We have designed a transgene that encodes a scFv(G250) chimeric receptor, which is specific for carboxyanhydrase IX (G250-ligand, G250L), a molecule overexpressed by renal cell cancer (RCC). Retroviral transduction of this transgene into primary human T lymphocytes confers these cells with specific functional responses towards G250L-positive RCC cells. In preparation of a clinical phase (I/II) study in RCC patients, we set up a protocol for gene transduction and expansion of primary human T cells. For this purpose, we directly compared two packaging cell lines, that is, the GALV-pseudotyped MLV producing cell line PG13, and the MLV-A-producing cell line Phi-NX-Ampho (a.k.a. Phoenix-A). We generated and characterized stable scFv(G250)-positive clones of both PG13 and Phoenix cells and optimized the retrovirus production conditions. Transductions of primary human T cells yielded 30–60% scFv(G250) þ T cells using PG13-derived retrovirus versus up to 90% scFv(G250) þ T cells using Phoenix-derived retrovirus. The median number of transgene integrations per scFv(G250) þ T cell differed only 1.5-fold as determined by real-time PCR (mean number of integrations per T cell 2.6 and 3.7 for PG13 and Phoenix-based transductions, respectively). In addition, T cells transduced with Phoenix-derived retrovirus showed, on a per cell basis, 10–30% higher levels of scFv(G250)-mediated TNFa production and cytolysis of G250L þ RCC cells than T cells ransduced with PG13-derived retrovirus. The improved functional transduction efficiency together with a limited increase in the number of integrations per recipient cell, made us select Phoenix clone 58 for our clinical immunogene therapy study.

Cancer, Cell Line, Expansion, G250, GMP production, Gene Therapy, Human, Lymphocytes, PCR, PG13, Packaging Cells, Packaging cell, Phoenix, Receptor, T Cell, T cells, T-Lymphocytes, Transgene, immunogene therapy, immunology, therapy
dx.doi.org/10.1038/sj.cgt.7700916, hdl.handle.net/1765/21351
Cancer Gene Therapy
Erasmus MC: University Medical Center Rotterdam

Lamers, C.H.J, Willemsen, R.A, van Elzakker, P.M.M.L, van Krimpen, B.A, Gratama, J.W, & Debets, J.E.M.A. (2006). Phoenix-ampho outperforms PG13 as retroviral packaging cells to transduce human T cells with tumor-specific receptors: implications for clinical immunogene therapy of cancer. Cancer Gene Therapy, 13(5), 503–509. doi:10.1038/sj.cgt.7700916