Immune Checkpoint Assays

Immune checkpoint molecules play an important role in the elimination of cells that express foreign antigens while maintaining tolerance to self-antigens. Given their capacity to regulate immune responses, immune checkpoint receptors have emerged as promising new immunotherapy targets for a variety of diseases, including cancer and autoimmune disorders.

In general, immune checkpoints are divided into two categories: activating receptors and inhibitory receptors. The two key mechanisms through which an immunotherapeutic compound or biologic acts are either by blocking the immune pathway (antagonists) or by enhancing the immune pathway response (agonists). Using novel therapeutics to target these immune response pathways has proven to be an effective treatment strategy against multiple diseases. Reaction Biology's scientists have developed a comprehensive portfolio of immune checkpoint assays, ready to be used in a variety of settings, from the screening and characterization of biologics (antibodies) in cell-based assay formats to quantifying potency and binding of small and large molecules in biochemical assay formats.

Please reach out today to discuss how we can best assist you with your immuno-therapy research. 

Immune Checkpoint Strategies for Immunotherapy

  • Promoting immune cell responses and activation
  • Stimulating immune cell proliferation and survival
  • Downregulation of the harmful immune response to prevent tissue damage and autoimmunity
  • Helping immune cells recognize and infiltrate the tumor
  • Co-stimulation and activation of cell-signaling molecules such as cytokines
Immune Checkpoint Strategies

Immune Checkpoint Bioassays Available at Reaction Biology

PD-1/PD-L1 Blockade Bioassay

PD-1 is an immune inhibitory receptor that is expressed on activated T and B cells and regulates immune responses to tumor antigens and autoantigens. Engagement of PD-1 on an adjacent cell by either of its ligands, PD-L1 or PD-L2, inhibits TCR signaling and TCR-mediated proliferation, transcriptional activation, and cytokine production. As a result, therapeutic antibodies designed to inhibit the PD-1/PD-L1 interaction are showing promising results in clinical trials for the treatment of a variety of cancers.

Goal:
To evaluate the potency and stability of antibodies and other biologics designed to block the PD-1/PD-L1 interaction.

Method:
PD-1 effector cells and PD-L1 aAPC/CHO-K1 cells are cocultured, and the interaction of these two cells inhibits TCR signaling and NFAT-RE-mediated luminescence. The addition of either an anti-PD-1 or anti-PD-L1 antibody blocks the PD-1/PD-L1 interaction and releases the inhibitory signal, resulting in TCR activation and NFAT-RE-mediated luminescence. 

 

PD-1/PD-L1 Blockade Bioassay


The PD-1/PD-L1 blockade bioassay demonstrating anti-PD-1 antibody potency.

CTLA-4 Blockade Bioassay

CTLA-4 is an immune inhibitory receptor that is expressed on regulatory T cells and activated T cells. CTLA-4 is essential for controlling immune responses to tumor antigens and autoantigens. When CTLA-4 expression on the surface of T cells increases, the T cells bind B7 with greater avidity, out-competing the positive co-stimulatory signal from CD28. CTLA-4 engagement by either of its ligands, CD80 (B7-1) or CD86 (B7-2) on a neighboring antigen-presenting cell inhibits CD28 co-stimulation of T cell activation, cell proliferation, and cytokine production. Thus, therapeutic antibodies designed to block CTLA-4 and its ligands CD80 and CD86 show promising results in clinical trials for the treatment of various cancers.

Goal:
To evaluate the potency and stability of antibodies and other biologics targeting CTLA4.


Method:
Co-culturing of CTLA-4 effector cells (Jurkat T cells expressing human CTLA-4 and a luciferase reporter driven by a native promoter that responds to TCR/CD28 activation) and aAPC/Raji cells inhibits CD28 pathway activation and promoter-mediated luminescence. The addition of an anti-CTLA-4 antibody blocks the interaction of CTLA-4 with its ligands CD80 and CD86, ultimately resulting in promoter-mediated luminescence.

 

CTLA-4 Blockade Bioassay


The CTLA-4 Blockade Bioassay measuring the inhibitory activity of anti-CTLA-4 blocking antibody.

CD40 Bioassay

The cell surface molecule CD40, expressed by B cells, dendritic cells and monocytes, is a member of the tumor necrosis factor receptor superfamily. CD40 ligand (CD154) is the primary ligand for CD40 and is expressed by activated T cells, which are critical regulators of cellular and humoral immunity. Signaling via CD40 triggers activation of antigen-presenting cells (APC). Agonist CD40 antibodies were found to mimic the signal of CD40 ligand and were capable of substituting for the function of CD4+ helper T cells in murine models of T cell-mediated immunity.

Therefore, agonist CD40 antibodies can rescue the function of APC in tumor-bearing hosts and restore effective immune responses against tumor antigens. Subsequent data from multiple preclinical models has demonstrated synergistic enhancement from combining CD40 agonists with cytotoxic drugs, especially chemotherapy.

Goal: 
The goal is to measure the potency and stability of ligands or agonist antibodies and other biologics that can bind and activate CD40.

Method: 
Genetically engineered cell line that expresses human CD40 and a luciferase reporter driven by a response element that can respond to CD40 ligand/agonist antibody stimulation.

CD40 Bioassay


The CD40 Bioassay showing specificity of CD40 ligand/agonist biomolecules.

OX40 Bioassay

OX40 is a stimulatory immune checkpoint receptor that has a significant role in cancer progression and autoimmune disease. Activating OX40 with its ligands or agonist antibodies has emerged as the next generation of an immunotherapeutic strategy to enhance anti-tumor immune responses and promote immune-mediated tumor rejection. When OX40 is present on the cell surface, OX40 interacts with OX40 ligand (OX40L), and induces subsequent cell proliferation, survival, and the production of cytokines, particularly in T cells. 

Goal:
The goal is to measure the potency and stability of ligands or agonist antibodies and other biologics that can bind and activate OX40.

Method:
Genetically engineered Jurkat T cell line that expresses human OX40 and a luciferase reporter driven by a response element that can respond to OX40 ligand/agonist antibody stimulation. The OX40 bioassay reflects the mechanism of action (MOA) of biologics designed to activate OX40.

OX40 Bioassay


The OX40 bioassays resemble the mechanism of action and demonstrate the specificity of biologics designed to activate OX40.

Immune Checkpoint Biochemical Assays Available at Reaction Biology

CTLA4-CD80 Biochemical Assay

Cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), also known as CD152, is a key regulator of T-cell immunity by maintaining activation and inhibition of T-cell immune responses. CTLA4 and CD28 are co-stimulatory and co-inhibitory cell surface signaling proteins that interact with the same ligands (CD80 and CD86), with CTLA4 displaying a greater affinity than CD28 for both, thus creating effective ligand binding competition. Studies have shown that functional blockage of CTL4 by anti-CTL4 binding by biologics and small molecules with high affinity results in enhanced T cell responses, ultimately resulting in more effective immune responses targeting many cancers.


View data sheet
 

PD1-PDL1 Biochemical Assay

PD-1 is an immune inhibitory receptor that is expressed on activated T and B cells and regulates immune responses to tumor antigens and autoantigens. Engagement of PD-1 on an adjacent cell by either of its ligands, PD-L1 or PD-L2, inhibits TCR signaling and TCR-mediated proliferation, transcriptional activation, and cytokine production. As a result, therapeutic antibodies designed to inhibit the PD-1/PD-L1 interaction are showing promising results in clinical trials for the treatment of a variety of cancers.


View data sheet