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Turkish Journal of Biology

Abstract

Background/aim: Targeted therapies with monoclonal antibodies provide cancer patients with better prognosis and diseasefree survival. The blockade of immune checkpoints, including programmed cell death protein-1 (PD-1) and its ligand PD-L1, with monoclonal antibodies may boost immune responses against tumors and is regarded as an effective strategy in cancer immunotherapy. We describe the generation of anti-PD-L1 monoclonal antibodies with high affinity and specificity, and we assess their potential for therapeutic use in cancer.

Materials and methods: Hybridomas were selected for PD-L1 specificity and cross-reactivity with other immune checkpoint proteins and PD-L1 orthologs using indirect ELISA. Immunofluorescence and Western blotting assays were conducted for further characterization of the antibodies. The affinities of the antibodies for PD-L1 were determined using surface plasmon resonance. Receptor blocking activities were examined through competitive ELISA and cell-based luciferase reporter assays. Sequences of variable regions of the selected antibodies were determined by Sanger sequencing and subjected to BLAST analysis.

Results: A total of 25 PD-L1-specific monoclonal antibodies were generated. While most clones reacted with PD-L1 from cynomolgus monkeys, none of the antibodies displayed cross-reactivity with other checkpoint proteins. Immunofluorescence assays showed that the selected clones stained PD-L1-expressing cell membranes specifically, but not those of PD-L1-negative cells. Western blotting revealed that most of the clones recognized both glycosylated and nonglycosylated PD-L1, and a few reacted with the glycosylated form only. Only two clones with subnanomolar affinity for human PD-L1 were effective at blocking PD-1/PD-L1 and CD80/PD-L1 interactions. Sequence analysis of their variable regions revealed their unique specificity.

Conclusion: Of the 25 monoclonal antibodies produced in this study, only one was identified as a potential therapeutic drug candidate thanks to its high capacity for checkpoint blockade and affinity, as well as its unique sequence specificity. These properties are comparable to those of anti-PD-L1 antibodies currently used in clinical practice.

Author ORCID Identifier

NURŞAH ERSEZEN: 0000-0002-7819-1420

MAİDE ŞEKER: 0000-0003-0013-2516

ARZU AYSAN: 0000-0001-7217-3347

DURMUŞ AKDOĞAN: 0009-0007-7284-9563

MEHMET AVCI: 0000-0002-1918-1134

ASLI KURDEN PEKMEZCİ: 0000-0002-4422-7229

SİBEL KALYONCU UZUNLAR: 0000-0003-2264-0757

MEHMET İNAN: 0000-0003-1806-7927

TAMER YAĞCI: 0000-0003-2050-7477

DOI

10.55730/1300-0152.2974

Keywords

PD-L1, immunotherapy, immune checkpoint protein, monoclonal antibody, hybridoma technology

First Page

306

Last Page

320

Publisher

The Scientific and Technological Research Council of Türkiye (TÜBİTAK)

Creative Commons License

Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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