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(H)CD3-V(L)PSMA and V(H)PSMA-V(L)CD3. 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 recombinant PSMA-specific single-chain immunotoxin has potent and selective toxicity against prostate cancer cells.

Prostate cancer is the most commonly diagnosed form of cancer and the second leading cancer-related death among men in the Western civilization. Since no effective therapy exists for this tumor after progression beyond resectable boundaries, there is an urgent need for new treatment strategies. Prostate specific membrane antigen (PSMA) represents an excellent target on prostate cancer cells, and therefore specific immunotherapy may be a novel therapeutic option for the management of this tumor. We constructed a fully recombinant immunotoxin (A5-PE40) from a single-chain antibody fragment (scFv) against cell-adherent PSMA and a truncated form of Pseudomonas exotoxin A (PE40) lacking its natural binding domain Ia. The scFv A5 was obtained from a mAb elicited with native PSMA by phage display technology and direct selection on cells carrying the antigen. The bacterially expressed and purified immunotoxin A5-PE40 specifically binds to PSMA-positive prostate cancer cells and induces a 50% reduction of viability (IC50) at a concentration of 20 pM, while PSMA-negative cells remain unaffected. Due to its high and specific toxicity this recombinant immunotoxin is a promising candidate for therapeutic applications in patients with prostate cancer.

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

BACKGROUND: 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. METHODS: TIL were isolated from mechanically disaggregated tumor material by gradient centrifugation. The cells of the interphase were depleted from epithelial cells with anti-human epithelial antigen magnetic beads and then CD3(+)- lymphocytes were selected with magnetic beads against this determinant. In these pure lymphocyte preparations the mRNA expression of IL-1, IL-10, IFN-gamma, TNF-alpha, Fas and Fas ligand was determined by using a semiquantitative RT-PCR. Contamination with tumor cells was excluded by a PCR for PSA and PSMA. RESULTS: The CD3(+)-TIL from 21 patients with PCa and 20 patients with BPH expressed significantly higher levels of IL-10- and Fas ligand-mRNA compared to the autologous CD3(+)- PBL, whereas the expression of IL-1-, TNF-alpha- and Fas-mRNA was not different in either cell population. In contrast, the mRNA levels of IFN-gamma were significantly higher only in the CD3(+)-TIL from the carcinomas but not from the BPH compared to autologous CD3(+)-PBL. CONCLUSIONS: Since high levels of IFN-gamma have been reported to be produced by specifically lytic lymphocytes, our results suggest the presence of specifically activated TIL in the prostate carcinomas but not in the BPH, whereas inflammatory activated TIL are present both in the carcinomas and the BPH.

Construction and in vivo evaluation of an anti-PSA x anti-CD3 bispecific antibody for the immunotherapy of prostate cancer.

BACKGROUND: Cross-linking of tumor antigens with the T-cell associated CD3 antigen can be effectively achieved by bispecific monoclonal antibodies and lead to an increase in antigen-specific cytotoxicity in T cells. Because of the high organ specificity of the prostate specific antigen (PSA), a bispecific antibody (BiAb) directed against this antigen and CD3 may be a tool for a highly specific immune therapy of prostate cancer. METHODS: For generating BiAb, the quadroma technique was used. Binding properties both to CD3 and PSA were shown by flow cytometry with the CD3 expressing Jurkat cell line and fluorescein-labeled PSA. Specific tumor cell lysis was tested with the PSA expressing prostate carcinoma cell line LNCaP as target and interleukin-2 activated human peripheral blood lymphocytes as effector cells in a chromium-51-release assay. For in vivo evaluation of the BiAb, a nude mouse model was used. The mice were inoculated with LNCaP prostate carcinoma cells. Animals with growing tumors were treated with 100 micrograms BiAb and 5 x 10(6) effector cells. RESULTS: Three stable quadromas producing anti-CD3 x anti-PSA BiAb were established. From the culture supernatant of one quadroma, BiAb was separated by affinity chromatography and tested in vitro and in vivo for its ability to target effector T lymphocytes against appropriate tumor cells. In vitro, a specific lysis of PSA expressing prostate carcinoma cells was demonstrated. In vivo, a significant reduction in tumor growth (p < 0.05) could be shown in nude mice treated with BiAb and effector cells as compared to a group treated only with effector cells and an untreated control group. CONCLUSION: In the present study, an anti-CD3 x anti-PSA-BiAb was demonstrated to be effective against prostate carcinoma cells in vitro and in vivo. Therefore this BiAb may be a tool for the immunotherapy of prostate cancer.

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

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 gamma (IFNgamma), and tumor necrosis factor alpha (TNFalpha) was determined by using a polymerase-chain-reaction-assisted mRNA amplification assay. In the CD3+ TIL, levels of mRNA for IFNgamma, IL-10, IL-1 and TNFalpha 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 IFNgamma 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.