Dorothee Gierschner

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.

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.

Target-dependent T-cell Activation by Coligation With a PSMA x CD3 Diabody Induces Lysis of Prostate Cancer Cells

Recently, we have described a bispecific PSMA×CD3 diabody with one binding site for the T-cell antigen receptor (TCR-CD3) and another for the Prostate Specific Membrane Antigen (PSMA). It effectively eliminates human prostate cancer cells by redirecting T-lymphocytes in vitro and in vivo. Here, we show that activation of the T-cells and killing of the tumor cells, only occurred when the T-cells were coincubated with PSMA-positive tumor cells and the PSMA×CD3 diabody. Both CD4+ and CD8+ human T-lymphocytes were activated. Surprisingly, they were equally potent in their cytotoxic activity, proliferation, and up-regulation of activation markers. Both, CD4+, and CD8+ T-cells mainly used the perforin-granzyme– based pathway and to a somewhat lesser extent the FasL pathway to lyse tumor cells. When Jurkat T-cells were stimulated with the diabody alone, the TCR-CD3 was not triggered. In contrast, when the diabody was clustered with a secondary antibody the TCR-CD3 was stimulated as detected by Ca2+-influx and Erk, IκB, and linker of activated T-cell phosphorylation. Clustering of the diabody could also be achieved by the dimeric PSMA antigen expressed on tumor cells. Thus, although the diabody binds to all T-cells, only those in contact with PSMA-expressing cancer cells are activated. In conclusion, the PSMA×CD3 diabody is suitable for a controlled polyclonal T-cell therapy of prostate cancer.

Semiquantitative analysis of Th1 and Th2 cytokine expression in CD3+, CD4+, and CD8+ renal-cell-carcinoma-infiltrating lymphocytes.

Abstract The mRNA expression of Th1 and Th2 cytokines was compared in freshly isolated CD3+ tumor-infiltrating lymphocytes (CD3+ TIL) and in autologous CD3+ peripheral blood lymphocytes (CD3+ PBL) obtained simultaneously from 20 patients with renal cell carcinomas (RCC). In addition cytokine expression was compared in CD4+ TIL and CD8+ TIL from another group of 20 patients with RCC. TIL were isolated from mechanically disaggregated tumor material and PBL from peripheral blood by gradient centrifugation and subsequent selection with anti-CD3, anti-CD4 or anti-CD8 magnetic beads. In these pure lymphocyte preparations the constitutive expression of interleukin-1 (IL-1), IL-2, IL-10, interferon γ (IFN), and tumor necrosis factor α (TNF) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3+ TIL, levels of mRNA for IFN, IL-10, IL-1 and TNF were significantly higher than in the autologous CD3+ PBL whereas IL-2 expression was rather low and did not differ in the two populations. Comparison of cytokine mRNA expression in CD4+ TIL and simultaneously obtained CD8+ TIL revealed a significantly higher expression of IFN in the CD8+ cells. These data reflect an in vivo activation of RCC-infiltrating lymphocytes at the mRNA level with respect to the Th1 as well as the Th2 immune response. Th1 activation seems to be most evident in the CD8+ TIL.

Comparison of the activation status of tumor infiltrating and peripheral lymphocytes of patients with adenocarcinomas and benign hyperplasia of the prostate

The presence of lymphocytic infiltration in prostate carcinomas has been shown to have prognostic relevance. However, it is not yet clear if this infiltrate represents a tumor‐specific activated cell population or not. Therefore, the aim of the present study was to characterize the activation status of freshly isolated tumor infiltrating lymphocytes (TIL) from prostate carcinomas (PCa) and benign hyperplasia (BPH) with respect to the mRNA expression of cytokines and apoptotic factors.

Preclinical evaluation of a recombinant anti-prostate specific membrane antigen single-chain immunotoxin against prostate cancer

The prostate-specific membrane antigen (PSMA) is abundantly expressed on prostate cancer epithelial cells and its expression correlates with tumor progression. Therefore, a specific immunotherapy against this antigen may be a novel therapeutic option for the management of prostate cancer. We generated an anti-PSMA single-chain antibody fragment (scFv), called D7, by phage display from the monoclonal antibody 3/F11. By C-terminal ligation of the toxic domain of Pseudomonas Exotoxin A (PE40) to the genes of D7, the immunotoxin D7-PE40 was generated. D7 and D7-PE40 specifically bound to PSMA transfectants and to the PSMA expressing prostate cancer cell line C4-2. In addition, D7-PE40 showed a high serum stability and induced a 50% reduction of viability (IC50) in C4-2 cells at a concentration of 140 pM. In vivo, D7-PE40 was well tolerated in SCID mice up to a single dose of 20 μg, whereas higher doses induced severe hepatotoxicity with deaths of the animals. Immunotoxin treatment of mice bearing C4-2 tumor xenografts caused a significant inhibition of tumor growth, whereas mice with PSMA-negative DU 145 tumors remained unaffected. Owing to its high and specific cytotoxicity and its capability to inhibit prostate tumor growth in vivo the immunotoxin D7-PE40 represents a promising candidate for the immunotherapy of prostate cancer.

Antitumor activities of PSMA×CD3 diabodies by redirected T-cell lysis of prostate cancer cells.

AIM Although prostate cancer is one of the most commonly diagnosed malignancies in men, there is no effective curative therapy for the advanced disease. Therefore, the aim of the present study was to generate prostate-specific membrane antigen (PSMA)×CD3 diabodies as a novel treatment option for this tumor. METHODS A PSMA×CD3 diabody and a covalently linked single-chain diabody were constructed from the anti-PSMA single-chain Fv fragment D7 and an anti-CD3 single-chain Fv fragment. The fusion proteins were periplasmatically expressed in Escherichia coli. The binding properties were tested on PSMA-expressing C4-2 prostate cancer cells and CD3(+) Jurkat cells by flow cytometry. For in vitro functional analysis, a cell viability assay was used. T-cell activation was determined by flow cytometry. In vivo activity of the diabody was tested in SCID mice reconstituted with human peripheral blood lymphocytes bearing C4-2 tumor xenografts. RESULTS Bacterial expression levels were significantly higher for the diabody (1-1.5 mg/l culture) compared with the single-chain diabody (0.2-0.4 mg/l culture). Specific binding on CD3-expressing Jurkat cells and PSMA-expressing C4-2 cells was shown with both diabody formats. In vitro, both diabodies proved to be potent agents for retargeting human CD4(+) and CD8(+) lymphocytes to lyse C4-2 prostate cancer cells. The formation of conjugates between T cells and target cells with clustering of the diabody at sites of interaction could be shown. SCID mice reconstituted with human peripheral blood lymphocytes bearing C4-2 tumor xenografts with the diabody showed an efficient inhibition of tumor growth. CONCLUSION Both diabody formats showed a highly efficient and specific T cell-mediated killing of prostate cancer cells and are encouraging for further development in preclinical and clinical studies.

Pharmacokinetics and PET imaging properties of two recombinant anti‐PSMA antibody fragments in comparison to their parental antibody

Radioimmunoimaging with disease‐specific tracers can be advantageous compared to that with nonspecific tracers for the imaging of glucose metabolism and cell proliferation. Monoclonal antibodies (mAbs) or their fragments are excellent tools for immuno‐positron emission tomography (PET). In this study, PSMA‐specific mAb 3/F11 and its recombinant fragments were compared for the imaging of prostate cancer in xenografts.

Comparison of gene expression in LNCaP prostate cancer cells after treatment with bicalutamide or 5-alpha-reductase inhibitors.

Introduction: Androgen deprivation is the preferred treatment for disseminated prostate cancer. However, it mostly leads to the development of incurable androgen-independent disease. The aim of the present study was to compare gene expression changes that occur after treatment with either the antiandrogen bicalutamide or the 5-α-reductase inhibitors finasteride (MK906) and MK386. Materials and Methods: LNCaP cells of low passages were treated with MK906 and MK386 at 5 µM each or with bicalutamide at 10 µM for 48 h. In these cultures we analyzed the expression of 22,500 transcripts on the Affymetrix Human U133+ 2.0 GeneChip platform. Gene expression was verified by real-time quantitative Taqman PCR. Results: Our studies revealed 312 differentially regulated genes upon bicalutamide treatment and 68 differentially regulated genes upon treatment with the 5-α-reductase inhibitors. There were 35 genes equally regulated by both drugs. This subset of genes included those with the highest fold change in both treatment groups. In the subset KlK2, TMPRSS2, TRGC2, PMEPA1 and TM4SF1 were downregulated, whereas EGR1, DDC and OPRK1 were upregulated. Conclusions: A cohort of interesting genes that are differentially expressed after androgen withdrawal could be found in this study. Investigation into these genes could contribute to a better understanding of antiandrogen treatment and development of androgen-independent prostate cancer.