Designing and generating a single-chain fragment variable (scFv) antibody against IL2Rα (CD25): An in silico and in vitro study

Document Type : Original Article


1 Department of Immunology, Faculty of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

2 Department of Cell and Molecular Biology, Faculty of Biological Science and Technology, University of Isfahan, Isfahan, Iran

3 Division of Genetics, Department of Cell and Molecular Biology and Microbiology, Faculty of Biological Sciences and Technologies, University of Isfahan, Isfahan, Iran


Objective(s): IL-2Rα plays a critical role in maintaining immune function. However, expression and secretion of CD25 in various malignant disorders and autoimmune diseases are now well established. Thus, CD25 is considered an important target candidate for antibody-based therapy. This study aimed to find the most suitable linker peptide to construct a functional anti-CD25 single-chain fragment variable (scFv) by bioinformatics studies and its production in a bacterial expression system.
Materials and Methods: Here, the 3D structures of the scFvs with different linkers were predicted and molecular dynamics simulation was performed to compare their structures and dynamics. Then, interactions between five models of scFv and human CD25 were calculated via molecular docking. According to MD and docking results, the anti-CD25 scFvs with (Gly4Ser)3 linker were constructed and cloned into pET-22b(+). Then, recombinant plasmids were transformed into Escherichia coli Bl21 (DE3) for expression using IPTG and lactose as inducers. Anti-CD25 scFv was purified from the periplasm and detected by SDS-PAGE and Western blot. Afterward, functionality was evaluated using ELISA.
Results: In silico analysis showed that the model containing (Gly4Ser)3 as a linker has more stability compared with other linkers. The results of SDS-PAGE, Western blot, and ELISA confirmed the accuracy of anti-CD25 scFv production and its ability to bind to the human CD25.
Conclusion: Conclusively, our work provides a theoretical and experimental basis for production of an anti-CD25 scFv, which may be applied for various malignant disorders and autoimmune diseases.


1. Flynn MJ, Hartley JA. The emerging role of anti-CD 25 directed therapies as both immune modulators and targeted agents in cancer. Br J Haematol 2017; 179:20-35.
2. Du J, Yang H, Zhang D, Wang J, Guo H, Peng B, et al. Structural basis for the blockage of IL-2 signaling by therapeutic antibody basiliximab. J Immunol 2010; 184:1361-1368.
3. de Marco A. Biotechnological applications of recombinant single-domain antibody fragments. Microb Cell Fact 2011; 10:44-58.
4. Cheng J, Dul Q, Ren Y, Zhang B, Feng X. Study on the relationship between the structure and functions of anti-human cervical cancer single-chain antibody and the lengths of linkers. Eur J Gynaecol Oncol 2016; 37:171-177.
5. Pucca MB, Bertolini TB, Barbosa JE, Galina SVR, Porto GS. Therapeutic monoclonal antibodies: scFv patents as a marker of a new class of potential biopharmaceuticals. Braz J Pharm Sci 2011; 47:31-38.
6. Frenzel A, Frode D, Meyer T, Schirrmann T, Hust M. Generating recombinant antibodies for research, diagnostics and therapy using phage display. Curr Biotechnol 2012; 1:33-41.
7. Jordan E, Hust M, Roth A, Biedendieck R, Schirrmann T, Jahn D, et al. Production of recombinant antibody fragments in Bacillus megaterium. Microb Cell Fact 2007; 6:2-13.
8. Bird RE, Hardman KD, Jacobson JW, Johnson S, Kaufman BM, Lee SM, et al. Single-chain antigen-binding proteins. Science 1988; 242:423-426.
9. Bahara NHH, Tye GJ, Choong YS, Ong EBB, Ismail A, Lim TS. Phage display antibodies for diagnostic applications. Biologicals 2013; 41:209-216.
10.    Cheng J, Du Q, Zhang X, Ren Y, Zhang B, Feng X. Study on the relationship between the structure and functions of anti-human cervical cancer single-chain antibody and the lengths of linkers. Eur J Gynaecol Oncol 2016; 37:171-177.
11.    Fang M, Jiang X, Yang Z, Yu X, Yin C, Li H, et al. Effects of inter-peptide linkers to the biological activities of bispecific antibodies. Chin Sci Bull 2003; 48:1912-1918.
12.    Chen H, Wu B, Zhang T, Jia J, Lu J, Chen Z, et al. Effect of linker length and flexibility on the Clostridium thermocellum esterase displayed on Bacillus subtilis spores. Appl Biochem Biotechnol 2017; 182:168-180
13.    Abdurakhmonov IY. Bioinformatics: Basics, Development, and Future: InTech; 2016.
14.    Tang Z-J, Liao W-J, Yu B, Zhong J-X, Zhu H-F, Ye Q, et al. Modeling of scFv of human antibody against liver cancer. Acta Biophys Sin 2005; 21:195-199.
15.    Whitlow M, Bell BA, Feng SL, Filpula D, Hardman KD, Hubert SL, et al. An improved linker for single-chain Fv with reduced aggregation and enhanced proteolytic stability. Protein Eng Des Sel 1993; 6:989-995.
16.    Whitlow M, Filpula D. Single-chain Fv proteins and their fusion proteins. Methods 1991; 2:97-105.
17.    Lilly M, Fierobe HP, Van Zyl WH, Volschenk H. Heterologous expression of a Clostridium minicellulosome in Saccharomyces cerevisiae. FEMS Yeast Res 2009; 9:1236-1249.
18.    Yang H, Wang J, Du J, Zhong C, Zhang D, Guo H, et al. Structural basis of immunosuppression by the therapeutic antibody daclizumab. Cell Res 2010; 20:1361-1371.
19.    Kim DE, Chivian D, Baker D. Protein structure prediction and analysis using the Robetta server. Nucleic Acids Res 2004; 32:526-531.
20.    Guex N, Peitsch MC. SWISS-MODEL and the Swiss-Pdb Viewer: an environment for comparative protein modeling. Electrophoresis 1997; 18:2714-2723.
21.    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, et al. UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 2004; 25:1605-1612.
22.    Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph 1996; 14:33-38.
23.    Salomon-Ferrer R, Case DA, Walker RC. An overview of the Amber biomolecular simulation package. Wiley Interdiscip Rev Comput Mol Sci 2013; 3:198-210.
24.    Case DA, Cheatham III TE, Darden T, Gohlke H, Luo R, Merz Jr KM, et al. The Amber biomolecular simulation programs. J Comput Chem 2005; 26:1668-1688.
25.    Essmann U, Perera L, Berkowitz ML, Darden T, Lee H, Pedersen LG. A smooth particle mesh Ewald method. J Chem Phys 1995; 103:8577-8593.
26.    Loncharich RJ, Brooks BR, Pastor RW. Langevin dynamics of peptides: The frictional dependence of isomerization rates of N-acetylalanyl-N′-methylamide. Biopolymers 1992; 32:523-535.
27.    Baspinar A, Cukuroglu E, Nussinov R, Keskin O, Gursoy A. PRISM: a web server and repository for prediction of protein–protein interactions and modeling their 3D complexes. Nucleic Acids Res 2014; 42:285-289.
28.    Beatty JD, Beatty BG, Vlahos WG. Measurement of monoclonal antibody affinity by non-competitive enzyme immunoassay. J Immunol Methods 1987; 100:173-179.
29.    Dondelinger M, Filée P, Sauvage E, Quinting B, Muyldermans S, Galleni M, et al. Understanding the significance and implications of antibody numbering and antigen-binding surface/residue definition. Front Immunol 2018; 9:2278-2293-.
30.    Queen C, Schneider WP, Selick HE, Payne PW, Landolfi NF, Duncan JF, et al. A humanized antibody that binds to the interleukin 2 receptor. Proc Nat Acad Sci 1989; 86:10029-10033.
31.    AlDeghaither D, Smaglo BG, Weiner LM. Beyond peptides and mAbs—current status and future perspectives for biotherapeutics with novel constructs. J Clin Pharmacol 2015; 55:4-20.
32.    Brinkmann U, Kontermann RE, Editors. The making of bispecific antibodies. MAbs; 2017: Taylor & Francis.
33.    Frenzel A, Hust M, Schirrmann T. Expression of recombinant antibodies. Front Immunol 2013; 4: 217-265.
34.    Ahmad ZA, Yeap SK, Ali AM, Ho WY, Alitheen NBM, Hamid M. scFv antibody: principles and clinical application. Clin  Dev Immunol 2012;2012:980250.
35.    Kim G-B, Wang Z, Liu YY, Stavrou S, Mathias A, Goodwin KJ, et al. A fold-back single-chain diabody format enhances the bioactivity of an anti-monkey CD3 recombinant diphtheria toxin-based immunotoxin. Protein Eng Des Sel 2007; 20:425-432.
36.    Miller BR, Demarest SJ, Lugovskoy A, Huang F, Wu X, Snyder WB, et al. Stability engineering of scFvs for the development of bispecific and multivalent antibodies. Protein Eng Des Sel 2010; 23:549-557.
37.    Vaks L, Benhar I. Production of stabilized scFv antibody fragments in the E. coli bacterial cytoplasm.  Human Monoclonal Antibodies: Springer; 2014. p. 171-184.
38.    Schmidt F. Recombinant expression systems in the pharmaceutical industry. Appl Microbiol Biotechnol 2004; 65:363-372.
39.    Miller KD, Weaver-Feldhaus J, Gray SA, Siegel RW, Feldhaus MJ. Production, purification, and characterization of human scFv antibodies expressed in Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Protein Expr 2005; 42:255-267.
40.    Verma R, Boleti E, George A. Antibody engineering: comparison of bacterial, yeast, insect and mammalian expression systems. J Immunol Methods 1998; 216:165-181.
41.    Liu A, Ye Y, Chen W, Wang X, Chen F. Expression of V H-linker-V L orientation-dependent single-chain Fv antibody fragment derived from hybridoma 2E6 against aflatoxin B 1 in Escherichia coli. J Indust Microbiol Biotechnol 2015; 42:255-262.
42.    Spadiut O, Capone S, Krainer F, Glieder A, Herwig C. Microbials for the production of monoclonal antibodies and antibody fragments. Trends Biotechnol 2014; 32:54-60.
43.    Lefranc M-P, Giudicelli V, Kaas Q, Duprat E, Jabado-Michaloud J, Scaviner D, et al. IMGT, the international ImMunoGeneTics information system. Nucleic Acids Res 2005; 33:593-597.
44.    Lee YJ, Jeong KJ. Challenges to production of antibodies in bacteria and yeast. J Biosci Bioeng2015; 120:483-490.
45.    Butler M, Spearman M. The choice of mammalian cell host and possibilities for glycosylation engineering. Curr Opin Biotechnol 2014; 30:107-112.
46.    Vendel MC, Favis M, Snyder WB, Huang F, Capili AD, Dong J, et al. Secretion from bacterial versus mammalian cells yields a recombinant scFv with variable folding properties. Arch Biochem Biophys 2012; 526:188-193.
47.    Joosten V, Lokman C, Van den Hondel CA, Punt PJ. The production of antibody fragments and antibody fusion proteins by yeasts and filamentous fungi. Microb Cell Fact 2003; 2:1-15.
48.    Shi J, Wan Y, Shi S, Zi J, Guan H, Zhang Y, et al. Expression, purification, and characterization of scar tissue neovasculature endothelial cell-targeted rhIL10 in Escherichia coli. Appl biochem Biotechnol 2015; 175:625-634.
49.    Montoliu-Gaya L, Martínez JC, Villegas S. Understanding the contribution of disulfide bridges to the folding and misfolding of an anti-Aβ scFv. Protein Sci 2017; 26:1138-1149.
50.    Guglielmi L, Martineau P. Expression of single-chain Fv fragments in Escherichia coli cytoplasm. Antibody Phage Display: Springer; 2009. p. 215-224.
51.    Miethe S, Meyer T, Wöhl-Bruhn S, Frenzel A, Schirrmann T, Dübel S, et al. Production of single chain fragment variable (scFv) antibodies in Escherichia coli using the LEX™ bioreactor. J biotechnol 2013; 163:105-111.
52.    Chi WJ, Kim H, Yoo H, Kim YP, Hong SK. Periplasmic expression, purification, and characterization of an anti-epidermal growth factor receptor antibody fragment in Escherichia coli. Biotechnol Bioprocess Eng 2016; 21:321-330.
53.    Dewi K, Retnoningrum D, Riani C, Fuad A. Construction and periplasmic expression of the anti-EGFRvIII scFv antibody gene in Escherichia coli. Sci Pharm 2016; 84:141-152.
54.    Singh A, Upadhyay V, Upadhyay AK, Singh SM, Panda AK. Protein recovery from inclusion bodies of Escherichia coli using mild solubilization process. Microb Cell Fact 2015; 14:1-10.
55.    Lin L, Li L, Zhou C, Li J, Liu J, Shu R, et al. A HER2 bispecific antibody can be efficiently expressed in Escherichia coli with potent cytotoxicity. Oncol lett 2018; 16:1259-1266.
56.    Reusch U, Harrington KH, Gudgeon CJ, Fucek I, Ellwanger K, Weichel M, et al. Characterization of CD33/CD3 tetravalent bispecific tandem diabodies (TandAbs) for the treatment of acute myeloid leukemia. Clin Cancer Res 2016; 22:5829-5838.
57.    Renaut L, Monnet C, Dubreuil O, Zaki O, Crozet F, Bouayadi K, et al. Affinity maturation of antibodies: optimized methods to generate high-quality ScFv libraries and isolate IgG candidates by high-throughput screening.  Antibody engineering: Springer; 2012. p. 451-461.
58.    Tsurushita N, Hinton PR, Kumar S. Design of humanized antibodies: from anti-Tac to Zenapax. Methods 2005; 36:69-83.