Experimental model of ligands to immune checkpoint receptor interaction on the example of PD1 and PD-L1
AbstractIntroduction. Blockade of immune checkpoint receptors (immune checkpoint blockade, ICB) is one of the effective approaches of modern immunotherapy for patients with malignant neoplasms. Clinicians use blocking antibodies to the PD1, PD-L1 and LAG3 receptor proteins exposed on various types of cells, including T-, NK-, B-cells, as well as myeloid cells of the tumor microenvironment and malignant cells. Extensive experience with ICB has shown significant differences in the clinical effectiveness of different checkpoint blocking drugs and their combinations. The need of development of a wide range of new drugs for ICB has become obvious. In turn, the development of new checkpoint blockers requires convenient and efficient experimental models suitable for the screening of existing and newly developed drugs.
Aim. This paper is devoted to the development and detailed study of two experimental models for the detection and characterization of new checkpoint blockers.
Material and methods. To analyze the blockade of the PD1 → PD-L1 signaling axis, transduced HEK293-PD1 cells that stably express the PD1 receptor protein on their surface were used. One of two experimental models developed examines the inhibition of the interaction of soluble recombinant biotin-labeled PD-L1-Fc protein with HEK293-PD1 cells. In a second experimental model, we analyze the inhibition of interaction of a fluorochrome-labeled anti-PD1 antibody with HEK293-PD1 cells. The labeled ligands to cell binding was studied by flow cytometry. Inhibition of binding was assessed according to changes in the fluorescence intensity of labeled cells.
Results. In the developed experimental models, the blocking properties of therapeutic antibodies to PD1 and PD-L1, as well as soluble recombinant PD-L1-Fc and PD1-Fc proteins were studied. The proposed experimental models shown to be highly sensitive, detecting not only the blocking properties of any substance with respect to the PD-L1-to-PD1 interaction, but also determining the specific activity of PD-L1 and PD1 blockers with half-maximal inhibition constant (K50) in the range of nanomolar concentrations of the test substance. It is claimed that the proposed approach can be used to create experimental models with any cellular receptors as molecular targets of inhibition.
Keywords: PD1-positive cell line; soluble PD1 and PD-L1; antibody; inhibition; cytometry
For citation: Vasileva T.V., Ivanov S.V., Ushakova E.I., Al Khudhur S.А., Lebedeva E.S., Pichugin A.V., Ataullakhanov R.I. Experimental model of ligands to immune checkpoint receptor interaction on the example of PD1 and PD-L1. Immunologiya. 2024; 45 (4): 473–85. DOI: https://doi.org/10.33029/1816-2134-2024-45-4-473-485 (in Russian)
Funding. The study was supported by State assignment for 2024-2025 (agreement No. 388-03-224-156 from 19.02.2024 with Federal Medical-Biological Agency). Open publication of the research results is allowed.
Conflict of interests. The authors declare no conflict of interests.
Authors’ contribution. The idea of the study – Ataullakhanov R.I.; design of experiments – Ataullakhanov R.I., Pichugin A.V.; obtaining the HEK293-PD1 cell line and recombinant proteins PD1-Fc and PD-L1-Fc – Ivanov S.V.; conducting experiments in cell cultures – Vasilyeva T.V.; cell cytometry – Vasilyeva T.V., Pichugin A.V.; statistical processing of results – Vasilyeva T.V.; analysis of results – Ataullakhanov R.I., Pichugin A.V., Lebedeva E.S., Vasilyeva T.V.; concept of the article – Ataullakhanov R.I.; writing the article – Ataullakhanov R.I., Lebedeva E.S., Vasilyeva T.V.; technical design of the article – Ushakova E.I., Al Khudur S.A.
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