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An anti-MUC1-antibody-interleukin-2 fusion protein that activates resting NK cells to lysis of MUC1-positive tumour cells.

Heuser C, Ganser M, Hombach A, Brand H, Denton G, Hanisch FG, Abken H - Br. J. Cancer (2003)

Bottom Line: MUC1 mucin is aberrantly glycosylated and overexpressed in a number of epithelial malignancies and is therefore a promising tumour-associated antigen for target-directed immunotherapy of a panel of malignant diseases.To activate lymphocytes and natural killer (NK) cells, we here generated an anti-MUC1-scFv-IL2 fusion protein (C595scFv-Fc-IL2) that contains the C595 single-chain antibody for MUC1 binding, the human IgG1 CH2CH3 domain for protein dimerisation, and interleukin-2 (IL2) for activation of immunological effector cells.Bound to MUC1, the C595scFv-Fc-IL2 fusion protein stimulates proliferation of human activated lymphocytes in vitro.

View Article: PubMed Central - PubMed

Affiliation: Lab. Tumorgenetik, Klinik I für Innere Medizin, Klinikum der Universität zu Köln, Joseph-Stelzmann-Str. 9, D-50931 Köln, Germany.

ABSTRACT
MUC1 mucin is aberrantly glycosylated and overexpressed in a number of epithelial malignancies and is therefore a promising tumour-associated antigen for target-directed immunotherapy of a panel of malignant diseases. In MUC1-positive tumours, MHC class I expression is frequently downregulated and MUC1-specific cytotoxic T cells (CTLs) are either not available or in a state of anergy allowing tumour growth without limitation by CTL control. To activate lymphocytes and natural killer (NK) cells, we here generated an anti-MUC1-scFv-IL2 fusion protein (C595scFv-Fc-IL2) that contains the C595 single-chain antibody for MUC1 binding, the human IgG1 CH2CH3 domain for protein dimerisation, and interleukin-2 (IL2) for activation of immunological effector cells. The fusion protein binds to MUC1-derived peptides and to MUC1-positive tumour cells with the same specificity as does the C595 monoclonal antibody. Bound to MUC1, the C595scFv-Fc-IL2 fusion protein stimulates proliferation of human activated lymphocytes in vitro. Upon binding to MUC1-positive MCF7 breast carcinoma cells, moreover, the fusion protein activates resting NK cells to tumour cell lysis. These properties make the C595scFv-Fc-IL2 fusion protein a suitable candidate for the immunotherapy of MUC1-positive tumours.

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The fusion protein C595scFv-Fc-IL2 activates resting NK cells to tumour cell lysis (A). MCF7 cells (MUC1+) (5 × 104 cells per well) were cocultured with resting NK cells (1x105 cells per well) in the presence of C595scFv-Fc-IL2 fusion protein or IL2 (400 U ml−1). As control, MCF7 cells were cocultured with NK cells in cell culture medium without additives. (B) MCF7 and T-47D tumour cells (MUC1+) were preincubated with cell culture supernatants containing C595scFv-Fc, C595scFv-Fc-IL2, or IL2 (400 U ml−1), respectively. After four rounds of washings, cells were seeded into round-bottom microtiter plates (5 × 104 cells per well) and cocultured with resting NK cells (1 × 105 cells per well). The viability of MCF7 and T-47D cells was determined by an XTT-based viability assay as described in Material and Methods. The assays were performed in triplicate; data are presented as the mean±s.e.m. Statistical significance was determined utilising Student's t-test.
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fig9: The fusion protein C595scFv-Fc-IL2 activates resting NK cells to tumour cell lysis (A). MCF7 cells (MUC1+) (5 × 104 cells per well) were cocultured with resting NK cells (1x105 cells per well) in the presence of C595scFv-Fc-IL2 fusion protein or IL2 (400 U ml−1). As control, MCF7 cells were cocultured with NK cells in cell culture medium without additives. (B) MCF7 and T-47D tumour cells (MUC1+) were preincubated with cell culture supernatants containing C595scFv-Fc, C595scFv-Fc-IL2, or IL2 (400 U ml−1), respectively. After four rounds of washings, cells were seeded into round-bottom microtiter plates (5 × 104 cells per well) and cocultured with resting NK cells (1 × 105 cells per well). The viability of MCF7 and T-47D cells was determined by an XTT-based viability assay as described in Material and Methods. The assays were performed in triplicate; data are presented as the mean±s.e.m. Statistical significance was determined utilising Student's t-test.

Mentions: We assayed the bioactivitiy of the C595scFv-Fc-IL2 fusion protein bound to MUC1-positive target cells by monitoring the activation of resting NK cells to lyse target cells. MUC1-positive MCF7 tumour cells were incubated with C595scFv-Fc-IL2 fusion protein and IL2, respectively, and cocultured with resting NK cells. Tumour cell viability was monitored by an XTT-based colorimetric assay. As shown in Figure 9AFigure 9


An anti-MUC1-antibody-interleukin-2 fusion protein that activates resting NK cells to lysis of MUC1-positive tumour cells.

Heuser C, Ganser M, Hombach A, Brand H, Denton G, Hanisch FG, Abken H - Br. J. Cancer (2003)

The fusion protein C595scFv-Fc-IL2 activates resting NK cells to tumour cell lysis (A). MCF7 cells (MUC1+) (5 × 104 cells per well) were cocultured with resting NK cells (1x105 cells per well) in the presence of C595scFv-Fc-IL2 fusion protein or IL2 (400 U ml−1). As control, MCF7 cells were cocultured with NK cells in cell culture medium without additives. (B) MCF7 and T-47D tumour cells (MUC1+) were preincubated with cell culture supernatants containing C595scFv-Fc, C595scFv-Fc-IL2, or IL2 (400 U ml−1), respectively. After four rounds of washings, cells were seeded into round-bottom microtiter plates (5 × 104 cells per well) and cocultured with resting NK cells (1 × 105 cells per well). The viability of MCF7 and T-47D cells was determined by an XTT-based viability assay as described in Material and Methods. The assays were performed in triplicate; data are presented as the mean±s.e.m. Statistical significance was determined utilising Student's t-test.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376954&req=5

fig9: The fusion protein C595scFv-Fc-IL2 activates resting NK cells to tumour cell lysis (A). MCF7 cells (MUC1+) (5 × 104 cells per well) were cocultured with resting NK cells (1x105 cells per well) in the presence of C595scFv-Fc-IL2 fusion protein or IL2 (400 U ml−1). As control, MCF7 cells were cocultured with NK cells in cell culture medium without additives. (B) MCF7 and T-47D tumour cells (MUC1+) were preincubated with cell culture supernatants containing C595scFv-Fc, C595scFv-Fc-IL2, or IL2 (400 U ml−1), respectively. After four rounds of washings, cells were seeded into round-bottom microtiter plates (5 × 104 cells per well) and cocultured with resting NK cells (1 × 105 cells per well). The viability of MCF7 and T-47D cells was determined by an XTT-based viability assay as described in Material and Methods. The assays were performed in triplicate; data are presented as the mean±s.e.m. Statistical significance was determined utilising Student's t-test.
Mentions: We assayed the bioactivitiy of the C595scFv-Fc-IL2 fusion protein bound to MUC1-positive target cells by monitoring the activation of resting NK cells to lyse target cells. MUC1-positive MCF7 tumour cells were incubated with C595scFv-Fc-IL2 fusion protein and IL2, respectively, and cocultured with resting NK cells. Tumour cell viability was monitored by an XTT-based colorimetric assay. As shown in Figure 9AFigure 9

Bottom Line: MUC1 mucin is aberrantly glycosylated and overexpressed in a number of epithelial malignancies and is therefore a promising tumour-associated antigen for target-directed immunotherapy of a panel of malignant diseases.To activate lymphocytes and natural killer (NK) cells, we here generated an anti-MUC1-scFv-IL2 fusion protein (C595scFv-Fc-IL2) that contains the C595 single-chain antibody for MUC1 binding, the human IgG1 CH2CH3 domain for protein dimerisation, and interleukin-2 (IL2) for activation of immunological effector cells.Bound to MUC1, the C595scFv-Fc-IL2 fusion protein stimulates proliferation of human activated lymphocytes in vitro.

View Article: PubMed Central - PubMed

Affiliation: Lab. Tumorgenetik, Klinik I für Innere Medizin, Klinikum der Universität zu Köln, Joseph-Stelzmann-Str. 9, D-50931 Köln, Germany.

ABSTRACT
MUC1 mucin is aberrantly glycosylated and overexpressed in a number of epithelial malignancies and is therefore a promising tumour-associated antigen for target-directed immunotherapy of a panel of malignant diseases. In MUC1-positive tumours, MHC class I expression is frequently downregulated and MUC1-specific cytotoxic T cells (CTLs) are either not available or in a state of anergy allowing tumour growth without limitation by CTL control. To activate lymphocytes and natural killer (NK) cells, we here generated an anti-MUC1-scFv-IL2 fusion protein (C595scFv-Fc-IL2) that contains the C595 single-chain antibody for MUC1 binding, the human IgG1 CH2CH3 domain for protein dimerisation, and interleukin-2 (IL2) for activation of immunological effector cells. The fusion protein binds to MUC1-derived peptides and to MUC1-positive tumour cells with the same specificity as does the C595 monoclonal antibody. Bound to MUC1, the C595scFv-Fc-IL2 fusion protein stimulates proliferation of human activated lymphocytes in vitro. Upon binding to MUC1-positive MCF7 breast carcinoma cells, moreover, the fusion protein activates resting NK cells to tumour cell lysis. These properties make the C595scFv-Fc-IL2 fusion protein a suitable candidate for the immunotherapy of MUC1-positive tumours.

Show MeSH
Related in: MedlinePlus