CTLA-4 blockade cell-based assay

Similar to the PD-1/PD-L1 assay, and here also to complete our in vivo services, we operate a 96-well plate luminescent bioassay to address the potency of candidate drugs capable of blocking the interaction between CD80/CD86 exposed at the surface of engineered Raji cells and CTLA-4 expressed by modified Jurkat cells.

Assay Principle

 

  • Raji Antigen Presenting Cells exposing MHC and CD80/86 are plated in 96-well plates with effector T Cells (Jurkat) expressing CD28, CTLA4 and TCR which controls the expression of Luciferase reporter gene.
  • Concomitant T Cell Receptor and CD28 engagement induces luciferase activity in the effector T cell (Jurkat) while co-engagement of an immune checkpoint receptor-ligand pair inhibits the luciferase activity
  • Antibody-mediated blockade of the immune checkpoint inhibitory signal restores luciferase activity which is detected using an appropriated luminescence plate reader

anti ctla-4 cell-based assay immune checkpoint inhibitor screening CRO service

Representative Results

Blocake of anti-CTLA4 restores TCR signaling and luciferase expression in a Promega assay

CTLA4 expressing effector Cells and APC cells were incubated for 16 hrs at 37°C with increasing concentrations of  anti-CTLA-4 antibodies. Ipilimumab was found to abolish the immune checkpoint inhibitory signal thus resulting in the restoration of luciferase activity.

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Explicyte immuno-oncology offers to test novel cancer immunotherapies for their ability to induce CTLA-4 blockade. Our team has validated an anti-CTLA-4 cell-based assay for the screening of novel inhibitors.