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

BACKGROUND: 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. METHODS: The PSMA × CD3 bsc diabody was generated from an anti-CD3 single chain Fv fragment (scFv) and the anti-PSMA scFv D7. It was expressed in E. coli and purified from the periplasmic extract and culture supernatant 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-1) was used and activation of T-cells was determined by measuring the surface marker expression of CD25 and CD69. For in vivo evaluation, the diabody was administered in combination with human peripheral blood lymphocytes (Ly) in a C4-2 xenograft-SCID mouse model. RESULTS: Specific binding of the PSMA × CD3 bsc diabody both to CD3-positive Jurkat cells and PSMA-expressing C4-2 cells was shown by flow cytometry. In vitro, the PSMA × CD3 bsc diabody proved to be a potent agent for retargeting CD4+ and CD8+ human lymphocytes to lyse C4-2 prostate cancer cells. Treatment of SCID mice bearing C4-2 tumor xenografts with the diabody and human lymphocytes efficiently inhibited tumor growth. CONCLUSIONS: The PSMA × CD3 bsc diabody bears a high potential for the immunotherapy of prostate cancer.

Large cell neuroendocrine lung carcinoma induces peripheral T-cell repertoire alterations with predictive and prognostic significance.

OBJECTIVES: This study was performed to evaluate for a potentially important role of T cells in the pathophysiology and treatment sensitivity of large cell neuroendocrine lung carcinoma (LCNEC), an orphan disease with poor prognosis and scarce data to guide novel therapeutic strategies. MATERIALS AND METHODS: We performed T-cell receptor (TCR) ß-chain spectratyping on blood samples of patients treated within the CRAD001KDE37 trial (n = 35) using age-matched current or former (n = 11) and never smokers (n = 10) as controls. The data were analyzed in conjunction with the complete blood counts of the probands as well as the data about response to treatment and overall survival in the clinical trial. RESULTS AND CONCLUSION: Untreated stage IV LCNEC patients had significant T-cell repertoire alterations (p < 0.001) compared to age-matched smokers. These changes correlated positively with blood lymphocyte counts (r = 0.49, p < 0.01), suggesting antigen-induced T-cell proliferation as the causative mechanism. At the same time, LCNEC patients showed mild lymphopenia (1.54 vs. 2.51/nl in median, p < 0.01), which reveals a second, antigen-independent mechanism of systemic immune dysregulation. More pronounced T-cell repertoire alterations and higher blood lymphocyte counts at diagnosis were associated with a better treatment response by RECIST and with a longer overall survival (441 vs. 157 days in median, p = 0.019). A higher degree of T-cell repertoire normalization after 3 months of therapy also distinguished a patient group with more favourable prognosis (median overall survival 617 vs. 316 days, p = 0.036) independent of radiological response. Thus, LCNEC induces clinically relevant changes of the T-cell repertoire, which are measurable in the blood and could be exploited for prognostic, predictive and therapeutic purposes. Their pathogenesis appears to involve antigen-induced oligoclonal T-cell expansions superimposed on TCR-independent lymphopenia.

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.

An antibody fusion protein for cancer immunotherapy mimicking IL-15 trans-presentation at the tumor site.

Cytokines driving the immune response are powerful tools for cancer immunotherapy, but their application is generally limited by severe systemic toxicity. Targeted approaches by means of antibody-cytokine fusion proteins might enable focus on the cytokine activity to the tumor site, thereby reducing unwanted side effects. Here, we investigated the possibility to improve the efficiency of interleukin (IL)-15 presentation in a targeted approach by the incorporation of an IL-15Rα chain fragment, mimicking physiologic trans-presentation. Therefore, an antibody cytokine fusion protein (scFv_RD_IL-15) composed of an antibody moiety targeting the tumor stromal fibroblast activation protein (FAP), an extended IL-15Rαsushi domain (RD) and IL-15 was generated, exhibiting antibody-mediated specific binding and cytokine activity in soluble and targeted form. Comparative analysis with a corresponding antibody fusion protein devoid of RD (scFv_IL-15) showed for scFv_RD_IL-15 in solution enhanced stimulatory activity on Mo7e (IL-15Rßγ) cells and reduced proliferation response on CTLL-2 (IL-15Rαßγ) cells, while in FAP-targeted, that is, membrane-bound form, comparable proliferation of CTLL-2 (IL-15Rαßγ) cells was obtained. In addition, scFv_RD_IL-15 achieved in its soluble and target-bound form stronger proliferation and cytotoxicity on unstimulated and activated T cells, respectively. Furthermore, in vivo analysis in a lung metastasis tumor mouse model revealed a superior antitumor effect for scFv_RD_IL-15 in comparison with that obtained by an untargeted or RD missing version of IL-15 fusion protein. Thus, tumor-directed trans-presentation of IL-15 in association with RD in form of an antibody fusion protein seems to be a promising approach to further improve the antitumor effect of IL-15.