Phoenix-ampho outperforms PG13 as retroviral packaging cells to transduce human T cells with tumor-specific receptors: implications for clinical immunogene therapy of cancer.

Unit of Clinical and Tumor Immunology, Department of Medical Oncology, Erasmus MC-Daniel den Hoed Cancer Center, Rotterdam, The Netherlands.
Cancer Gene Therapy (Impact Factor: 2.55). 06/2006; 13(5):503-9. DOI: 10.1038/sj.cgt.7700916
Source: PubMed

ABSTRACT 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)+ T cells using PG13-derived retrovirus versus up to 90% scFv(G250)+ T cells using Phoenix-derived retrovirus. The median number of transgene integrations per scFv(G250)+ 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 TNFalpha production and cytolysis of G250L+ RCC cells than T cells transduced 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.


Available from: Reno Debets, Jun 09, 2014
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    ABSTRACT: TCR gene therapy represents a feasible and promising treatment for patients with cancer and virus infections. Currently, this treatment rationale is hampered by diluted surface expression of the TCR transgene and generation of potentially self reactive T-cells, both a direct consequence of mis-pairing with endogenous TCR chains. As we reported previously (Gene Ther 16:1369, 2000; J Immunol 180:7736, 2008), TCR mis-pairing can be successfully addressed by a TCR:CD3 fusion protein (i.e., TCR:). Here, we set out to minimize the content of CD3 in TCR:, specific for MAGE-A1/HLA-A1, without compromising TCR pairing and function. Domain-exchange and 3D-modeling strategies defined a set of minimal TCR: variants, which, together with a murinized and cysteine-modified TCR (TCR:mu+cys), were tested for functional TCR expression and TCR pairing. Our data with Jurkat T cells show that the CD3 transmembrane domain is important for cell-surface expression, whereas the CD3 intracellular domain is crucial for T-cell activation. Notably, inability of TCR: to mis-pair was not observed for TCR:mu+cys, which depended exclusively on the transmembrane domain of CD3 and could not be recapitulated by a limited number of structurally defined CD3 transmembrane amino acids. The extracellular CD3 domain was dispensable for TCR:'s ability to prevent TCR mis-pairing, bind pMHC and mediate NFAT activation. In primary human T cells, however, minimal TCR: without CD3's extracellular domain but not TCR: nor TCR:mu+cys revealed compromised cell surface expression and T cell function. Taken together, our study demonstrates that CD3's transmembrane domain dictates TCR:'s inability to TCR mis-pair, but only TCR coupled to complete CD3 and not its minimal variants were functionally expressed in primary T cells.