The programmed cell death protein 1 (PD-1) receptor (CD279), expressed on the surface of activated T cells, and its ligand PD-L1 (CD274), expressed on the surface of dendritic cells or macrophages, are important targets in multiple cancers. The PD-1 and PD-L1 interaction functions as an immune checkpoint that helps maintain immune homeostasis and inhibits autoimmunity. In cancer, immune checkpoint mechanisms often suppress the nascent anti-tumour immune response. Blocking the PD-1 - PD-L1 interaction is promising for cancer immunotherapies.
Monoclonal antibodies (e. g. rituximab, nivolumab) have been approved for treatment of various cancers. However, to date there have been no approved small molecule PD-1/PD-L1 inhibitors. Small molecules have advantages over monoclonal antibodies, including simpler manu-facturing, more stability, and reduced likelihood of immunogenicity. A PD-1/PD-L1 assay was developed to screen small molecules from the BioAscent diversity library for inhibitors that are able to block the PD-1/PD-L1 interaction and serve as suitable starting points for drug development.
Homogenous Time Resolved Fluorescence (HTRF) uses a long ﬂuorescence lifetime FRET pair to assess molecular interactions. Donor and acceptor molecules are coupled to either protein, PD-L1 or PD-1, via tags or antibodies. If PD-1 and PD-L1 are in close proximity then excitation, at 337 nm, causes energy transfer between donor and acceptor molecules and results in a FRET signal (read at 620 nm and 665 nm). If the binding partners are not in close proximity (PD-1 and PD-L1 not bound together) then FRET does not occur.
Materials & Methods
- White small volume 384 well plate (Greiner) Human 6His-PD-L1 (Abcam)
- Human IgG-PD-1 (R&D Systems)
- pAb Anti-human IgG-Tb (CisBio)
- mAb Anti 6His-d2 (CisBio)
- 80 nL of compound at 100x ﬁnal concentration is added to white 384 well plates using an ECHO acoustic dispenser (Labcyte) – 80 nL of reference to 0.1 mM. compound or DMSO is added to positive and negative control wells respectively.
- 4 μL of PD-1 (at 2x ﬁnal concentration) is added to all plates using a Preddator bulk reagent dispenser (Redd & Whyte).
- After 30 min at room temp (in the dark), 4 μL of PD-L1/HTRF mixture (at 4x ﬁnal concentration) is added to all plates using the Preddator.
- The plates are then incubated for 30-60 min before reading on the PHERAstar.
||Time resolved fluorescence, endpoint Top optic|
|Optic module||HTRF 337 665 620|
|Excitation source||Flash lamp|
|Integration start||50 µs|
|Integration time||200 µs|
|General settings||Number of flashes||200 with flash lamp|
|Settling time||0.5 s|
False positive - speciﬁc considerations for HTRF
HTRF false positive can be due to:
- Auto-ﬂuorescence or quenching of ﬂuorescence
- Compounds acting as ﬂuorescence donors
- Tag/antibody binders
- Common/target speciﬁc false positive modalities, e. g. aggregators, denaturants, redox, metal contaminants
Two methods were used to identify false positives prior to orthogonal testing:
- HTRF/PD-1/PD-L1 complex was equilibrated for 30 min. Test compounds were then added and the effect was measured at time = 0 min and time = 60 min - compounds binding to the PD-1/PD-L1 interaction surface should show little activity at 0 min due to the time required to reach a new equilibrium.
- Assessment of the raw emission wavelength (620 nm and 665 nm) for each compound - genuine positives should only affect the 665 nm data.
Results & Discussion
A selection of 5168 BioAscent Library compounds were screened at 20 μM for PD-1/PD-L1 inhibition with 112 compounds producing ≥ 20 % effect relative to controls, which were further screened for potency.
28 of the 112 hit compounds produced a dose response curve and were tested for false positive activity using the two methods as described. Thereof, 5 compounds did not show false positive behaviour and are most likely to genuinely interrupt the PD-1/PD-L1 interaction. The remaining compounds interfered with the HTRF technology and were excluded from further testing.
The 5 most promising compounds identiﬁed from this screen were further tested in orthogonal biophysical assays (Differential light scattering and Microscale Thermophoresis) and formed the backbone of a medicinal chemistry synthesis campaign for PD-1/PD-L1 inhibitors.
This work was carried out with the aid of the SULSA assay development fund. The SULSA Assay Development Fund was funded through the Scottish Universities Life Sciences Alliance by a grant award from the Scottish Funding Council (SFC Ref: 240599989).