Abstract
Objective
To investigate the feasibility of producing human IgG1 Fc fragment fused factor IX (FIX-Fc) in the milk of transgenic animals, for an alternative possible solution to the unmet need of FIX-Fc products for hemophilia B treatment.
Results
Six founder lines of transgenic mice harboring FIX-Fc cassette designed to be expressed specifically in the mammary gland were generated. FIX-Fc protein was secreted into the milk of transgenic mice with preserved biological activity (with the highest value of 6.2 IU/mL), similar to that of the non-fused FIX transgenic milk. RT-PCR and immunofluorescence analysis confirmed that FIX-Fc was specifically expressed in the mammary gland. The blood FIX clotting activities were unchanged, and no apparent health defects were observed in the transgenic mice. Moreover, the stability of FIX protein in milk was increased by the Fc fusion.
Conclusions
It is feasible to produce biologically functional FIX-Fc in the mammary gland of transgenic mice. Our preliminary results provide a foundation for the potential scale-up production of FIX-Fc in the milk of dairy animals.
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References
Amiri Yekta A, Dalman A, Eftekhari-Yazdi P et al (2013) Production of transgenic goats expressing human coagulation factor IX in the mammary glands after nuclear transfer using transfected fetal fibroblast cells. Transgenic Res 22:131–142. https://doi.org/10.1007/s11248-012-9634-y
Bertolini LR, Meade H, Lazzarotto CR et al (2016) The transgenic animal platform for biopharmaceutical production. Transgenic Res 25:329–343. https://doi.org/10.1007/s11248-016-9933-9
Björkman S (2013) Population pharmacokinetics of recombinant factor IX: implications for dose tailoring. Haemophilia 19:753–757. https://doi.org/10.1111/hae.12188
Carcao M (2014) Changing paradigm of prophylaxis with longer acting factor concentrates. Haemophilia 20:99–105. https://doi.org/10.1111/hae.12405
Caroprese M, Schena L, Marzano A et al (2007) Contribution of macrophages to plasmin activity in ewe bulk milk. Ital J Anim Sci 6:545–547. https://doi.org/10.3168/jds.2006-691
Carter PJ (2011) Introduction to current and future protein therapeutics: a protein engineering perspective. Exp Cell Res 317:1261–1269. https://doi.org/10.1016/j.yexcr.2011.02.013
Chang J, Jin J, Lollar P et al (1998) Changing residue 338 in human factor IX from arginine to alanine causes an increase in catalytic activity. J Biol Chem 273:12089–12094. https://doi.org/10.1074/jbc.273.20.12089
Clark AJ, Bessos H, Bishop JO et al (1989) Expression of human anti-hemophilic factor ix in the milk of transgenic sheep. Bio/Technology 7:487–492. https://doi.org/10.1038/nbt0589-487
Czajkowsky DM, Hu J, Shao Z, Pleass RJ (2012) Fc-fusion proteins: new developments and future perspectives. EMBO Mol Med 4:1015–1028. https://doi.org/10.1002/emmm.201201379
Green KA, Nielsen BS, Castellino FJ et al (2006) Lack of plasminogen leads to milk stasis and premature mammary gland involution during lactation. Dev Biol 299:164–175. https://doi.org/10.1016/j.ydbio.2006.07.021
Houdebine L-M (2009) Production of pharmaceutical proteins by transgenic animals. Comp Immunol Microbiol Infect Dis 32:107–121. https://doi.org/10.1016/j.cimid.2007.11.005
Ismail B, Nielsen SS (2010) Invited review: plasmin protease in milk: current knowledge and relevance to dairy industry. J Dairy Sci 93:4999–5009. https://doi.org/10.3168/jds.2010-3122
Kong Q, Wu M, Huan Y et al (2009) Transgene expression is associated with copy number and cytomegalovirus promoter methylation in transgenic pigs. PLoS ONE 4:e6679. https://doi.org/10.1371/journal.pone.0006679
Lee MH, Lin YS, Tu CF, Yen CH (2014) Recombinant human factor IX produced from transgenic porcine milk. Biomed Res Int 2014:1–8. https://doi.org/10.1155/2014/315375
Lisauskas SFC, Cunha NB, Vianna GR et al (2008) Expression of functional recombinant human factor IX in milk of mice. Biotechnol Lett 30:2063–2069. https://doi.org/10.1007/s10529-008-9818-y
Maksimenko OG, Deykin AV, Khodarovich YM, Georgiev PG (2013) Use of transgenic animals in biotechnology: prospects and problems. Acta Naturae 5:33–46
Nuttall GA (2007) Hemostasis and thrombosis: basic principles and clinical practice, 5th ed. Lippincott Williams & Wilkins, Philadelphia
Paidas MJ, Forsyth C, Quéré I et al (2014) Perioperative and peripartum prevention of venous thromboembolism in patients with hereditary antithrombin deficiency using recombinant antithrombin therapy. Blood Coagul Fibrinolysis 25:444–450. https://doi.org/10.1097/MBC.0000000000000076
Powell JS, Pasi KJ, Ragni MV et al (2013) Phase 3 study of recombinant factor IX Fc fusion protein in hemophilia B. N Engl J Med 369:2313–2323. https://doi.org/10.1056/NEJMoa1305074
Qian X, Kraft J, Ni Y, Zhao FQ (2014) Production of recombinant human proinsulin in the milk of transgenic mice. Sci Rep 4:1–8. https://doi.org/10.1038/srep06465
Riedl MA, Grivcheva-Panovska V, Moldovan D et al (2017) Recombinant human C1 esterase inhibitor for prophylaxis of hereditary angio-oedema: a phase 2, multicentre, randomised, double-blind, placebo-controlled crossover trial. Lancet 390:1595–1602. https://doi.org/10.1016/S0140-6736(17)31963-3
Samis JA, Ramsey GD, Walker JB et al (2000) Proteolytic processing of human coagulation factor IX by plasmin. Blood 95:943–951
Santagostino E, Martinowitz U, Lissitchkov T et al (2016) Long acting recombinant coagulation factor IX albumin fusion protein (rIX-FP) in hemophilia B: results of a phase 3 trial. Blood 127:1–33. https://doi.org/10.1182/blood-2015-09-669234
Tortella BJ, Alvir J, McDonald M et al (2018) Real-world analysis of dispensed IUs of coagulation factor IX and resultant expenditures in hemophilia B patients receiving standard half-life versus extended half-life products and those switching from standard half-life to extended half-life products. J Manag Care Spec Pharm 24:643–653. https://doi.org/10.18553/jmcp.2018.17212
von Mackensen S, Kalnins W, Krucker J et al (2017) Haemophilia patients’ unmet needs and their expectations of the new extended half-life factor concentrates. Haemophilia 23:566–574. https://doi.org/10.1111/hae.13221
Yan J-B, Wang S, Huang W-Y et al (2006) Transgenic mice can express mutant human coagulation factor IX with higher level of clotting activity. Biochem Genet 44:347–358. https://doi.org/10.1007/s10528-006-9034-1
Zhang R, Cui D, Wang H et al (2012) Functional recombinant human anti-HBV antibody expressed in milk of transgenic mice. Transgenic Res 21:1085–1091. https://doi.org/10.1007/s11248-012-9589-z
Zhao J, Xu W, Ross JW et al (2015) Engineering protein processing of the mammary gland to produce abundant hemophilia B therapy in milk. Sci Rep 5:1–13. https://doi.org/10.1038/srep14176
Acknowledgements
This work was supported by the National Key Research and Development Program (2016YFC0905102, 2016YFC1000503, 2019YFA0801402), National Natural Science Foundation (81500108), National Basic Research Program (2014CB964703), National Science and Technology Major Project of China (2016ZX08012-002), Clinical Medical Center and Key Disciplines Construction Grogram of Shanghai (2017ZZ02019), Shanghai Science and Technology Support Program (13431901300), and KingMed Diagnostics Project of Academician Workstation (2017B090904030).
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Yan, H., Gong, X., Xu, M. et al. Production of biologically active human factor IX-Fc fusion protein in the milk of transgenic mice. Biotechnol Lett 42, 717–726 (2020). https://doi.org/10.1007/s10529-020-02808-1
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DOI: https://doi.org/10.1007/s10529-020-02808-1