Skip to main content

Advertisement

SpringerLink
Log in

Isolation and characterization of a novel single-chain variable fragment (scFv) against Lymphocyte function-associated antigen-1 (LFA-1) using phage display method

  • Original Paper
  • Published:
Medical Oncology Aims and scope Submit manuscript

Abstract

Lymphocyte function-associated antigene-1 (LFA-1) is a well-described integrin found on lymphocytes and other leukocytes, which is known to be overexpressed in leukemias and lymphomas. This receptor plays a significant role in immune responses such as T-cell activation, leukocyte cell–cell interactions, and trafficking of leukocyte populations. Subsequently, binders of LFA-1 emerge as potential candidates for cancer and autoimmune therapy. This study used the phage display technique to construct and characterize a high-affinity single-chain fragment variable (scFv) antibody against LFA-1. After expression, purification, dialysis, and concentration of the recombinant LFA-1 protein, four female BALB/c mice were immunized, splenocyte’s mRNA was extracted, and cDNA was synthesized. A scFv library was constructed by linking the amplified VH/Vκ fragments through a 72-bp linker using SOEing PCR. Next, the scFv gene fragments were cloned into the pComb-3XSS phagemid vector; thus, the phage library was developed. The selection process involved three rounds of phage-bio-panning, polyclonal, and monoclonal phage ELISA. AF17 was chosen and characterized among the positive clones through SDS-PAGE, Western blotting, indirect ELISA, and in-silico analyses. The results of the study showed the successful construction of a high-affinity scFv library against LFA-1. The accuracy of the AF17 production and its ability to bind to the LFA-1 were confirmed through SDS-PAGE, Western blot, and ELISA. This study highlights the potential application of the high-affinity AF17 against LFA-1 for targeting T lymphocytes for therapeutic purposes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Data availability

Certain data are confidential but might be available from the corresponding author upon reasonable request.

References

  1. Li D, Molldrem JJ, Ma Q. LFA-1 regulates CD8+ T cell activation via T cell receptor-mediated and LFA-1-mediated Erk1/2 signal pathways. J Biol Chem. 2009;284(31):21001–10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Pflugfelder SC, Stern M, Zhang S, Shojaei A. LFA-1/ICAM-1 interaction as a therapeutic target in dry eye disease. J Ocul Pharmacol Ther. 2017;33(1):5–12.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Kozani PS, Shabani S. Adverse events and side effects of chimeric antigen receptor (CAR) T cell therapy in patients with hematologic malignancies. Trends Med Sci. 2021;1(1):e116301.

    Google Scholar 

  4. Reina M, Espel E. Role of LFA-1 and ICAM-1 in cancer. Cancers. 2017;9(11):153.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Phongpradist R, Chittasupho C, Okonogi S, Siahaan T, Anuchapreeda S, Ampasavate C, Berkland C. LFA-1 on leukemic cells as a target for therapy or drug delivery. Curr Pharm Des. 2010;16(21):2321–30.

    Article  CAS  PubMed Central  Google Scholar 

  6. Hust M, Meyer T, Voedisch B, Rülker T, Thie H, El-Ghezal A, Kirsch MI, Schütte M, Helmsing S, Meier D, Schirrmann T. A human scFv antibody generation pipeline for proteome research. J Biotechnol. 2011;152(4):159–70.

    Article  CAS  PubMed  Google Scholar 

  7. Kumar R, Parray HA, Shrivastava T, Sinha S, Luthra K. Phage display antibody libraries: a robust approach for generation of recombinant human monoclonal antibodies. Int J Biol Macromol. 2019;135:907–18.

    Article  CAS  Google Scholar 

  8. Kaboli PJ, Shabani S, Sharma S, Nasr MP, Yamaguchi H, Hung MC. Shedding light on triple-negative breast cancer with Trop2-targeted antibody-drug conjugates. Am J Cancer Res. 2022;12(4):1671.

    CAS  Google Scholar 

  9. Nikolova G, Georgieva Y, Atanasova A, Radulova G, Kapogianni A, Tsacheva I. Autoinduction as means for optimization of the heterologous expression of recombinant single-chain Fv (scFv) antibodies. Mol Biotechnol. 2021;63(11):1049–56.

    Article  CAS  Google Scholar 

  10. Accardi L, Di Bonito P. Antibodies in single-chain format against tumour-associated antigens: present and future applications. Curr Med Chem. 2010;17(17):1730–55.

    Article  CAS  PubMed  Google Scholar 

  11. Kuhn P, Fühner V, Unkauf T, Moreira GM, Frenzel A, Miethe S, Hust M. Recombinant antibodies for diagnostics and therapy against pathogens and toxins generated by phage display. Proteom Clin Appl. 2016;10(9–10):922–48.

    Article  CAS  Google Scholar 

  12. Jeong KJ, Jang SH, Velmurugan N. Recombinant antibodies: engineering and production in yeast and bacterial hosts. Biotechnol J. 2011;6(1):16–27.

    Article  CAS  Google Scholar 

  13. Miethe S, Meyer T, Wöhl-Bruhn S, Frenzel A, Schirrmann T, Dübel S, Hust M. Production of single chain fragment variable (scFv) antibodies in Escherichia coli using the LEX™ bioreactor. J Biotechnol. 2013;163(2):105–11.

    Article  CAS  Google Scholar 

  14. Shukra AM, Sridevi NV, Chandran D, Maithal K. Production of recombinant antibodies using bacteriophages. Eur J Microbiol Immunol. 2014;4(2):91–8.

    Article  CAS  Google Scholar 

  15. Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NB, Hamid M. scFv antibody: principles and clinical application. J Immunol Res. 2012;2012.

  16. Fath MK, Naderi M, Hamzavi H, Ganji M, Shabani S, Khalesi B, Pourzardosht N, Hashemi ZS, Khalili S. Molecular Mechanisms and therapeutic effects of different vitamins and minerals in COVID-19 patients. J Trace Elem Med Biol. 2022;73:127044.

    Article  CAS  PubMed Central  Google Scholar 

  17. André AS, Moutinho I, Dias JN, Aires-da-Silva F. In vivo Phage Display: a promising selection strategy for the improvement of antibody targeting and drug delivery properties. Front Microbiol. 2022;13:962124.

    Article  PubMed  PubMed Central  Google Scholar 

  18. Shabani S, Moghadam MF, Gargari SL. Isolation and characterization of a novel GRP78-specific single-chain variable fragment (scFv) using ribosome display method. Med Oncol. 2021;38(9):115.

    Article  CAS  PubMed  Google Scholar 

  19. Ledsgaard L, Kilstrup M, Karatt-Vellatt A, McCafferty J, Laustsen AH. Basics of antibody phage display technology. Toxins. 2018;10(6):236.

    Article  PubMed  PubMed Central  Google Scholar 

  20. Aghebati-Maleki L, Bakhshinejad B, Baradaran B, Motallebnezhad M, Aghebati-Maleki A, Nickho H, Yousefi M, Majidi J. Phage display as a promising approach for vaccine development. J Biomed Sci. 2016;23:1–8.

    Article  Google Scholar 

  21. Sioud M. Phage display libraries: from binders to targeted drug delivery and human therapeutics. Mol Biotechnol. 2019;61(4):286–303.

    Article  CAS  PubMed  Google Scholar 

  22. Yu H, Yang Z, Li F, Xu L, Sun Y. Cell-mediated targeting drugs delivery systems. Drug Deliv. 2020;27(1):1425–37.

    Article  CAS  PubMed Central  Google Scholar 

  23. Hashemi A, Bigdeli R, Shahnazari M, Oruji F, Fattahi S, Panahnejad E, Ghadri A, Movahedi-Asl E, Mahdavi-Ourtakand M, Asgary V, Baghbani-Arani F. Evaluation of inflammasome activation in peripheral blood mononuclear cells of hemodialysis treated patients with glomerulonephritis. Iran J Pharm Res. 2021;20(3):609.

    CAS  PubMed  PubMed Central  Google Scholar 

  24. Oruji F, Baghbani Arani F, Mahdavi OM. Evaluation of the gene expression of IL-1β and Casp-1 related to inflammation process in glomerulonephritis patients. J Anim Environ. 2018;10(3):477–82.

    Google Scholar 

  25. Ohashi Y, Minegishi M, Tsuchiya S, Konno T. Three monoclonal antibodies against human LFA-1 α and β chains with different biological activities. Tohoku J Exp Med. 1992;168(4):599–610.

    Article  CAS  Google Scholar 

  26. Leonardi CL. Efalizumab: an overview. J Am Acad Dermatol. 2003;49(2):98–104.

    Article  Google Scholar 

  27. Beck A, Wurch T, Bailly C, Corvaia N. Strategies and challenges for the next generation of therapeutic antibodies. Nat Rev Immunol. 2010;10(5):345–52.

    Article  CAS  Google Scholar 

  28. Gaughan CL. The present state of the art in expression, production and characterization of monoclonal antibodies. Mol Divers. 2016;20(1):255–70.

    Article  CAS  PubMed  Google Scholar 

  29. Mehralizadeh H, Nazari A, Oruji F, Roostaie M, Hosseininozari G, Yazdani O, Esbati R, Roudini K. Cytokine sustained delivery for cancer therapy; special focus on stem cell-and biomaterial-based delivery methods. Pathol Res Pract. 2023;9:154528.

    Article  Google Scholar 

  30. Reader RH, Workman RG, Maddison BC, Gough KC. Advances in the production and batch reformatting of phage antibody libraries. Mol Biotechnol. 2019;61:801–15.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Bashir S, Paeshuyse J. Construction of antibody phage libraries and their application in veterinary immunovirology. Antibodies. 2020;9(2):21.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kaplon H, Chenoweth A, Crescioli S, Reichert JM. Antibodies to watch in 2022. In: MAbs 2022 (Vol. 14, No. 1, p. 2014296).

  33. Anand T, Virmani N, Bera BC, Vaid RK, Vashisth M, Bardajatya P, Kumar A, Tripathi BN. Phage display technique as a tool for diagnosis and antibody selection for coronaviruses. Curr Microbiol. 2021;78(4):1124–34.

    Article  CAS  PubMed Central  Google Scholar 

  34. Dong Y, Meng F, Wang Z, Yu T, Chen A, Xu S, Wang J, Yin M, Tang L, Hu C, Wang H. Construction and application of a human scFv phage display library based on Cre-LoxP recombination for anti-PCSK9 antibody selection. Int J Mol Med. 2021;47(2):708–18.

    Article  CAS  Google Scholar 

  35. Sheets MD, Amersdorfer P, Finnern R, Sargent P, Lindqvist E, Schier R, Hemingsen G, Wong C, Gerhart JC, Marks JD. Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens. Proc Natl Acad Sci. 1998;95(11):6157–62.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Pardon E, Laeremans T, Triest S, Rasmussen SG, Wohlkönig A, Ruf A, Muyldermans S, Hol WG, Kobilka BK, Steyaert J. A general protocol for the generation of Nanobodies for structural biology. Nat Protoc. 2014;9(3):674–93.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Beatty JD, Beatty BG, Vlahos WG. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods. 1987;100(1–2):173–9.

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

This paper has been extracted from a M.Sc. thesis. The authors would like to acknowledge Tarbiat Modares University for funding this project. I also wish to thank Dr. Faezeh Noorabad ghahroodi as well as Miss. Shima Shabani for their kind consulting.

Author information

Authors and Affiliations

  1. Department of Medical Biotechnology, Faculty of Medical Sciences, Tarbiat Modares University, P.O. Box 14115-111, Tehran, Iran

    Fatemeh Afsharnoori & Mehdi Forouzandeh Moghadam

Authors
  1. Fatemeh Afsharnoori
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Mehdi Forouzandeh Moghadam
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mehdi Forouzandeh Moghadam.

Ethics declarations

Conflict of interest

Fatemeh Afsharnoori and Mehdi Forouzandeh-Moghadam declare that they have no conflict of interest.

Ethical approval

All the experiments reported herein have been performed completely in accordance with standard animal welfare regulations as approved by Tarbiat Modares University.

Additional information

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Afsharnoori, F., Forouzandeh Moghadam, M. Isolation and characterization of a novel single-chain variable fragment (scFv) against Lymphocyte function-associated antigen-1 (LFA-1) using phage display method. Med Oncol 41, 15 (2024). https://doi.org/10.1007/s12032-023-02242-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1007/s12032-023-02242-z

Keywords

Search

Navigation