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Protocol for gene transduction and expansion of human T lymphocytes for clinical immunogene therapy of cancer

Abstract

In preparation of a clinical phase I/II study in renal cell carcinoma (RCC) patients, we developed a clinically applicable protocol that meets good clinical practice (GCP) criteria regarding the gene transduction and expansion of primary human T lymphocytes. We previously designed a transgene that encodes a single chain (sc) FvG250 antibody chimeric receptor (ch-Rec), specific for a RCC tumor-associated antigen (TAA), and that genetically programs human T lymphocytes with RCC immune specificity. Here we describe the conditions for activation, gene transduction, and proliferation for primary human T lymphocytes to yield: (a) optimal functional expression of the transgene; (b) ch-Rec–mediated cytokine production, and (c) cytolysis of G250-TAAPOS RCC by the T-lymphocyte transductants. Moreover, these parameters were tested at clinical scale, i.e., yielding up to 5–10×109 T-cell transductants, defined as the treatment dose according to our clinical protocol. The following parameters were, for the first time, tested in an interactive way: (1) media compositions for production of virus by the stable PG13 packaging cell; (2) T-lymphocyte activation conditions and reagents (anti-CD3 mAb; anti-CD3+anti-CD28 mAbs; and PHA); (3) kinetics of T-lymphocyte activation prior to gene transduction; (4) (i) T-lymphocyte density, and (ii) volume of virus-containing supernatant per surface unit during gene transduction; and (5) medium composition for T-lymphocyte maintenance (i) in-between gene transduction cycles, and (ii) during in vitro T-lymphocyte expansion. Critical to gene transduction of human T lymphocytes at clinical scale appeared to be the use of the fibronectin fragment CH-296 (Retronectin™) as well as Lifecell® X-fold™ cell culture bags. In order to comply with GCP requirements, we used: (a) bovine serum-free human T-lymphocyte transduction system, i.e., media supplemented with autologous patients' plasma, and (b) a closed cell culture system for all lymphocyte processing. This clinical protocol routinely yields 30–65% scFvG250 ch-RecPOS T lymphocytes in both healthy donors and RCC patients.

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Acknowledgements

The authors thank Brigitte van Krimpen and Pascal van Elzakker for their technical assistance; Nexell International for kindly providing Lifecell® X-fold™ culture bags; and Takara Shuzo (Otsu, Shiga, Japan) for kindly providing fibronectin fragment CH-296 (Retronectin™). The work was supported by the Dutch Technology Foundation STW (project RGN.3498), the Dutch Cancer Society (Nederlandse Kankerbestrijding project DDHK99-1865), the Cancer Research Institute (New York, NY, USA), and the EU Grant QLK3-1999-01-262.

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Lamers, C., Willemsen, R., Luider, B. et al. Protocol for gene transduction and expansion of human T lymphocytes for clinical immunogene therapy of cancer. Cancer Gene Ther 9, 613–623 (2002). https://doi.org/10.1038/sj.cgt.7700477

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