PD-1 functions as an immune checkpoint and is involved in tumor progression. The binding of PD-1 to PD-L1 could inhibit the proliferation and activation of T cells, downregulate the secretion of cytokines, and ultimately inhibit the immune function of T cells, leading to tumor immune escape. Therefore, the blockade of PD-1/PD-L1 by inhibitors has a wide range of anti-tumor applications in basic research.
Fig. 1 The mechanism of PD1/PDL1 blockade. (Lei, et al, 2020)
PD-1 is mainly expressed on the surface of activated T cells and PD-L1 is frequently observed on the surface of tumor cells. PD-1 and PD-L1 play a role in the modulating of T cell-mediated immune responses. Blocking the binding of PD-1 to PD-L1 is a promising strategy for cancer immunotherapy. Creative Biolabs has developed robust and consistent bioassays to evaluate the efficacy of antibodies to block the interaction between PD-1 and PD-L1, such as in vitro cell-based assays and in vivo tumor xenograft models.
The binding of PD-1 to PD-L1 on neighboring cells can inhibit the transduction of TCR signaling pathways and TCR-mediated effects such as cell proliferation, transcriptional activation, and cytokine production. Therefore, blockade of this interaction can be detected in vitro by measuring proliferation, luciferase reporter expression, or IFN-γ/IL-2 release.
Fig. 2 Proliferation of PBMCs was induced by OKT3. (Creative Biolabs)
Peripheral blood mononuclear cells (PBMCs) were cultured and stimulated with the anti-CD3 antibody OKT3 (V3S-1222-YC1222, Creative Biolabs) at a series of concentrations (0 μg/mL, 0.01 μg/mL, 0.05 μg/mL, 1 μg/mL, and 5 μg/mL).
Fig. 3 Blockade of PD-1/PD-L1 interaction by anti-PD-1 antibodies resulted in an increase of IFN-γ production. (Creative Biolabs)
PBMCs were stimulated with 2 μg/mL anti-CD3 antibody OKT3 (V3S-1222-YC1222, Creative Biolabs) and cultured for 144 hours with PD-1 antibody Nivolumab or PD-1 antibody (V3S-1022-YC6030, Creative Biolabs). Human IgG4 antibody as an isotype control. After cultivation, the supernatant was collected and the IFN-γ secretion level was analyzed using a human IFN gamma ELISA kit. The assay was performed in three parallel wells.
Blockade of the PD-1/PD-L1 interaction can also be assessed using a luciferase bioassay, the Jurkat NFAT luciferase PD-1/PD-L1 reporter assay. Two genetically engineered cell lines are required for this assay.
PD-1 effector cells: Jurkat T cells stably express human PD-1 and the NFAT-RE-induced luciferase reporter.
APCCHO-PD-L1 cells: aAPC/CHO-K1 cells stably express human PD-L1 and anti-CD3 scFv antibody.
Fig.4 Schematic diagram of PD-1/PD-L1 blockade bioassay. (Creative Biolabs)
When PD-1 effector Jurkat cells are co-cultured directly with PD-L1 artificial antigen-presenting CHO cells (APCCHO-PD-L1 cells), the interaction between PD-1/PD-L1 inhibits TCR signal transduction and NFAT-RE-induced luminescence, as shown in Figure 4 (left). PD-1 or PD-L1 antibodies with blocking activity could inhibit the signaling pathway, resulting in TCR activation and NFAT-RE-induced luminescence, as shown in Figure 4 (right).
Fig.5 Inhibition of PD-1 blockade in PD-1/PD-L1 NFAT reporter assay. (Parthiban, et al, 2019)
PD-1 effector Jurkat cells were co-cultured with aAPC/CHO-K1 cells presenting PD-L1 in the presence or absence of anti-PD-1 blocking antibodies (K5T8-9, K5E3-61, nivolumab and 331_1_C08). The luminescence signal is plotted against the concentration of these antibodies.
Creative Biolabs has comprehensive reporter platforms for detecting the blockade activity of PD-1/PD-L1 detection. With years of experience in process optimization, we provide one-stop services from neutralizing/blocking antibodies to rapid PD-1/PD-L1 blockade activity detection in the mass assay.
Fig.6 Workflow of PD-1/PD-L1 blockade bioassays. (Creative Biolabs)
Creative Biolabs offers customized services and guarantees to meet project timelines. Our team has hundreds of successful experiences in functional assays of immune cells against various targets. We also have positive and negative antibodies available for our clients. For more information about the PD-1/PD-L1 blockade bioassay platform, please feel free to contact us.