FcRn – Ypatent

The MHC class I-related neonatal Fc receptor (FcRn) is also known as the Brambell receptor. The (Abrahmsen, WO/2009/016043).

Neonatal Fcr (FcRn) (aka “MHC Class I-related receptor”) Structure

Unlike FcgammaRs which belong to the immunoglobulin superfamily, human FcRns strcturally resemble polypeptides of MHC class I. FcRn is typically expressed as a heterodimer consisting of a transmembrane a or heavy chain in complex with a solbue beta or light chain. FcRn shares 22-29% sequence identity with Class I MHC molecules. (Farrington US2007/0148164). FcRn functions to salvage IgG from the lysosomal degradation pathway, resulting in reduced clearance and long half-life (Falkenstein, US 14/378808).

FcRN is a heterodimeric protein consisting of two polypeptides: a 50 kDa class I major histocompatibility complex like protein (alpha-FcRn) and a 15 kDa beta2-microglobulin (beta2m). (Falkenstein, US 14/378808)

FcRn is a heterodimeric protein consisting of two polypeptides: a 50 kDa class I major histocompatbility complex-life prtoein (a-FcRn) and a 15 kDa p2-microglobulin (beta2 ). (Schlothauer, WO 2016/071377)

Where is FcRn expressed

FcRn expression in endothelial cells is primarily in intracellular organelles, with limited cell surface expression. (Ober J immunol 2004, 172: 2021-2029)

FcRn is also expressed in podocytes. FcRn deficient mice have an impaired clearance of IgG from the glomerular basement membrane (GBM) resulting in accumulation of IgG in the glomeruli of FcRn knockout mice as they age. (Shaw “Podocytes use FcRn to clear IgG from the glomerular basement membrane” PNAS, 2008, 105(3)).

Where does FcRn bind Antibodies:

FcRn binds with high affinity to the CH2-CH3 protion of the Fc region of an antibdoy of the IgG class. The itneraction between an antibody of the IgG class and FcRn is pH dependent and occurs in a 1:2 stoichiometry. Thus one IgG antibody molecule can interact with two FcRn molecuels via its two heavy chain Fc-region polypeptides. Different mutations that influence the FcRn binding and thus the half-life in the blood are known. (Schlothauer, WO 2016/071377)

Functions of FcRn

Protection of albumin from degradation

Albumin like IgG bind noncooperatively to distinct sties on FcRnb in a pH dependent manner which suggests that the mechanism by which albumin interacts with FcRn and thus is protected from degradation is identical to that of IgG. (Lasters, US 2010/0216187).

Salvage of IgG:

The protection of IgGs from degradation as well as the maternal fetal transfer of IgG from mother to young are tighly controlled processes in mammals. The noenatal FcR (FcRN) plays a central role in these processes.

The neonatal Fc-receptor (FcRn) is iportant for teh metabolic fate of antibodies of the IgG class. FcRn functions to salvage IgG form the lysosomal degredation pathway, resulting in reduced clearance and increased healf-life. (Schlothauer, WO 2016/071377)

It has been known for some time that FcRn-IgG recylcing is mediated by the Fc region of the antibody, as isolated Fc has a long half-life, whereas Fab is cleared relatively rapidly. Also, coadminsitering Fc with IgG can result in reduced IgG half-life in a similar manner to increasing the IgG dose. The receptor binding site is located at the interace reigon between the CH2 and CH3 domains and overlaps with the bind site of Staphylococcal protein A, commonly used for purifciation of IgG. Thus protein A complexes are rapidly cleared due to competition with the receptor-mediated process, and isolated Fc fragments have the same T1/2 as the whole IgG molecule. FcRn is the same receptor responsible for transfer of IgG from mother to the neonate which provides immunity for the first few weeks after birth. Mice deficient in this receptor are unable to acquire IgG form the mother, have reduced plasma IgG levels and clear adminsitered IgG or Fc with an abnormally short half-life. (King, “Applications and engineering of monoclonal antibodies, 1998).

The MHC class I-related neonatal Fc receptor (FcRn) mediates cellular trafficking and recycling of albumin and IgG. It specifically binds albumin and IgG at low endosomal pH and thus protects pinocytosed proteins form lysosomal degradation by transportation to the cell surface and release at neutral pH. The FcRN has a good affinity for both alumin and IgG at pH 5-6, while showing from poor to no affinity at neutral pH. (Abrahmsen, WO/2009/016043).

This pH dependence is mediated by the interaction of conserved IgG histidines located at the CH2-CH3 domain interface of the Fc region with acidic residues on FcRn. pH dependent binding is consistent with the model that IgGs are taken up by FcRn expressing cells and enter acidic endosomes where the FcRn-IgG interaction can occur. (Ober J immunol 2004, 172: 2021-2029)

Release of antigen in endosome

The FcRn mediated pathway for maintaining high IgG levels is thought to operate through its pH dependence. IgG is strongly boudn to FcRn at low pH and dissociates at high pH. IgG is internalised into cells and salvaged form the endosome during acidification due to binding to FcRn at low pH. Low pH may also strip any bound antigen form the antibody which would then be degraded in the lysosome. Antiboy is then recylced to the cell surface where the higher pH of the extracellular fluid promotes dissociation from FcRn and retun of the IgG to the circulation. (King, “Applications and engineering of monoclonal antibodies, 1998)

IgG Recycling to the maternal membrane

The pH dependent binding of IgG to FcRn allows for IgG transport through a cell layer and down a concentration gradient of IgG. In human, the syncytiotrophoblast internalizes fluid containing maternal IgG into endosomes; the IgG-containing endosome is then gradually acidified thereby allowing IgG to bind tightly to FcRn present in this compartment. The vesicle then fuses with the membrane on the fetal side of the syncytiotrophoblast, wehre the physiological pH promotes the dissociation of IgG from FcRn. The FcRn molecule may then be recycled to the maternal membrane to perform additional rounds of transcytosis. (Roopernian NatRev Immunol., 7(9): 715-25 (2007).

Affinity of IgG with FcRn:

The Fc portion of IgG binds FcRn with a high affinity at pH 6 and is released at pH 7.2. FcRn acts as a salvage receptor, binding and transporting pinocytosed IgGs in intact form both within and across cells, and rescuing them from a default degradative pathway.

Human FcRn plays a role in adult salvage of IgGs through its occurrence in the pathway of endocytosis in endothelia cells. FcRn receptors lcocated in the acidic endosomes bind and recycle internalized IgGs to the cell surface . IgGs are released from FcRn receptors at the basic pH of blood, thereby escaping lysosomal degradation. This explains the greater half-life of IgGs in the blood compared to other isotypes. FcRn forms a 2:1 complex with IgGs, two FcRn molecules bind to one Fc region of each of the two heavy chains or each of the two heavy chains form the Fc region bind on one molecule of FcRn. FcRn binds to the Fc region of IgGs at the junction between the CH2 and CH3 domains. Critical to the funciton of FcRN is its pH depenent binding of IgG: at pH 6.0, FcRn binds IgG whereasin it does not bind at pH 7.5 The strict pH dependent of the FcRn/IgG interaciton suggests invovlement of the imidazole side chains of histidines which deprotonat over the pH range of 6.0-6.5. Mutation of the surface accessible histidine residues at positions 310 and 435 of the CH2 and CH3 domains severely reduced or eliminated IgG binding to FcRn. (Blein, US 14/431207).

FcRn binds to the Fc porition of IgG at a site that is distinct from the binding sites of classical FcgammaRs or the C1q component of complement. FcRn binds to the CH2-CH3 hinge region of IgG and also binds to this Fc region in a strictly pH dependent manner. At physiological pH 7.4, FcRn does not bind IgG, but at the acidic pH of the endosome (pH 6-6.5), FcRn has a low micromolar to nanomlar affinity for the Fc region of IgG. (Roopernian NatRev Immunol., 7(9): 715-25 (2007).

Mouse models for FcRn:

FcRn plays a pivotal role in modulating the pharmacokinetics of IgG antibodies, which have a high degree of target specificity. Howe er, mouse and rat FcRn do not bind human IgG with the same affinity as human FcRn and thus should not be used to make clinical predictions. Two models in particular, in which the endogenous mouse gene encoding FcRn (Fcgrt) is deleted, and carrying the “Tg32” or “Tg276) alleles encoding the human FcRn, have now been sued to measure and predict the half-life of hundreds of antibodies. (see Jackson Laboratory)