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

BACKGROUND: Although 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. METHODS: A 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 V_HCD3-V_LPSMA and V_HPSMA-V_LCD3. 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. RESULTS: By 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. CONCLUSIONS: The PSMAxCD3 diabody bears the potential for facilitating immunotherapy of prostate cancer and for the elimination of minimal residual disease

A wirelessly actuated robotic arm for endoscopy

Endoscopy enables a number of important minimally invasive medical procedures. Current commercial flexible endoscopes with small diameter often have only one bending section near the tip with only one degree of freedom (DoF). This strongly limits the area that can be reached by the endoscope. Researchers have made many efforts to develop multi-DoF miniaturized robotic endoscopes that can be controlled in multiple bending sections and that still possess an overall size small enough to enter the body through a single-port [1]. Robotic endoscopes are normally actuated by tendons [2], pneumatics [3] or the rotation of concentric tubes [4]. In comparison to these tethered approaches, wireless actuation allows for more flexibility and easier miniaturization. Ultrasound is a promising way to transfer power wirelessly in vivo. Recently, we reported an active surface actuator that directly converts ultrasound power into mechanical work via acoustic streaming from an array of micro-bubbles [5]. Here, we apply such wireless actuators to a miniaturized robotic arm, which works as an endoscopic tip (Fig. 1). The active surfaces consisting of arrays of micro-bubbles are attached to the arm and generate streaming of the adjacent fluid under ultrasound excitation. The recoil force actuates the arm. Different bubble sizes are addressed by different ultrasound frequencies, thus multiple DoFs are realized by the arm and require only one tunable ultrasound source

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 x 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 x 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 x CD3 diabody is suitable for a controlled polyclonal T-cell therapy of prostate cancer