Phase I dose-escalation study of F60008, a novel apoptosis inducer, in patients with advanced solid tumours.

Resistance of cancer cells to cytotoxic therapy can be caused by the activation of strong anti-apoptotic effectors, for example NF-kappaB. Therefore, compounds that inhibit NF-kappaB stimulation might overcome chemotherapy resistance. F60008, a semi-synthetic derivate of triptolide, is converted to triptolide in vivo and activates apoptosis in human tumour cells. We performed a phase I and pharmacological study of F60008 given intravenously as a weekly infusion for 2 weeks every 3 weeks in patients with advanced solid tumours. Twenty patients were enrolled, and a total of 35 cycles were administered. The most frequent haematological side-effect was mild grade 1-2 anaemia. Non-haematological toxicities included fatigue, nausea, vomiting, diarrhoea and constipation, all grade 1-2. Two lethal events were observed in which an increase in caspase-3 activity and overt apoptosis in monocytes and neutrophils could be seen. Pharmacokinetic studies showed high inter-individual variability and rendered F60008 a far from optimal derivate of triptolide.

Retronectin-assisted retroviral transduction of primary human T lymphocytes under good manufacturing practice conditions: tissue culture bag critically determines cell yield.

BACKGROUND: For our clinical immunogene therapy study for the treatment of renal cell carcinoma (RCC) patients, we had developed a protocol for gene transduction and expansion of human T cells in compliance with good manufacturing practice (GMP) criteria. Critical to our successful clinical-scale transductions of patient T cells was the use of Retronectin in combination with Lifecell X-foldtrade mark cell culture bags. METHODS: In our current study, we evaluated two alternative types of bags for the Retronectin-mediated retroviral transduction of human T cells: the Miltenyi DC-generation bag and the Takara CultiLife Spin bag. RESULTS: In static transductions, but not in spinoculation, the DC-generation bags and CultiLife Spin bags performed as well as Lifecell X-foldtrade mark bags in Retronectin-assisted retroviral transduction of human T cells with respect to transduction efficiency, lymphocyte subset composition and lymphocyte function. However, both types of bags performed less well than Lifecell X-foldtrade mark cell culture bags in terms of cell yield. DISCUSSION: Adjusted numbers of cells at the start of transduction should be used when using the Miltenyi or Takara bags in order to compensate for the lower cell yield following transduction.

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

Recombinant retroviruses are one of the most commonly used gene transfer vehicles for therapeutic gene delivery. The stability of viral vectors upon long-term storage, anticipated to be short lived, is expected to impact timeline and financial course of clinical immunogene therapy. However, to date little is known about vector stability. Therefore, we analyzed the stability of retroviral vectors produced in culture supernatants (RTVsup) for ex vivo gene therapy upon long-term storage. We have generated RTVsups derived from two packaging cell lines, PG13 and Phoenix(Ampho). Both lines produced murine leukemia virus-derived SFG-scFv(G250)-CD4gamma vector, which were pseudotyped with the gibbon ape leukemia virus envelope and amphotropic envelope, respectively. The supernatants were stored at -80 or -196 degrees C. To date, the PG13-derived RTVsups have been evaluated over a period of 9 years (1998-2007). In addition, a clinical batch of Phoenix(Ampho)-derived RTVsup has been evaluated over a period of 5 years (2002-2007). Here, we show that both RTVsups, when stored up to 9 and 5 years, respectively, do not show any sign of decay in their capacity to functionally transduce primary human T cells. These data provide evidence that in terms of 'life expectancy' the production and storage of clinical batches of RTVsup for gene therapy warrants the corresponding professional and financial risks.

Bone-marrow transplantation fails to halt intrathecal lymphocyte activation in multiple sclerosis.

BACKGROUND: Given the presumed key role for autoreactive lymphocytes in multiple sclerosis (MS), treatment strategies have been developed to ablate lymphocyte activity. Intrathecal lymphocyte activation can be measured by CSF-soluble(s)CD27. OBJECTIVE: To determine the effect of maximum whole-body immune ablation on two different markers that detect lymphocyte activation in CSF-oligoclonal IgG bands and levels of CSF-sCD27. DESIGN, SETTING AND PATIENTS: The study quantified sCD27 levels and assessed the presence of oligoclonal IgG bands in CSF samples of secondary progressive patients with MS treated by autologous bone-marrow transplantation. In eight individuals, CSF was taken before and 6-9 months after conditioning. CSF-sCD27 levels were compared with other MS and non-inflammatory neurological disease controls. Regarding the effect of stem-cell transplantation on CSF oligoclonal bands, the study analysed pooled data of this and four other international studies on stem-cell transplantation in MS. RESULTS: CSF-sCD27 was significantly lower after the extremely immunoablative protocol. However, levels remained elevated compared with non-inflammatory controls and stayed within the range observed in other MS controls. The joint analysis of CSF oligoclonal bands demonstrated persistence of this immune abnormality in 88% of the reported cases (n = 34). CONCLUSIONS: The persistence of CSF lymphocyte activation markers sCD27 and intrathecal oligoclonal IgG bands after maximum immunoablative treatment indicates that complete eradication of activated lymphocytes from the CNS has not been established. This is paralleled by disease progression observed in several studies on the effect of stem-cell transplantation in MS.

Process validation and clinical evaluation of a protocol to generate gene-modified T lymphocytes for imunogene therapy for metastatic renal cell carcinoma: GMP-controlled transduction and expansion of patient's T lymphocytes using a carboxy anhydrase IX-specific scFv transgene.

BACKGROUND: Adoptive transfer of autologous T cells that are gene-transduced to express Ag-specific receptors represents an experimental strategy to provide tumor-specific immunity to cancer patients. We studied this concept in patients with metastatic renal cell cancer (RCC) using retroviral transduction of T cells with a single-chain Ab-G250 chimeric receptor [scFv(G250)]. We describe the validation of our clinical protocol for gene transduction and expansion of human T lymphocytes. METHODS: A batch of scFv(G250) transgene-containing retrovirus was produced under conditions of good manufacturing practice (GMP). In addition to quality control and safety testing of the virus batch, extensive potency testing was performed, i.e. assessment of its functional transduction efficiency in primary human T cells. Subsequently, the clinical gene transduction and cell-expansion protocol was subjected to a series of process validations and a clinical evaluation using T cells obtained from healthy donors and three RCC patients. RESULTS: The clinical batch of scFv(G250) transgene-containing retrovirus met the quality and safety control criteria. Small-scale transductions yielded 62-92% scFv(G250)+ T cells and, at a clinical scale, 50-84% transduction efficiencies were obtained. Patient and healthy donor T cells showed similar expansion potencies, and also yielded similar levels of scFv(G250)-mediated immune functions, i.e. specific cytolysis of G250-ligand expressing RCC cells and production of IFN-gamma upon stimulation with such cells. All T cell cultures were free of replication competent retroviruses. DISCUSSION: We have shown that the validated batch of scFv(G250) transgene-containing retrovirus in combination with our GMP T-cell transduction and expansion protocol successfully generates clinically relevant numbers of functional scFv(G250) gene-modified T cells for patient treatment.

T-lymphocyte reconstitution following rigorously T-cell-depleted versus unmodified autologous stem cell transplants.

We compared the kinetics of T-cell recovery after extensive ex vivo and in vivo T-cell depleted autologous stem cell transplantation (SCT) for multiple sclerosis (MS; n=8) with unmodified SCT for hematological malignancies (HM; n=39). Both patient group showed a very protracted recovery of 'naive' CD4(+), 45R0(-) ( approximately CD45RA(+)) T-cells. Within the 'primed' CD4(+), 45R0(+) T-cells, the 'central memory' cells expressing the CD62L and CD27 markers were the slowest to recover. The repopulating T-cells were highly activated, as shown by increased expression of HLA-DR and the apoptosis marker CD95. The capability of CD4(+) and CD8(+) T-cells to produce IFN-gamma, IL-2 and TNF-alpha had reached normal ranges from 2 months post SCT onwards. Unexpectedly, the kinetics of T-cell recovery between 3 and 12 months post transplant was similar in T-depleted and unmodified SCT. Before SCT, the HM patients showed lymphopenia of all T-cell subsets, upregulated HLA-DR and CD95 expression and increased cytokine responses. We suggest that the similar kinetics of T-cell recovery in the two patient groups may be explained by the susceptibility to apoptosis of the activated CD4(+) T-cells in the autografts of the HM patients. This susceptibility to apoptosis would interfere with a swift and sustained CD4(+) T-cell regeneration post SCT.

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.

Parallel detection of transduced T lymphocytes after immunogene therapy of renal cell cancer by flow cytometry and real-time polymerase chain reaction: implications for loss of transgene expression.

We have started a phase I/II immunogene therapy study of metastatic renal cell cancer (RCC), using autologous T lymphocytes transduced ex vivo with a gene encoding a single-chain receptor based on the monoclonal antibody (mAb) G250 [scFv(G250)]. G250 recognizes carbonic anhydrase IX, which is overexpressed by RCC cells. We have developed and validated flow cytometric and real-time polymerase chain reaction (PCR) assays to quantitatively detect transduced T cells in patient blood. The flow assay was based on staining with the anti-G250 idiotype mAb NuH82 and showed a sensitivity of 0.06% scFv(G250)(1) cells within CD3(1) T cells. The real-time PCR method showed a sensitivity of 14 copies of scFv(G250) DNA per 100 ng of total DNA, which enabled detection of 0.008% scFv(G250)(1) T cells within leukocytes. Both assays were further validated for their specificity and reproducibility. When applied to blood samples from three RCC patients treated with intravenous infusions of scFv(G250)(1) T cells, the kinetics of scFv(G250)(1) T cell counts as detected by flow cytometry were similar to those detected by real-time PCR, although PCR allowed detection of transduced T cells over a longer period of time (i.e., for patient 3, 7 versus 32 days, respectively). Interestingly, follow-up studies of patient 3 demonstrated that the number of circulating scFv(G250)(1) T cells remained fairly constant during the first 7 days posttreatment, whereas the number of gene copies increased during the same period of time. These results suggest loss of scFv(G250) membrane expression on adoptive transfer, which would have important implications for the antitumor efficacy of this form of immunogene therapy.

Adoptive immuno-gene therapy of cancer with single chain antibody [scFv(Ig)] gene modified T lymphocytes.

Adoptive transfer of antigen-specific T cells has recently shown therapeutic successes in the treatment of viral infections and tumors. T cells specific for the antigen of interest can be generated in vitro, and adoptively transferred back to provide patients with large numbers of immune-competent T cells. Adoptive T cell therapy, however, is a patient-tailored treatment that unfortunately is not universally applicable to treat viral infections and tumors. We and others have demonstrated that the transfer of genes encoding antigen-specific receptors into T cells (i.e., genetic retargeting) represents an attractive alternative to induce antigen-specific immunity. Currently, we evaluate this concept in a clinical protocol to treat patients with metastatic renal cell cancer (RCC) using autologous RCC-specific gene-modified T lymphocytes.

Increased levels of soluble CD27 in the cerebrospinal fluid are not diagnostic for leptomeningeal involvement by lymphoid malignancies.

Soluble CD27 (sCD27) reportedly is a sensitive and specific marker for leptomeningeal involvement (LI) of CD27-expressing lymphoproliferations such as B-cell non-Hodgkin's lymphoma (B-NHL) or chronic B-lymphocytic leukemia (B-CLL). Because morphological analysis of cerebrospinal fluid (CSF) in patients suspected of LI is false negative in one-third of patients, a diagnostic marker for LI by B-NHL or B-CLL would be very valuable. sCD27 was determined in the first CSF sample from each of 102 unselected patients submitted for (immuno)morphologic detection of malignant cells. The patients were considered to have LI if either (immuno)morphologic analyses showed tumor cells or if neuroradiological evaluation showed typical abnormalities consistent with LI. Patients were suspected of having LI if CSF samples revealed atypical lymphocytes and/or if clinical symptoms and signs suggestive of LI were present, but clinical follow-up was shorter than 3 months because of deterioration of the patient. LI was considered absent if (immuno)morphologic analyses of CSF samples were negative without evidence for LI during 3 months of clinical follow-up. In patients with chronic lymphoproliferative disorders [mainly B-non-Hodgkin's lymphoma (NHL)], sCD27 concentrations were significantly higher in the CSF samples of 16 patients with confirmed or suspected LI than in those of 46 patients without LI. However, sCD27 was also increased in a variety of other predominantly inflammatory neurological disorders including herpes simplex and zoster infections. The positive predictive value of sCD27 determination for LI was only 54%, but the negative predictive value was 92%. Normal sCD27 concentrations in CSF samples of patients with chronic lymphoproliferation makes LI unlikely, but the determination of CSF sCD27 is not sufficiently specific to serve as a reliable tumor marker.