RESUMO
Serum albumin shows slow clearance from circulation due to neonatal Fc receptor (FcRn)-mediated recycling and has been used for half-life extension. We report here fusions to a high-affinity DARPin, binding to Epithelial Cell Adhesion Molecule (EpCAM). We developed a novel, efficient expression system for such fusion proteins in Pichia pastoris with titers above 300 mg/L of lab-scale shake-flask culture. Since human serum albumin (HSA) does not bind to the murine FcRn, half-lives of therapeutic candidates are frequently measured in human FcRn transgenic mice, limiting useable tumor models. Additionally, serum albumins with extended half-life have been designed. We tested HSA7, motivated by its previously claimed extraordinarily long half-life in mice, which we could not confirm. Instead, we determined a half-life of only 29 h for HSA7, comparable to MSA. The fusion of HSA7 to a DARPin showed a similar half-life. To rationalize these findings, we measured binding kinetics and affinities to murine and human FcRn. Briefly, HSA7 showed affinity to murine FcRn only in the micromolar range, comparable to MSA to its cognate murine FcRn, and an affinity in the nanomolar range only to the human FcRn. This explains the comparable half-life of MSA and HSA7 in mice, while wild-type-HSA has a half-life of only 21 h, as it does not bind the murine FcRn and is not recycled. Thus, HSA-fusions with improved FcRn-affinity, such as HSA7, can be used for preclinical experiments in mice when FcRn transgenes cannot be used, as they reflect better the complex FcRn-mediated recycling and distribution mechanisms.
Assuntos
Proteínas de Repetição de Anquirina Projetadas/metabolismo , Antígenos de Histocompatibilidade Classe I/metabolismo , Receptores Fc/metabolismo , Albumina Sérica/metabolismo , Animais , Feminino , Meia-Vida , Antígenos de Histocompatibilidade Classe I/genética , Humanos , Camundongos , Camundongos Transgênicos , Receptores Fc/genética , Saccharomycetales/metabolismo , Albumina Sérica Humana/metabolismoRESUMO
The neonatal Fc receptor (FcRn) was first recognized for its role in transfer of maternal IgG to the foetus or newborn, providing passive immunity early in life. However, it has become clear that the receptor is versatile, widely expressed and plays an indispensable role in both immunological and non-immunological processes throughout life. The receptor rescues immunoglobulin G (IgG) and albumin from intracellular degradation and shuttles the ligands across polarized cell barriers, in all cases via a pH-dependent binding-and-release mechanism. These processes secure distribution and high levels of both IgG and albumin throughout the body. At mucosal sites, FcRn transports IgG across polarized epithelial cells where it retrieves IgG in complex with luminal antigens that is delivered to tissue-localized immune cells. In dendritic cells (DCs), FcRn orchestrates processing of IgG-opsonized immune complexes (ICs) in concert with classical Fcγ receptors, which results in antigen presentation and cross-presentation of antigenic peptides on MHC class II and I to CD4+ and CD8+ T cells, respectively. Hence, FcRn regulates transport of the ligands within and across different types of cells, but also processing of IgG-ICs by immune cells. As such, the receptor is involved in immune surveillance and protection against infections. In this brief review, we highlight how FcRn expressed by hematopoietic and non-hematopoietic cells contributes to immune regulation at mucosal barriers-biology that can be utilized in development of biologics and subunit vaccines for non-invasive delivery.