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Quantification of PD-1/PD-L1 Interaction between Membranes from PBMCs and Melanoma Samples Using Cell Membrane Microarray and Time-Resolved Förster Resonance Energy Transfer

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Research and Development Division, FASTBASE Solutions, 48160 Derio, Spain
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Research and Development Division, IMG Pharma Biotech, 48160 Derio, Spain
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School of Medicine and Nursing, University of the Basque Country UPV/EHU, 48940 Leioa, Spain
*
Author to whom correspondence should be addressed.
Academic Editors: Marcello Locatelli, Victoria Samanidou, Roberto Mandrioli and Thomas W. Bocklitz
Analytica 2021, 2(4), 156-170; https://doi.org/10.3390/analytica2040015
Received: 30 July 2021 / Revised: 5 October 2021 / Accepted: 7 October 2021 / Published: 13 October 2021
Melanoma is a carcinoma known to evade the host immune defenses via the downregulation of the immune response. One of the molecules involved in this mechanism is programmed cell death ligand 1 (PD-L1), which interacts with its receptor, programmed cell death protein 1 (PD-1), expressed on T cells, leading to a reduction in cytokine release and cytotoxic activity, as well as a halt in T-cell proliferation. The approved therapeutic monoclonal antibodies, such as pembrolizumab, target the PD-1/PD-L1 interaction and are revolutionizing cancer treatments. We developed an assay that provides a quantitative readout of PD-1/PD-L1 interactive states between cell membranes of human immune cells (peripheral blood mononuclear cells, PBMCs) and PD-L1-expressing samples. For this purpose, cell membrane microarrays (CMMAs) were developed from membranes isolated from a HT144 cell line and melanoma samples, and PD-L1 expression was quantified using immunofluorescence (IF). CMMAs were incubated with cell membranes of PBMCs expressing PD-1, and the interaction with PD-L1 was quantified by time-resolved Förster resonance energy transfer, in the presence and absence of pembrolizumab as a blocking drug. The developed assay was able to quantify the PD-1/PD-L1 interaction, and this engagement was disrupted in the presence of the blocking antibody. This demonstrates the potential of the method to analyze monoclonal antibody drugs, as well as the functional states of immune checkpoint regulators. Furthermore, our findings provide evidence to support the future implementation of this methodology for both drug discovery and immune system monitoring in cancer, transplantation, and inflammatory and autoimmune diseases. View Full-Text
Keywords: immunotherapy; protein–protein interaction; melanoma; microarray; amplified FRET; PD-L1/PD-1; immune checkpoint inhibitors immunotherapy; protein–protein interaction; melanoma; microarray; amplified FRET; PD-L1/PD-1; immune checkpoint inhibitors
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MDPI and ACS Style

Sánchez-Magraner, L.; de la Fuente, M.; Evans, C.; Miles, J.; Elexpe, A.; Rodriguez-Astigarraga, M.; Astigarraga, E.; Barreda-Gómez, G. Quantification of PD-1/PD-L1 Interaction between Membranes from PBMCs and Melanoma Samples Using Cell Membrane Microarray and Time-Resolved Förster Resonance Energy Transfer. Analytica 2021, 2, 156-170. https://doi.org/10.3390/analytica2040015

AMA Style

Sánchez-Magraner L, de la Fuente M, Evans C, Miles J, Elexpe A, Rodriguez-Astigarraga M, Astigarraga E, Barreda-Gómez G. Quantification of PD-1/PD-L1 Interaction between Membranes from PBMCs and Melanoma Samples Using Cell Membrane Microarray and Time-Resolved Förster Resonance Energy Transfer. Analytica. 2021; 2(4):156-170. https://doi.org/10.3390/analytica2040015

Chicago/Turabian Style

Sánchez-Magraner, Lissete, Miguel de la Fuente, Charles Evans, James Miles, Ane Elexpe, Maddalen Rodriguez-Astigarraga, Egoitz Astigarraga, and Gabriel Barreda-Gómez. 2021. "Quantification of PD-1/PD-L1 Interaction between Membranes from PBMCs and Melanoma Samples Using Cell Membrane Microarray and Time-Resolved Förster Resonance Energy Transfer" Analytica 2, no. 4: 156-170. https://doi.org/10.3390/analytica2040015

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