Validation of pharmacodynamic assays to evaluate the clinical efficacy of an antisense compound (AEG 35156) targeted to the X-linked inhibitor of apoptosis protein XIAP

Abstract

The inhibitor of apoptosis protein, XIAP, is frequently overexpressed in chemoresistant human tumours. An antisense oligonucleotide (AEG 35156/GEM 640) that targets XIAP has recently entered phase I trials in the UK. Method validation data are presented on three pharmacodynamic assays that will be utilised during this trial. Quantitative RT-PCR was based on a Taqman assay and was confirmed to be specific for XIAP. Assay linearity extended over four orders of magnitude. MDA-MB-231/U6-E1 cells and clone X-G4 stably expressing an RNAi vector against XIAP were chosen as high and low XIAP expression quality controls (QCs). Within-day and between-day coefficients of variation (CVs) in precision for cycle threshold (CT) and delta CT values (employing GAPDH and beta 2 microglobulin as housekeepers) were always less than 10%. A Western blotting technique was validated using a GST-XIAP fusion protein as a standard and HeLa cells and SF268 (human glioblastoma) cells as high and low XIAP expression QCs. Specificity of the final choice of antibody for XIAP was evaluated by analysing a panel of cell lines including clone X-G4. The assay was linear over a 29-fold range of protein concentration and between-day precision was 29% for the low QC and 23% for the high QC when normalised to GAPDH. XIAP protein was also shown to be stable at -80 degrees C for at least 60 days. M30-Apoptosense plasma Elisa detects a caspase-cleaved fragment of cytokeratin 18 (CK18), believed to be a surrogate marker for tumour cell apoptosis. Generation of an independent QC was achieved through the treatment of X-G4 cells with staurosporine and collection of media. Measurements on assay precision and kit-to-kit QC were always less than 10%. The M30 antigen (CK18-Asp396) was stable for 3 months at -80 degrees C, while at 37 degrees C it had a half-life of 80-100 h in healthy volunteer plasma. Results from the phase I trial are eagerly awaited.

Figure 1

Dynamic range of a qRT-PCR method for XIAP evaluated using a purified pCI plasmid containing full-length human XIAP cDNA as the template.

Figure 2

Stability of XIAP mRNA determined by qRT-PCR in replicate pellets of the high and low XIAP expression QC cell lines stored at −80°C over 106 days. At the time intervals indicated, three replicates were removed from the freezer and analysed. Each time point represents the mean±s.d. Results are expressed relative to GAPDH as delta CT values.

Figure 3

Calibration of a Western blotting technique for the determination of XIAP using a GST–XIAP protein standard. The fusion protein diluted in MDA-MB-231/X-G4 lysate was analysed within a concentration range of 0.06–135 pg μg⁻¹. Lanes are identified as follows: GST–XIAP (pg μg⁻¹) at 135, lane 1; 110, lane 2; 80, lane 3; 55, lane 4; 28, lane 5; 5.5, lane 6; 2.8, lane 7; 0.55, lane 8; 0.28, lane 9 and 0.06, lane 10. Also included on the blot are the XIAP-deficient cell line MDA-MB-231/X-G4, lane 11; the low QC cell line SF268, lane 12 and the high-QC cell line HeLa, lane 13. The blot was also probed for GAPDH, which acted as a housekeeper protein.

Figure 4

Western blot analysis of XIAP in peripheral blood mononuclear cells harvested from a healthy volunteer. Each lane was assayed at a different level of total protein loading. Lanes are identified as follows: 1, 5 μg; 2, 10 μg; 3, 20 μg and 4, 40 μg. The blot was also probed for GAPDH, which acted as a housekeeper protein.

Figure 5

Stability of XIAP protein determined by Western blot in replicate pellets of the high and low XIAP expression QC cell lines stored at −80°C over 60 days. At the time intervals indicated, three replicates were removed from the freezer and analysed. Each time point represents the mean±s.d.

Figure 6

Effect of the treatment of MDA-MB-231/X-G4 cells with staurosporine plus or minus the general caspase inhibitor zVAD on the levels of CK18-asp³⁹⁶ NE antigen detected in the culture medium by the M30 Apoptosense Elisa assay. Cells were incubated for 24 h with either 10 or 100 nM staurosporine (Stauro, S) in the presence or absence of 50 μM zVAD. Each bar represents the mean value±s.d., n=3.

Figure 7

Stability of CK18-asp³⁹⁶ NE antigen in tissue culture medium stored at −80°C. Each time point represents the mean value±s.d., n=3.

Figure 8

Stability of CK18-asp³⁹⁶ NE antigen in healthy volunteer plasma after incubation at 37°C in the dark. Blood was collected from three different subjects and each time point represents the mean value±s.d., n=3.