Hinge-Deficient IgG1 Fc Fusion: Application to Human Lactoferrin

• Published in: Molecular pharmaceutics 2017-09, Vol.14 (9), p.3025-3035
• Main Authors: Shiga, Yuki, Murata, Daisuke, Sugimoto, Akinori, Oshima, Yuta, Tada, Minoru, Ishii-Watabe, Akiko, Imai, Kenichiro, Tomii, Kentaro, Takeuchi, Takashi, Kagaya, Shinji, Sato, Atsushi
• Format: Article
• Language: English
• Subjects: Animals; Caco-2 Cells; CHO Cells; Chromatography, Gel; Circular Dichroism; Cricetinae; Cricetulus; Electrophoresis, Polyacrylamide Gel; Enzyme-Linked Immunosorbent Assay; Humans; Immunoglobulin Fc Fragments - genetics; Immunoglobulin Fc Fragments - metabolism; Immunoglobulin G - genetics; Immunoglobulin G - metabolism; Lactoferrin - genetics; Lactoferrin - metabolism; Protein Binding
• ISSN: 1543-8384; 1543-8392
• DOI: 10.1021/acs.molpharmaceut.7b00221

Fusion of therapeutic proteins with the antibody Fc domain is a strategy widely applied to increase protein half-life in plasma. In our previous study, we generated a recombinant human lactoferrin (hLF)-immunoglobulin G1 Fc fusion protein (hLF-hinge-CH2-CH3) with improved stability, biological activity, and pharmacokinetics ( Shiga, Y. et al. Eur J Pharm Sci., 2015, 67, 136 –143 ). However, the Fc domain in fusion proteins can potentially induce antibody-dependent and complement-dependent cytotoxicity and serious side effects. To overcome these drawbacks, we engineered an hLF-Fc fusion protein (hLF-CH2-CH3) without the Fc hinge region which is essential for engaging Fc receptors on immune cells and inducing complement-mediated cell lysis. The hLF-CH2-CH3 protein was stably expressed in Chinese hamster ovary (CHO) DG44 cells and compared for in vitro activities, thermal stability, pharmacokinetics, and attenuation of Fc-mediated immune effector functions with the conventional hinge-containing Fc fusion protein. Both hLF-hinge-CH2-CH3 and hLF-CH2-CH3 exhibited iron-binding activity, superior uptake by Caco-2 cells, similar thermal stability, and longer plasma half-life compared to recombinant hLF. However, in contrast to conventional hLF-hinge-CH2-CH3, hinge-deficient hLF-CH2-CH3 did not elicit Fc-mediated effector response potentially damaging for the target cells. Our findings demonstrate that conjugation of hinge-deficient Fc to therapeutic proteins is a promising strategy for improving their pharmacokinetic properties without enhancing effector functions. Cell-expressed hinge-deficient hLF-CH2-CH3 is a potential drug candidate with improved plasma half-life for parenteral administration.