Immune checkpoint assays

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

Cell-based immune checkpoint assay formats to quantify the potency and binding of novel immunotherapeutic small molecules and biologics.

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.

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

PD-1/PD-L2 Blockade Bioassay

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

Goal:
To measure the potency and stability of antibodies and other biologics designed to block the PD-1/PD-L2 interaction.

The PD-1/PD-L2 blockade bioassay demonstrating anti-PD-1/PD-L2 antibody potency designed to block the PD-1/PD-L2 interaction.

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.

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.

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.

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

ICOS Blockade Bioassay

ICOS (CD278) binds to its ligand ICOSL (B7-H2, CD275), which is constitutively expressed on B cells, monocytes, and dendritic cells and can be induced on endothelial and epithelial cells during inflammation. ICOS co-stimulation induces the production of effector T cell cytokines such as interferon (IFN)-γ, interleukin (IL)-4, and IL-10. Blockade of ICOS or ICOSL has been investigated in preclinical models of allergy, autoimmunity, and alloimmunity. ICOS blockade reduces the severity of experimental graft-versus-host disease, graft rejection, experimental autoimmune arthritis, and experimental allergic encephalomyelitis.

Goal:
The goal is to measure the potency and stability of antibodies and other biologics that block ICOS/ICOSL.

Method:
Co-culturing of ICOS effector cells (Jurkat T cells expressing ICOS and endogenous TCR/CD3 and a NanoLuc luciferase reporter) and antigen-presenting cells (CHO-K1 cells expressing an engineered cell surface protein) activates TCR/CD3 and ICOS to induce maximum promoter-mediated luminescence. Adding a biologic that blocks ICOS/ICOSL inhibits co-stimulation by ICOS and results in decreased promoter-mediated luminescence.

The ICOS blockade bioassay demonstrating potency of anti-ICOS/ICOSL biologic ultimately resulting in decreased promoter-mediated luminescence.

Immune Checkpoint Biochemical Assays Available at Reaction Biology

Biochemical immune checkpoint assay formats to quantify the potency and binding of novel immunotherapeutic small molecules and biologics.

CD47-SIRPA Biochemical Assay

Signal-regulatory protein alpha (SIRP-alpha), also known as CD172a, is a cell surface protein expressed on all myeloid cells, including monocytes, macrophages, and neutrophils. These cells are typically the first in line immune cells to react against infectious pathogens and tumor cells. SIRPa binds to its ligand, CD47, which is ubiquitously expressed on the surface of all cells and has been found to be overexpressed in some cancers. The binding of SIRPa to CD47 initiates a cascade of SIRPa-phosphatase enzyme coupling, which inhibits phagocytosis by phagocytes such as macrophages. Tumor cells strategically misuse CD47 overexpression to evade myeloid cell-mediated elimination. Targeting the SIPRa/CD47 interaction has become a promising therapeutic strategy to treat many cancers by promoting tumor elimination through innate immunity.

Assay readout : HTRF

View data sheet

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.

Assay readout : HTRF

View data sheet

CTLA4-CD86 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.

Assay readout : HTRF

View data sheet

LAG3-MHCII Biochemical Assay

Lymphocyte activation gene-3 (LAG-3), also known as CD223 or FDC protein, is a novel immune checkpoint receptor expressed on CD4+, CD8+Tcells and natural killer (NK) cells and plays an important role in regulating the immune response against tumor antigens and autoantigens. The binding of LAG-3 to its functional ligand, the MHCII complex, results in cytokine production, activated T cells proliferation, and inhibition of TCR signaling. Therapeutics designed to exploit the LAG-3/MHCII binding have shown encouraging results as a potential treatment for many human diseases, including autoimmunity and cancer.

Assay readout : HTRF

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.

Assay readout : HTRF

View data sheet

Immune Checkpoint Strategies for Immunotherapy

Immune Checkpoint Bioassays Available at Reaction Biology

Immune Checkpoint Biochemical Assays Available at Reaction Biology

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