Immune Checkpoint Blockade in Oncology Drug Discovery
In recent years, immune checkpoint blockade has emerged as a groundbreaking therapeutic strategy in immuno-oncology. Programmed cell death 1 receptor (PD-1), and its ligand programmed cell death ligand 1 (PD-L1) are immune checkpoint proteins found on the cell surface of T cells. PD-1 is expressed on various hematopoietic cells, including T cells, B cells, natural killer (NK) cells, monocytes, macrophages and dendritic cells (DCs) following their activation. PD-L1 is the ligand of PD-1 and is expressed by antigen-presenting cells and tissue cells, including cancer cells.
The PD-1/PD-L1 pathway negatively regulates the immune response by inhibiting the activation and proliferation of T lymphocytes, reducing the production of cytokines, and enhancing the exhaustion of CD8+ T lymphocytes. During cancer, expression of both PD-1 and PD-1 ligands is abundant, and as such PD-1 mediated T cell dysfunction strongly weakens the antitumor immune response (Figure 1).
As such interfering with the PD-1 PD-L1 pathway has shown significant promise in restoring immune responses, enabling the immune system to target tumors more effectively (Figure 1). These discoveries have sparked intense research efforts to investigate the efficacy of PD-1 PD-L1 pathway inhibitors as therapeutic anticancer drugs. Indeed, many preclinical and clinical studies have now shown that inhibition of the PD-1/PD-L1 pathway provides effective cancer treatments. To date, many drugs that target the PD-1 PD-L1 pathway have been FDA and EMA-approved for clinical cancer treatment, with more drugs being discovered or tested in the preclinical, and clinical phases of drug discovery.
FIGURE 1. Simplified schematic representation of the PD-1 PD-L1 pathway. Programmed death protein 1 (PD-1) is a type 1 cell surface receptor expressed by activated CD4+ and CD8+ T cells, natural killer (NK) T cells, and antigen-presenting cells (APCs). The PD-1 receptor has two ligands, programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), which are expressed on APCs as well as cancer cells. PD-1 PD-L1 binding inhibits TCR-mediated positive signaling, leading to reduced proliferation, reduced cytokine secretion, and reduced survival; this results in a downregulation of the immune response. Interfering with PD-L1 and PD1 binding (such as via antibody PD-1 PD-L1 mediated blockade) inhibits TCR-mediated signaling, leading to increased T-cell proliferation, increased cytokine secretion and increased cell survival.
At InnoSer, our in vitro and in vivo oncology services include specialized assays designed to evaluate the potency, mechanism of action (MoA), as well as therapeutic efficacy of compounds targeting the PD-1 PD-L1 immune checkpoint pathway.
In this blog post, we showcase an example of a cost-effective in vitro PD-1 PD-L1 blockade assay that can be utilized to obtain quick, but essential MoA and potency in vitro data prior to progressing to in vivo animal pharmacology, toxicology and/or efficacy studies.
Key Applications of the PD-1 PD-L1 Blockade Assay
InnoSer’s research services using the PD-1 PD-L1 blockade assay have several applications in early-stage drug discovery and development.
- Suitable for compounds targeting the PD-1 PD-L1 pathway: This assay is highly suitable to test antibodies, Fc-fusion proteins, small molecule antagonists or other biologics aimed at PD-1 PD-L1 inhibition.
- Lead compounds screening: InnoSer helps you efficiently incorporate the PD-1 PD-L1 blockade assay in your lead compound screening program; helping you identify lead therapeutic candidates during your early drug discovery phases. Indeed, testing of multiple novel lead compounds without the involvement of animal models proves to be a strategic tool to gain early insights into predicted compound efficacy.
- MoA studies: Using this assay, our dedicated research team helps you uncover key insights into how your compounds affect immune checkpoint interactions, by directly testing the ability of novel compounds to block PD-1 signaling cascade.
- Potency testing: PD-1 PD-L1 blockade assay evaluates antibody’s potency allowing you to calculate EC50 values. In this assay, the fluorescence is directly correlated to the compound’s potency; one of key compound’s characteristics that provides predictive basis for in vivo efficacy.
These initial results can provide you with a basis for either further optimization of the lead compounds or confirmation of their suitability in further preclinical testing. This allows you to make more informed decisions, rendering your overall drug development process more efficient.
PD-1 PD-L1 Blockade Aassay Principles
The PD-1 PD-L1 blockade assay offered by InnoSer is an in vitro based assay that enables you to assess your PD-1 PD-L1 therapeutics’ MoA. We conduct the assay using two genetically engineered cell lines:
- PD-1 Effector Cells: Jurkat T cells that express the human PD-1 receptor and contain a luciferase reporter driven by the NFAT response element (NFAT-RE).
- PD-L1 aAPC/CHO-K1 Cells: CHO-K1 cells express human PD-L1, along with a cell surface protein designed to activate TCRs independently of antigens.
When these two cell types are cocultured, the PD-1/PD-L1 interaction inhibits TCR-mediated luminescence. However, the addition of therapeutic compounds —either anti-PD-1 or anti-PD-L1—blocks this interaction, activating TCR signaling, which in turn triggers luminescence via the NFAT pathway. Luminescence is then quantified by addition of a Bio-Glo™ reagent and a luminometer.
InnoSer offers the PD-1/PD-L1 bioassay in 96-well format, providing scalability for different research needs. Our team has also fine-tuned the assay conditions—such as plate layout, background signal correction, and controls (positive and negative) setup—to ensure you obtain the most accurate results. Key elements of the assay set-up include:
- Background signal subtraction: We ensure luminescence readings are accurate by subtracting the signal from wells containing only assay medium.
- Positive and negative controls: To ensure your accuracy of your observed effects, experts at InnoSer work with validated positive and negative controls.
- Reference antibody dilution curves: We construct reference antibody dilution curves to benchmark test antibody performance. Control antibody shows blocking activity of the PD-1 with its PD-L1 ligand (Figure 2).
- Test compound dilution curves: To assess dose-dependent blockade effects of your compound, experts at InnoSer perform serial dilutions.
FIGURE 2. Representative dose-response curve for reference antibody expressed as fold induction.
Partnering with InnoSer for custom in vitro assay development for oncology research
PD-1 PDL-1 blockade assay forms part of InnoSer’s in vitro oncology assay portfolio. Other relevant immuno-oncology assays include cytotoxicity assays, flow cytometry-based screening assays as well as in vitro screening using organoids. To read more about our in vitro oncology assays, please visit our overview webpage here. In addition to this, InnoSer regularly performs and validates in vitro assays in close collaboration with our clients to closely match their study-specific needs, taking into account different compound types and MoAs, timelines as well as available budgets for outsourced studies.
Seamlessly progress from in vitro to in vivo research with InnoSer
While in vitro assays provide valuable insights, it is essential to confirm these findings in vivo to fully understand a candidate drug’s therapeutic efficacy and safety. InnoSer’s immuno-oncology platform offers you both in vitro assay expertise as well as in vivo experiments including pharmacology (e.g., PK/PD profiling, ADME), early-stage toxicology (e.g., MTD) as well as efficacy in multiple mouse models.
Recommended in vivo efficacy experimental set-ups that InnoSer can carry-out include testing of anti-PD1/PD-L1 compounds in syngeneic tumor models with intact immune system. One such validated syngeneic mouse model at InnoSer that is anti-PD-1 responsive is the MC38 colorectal carcinoma model. To learn more about the different possible in vivo tumor models that InnoSer can work with, please visit our oncology CRO services webpage overview here.
Curious to learn more? Contact us today to explore how our tailored preclinical CRO solutions can meet your unique research needs.