Lamers, CH, Willemsen, RA, van Elzakker, P, van Krimpen, BA, Gratama, JW and Debets, R. 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 Ther 13: 503-509

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: 2.42). 06/2006; 13(5):503-9. DOI: 10.1038/sj.cgt.7700916
Source: PubMed


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.

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    • "Nonspecific cytolysis was assessed using the activated kill (AK)-sensitive Burkitt's lymphoma cell line Daudi. The percentage of specific cytolysis was expressed as weighted mean of specific lysis (WMSL) at effector cell-to-target cell ratio of 20:1, as described (Lamers et al., 2006c). "
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    ABSTRACT: Therapy with autologous T cells that have been gene-engineered to express chimeric antigen receptors (CAR) or T cell receptors (TCR) provides a feasible and broadly applicable treatment for cancer patients. In a clinical study in advanced renal cell carcinoma (RCC) patients with CAR T cells specific for Carbonic Anhydrase IX (CAIX) we observed toxicities which (most likely) indicated in vivo function of CAR T cells as well as low T cell persistence and clinical response rates. The latter observations were confirmed by later clinical trials in other solid tumor types and other gene-modified T cells. To improve the efficacy of T cell therapy, we have re-defined in vitro conditions to generate T cells with young phenotype, a key correlate with clinical outcome. For their impact on gene modified T cell phenotype and function, we have tested various anti-CD3/CD28 mAb-based T cell activation and expansion conditions as well as several cytokines prior to and/or post gene transfer using two different receptors: CAIX CAR and MAGE-C2(ALK)/HLA-A2 TCR. In a total set of 16 healthy donors, we observed that T cell activation with soluble anti-CD3/CD28 mAbs in the presence of both IL15 and IL21 prior to TCR gene transfer resulted in enhanced proportions of gene-modified T cells with a preferred in vitro phenotype and better function. T cells generated according to these processing methods demonstrated enhanced binding of pMHC, and an enhanced proportion of CD8+,CD27+,CD62L+,CD45RA+ T cells. These new conditions will be translated into a GMP protocol in preparation of a clinical adoptive therapy trial to treat patients with MAGE-C2-positive tumors.
    Human Gene Therapy Methods 11/2014; 25(6). DOI:10.1089/hgtb.2014.051 · 2.44 Impact Factor
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    • "Human T lymphocytes of healthy donors were activated with anti- CD3 mAb and transduced with retrovirus harboring either MAGE-specific or control TCRα and β transgenes. The transduction procedure was described by Lamers and colleagues [50] except that in the current study TCR-encoding retroviruses were produced by a coculture of 293T and Phoenix- Ampho packaging cells. T cells were FACSorted using the corresponding p/MHC multimer prior to functional assays. "
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    ABSTRACT: Adoptive therapy with TCR gene-engineered T cells provides an attractive and feasible treatment option for cancer patients. Further development of TCR gene therapy requires the implementation of T-cell target epitopes that prevent "on-target" reactivity towards healthy tissues and at the same time direct a clinically effective response towards tumor tissues. Candidate epitopes that meet these criteria are MAGE-C2(336-344)/HLA-A2 (MC2/A2) and MAGE-A3(243-258)/HLA-DP4 (MA3/DP4). We molecularly characterized TCRαβ genes of an MC2/A2-specific CD8 and MA3/DP4-specific CD4 T-cell clone derived from melanoma patients who responded clinically to MAGE vaccination. We identified MC2/A2 and MA3/DP4-specific TCR-Vα3/Vβ28 and TCR-Vα38/Vβ2 chains and validated these TCRs in vitro upon gene transfer into primary human T cells. The MC2 and MA3 TCR were surface-expressed and mediated CD8 T-cell functions towards melanoma cell lines and CD4 T-cell functions towards dendritic cells, respectively. We intend to start testing these MAGE-specific TCRs in phase I clinical trial.
    Clinical and Developmental Immunology 02/2012; 2012(5-6):586314. DOI:10.1155/2012/586314 · 2.93 Impact Factor
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    • "4.5. Retroviral gene transduction of CARs into human T cells Primary human T cells were retrovirally transduced according to a protocol optimized and reported previously (Lamers et al., 2006b), with the exception that transiently transected packaging cells instead of a stably transduced packaging clone were used as a source of virus particles. In short, 24 well culture plates were coated with RetroNectin and pre-treated with CAR-positive and MLV-enveloped retroviral particles, derived from a 1:1 mixture of transfected 293T and Phoenix cells. "
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    ABSTRACT: Gene-engineered T cell therapy represents a promising strategy to treat cancers. To enable pre-selection of patients sensitive to this type of treatment we have setup and validated a T cell activation assay to test antigen expression on patient-derived tumor tissues. Chimeric antibody-based receptor (CAR) directed against CAIX, currently used in a clinical trial to treat RCC patients, was used as a model receptor. Primary human T cells expressing CAIX CAR were able to respond to CAIX-positive but not CAIX-negative tumor tissue and showed an increased production of IFNgamma, TNFalpha, IL-10 and IL-4, but not IL-2 or IL-5. Tumor tissue driven responses of primary T cells were paralleled by NFAT activation measured in CAR-transduced Jurkat T cells, which was shown to be triggered in a CAR and antigen-specific manner. Next, the reporter gene assay was applied to two independent PSMA CARs, which both mediated NFAT activation in response to tumor tissue. Taken together, a sensitive and donor-independent assay was established to measure T cell activation upon exposure to patient-derived tumor tissue, which may facilitate pre-selection of patients for clinical adoptive T cell therapy.
    Journal of immunological methods 05/2010; 359(1-2):11-20. DOI:10.1016/j.jim.2010.04.006 · 1.82 Impact Factor
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