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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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Fusion proteins C595scFv-Fc and C595scFv-Fc-IL2 bind specifically to MUC1 positive tumour cells and TR5 peptide-coated beads. Cell culture supernatants from transfected 293 T cells containing the C595scFv-Fc and C595scFv-Fc-IL2 fusion protein (thick lines), respectively, and, as control, supernatant from 293 T cells transfected with an irrelevant DNA (thin lines), were incubated (A) with T-47D cells (MUC1+/CD25−), 293 T cells (MUC1−/CD25−), L540 cells (MUC1−/CD25+), TR5 peptide-coated beads or, as control, with human serum albumin-coated beads, and (B) with quiescent lymphocytes from the peripheral blood (PBL) or with PBL activated by preincubation with the anti-CD3 mAb OKT3 plus IL2. Bound fusion proteins were detected by an FITC-labelled anti-human IgG antibody. Expression of the IL2 receptor CD25 was monitored by incubation with a FITC-conjugated anti-CD25 mAb (anti-CD25).
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fig6: Fusion proteins C595scFv-Fc and C595scFv-Fc-IL2 bind specifically to MUC1 positive tumour cells and TR5 peptide-coated beads. Cell culture supernatants from transfected 293 T cells containing the C595scFv-Fc and C595scFv-Fc-IL2 fusion protein (thick lines), respectively, and, as control, supernatant from 293 T cells transfected with an irrelevant DNA (thin lines), were incubated (A) with T-47D cells (MUC1+/CD25−), 293 T cells (MUC1−/CD25−), L540 cells (MUC1−/CD25+), TR5 peptide-coated beads or, as control, with human serum albumin-coated beads, and (B) with quiescent lymphocytes from the peripheral blood (PBL) or with PBL activated by preincubation with the anti-CD3 mAb OKT3 plus IL2. Bound fusion proteins were detected by an FITC-labelled anti-human IgG antibody. Expression of the IL2 receptor CD25 was monitored by incubation with a FITC-conjugated anti-CD25 mAb (anti-CD25).

Mentions: The fusion proteins were furthermore assayed by flow cytometry for binding to MUC1-expressing tumour cells. As demonstrated in Figure 6AFigure 6


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)

Fusion proteins C595scFv-Fc and C595scFv-Fc-IL2 bind specifically to MUC1 positive tumour cells and TR5 peptide-coated beads. Cell culture supernatants from transfected 293 T cells containing the C595scFv-Fc and C595scFv-Fc-IL2 fusion protein (thick lines), respectively, and, as control, supernatant from 293 T cells transfected with an irrelevant DNA (thin lines), were incubated (A) with T-47D cells (MUC1+/CD25−), 293 T cells (MUC1−/CD25−), L540 cells (MUC1−/CD25+), TR5 peptide-coated beads or, as control, with human serum albumin-coated beads, and (B) with quiescent lymphocytes from the peripheral blood (PBL) or with PBL activated by preincubation with the anti-CD3 mAb OKT3 plus IL2. Bound fusion proteins were detected by an FITC-labelled anti-human IgG antibody. Expression of the IL2 receptor CD25 was monitored by incubation with a FITC-conjugated anti-CD25 mAb (anti-CD25).
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Related In: Results  -  Collection

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

fig6: Fusion proteins C595scFv-Fc and C595scFv-Fc-IL2 bind specifically to MUC1 positive tumour cells and TR5 peptide-coated beads. Cell culture supernatants from transfected 293 T cells containing the C595scFv-Fc and C595scFv-Fc-IL2 fusion protein (thick lines), respectively, and, as control, supernatant from 293 T cells transfected with an irrelevant DNA (thin lines), were incubated (A) with T-47D cells (MUC1+/CD25−), 293 T cells (MUC1−/CD25−), L540 cells (MUC1−/CD25+), TR5 peptide-coated beads or, as control, with human serum albumin-coated beads, and (B) with quiescent lymphocytes from the peripheral blood (PBL) or with PBL activated by preincubation with the anti-CD3 mAb OKT3 plus IL2. Bound fusion proteins were detected by an FITC-labelled anti-human IgG antibody. Expression of the IL2 receptor CD25 was monitored by incubation with a FITC-conjugated anti-CD25 mAb (anti-CD25).
Mentions: The fusion proteins were furthermore assayed by flow cytometry for binding to MUC1-expressing tumour cells. As demonstrated in Figure 6AFigure 6

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