Good manufacturing practice production of self-complementary serotype 8 adeno-associated viral vector for a hemophilia B clinical trial.

To generate sufficient clinical-grade vector to support a phase I/II clinical trial of adeno-associated virus serotype 8 (AAV8)-mediated factor IX (FIX) gene transfer for hemophilia B, we have developed a large-scale, good manufacturing practice (GMP)-compatible method for vector production and purification. We used a 293T-based two-plasmid transient transfection system coupled with a three-column chromatography purification process to produce high-quality self-complementary AAV2/8 FIX clinical-grade vector. Two consecutive production campaigns using a total of 432 independent 10-stack culture chambers produced a total of ∼2 × 10(15) vector genomes (VG) by dot-blot hybridization. Benzonase-treated microfluidized lysates generated from pellets of transfected cells were purified by group separation on Sepharose beads followed by anion-exchange chromatography. The virus-containing fractions were further processed by gel filtration and ultrafiltration, using a 100-kDa membrane. The vector was formulated in phosphate-buffered saline plus 0.25% human serum albumin. Spectrophotometric analysis suggested ∼20% full particles, with only low quantities of nonviral proteins were visible on silver-stained sodium dodecyl sulfate-polyacrylamide gels. A sensitive assay for the detection of replication-competent AAV was developed, which did reveal trace quantities of such contaminants in the final product. Additional studies have confirmed the long-term stability of the vector at -80°C for at least 24 months and for at least 24 hr formulated in the clinical diluent and stored at room temperature within intravenous bags. This material has been approved for use in clinical trials in the United States and the United Kingdom.

Reactive oxygen species regulate caspase activation in tumor necrosis factor-related apoptosis-inducing ligand-resistant human colon carcinoma cell lines.

The effects of reactive oxygen species (ROS) on tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in solid cancers have yet to be clearly defined. In this study, we found that the classic uncoupler of oxidative phosphorylation, carbonyl cyanide m-chlorophenylhydrazone (CCCP), induced a reduction in DeltaPsim and generation of ROS. This uncoupling effect enhanced TRAIL-induced apoptosis in TRAIL-resistant human colon carcinoma cell lines (RKO, HT29, and HCT8). Sensitization was inhibited by benzyloxycarbonyl-valine-alanine-aspartate fluoromethylketone, indicating the requirement for caspase activation. CCCP per se did not induce apoptosis or release of proapoptotic factors from mitochondria. Generation of ROS by CCCP was responsible for TRAIL-induced Bax and caspase activation because scavenging ROS completely abrogated apical caspase-8 activation and further downstream events leading to cell death. Overexpression of Bcl-2 did not prevent the initial loss of DeltaPsim and ROS generation following CCCP treatment, but did prevent cell death following TRAIL and CCCP exposure. Uncoupling of mitochondria also facilitated TRAIL-induced release of proapoptotic factors. X-linked inhibitor of apoptosis overexpression abrogated TRAIL-induced apoptosis in the presence of CCCP and decreased initiator procaspase-8 processing, indicating that additional processing of caspase-8 required initiation of a mitochondrial amplification loop via effector caspases. Of interest, depletion of caspase-9 in RKO cells did not protect cells from TRAIL/CCCP-induced apoptosis, indicating that apoptosis occurred via a caspase-9-independent pathway. Data suggest that in the presence of mitochondrial-derived ROS, TRAIL induced mitochondrial release of Smac/DIABLO and inactivation of X-linked inhibitor of apoptosis through caspase-9-independent activation of caspase 3.

Interferon-gamma pharmacokinetics and pharmacodynamics in patients with colorectal cancer.

PURPOSE: The study objectives were to define subcutaneous (s.c.) interferon gamma (IFN-gamma) disposition in patients with gastrointestinal malignancies receiving 5-fluorouracil (5-FU) and leucovorin (LV) and to examine the relationship between IFN-gamma exposures and Fas upregulation in vivo and in vitro. METHODS: Patients received IFN-gamma (10, 25, 50, 75, and 100 microg/m(2)) with LV and 5-FU, and serial samples were collected after the first dose. IFN-gamma concentrations were measured by ELISA. A linear one-compartment model with a lag was fitted to the IFN-gamma plasma concentration-time data. To examine the relationship between IFN-gamma systemic exposure and biological activity in vivo, cell surface Fas upregulation was assessed in peripheral blood mononuclear cell (PBMC) subcompartments. RESULTS: The median (range) apparent IFN-gamma clearance was 46 l/m(2) per hour (2.6-92 l/m(2) per hour). With increasing IFN-gamma dosages, the area under the concentration-time curve (AUC(0--> infinity )) and C(max) increased; however, significant interpatient variability was observed. IFN-gamma AUC(0--> infinity ) and time above 33.3 pg/ml significantly correlated with Fas upregulation in several PBMC compartments, but dosage was significantly correlated with this pharmacodynamic marker only in CD4(+) and CD56(+) cells. In vitro studies in HT29 cells demonstrated that clinically relevant IFN-gamma concentrations (1 to 10 U/ml for 6.5 h) with 5-FU/LV upregulated Fas expression 3.5-fold, similar to that in PBMC in vivo. CONCLUSIONS: We characterized IFN-gamma disposition and developed a limited sampling model for use in future pharmacokinetic studies. Our results showed that IFN-gamma upregulates Fas in PBMC in vivo and in HT29 cells in vitro at tolerable, clinically relevant exposures and that monitoring IFN-gamma pharmacokinetics/pharmacodynamics may be warranted in IFN-gamma clinical use.

Interferon-gamma-induced sensitization of colon carcinomas to ZD9331 targets caspases, downstream of Fas, independent of mitochondrial signaling and the inhibitor of apoptosis survivin.

We have demonstrated previously a Fas-dependent component in thymineless death of human colon carcinoma cells. Importantly, the cytotoxic effects of thymidine deprivation induced by 5-fluorouracil (FUra) combined with leucovorin (LV) was enhanced by IFN-gamma, and the synergism was shown to be dependent on Fas, FUra-induced DNA damage, and independent of p53. Subsequently we examined the potential for synergistic interactions between IFN-gamma and the specific thymidylate synthase inhibitor, ZD9331. IFN-gamma sensitized colon carcinomas to ZD9331-induced apoptosis and loss in clonogenic survival, also dependent on ZD9331-induced DNA damage, independent of p53. Synergism occurred in HCT116, demonstrating previously RNA-mediated FUra/LV cytotoxicity that could not be potentiated by IFN-gamma. Manipulation of the Fas death receptor pathway from the level of the receptor (Nok1/Nok2, Fas overexpression, and DN-FADD) to the mitochondria (Bcl-xL and Bcl-2) did not modulate ZD9331 +/- IFN-gamma-induced cytotoxicity in HT29, with the exception that Nok1/Nok2-blocking antibodies partially protected HT29 from the cytotoxic activity of ZD9331 alone. However, IFN-gamma alone (but not ZD9331) up-regulated the expression of caspases -3, -4, -7, and -8, and in combination with ZD9331 demonstrated enhanced caspase activation and cleavage of poly(ADP-ribose) polymerase that was not prevented by overexpression of Bcl-2. Additionally, IFN-gamma increased the activity of the proteasome in HT29, leading to selective down-regulation of the antiapoptotic protein survivin, whereas simultaneously increasing Fas expression. However, reduction in the survivin:Fas ratio by transfection of survivin small interfering RNA and/or overexpression of Fas did not affect sensitivity of HT29 to ZD9331 +/- IFN-gamma. Data demonstrate that IFN-gamma combined with ZD9331 is synergistic in additional cell lines that demonstrate RNA-mediated FUra/LV cytotoxicity, and that a major target of interaction is at the level of caspases, downstream of Fas, and independent of involvement of either the mitochondria or survivin.

Rottlerin sensitizes colon carcinoma cells to tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis via uncoupling of the mitochondria independent of protein kinase C.

Signaling pathways involved in survival responses may attenuate the apoptotic response to the cytotoxic tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in human colon carcinomas. In six lines examined, three were sensitive (GC(3)/c1, VRC(5)/c1, HCT116), HT29 demonstrated intermediate sensitivity, and RKO and HCT8 were resistant to TRAIL-induced apoptosis. Calphostin c [an inhibitor of classic and novel isoforms of protein kinase C (PKC)] sensitized five of six cell lines to TRAIL, whereas Go6976, (inhibitor of classic PKC isoforms), did not influence TRAIL sensitivity. Rottlerin, an inhibitor of novel isoforms of PKC, specifically PKC delta, sensitized five of six cell lines to TRAIL-induced apoptosis, suggesting that PKC delta may be involved in the mechanism of TRAIL resistance. Transfection of HCT116 with a proapoptotic cleaved fragment of PKC delta or an antiapoptotic full-length PKC delta did not influence the sensitivity of HCT116 to TRAIL. Furthermore, the incubation of HCT116 or RKO with phorbol myristate acetate for 16 h, which down-regulated the expression of novel PKC isoforms, also did not influence sensitivity to TRAIL either in the absence or presence of rottlerin. However, after 15-min incubation with rottlerin, mitochondrial membrane potential (Delta psi m) was dramatically reduced in RKO cells, and, in cells subsequently treated with TRAIL, rapid apoptosis was evident within 8 h. Calphostin c, but not Go6976, also caused a decrease in Delta psi m. In RKO, rottlerin induced the release of cytochrome c, HtrA2/Omi, Smac/DIABLO, and AIF from the mitochondria, potentiated in combination with TRAIL, with concomitant caspase activation and down-regulation of XIAP. In HT29, the release of proapoptotic factors was demonstrated only when rottlerin and TRAIL were combined, and Bcl-2 overexpression inhibited this release and the induction of apoptosis. TRAIL-induced apoptosis was not influenced by rottlerin or Bcl-2 overexpression in type I (GC(3)/c1) cells. Data suggest that rottlerin affects mitochondrial function independent of PKC delta, thereby sensitizing cells to TRAIL, and that mitochondria constitute an important target in overcoming inherent resistance to TRAIL in colon carcinomas.

Modulation of the Fas signaling pathway by IFN-gamma in therapy of colon cancer: phase I trial and correlative studies of IFN-gamma, 5-fluorouracil, and leucovorin.

Potentiation of 5-fluorouracil/leucovorin (FUra/LV) cytotoxicity by IFN-gamma in colon carcinoma cells is dependent on FUra-induced DNA damage, the Fas death receptor, and independent of p53 and RNA-mediated FUra toxicity, which occurs in normal gastrointestinal tissues. This provides a rationale for enhancing the selective action of FUra/LV by IFN-gamma in the treatment of colorectal carcinoma. Based on results from our preclinical studies we designed a Phase I trial combining FUra (370 mg/m2) and LV (200 mg/m2), i.v. bolus daily x 5 days, with escalating doses of IFN-gamma (10-100 micro g/m2) s.c. on days 1, 3, and 5, every 28 days. Twenty-five patients with carcinomas were enrolled; 6 patients received IFN-gamma on days 1 and 3 only. The dose-limiting toxicity, stomatitis, occurred most frequently at 100 micro g/m2 IFN-gamma. Minor response or SD was observed in 2 of 9 patients and in 4 of 12 patients at dose levels of < or =50 micro g/m2 and > or =75 micro g/m2 IFN-gamma, respectively. Three evaluable chemonaive patients demonstrated partial response (2) or complete response (1). Serial plasma samples revealed peak FUra concentrations of >100 micro M; at 100 micro g/m2 IFN-gamma plasma concentrations >5 units/ml persisted for 6.5 h and >1 unit/ml for 28.5 h. The pharmacokinetic parameters of IFN-gamma correlated with a 2-3-fold up-regulation of Fas expression at 24 h in CD15+ cells in peripheral blood samples. Furthermore, clinically relevant IFN-gamma concentrations up-regulated Fas expression and sensitized HT29 colon carcinoma cells in vitro to FUra/LV cytotoxicity. On the basis of the modulation of Fas signaling, FUra/LV combined with IFN-gamma has shown activity in a Phase I trial in colorectal carcinoma and warrants additional evaluation in Phase II.