Ursula Elsässer-Beile

Evaluation of a test system for measuring cytokine production in human whole blood cell cultures

A simple and reproducible method is described for the measurement of mitogen-induced cytokine production in cultures of both human peripheral blood mononuclear cells (PBMC) and whole blood. In the culture supernatants the cytokines interleukin-1 alpha (IL-1 alpha), interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) were determined by a rapid and sensitive immunoassay using various monoclonal and polyclonal antibodies. Comparing the PBMC cultures with the whole blood system a good correlation was obtained if the cell number was taken into account. In the post-induction supernatants the cytokine values were found to follow typical kinetic curves. The protocol was evaluated by screening 60 cancer patients with primary disease and 60 healthy controls. A markedly reduced secretion of IFN-gamma and IL-1 alpha was found in the cancer patients compared to controls, although leukocyte and lymphocyte counts were almost identical in both groups.

A bispecific diabody directed against prostate-specific membrane antigen and CD3 induces T-cell mediated lysis of prostate cancer cells

BackgroundAlthough cancer of the prostate is one of the most commonly diagnosed cancers in men, no curative treatment currently exists after its progression beyond resectable boundaries. Therefore, new agents for targeted treatment strategies are needed. Cross-linking of tumor antigens with T-cell associated antigens by bispecific monoclonal antibodies have been shown to increase antigen-specific cytotoxicity in T-cells. Since the prostate-specific membrane antigen (PSMA) represents an excellent tumor target, immunotherapy with bispecific diabodies could be a promising novel treatment option for prostate cancer.MethodsA heterodimeric diabody specific for human PSMA and the T-cell antigen CD3 was constructed from the DNA of anti-CD3 and anti-PSMA single chain Fv fragments (scFv). It was expressed in E. coli using a vector containing a bicistronic operon for co-secretion of the hybrid scFv VHCD3-VLPSMA and VHPSMA-VLCD3. The resulting PSMAxCD3 diabody was purified from the periplasmic extract by immobilized metal affinity chromatography (IMAC). The binding properties were tested on PSMA-expressing prostate cancer cells and PSMA-negative cell lines as well as on Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability test (WST) was used. For in vivo evaluation the diabody was applied together with human peripheral blood lymphocytes (PBL) in a C4-2 xenograft-SCID mouse model.ResultsBy Blue Native gel electrophoresis, it could be shown that the PSMAxCD3 diabody is mainly a tetramer. Specific binding both to CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the diabody proved to be a potent agent for retargeting PBL to lyze C4-2 prostate cancer cells. Treatment of SCID mice inoculated with C4-2 tumor xenografts with the diabody and PBL efficiently inhibited tumor growth.ConclusionsThe PSMAxCD3 diabody bears the potential for facilitating immunotherapy of prostate cancer and for the elimination of minimal residual disease.

PET imaging of prostate cancer xenografts with a highly specific antibody against the prostate-specific membrane antigen.

Prostate-specific membrane antigen (PSMA), a transmembrane glycoprotein, is highly expressed by virtually all prostate cancers and is currently the focus of several diagnostic and therapeutic strategies. We have previously reported on the generation of several monoclonal antibodies (mAb) and antibody fragments that recognize and bind with high affinity to the extracellular domain of cell-adherent PSMA. This article reports the in vivo behavior and tumor uptake of the radiolabeled anti-PSMA mAb 3/A12 and its potential as a tracer for PET. Methods: The mAb 3/A12 was conjugated with the chelating agent 1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA) and radiolabeled with 64Cu. Severe combined immunodeficient mice bearing PSMA-positive C4-2 prostate carcinoma xenografts were used for small-animal PET imaging. Mice with PSMA-negative DU 145 tumors served as controls. For PET studies, each animal received 20–30 μg of radiolabeled mAb corresponding to an activity of 7.6–11.5 MBq. Imaging was performed 3, 24, and 48 h after injection. After the last scan, the mice were sacrificed and tracer in vivo biodistribution was measured by γ-counting. Results: Binding of the mAb 3/A12 on PSMA-expressing C4-2 cells was only minimally influenced by DOTA conjugation. The labeling efficiency using 64Cu and DOTA-3/A12 was 95.3% ± 0.3%. The specific activity after 64Cu labeling was between 327 and 567 MBq/mg. After tracer injection, static small-animal PET images of mice with PSMA-positive tumors revealed a tumor-to-background ratio of 3.3 ± 1.3 at 3 h, 7.8 ± 1.4 at 24 h, and 9.6 ± 2.7 at 48 h. In contrast, no significant tracer uptake occurred in the PSMA-negative DU 145 tumors. These results were confirmed by direct counting of tissues after the final imaging. Conclusion: Because of the high and specific uptake of 64Cu-labeled mAb 3/A12 in PSMA-positive tumors, this ligand represents an excellent candidate for prostate cancer imaging and potentially for radioimmunotherapy.

Biorecognition and subcellular trafficking of HPMA copolymer-anti-PSMA antibody conjugates by prostate cancer cells.

A new generation of antibodies against the prostate specific membrane antigen (PSMA) has been proven to bind specifically to PSMA molecules on the surface of living prostate cancer cells. To explore the potential of anti-PSMA antibodies as targeting moieties for macromolecular therapeutics for prostate cancer, fluorescently labeled HPMA (N-(2-hydroxypropyl)methacrylamide) copolymer-anti-PSMA antibody conjugates (P-anti-PSMA) were synthesized and the mechanisms of their endocytosis and subcellular trafficking in C4-2 prostate cancer cells were studied. Radioimmunoassays showed the dissociation constants of P-anti-PSMA for C4-2 prostate cancer cells in the low nanomolar range, close to values for free anti-PSMA. It indicated that conjugation of anti-PSMA to HPMA copolymers did not compromise their binding affinity. The rate of endocytosis of P-anti-PSMA was much faster than that of control HPMA copolymer conjugates containing nonspecific IgG. Selective pathway inhibitors of clathrin-mediated endocytosis and of macropinocytosis inhibited the internalization of P-anti-PSMA. Inhibition of clathrin-mediated endocytosis was further evidenced by down-regulation of clathrin heavy chain expression by siRNA. Using a dominant-negative mutant of dynamin (Dyn K44A) to abolish the clathrin-, caveolae-independent endocytic pathway, we found that some of P-anti-PSMA adopted this pathway to be endocytosed into C4-2 cells. Thus multiple receptor-mediated endocytic pathways, including clathrin-mediated endocytosis, macropinocytosis, and clathrin-, caveolae-independent endocytosis, were involved in the internalization of P-anti-PSMA. The extent of the participation of each pathway in P-anti-PSMA endocytosis was estimated. Membrane vesicles containing P-anti-PSMA rapidly colocalized with membrane vesicles overexpressing Rab7, a late endosome localized protein, demonstrating that a part of P-anti-PSMA was transported to late endosomes.

Three conformational antibodies specific for different PSMA epitopes are promising diagnostic and therapeutic tools for prostate cancer.

The prostate specific membrane antigen (PSMA) represents an attractive antigen for antibody‐based diagnostic and therapeutic intervention in prostate cancer, since it is highly restricted to the prostate and overexpressed in all tumor stages. The present work describes the in vitro characterization of the three anti‐PSMA monoclonal antibodies (mAbs) 3/A12, 3/E7, and 3/F11 in comparison to the mAb J591.

Impaired cytokine production in whole blood cell cultures of patients with gynaecological carcinomas in different clinical stages

The production of the cytokines IFN-gamma, IL-1-alpha, IL-2 and TNF-alpha was investigated in mitogen-stimulated, whole blood cell culture from 239 untreated patients with primary gynaecological carcinomas (breast, cervix, ovary, endometrium), and 191 healthy female controls. The cytokines were measured in the 4-day post-induction supernatants by a sensitive enzymoimmunological assay. In the blood cell cultures of all four groups of cancer patients, significantly lower values of IFN-gamma (P < or = 0.001), IL-2 (P < or = 0.01) and IL-1 alpha (P < or = 0.01) were found as compared to the controls, although lymphocyte and monocyte counts were almost identical. Grouping the tumour patients into different clinical stages we could show in the four groups of carcinomas a gradual depression of the cytokine production according to growing tumour burden.

Th1 and Th2 Cytokine Response Patterns in Leukocyte Cultures of Patients with Urinary Bladder, Renal Cell and Prostate Carcinomas

As a decreased production of Th1 cytokines by stimulated peripheral blood leukocytes has recently been shown in patients with various carcinomas, the present study was performed to determine whether these patients also exhibit a Th1/Th2 imbalance compared to healthy controls. We measured the production of the Th1 cytokines IL-2 and IFN-γ as well as the Th2 cytokines IL-4, IL-6 and IL-10 in mitogen-stimulated peripheral blood mononuclear cell (PBMC) cultures of patients with urinary bladder carcinomas (n = 47), prostate carcinomas (n = 111) and renal cell carcinomas (n = 67) as compared to 40 age-matched healthy controls. In the PBMC cultures of the tumor patients, the levels of the Th1 cytokines IL-2 and IFN-γ were lower as compared to the controls. For IFN-γ, the differences were highly significant and in the patients with renal cell carcinomas it could be shown that the values decreased with increasing tumor mass. In contrast, the levels of the Th2 cytokines IL-4, IL-6 and IL-10 were comparable in the PBMC cultures of tumor patients and controls. From these results, it is concluded that there is only a malfunction in Th1 cells but no switch from a Th1 type to a Th2 type cytokine profile in the PBMCs of cancer patients.

Biological effects of natural and recombinant mistletoe lectin and an aqueous mistletoe extract on human monocytes and lymphocytes in vitro.

Mistletoe lectin is thought to constitute the active principle in extract preparations from mistletoe, which are widely used as immunomodulators in adjuvant tumor therapy. However, no study exists which compares the immunological potency of different well‐defined mistletoe lectin preparations on human immune cells. Therefore, in the present study the biological effects of an aqueous mistletoe extract, standardized for mistletoe lectin I (eML), the isolated natural mistletoe lectin (nML), and the recombinant form of this lectin (rML) on human peripheral blood monocytes and lymphocytes were compared with respect to cell viability and cytokine induction. After 48‐hr incubation of peripheral blood mononuclear cells (PBMC) with rML, nML, and eML, a continuous concentration‐dependent decrease in cell viability was found with an IC50 of about 3 ng/ml for rML and nML and 10 ng/ml for eML, respectively. This effect also was seen when isolated lymphocytes and monocytes were separately incubated with the lectin preparations. After incubation of PBMC and isolated monocytes of 5/10 blood donors with eML, an increase in cell viability was found at lectin concentrations between 10 and 1,000 pg/ml. This effect was not seen with the pure lectin preparations nML and rML. After 48‐hr incubation of PBMC with rML, nML, and eML, induction of IL‐1‐β, TNF‐α, IL‐2, IL‐6, and IL‐10 but not IFN‐γ was measured. For IL‐1‐β it could be shown that cytokine induction took place at a broad lectin concentration range (0.1–100 ng/ml). Cytokine levels varied greatly in the PBMC cultures of the different blood donors. When monocytes were separately incubated with eML, nML, and rML for 48 hr, high levels of IL‐1‐β were found. In contrast, in cultures of separated lymphocytes from the same donors only a minimal production of IL‐1‐β and no production of IFN‐γ was found after incubation with rML, nML, and eML. It is concluded that there are quantitative differences in the immunomodulatory effects of the mistletoe lectin preparations on human monocytes and lymphocytes. Therefore, measurement of cell viability and cytokine induction may be a diagnostic laboratory tool to determine the immunological potency of various mistletoe preparations and may help to clarify the clinical benefit of therapies with these substances. J. Clin. Lab. Anal. 14:255–259, 2000.

Cytokine levels in whole blood cell cultures as parameters of the cellular immunologic activity in patients with malignant melanoma and basal cell carcinoma

For the determination of cellular immunity status, mitogen‐induced lymphocyte proliferation tests are used, along with measurements of cytokine secretion. The authors have established a test system with whole blood cell cultures in which they measured the following cytokines: alpha‐interleukin‐1 (α‐IL‐1), interleukin‐2 (IL‐2), gamma‐interferon (τ‐IFN), and alpha‐tumor necrosis factor (α‐TNF) in the supernatants by enzymoimmunologic methods. With this system, the authors tested blood samples of 72 patients with malignant melanoma, 38 patients with basal cell carcinoma, and 315 healthy control subjects. In the blood cell cultures of the patients with melanoma, significantly lower values of the lymphokines τ‐IFN and IL‐2 were found, compared with those of the control subjects, and the levels of the monokines α‐IL‐1 and α‐TNF were reduced. τ‐IFN values correlated with different clinical stages. In contrast, the patients with basal cell carcinoma had equal values for all four cytokines as an age‐matched control group. Cancer 1993; 71:231‐6.

Effective targeting of prostate cancer by lymphocytes redirected by a PSMA × CD3 bispecific single-chain diabody

For redirecting T‐lymphocytes to induce prostate cancer cell lysis, we constructed a novel bispecific single‐chain (bsc) diabody directed to the prostate specific membrane antigen (PSMA) and the T‐cell receptor (TCR)‐associated CD3 molecule on T‐cells.