Abstract
Interaction of antibodies with the neonatal FcRn receptor controls largely the length of their life cycle. Altering the serum persistence of antibodies through modulation of their interaction with the FcRn may be advantageous for achieving high contrast images shortly after application of the radiolabeled antibody tracer or more efficient therapy with fewer administrations. This protocol describes the steps required for producing antibodies, antibody fragments or antibody Fc fusion proteins with altered in vivo pharmacokinetics through introduction of specific mutations in the antibody Fc region. Included are also protocols for evaluation of binding to the FcRn in vitro and determination of serum half-life through radioiodinating the antibody Fc variants and performing biodistribution studies in mice.
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Akilesh S, Christianson GJ, Roopenian DC, Shaw AS (2007) Neonatal FcR expression in bone marrow-derived cells functions to protect serum IgG from catabolism. J Immunol 179:4580–4588
Akilesh S, Huber TB, Wu H, Wang G, Hartleben B, Kopp JB, Miner JH, Roopenian DC, Unanue ER, Shaw AS (2008) Podocytes use FcRn to clear IgG from the glomerular basement membrane. Proc Natl Acad Sci USA 105:967–972
Andersen JT, Justesen S, Berntzen G, Michaelsen TE, Lauvrak V, Fleckenstein B, Buus S, Sandlie I (2008a) A strategy for bacterial production of a soluble functional human neonatal Fc receptor. J Immunol Methods 331:39–49
Andersen JT, Justesen S, Fleckenstein B, Michaelsen TE, Berntzen G, Kenanova VE, Daba MB, Lauvrak V, Buus S, Sandlie I (2008b) Ligand binding and antigenic properties of a human neonatal Fc receptor with mutation of two unpaired cysteine residues. Febs J 275:4097–4110
Baselga J, Tripathy D, Mendelsohn J, Baughman S, Benz CC, Dantis L, Sklarin NT, Seidman AD, Hudis CA, Moore J, Rosen PP, Twaddell T, Henderson IC, Norton L (1999) Phase II study of weekly intravenous trastuzumab (Herceptin) in patients with HER2/neu-overexpressing metastatic breast cancer. Semin Oncol 26:78–83
Bauer RJ, Dedrick RL, White ML, Murray MJ, Garovoy MR (1999) Population pharmacokinetics and pharmacodynamics of the anti-CD11a antibody hu1124 in human subjects with psoriasis. J Pharmacokinet Biopharm 27:397–420
Baxter LT, Zhu H, Mackensen DG, Jain RK (1994) Physiologically based pharmacokinetic model for specific and nonspecific monoclonal antibodies and fragments in normal tissues and human tumor xenografts in nude mice. Cancer Res 54:1517–1528
Berntzen G, Lunde E, Flobakk M, Andersen JT, Lauvrak V, Sandlie I (2005) Prolonged and increased expression of soluble Fc receptors, IgG and a TCR-Ig fusion protein by transiently transfected adherent 293E cells. J Immunol Methods 298:93–104
Burmeister WP, Huber AH, Bjorkman PJ (1994) Crystal structure of the complex of rat neonatal Fc receptor with Fc. Nature 372:379–383
Covell DG, Barbet J, Holton OD, Black CD, Parker RJ, Weinstein JN (1986) Pharmacokinetics of monoclonal immunoglobulin G1, F(ab')2, and Fab' in mice. Cancer Res 46:3969–3978
Dall’Acqua WF, Woods RM, Ward ES, Palaszynski SR, Patel NK, Brewah YA, Wu H, Kiener PA, Langermann S (2002) Increasing the affinity of a human IgG1 for the neonatal Fc receptor: biological consequences. J Immunol 169:5171–5180
Dall'Acqua WF, Kiener PA, Wu H (2006) Properties of human IgG1s engineered for enhanced binding to the neonatal Fc receptor (FcRn). J Biol Chem 281:23514–23524
Flessner MF, Lofthouse J, Zakaria el-R (1997) In vivo diffusion of immunoglobulin G in muscle: effects of binding, solute exclusion, and lymphatic removal. Am J Physiol 273:H2783–H2793
Ghetie V, Popov S, Borvak J, Radu C, Matesoi D, Medesan C, Ober RJ, Ward ES (1997) Increasing the serum persistence of an IgG fragment by random mutagenesis. Nat Biotechnol 15:637–640
Gillies SD, Lo KM, Burger C, Lan Y, Dahl T, Wong WK (2002) Improved circulating half-life and efficacy of an antibody-interleukin 2 immunocytokine based on reduced intracellular proteolysis. Clin Cancer Res 8:210–216
Haymann JP, Levraud JP, Bouet S, Kappes V, Hagege J, Nguyen G, Xu Y, Rondeau E, Sraer JD (2000) Characterization and localization of the neonatal Fc receptor in adult human kidney. J Am Soc Nephrol 11:632–639
Hinton PR, Xiong JM, Johlfs MG, Tang MT, Keller S, Tsurushita N (2006) An engineered human IgG1 antibody with longer serum half-life. J Immunol 176:346–356
Kenanova V, Olafsen T, Crow DM, Sundaresan G, Subbarayan M, Carter NH, Ikle DN, Yazaki PJ, Chatziioannou AF, Gambhir SS, Williams LE, Shively JE, Colcher D, Raubitschek AA, Wu AM (2005) Tailoring the pharmacokinetics and positron emission tomography imaging properties of anti-carcinoembryonic antigen single-chain Fv-Fc antibody fragments. Cancer Res 65:622–631
Kim JK, Tsen MF, Ghetie V, Ward ES (1994) Localization of the site of the murine IgG1 molecule that is involved in binding to the murine intestinal Fc receptor. Eur J Immunol 24:2429–2434
Kuus-Reichel K, Grauer LS, Karavodin LM, Knott C, Krusemeier M, Kay NE (1994) Will immunogenicity limit the use, efficacy, and future development of therapeutic monoclonal antibodies? Clin Diagn Lab Immunol 1:365–372
Lobo ED, Hansen RJ, Balthasar JP (2004) Antibody pharmacokinetics and pharmacodynamics. J Pharm Sci 93:2645–2668
Mager DE, Mascelli MA, Kleiman NS, Fitzgerald DJ, Abernethy DR (2003) Simultaneous modeling of abciximab plasma concentrations and ex vivo pharmacodynamics in patients undergoing coronary angioplasty. J Pharmacol Exp Ther 307:969–976
Medesan C, Radu C, Kim JK, Ghetie V, Ward ES (1996) Localization of the site of the IgG molecule that regulates maternofetal transmission in mice. Eur J Immunol 26:2533–2536
Medesan C, Matesoi D, Radu C, Ghetie V, Ward ES (1997) Delineation of the amino acid residues involved in transcytosis and catabolism of mouse IgG1. J Immunol 158:2211–2217
Meier W, Gill A, Rogge M, Dabora R, Majeau GR, Oleson FB, Jones WE, Frazier D, Miatkowski K, Hochman PS (1995) Immunomodulation by LFA3TIP, an LFA-3/IgG1 fusion protein: cell line dependent glycosylation effects on pharmacokinetics and pharmacodynamic markers. Ther Immunol 2:159–171
Meijer RT, Koopmans RP, ten Berge IJ, Schellekens PT (2002) Pharmacokinetics of murine anti-human CD3 antibodies in man are determined by the disappearance of target antigen. J Pharmacol Exp Ther 300:346–353
Montoyo HP, Vaccaro C, Hafner M, Ober RJ, Mueller W, Ward ES (2009) Conditional deletion of the MHC class I-related receptor FcRn reveals the sites of IgG homeostasis in mice. Proc Natl Acad Sci USA 106:2788–2793
Newkirk MM, Novick J, Stevenson MM, Fournier MJ, Apostolakos P (1996) Differential clearance of glycoforms of IgG in normal and autoimmune-prone mice. Clin Exp Immunol 106:259–264
Ober RJ, Radu CG, Ghetie V, Ward ES (2001) Differences in promiscuity for antibody-FcRn interactions across species: implications for therapeutic antibodies. Int Immunol 13:1551–1559
Petkova SB, Akilesh S, Sproule TJ, Christianson GJ, Al Khabbaz H, Brown AC, Presta LG, Meng YG, Roopenian DC (2006) Enhanced half-life of genetically engineered human IgG1 antibodies in a humanized FcRn mouse model: potential application in humorally mediated autoimmune disease. Int Immunol 18:1759–1769
Qiao SW, Kobayashi K, Johansen FE, Sollid LM, Andersen JT, Milford E, Roopenian DC, Lencer WI, Blumberg RS (2008) Dependence of antibody-mediated presentation of antigen on FcRn. Proc Natl Acad Sci USA 105:9337–9342
Raghavan M, Bonagura VR, Morrison SL, Bjorkman PJ (1995) Analysis of the pH dependence of the neonatal Fc receptor/immunoglobulin G interaction using antibody and receptor variants. Biochemistry 34:14649–14657
Roopenian DC, Akilesh S (2007) FcRn: the neonatal Fc receptor comes of age. Nat Rev Immunol 7:715–725
Waldmann TA, Strober W (1969) Metabolism of immunoglobulins. Prog Allergy 13:1–110
Wawrzynczak EJ, Cumber AJ, Parnell GD, Jones PT, Winter G (1992) Blood clearance in the rat of a recombinant mouse monoclonal antibody lacking the N-linked oligosaccharide side chains of the CH2 domains. Mol Immunol 29:213–220
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Kenanova, V.E., Olafsen, T., Andersen, J.T., Sandlie, I., Wu, A.M. (2010). Engineering of the Fc Region for Improved PK (FcRn Interaction). In: Kontermann, R., Dübel, S. (eds) Antibody Engineering. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-01144-3_27
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DOI: https://doi.org/10.1007/978-3-642-01144-3_27
Publisher Name: Springer, Berlin, Heidelberg
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