Nature Biotechnology: doi: /nbt.1691

Transcription

1 a b Supplementary Figure 1. Effect of tocilizumab (TCZ) on hil-6r transgenic mice hil-6 induced SAA model. (a), (b) In vivo study of tocilizumab (TCZ) in hil-6r transgenic mice. TCZ were intravenously administered at single doses of 25 mg/kg. 20 mg/kg of MR16-1 was intravenously administered at 0 h and 47 h to inhibit the effect of hil-6 binding to mouse IL-6R. 4 μg/kg of hil-6 was intraperitoneally administered at 24 h, 48 h and 72 h for TCZ and hil-6 control (IL-6 + vehicle) group. Vehicle group was injected with buffer, instead of antibody and hil-6 (vehicle + vehicle). SAA concentration was measured as a marker of neutralization of hil-6r. Time profiles of plasma antibody concentration (a) and plasma SAA concentration (b) are shown. Each data point represents the mean±s.d. for antibody concentration and mean±s.e.m for SAA concentration (n = 3-4 each).

2 a b Supplementary Figure 2. Proposed differences between conventional and ph-dependent binding antibody on mil-6r and sil-6r. (a) Conventional antibody (TCZ) bound to mil-6r (i) is internalized into endosome (ii-iii) and transferred into lysosome and degraded (iv), resulting in single antigen-binding site binding to only one mil-6r molecule. (b) Antibody binding ph-dependently (PH2) bound to mil-6r (i) is internalized into endosome (ii). Antibody is dissociated from mil-6r in acidic ph (iii). mil-6r is transferred into lysosome and degraded (iv), whereas dissociated antibody is recycled back to plasma (v). Recycled free antibody can be reused for binding to another mil-6r (vi), allowing single antigen-binding site to bind to multiple mil-6r molecules. Red circle, blue circle and yellow triangle represent mil-6r, FcRn and lysozyme, respectively.

3 c d Supplementary Figure 2 (Continued). Proposed differences between conventional and ph-dependent binding antibody on mil-6r and sil-6r. (c) Conventional antibody (TCZ) bound to sil-6r (i) is non-specifically taken up by pinocytosis (ii), and binds to FcRn in acidic endosome (iii). Antibody as well as sil-6r is recycled back to plasma by FcRn (iv) as an antibody/sil-6r complex (v) resulting in single antigen-binding site binding to only one sil-6r molecule. (d) Antibody binding ph-dependently (PH2) bound to sil-6r (i) is non-specifically taken up by pinocytosis (ii), and binds to FcRn in acidic endosome and sil-6r is dissociated from antibody in acidic ph (iii). sil-6r is transferred into lysosome and degraded (iv), whereas dissociated antibody is recycled back to plasma by FcRn (iv). Recycled free antibody can be reused for binding to another sil-6r (vi), allowing single antigen-binding site to bind to multiple sil-6r molecules. Red circle, blue circle and yellow triangle represent sil-6r, FcRn and lysozyme, respectively.

4 a b Supplementary Figure 3. Effect of TCZ and PH2-FcRn on hil-6r transgenic mice hil-6 induced SAA model. (a), (b) In vivo study of TCZ and PH2-FcRn in hil-6r transgenic mice. TCZ and PH2-FcRn were intravenously administered at single doses of 25 mg/kg. 20 mg/kg of MR16-1 was intravenously administered at 23 h and 71 h to inhibit the effect of hil-6 binding to mouse IL-6R. 4 μg/kg of hil-6 was intraperitoneally administered at 48 h, 72 h and 96 h for TCZ, PH2-FcRn and hil-6 control (IL-6 + vehicle) group. Vehicle group was injected with buffer, instead of antibody and hil-6 (vehicle + vehicle). SAA concentration was measured as a marker of neutralization of hil-6r. Time profiles of plasma antibody concentration (a) and plasma SAA concentration (b) are shown. Each data point represents the mean± s.d. for antibody concentration and mean±s.e.m for SAA concentration (n = 4 each).

5 Supplementary Figure 4. Antigen-mediated clearance and recycling model. Antibody is injected intravenously to central compartment and distributes to peripheral compartment. Antibody in the central compartment binds to mil-6r and forms antibody/mil-6r complex. Antibody/mIL-6R complex is internalized and fraction of internalized antibody is recycled back to the central compartment, whereas the remaining fraction is degraded by lysosome. Antibody in central compartment is eliminated in a non-antigen-mediated manner. Parameters used in this model are R total (total amount of mil-6r), R free (amount of free mil-6r), DR complex (amount of antibody/mil-6r complex), k int (internalization rate constant of mil-6r), F rec (fraction recycled from endosome (relative recycling ratio of the antibody from the endosome)), k el (elimination rate constant (non-antigen-mediated elimination)), k a (association rate constant), k d (dissociation rate constant), k 12 (transfer rate constant from central to peripheral compartment) and k 21 (transfer rate constant from peripheral to central compartment).

6 Dose T1/2 AUCinf CL MRT Vdss mg/kg day µg*day/ml ml/day/kg day ml/kg TCZ mean s.d PH1 mean s.d PH2 mean s.d Supplementary Table 1. Pharmacokinetic parameters of TCZ, PH1 and PH2 in hil-6r transgenic mice. TCZ, PH1 and PH2 were intravenously injected at single doses of 25 mg/kg. Time-points for T1/2 calculation were automatically set by WinNonlin on the basis of the result of the individual time-profile in plasma concentrations. Time-points for T1/2 calculation were 78 h to 120 h for TCZ (n = 4), 54 h to 120 h (n = 3) and 24 h to 102 h (n = 1) for PH1, and 54 h to 120 h for PH2 (n = 4). Calculated parameters are T1/2 (terminal phase half life), AUCinf (area under the curve to infinity), CL (clearance), MRT (mean residence time) and Vdss (volume of distribution at steady state).

7 Dose T1/2 AUCinf CL MRT Vdss mg/kg day µg*day/ml ml/day/kg day ml/kg TCZ mean s.d PH2 mean s.d Supplementary Table 2. Pharmacokinetic parameters of TCZ and PH2 in normal mice. TCZ and PH2 were intravenously injected at single doses of 1 mg/kg. Time-points for T1/2 calculation were automatically set by WinNonlin on the basis of the result of the individual time-profile in plasma concentrations. Calculated parameters are T1/2 (terminal phase half life), AUCinf (area under the curve to infinity), CL (clearance), MRT (mean residence time) and Vdss (volume of distribution at steady state).

8 Dose T1/2 AUCinf CL MRT Vdss ug/kg hr ng*hr/ml ml/hr/kg hr ml/kg hsil-6r (-antibody) mean s.d hsil-6r + TCZ mean s.d hsil-6r + PH2 mean s.d Supplementary Table 3. Pharmacokinetic parameters of hsil-6r in normal mice. 50 μg/kg of hsil-6r were intravenously injected either alone, or with TCZ or PH2 at single doses of 1 mg/kg. Time-points for T1/2 calculation were automatically set by WinNonlin on the basis of the result of the individual time-profile in plasma concentrations. Calculated parameters are T1/2 (terminal phase half life), AUCinf (area under the curve to infinity), CL (clearance), MRT (mean residence time) and Vdss (volume of distribution at steady state).

9 Dose T1/2 AUCinf CL MRT Vdss mg/kg day µg*day/ml ml/day/kg day ml/kg TCZ mean s.d TCZ-FcRn mean s.d AM-FcRn mean s.d PH2-FcRn mean s.d Supplementary Table 4. Pharmacokinetic parameters of TCZ, TCZ-FcRn, PH2-FcRn and AM-FcRn in cynomolgus monkey after i.v. injection. TCZ, TCZ-FcRn, PH2-FcRn and AM-FcRn were intravenously injected at single doses of 1 mg/kg. Time-points for T1/2 calculation were automatically set by WinNonlin on the basis of the result of the individual time-profile in plasma concentrations. Time-points for T1/2 calculation were day 7 to day 9 (n = 1) and day 6 to day 8 (n = 2) for TCZ, day 8 to day 10 (n = 1) and day 7 to day 9 (n = 2) for TCZ-FcRn, day 15 to day 20 (n = 3) and day 17 to day 20 (n = 1) for PH2-FcRn, and day 11 to day13 (n = 1), day 7 to day 9 (n = 1), day 8 to day 10 (n = 1) and day 9 to day 11 (n = 1) for AM-FcRn. Calculated parameters are T1/2 (terminal phase half life), AUCinf (area under the curve to infinity), CL (clearance), MRT (mean residence time) and Vdss (volume of distribution at steady state).

10 Dose T1/2 AUCinf CL/F MRT Vz/F mg/kg day µg*day/ml ml/day/kg day ml/kg TCZ mean s.d PH2-FcRn mean s.d Supplementary Table 5. Pharmacokinetic parameters of TCZ and PH2-FcRn in cynomolgus monkey after s.c. injection. TCZ and PH2-FcRn were subcutaneously injected at single doses of 2 mg/kg. Time-points for T1/2 calculation were automatically set by WinNonlin on the basis of the result of the individual time-profile in plasma concentrations. Time-points for T1/2 calculation were day 6 to day 10 (n =2 ) and day 6 to day 12 (n = 1) for TCZ, and day 16 to day 24 (n = 1), day 18 to day 28 (n = 1) and day 6 to day 28 (n = 1) for PH2-FcRn. Calculated parameters are T1/2 (terminal phase half life), AUCinf (area under the curve to infinity), CL/F (total clearance), MRT (mean residence time) and Vz/F (apparent volume of distribution at elimination phase).

11 TCZ PH2-FcRn F rec R total nmol/kg V d L/kg k 12 1/h k 21 1/h k el 1/h Supplementary Table 6. Fitted parameters of TCZ and PH2-FcRn in antigen-mediated clearance and recycling model in cynomolgus monkey. Plasma concentration time profiles of TCZ and PH2-FcRn at various i.v. doses were fitted to an antigen-mediated clearance and recycling model. Fitted values of V d (volume of distribution), k el (elimination rate constant), k 12 (transfer rate constant from central to peripheral compartment), k 21 (transfer rate constant from peripheral to central compartment), R total (total amount of mil-6r) and F rec (fraction recycled from endosome) were obtained.