Seijsing Johan, Lindborg Malin, Höidén-Guthenberg Ingmarie, Bönisch Heiko, Guneriusson Elin, Frejd Fredrik Y, Abrahmsén Lars, Ekblad Caroline, Löfblom John, Uhlén Mathias, Gräslund Torbjörn
Division of Protein Technology, School of Biotechnology, KTH Royal Institute of Technology, SE-106 91 Stockholm, Sweden;
Affibody AB, SE-171 63 Solna, Sweden;
Proc Natl Acad Sci U S A. 2014 Dec 2;111(48):17110-5. doi: 10.1073/pnas.1417717111. Epub 2014 Nov 18.
Proteins endocytosed from serum are degraded in the lysosomes. However, serum albumin (SA) and IgG, through its Fc part, bind to the neonatal Fc receptor (FcRn) at low pH in the endosome after endocytosis, and are transported back to the cellular surface, where they are released into the bloodstream, resulting in an extended serum circulation time. Association with Fc or SA has been used to prolong the in vivo half-life of biopharmaceuticals, using the interaction with FcRn to improve treatment regimens. This has been achieved either directly, by fusion or conjugation to Fc or SA, or indirectly, using SA-binding proteins. The present work takes this principle one step further, presenting small affinity proteins that bind directly to FcRn, mediating extension of the serum half-life of fused biomolecules. Phage display technology was used to select affibody molecules that can bind to FcRn in the pH-dependent manner required for rescue by FcRn. The biophysical and binding properties were characterized in vitro, and the affibody molecules were found to bind to FcRn more strongly at low pH than at neutral pH. Attachment of the affibody molecules to a recombinant protein, already engineered for increased half-life, resulted in a nearly threefold longer half-life in mice. These tags should have general use as fusion partners to biopharmaceuticals to extend their half-lives in vivo.
从血清中内吞的蛋白质在溶酶体中被降解。然而，血清白蛋白（SA）和IgG通过其Fc部分，在胞吞作用后于内体的低pH条件下与新生儿Fc受体（FcRn）结合，并被转运回细胞表面，在那里它们被释放到血液中，从而延长了血清循环时间。与Fc或SA的结合已被用于延长生物制药的体内半衰期，利用与FcRn的相互作用来改善治疗方案。这可以通过直接将其与Fc或SA融合或偶联来实现，也可以通过使用SA结合蛋白间接实现。目前的工作在此基础上更进一步，提出了直接与FcRn结合的小亲和力蛋白，介导融合生物分子血清半衰期的延长。噬菌体展示技术被用于筛选能够以FcRn拯救所需的pH依赖性方式与FcRn结合的亲和体分子。对其生物物理和结合特性进行了体外表征，发现亲和体分子在低pH下比在中性pH下与FcRn的结合更强。将亲和体分子连接到一个已经设计用于延长半衰期的重组蛋白上，在小鼠体内导致其半衰期延长了近三倍。这些标签作为生物制药的融合伴侣在体内延长其半衰期应具有广泛的用途。
