Abstract
1206
Objectives: Targeting immune checkpoint proteins is changing the landscape of cancer therapy. Recently, the phase III PACIFIC trial demonstrated clear overall survival benefit in the Durvalumab treated cohort in patients with stage III non-small cell lung cancer.[1] Remarkably, treatment benefit was also seen in patients with tumours with low PD-L1 expression. However, PD-L1 characterisation was performed on single, pre-treatment tumour biopsies. PD-L1 expression can be up-regulated over the course of chemo- and radiotherapy treatment. The Immuno-PET study aims to examine the dynamics of PD-L1 expression via 89Zr-Durvalumab PET imaging before, during and after chemoradiotherapy. To support the large number of clinical productions required for this study, we have developed a fully automated protocol for the radiosynthesis of [89Zr]Zr-DFOSq-Durvalumab.
Methods: Automated radiolabelling of DFOSq-Durvalumab with zirconium-89 was optimized on the disposable cassette based MultiSyn module using a modified kit. The system was programmed using an Excel based step list and synthesis progression was monitored by the built-in radioactivity detectors. Purification was performed interactively by observing the characteristic radioactivity trace recorded by one of the radioactivity detectors located at the position of the PD-10 column. Activity losses were tracked using a dose calibrator and minimized by optimizing fluid transfers, reaction buffer, antibody formulation additives and pH. [89Zr]Zr-DFOSq-Durvalumab was reformulated and sterile filtered using the built-in syringe drives. Quality control was performed on the formulated product with respect to radiochemical purity (iTLC), specific activity, protein integrity (SEC-HPLC), immunoreactive fraction, and stability over time when incubated in human serum.
Results: DFOSq-Durvalumab with an average chelator-to-antibody ratio of 3.69 was used for radiolabelling experiments and the MultiSyn module was setup following the schematic in Figure 1. Reaction kinetics in succinate pH 6 were significantly faster compared to HEPES pH 7.2 with 90% conversion observed in 15 and 60 minutes, respectively. This buffer and pH change also reduced the amount of residual radioactivity in the Zr-89 isotope vial from 24% to 0.5% ± 0.2% (n=5). Further radioactivity losses were seen in the reactor vial which could not be removed by rinsing. Additives in the reaction buffer such as 1% HSA or 0.02% Tween 80 were both effective at reducing the amount of residual [89Zr]Zr-DFOSq-Durvalumab. The latter reduced losses in the reactor from 36% ± 6% (n=4) to 0.2% ± 0.0% (n=2). Interactive peak collection via the PD-10 column radiation profile (supplementary figure) allowed reproducible collection of the product fraction with low amounts of radioactivity remaining on the PD-10 column (3.0% ± 1.2%, n=3). Residual on the sterile filter was 6.2% ± 1.9% (n=3) and the remaining kit components such as manifolds, transfer syringes, and tubing accounted for 4.3% ± 1.4% (n=3) of radioactivity losses. In summary, the total process yield was improved from 13% to 74.5% ± 5.4% (n=3) and the total process time was 45 minutes. Typically, 164 MBq of product was formulated in a volume of 2.1 mL of 0.5% sodium gentisate in PBS + 0.02% Tween 80 with a specific activity of 182 MBq/mg. Radiochemical purity and immunoreactive fraction were always >99% and >93% at end-of-synthesis, respectively, and dropped to 90% and 74% after incubation in human serum for 7 days at 37°C. SEC-HPLC analysis of formulated [89Zr]Zr-DFOSq-Durvalumab showed very good antibody integrity with levels of aggregation at 3.9% ± 0.8% (n=3). Conclusion: Fully automated production of [89Zr]Zr-DFOSq-Durvalumab for clinical use was achieved with minimal exposure to the operator. The cassette based approach allows for multiple consecutive productions on the same day which will have clinical impact considering the growing number of clinical trials investigating 89Zr-labelled antibodies.
