CTLA-4, also known as CD152, is an immune inhibitory receptor constitutively expressed on regulatory T cells (Tregs) and upregulated in activated T cells. CTLA-4 plays a critical role in regulating immune responses to tumor antigens and autoantigens. When CTLA-4 expression is upregulated on the surface of T cells, the T cells bind B7 with a higher avidity, and thus out-compete the positive co-stimulatory signal from CD28. In addition, engagement of CTLA-4 by either of its ligands, CD80 (B7-1) or CD86 (B7-2) on an adjacent antigen presenting cell (APC) inhibits CD28 co-stimulation of T cell activation, cell proliferation and cytokine production. Following the success of CTLA-4 immunotherapy drug ipilimumab (marketed as YERVOY for metastatic melanoma), other new therapeutic antibodies and Fc fusion proteins designed to block the CTLA-4/CD80 and CD86 interaction have been launched as drugs or have shown promising results in clinical trials for the treatment of a variety of cancers. The CTLA-4 Blockade Bioassay reflects the mechanism of action (MOA) of biologics designed to block the interaction of CTLA-4 with its ligands, CD80 and CD86. Specifically, CD28-mediated luminescence is detected following the addition of anti-CTLA-4 blocking antibodies but not following addition of a non-specific anti-HER2, anti-PD-1 or anti-TIGIT blocking antibody. The assay consists of two genetically engineered cell lines: 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, aAPC/Raji Cells: Raji cells expressing an engineered cell surface protein designed to activate cognate TCRs in an antigen-independent manner and endogenously expressing CTLA-4 ligands CD80 and CD86. When the two cell types are co-cultured, CTLA-4 competes with CD28 for their shared ligands, CD80 and CD86, and thus inhibits CD28 pathway activation and promoter-mediated luminescence. Addition of an anti-CTLA-4 antibody blocks the interaction of CTLA-4 with its ligands CD80 and CD86 and results in promoter-mediated luminescence. The CTLA-4 Blockade Bioassay is available in 1X and 5X sizes. Control Ab, Anti-CTLA-4, is available separately. CTLA-4 Negative Cells are available separately.
