Document Type : Original Article


1 Venom and Biotherapeutics Molecules Laboratory, Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

2 Department of Mycobacteriology and Pulmonary Research, Pasteur Institute of Iran, Tehran, Iran

3 Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran

4 Human Genetics Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran

5 Zoonoses Research Center, Pasteur Institute of Iran, Amol, Iran


Programmed death ligand-1 (PD-L1, CD274 and B7-H1) has been described as a ligand for immune inhibitory receptor programmed death protein 1 (PD-1). With binding to PD-1 on activated T cells, PD-L1 can prevent T cell responses via motivating apoptosis. Consequently, it causes cancers immune evasion and helps the tumor growth; hence, PD-L1 is regarded as a therapeutic target for malignant cancers. The anti-PD-L1 monoclonal antibody targeting PD-1/PD-L1 immune checkpoint has attained remarkable outcomes in clinical application and has turned to one of the most prevalent anti-cancer drugs. The present study aimed to develop polyclonal heavy chain antibodies targeting PD-L1via Camelus dromedarius immunization. The extra-cellular domain of human PD-L1 (hPD-L1) protein was cloned, expressed, and purified. Afterwards, this recombinant protein was utilized as an antigen for camel immunization to acquire polyclonal camelid sera versus this protein. Our outcomes showed that hPD-L1 protein was effectively expressed in the prokaryotic system. The antibody-based techniques, such as enzyme-linked immunosorbent assay, western blotting, and flow cytometry displayed that the hPD-L1 protein was detected by generated polyclonal antibody. Due to the advantages of multi-epitope-binding ability, our study exhibited that camelid antibody is effective to be applied significantly for detection of PD-L1 protein in essential antibody-based studies.


  1. Marshall JS, Warrington R, Watson W, et al. An introduction to immunology and immunopathology. Allergy Asthma Clin Immunol 2018; 14(Suppl 2): 49. doi: 10.1186/s13223-018-0278-1.
  2. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012; 12(4): 252-264.
  3. Ostrand-Rosenberg S, Horn LA, Haile ST. The programmed death-1 immune suppressive pathway: barrier to antitumor immunity. J Immunol 2014; 193(8): 3835-3841.
  4. Francisco LM, Sage PT, Sharpe AH. The PD‐1 pathway in tolerance and autoimmunity. Immunol Rev 2010; 236: 219-242.
  5. Hudson K, Cross N, Jordan-Mahy N, et al. The extrinsic and intrinsic roles of PD-L1 and its receptor PD-1: implications for immunotherapy treatment. Front Immunol 2020; 11: 568931. doi: 10.3389/fimmu.2020.568931.
  6. Han Y, Liu D, Li L. PD-1/PD-L1 pathway: current researches in cancer. Am J Cancer Res 2020; 10(3): 727-742.
  7. Wu X, Gu Z, Chen Y, et al. Application of PD-1 blockade in cancer immunotherapy. Comput Struct Biotechnol J 2019; 17: 661-674.
  8. Zhan MM, Hu XQ, Liu XX, et al. From monoclonal antibodies to small molecules: the development of inhibitors targeting the PD-1/PD-L1 pathway. Drug Discov Today 2016; 21(6): 1027-1036.
  9. Arbabi-Ghahroudi M. Camelid single-domain antibodies: historical perspective and future outlook. Front Immunol 2017; 8: 1589. doi: 10.3389/fimmu. 2017.01589.
  10. Muyldermans S. A guide to: generation and design of nanobodies. FEBS J 2021; 288(7): 2084-2102.
  11. Shoari A, Tahmasebi M, Khodabakhsh F, et al. Angiogenic biomolecules specific nanobodies application in cancer imaging and therapy; review and updates. Int Immunopharmacol. 2022; 105: 108585. doi: 10.1016/j.intimp.2022.108585.
  12. Riechmann L, Muyldermans S. Single domain anti-bodies: comparison of camel VH and camelised human VH J Immunol Methods 1999; 231(1-2): 25-38.
  13. Bagheri M, Babaei E, Shahbazzadeh D, et al. Development of a recombinant camelid specific diabody against the heminecrolysin fraction of Hemiscorpius lepturus Toxin Rev 2017; 36(1): 7-11.
  14. Kaur J, Kumar A, Kaur J. Strategies for optimization of heterologous protein expression in E. coli: Roadblocks and reinforcements. Int J Biol Macromol 2018; 106: 803-822.
  15. Mirzaei H, Kazemi B, Bandehpour M, et al. Computational and nonglycosylated systems: a simpler approach for development of nanosized PEGylated proteins. Drug Des Devel Ther 2016; 10: 1193-1200.
  16. Baharlou R, Tajik N, Behdani M, et al. An antibody fragment against human delta-like ligand-4 for inhibition of cell proliferation and neovascularization. Immunopharmacol Immunotoxicol 2018; 40(5): 368-374.
  17. Mousavi A, Sabouri A, Hassanzadeh Eskafi A, et al. In vivo tumor therapy with novel immunotoxin containing programmed cell death protein-1 and diphtheria toxin. Monoclon Antib Immunodiagn Immunother 2021; 40(3): 113-117.
  18. Beatty GL, Gladney WL. Immune escape mechanisms as a guide for cancer immunotherapy. Clin cancer Res 2015; 21(4): 687-692.
  19. Juneja VR, McGuire KA, Manguso RT, et al. PD-L1 on tumor cells is sufficient for immune evasion in immunogenic tumors and inhibits CD8 T cell cytotoxicity. J Exp Med 2017; 214(4): 895-904.
  20. Chen Y, Pei Y, Luo J, et al. Looking for the optimal PD-1/PD-L1 inhibitor in cancer treatment: a comparison in basic structure, function, and clinical practice. Front Immunol 2020; 11: 1088. doi: 10.3389/fimmu. 2020.01088.
  21. Marin-Acevedo JA, Kimbrough EO, Lou Y. Next generation of immune checkpoint inhibitors and beyond. J Hematol Oncol 2021; 14(1): 45. doi: 10.1186/s13045-021-01056-8.
  22. Duan J, Wang Y, Jiao S. Checkpoint blockade‐based immunotherapy in the context of tumor micro-environment: opportunities and challenges. Cancer Med 2018; 7(9): 4517-4529.
  23. Fernandes CFC, Pereira SDS, Luiz MB, et al. Camelid single-domain antibodies as an alternative to overcome challenges related to the prevention, detection, and control of neglected tropical diseases. Front Immunol 2017; 8: 653. doi: 10.3389/fimmu.2017.00653.
  24. Blank C, Gajewski TF, Mackensen A. Interaction of PD-L1 on tumor cells with PD-1 on tumor-specific T cells as a mechanism of immune evasion: implications for tumor immunotherapy. Cancer Immunol Immunother 2005; 54(4): 307-314.
  25. Ghebeh H, Mansour FA, Colak D, et al. Higher PD-L1 immunohistochemical detection signal in frozen compared to matched paraffin-embedded formalin-fixed tissues. Antibodies (Basel) 2021; 10(3): 24. doi: 10.3390/antib10030024.
  26. Califano R, Lal R, Lewanski C, et al. Patient selection for anti-PD-1/PD-L1 therapy in advanced non-small-cell lung cancer: implications for clinical practice. Futur Oncol 2018; 14(23): 2415-2431.
  27. Ahadi M, Ghasemian H, Behdani M, et al. Oligoclonal selection of nanobodies targeting vascular endothelial growth factor. J Immunotoxicol 2019; 16(1): 34-42.