Background
Over the last decade, immune checkpoint blockade has emerged as a pillar of cancer immunotherapy. A key checkpoint is the interaction between Programmed Death 1 (PD-1) protein and Programmed Death Ligand 1 (PD-L1) which leads to self-tolerance and results in tumors escape from immune surveillance. Disruption of this interaction has proven clinically beneficial with seven monoclonal antibodies (mAb) currently approved by the US Food and Drug Administration and many more in development.
Methods
The syntheses of compounds used in this study are described in the patent literature. The compounds’ naming corresponds to the location of their description in respective patent application: BMS-10034 from Bristol Myers Squibb, INCY-11 from Incyte Corporation, and GS-4224 from Gilead Sciences Inc.The PD-L1 blocking antibody, (Durvalubamab analogue) was produced in-house. The heavy and light chain sequences of the antibody were downloaded from Drugbank. DNA encoding heavy chain and light chain sequences was synthesized by GeneArt and cloned into pcDNA3.1 expression vector. Expression of the Durvalumab analogue, was performed in Expi293F cells (Thermo Fisher Scientific) according to manufacturer’s protocol, and purification was performed following standard protocols.
Results
The application of stable isotope labelling with amino acids in cell culture (SILAC) technology for pulse chase experiments to determine protein turnover and degradation rates has been demonstrated in multiple models. In this study, the SILAC method was used to evaluate whether the inhibitors of PD-1/PD-L1 interaction, known to induce PD-L1 dimerization, influenced the rate of protein degradation. Previous reports have estimated that the half-life of PD-L1 is around 20 h (h). To more precisely determine the relative degradation rate of PD-L1, we chose to perform 48 h pulse chase experiments and apply a mathematical correction for each cell doubling event. Such a model makes general assumptions about the steady state level of PD-L1 and cellular growth rate. However, it is a suitable approximation for the purpose of a direct comparison between equivalent samples subjected to different small molecule treatments.
Conclusions
PD-1/PD-L1 checkpoint blockade for oncology indications has been a success story of the last decade with seven antibody treatments approved for clinical use and many more in the pipeline. Along with indisputable benefits of this treatment, opportunities for improvement also became apparent. Two notable issues are resistance or low objective response rate in certain patient populations and the general cost associated with antibody therapies.