Oliver Manzke

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.

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.

Rapid tumor lysis in a patient with B-cell chronic lymphocytic leukemia and lymphocytosis treated with an anti-CD20 monoclonal antibody (IDEC-C2B8, rituximab)

SummaryIn this report we present a patient with B-cell chronic lymphocytic leukemia who developed an acute tumor lysis syndrome after administration of the human anti-CD20 antibody IDEC-C2B8 (RITUXIMAB) in standard dose of 375 mg/m2. IDEC-C2B8 has been demonstrated to have only mild and tolerable side effects in patients with follicular lymphoma. In these trials patients with lymphocytosis >5000/μl were excluded. Physicians must be aware of this hitherto unreported phenomenon in patients with high CD20-positive blood counts.

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.

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.

Locoregional treatment of low‐grade B‐cell lymphoma with CD3×CD19 bispecific antibodies and CD28 costimulation: II. Assessment of cellular immune responses

Ten patients with advanced B‐cell lymphoma were treated with a single locoregional injection of CD3×CD19 bispecific and costimulating CD28 monospecific antibodies to activate tumor‐infiltrating T‐lymphocytes. Antibodies were administered at 4 different dose levels (30 μg, 270 μg, 810 μg, 1,600 μg of each antibody) either by intratumoral or intralymphatic injection. Most patients developed responses within different compartments of the immune systems (T cells, NK cells) subsequent to the antibody application. Comparative studies in 2 patients of which treated as well as untreated lymph nodes were available revealed the up‐regulation of T‐cell activation markers induced by the antibody injection. Additionally, in 1 patient the induction of apoptosis of lymphoma B cells in the antibody‐treated lymph node was observed. Specificity analyses of peripheral blood T cells by means of IFN‐γ ELISpot measurement indicated the recruitment of idiotype‐specific T cells, as in 1 out of 3 investigated patients an increased T‐cell response toward autologous idiotype peptides could be demonstrated. We conclude that a single injection of CD3×CD19 bispecific antibodies is capable to induce an activation of autologous T lymphocytes if simultaneous costimulatory signaling by CD28 antibodies is provided. Furthermore, our data suggest that at least in some patients lymphoma‐specific T cells can be recruited by this immunotherapeutic approach toward B‐cell lymphoma.

Locoregional treatment of low-grade B-cell lymphoma with CD3×CD19 bispecific antibodies and CD28 costimulation: I. Clinical phase I evaluation

We describe the first clinical application of T‐cell‐recruiting bispecific antibodies directly into the tumor without the need to preactivate the effector cells. In a Phase I clinical trial, 10 patients with low‐grade B‐cell lymphoma were treated by a single locoregional injection of CD3×CD19 bispecific antibodies. Costimulatory signaling, which is required for the optimal activation of resting T cells, was provided by the simultaneous administration of CD28 antibodies. Equal amounts of both antibodies were injected together at 4 different dose levels (30 μg: 3 patients; 270 μg: 3 patients; 810 μg: 3 patients; 1,600 μg: 1 patient). The injection was well tolerated with mild to moderate adverse effects (2/10 patients) consisting of erythema and fever at the third dose level. The maximum tolerated dose was not reached at 810 μg of injected antibodies. Three patients showed a serum peak of TNFα on day 2 or 3 after the antibody application, reflecting rather an activation of CD4‐positive T cells than an FcR‐mediated effect. Five patients developed anti‐mouse antibodies after injection of the murine immunoglobulins. Nine patients were evaluable for restaging examinations 6 weeks after the antibody application, with 2 of them (22%) showing a local clinical response. We found that a single locoregional injection of CD3×CD19+CD28 antibodies is feasible up to a dose of at least 1,600 μg of each antibody. However, the development of human anti‐mouse antibodies points toward the requirement for new formats of bispecific proteins with reduced immunogenicity.

CD3xCD19 bispecific antibodies and CD28 bivalent antibodies enhance T-cell reactivity against autologous leukemic cells in pediatric B-ALL bone marrow

Bispecific CD3xCD19 antibodies and CD28 co‐stimulating antibodies were used to activate T cells in bone marrow aspirates (n = 8) of children with B cell–derived acute lymphoblastic leukemia. Bone marrow specimens were incubated for 10 days with CD3xCD19 bispecific and CD28 antibodies. Changes in the numbers of T lymphocytes and tumoral B cells as well as surface expression of T cell–activation markers were determined by flow cytometry, and cytokines (human IFN‐γ, IL‐2, IL‐4 and IL‐12) were measured in the cell culture supernatant. In 7 of 8 bone marrow samples, an increase in the number of CD4‐ and CD8‐positive T lymphocytes was found, which correlated with an up‐regulation of T cell–activation markers. Additionally, we demonstrated a decrease of tumoral B cells in 3 samples and enhanced cytotoxic T‐cell activity against autologous malignant B cells. ELISpot analyses in an autologous Epstein‐Barr virus model showed that bispecific antibodies (CD3xCD19+CD28) were more potent at generating T‐cell responses against autologous and allogeneic tumoral targets than a combination of monospecific antibodies (CD3+CD28). Thus, T‐cell targeting by CD3xCD19 bispecific and CD28 antibodies may be used to eliminate leukemic B cells ex vivo and reconstitute immunological control of residual malignant disease by the induction of anti‐tumoral T‐cell responses. Int. J. Cancer 80:715–722, 1999.

Differentiation of cytotoxicity using target cells labelled with europium and samarium by electroporation

We report the simultaneous use of europium-DTPA (Eu-DTPA) and samarium-DTPA (Sm-DTPA) in cytotoxicity experiments to analyze simultaneously LAK and NK cell lysis and to differentiate between specific target lysis and bystander killing. The target cells were either labelled with Eu-DTPA or Sm-DTPA chelates by electroporation, which permits the use of target cell lines or primary leukemic B cells (B-CLL) that cannot be labelled by the conventional dextran-sulphate method. The release of europium and samarium reaches a maximum at comparable time intervals (2-3 h). Due to the shorter counting interval within the samarium window the labelling efficiency is about ten times less efficient compared to europium. Using europium as label for the LAK target Daudi and samarium as label for the NK sensitive cell line K562 the differentiation of LAK versus NK activity can be performed in a single culture assay. Also, the killing of B cells and bystander cells by cytotoxic T cells was analyzed in a system where T cells were redirected to B cells through CD3 x CD19 bispecific antibodies. In fact, no bystander killing was noted when bispecific antibodies were used to bridge cytotoxic T cells to the B cells. This approach provides a simple non-radioactive method for evaluating cytotoxicity against two different cells in a single culture well.

Dendritic cells are significantly reduced in non-Hodgkin's lymphoma and express less CCR7 and CD62L.

Despite the lack of tumor control, infiltration of immune cells has been demonstrated for several malignancies including non-Hodgkins lymphoma. Since dendritic cells play a pivotal role in the initiation and control of the immune response, the frequency and phenotype of recently described sub-types of dendritic cells in non-Hodgkins lymphoma were characterized. Myeloid and plasmacytoid dendritic cells were analysed in 55 non-Hodgkins lymphoma and 33 reactive lymph nodes by flow cytometry and immunohistochemistry. Overall frequency of dendritic cells in reactive lymph nodes was higher than in non-Hodgkins lymphoma while the pDC/mDCs ratio was comparable. The low frequency of dendritic cells in infiltrated lymph nodes was confirmed by immunohistochemistry; however, no significant difference in the distribution within lymphoid and tumor tissue was detected. For further characterization of the dendritic cells in non-Hodgkins lymphoma, the expressions of adhesion molecules, costimulatory molecules, chemokine receptors and activation markers were assessed. Interestingly, a significantly decreased expression of CD62L and CCR7, receptors necessary for homing to lymph nodes, was identified in dendritic cells in non-Hodgkins lymphoma, potentially explaining the lack of these cells. Taken together, dendritic cells are phenotypically altered and reduced in number in NHL, potentially contributing to the loss of tumor control in these patients.