Volume 27, Issue 5 (9-2023)                   IBJ 2023, 27(5): 269-279 | Back to browse issues page

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Mohammadkhani N, Rahimpour A, Hoseinpoor R, Rajabibazl M. Development of Stable CHO-K1 Cell Lines Overexpressing Full Length Human CD20 Antigen. IBJ 2023; 27 (5) :269-279
URL: http://ibj.pasteur.ac.ir/article-1-3916-en.html
Background: CD20 is a differentiation-related antigen exclusively expressed on the membrane of B lymphocytes. CD20 amplification is observed in numerous immune-related disorders, making it an ideal target for immunotherapy of hematological malignancies and autoimmune diseases. MAb-based therapies targeting CD20 have a principal role in the treatment of several immune-related disordes and cancers, including CLL. Fc gamma receptors mediate CD20 internalization in hematopoietic cells; therefore, this study aimed to establish non-hematopoietic stable cell lines overexpressing full-length human CD20 antigen as an in vitro model for CD20-related studies.
Methods: CD20 gene was cloned into the transfer vector. The lentivirus system was transfected to packaging HEK 293T cells, and the supernatants were harvested. CHO-K1 cells were transduced using recombinant viruses, and a stable cell pool was developed by the antibiotic selection. CD20 expression was confirmed at the mRNA and protein levels.
Results: Simultaneous expression of GFP protein facilitated the detection of CD20-expressing cells. Immunophenotyping analysis of stable clones demonstrated expression of CD20 antigen. In addition, the mean fluorescence intensity was significantly higher in the CD20-CHO-K1 clones than the wild-type CHO-K1 cells.
Conclusion: This study is the first report on using second-generation lentiviral vectors for the establishment of a non-hematopoietic cell-based system, which stably expresses full-length human CD20 antigen. Results of stable CHO cell lines with different levels of CD20 antigen are well suited to be
used for CD20-based investigations, including binding and functional assays.

1. Kuijpers TW, Bende RJ, Baars PA, Grummels A, Derks IAM, Dolman KM, Beaumont T, Tedder TF, van Noesel CJM, Eldering E, van Lier RAW. CD20 deficiency in humans results in impaired T cell-independent antibody responses. The journal of clinical investigation 2010; 120(1): 214-222. [DOI:10.1172/JCI40231]
2. Casan JML, Wong J, Northcott MJ, Opat S. Anti-CD20 monoclonal antibodies: reviewing a revolution. Human vaccines & immunotherapeutics 2018; 14(12): 2820-2841. [DOI:10.1080/21645515.2018.1508624]
3. Craig FE, Foon KA. Flow cytometric immunophenotyping for hemato logic neoplasms. Blood 2008; 111(8): 3941-3967. [DOI:10.1182/blood-2007-11-120535]
4. Naeim F, Rao PN, Grody WW. Hematopathology: Morphology, Immunophenotype, Cytogenetics, and Molecular Approaches: Academic Press, 2009.
5. Tomita A. Genetic and epigenetic modulation of CD20 expression in B-cell malignancies: molecular mechanisms and significance to rituximab resistance. Journal of clinical and experimental hematopathology 2016; 56(2): 89-99. [DOI:10.3960/jslrt.56.89]
6. Anolik J, Looney RJ, Bottaro A, Sanz I, Young F. Down-regulation of CD20 on B cells upon CD40 activation. European journal of immunology 2003; 33(9): 2398-2409. [DOI:10.1002/eji.200323515]
7. Mueller AL, Payandeh Z, Mohammadkhani N, Mubarak SMH, Zakeri A, Alagheband Bahrami A, Brockmueller A, Shakibaei M. Recent Advances in understanding the pathogenesis of rheumatoid arthritis: new treatment strategies. Cells 2021; 10(11): 3017. [DOI:10.3390/cells10113017]
8. Payandeh Z, Mohammadkhani N, Nabi Afjadi M, Khalili S, Rajabibazl M, Houjaghani Z, Dadkhah M. The immunology of SARS-CoV-2 infection, the potential antibody based treatments and vaccination strategies. Expert review of anti-infective therapy 2021; 19(7): 899-910. [DOI:10.1080/14787210.2020.1863144]
9. Leandro MJ. B-cell subpopulations in humans and their differential susceptibility to depletionwith anti-CD20 monoclonal antibodies. Arthritis research and therapy 2013; 15(S1): S3. [DOI:10.1186/ar3908]
10. Pescovitz MD. Rituximab, an anti-CD20 monoclonal antibody: history and mechanism of action. American journal of transplantation 2006; 6(5p1): 859-866. [DOI:10.1111/j.1600-6143.2006.01288.x]
11. Kozlova V, Ledererova A, Ladungova A, Peschelova H, Janovska P, Slusarczyk A, Domagala J, Kopcil P, Vakulova V, Oppelt J, Bryja V, Doubek M, Mayer J, Pospisilova S, Smida M. CD20 is dispensable for B-cell receptor signaling but is required for proper actin polymerization, adhesion and migration of malignant B cells. Plos one 2020; 15(3): e0229170. [DOI:10.1371/journal.pone.0229170]
12. Pavlasova G, Borsky M, Seda V, Cerna K, Osickova J, Doubek M, Mayer J, Calogero R, Trbusek M, Pospisilova S, Davids MS, Kipps TJ, Brown JR, Mraz M. Ibrutinib inhibits CD20 upregulation on CLL B cells mediated by the CXCR4/SDF-1 axis. Blood 2016; 128(12): 1609-1613. [DOI:10.1182/blood-2016-04-709519]
13. Boross P, Leusen JHW. Mechanisms of action of CD20 antibodies. American journal of cancer research 2012; 2(6): 676-690.
14. Hofmann K, Clauder A-K, Manz RA. Targeting B cells and plasma cells in autoimmune diseases. Frontiers in immunology 2018; 9: 835. [DOI:10.3389/fimmu.2018.00835]
15. Payandeh Z, Bahrami AA, Hoseinpoor R, Mortazavi Y, Rajabibazl M, Rahimpour A, Taromchi AH, Khalil S. The applications of anti-CD20 antibodies to treat various B cells disorders. Biomedicine & pharmacotherapy 2019; 109: 2415-2426. [DOI:10.1016/j.biopha.2018.11.121]
16. Egle A, Pleyer L, Melchardt T, Hartmann TN, Greil R. Remission maintenance treatment options in chronic lymphocytic leukemia. Cancer treatment reviews 2018; 70: 56-66. [DOI:10.1016/j.ctrv.2018.08.003]
17. Montalban X, Hauser SL, Kappos L, Arnold DL, Bar-Or A, Comi G, De Seze J, Giovannoni G, Hartung H-P, Hemmer B, Lublin F, Rammohan KW, Selmaj K, Traboulsee A, Sauter A, Masterman D, Fontoura P, Belachew S, Garren H, Mairon N, Chin P, Wolinsky JS. Ocrelizumab versus placebo in primary progressive multiple sclerosis. New england journal of medicine 2017; 376(3): 209-220. [DOI:10.1056/NEJMoa1606468]
18. Beers SA, French RR, Chan HTC, Lim SH, Jarrett TC, Vidal RM, Wijayaweera SS, Dixon SV, Kim H, Cox KL, Kerr JP, Johnston DA, Johnson PWM, Verbeek JS, Glennie MJ, Cragg MS. Antigenic modulation limits the efficacy of anti-CD20 antibodies: implications for antibody selection. Blood 2010; 115(25): 5191-5201. [DOI:10.1182/blood-2010-01-263533]
19. Hiraga J, Tomita A, Sugimoto T, Shimada K, Ito M, Nakamura S, Kiyoi H, Kinoshita T, Naoe T. Down-regulation of CD20 expression in B-cell lymphoma cells after treatment with rituximab-containing combination chemotherapies: its prevalence and clinical significance. Blood 2009; 113(20): 4885-4893. [DOI:10.1182/blood-2008-08-175208]
20. Beum PV, Peek EM, Lindorfer MA, Beurskens FJ, Engelberts PJ, Parren PWHI, Jan G J van de Winkel, Taylor RP. Loss of CD20 and bound CD20 antibody from opsonized B cells occurs more rapidly because of trogocytosis mediated by Fc receptor-expressing effector cells than direct internalization by the B cells. The journal of immunology 2011; 187(6): 3438-3447. [DOI:10.4049/jimmunol.1101189]
21. Lim SH, Vaughan AT, Ashton-Key M, Williams EL, Dixon SV, Chan HTC, Beers SA, French RR, Cox KL, Davies AJ, Potter KN, Mockridge CI, Oscier DG, Johnson PWM, Cragg MS, Glennie MJ. Fc gamma receptor IIb on target B cells promotes rituximab internalization and reduces clinical efficacy. Blood 2011; 118(9): 2530-2540. [DOI:10.1182/blood-2011-01-330357]
22. Vaughan AT, Iriyama C, Beers SA, Chan CHT, Lim SH, Williams EL, Shah V, Roghanian A, Frendéus B, Glennie MJ, Cragg MS . Inhibitory FcγRIIb (CD32b) becomes activated by therapeutic mAb in both cis and trans and drives internalization according to antibody specificity. Blood 2014; 123(5): 669-677. [DOI:10.1182/blood-2013-04-490821]
23. Dull T, Zufferey R, Kelly M, Mandel RJ, Nguyen M, Trono D, Naldini L. A third-generation lentivirus vector with a conditional packaging system. Journal of virology 1998; 72(11): 8463-8471. [DOI:10.1128/JVI.72.11.8463-8471.1998]
24. Roychoudhury A, Basu S, Sengupta DN. Analysis of comparative efficiencies of different transformation methods of E. coli using two common plasmid vectors. Indian journal of biochemistry & biophysics 2009; 46(5): 395-400.
25. Zufferey R, Nagy D, Mandel RJ, Naldini L, Trono D. Multiply attenuated lentiviral vector achieves efficient gene delivery in vivo. Nature biotechnology 1997; 15(9): 871-875. [DOI:10.1038/nbt0997-871]
26. Roghanian A, Teige I, Mårtensson L, Cox KL, Kovacek M, Ljungars A, Mattson J, Sundberg A, Vaughan AT, Shah V, Smyth NR, Sheth B, Chan HTC, Li ZC, Williams EL, Manfredi G, Oldham RJ, Mockridge CI, James SA, Dahal LN, Hussain K, Nilsson B, Verbeek JS, Juliusson G, Hansson M, Jerkeman M, Johnson PWM, Davies A, Beers SA, Glennie MJ, Frendéus B, Cragg MS. Antagonistic human FcγRIIB (CD32B) antibodies have anti-tumor activity and overcome resistance to antibody therapy in vivo. Cancer cell 2015; 27(4): 473-488. [DOI:10.1016/j.ccell.2015.03.005]
27. Behiels E, Elegheert J. High-level production of recombinant eukaryotic proteins from mammalian cells using lentivirus. Methods in molecular biology 2021; 2305: 83-104. [DOI:10.1007/978-1-0716-1406-8_4]
28. Wolff JH, Mikkelsen JG. Delivering genes with human immunodeficiency virus-derived vehicles: still state-of-the-art after 25 years. Jornal of biomedical science 2022; 29: 79. [DOI:10.1186/s12929-022-00865-4]
29. Milone MC, O'Doherty U. Clinical use of lentiviral vectors. Leukemia 2018; 32(7): 1529-1541. [DOI:10.1038/s41375-018-0106-0]
30. Ansorge S, Henry O, Kamen A. Recent progress in lentiviral vector mass production. Biochemical engineering journal 2010; 48(3): 362-377. [DOI:10.1016/j.bej.2009.10.017]
31. Jordan M, Wurm F. Transfection of adherent and suspended cells by calcium phosphate. Methods 2004; 33(2): 136-143. [DOI:10.1016/j.ymeth.2003.11.011]
32. Mitta B, Rimann M, Fussenegger M. Detailed design and comparative analysis of protocols for optimized production of high-performance HIV-1-derived lentiviral particles. Metabolic engineering 2005; 7(5-6): 426-436. [DOI:10.1016/j.ymben.2005.06.006]
33. Jiang W, Hua R, Wei M, Li C, Qiu Z, Yang X, Zhang C. An optimized method for high-titer lentivirus preparations without ultracentrifugation. Scientific Reports 2015; 5: 13875. [DOI:10.1038/srep13875]
34. Zufferey R, Donello JE, Trono D, Hope TJ. Woodchuck hepatitis virus posttranscriptional regulatory element enhances expression of transgenes delivered by retroviral vectors. Journal of virology 1999; 73(4): 2886-2892. [DOI:10.1128/JVI.73.4.2886-2892.1999]
35. Barde I, Salmon P, Trono D. Production and titration of lentiviral vectors. Current protocols in neuroscience 2010; Chapter 4: Unit 4.21. [DOI:10.1002/0471142301.ns0421s53]
36. Payandeh Z, Rajabibazl M, Mortazavi Y, Rahimpour A, Taromchi AH, Dastmalchi S. Affinity maturation and characterization of the ofatumumab monoclonal antibody. Journal of cellular biochemistry 2019; 120(1): 940-950. [DOI:10.1002/jcb.27457]
37. Mohammadian O, Rajabibazl M, Pourmaleki E, Bayat H, Ahani R, Rahimpour A. Development of an improved lentiviral based vector system for the stable expression of monoclonal antibody in CHO cells. Preparative biochemistry and biotechnology 2019; 49(8): 822-829. [DOI:10.1080/10826068.2019.1621893]
38. Oberbek A, Matasci M, Hacker DL, Wurm FM. Generation of stable, high-producing cho cell lines by lentiviral vector-mediated gene transfer in serum-free suspension culture. Biotechnology and bioengineering 2011; 108(3): 600-610. [DOI:10.1002/bit.22968]
39. Moritz B, Becker PB, Göpfert U. CMV promoter mutants with a reduced propensity to productivity loss in CHO cells. Scientific reports 2015; 5: 16952. [DOI:10.1038/srep16952]
40. Elegheert J, Behiels E, Bishop B, Scott S, Woolley RE, Griffiths SC, Byrne EFX, Chang VT, Stuart DI, Jones EY, Siebold C, Aricescu AR. Lentiviral transduction of mammalian cells for fast, scalable and high-level production of soluble and membrane proteins. Nature protocols 2018; 13(12): 2991-3017. [DOI:10.1038/s41596-018-0075-9]
41. Zhang Y, Na D, Zhang W, Liu X, Miao S, Tan WS, Zhao L. Development of stable HEK293T cell pools expressing CSFV E2 protein: A potential antigen expression platform. Vaccine 2023; 41(9): 1573-1583. [DOI:10.1016/j.vaccine.2023.01.038]
42. Yamagata Y, Parietti V, Stockholm D, Corre G, Poinsignon C, Touleimat N, Delafoy D, Besse C, Tost J, Galy A, Paldi A. Lentiviral transduction of CD34(+) cells induces genome-wide epigenetic modifications. PLoS one 2012; 7(11): e48943. [DOI:10.1371/journal.pone.0048943]
43. Fus-Kujawa A, Prus P, Bajdak-Rusinek K, Teper P, Gawron K, Kowalczuk A, Sieron AL. An overview of methods and tools fortransfection of eukaryotic cells in vitro. Frontiers in bioengineering and biotechnology 2021; 9: 701031. [DOI:10.3389/fbioe.2021.701031]
44. Cereseto A, Giacca M. Integration site selection by retroviruses. AIDS reviews 2004; 6(1): 13-21
45. Ciuffi A. Mechanisms governing lentivirus integration site selection. Current gene therapy 2008; 8(6): 419-429. [DOI:10.2174/156652308786848021]
46. Chong ZX, Yeap SK, Ho WY. Transfection types, methods and strategies: a technical review. PeerJ 2021; 9: e11165. [DOI:10.7717/peerj.11165]
47. Ayyar BV, Arora S, Ravi SS. Optimizing antibody expression: the nuts and bolts. Methods 2017; 116: 51-62. [DOI:10.1016/j.ymeth.2017.01.009]
48. Wurm FM, Pallavicini MG, Arathoon R. Integration and stability of CHO amplicons containing plasmid sequences. Developments in biological standardization 1992; 76: 69-82.
49. Tati K, Yazdanpanah-Samani M, Ramezani A, Mahmoudi Maymand E, Ghaderi A. Establishment a CHO cell line expressing human CD52 molecule. Reports of biochemistry & molecular biology 2016; 5(1): 56-61.
50. Hedayatizadeh-Omran A, Valadan R, Rafiei A, Ajami A, Tehrani M, Alizadeh-Navaei R. Generation of CHO stable cell line overexpressing HER2: an in vitro model for breast cancer. Research in molecular medicine 2015; 3(3): 6-10.

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