The aim of this study was to create and evaluate a cell model designed for in vitro and in vivo testing of anti-human PD-L1 therapeutic and diagnostic agents’ specificity. Materials and Methods. Genetically modified cells expressing human PD-L1 (strain CT26-PD-L1) were obtained by retroviral transduction of murine CT26 carcinoma cells. PD-L1 gene activity was assessed by real-time PCR, and PD-L1 expression on cells was identified by flow cytometry. Cells were tested using recombinant single-domain human anti-PD-L1 antibodies (nanoantibodies) conjugated with radioisotopes68 Ga or177 Lu. Immunoreactive fraction and cell internalization of the radioconjugates were evaluated in vitro. For in vivo experiments CT26-PD-L1 cells were transplanted into mice, radioimmunoconjugates were injected 9–14 days later, in 1–48 h the tumors were retrieved and subjected to direct radiometry. Intact CT26 cells not expressing the antigen served as a control. Results. CT26-PD-L1 strain of murine tumor cells expressing human membrane PD-L1 was created. When transplanted into intact BALB/c mice or sublethally irradiated F1(DBA×BALB/c) mice, these cells formed tumors. Thus, a significant advantage of the model was the possibility of in vivo testing of human PD-L1-affinity agents using animals under conventional vivarium conditions. When radioimmunoconjugates were administered to tumor bearing mice, radionuclides accumulated in tumors generated from the transplanted CT26-PD-L1 cells, but not CT26 cells. CT26-PD-L1 cells internalized anti-PD-L1 nanobodies in vitro. Due to a high density of target molecules, CT26-PD-L1 cells allowed both to confirm pharmaceuticals’ specificity and to quantify the target-binding fraction of conjugates in a single test. Conclusion. The created cells are the first genetically engineered cells designed to evaluate affinity of anti-human PD-L1 therapeutic and diagnostic agents in Russia. Test results confirmed the model suitability for in vitro and in vivo testing of the specificity of pharmaceuticals targeting human PD-L1. © 2024, Privolzhsky Research Medical University. All rights reserved.
