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Redirecting mouse T hybridoma against human breast and ovarian carcinomas: in vivo activity against HER-2/neu expressing cancer cells.

Gritzapis AD, Mamalaki A, Kretsovali A, Papamatheakis J, Belimezi M, Perez SA, Baxevanis CN, Papamichail M - Br. J. Cancer (2003)

Bottom Line: The scFv(anti-HER-2/neu)/zeta chimeric gene was successfully expressed as a functional surface receptor in the MD.45 CTL hybridoma (MD.45-HER/zeta).The MD.45-HER/zeta-transduced cells also lysed HER-2/neu(+) target cells in vitro with high specificity.The adoptively transferred MD.45-HER/zeta cells both slowed significantly the growth of human FM3 melanoma or murine ALC leukaemic cells both transfected to express HER-2/neu.

View Article: PubMed Central - PubMed

Affiliation: Saint Savas Cancer Hospital, Cancer Immunology and Immunotherapy Center, Athens, Greece.

ABSTRACT
Chimeric receptors comprising of the T-cell receptor-zeta cytoplasmic signalling chain fused to an extracellular ligand-binding domain of a single-chain antibody (scFv) have served as effective tools for redirecting cytotoxic T lymphocytes (CTL) against tumour cells. In this report, we constructed a chimeric scFv/zeta gene composed of the variable regions of an HER-2/neu-specific monoclonal antibody (MAb) joined to the TCR-zeta chain. The scFv(anti-HER-2/neu)/zeta chimeric gene was successfully expressed as a functional surface receptor in the MD.45 CTL hybridoma (MD.45-HER/zeta). More importantly, the scFv(anti-HER-2/neu)/zeta receptor was functionally active, since it triggered cytokine secretion by the MD.45-HER/zeta cells upon recognition of HER-2/neu-positive (+) tumour cell lines, or primary tumour cells from patients with HER-2/neu(+) cancers. The MD.45-HER/zeta-transduced cells also lysed HER-2/neu(+) target cells in vitro with high specificity. We tested the antitumour efficacy of scFv(anti-HER-2/neu)/zeta expressing MD.45 cells in severe combined immunodeficiency disease mice/human and murine tumour models. The adoptively transferred MD.45-HER/zeta cells both slowed significantly the growth of human FM3 melanoma or murine ALC leukaemic cells both transfected to express HER-2/neu. Our data demonstrate the feasibility of redirecting MD.45 CTL with the scFv(anti-HER-2/neu)/zeta chimeric receptor to respond specifically against HER-2/neu expressing tumour cells in vitro and in vivo. Moreover, they make it likely that T cells transduced with the same chimeric gene might be utilised in the treatment of patients with HER-2/neu(+) tumours.

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FACS analysis of immunofluorescence staining of MD.45 hybridoma transduced with pLRNLscFv(anti-HER-2/neu)/ζ (MD.45-HER/ζ) or with the vector alone (MD.45-mock). Expression of scFv(anti-HER-2/neu)/ζ was detected with an anti-Flag MAb plus FITC-labelled anti-mouse Fab′ (solid lines). Dotted lines: staining with FITC-labelled anti-mouse Fab′ alone.
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fig3: FACS analysis of immunofluorescence staining of MD.45 hybridoma transduced with pLRNLscFv(anti-HER-2/neu)/ζ (MD.45-HER/ζ) or with the vector alone (MD.45-mock). Expression of scFv(anti-HER-2/neu)/ζ was detected with an anti-Flag MAb plus FITC-labelled anti-mouse Fab′ (solid lines). Dotted lines: staining with FITC-labelled anti-mouse Fab′ alone.

Mentions: Expression of the chimeric scFv(anti-HER-2/neu)/ζ receptor selective for HER-2/neu+ tumour cells was performed by constructing one continuous molecule comprising gene segments of the variable region of the murine anti-HER-2/neu MAb produced by the HB8696 hybridoma and the signal-transducing human TCR-ζ chain transmembrane and intracellular region. Introduction of the chimeric scFv(anti-HER-2/neu)/ζ gene into the MD.45-murine CTL hybridoma, resulted in the expression of the chimeric molecule on the cell surface of selected clones as revealed after staining with the anti-Flag-FITC MAb (Figure 3Figure 3


Redirecting mouse T hybridoma against human breast and ovarian carcinomas: in vivo activity against HER-2/neu expressing cancer cells.

Gritzapis AD, Mamalaki A, Kretsovali A, Papamatheakis J, Belimezi M, Perez SA, Baxevanis CN, Papamichail M - Br. J. Cancer (2003)

FACS analysis of immunofluorescence staining of MD.45 hybridoma transduced with pLRNLscFv(anti-HER-2/neu)/ζ (MD.45-HER/ζ) or with the vector alone (MD.45-mock). Expression of scFv(anti-HER-2/neu)/ζ was detected with an anti-Flag MAb plus FITC-labelled anti-mouse Fab′ (solid lines). Dotted lines: staining with FITC-labelled anti-mouse Fab′ alone.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2747561&req=5

fig3: FACS analysis of immunofluorescence staining of MD.45 hybridoma transduced with pLRNLscFv(anti-HER-2/neu)/ζ (MD.45-HER/ζ) or with the vector alone (MD.45-mock). Expression of scFv(anti-HER-2/neu)/ζ was detected with an anti-Flag MAb plus FITC-labelled anti-mouse Fab′ (solid lines). Dotted lines: staining with FITC-labelled anti-mouse Fab′ alone.
Mentions: Expression of the chimeric scFv(anti-HER-2/neu)/ζ receptor selective for HER-2/neu+ tumour cells was performed by constructing one continuous molecule comprising gene segments of the variable region of the murine anti-HER-2/neu MAb produced by the HB8696 hybridoma and the signal-transducing human TCR-ζ chain transmembrane and intracellular region. Introduction of the chimeric scFv(anti-HER-2/neu)/ζ gene into the MD.45-murine CTL hybridoma, resulted in the expression of the chimeric molecule on the cell surface of selected clones as revealed after staining with the anti-Flag-FITC MAb (Figure 3Figure 3

Bottom Line: The scFv(anti-HER-2/neu)/zeta chimeric gene was successfully expressed as a functional surface receptor in the MD.45 CTL hybridoma (MD.45-HER/zeta).The MD.45-HER/zeta-transduced cells also lysed HER-2/neu(+) target cells in vitro with high specificity.The adoptively transferred MD.45-HER/zeta cells both slowed significantly the growth of human FM3 melanoma or murine ALC leukaemic cells both transfected to express HER-2/neu.

View Article: PubMed Central - PubMed

Affiliation: Saint Savas Cancer Hospital, Cancer Immunology and Immunotherapy Center, Athens, Greece.

ABSTRACT
Chimeric receptors comprising of the T-cell receptor-zeta cytoplasmic signalling chain fused to an extracellular ligand-binding domain of a single-chain antibody (scFv) have served as effective tools for redirecting cytotoxic T lymphocytes (CTL) against tumour cells. In this report, we constructed a chimeric scFv/zeta gene composed of the variable regions of an HER-2/neu-specific monoclonal antibody (MAb) joined to the TCR-zeta chain. The scFv(anti-HER-2/neu)/zeta chimeric gene was successfully expressed as a functional surface receptor in the MD.45 CTL hybridoma (MD.45-HER/zeta). More importantly, the scFv(anti-HER-2/neu)/zeta receptor was functionally active, since it triggered cytokine secretion by the MD.45-HER/zeta cells upon recognition of HER-2/neu-positive (+) tumour cell lines, or primary tumour cells from patients with HER-2/neu(+) cancers. The MD.45-HER/zeta-transduced cells also lysed HER-2/neu(+) target cells in vitro with high specificity. We tested the antitumour efficacy of scFv(anti-HER-2/neu)/zeta expressing MD.45 cells in severe combined immunodeficiency disease mice/human and murine tumour models. The adoptively transferred MD.45-HER/zeta cells both slowed significantly the growth of human FM3 melanoma or murine ALC leukaemic cells both transfected to express HER-2/neu. Our data demonstrate the feasibility of redirecting MD.45 CTL with the scFv(anti-HER-2/neu)/zeta chimeric receptor to respond specifically against HER-2/neu expressing tumour cells in vitro and in vivo. Moreover, they make it likely that T cells transduced with the same chimeric gene might be utilised in the treatment of patients with HER-2/neu(+) tumours.

Show MeSH
Related in: MedlinePlus