Department of Medical Oncology, Subdivision of Clinical and Tumor Immunology, Erasmus Medical Center-Daniel, Rotterdam, The Netherlands

Correspondence to: Dr Cor H J Lamers, Department of Medical Oncology, Subdivision of Clinical and Tumor Immunology, Erasmus Medical Center-Daniel, PO Box 5201, 3008 AE Rotterdam, The Netherlands. E-mail: lamers@immh.azr.nl

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-TAA^POS 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 (RetronectinÔ) as well as LifecellÒ X-foldÔ 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-Rec^POS T lymphocytes in both healthy donors and RCC patients. Cancer Gene Therapy (2002) 9, 613-623 doi:10.1038/sj.cgt.7700477

clinical protocol; immunogene therapy; human T lymphocytes; single chain chimeric receptor; renal cell carcinoma