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T lymphocytes isolated from patients with advanced colorectal cancer are suitable for gene immunotherapy approaches.

Sheen AJ, Sherlock DJ, Irlam J, Hawkins RE, Gilham DE - Br. J. Cancer (2003)

Bottom Line: Retroviral gene transfer methods have been shown to transduce primary human T lymphocytes effectively resulting in the expression of therapeutic genes.In each case, functional-specific cytotoxic activity was observed.Negligible activity was found in control cultures.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Department of Medical Oncology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, University of Manchester, UK.

ABSTRACT
Despite improvements in treatment, the 5-year survival for metastatic colorectal cancer remains poor. Novel approaches such as gene immunotherapy are being investigated to improve treatment. Retroviral gene transfer methods have been shown to transduce primary human T lymphocytes effectively resulting in the expression of therapeutic genes. However, a number of defects have been identified in T lymphocytes isolated from patients bearing tumour, which may have critical implications for the development of gene-targeted T cells as an anticancer therapy. To address this issue, primary T lymphocytes were isolated from patients with advanced colorectal cancer and tested for their ability to be transduced and to express subsequently a chimeric immune receptor consisting of a single-chain antibody fragment antigen-binding moiety specific for carcinoembryonic antigen (CEA) fused to the T cell receptor (TCR) CD3zeta chain. In 10 out of 10 patients, T lymphocytes were transduced, expanded in the absence of selection and tested for functional activity against CEA-expressing tumour cells. In each case, functional-specific cytotoxic activity was observed. Negligible activity was found in control cultures. This study highlights the feasibility of patient-derived T lymphocytes as a source of immune cells for autologous gene immunotherapy approaches.

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Expression of chimeric receptor proteins in primary human T lymphocytes derived from patients with advanced colorectal cancer. (A) The specificity of the scFv used in these studies. (B) Chimeric receptor transduced and mock patient T lymphocytes, at time of cytotoxicity assays, were lysed with RIPA and subjected to reducing or nonreducing SDS–PAGE and transferred to nitrocellulose filters. Blots were probed with anti-CD3ζ Mab (1 : 1000 dilution, Pharmingen, Oxford, UK) with a secondary antibody of anti-mouse Fc-HRP conjugate (1 : 2000) and immunoreactive bands visualised using ECL™ (Amersham, UK). Under reducing conditions, the endogenous CD3ζ protein was identified as a 16 kDa band in all samples with the chimeric receptors clearly identified as single immunoreactive bands at the predicted molecular mass of 43 kDa. Chimeric homodimers (86 kDa) and heterodimers (60 kDa) formed with the endogenous CD3ζ were identified along with CD3ζ homodimers (32 kDa) under nonreducing conditions.
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fig1: Expression of chimeric receptor proteins in primary human T lymphocytes derived from patients with advanced colorectal cancer. (A) The specificity of the scFv used in these studies. (B) Chimeric receptor transduced and mock patient T lymphocytes, at time of cytotoxicity assays, were lysed with RIPA and subjected to reducing or nonreducing SDS–PAGE and transferred to nitrocellulose filters. Blots were probed with anti-CD3ζ Mab (1 : 1000 dilution, Pharmingen, Oxford, UK) with a secondary antibody of anti-mouse Fc-HRP conjugate (1 : 2000) and immunoreactive bands visualised using ECL™ (Amersham, UK). Under reducing conditions, the endogenous CD3ζ protein was identified as a 16 kDa band in all samples with the chimeric receptors clearly identified as single immunoreactive bands at the predicted molecular mass of 43 kDa. Chimeric homodimers (86 kDa) and heterodimers (60 kDa) formed with the endogenous CD3ζ were identified along with CD3ζ homodimers (32 kDa) under nonreducing conditions.

Mentions: Our aim is to utilise the power of the immune system as a novel form of cancer therapy. Gene therapy techniques have been developed to modify T lymphocytes in order to target and lyse colorectal tumour cells through the development of chimeric immune receptors (CIRs) (Hombach et al, 1999; Nolan et al, 1999; Beecham et al, 2000; Daly et al, 2000). CIRs are composed of antigen binding domains, usually derived from single-chain antibody fragments (scFv) (Hawkins et al, 1998), fused to the cytosolic domains of important signalling receptors (Gross et al, 1989) (Figure 1AFigure 1


T lymphocytes isolated from patients with advanced colorectal cancer are suitable for gene immunotherapy approaches.

Sheen AJ, Sherlock DJ, Irlam J, Hawkins RE, Gilham DE - Br. J. Cancer (2003)

Expression of chimeric receptor proteins in primary human T lymphocytes derived from patients with advanced colorectal cancer. (A) The specificity of the scFv used in these studies. (B) Chimeric receptor transduced and mock patient T lymphocytes, at time of cytotoxicity assays, were lysed with RIPA and subjected to reducing or nonreducing SDS–PAGE and transferred to nitrocellulose filters. Blots were probed with anti-CD3ζ Mab (1 : 1000 dilution, Pharmingen, Oxford, UK) with a secondary antibody of anti-mouse Fc-HRP conjugate (1 : 2000) and immunoreactive bands visualised using ECL™ (Amersham, UK). Under reducing conditions, the endogenous CD3ζ protein was identified as a 16 kDa band in all samples with the chimeric receptors clearly identified as single immunoreactive bands at the predicted molecular mass of 43 kDa. Chimeric homodimers (86 kDa) and heterodimers (60 kDa) formed with the endogenous CD3ζ were identified along with CD3ζ homodimers (32 kDa) under nonreducing conditions.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2376387&req=5

fig1: Expression of chimeric receptor proteins in primary human T lymphocytes derived from patients with advanced colorectal cancer. (A) The specificity of the scFv used in these studies. (B) Chimeric receptor transduced and mock patient T lymphocytes, at time of cytotoxicity assays, were lysed with RIPA and subjected to reducing or nonreducing SDS–PAGE and transferred to nitrocellulose filters. Blots were probed with anti-CD3ζ Mab (1 : 1000 dilution, Pharmingen, Oxford, UK) with a secondary antibody of anti-mouse Fc-HRP conjugate (1 : 2000) and immunoreactive bands visualised using ECL™ (Amersham, UK). Under reducing conditions, the endogenous CD3ζ protein was identified as a 16 kDa band in all samples with the chimeric receptors clearly identified as single immunoreactive bands at the predicted molecular mass of 43 kDa. Chimeric homodimers (86 kDa) and heterodimers (60 kDa) formed with the endogenous CD3ζ were identified along with CD3ζ homodimers (32 kDa) under nonreducing conditions.
Mentions: Our aim is to utilise the power of the immune system as a novel form of cancer therapy. Gene therapy techniques have been developed to modify T lymphocytes in order to target and lyse colorectal tumour cells through the development of chimeric immune receptors (CIRs) (Hombach et al, 1999; Nolan et al, 1999; Beecham et al, 2000; Daly et al, 2000). CIRs are composed of antigen binding domains, usually derived from single-chain antibody fragments (scFv) (Hawkins et al, 1998), fused to the cytosolic domains of important signalling receptors (Gross et al, 1989) (Figure 1AFigure 1

Bottom Line: Retroviral gene transfer methods have been shown to transduce primary human T lymphocytes effectively resulting in the expression of therapeutic genes.In each case, functional-specific cytotoxic activity was observed.Negligible activity was found in control cultures.

View Article: PubMed Central - PubMed

Affiliation: Cancer Research UK Department of Medical Oncology, Paterson Institute for Cancer Research, Christie Hospital NHS Trust, University of Manchester, UK.

ABSTRACT
Despite improvements in treatment, the 5-year survival for metastatic colorectal cancer remains poor. Novel approaches such as gene immunotherapy are being investigated to improve treatment. Retroviral gene transfer methods have been shown to transduce primary human T lymphocytes effectively resulting in the expression of therapeutic genes. However, a number of defects have been identified in T lymphocytes isolated from patients bearing tumour, which may have critical implications for the development of gene-targeted T cells as an anticancer therapy. To address this issue, primary T lymphocytes were isolated from patients with advanced colorectal cancer and tested for their ability to be transduced and to express subsequently a chimeric immune receptor consisting of a single-chain antibody fragment antigen-binding moiety specific for carcinoembryonic antigen (CEA) fused to the T cell receptor (TCR) CD3zeta chain. In 10 out of 10 patients, T lymphocytes were transduced, expanded in the absence of selection and tested for functional activity against CEA-expressing tumour cells. In each case, functional-specific cytotoxic activity was observed. Negligible activity was found in control cultures. This study highlights the feasibility of patient-derived T lymphocytes as a source of immune cells for autologous gene immunotherapy approaches.

Show MeSH
Related in: MedlinePlus