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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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Tumour target cell survival after coculture with targeted and nontargeted T lymphocytes. Adherent tumour cell lines (5000) were cultured with either MFE23.CD3ζTDGA (▪) or the D29.CD3ζTDGA (▴) chimeric receptors or control mock-transduced T cells (♦), generated from Patient 10, at the indicated effector : target ratios (The transduced effector : target ratio based upon EGFP fluorescence is also presented.) in wells of flat-bottomed 96-well plates in a final volume of 200 μl. Interleukin-2 was added to some wells at a final concentration of 100 IU ml−1 (E–H). After 24 h, media and nonadherent cells were removed and replaced with target cell culture medium without T-cell cytokines. After a further 5 days of culture, the wells were aspirated, washed with PBS and 100 μl of DMEM/10% FBS containing a 1 : 40 dilution of wst-1 reagent (Roche, Surrey, UK). The optical density of the wells was assessed at 450–650 nm using an ELISA plate reader (Molecular Devices, Sunnydale, CA, USA). Maximal tumour cell growth was assessed from the optical density of wells containing tumour cells only and minimal growth determined from wells containing tumour cells lysed with 2% Triton X-100. Target cell lines used were MKN45 K (A), LS174 T (B), SK-N-BE (C) and HeLa (D). The results are presented as the mean±s.d. values of quadruplicate wells.
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fig4: Tumour target cell survival after coculture with targeted and nontargeted T lymphocytes. Adherent tumour cell lines (5000) were cultured with either MFE23.CD3ζTDGA (▪) or the D29.CD3ζTDGA (▴) chimeric receptors or control mock-transduced T cells (♦), generated from Patient 10, at the indicated effector : target ratios (The transduced effector : target ratio based upon EGFP fluorescence is also presented.) in wells of flat-bottomed 96-well plates in a final volume of 200 μl. Interleukin-2 was added to some wells at a final concentration of 100 IU ml−1 (E–H). After 24 h, media and nonadherent cells were removed and replaced with target cell culture medium without T-cell cytokines. After a further 5 days of culture, the wells were aspirated, washed with PBS and 100 μl of DMEM/10% FBS containing a 1 : 40 dilution of wst-1 reagent (Roche, Surrey, UK). The optical density of the wells was assessed at 450–650 nm using an ELISA plate reader (Molecular Devices, Sunnydale, CA, USA). Maximal tumour cell growth was assessed from the optical density of wells containing tumour cells only and minimal growth determined from wells containing tumour cells lysed with 2% Triton X-100. Target cell lines used were MKN45 K (A), LS174 T (B), SK-N-BE (C) and HeLa (D). The results are presented as the mean±s.d. values of quadruplicate wells.

Mentions: Multiple cell lines were used in order to examine the activity of the anti-CEA-targeted T cells. The CEA-expressing human colon adenocarcinoma lines (LS174 T and LoVo) as well as the gastric carcinoma cell line (MKN45 K) were tested as targets along with HeLa cells used as a control since they do not express CEA. This subsequent work utilised T lymphocytes generated from four further patients (7–10, Table 1). The results depicted in Figure 4Figure 4


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)

Tumour target cell survival after coculture with targeted and nontargeted T lymphocytes. Adherent tumour cell lines (5000) were cultured with either MFE23.CD3ζTDGA (▪) or the D29.CD3ζTDGA (▴) chimeric receptors or control mock-transduced T cells (♦), generated from Patient 10, at the indicated effector : target ratios (The transduced effector : target ratio based upon EGFP fluorescence is also presented.) in wells of flat-bottomed 96-well plates in a final volume of 200 μl. Interleukin-2 was added to some wells at a final concentration of 100 IU ml−1 (E–H). After 24 h, media and nonadherent cells were removed and replaced with target cell culture medium without T-cell cytokines. After a further 5 days of culture, the wells were aspirated, washed with PBS and 100 μl of DMEM/10% FBS containing a 1 : 40 dilution of wst-1 reagent (Roche, Surrey, UK). The optical density of the wells was assessed at 450–650 nm using an ELISA plate reader (Molecular Devices, Sunnydale, CA, USA). Maximal tumour cell growth was assessed from the optical density of wells containing tumour cells only and minimal growth determined from wells containing tumour cells lysed with 2% Triton X-100. Target cell lines used were MKN45 K (A), LS174 T (B), SK-N-BE (C) and HeLa (D). The results are presented as the mean±s.d. values of quadruplicate wells.
© Copyright Policy
Related In: Results  -  Collection

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fig4: Tumour target cell survival after coculture with targeted and nontargeted T lymphocytes. Adherent tumour cell lines (5000) were cultured with either MFE23.CD3ζTDGA (▪) or the D29.CD3ζTDGA (▴) chimeric receptors or control mock-transduced T cells (♦), generated from Patient 10, at the indicated effector : target ratios (The transduced effector : target ratio based upon EGFP fluorescence is also presented.) in wells of flat-bottomed 96-well plates in a final volume of 200 μl. Interleukin-2 was added to some wells at a final concentration of 100 IU ml−1 (E–H). After 24 h, media and nonadherent cells were removed and replaced with target cell culture medium without T-cell cytokines. After a further 5 days of culture, the wells were aspirated, washed with PBS and 100 μl of DMEM/10% FBS containing a 1 : 40 dilution of wst-1 reagent (Roche, Surrey, UK). The optical density of the wells was assessed at 450–650 nm using an ELISA plate reader (Molecular Devices, Sunnydale, CA, USA). Maximal tumour cell growth was assessed from the optical density of wells containing tumour cells only and minimal growth determined from wells containing tumour cells lysed with 2% Triton X-100. Target cell lines used were MKN45 K (A), LS174 T (B), SK-N-BE (C) and HeLa (D). The results are presented as the mean±s.d. values of quadruplicate wells.
Mentions: Multiple cell lines were used in order to examine the activity of the anti-CEA-targeted T cells. The CEA-expressing human colon adenocarcinoma lines (LS174 T and LoVo) as well as the gastric carcinoma cell line (MKN45 K) were tested as targets along with HeLa cells used as a control since they do not express CEA. This subsequent work utilised T lymphocytes generated from four further patients (7–10, Table 1). The results depicted in Figure 4Figure 4

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