Background

The identification of compounds that might be able to modulate the affinity of the complex formed between PD-1 and PD-L1 represents a major advance in the development of new treatments targeting immune escape for oncology. This protocol involves the overexpression of an eGFP fusion protein, which is then extracted from the cell lysate, enabling the determination of the affinity constant between PD-1 and PD-L1 without any purification steps. Thus, the development of this protocol required the production of eGFP fusion proteins. This protocol was designed to quantitatively accelerate the characterization of protein interactions by avoiding tedious purification steps. This protocol could also be used to perform high-throughput screening with PD-1/PD-L1 inhibitors to evaluate the ability of these recently developed molecules to modulate the affinity of the PD-1 protein for its ligand, as the small-molecule “blocking pathway” represents a major challenge for the development of new strategies targeting immune escape for oncology.

Microscale thermophoresis (MST) is a biophysical technique that measures the strength of the interaction between two molecules by detecting variations in the fluorescence signal as a result of an IR laser-induced temperature change. The range of the variation in the fluorescence signal correlates with the binding of a ligand to the fluorescent target. Overall, MST is based on the movement of a fluorescent molecule within a temperature gradient. MST also has broad applications at concentrations ranging from picomolar to molar and involves very little sample consumption, requiring only a few microliters; in addition, no immobilization of the protein of interest is required. You can either add a fluorescent tag to your protein of interest or exploit the intrinsic fluorescence of the target. In any case, one binding partner is fluorescent, while the other binding partner remains label-free. MST has also been used to characterize protein-small molecule interactions such as those involving aptamers (Entzian and Schubert, 2016) and PD-L1 inhibitors by BMS (Ganesan et al., 2019) or for high-throughput screening to identify hits (Rainard et al., 2018). Here, we describe a protocol using MST to determine the binding affinity of the PD-1/PD-L1 complex, which is involved in tumor escape processes, without purification of the target protein from cell lysates (Khavrutskii et al., 2013). This method requires the overexpression of fluorescent proteins in CHO-K1 cells and describes the optimal conditions for determining the dissociation constant. The protocol has a variety of potential applications for studying the interactions of these proteins with small molecules and demonstrates that MST is a valuable method for studying the PD-1/PD-L1 pathway.