Volume 25, Issue 4 (7-2021)                   IBJ 2021, 25(4): 275-283 | Back to browse issues page

PMID: 34217158


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Abstract:  
Background: Bispecific antibodies represent an important class of monoclonal antibodies (mAbs), with great therapeutic potentials due to their ability to target simultaneously two distinct epitopes. The generation of functional bispecific antibodies with the highest possible yields is particularly critical for the production of these compounds on industrial scales. Anti-CD3 × CD19 bispecific antibody (bsAb) is a bispecific T-cell engager currently used for treating ALL. Herein, we have tried to optimize the expression level of this antibody in mammalian hosts.  Methods: Woodchuck hepatitis virus post-transcriptional regulation (WPRE) sequence was incorporated at the 3’ end of the expression cassette. This modification resulted in a notable about two-fold increase in the expression of the bsAb in the Expi293 cell line. Results & Conclusion: Follow-up flow cytometry analysis demonstrated the binding properties of the produced antibody at acceptable levels, and in vitro bioactivity assays showed that this product is potent enough for targeting and destroying CD19-positive cells. Our findings show that WPRE enhances the expression of this type of bispecific mAbs in human embryonic kidney-293 family cell lines. This approach can be used in biopharma industry for the mass production of anti-CD3 × CD19 bispecific antibody.

References
1. Goebeler ME, Bargou R. Blinatumomab: a CD19/CD3 bispecific T cell engager (BiTE) with unique anti-tumor efficacy. Leukemia and lymphoma 2016; 57(5): 1021-1032. [DOI:10.3109/10428194.2016.1161185]
2. Ventola CL. Cancer immunotherapy, part 1: Current strategies and agents. Pharmacy and therapeutics 2017; 42(6): 375-383.
3. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nature reviews cancer 2012; 12: 278-287. [DOI:10.1038/nrc3236]
4. Chames P, Van Regenmortel M, Weiss E, Baty D. Therapeutic antibodies: Successes, limitations and hopes for the future. British journal of pharmacology 2009; 157(2): 220-233. [DOI:10.1111/j.1476-5381.2009.00190.x]
5. Beckman RA, Weiner LM, Davis HM. Antibody constructs in cancer therapy: protein engineering strategies to improve exposure in solid tumors. Cancer 2007; 109(2): 170-179. [DOI:10.1002/cncr.22402]
6. Cruz E, Kayser V. Monoclonal antibody therapy of solid tumors: Clinical limitations and novel strategies to enhance treatment efficacy. Biologics: targets and therapy 2019; 13: 33-51. [DOI:10.2147/BTT.S166310]
7. Nunez-Prado N, Compte M, Harwood S, Álvarez-Méndez A, Lykkemark S, Sanz L, Álvarez-Vallina L. The coming of age of engineered multivalent antibodies. Drug discovery today 2015; 20(5): 588-594. [DOI:10.1016/j.drudis.2015.02.013]
8. Krishnamurthy A, Jimeno A. Bispecific antibodies for cancer therapy: A review. Pharmacology and therapeutics 2018; 185: 122-134. [DOI:10.1016/j.pharmthera.2017.12.002]
9. May C, Sapra P, Gerber HP. Advances in bispecific biotherapeutics for the treatment of cancer. Biochemical pharmacology 2012; 84(9): 1105-1112. [DOI:10.1016/j.bcp.2012.07.011]
10. Thakur A, Lum LG. "NextGen" biologics: Bispecific antibodies and emerging clinical results. Expert opinion on biological therapy 2016; 16(5): 675-688. [DOI:10.1517/14712598.2016.1150996]
11. Husain B, Ellerman D. Expanding the boundaries of biotherapeutics with bispecific antibodies. Biodrugs 2018; 32(5): 441-464. [DOI:10.1007/s40259-018-0299-9]
12. Fan D, Li W, Yang Y, Zhang X, Zhang Q, Yan Y, Yang M, Wang J, Xiong D. Redirection of CD4+ and CD8+ T lymphocytes via an anti-CD3× anti-CD19 bi-specific antibody combined with cytosine arabinoside and the efficient lysis of patient-derived B-ALL cells. Journal of hematology and oncology 2015; 8: 108. [DOI:10.1186/s13045-015-0205-6]
13. Ribera JM. Efficacy and safety of bispecific T-cell engager blinatumomab and the potential to improve leukemia-free survival in B-cell acute lymphoblastic leukemia. Expert review of hematology 2017; 10(12): 1057-1067. [DOI:10.1080/17474086.2017.1396890]
14. Brandl C, Haas C, d'Argouges S, Fisch T, Kufer P, Brischwein K, Prang N, Bargou R, Suzich J, Baeuerle PA, Hofmeister R. The effect of dexamethasone on polyclonal T cell activation and redirected target
15. cell lysis as induced by a CD19/CD3-bispecific single-chain antibody construct. Cancer immunology, immunotherapy 2007; 56(10): 1551-1563. [DOI:10.1007/s00262-007-0298-z]
16. Hoffman L, Gore L. Blinatumomab, a bi-specific anti-CD19/CD3 BiTE® antibody for the treatment of acute lymphoblastic leukemia: Perspectives and current pediatric applications. Frontiers in oncology 2014; 4: 63. [DOI:10.3389/fonc.2014.00063]
17. Nagorsen D, Baeuerle PA. Immunomodulatory therapy of cancer with T cell-engaging BiTE antibody blinatumomab. Experimental cell research 2011; 317(9): 1255-1260. [DOI:10.1016/j.yexcr.2011.03.010]
18. May MB, Glode A. Blinatumomab: A Novel, Bispecific, T-cell Engaging Antibody. Oxford University Press, 2016. [DOI:10.2146/ajhp150134]
19. Portell CA, Wenzell CM, Advani AS. Clinical and pharmacologic aspects of blinatumomab in the treatment of B-cell acute lymphoblastic leukemia. Clinical pharmacology: advances and applications 2013; 5 (Suppl 1): 5-11. [DOI:10.2147/CPAA.S42689]
20. Buie LW, Pecoraro JJ, Horvat TZ, Daley RJ. Blinatumomab: a first-in-class bispecific T-cell engager for precursor B-cell acute lymphoblastic leukemia. Annals of pharmacotherapy 2015; 49(9): 1057-1067. [DOI:10.1177/1060028015588555]
21. Kuo S-R, Wong L, Liu J-S. Engineering a CD123xCD3 bispecific scFv immunofusion for the treatment of leukemia and elimination of leukemia stem cells. Protein engineering, design and selection 2012; 25(10): 561-569. [DOI:10.1093/protein/gzs040]
22. Wang Q, Chen Y, Park J, Liu X, Hu Y, Wang T, McFarland K, Betenbaugh MJ. Design and production of bispecific antibodies. Antibodies 2019; 8(3): 43. [DOI:10.3390/antib8030043]
23. Regula JT, von Leithner PL, Foxton R, Barathi VA, Cheung CM, Bo Tun SB, Wey YS, Iwata D, Dostalek M, Moelleken J, Stubenrauch KG, Nogoceke E, Widmer G, Strassburger P, Koss MJ, Klein C, Shima DT, Hartmann G. Targeting key angiogenic pathways with a bispecific CrossMAb optimized for neovascular eye diseases. EMBO molecular medicine 2016; 8(11): 1265-1288. [DOI:10.15252/emmm.201505889]
24. Huang Y, Yu J, Lanzi A, Yao X, Andrews CD, Tsai L, Gajjar MR, Sun M, Seaman MS, Padte NN, Ho DD. Engineered bispecific antibodies with exquisite HIV-1-neutralizing activity. Cell 2016; 165(7): 1621-1631. [DOI:10.1016/j.cell.2016.05.024]
25. Baldi L, Hacker DL, Adam M, Wurm FM. Recombinant protein production by large-scale transient gene expression in mammalian cells: State of the art and future perspectives. Biotechnology letters 2007; 29(5): 677-684. [DOI:10.1007/s10529-006-9297-y]
26. Wurm F, Bernard A. Large-scale transient expression in mammalian cells for recombinant protein production. Current opinion in biotechnology 1999; 10(2): 156-159. [DOI:10.1016/S0958-1669(99)80027-5]
27. Matasci M, Hacker DL, Baldi L, Wurm FM. Recombinant therapeutic protein production in cultivated mammalian cells: current status and future prospects. Drug discovery today: technologies 2008; 5(2-3): e37-e42. [DOI:10.1016/j.ddtec.2008.12.003]
28. Mariati, Ho SC, Yap MG, Yang Y. Evaluating post-transcriptional regulatory elements for enhancing transient gene expression levels in CHO K1 and HEK293 cells. Protein expression and purification 2010; 69(1): 9-15. [DOI:10.1016/j.pep.2009.08.010]
29. Brun S, Faucon-Biguet N, Mallet J. Optimization of transgene expression at the posttranscriptional level in neural cells: implications for gene therapy. Molecular therapy 2003; 7(6): 782-789. [DOI:10.1016/S1525-0016(03)00097-2]
30. Xu ZL, Mizuguchi H, Mayumi T, Hayakawa T. Woodchuck hepatitis virus post-transcriptional regulation element enhances transgene expression from adenovirus vectors. Biochimica et biophysica acta 2003; 1621(3): 266-271. [DOI:10.1016/S0304-4165(03)00078-3]
31. 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]
32. Kim KS, Kim MS, Moon JH, Jeong MS, Kim J, Lee GM, Myung PK. Enhancement of recombinant antibody production in HEK 293E cells by WPRE. Biotechnology and bioprocess engineering 2009; 14: 633. [DOI:10.1007/s12257-008-0221-2]
33. Kokkinopoulos D, Perez S, Sotiriadou R, Stinios J, Papamichail M. The use of nylon wool for the isolation of T lymphocyte subpopulations. Journal of immunological methods 1992; 154(1): 1-6. [DOI:10.1016/0022-1759(92)90205-8]
34. Wang XM, Terasaki PI, Rankin Jr GW, Chia D, Zhong HP, Hardy S. A new microcellular cytotoxicity test based on calcein AM release. Human immunology 1993; 37(4): 264-270. [DOI:10.1016/0198-8859(93)90510-8]
35. Loeb JE, Cordier WS, Harris ME, Weitzman MD, Hope TJ. Enhanced expression of transgenes from adeno-associated virus vectors with the woodchuck hepatitis virus posttranscriptional regulatory element: Implications for gene therapy. Human gene therapy 1999; 10(14): 2295-2305. [DOI:10.1089/10430349950016942]
36. Hlavaty J, Schittmayer M, Stracke A, Jandl G, Knapp E, Felber BK, Salmons B, Günzburg WH, Renner M. Effect of posttranscriptional regulatory elements on transgene expression and virus production in the context of retrovirus vectors. Virology 2005; 341(1): 1-11. [DOI:10.1016/j.virol.2005.06.037]
37. Schambach A, Wodrich H, Hildinger M, Bohne J, Kräusslich HG, Baum C. Context dependence of different modules for posttranscriptional enhancement of gene expression from retroviral vectors. Molecular therapy 2000; 2(5): 435-445. [DOI:10.1006/mthe.2000.0191]
38. Klein R, Ruttkowski B, Knapp E, Salmons B, Günzburg WH, Hohenadl C. WPRE-mediated enhancement of gene expression is promoter and cell line specific. Gene 2006; 372: 153-161. [DOI:10.1016/j.gene.2005.12.018]
39. Dreier T, Lorenczewski G, Brandl C, Hoffmann P, Syring U, Hanakam F, Kufer P, Riethmuller G, Bargou R, Baeuerle PA. Extremely potent, rapid and costimulation‐independent cytotoxic T‐cell response against lymphoma cells catalyzed by a single‐chain bispecific antibody. International journal of cancer 2002; 100(6): 690-697. [DOI:10.1002/ijc.10557]
40. Hoffmann P, Hofmeister R, Brischwein K, Brandl C, Crommer S, Bargou R, Itin C, Prang N, Baeuerle PA. Serial killing of tumor cells by cytotoxic T cells redirected with a CD19‐/CD3‐bispecific single‐chain antibody construct. International journal of cancer 2005; 115(1): 98-104. [DOI:10.1002/ijc.20908]
41. Gruber M, Schodin BA, Wilson ER, Kranz DM. Efficient tumor cell lysis mediated by a bispecific single chain antibody expressed in Escherichia coli. The journal of immunology 1994; 152(11): 5368-5374.

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