In vivo evaluation of human IgG and human serum albumin (HSA) variants with altered binding properties to the neonatal Fc-receptor (FcRn)

The two most abundant proteins in blood, albumin and immunoglobulin G (IgG) have unusual long half-lives (three weeks in humans), because they are protected from degradation inside cells by a receptor, named FcRn. This unique property is being exploited to improve and extend the serum half-life of drugs, as many therapeutic peptides, proteins and chemical drugs on the market have limited effect because of rapid clearance from the circulation. For optimal use of albumin and IgG-based therapeutics, we are studying the relationship between FcRn and the two proteins in great detail. Through this work we have developed human serum albumin (HSA) and human IgG variants that show a range of different binding strengths to FcRn. We plan to test if binding strength to FcRn correlates with serum half-life in mice; find out whether stronger binding to the receptor prolongs serum half-life. Our study will reveal whether modifying the binding strength can be used as a strategy to tailor the half-life of couple drugs, which would allow for more favorable dosing regimes. We also aim to investigate whether human IgG and HSA, and coupled drugs, may be delivered to the circulation via FcRn expressed in the mucosal epithelium of the lungs, which may offer a needle-free route of delivery of drugs. The study will include 42 C57BL/6J mice and 42 BALB/c, 126 Tg32 mice (human FcRn (hFcRn) transgenic) and 288 Tg32-ALB KO mice (hFcRn transgenic and mouse serum albumin (MSA) knock out). The use of three different mouse strains is necessary because there are cross-species differences in binding of FcRn to albumin and IgG. The animals may experience brief fear and pain while being handled for administration of test substance and for collection of blood. We will minimize stress by handing the mice gently, quietly, and as little as possible. By thoroughly characterization using biochemical and cellular methods, we have limited the number of HSA and human IgG variants, yet an in vivo model is necessary to determine serum half-life,which is dependent on many factors.