title: 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-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.
authors.affiliation: Department of Medical Oncology, Subdivision of Clinical and Tumor Immunology, Erasmus Medical Center-Daniel, Rotterdam, The Netherlands. lamer@immh.azr.nl
authors.forename: Cor H J
authors.initials: CH
authors.lastname: Lamers
authors.forename: Ralph A
authors.initials: RA
authors.lastname: Willemsen
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Luider
authors.forename: Reno
authors.initials: R
authors.lastname: Debets
authors.forename: Reinder L H
authors.initials: RL
authors.lastname: Bolhuis
mesh_terms: D000911:Antibodies, Monoclonal; D018106:CD28 Antigens / Q000235:genetics; D017252:CD3 Complex / Q000235:genetics; D015704:CD4 Antigens / Q000235:genetics; D002292:Carcinoma, Renal Cell / Q000628:therapy; D002455:Cell Division; D016895:Culture Media, Serum-Free; D005434:Flow Cytometry; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods*; D006801:Humans; D007167:Immunotherapy / Q000379:methods*; D007680:Kidney Neoplasms / Q000628:therapy; D007963:Leukocytes, Mononuclear / Q000166:cytology; D009369:Neoplasms / Q000628:therapy*; D012190:Retroviridae / Q000235:genetics; D013050:Spectrometry, Fluorescence; D013601:T-Lymphocytes / Q000378:metabolism*; D013997:Time Factors; D014161:Transduction, Genetic; D019076:Transgenes; D014407:Tumor Cells, Cultured
chemical_list: D000911:Antibodies, Monoclonal; D018106:CD28 Antigens; D017252:CD3 Complex; D015704:CD4 Antigens; D016895:Culture Media, Serum-Free