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

摘要: In preparation of a clinical phase I/II study in renal cell carcinoma (RCC) patients, we developed clinically applicable protocol that meets good practice (GCP) criteria regarding the gene transduction and expansion primary human T lymphocytes. We previously designed transgene encodes single chain (sc) FvG250 antibody chimeric receptor (ch-Rec), specific for RCC tumor-associated antigen (TAA), genetically programs lymphocytes with immune specificity. Here describe conditions activation, transduction, proliferation to yield: (a) optimal functional expression transgene; (b) ch-Rec-mediated cytokine production, (c) cytolysis G250-TAA(POS) by T-lymphocyte transductants. Moreover, these parameters were tested at scale, i.e., yielding up 5-10 x 10(9) T-cell transductants, defined as treatment dose according our protocol. The following were, first time, an interactive way: (1) media compositions production virus stable PG13 packaging cell; (2) activation reagents (anti-CD3 mAb; anti-CD3+anti-CD28 mAbs; PHA); (3) kinetics prior transduction; (4) (i) density, (ii) volume virus-containing supernatant per surface unit during (5) medium composition maintenance in-between cycles, vitro expansion. Critical scale appeared be use fibronectin fragment CH-296 (Retronectin) well Lifecell) X-fold culture bags. order comply GCP requirements, used: bovine serum-free system, supplemented autologous patients' plasma, closed system all lymphocyte processing. This routinely yields 30-65% scFvG250 ch-Rec(POS) both healthy donors patients.