Article Figures & Data
Figures
- Fig. 1.
Effect of charge (A) and hydrophobicity (B) on Nav1.7 peptide-antibody conjugate pharmacokinetics. A reduction in clearance was observed for peptide-antibody conjugates with a peptide charge of +1 or +2 as compared with a peptide charge of +6, whereas a Pearson correlation (r = 0.8005) was observed between the HIC retention time and in vivo clearance. Mouse data are denoted with closed circles, whereas cynomolgus monkey data are denoted by open circles.
- Fig. 2.
64Cu-NOTA–labeled imaging studies of the unconjugated antibody (aDNP) and three peptide-antibody conjugates. At 3 hours postdose, aDNP and a representative symmetrical divalent conjugate (P2S-aDNP) were confined primarily to the central compartment, while the asymmetric divalent conjugate (P2A-aDNP) and a tetravalent peptide-antibody conjugate (P4S-aDNP) distributed rapidly to the liver. B, bladder; H, heart; %ID/g, percent injected dose per gram; L, liver.
- Fig. 3.
Percentage of peptide-antibody conjugates remaining in the medium after 2.5 hours in plated mouse hepatocyte assays versus in vivo clearance from intravenous pharmacokinetic studies (A) or HIC retention time (B). Peptide conjugates exhibiting extensive association with hepatocytes (lower percentage remaining in media) demonstrated higher in vivo clearance values than those with decreased association (higher percentage remaining in media; Pearson r = −0.5525). A correlation was also observed between HIC retention time and percentage remaining in the hepatocyte media (Pearson r = −0.7185). Mouse data are denoted with closed circles, whereas cynomolgus monkey data are denoted by open circles.
- Fig. 4.
Observed correlation between FcRn binding affinity of peptide conjugates at pH 7.4 or 5.5 (RUs) and in vivo clearance (A and B), HIC retention time (C and D), or percentage remaining in hepatocyte media (E and F). Peptide conjugates with high affinity for FcRn tended to exhibit higher in vivo clearances, HIC retention times, and depletion rates from hepatocyte media (Pearson r values noted in Table 3). Mouse data are denoted with closed circles, whereas cynomolgus monkey data are denoted by open circles.
- Fig. 5.
Depletion of unconjugated antibody (aDNP) or conjugate 4 from media of plated mouse hepatocyte assays. aDNP demonstrated limited uptake into mouse hepatocytes, whereas greater than 60% of conjugate 4 was removed from the media over 2.5 hours. Uptake of conjugate 4 into mouse hepatocytes was attenuated in FcRn knockout hepatocytes or by the addition of acetylated LDL (acLDL; Stab/Scarb1) or ovalbumin (mannose/galactose receptors) to wild-type hepatocytes.
- Fig. 6.
IC50 determination for the inhibition of conjugate 4 association with mouse hepatocytes by acetylated LDL (acLDL; Stab/Scarb1 receptor) or ovalbumin (mannose/galactose receptors). Addition of acetylated LDL to wild-type mouse hepatocytes resulted in complete inhibition of conjugate 4 uptake (IC50 = 0.024 mg/ml), whereas addition of ovalbumin reached a maximum inhibition of 64% of control uptake (IC50 = 1.91 mg/ml).
- Fig. 7.
In vivo pharmacokinetics of aDNP and conjugate 4 in wild-type (WT), FcRn knockout , LDL receptor (LDLr) knockout, mannose receptor knockout, or ASGR1 knockout mouse models following 2-mg/kg intravenous administration. Both aDNP and conjugate 4 exhibited higher in vivo clearance values in FcRn knockout mice as opposed to wild-type controls. Conjugate 4 also exhibited higher in vivo clearance values in mannose receptor knockout mice. No difference was observed between wild-type and LDL receptor or ASGR1 knockout mice.
Tables
Conjugate Ab Core Linkera Peptide Peptide Charge In Vivo Clearance AUCintact/AUCtotal Percentage Remaining in Hep Media FcRn at pH 7.4 FcRn at pH 5.5 HIC Retention Time l/h/kg min aDNP aDNP D88C N/A N/A N/A 0.0006 N/A 93 21.5 0.8 3.90 1 aDNP D88C PEG11 AM-0422 6 0.0205 0.1 57.7 2 aDNP E384C bisPEG23 AM-0422 6 0.079 ND 38 3 aDNP D88CC PEG11 AM-6122 2 0.00175 0.33 74 92.5 25.1 4 aDNP D88C GGS AM-2752 2 0.0044 0.42 47.8 147 30.9 5.18 5 aDNP E384C GGS AM-2752 2 0.0032 0.76 88 47.7 239.2 5.69 6 aDNP D88C GGS AM-5454 1 0.0062 0.51 76 5.43 7 aDNP D88C GGS AM-5759 1 0.0024 0.27 74.9 5.45 8 aDNP D88C GGS AM-5458 1 0.0045 0.61 74.8 5.42 9 aDNP-3A4-F D88C GGS AM-6918 1 0.0037 0.36 72.6 5.61 10 aDNP-3A4-F D88C GGS AM-6262 1 0.0083 0.26 39 228.1 337 6.46 11 3B1HC-3A4LC D88C GGS AM-2752 2 0.0033 0.44 63.8 79.7 93.7 5.71 12 3B1HC-3A4LC D88C GGS AM-2752 2 0.0013 0.4 73.4 73.1 34.6 4.92 13 aDNP-3A4-F D88C GGS AM-8960 1 0.0013 0.24 75.6 5.43 14 aDNP-3A4-F D88C PEG11 AM-8248 1 0.0118 0.28 37.6 6.65 Ab, antibody; bisPEG23, bis-23-unit polyethylene glycol; GGS, glycine-glycine-serine; Hep, %ID/g, percent injected dose per gram; N/A, not applicable; ND, data not collected; PEG11, 11-unit polyethylene glycol.
- TABLE 2
In vivo pharmacokinetic parameters following 2-mg/kg intravenous administration of aDNP or conjugate 4 to wild-type mice and receptor knockout mouse models
Mouse Strain T1/2 AUC0–168 Cl Vss AUCKO/AUCWT ClKO/ClWT h μM *h l/h/kg l/kg aDNP WT 191 11.4 0.0006 0.150 — — FcRn KO 4.98 1.36 0.0092 0.063 0.12 15.3 LDL KO 183 8.45 0.0007 0.199 0.74 1.16 MR KO 240 12.3 0.0005 0.150 1.08 0.83 ASGR1 KO 333 14.2 0.0009 0.142 1.24 1.50 Conjugate 4 (total) WT 164 2.79 0.0027 0.544 — — FcRn KO 1.19 0.096 0.133 0.237 0.03 49.3 LDL KO 118 4.67 0.0028 0.376 1.67 1.04 MR KO 104 1.55 0.0059 0.759 0.56 2.18 ASGR1 KO 197 3.57 0.0036 0.456 1.28 1.33 Conjugate 4 (intact) WT 26.3 0.943 0.0131 0.277 — — FcRn KO 1.27 0.149 0.0892 0.164 0.16 6.81 LDL KO 38.4 1.30 0.0103 0.286 1.38 0.79 MR KO 24.7 1.12 0.0117 0.418 1.19 0.89 ASGR1 KO 19.9 1.32 0.0092 0.174 1.39 0.70 Cl, clearance; KO, knockout; MR, mannose receptor; WT, wild-type.
- TABLE 3
Summary of Pearson correlation results for in vivo clearance (Cl), percentage remaining in hepatocyte media, HIC retention time, and FcRn binding at pH 7.4 or 5.5
x-Axis Data y-Axis Data Pearson r N P Value Significant HIC RT In vivo Cl 0.8005 12 0.0018 Yes Percentage remaining in media In vivo Cl −0.5525 15 0.0327 Yes HIC RT Percent remaining in media −0.7185 15 0.0085 Yes FcRn (pH 7.4) In vivo Cl 0.9107 7 0.0044 Yes FcRn (pH 5.5) In vivo Cl 0.8088 7 0.0276 Yes FcRn (pH 7.4) HIC RT 0.7144 6 0.1129 No FcRn (pH 5.5) HIC RT 0.8449 6 0.0342 Yes FcRn (pH 7.4) Percent remaining in media −0.9455 7 0.0013 Yes FcRn (pH 5.5) Percent remaining in media −0.3902 7 0.3868 No RT, retention time.
Data Supplement
- Supplemental Table -
Surrogate Peptide Sequences and MRM Ion Transitions
- Supplemental Table -
In this issue
PK Characterization of Nav1.7 Peptide-Antibody Conjugates
