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

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) 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 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.

Gene-modified T cells for adoptive immunotherapy of renal cell cancer maintain transgene-specific immune functions in vivo

“…K562) to the test. The speciWcity of the scFv(G250)-mediated eVector functions was veriWed by blocking CAIX on the target cells by adding G250 mAb (at a saturating Wnal concentration of 25 g/ml) to the target cells 15 min before addition of the eVector cells [10,11].…”

“…speciWc cytotoxic activity and cytokine production, using the CAIX + RCC cell line SKRC17-MW1 (clone 4) [10,11]. A scFv(G250)-chimeric receptorexpressing T-cell clone (Clone 46) was used as positive control in these assays.…”

“…Autologous T lymphocytes were transduced with the scFv(G250)-chimeric receptor using retroviral transduction [11,12]. The treatment schedule consisted of i.v.…”

Retroviral vectors for clinical immunogene therapy are stable for up to 9 years

“…14 To this end, we have constructed two stable packaging cell lines that produce a SFG-scFv(G250)-CD4g retrovirus. 15,16 In the current paper, we have performed a long-term functional potency (that is, stability) study of RTVsup. RTVsups derived from the virus-producing PG13 clone 1.2, 15 containing the gibbon ape leukemia virus (GaLV)-enveloped SFG-scFv(G250)-CD4g retrovirus were tested for their functional transduction capacity on primary human T lymphocytes over a period of 9 years (1998-2007).…”

“…RTVsups derived from the virus-producing PG13 clone 1.2, 15 containing the gibbon ape leukemia virus (GaLV)-enveloped SFG-scFv(G250)-CD4g retrovirus were tested for their functional transduction capacity on primary human T lymphocytes over a period of 9 years (1998-2007). Phoenix(Ampho)-derived SFG-scFv(G250)-CD4g retrovirus, containing the MLV envelope, showed improved functional transduction efficiency when compared with PG13-derived retrovirus 16 and was selected for our clinical immunogene therapy study.…”