells (DC‐14). These were obtained after a 10 day stimulation period in GM‐CSF, IL‐4 and monocyte conditioned medium. DC‐14 were found to express high amounts of MHC class II, B7, CD40 as well as CD83. The functional analysis, using single cell Ca2+ imaging, demonstrated the expression of at least three subtypes of P2Y receptors. We further found using patch‐clamp measurements that ATP evoked a pertussis toxin insensitive non‐selective cation current with a peak current amplitude of −276±43 pA (holding potential −80 mV, n=23). This current was not Ca2+‐activated, since it was still observed under conditions of high intracellular Ca2+ buffering and could be blocked by Gd3+ (0.5 mM). In addition, intracellular application of GTP‐γ‐S (0.3 mM) also activated the current. Interestingly, DC‐14 redirected the orientation of their dendrites as well as cell shape towards a pipette containing ATP as observed with time lapse microscopy. These data suggest that in human DCs, ATP acts via P2Y receptors and induces chemokine effects.