Antibody Fc engineering for enhanced neonatal Fc receptor binding and prolonged circulation half-life

Abstract

The neonatal Fc receptor (FcRn) promotes antibody recycling through rescue from normal lysosomal degradation. The binding interaction is pH-dependent with high affinity at low pH, but not under physiological pH conditions. Here, we combined rational design and saturation mutagenesis to generate novel antibody variants with prolonged half-life and acceptable development profiles. First, a panel of saturation point mutations was created at 11 key FcRn-interacting sites on the Fc region of an antibody. Multiple variants with slower FcRn dissociation kinetics than the wildtype (WT) antibody at pH 6.0 were successfully identified. The mutations were further combined and characterized for pH-dependent FcRn binding properties, thermal stability and the FcγRIIIa and rheumatoid factor binding. The most promising variants, YD (M252Y/T256D), DQ (T256D/T307Q) and DW (T256D/T307W), exhibited significantly improved binding to FcRn at pH 6.0 and retained similar binding properties as WT at pH 7.4. The pharmacokinetics in human FcRn transgenic mice and cynomolgus monkeys demonstrated that these properties translated to significantly prolonged plasma elimination half-life compared to the WT control. The novel variants exhibited thermal stability and binding to FcγRIIIa in the range comparable to clinically validated YTE and LS variants, and showed no enhanced binding to rheumatoid factor compared to the WT control. These engineered Fc mutants are promising new variants that are widely applicable to therapeutic antibodies, to extend their circulation half-life with obvious benefits of increased efficacy, and reduced dose and administration frequency.

Pharmacokinetics of novel Fc-engineered monoclonal and multispecific antibodies in cynomolgus monkeys and humanized FcRn transgenic mouse models

“…The usefulness of transgenic mouse models for PK profiling of mAbs has been shown in a series of different studies. 18 , 30–32 , 39 We report here for the first time that hFcRn transgenic mouse models – in particular, Tg32 mice – are highly sensitive for differentiating PK of mAbs and multispecific antibodies compared with a NHP model. Our results confirm literature reports of a differential impact in hFcRn transgenic mice of various antibodies on PK behavior depending on the antibodies’ antigen-binding fragments (Fabs).…”

“…Variants with the LS, DQ, DW, and YD mutations were previously developed in-house. 18 Four multispecific antibodies were included in our study: 2 bi-Abs and 2 tri-Abs. Of the multispecific antibodies, 2 were WT and 2 were engineered through the Fc region.…”

“…Based on their FcRn binding properties alone, NNAS would be expected to maintain a WT-like PK profile in the in vivo models, while DQ, DW, YD, LS, and the other FcRn-binding variants would contribute to a prolonged plasma PK. 18 In summary, the Tg32 hFcRn mouse model is well suited to screen the absence of impact of the NNAS mutation on the PK profile of mAbs as also seen in cynomolgus monkeys.…”

“…Recently, our group engineered a panel of novel Fc variants, namely, YD (M252Y/T256D), DQ (T256D/T307Q), and DW (T256D/T307W), that showed improved binding to FcRn and prolonged half-lives versus their wild-type (WT) counterparts, in both Tg32 mouse and cynomolgus monkey models. 18 Long circulating half-life is paramount to the clinical success of mAbs, 19 enabling lower dosing and less frequent administration, which may result in better compliance and reduced costs. 20 …”

“… 27–29 The relevance of the Tg32 mouse model to demonstrate in vivo half-life prolongation of engineered or native mAbs and to predict NHP or human PK has been recently reported. 18 , 30–32 …”