Original Article

Construction and Characterization of 1F5 Chimeric Anti-CD20 Monoclonal Antibodies: An Efficient Splicing by Overlap Extension-polymerase Chain Reaction Technique

Construction of 1F5 Chimeric mAbs Using an Efficient SOE-PCR Technique


Despite the unparalleled success of anti-CD20-targeted immunotherapy, the currently available mAbs are not sufficiently efficacious in the treatment of lymphoma. 1F5 is one of a panel of anti-CD20 mAbs that was used in the B-cell lymphoma serotherapy. Despite the efficacy of murine 1F5 mAbs in lymphoma patients, the 1F5 chimeric antibodies with human effector functionality are yet to be approved and widely used in the treatment of lymphoma. In this study, the conversion of 1F5 mAb from mouse IgG2a to human-mouse chimeric IgG1 was achieved and the chimeric antibody was partially characterized.
We constructed the 1F5 chimeric mouse-human anti-CD20 antibody genes using an efficient Splicing by overlap extension-polymerase chain reaction (SOE-PCR) technique and cloned the chimeric heavy and light genes in pBudCE4.1mammalian expression vector, followed by purification of the expressed chimeric 1F5 mAbs using affinity chromatography. Our investigation also included the biological properties of purified chimeric antibodies.
The generated 1F5 chimeric mAbs mediate complement-dependent cytotoxicity (CDC) and antibody-dependent cellular cytotoxicity (ADCC) against Raji and Daudi Burkitt's lymphoma cell lines, which were comparable with rituximab and exhibit superior reduction in cell viability in vitro, compared to rituximab.
The current study indicated that the generated chimeric 1F5 mAbs has potential CDC and ADCC activity which was comparable with rituximab whereas it exhibits a superior reduction in cell viability, compared to rituximab. Our work contributes to future studies involving in vivo biological functions and the application of the 1F5 chimeric antibody.

1. Singh V, Gupta D, Almasan A. Development of Novel Anti-Cd20 Monoclonal Antibodies and Modulation in Cd20 Levels on Cell Surface: Looking to Improve Immunotherapy Response. J Cancer SciTher. 2015;7(11):347-58.
2. Czuczman MS, Gregory SA. The future of CD20 monoclonal antibody therapy in B-cell malignancies. Leuk Lymphoma. 2010;51(6):983-94.
3. Maloney DG, Grillo-Lopez AJ, White CA, Bodkin D, Schilder RJ, Neidhart JA, et al. IDEC-C2B8 (Rituximab) anti-CD20 monoclonal antibody therapy in patients with relapsed low-grade non-Hodgkin's lymphoma. Blood. 1997;90(6):2188-95.
4. Kaminski MS, Zasadny KR, Francis IR, Fenner MC, Ross CW, Milik AW, et al. Iodine-131-anti-B1 radioimmunotherapy for B-cell lymphoma. J Clin Oncol. 1996;14(7):1974-81.
5. Du FH, Mills EA, Mao-Draayer Y.Next-generation anti-CD20 monoclonal antibodies in autoimmune disease treatment.Auto Immun Highlights. 2017;8(1):12.
6. Myhr KM, Torkildsen Ø, Lossius A, Bø L, Holmøy T.B cell depletion in the treatment of multiple sclerosis.Expert Opin Biol Ther. 2019;19(3):261-71.
7. Kadavakolan S, Puri S, Sahay S, Joshi J. An update on newer monoclonal antibodies in lymphoma therapy. Asian J Oncol. 2016;2(1):3-7.
8. Robak T, Robak E. New Anti-CD20 Monoclonal Antibodies for theTreatment of B-Cell Lymphoid Malignancies. Biodrugs. 2011;25(1):13-25.
9. Cragg MS, Walshe CA, Ivanov AO, Glennie MJ. The biology of CD20 and its potential as a target for mAb therapy. Curr Dir Autoimmun 2005;8:140-74.
10. Stashenko P, Nadler LM, Hardy R, Schlossman SF. Characterization of a human B lymphocyte-specific antigen. J Immunol 1980;125(4):1678-85.
11. Klein C, Lammens A, Schafer W, Georges G, Schwaiger M, Mossner E, et al. Epitope interactions of monoclonal antibodies targeting CD20 and their relationship to functional properties. mAbs 2013;5(1):22-33.
12. Glennie MJ, French RR, Cragg MS, Taylor RP. Mechanisms of killing by anti-CD20 monoclonal antibodies. Mol immunol. 2007;44(16):3823-37.
13. Lim SH, Beers SA, French RR, Johnson PW, Glennie MJ, Cragg MS. Anti-CD20 monoclonal antibodies: historical and future perspectives. Haematologica. 2010;95(1):135-43.
14. Cragg MS, Morgan SM, Chan HT, Morgan BP, Filatov AV, Johnson PW, et al. Complement-mediated lysis by anti-CD20 mAb correlates with segregation into lipid rafts. Blood. 2003;101(3):1045-52.
15. Cragg MS, Glennie MJ. Antibody specificity controls in vivo effector mechanisms of anti-CD20 reagents. Blood. 2004;103(7):2738-43.
16. Chan HTC, Hughes D, French RR, Tutt AL, Walshe CA, Teeling JL, et al. CD20-induced lymphoma cell death is independent of both caspases and its redistribution into triton X-100 insoluble membrane rafts. Cancer Res. 2003;63(17):5480-9.
17. Khademi F, Mostafaie A, Parvaneh S, Gholami Rad F, Mohammadi P, Bahrami G, Construction and characterization of monoclonal antibodies against the extracellular domain of B-lymphocyte antigen CD20 using DNA immunization method. Int Immunopharmacol.2017;43:23-32.
18. Press OW, Appelbaum F, Ledbetter JA, Martin PJ, Zarling J, Kidd P, et al. Monoclonal antibody 1F5 (anti-CD20) serotherapy of human B cell lymphomas. Blood. 1987;69(2):584-91.
19. Horton RM, Hunt HD, Ho SN, Pullen JK, Pease LR. Engineering hybrid genes without the use of restriction enzymes: gene splicing by overlap extension. Gene. 1989;77(1):61-8.
20. Jones ML, Barnard RT.Chimerization of multiple antibody classes using splice overlap extension PCR. BioTechniques. 2005;38(2):181-2.
21. Khademi F, Mohammadi P, Yari Kh, Mostafaie A. Resistance of Cloned 1F5 Chimeric Anti-CD20 Antibody Heavy-Chain Gene to DNA Polymerase due to a Predicted Hairpin Structure. Res Mol Med. 2016;4(4):28-37
22. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, et al. Current protocols in molecular biology. J Wiley; New York. 2001.
23. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680-5.
24. Towbin H, Staehelin T, Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979;76:4350-4.
25. Nechansky A, Szolar OH, Siegl P, Zinoecker I, Halanek N, Wiederkum S,et al. Complement dependent cytotoxicity (CDC) activity of a humanized anti Lewis-Y antibody: FACS-based assay versus the 'classical' radioactive method - qualification, comparison and application of the FACS-based approach. J Pharm Biomed Anal. 2009;49(4):1014-20.
26. Broussas M, Broyer L, Goetsch L. Evaluation of antibody-dependent cell cytotoxicity using lactate dehydrogenase (LDH) measurement. Methods Mol Biol. 2013;988:305-17.
27. 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. Biomed. Pharmacother. 2019;109:2415–2426.
28. Mohammed R, MilneA, Kayani K, and Ojha U. How the discovery of rituximab impacted the treatment of B-cell non-Hodgkin’s lymphomas. J Blood Med. 2019;10:71–84.
29. Di Gaetano N, Cittera E, Nota R, Vecchi A, Grieco V, Scanziani E, et al. Complement activation determines the therapeutic activity of rituximab in vivo. J immunol. 2003;171(3):1581-7.
30. Treon SP, Mitsiades C, Mitsiades N, Young G, Doss D, Schlossman R, et al. Tumor cell expression of CD59 is associated with resistance to CD20 serotherapy in patients with B-cell malignancies. J Immunother 2001;24(3):263-271.
31. Nimmerjahn F, Ravetch JV. Antibodies, Fc receptors and cancer.Curr Opin Immunol 2007;19(2):239-245.
32. Stewart LM, Young S, Watson G, Mather SJ, Bates PA, Band HA, et al. Humanisation and characterisation of PR1A3, a monoclonal antibody specific for cell-bound carcinoembryonic antigen. Cancer Immunol Immunother 1999;47(6):299-306.
IssueVol 20 No 2 (2021) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijaai.v20i2.6054
CD20 Cell proliferation Cytotoxicity tests Monoclonal antibody

Rights and permissions
Creative Commons License This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
How to Cite
Khademi F, Mohammadi P, Mostafaei A. Construction and Characterization of 1F5 Chimeric Anti-CD20 Monoclonal Antibodies: An Efficient Splicing by Overlap Extension-polymerase Chain Reaction Technique. Iran J Allergy Asthma Immunol. 20(2):205-220.