Advances on biological evaluation methods of programmed cell death protein-1/ligand-1 inhibitors

Immuno-oncology represents a groundbreaking and well-established field within cancer treatment. Among the various immuno-oncology targets, the exploration of programmed cell death-1/ligand-1 for drug discovery has proven to be one of the most successful endeavors. Remarkably, it took nearly 30 years from the initial target identification to the clinical approval of monoclonal antibodies. Providing suitable and reliable bioassays for drug candidate evaluation is of paramount importance throughout the early stages of drug discovery, from lead compound identification to in vivo efficacy testing. This assay review aims to shed light on diverse assays reported in the literature for testing antagonism activity and efficacy of programmed cell death-1/ligand-1 inhibitors. Each of these assays possesses inherent advantages and can be applied in different research scenarios. The insights presented in this summary can serve as a valuable resource for scientists in this field, aiding in the selection of appropriate assays for their specific investigations.


Introduction
The programmed cell death protein-1/ligand-1 (PD-1/PD-L1) pathway is one of the most successful targets in the field of tumor immunotherapy [1,2].Till now, 10 PD-1 and 3 PD-L1 monoclonal antibody drugs have been approved by FDA [3].As an immuno-related protein-protein interaction (PPI) based target, biological evaluation assay plays a very important role in PD-1/PD-L1 related drug discovery.Here, we summarized reported assays for PD-1/PD-L1 inhibitor evaluation.

Cell-free assay
Currently, there have been some mature methods for measuring antagonistic activity of PD-1/PD-L1 inhibitors [4][5][6][7][8][9].Because the PD-L1 binding interface with PD-1 is the largest binding site of PD-L1, also no other known binding pocket of PD-L1 protein, binding affinity between ligands and PD-L1 protein can be measured either with or without PD-1 protein.When without PD-1, binding affinity between PD-1 and PD-L1 can be described by equilibrium dissociation constant (K D ).While IC 50 values are used when with the presence of both PD-1 and PD-L1.

PD-1/PD-L1 antagonizing assay
Homogeneous Time Resolved Fluorescence (HTRF) assay is based on proximity induced fluorescence and can identify candidate compounds that can hinder interaction between hPD-1/hPD-L1 (both proteins need to be tagged).Briefly, this working system is constructed with addition of recombinant proteins, Tag1-PD-L1 and Tag2-PD-1, to 384-well plate.Anti-Tag1-EuK, anti-Tag2-XL665 and diluted solution of small-molecule compounds are added to corresponding wells and incubated at room temperature.Then microplate reader is used to detect the absorbance values at 665 nm and 620 nm at specified time points.The inhibitory ability of the compounds against PD-1/PD-L1 is calculated based on the ratio of readout values of 665 nm/620 nm at different concentrations [10].
Bio-layer interferometry (BLI) assay can analyze light interference induced by interactions between compounds or protein and a biosensor tip.Binding and dissociation protein and ligand will result to mass or thickness change of biolayer on sensor chips, which can further induce relectance interference wave that can be monitored by BLI instrument.Hence, PD-1 need to be immobilized on amine-reactive biosensors via chemical coupling method in prior of BLI test.After protein loading, followed by loading quench and active site block, PD-L1 and test compounds in running buffer are applied on the chips to test in what extent PD-1/PD-L1 interaction is blocked by ligands [11].
Nuclear magnetic resonance (NMR) is another method to trace ligand-protein interaction in an active manner.NMR assay for testing of binding between PD-L1 and ligand require the use of 15 N isotopically enriched, PD-1 binding domain of human PD-L1.One typical assay is that PD-1 protein is dissolved in PBS pH 7.4 buffer with 10% D 2 O for assistance of locking.Then PD-L1 and test compound are subsequently titrated in PD-1 solution in sequence, and at the same time, 1 H proton NMR and 1 H-15 N 2D SOFAST-HMQC NMR are measured by allelic inhibition of displacement activity assay. 1 H and 1 H- 15 N cross correlation signals of isotopically enriched PD-1 are first monitored upon titration with PD-L1, which indicates complex formation.Signal recovery induced by ligands titration indicates blocking of interactions between PD-1 and PD-L1 [12].

PD-L1 binding assay
Microscale thermophoresis (MST) assay is to detect changes of hydration shell, charge or size of target protein via monitoring molecular dynamic pattern under microscopic temperature fluctuation.For PPI investigation with MST, one protein needed to be labeled by fluorophore.While other binding partners could remain in their natural form.For PD-L1 sample preparation, green-fluorescent protein-labelled PD-L1 is extracted from engineered CHO-K1 cells.Cell lysate is diluted in buffer and diluted in different concentration gradients for testing of different inhibition level of compounds.And then solution of compounds was added in corresponding wells containing green-fluorescent protein-PD-L1 sample in prior of MST test.For measurement K D between PD-1/PD-L1, typical MST assay protocol can be performed on a NanoTemper Monolith NT.115.Samples need to be dissolved in several recommended MST buffer to find the optimized buffer.The buffer mixture is then loaded into treated capillaries and tested in different MST power level.K D values are measured based on binding curves between proteins under equilibrium condition.The fitting function is derived from the law of mass action: Where [LP] represents bound complex concentration, [P tot ] and [L tot ] represent total protein and total ligand concentration, respectively [13].
Surface plasmon resonance (SPR) assay is to analyze refractive index caused by interactions between mobile ligands and immobilized proteins.During ligand-protein interaction, angle of extinction of light, generating from reflection of polarized light on a film will be monitored.Mass is the only parameter of protein involves in this assay, so no further labelling techniques need to be used.For SPR Figure 1 A diagram for reported PD-1/PD-L1 evaluation assay.PD-1, programmed cell death-1; PD-L1, programmed cell death ligand-1.Submit a manuscript: https://www.tmrjournals.com/pmrexperiment, extracellular domain of PD-L1 (residues 18-239) is expressed and immobilized through amino coupling method on a CM5 sensor chip.Then, blank control of concentration gradients are used for solvent calibration to deduct the errors caused by the solvent Dimethyl sulfoxide.Then, binding response of small molecule compounds are measured under different concentration gradients [14].
Differential scanning fluorimetry (DSF) assay can evaluate the binding efficacy through measuring the ability to stablize PD-L1 protein of ligands.Compounds in high concentration are incubated with PD-L1 first to reach complete binding with PD-L1.After incubation at room temperature, SyproOrange dye is added as a fluorescent indicator.It is recommended to run DSF assay shortly after dye addition to avoid possible photobleaching.Thermal scan can be conducted from 25 to 95 °C with 0.5 degree increase per minute.Melting points (T m ) are calculated based on fluorescence data using Boltzmann equation with software [15].
The characteristics and comparison of cell-free assay methods are shown in Table 1 below.

Flow cytometry (FC)
FC is also a method to determine PD-1/PD-L1 PPI.Construction an incubation system includes Jurkat T cell line with stable and high expression of hPD-1, candidate compounds, hPD-L1-His protein solution and APC anti-His tag antibody.All of the reagents are mixed and co-incubated at 4 °C in the dark.Samples are then monitored by flow cytometry and calculation of PD-1/PD-L1 inhibition blocking efficiency according to the formula: Inhibitory rate (%) = 100% × (1-positive rate treatment /positive rate positive control ) [14]

Downstream pathway activation
This assay is to monitor NFAT pathway activation-related fluorescence for PD-1/PD-L1 inhibitor evaluation.This assay consists of two genetically engineered cell lines: PD-1 effector cells (Jurkat T cells expressing human PD-1 and NFAT luciferase reporter), and PD-L1 aAPC/CHO-K1 cells (CHO-K1 cells expressing T cell receptor ligand, functioning as T cell activator and human PD-L1).APC cells are seeded in wells of plates and incubate the cells.Serial dilutions of test compounds are firstly prepared in assay buffer and added to the wells containing PD-L1 aAPC/CHO-K1 cells.Compounds dissolve in DMSO and added into the well, and immediately followed by addition of PD-1 effector cells.After incubation, Bio-Glo reagent is added to induce luminescence and relative light units (RLU) are normalized to positive control.Then dose-response curves and half maximal effective concentration values are calculated [16].This method has been successfully applied on evaluation of peptide and antibody, but may not be used on small molecule compounds because of toxicity issue [17,18].

Cytokine release
When interaction between PD-1 and PD-L1 is blocked, T cells will be rescued and re-activated.And cytokine can be expressed and released.Hence, expression level of cytokine can be indicators of PD-1/PD-L1 inhibitors.Interleukin-2 (IL-2) is one of the indicators.A typical protocol is Jurkat T cells (PD-1-expressed cell) and IFN-γ-pretreated LoVo cells (PD-L1-expressed cell) are co-incubated.Compounds can induce IL-2 expression, which is assessed by enzyme-linked immunosorbent assay (ELISA) [19].Besides, interferon-γ (IFN-γ) can also be an indicator of T cell rescue.One typical assay is PBMC-derived CD3 + T cells are co-cultured with engineered Hep3B cells with expression of human PD-L1 and anti-CD3 variable fragment, OS-8.Then different concentrations test compounds are added.IFN-γ expression levels are also detected by ELISA [17].

Co-incubation and cancer cell death
T cells can retrieve cell-killing ability when rescued by PD-1/PD-L1 inhibitor.So cancer cell death after co-incubation with T cells can also be an indicator.A typical protocol is that human peripheral blood mononuclear cells are pre-activated with CD3/CD28 antibody.Then, MDA-MB 231 cells are seeded in 96-well plates followed by different concentrations of compounds.Peripheral blood mononuclear cells, MDA-MB-231 cells and different concentration of compounds are co-incubated at 37 °C for 12 h.Cell death releases lactate dehydrogenase (LDH), which is monitored with an LDH cytotoxicity assay detection kit to evaluate the extent of cell death.Viability (%) is measured as: Where LD values refer to the detected LDH absorbency values.LD CC+TC+Cmpd means LDH absorbency of samples with all components including MDA-MB-231 cells, T cells and compound solution.LD TC+Cmpd means LDH absorbency of samples with only T cells and compound solutions.LD CC+Cmpd means LDH absorbency of samples with MDA-MB-231 cells and compound solutions.LD CC means the detected LDH absorbency value of samples with only MDA-MB-231 cells but still with full lysis at the end of co-incubation [10].
The characteristics and comparison of cell-based methods are shown in Table 2 below.[10,20].It was also reported that B16-F10 cell (mouse melanoma cell line) can also be used to build PDX model [21].

Humanized model
This model is constructed by injecting hPD-L1/MC38 (mouse PD-L1 knockout and human PD-L1 knock-in) cells into humanized immune system mouse.Construction of this model involves with transplantation human CD34 + hematopoietic progenitor into non-obese diabetic Cg-Prkdc scid IL2rg tm1Wjl /Sz (null; NCG) mice and monitored the development of human hematopoietic and immune systems.After 12 week, there are ＞ 25% CD45 + human T cells in mouse blood detected, which is considered as the successful establishment of humanized model.By applying this model, cancer cell line can be selected from human cell line, like HCT-116 [22,23] Compounds can be administered at the tumor volume with 50−100 mm 3 .The measurement, calculation and animal treatment are similar to the aforementioned method [7,24,25].

Summary and outlook
This review describes the biological evaluation methods of PD-1/PD-L1 inhibitors from protein level, cell level and animal level, respectively.Each of these assays possesses inherent advantages and can be applied in different research scenarios.The advantages and disadvantages of cell-free and cell assay were compared in Table 1 and Table 2.As for animal model test, despite the achievements of current approaches, tumor heterogeneity remains an obstacle.In addition, the innate immune system in mice is not identical to the human immune system, which is another obstacle to face.How to bridge the gap between preclinical screening models and clinical models is an issue that still needs to be discussed.It is still very important to develop a more complete drug screening system that is closer to the clinical model.

Table 2 Comparison among reported cell assay
), where L and W refer to the length and width dimensions of the tumor.TGI is calculated using the equation: TGI = [1 − (T final − T 0 )/(C final − C 0 )] × 100%, where T final and T 0 values are the average tumor volumes of test groups at the end of the treatment and on the first day of treatment, respectively; C final and C 0 are the average volumes of the control group at the end of the treatment and on the first day of treatment, respectively