Phoenix-ampho outperforms PG13 as retroviral packaging cells to transduce human T cells with tumor-specific receptors: implications for clinical immunogene therapy of cancer.
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.
Lamers
C H J
CH
Unit of Clinical and Tumor Immunology, Department of Medical Oncology, Erasmus MC-Daniel den Hoed Cancer Center, Rotterdam, The Netherlands. c.lamers@erasmusmc.nl
Willemsen
R A
RA
van Elzakker
P
P
van Krimpen
B A
BA
Gratama
J W
JW
Debets
R
R
D000951:Antigens, Neoplasm / Q000235:genetics*; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases / Q000235:genetics*; D002460:Cell Line; D002478:Cells, Cultured; D003131:Combined Modality Therapy; D003602:Cytotoxicity, Immunologic; D015316:Genetic Therapy / Q000379:methods; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D016219:Immunotherapy, Adoptive / Q000379:methods; D007680:Kidney Neoplasms / Q000628:therapy*; D011971:Receptors, Immunologic / Q000235:genetics*; D012190:Retroviridae / Q000235:genetics; D057127:Single-Chain Antibodies; D013601:T-Lymphocytes / Q000276:immunology / Q000637:transplantation; D014161:Transduction, Genetic / Q000379:methods*; D019065:Virus Assembly
D000951:Antigens, Neoplasm; D007135:Immunoglobulin Variable Region; D011971:Receptors, Immunologic; C511531:ScFv(G250) protein; D057127:Single-Chain Antibodies; C089226:CA9 protein, human; D000071231:Carbonic Anhydrase IX; D002256:Carbonic Anhydrases