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 x 10(9) 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-Rec(POS) T lymphocytes in both healthy donors and RCC patients

ANTIGEN, ANTIGENS, Antibodies, Antibodies,Monoclonal, Antigens,CD28, Antigens,CD3, Antigens,CD4, CD3, Cancer, Carcinoma,Renal Cell, Cell Culture, Cell Division, Culture Media, EXPANSION, EXPRESSION, Flow Cytometry, G250, GMP production, Gene Therapy, Gene Transfer Techniques, Human, Immunotherapy, In Vitro, Kidney Neoplasms, Kinetics, Leukocytes,Mononuclear, Lymphocytes, Neoplasms, Phoenix, RCR, RECEPTOR, Renal cell carcinoma, Retroviridae, Spectrometry,Fluorescence, Support,Non-U.S.Gov't, T-Lymphocytes, Time Factors, Transduction,Genetic, Transgenes, Tumor Cells,Cultured, carcinoma, cytology, genetics, immunogene therapy, immunology, metabolism, methods, therapy,
Cancer Gene Therapy
Erasmus MC: University Medical Center Rotterdam

Lamers, C.H.J, Willemsen, R.A, Luider, B.A, Debets, J.E.M.A, & Bolhuis, R.L.H. (2002). Protocol for gene transduction and expansion of human T lymphocytes for clinical immunogene therapy of cancer. Cancer Gene Therapy, 9(7), 613–623. doi:10.1038/sj.cgt.7700477