Cancer Immunotherapy: Checkpoint Inhibitors

Vivian Cao xCELLigence

By disrupting the signaling pathways that normally suppress immune cell activation, checkpoint inhibitors enable immune effector cells to attack cancer cells more aggressively. From mechanistic validation of novel checkpoint targets to comparing the relative efficacy of two different checkpoint-modulating antibody constructs, xCELLigence Real-Time Cell Analysis (RTCA) instruments can help you to efficiently answer your questions under conditions of maximal physiological relevance.

In the below example xCELLigence RTCA was used to monitor the impact of an anti-PD-1 antibody on the killing of prostate cancer PC3 cells by PBMCs. Target PC3 cells were seeded in ACEA’s patented biosensor plate (E-Plate) and allowed to attach and proliferate. Frozen PBMCs were thawed, activated by incubation with SEB superantigen, and then added on top of the PC3 cells in the presence or absence of anti-PD-1 antibody. The effector:target ratio was 5:1. As seen in the upper figure, by themselves PBMCs display a modest capacity for killing PC3 cells (blue trace), but this killing is much more robust in the presence of the anti-PD-1 antibody (orange trace). Using the xCELLigence RTCA software, the primary data is readily converted to a % Cytolysis plot (lower figure), which helps to elucidate the checkpoint inhibitor’s impact: earlier onset of target cell killing, increased rate of cytolysis, and a greater total extent of cell killing.

Key Benefits of Using xCELLigence for Checkpoint Inhibitor Studies:

  • Label-Free: Because 51Cr, luciferace, dyes, etc. aren’t needed, target cancer cells and immune effector cells can be used directly…maximizing physiological relevance.
  • Exquisite Sensitivity: Analyze checkpoint inhibitor efficacy at low, physiologically relevant effector:target ratios.
  • Unlimited Assay Window: The impact of checkpoint inhibitors on target cell killing can be monitored over assay windows spanning anywhere from minutes to days, elucidating both fast and slow killing kinetics. Differences in long term killing behavior are now observable.
  • Astonishingly Simply Workflow: Plate target cells, add effector cells and molecules, and start monitoring. High throughput comparisons of different constructs or combination therapies are easy to setup, run, and analyze.

Checkpoint Inhibitor Studies – Supporting Information:

Dual Purpose Single Plate Multi Plate High Throughput
RTCA DP RTCA SP RTCA MP RTCA HT
3×16 wells 1×96 wells 6×96 wells Up to 4×384 wells
Cancer Immunotherapy Supporting Information:

  • Checkpoint Inhibitor Publications:
    • Upregulation of PD-L1 by EML4-ALK fusion protein mediates the immune escape in ALK positive NSCLC: Implication for optional anti-PD-1/PD-L1 immune therapy for ALK-TKIs sensitive and resistant NSCLC patients. Hong S, Chen N, Fang W, Zhan J, Liu Q, Kang S, He X, Liu L, Zhou T, Huang J, Chen Y, Qin T, Zhang Y, Ma Y, Yang Y, Zhao Y, Huang Y, Zhang L. Oncoimmunology. 2015 Dec 21;5(3):e1094598.
    • PD-1 blockade enhances the vaccination-induced immune response in glioma. Antonios JP, Soto H, Everson RG, Orpilla J, Moughon D, Shin N, Sedighim S, Yong WH, Li G, Cloughesy TF, Liau LM, Prins RM. JCI Insight. 2016 Jul 7;1(10).
    • Immunosuppressive tumor-infiltrating myeloid cells mediate adaptive immune resistance via a PD-1/PD-L1 mechanism in glioblastoma. Antonios JP, Soto H, Everson RG, Moughon D, Orpilla JR, Shin NP, Sedighim S, Treger J, Odesa S, Tucker A, Yong WH, Li G, Cloughesy TF, Liau LM, Prins RM.Neuro Oncol. 2017 Jun 1;19(6):796-807.
    • PDL1 Signals through Conserved Sequence Motifs to Overcome Interferon-Mediated Cytotoxicity. Gato-Cañas M, Zuazo M, Arasanz H, Ibañez-Vea M, Lorenzo L, Fernandez-Hinojal G, Vera R, Smerdou C, Martisova E, Arozarena I, Wellbrock C, Llopiz D, Ruiz M, Sarobe P, Breckpot K, Kochan G, Escors D. Cell Rep. 2017 Aug 22;20(8):1818-1829.