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  • Özet
  • Özet
  • Giriş
  • Protokol
  • Sonuçlar
  • Tartışmalar
  • Açıklamalar
  • Teşekkürler
  • Malzemeler
  • Referanslar
  • Yeniden Basımlar ve İzinler

Özet

This protocol describes a blockade assay for PD-1/PD-L1 inhibitors using surface plasmon resonance technology. It employs a dual-step immobilization strategy and a tailored buffer system to accurately measure response units, facilitating the assessment of blockade rates for compounds or biologics. Additionally, it supports the high-throughput identification of PD-1/PD-L1 inhibitors.

Özet

The disruption of the PD-1/PD-L1 interaction is a promising strategy for cancer immunotherapy. Reliable screening platforms are essential for evaluating the efficacy of PD-1/PD-L1 inhibitors. A previously established human PD-1/PD-L1 blockade assay utilizing Surface Plasmon Resonance (SPR) technology (first-generation PD-1/PD-L1 inhibitor SPR screening platform) demonstrated results comparable to those obtained through Homogeneous Time-Resolved Fluorescence (HTRF) and cell-based assays, with potential for large-scale screening. Herein, an optimized version of this assay (second-generation PD-1/PD-L1 inhibitor SPR screening platform) is presented, featuring a dual-step coupling process that combines amine and bio-streptavidin coupling to enhance PD-1 orientation control on the chip and reduce PD-1 protein consumption. The updated platform was successfully validated using the PD-1/PD-L1 inhibitor BMS-1166, showing blockade effects comparable to the previous SPR-based method and other established techniques such as ELISA. These results confirm the reliability of the approach. This optimized SPR screening platform offers a high-throughput and reliable tool for identifying novel PD-1/PD-L1 inhibitors, advancing cancer immunotherapy research, and highlighting the potential of SPR in immune checkpoint inhibitor screening.

Giriş

Immune checkpoint blockade therapies, particularly those targeting Programmed Cell Death-1 (PD-1) and Programmed Cell Death-Ligand 1 (PD-L1), stand at the forefront of cancer immunotherapy strategies. Anti-PD-1/PD-L1 therapies have received approval for use in various cancer types, such as hematological, cutaneous, pulmonary, hepatic, urinary bladder, and renal cancers1. PD-1 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily, characterized by a single immunoglobulin variable (IgV)-like domain at the N-terminal, a roughly 20-amino acid stalk separating the IgV domain from the plasma membrane, a transmembrane domain, and a cytoplasmic tail containing tyrosine-based signaling motifs2. PD-L1, identified as one of the ligands for PD-1, is a type I transmembrane protein featuring a transmembrane region, two extracellular domains-immunoglobulin constant (IgC) and IgV-and a relatively short cytoplasmic domain that triggers intracellular signaling pathways3. The PD-1/PD-L1 inhibitory pathway serves as a critical immune checkpoint that regulates T cell activation and autoimmunity4. PD-1 is expressed on T cells, where it interacts with PD-L1, inhibits T cell receptor signaling, and blocks the stimulation of CD28 and CD80 molecules on antigen-presenting cells and T cells5. Cancer tissues exploit this physiological mechanism by overexpressing PD-L1 during the escape phase, thus creating an immunosuppressive environment that promotes tumor growth and progression6. Inhibitors of PD-1 and PD-L1 disrupt this interaction, enabling the immune system to evade tumor-induced suppression and reinitiate the T-cell-mediated tumor-cell death process7.

Building on the foundation laid by the prominent role of immune checkpoint blockade therapies, the development of PD-1/PD-L1 inhibitors has marked a significant advancement in cancer immunotherapy. The U.S. Food and Drug Administration (FDA) has endorsed nine immune checkpoint inhibitors that specifically target the PD-1/PD-L1 pathway. These include six PD-1 inhibitors-pembrolizumab, dostarlimab, nivolumab, cemiplimab, oripalimab, and tislelizumab-and three PD-L1 inhibitors-atezolizumab, avelumab, and durvalumab8,9. These therapies have been effectively utilized to treat a variety of cancers, such as melanoma, lung cancer, urothelial cancer, cervical cancer, gastric or gastroesophageal cancer, and other solid tumors10. Despite their efficacy, monoclonal antibody-based therapies face significant limitations, including low response rates, high costs, prolonged half-lives, severe immune-related adverse events, and restrictions to intravenous or subcutaneous delivery11,12,13. Consequently, research is increasingly focused on developing small-molecule inhibitors targeting the PD-1/PD-L1 axis. These small molecules offer distinct advantages, such as improved cellular penetration, modulation of diverse biological targets, enhanced oral bioavailability, and reduced costs, with the goal of achieving comparable therapeutic outcomes with fewer adverse effects14. However, the development of small molecule inhibitors targeting the PD-1/PD-L1 interaction is in its early stages, primarily due to the lack of a reliable high-throughput screening platform. Such platforms are essential for rapidly evaluating vast libraries of small molecules and identifying lead compounds for further validation and optimization. Overcoming this challenge is critical to advancing cancer immunotherapy.

Surface Plasmon Resonance (SPR) technology is extensively employed in detecting various biomolecules, including antibody antigens, enzymes, nucleic acids, and drugs, and is particularly effective in small molecule drug screening15,16. Unlike other biophysical techniques, SPR offers label-free detection, real-time kinetic data, and a broad detection range. In contrast, Isothermal Titration Calorimetry lacks real-time kinetic insights and requires larger sample volumes, limiting throughput. Microscale Thermophoresis is prone to buffer interference and cannot provide kinetic data, while Biolayer Interferometry has application-specific limitations based on molecular size and properties. Homogeneous Time-Resolved Fluorescence requires labeling and is susceptible to fluorescent interference. We acknowledge that HTRF is another suitable technology to explore PD-1/PD-L1 inhibitors. One inherent limitation of HTRF, compared to SPR, is fluorescence quenching caused by external interactions with the intramolecular excitation process (e.g., electron transfer, FRET, and bleaching), the sensitivity is too low in the drug screening process because of the small window range, and interference from fluorescent library compounds or biological proteins17. These features position SPR as a superior tool for drug discovery. Our previous studies have demonstrated that SPR is able to determine the blockade effect of small molecules against PD-1/PD-L1, which is advantageous over other techniques that require high labeling technology requirements, multiple steps, poor specificity, and high cost in the drug discovery process18.

This study introduces an optimized SPR-based platform, integrating a dual-step coupling process that utilizes both amine and bio-streptavidin coupling to enhance PD-1 orientation on the chip and minimize protein usage. This updated approach was successfully validated using the PD-1/PD-L1 inhibitor BMS-1166 as a positive control binder, demonstrating blockade effects comparable to both our previous SPR method and other established techniques such as ELISA19,20. This not only confirms the reliability and reproducibility of our protocol but also illustrates the effectiveness of our modified platform in facilitating high-throughput screening of PD-1/PD-L1 inhibitors. The incorporation of the bio-streptavidin capturing step provides site-directed rather than random protein orientation, allowing for reduced PD-1 concentration (40 µg/mL vs. 10 µg/mL) and cost savings by enabling the end user to immobilize streptavidin (SA) to a CM5 chip, a less expensive alternative to commercialized pre-immobilized SA chips. This makes it advantageous for large-scale, cost-effective screenings of compound/peptide libraries. Although additional characterization methods, including in silico, in vitro, and in vivo assays, are essential to evaluate the clinical potential of PD-1/PD-L1 inhibitors against cancer, our enhanced SPR-based screening platform stands out as an efficient tool for large-scale screening of PD-1/PD-L1 inhibitors.

Protokol

The reagents and equipment are listed in the Table of Materials.

1. Immobilization of the streptavidin (SA) protein on the CM5 chip

  1. Setup the immobilization method on the SPR instrument: Open/new wizard template, choose immobilization, set the chip type to CM5, and flow cells per cycle to 1. Check immobilize flow cell 1 and flow cell 2.
    1. Set Amine as the immobilization method. Set aim for immobilized level, ligand concentration: 40 µg/mL streptavidin, target level: 2000 RU, wash solution: 50 mM NaOH. Next, check prime before running.
  2. Prepare the following tubes: R2 B1 and R2 C1 - 40 µg/mL streptavidin; R2 B2 and R2 C2: 50 mM of NaOH; R2 B3 and R2 C3: EDC; R2 B4 and R2 C4: NHS; R2 B5 and R2 C5: Empty; R2 B6 and R2 C6: Ethanolamine.
    NOTE: EDC and NHS activate the carboxyl groups on the CM5 chip, allowing covalent coupling with amines on the ligand. Ethanolamine is used as a blocking buffer to prevent nonspecific binding during immobilization.
  3. Dilute 20 mL of HBS-EP+ buffer 10× in 180 mL of deionized (DI) water to prepare 200 mL of 1× HBS-EP+ running buffer solution.
  4. Add 1 mL of DNase-free water to 1 mg of streptavidin and incubate at room temperature for 30 min. Next, dilute the streptavidin solution to 40 µg/mL in acetate buffer (pH 4.5). Prepare the Amine Coupling reagent kit reagents according to the manufacturer's instructions and place all of the relevant reagent's layouts as indicated in step 1.2.
  5. Replace the maintenance sensor chip with the CM5 chip, then place Tube A into the prepared 1× HBS-EP+ buffer, reopen the immobilization method, and insert tubes according to step 1.2 layout. Run the method and save the results file.
  6. Post-run maintenance: replace the CM5 chip with the maintenance sensor chip, place Tube A into a bottle filled with deionized water, and run the prime. After taking out the CM5 chip, wash the chip with a few drops of DI water and air dry. Place the chip into a 50 mL tube at 4 °C.
    NOTE: The immobilized SA enables the CM5 chip to function as an SA chip.

2. Immobilization of PD-1 protein on the SA chip

  1. Set the immobilization method on the SPR instrument: Open/new wizard template, choose immobilization, set chip type to SA, and flow cells per cycle to 1. Check immobilize flow cell 1 and cell 2.
    1. Set SA-biotin capture as the method. For cell 1 set blank immobilization, for cell 2 set aim for immobilized level, 10 µg/mL PD-1 as the ligand, target level: 4000 RU, then check prime before running.
      NOTE: Prepare the following tubes - R2 B1 and R2 C1: 1 M of NaCl, 50 mM of NaOH; R2 B2 and R2 C2: 50% Isopropanol/50 mM of NaOH/1 M of NaCl; R2 C3: 10 µg/mL PD-1.
  2. Dissolve 58.44 mg of NaCl in 1 mL of 50 mM NaOH to prepare the 1 M of NaCl and 50 mM of NaOH solution. Dissolve 58.44 mg of NaCl and 4.0 mg of NaOH in 500 µL water, then add 500 µL of isopropanol to prepare the 50% Isopropanol/50 mM NaOH/1 M NaCl solution.
  3. Prepare PD-1 solution: Add 200 µL of DNase-free water to 100 µg of Biotinylated Human PD-1 (Fc and Avitagged) and stabilize at room temperature for 30 min, then dilute to 10 µg/mL in acetate buffer (pH 5.0).
  4. Place all the reagent layouts as described in step 2.1. Place Tube A into the 1× HBS-EP+ running buffer solution, then eject the maintenance sensor chip and insert the SA chip (CM5 chip coated by streptavidin protein from step 1). Reopen the immobilization method, insert reagent rack 2, and check the positions, then run the method for the estimated run time.
  5. Repeat the step 1.6.

3. Regeneration scouting for PD-1 and PD-L1

  1. Setup the regeneration scouting method: Open/new wizard template, choose Regeneration Scouting, set flow path: 2-1, 4-3, chip type to SA, check run conditioning cycle, record the solution as HBS-EP+, contact time as 30 s, number of injections as 3, solution as PD-L1, contact time: 30 s, flow rate: 30 µL/min.
    1. For regeneration parameters, the flow rate is 30 µL/min, and the stabilization period is 300 s. In the experimental design, set the number of conditions as 4, and the number of cycles for each condition as 2. Set the conditions as 4, regeneration solution: Glycine 1.5, 2.0, 2.5, 3.0, contact times: 30 s, then check prime before running.
  2. Set each PD-L1 concentration as a separate sample well position in a 96-well microplate layout: R1 A1 to R1 A9: 1 µM PD-L1; R1 A10 to R1 A12: HBS-EP+ buffer; R1 B1: Glycine 1.5; R1 B2: Glycine 2; R1 B3: Glycine 2.5; R1 B4: Glycine 3.
    NOTE: Glycine buffer with different pH is used as the regeneration buffer.
  3. Prepare PD-L1 solution: Add 200 µL of DNase-free water to 100 µg of Human PD-L1 Fc Tag protein (9.42 µM), then dilute to 1 µM in HBS-EP+ buffer.
  4. Place all the relevant reagents in the layout as described in step 3.2. Place Tube A into the 1× HBS-EP+ buffer, then replace the maintenance sensor chip with the SA chip. Reopen the Regeneration Scouting method, follow the tube position from step 3.2, insert the reagent rack, and then run the method for the estimated run time.
  5. Repeat the step 1.6.

4. Validation of PD-1/PD-L1 interaction

NOTE: For validation, a previously published report18 was followed with minor adjustments.

  1. Use the same parameters as the published report under General Settings, Assay Steps, and Cycle Types, set contact time to 60 s, dissociation time to 60 s, and flow rate to 30 µL/min. Under method variables, evaluation variables, and commands, use the same setting, except for using Glycine 2.0 as Regeneration, setup flow rate to 30 µL/min, goes through the flow path of 1, 2, 3, 4.
    1. Then set up run, and choose the flow path: 2-1, 4-3. Input PD-L1 with concentrations from 0 µM, 0.037 µM, 0.111 µM, 0.333 µM, and a molecular weight of 51,300 Da. Check all the assay steps for verification and select prime before the run.
    2. Then set each PD-L1 concentration as a separate sample well position in a Reagent Rack 2 layout: R2 B1: PD-L1 0 µM; R2 B2: PD-L1 0.037 µM; R2 B3: PD-L1 0.111 µM; R2 B4: PD-L1 0.333 µM; R2 A1: Glycine 2.0 as regeneration buffer; R2 A2: HBS-EP+ as startup buffer.
  2. Prepare 200 mL of 1× HBS-EP+ running buffer solution. Prepare PD-L1 concentrations: dilute PD-L1 protein at 9.42 µM to 0.333 µM, 0.111 µM, and 0.037 µM in HBS-EP+. Then place all the relevant reagents in the layout as described in step 4.1.2.
    1. Insert Tube A into the 1× HBS-EP+ running buffer solution, eject the maintenance sensor chip, and insert the SA chip. Reopen the regeneration scouting method, follow the tube positioning from step 4.1, insert reagent rack 2, and run the method for the estimated duration.
  3. Repeat the step 1.6.

5. PD-1/PD-L1 blockade assay with small molecule inhibitor: BMS-1166

NOTE: For the blockade assay, a previously published report18 was followed with minor adjustments.

  1. Use the same parameters as the published report under General Settings, Assay Steps, and Cycle Types, set contact time to 60 s, dissociation time to 60 s, and flow rate to 30 µL/min. Under method variables, evaluation variables, and commands also use the same setting, except for using Glycine 2.0 as Regeneration, setup flow rate to 30 µL/min, goes through the flow path of 1, 2, 3, 4.
    1. Then set up run, and choose flow path: 2-1, 4-3. Input the sample solution: PD-L1 (0.111 µM, molecular weight of 51,300 Da) with BMS-1166 at 0 µM, 0.125 µM, 0.625 µM, 3.125 µM. Check all the assay steps for verification and select prime before the run.
    2. Then set each PD-L1 concentration as a separate sample well position in a Reagent Rack 2 layout: R2 B1: PD-L1 (0.111 µM) + BMS-1166 0 µM R2 B2: PD-L1 (0.111 µM) + BMS-1166 0.125 µM; R2 B3: PD-L1 (0.111 µM) + BMS-1166 0.625 µM; R2 B4: PD-L1 (0.111 µM) + BMS-1166 3.125 µM; R2 A1: Glycine 2.0 for regeneration.
  2. Prepare 200 mL of 1× HBS-EP+ running buffer solution. Prepare BMS-1166/PD-L1 mixture: Dissolve 5 mg of BMS-1166 in 77.99 µL Dimethyl sulfoxide (DMSO) to prepare a 100 mM stock solution. Dilute the stock solution with PD-L1 protein (0.11 µM) to the target concentrations of BMS-1166 at 0 µM, 0.125 µM, 0.625 µM, and 3.125 µM in HBS-EP+. Place all the relevant reagents layout as described in step 5.1.2.
    1. Place Tube A into the 1× HBS-EP+ running buffer solution, then eject the maintenance sensor chip and insert the SA chip. Reopen the regeneration scouting method, follow the tube position from 5.1 and, insert reagent rack 2, then run the method for the estimated run time.
  3. Repeat the step 1.6.

Sonuçlar

Immobilization of SA on CM5 chip
Data were analyzed via the output from the SPR instrument and associated analysis software indicating successful achievement of the target RU (2000 RU) of SA protein on flow cell 1 and flow cell 2. Flow cells 1 and 2 were immobilized with SA (40 μg/mL) on the CM5 chip surface with a final response of 1902.3 RU on flow cell 1 (Figure 1A) and 1900.7 RU on flow cell 2 (Figure 1B).

Tartışmalar

Over the past few decades, various immunotherapy approaches-including cancer vaccines, immune checkpoint inhibitors, and CAR T-cell therapies-have significantly advanced cancer treatment21. Immune checkpoints play a crucial role in preventing immune cell-mediated collateral damage during pathogenic responses and in suppressing autoimmunity. A key example is the interaction between PD-L1 and PD-1, which forms a major immune checkpoint, allowing cancer cells to evade immune surveillance. Targeting t...

Açıklamalar

The authors have nothing to disclose.

Teşekkürler

The authors acknowledge the RI-INBRE core facility at the University of Rhode Island, supported by Grant P20GM103430 from the National Center for Research Resources (NCRR), a component of the National Institutes of Health (NIH). This research was supported by a Pilot Grant Award from the College of Pharmacy at the University of Rhode Island, a Small Grant Award from the Rhode Island Life Science Hub (RILSH), and a Rhode Island Foundation grant, all awarded to Chang Liu, Ph.D.

Malzemeler

NameCompanyCatalog NumberComments
50 mM NaOHCytiva Life Sciences100358
50 mM NaOHFisher Scientific905376
96-Well Polystyrene MicroplatesCytiva Life SciencesBR100503
Amine Coupling KitCytiva Life Sciences35120
Biacore T200 SPR System and Evaluation Software 3.2Cytiva Life Sciences28975001
Biotinylated Human PD-1 Fc, Avitag ProteinAcro BiosystemsPD1-H82F1
BMS1166MedChemExpressHY-102011
Dimethyl Sulfoxide (DMSO)Sigma-Aldrich276855
DNase Free WaterFisher Scientific188506
Glycine 1.5Cytiva Life SciencesBR100354
Glycine 2.0Cytiva Life SciencesBR100355
Glycine 2.5Cytiva Life SciencesBR100356
Glycine 3.0Cytiva Life SciencesBR100357
HBS-EP+ BufferCytiva Life SciencesBR100669
Human PD-L1 Fc Tag ProteinAcro BiosystemsPD-1-H5258
IsopropanolFisher ScientificBP2618-1
Microplate Foil, 96-Well Cytiva Life Sciences28975816
NaClSigma-Aldrich746398
Plastic Vials 7 mmCytiva Life SciencesBR100212
Rubber Caps, Type 3Cytiva Life SciencesBR100502
Series S Sensor Chip CM5Cytiva Life Sciences29149603
Sodium Acetate 4.5Cytiva Life Sciences100350
Sodium Acetate 5.0Cytiva Life Sciences100351
StreptavidinSigma-AldrichS4762

Referanslar

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