Subcutaneous bioavailability of therapeutic antibodies as a function 
of FcRn binding affinity in mice

Abstract

The neonatal Fc receptor (FcRn) plays an important and well-known role in immunoglobulin G (IgG) catabolism; however, its role in the disposition of IgG after subcutaneous (SC) administration, including bioavailability, is relatively unknown. To examine the potential effect of FcRn on IgG SC bioavailability, we engineered three anti-amyloid β monoclonal antibody (mAb) reverse chimeric mouse IgG2a (mIgG2a) Fc variants (I253A.H435A, N434H and N434Y) with different binding affinities to mouse FcRn (mFcRn) and compared their SC bioavailability to that of the wild-type (WT) mAb in mice. Our results indicated that the SC bioavailability of mIgG2a was affected by mFcRn-binding affinity. Variant I253A.H435A, which did not bind to mFcRn at either pH 6.0 or pH 7.4, had the lowest bioavailability (41.8%). Variant N434Y, which had the greatest increase in binding affinity at both pH 6.0 and pH 7.4, had comparable bioavailability to the WT antibody (86.1% vs. 76.3%), whereas Variant N434H, which had modestly increased binding affinity at pH 6.0 to mFcRn and affinity comparable to the WT antibody at pH 7.4, had the highest bioavailability (94.7%). A semi-mechanism-based pharmacokinetic model, which described well the observed data with the WT antibody and variant I253A.H435A, is consistent with the hypothesis that the decreased bioavailability of variant I253A.H435A was due to loss of the FcRn-mediated protection from catabolism at the absorption site. Together, these data demonstrate that FcRn plays an important role in SC bioavailability of therapeutic IgG antibodies.

Key words:

• monoclonal antibody
• FcRn
• binding affinity
• subcutaneous bioavailability
• semi-mechanism-based pharmacokinetic model

Disclosure of Potential Conflicts of Interest

R.D., G.M., K.H., J.L., Y.L., S.I., L.E.D., F.P.T., P.J.F. and S.P. are employees of Genentech, Inc., which supported the study financially.

Acknowledgments

Our sincere thanks to Camellia Adams for engineering the antibodies, Ernest Oh for measuring the mIgG2a Abs binding to mouse FcRn and Jose Imperio for conducting the mouse PK study.

Figures and Tables

Figure 1 Binding and dissociation of anti-Aβ WT mIgG2a (●) and its Fc variants I253A.H435A (○), N434H (▴) and N434Y (△) to mFcRn measured by ELISA. Serially diluted anti-Aβ antibodies were added to biotinylated mFcRn captured on NeutrAvidin-coated plate at pH 6.0. Bound mIgG2a was detected with anti-mouse IgG F(ab′)²-HRP (A). The mid-OD (the average OD reading of the highest and the lowest concentrations) of the titration curve of the WT was 0.213. The concentrations corresponding to this OD were 72.9, 5.57 and 0.950 ng/mL for the WT antibody, variants N434H and N434Y, respectively. The relative mFcRn binding of the variant to the WT was estimated by dividing the WT concentration with the variant concentration. Variants N434H and N434Y had 13.1 and 76.7-fold increased binding compared with the WT, respectively. To assess dissociation of bound antibodies from mFcRn, the mIgG2a-mFcRn complex on the ELISA plates was exposed to a pH 6.0 (B) or a 7.4 (C) buffer for 45 min before the bound mIgG2a was measured.

Figure 2 Mean (±SD) anti-Aβ mIgG2a mAbs serum concentration vs. time following single IV (A) or SC (B) administration of 5 mg/kg WT (●), I253A.H435A (○), N434H (▴) or N434Y (△) to SCID mice, n = 3 mice/timepoint.

Figure 3 Schematic representation of pharmacokinetic model for anti-Aβ WT and Fc variant I253A.H435A disposition after IV and SC administration in mice. Differential equations describing the change in mIgG2a mAbs concentrations with time for each compartment are described in the text. The symbols are also described in the text.

Figure 4 Observed [IV (●) and SC (○)] and model predicted [IV (-), SC (…)] concentration-time profile following single administration of 5 mg/kg anti-Aβ mIgG2a WT (A) and Fc variant I253A.H435A (B) in SCID mice. The concentrations of the WT and variant I253A.H435A were simultaneously fit to the model shown in Figure 3. Parameter values obtained from the fit are listed in Table 2.

Table 1 Non-compartmental pharmacokinetic parameters for anti-Aβ WT and its Fc variants I253A, H435A, N434H and N434Y in SCID mice

Table 2 Pharmacokinetic parameters for anti-Aβ WT and variant I253A.H435A in SCID mice by fitting data to the model shown in Figure 3