Abstract
Anti-HBV monoclonal antibodies (mAbs) have been successfully applied in clinic for the prevention and treatment of liver diseases caused by Hepatitis B virus (HBV). However, the mammalian cell culture technique used to produce mAbs provides low yield. Production capacity may be increased by manipulating the mammary gland of transgenic animals to produce mAbs which may be secreted in the milk and readily purified. In this study, we established a transgenic mouse model expressing anti-HBV mAbs and assessed the quantity and quality of such in milk secretions. Eight transgenic founders were generated by co-microinjection of two gene cassettes encoding the heavy- and light-chain of a neutralizing anti-HBV antibody. Both the heavy- and light-chain transgenes produced sufficient transcript expression in the transgenic mice. The expressed anti-HBV mAb was correctly assembled and modified in the mammary gland, as detected by western blotting. ELISA was used to determine the expression level of anti-HBV mAb, which reached up to 17.8 mg/mL, and the affinity of anti-HBV mAb for HBV surface antigen HBsAg, which reached up to 1,577 IU/mg. Therefore, our work confirms the concept of using transgenic animals for large-scale production of therapeutic anti-HBV mAb.
References
CDC (2008) Recommendations for identification and public health management of persons with chronic hepatitis B virus infection. Morb Mortal Wkly Rep 57(RR-08):1–20
Choi T, Huang M, Gorman C, Jaenisch R (1991) A generic intron increases gene expression in transgenic mice. Mol Cell Biol 11:3070–3074
Chung JH, Bell AC, Felsenfeld G (1997) Characterization of the chicken beta-globin insulator. Proc Natl Acad Sci USA 94:575–580
Dimitrov DS, Marks JD (2009) Therapeutic antibodies: current state and future trends—is a paradigm change coming soon? Methods Mol Biol 525:1–27
Dyck MK, Lacroix D, Pothier F, Sirard MA (2003) Making recombinant proteins in animals—different systems, different applications. Trends Biotechnol 21:394–399
Eren R, Ilan E, Nussbaum O, Lubin I, Terkieltaub D, Arazi Y, Ben-Moshe O, Kitchinzky A, Berr S, Gopher J, Zauberman A, Galun E, Shouval D, Daudi N, Eid A, Jurim O, Magnius LO, Hammas B, Reisner Y, Dagan S (2000) Preclinical evaluation of two human anti-hepatitis B virus (HBV) monoclonal antibodies in the HBV-trimera mouse model and in HBV chronic carrier chimpanzees. Hepatology 32:588–596
Farid SS (2007) Process economics of industrial monoclonal antibody manufacture. J Chromatogr B Anal Technol Biomed Life Sci 848:8–18
Galun E, Eren R, Safadi R, Ashour Y, Terrault N, Keeffe EB, Matot E, Mizrachi S, Terkieltaub D, Zohar M, Lubin I, Gopher J, Shouval D, Dagan S (2002) Clinical evaluation (phase I) of a combination of two human monoclonal antibodies to HBV: safety and antiviral properties. Hepatology 35:673–679
Guo Y, Liang M, Bi S (2004) Development of recombinant human-derived IgG antibody against HBsAg. Chin J Exp Clin Virol 18:105–108
Hogan B, Beddington R, Costantini F, Lacy E (1994) Manipulating the mouse embryo, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Kidd-Ljunggren K, Miyakawa Y, Kidd AH (2002) Genetic variability in hepatitis B viruses. J Gen Virol 83:1267–1280
Lavanchy D (2004) Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat 11:97–107
Marasco WA, Sui J (2007) The growth and potential of human antiviral monoclonal antibody therapeutics. Nat Biotechnol 25:1421–1434
McKnight RA, Shamay A, Sankaran L, Wall RJ, Hennighausen L (1992) Matrix-attachment regions can impart position-independent regulation of a tissue-specific gene in transgenic mice. Proc Natl Acad Sci USA 89:6943–6947
Ning D, Junjian X, Xunzhang W, Wenyin C, Qing Z, Kuanyuan S, Guirong R, Xiangrong R, Qingxin L, Zhouyao Y (2003) Expression, purification, and characterization of humanized anti-HBs Fab fragment. J Biochem 134:813–817
Okanoue T, Minami M (2006) Update of research and management of hepatitis B. J Gastroenterol 41:107–118
Pauwelyn K, Cassiman D, Laleman W, Verslype C, Monbaliu D, Aerts R, Van Steenbergen W, Pirenne J, Nevens F (2010) Outcomes of long-term administration of intravenous hepatitis B immunoglobulins for the prevention of recurrent hepatitis B after liver transplantation. Transplant Proc 42:4399–4402
Recillas-Targa F, Pikaart MJ, Burgess-Beusse B, Bell AC, Litt MD, West AG, Gaszner M, Felsenfeld G (2002) Position-effect protection and enhancer blocking by the chicken beta-globin insulator are separable activities. Proc Natl Acad Sci USA 99:6883–6888
Sambrook J, Russell DW (2001) Molecular cloning: a laboratory manual, 3rd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor
Shouval D, Samuel D (2000) Hepatitis B immune globulin to prevent hepatitis B virus graft reinfection following liver transplantation: a concise review. Hepatology 32:1189–1195
Whitelaw CB, Archibald AL, Harris S, McClenaghan M, Simons JP, Clark AJ (1991) Targeting expression to the mammary gland: intronic sequences can enhance the efficiency of gene expression in transgenic mice. Transgenic Res 1:3–13
Zhang W, Czupryn MJ (2002) Free sulfhydryl in recombinant monoclonal antibodies. Biotechnol Prog 18:509–513
Zhang P, Yu MY, Venable R, Alter HJ, Shih JW (2006) Neutralization epitope responsible for the hepatitis B virus subtype-specific protection in chimpanzees. Proc Natl Acad Sci USA 103:9214–9219
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This study was supported by a grant from the Chinese Universities Scientific Fund (2009-1-75).
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Zhang, R., Cui, D., Wang, H. et al. Functional recombinant human anti-HBV antibody expressed in milk of transgenic mice. Transgenic Res 21, 1085–1091 (2012). https://doi.org/10.1007/s11248-012-9589-z
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DOI: https://doi.org/10.1007/s11248-012-9589-z