Generation and characterization of a high affinity anti-human FcRn antibody, rozanolixizumab, and the effects of different molecular formats on the reduction of plasma IgG concentration

Abstract

Rozanolixizumab (UCB7665), a humanized high-affinity anti-human neonatal Fc receptor (FcRn) monoclonal antibody (IgG4P), has been developed to reduce pathogenic IgG in autoimmune and alloimmune diseases. We document the antibody isolation and compare rozanolixizumab with the same variable region expressed in various mono-, bi- and trivalent formats. We report activity data for rozanolixizumab and the different molecular formats in human cells, FcRn-transgenic mice, and cynomolgus monkeys. Rozanolixizumab, considered the most effective molecular format, dose-dependently and selectively reduced plasma IgG concentrations in an FcRn-transgenic mouse model (no effect on albumin). Intravenous (IV) rozanolixizumab dosing in cynomolgus monkeys demonstrated non-linear pharmacokinetics indicative of target-mediated drug disposition; single IV rozanolixizumab doses (30 mg/kg) in cynomolgus monkeys reduced plasma IgG concentration by 69% by Day 7 post-administration. Daily IV administration of rozanolixizumab (initial 30 mg/kg loading dose; 5 mg/kg daily thereafter) reduced plasma IgG concentrations in all cynomolgus monkeys, with low concentrations maintained throughout the treatment period (42 days). In a 13-week toxicology study in cynomolgus monkeys, supra-pharmacological subcutaneous and IV doses of rozanolixizumab (≤ 150 mg/kg every 3 days) were well tolerated, inducing sustained (but reversible) reductions in IgG concentrations by up to 85%, with no adverse events observed. We have demonstrated accelerated natural catabolism of IgG through inhibition of IgG:FcRn interactions in mice and cynomolgus monkeys. Inhibition of FcRn with rozanolixizumab may provide a novel therapeutic approach to reduce pathogenic IgG in human autoimmune disease. Rozanolixizumab is being investigated in patients with immune thrombocytopenia (NCT02718716) and myasthenia gravis (NCT03052751).

Keywords: FcRn; FcRn blockade; IgG catabolism; UCB7665; autoantibody; autoimmunity; immune thrombocytopenia; myasthenia gravis; rozanolixizumab; target-mediated drug disposition.

Figure 1.

Crystal structure of complex of human FcRn (deglycosylated extracellular domain) and 1519.g57 Fab’. (A) The binding epitope of 1519.g57 Fab’ on FcRn α-chain is shown in red; (B) PDB 4N0U – human IgG Fc domain interacting with FcRn, demonstrating the overlapping epitope with that of 1519.g57Fab’,²⁹ (C) FcRn α-chain sequence, showing residues involved in interaction with 1519.g57 Fab’ (in red). Residues involved in interaction between human FcRn and IgG (Fc domain) or albumin (as described by Oganesyan et al, 2014, highlighted in yellow or in blue, respectively) are also shown. The sequence of human FcRn α-chain ECD was taken from Swiss Prot (P55899-1); Residue F44, identified by Oganesyan et al.²⁹ as interacting with albumin, is residue E44 in this sequence and in the sequence of Schmidt et al.³⁰ Dark blue = FcRn α-chain; pale blue = β2 M; magenta = 1519.g57 heavy chain; orange = 1519.g57 light chain; yellow = IgG Fc domain; red = 1519.g57 Fab’ binding epitope on FcRn.

Figure 2.

Rozanolixizumab causes an increase in intracellular IgG AF647. (A) HUVECs incubated for 24 hours with AF647-conjugated IgG (green) in the presence of either rozanolixizumab or a control IgG4P antibody. Nuclei labelled with Hoechst (blue). (B) Quantification of vesicular area per cell from three independent experiments. Graph shows arithmetic mean ± SD. (c) Co-localization of Lysotracker (green) with internalized AF647-conjugated IgG (red). Nuclei labelled with Hoechst (blue). Areas of co-localization shown in yellow.

Figure 3.

Effect of a single dose of various 1519.g57 formats (100 mg/kg) on human plasma IgG concentration in human FcRn-transgenic mice. Group sizes (n) as indicated in the legend, combined from multiple experiments. For clarity, data are divided between two graphs: (A) and (B). Geometric mean and 95% confidence interval are shown.

Figure 4.

The PK of a single dose of various 1519.g57 formats in human FcRn-transgenic mice: the effect of molecular format on the concentration of anti-FcRn free in plasma. Group sizes (n) were as indicated in the legend, combined from multiple experiments. Geometric mean and 95% confidence interval are shown (one arm shown only, for greater clarity).

Figure 5.

(A) The effect of single IV doses of rozanolixizumab (5, 10 and 30 mg/kg) on plasma IgG concentration in cynomolgus monkeys, relative to baseline. Data are arithmetic mean ± standard errors. (B) The PK of single doses (IV) of rozanolixizumab in cynomolgus monkey plasma.

Figure 6.

The effect of daily IV and SC doses of rozanolixizumab on plasma IgG concentrations in cynomolgus monkeys. For IV dosing, a loading dose of 30 mg/kg on Day 1 was followed by a daily dose of 5 mg/kg for 41 days. For SC dosing, 5 mg/kg was administered daily for 60 days. Thick lines represent mean values; thin lines represent individual animals. *Animals received a loading dose of 30 mg/kg on Day 1.

Figure 7.

Individual percent change from baseline in plasma IgG concentration in cynomolgus monkeys following IV (A) or SC (B) rozanolixizumab dosing at 150 mg/kg every 3 days for 13 weeks. Lines represent individual animal data.