Rapid depletion of "catch-and-release" anti-ASGR1 antibody in vivo

Abstract

Targeting antigens with antibodies exhibiting pH/Ca²⁺-dependent binding against an antigen is an attractive strategy to mitigate target-mediated disposition and antigen buffering. Studies have reported improved serum exposure of antibodies exhibiting pH/Ca²⁺-binding against membrane-bound receptors. Asialoglycoprotein receptor 1 (ASGR1) is a membrane-bound receptor primarily localized in hepatocytes. With a high expression level of approximately one million receptors per cell, high turnover, and rapid recycling, targeting this receptor with a conventional antibody is a challenge. In this study, we identified an antibody exhibiting pH/Ca²⁺-dependent binding to ASGR1 and generated antibody variants with increased binding to neonatal crystallizable fragment receptor (FcRn). Serum exposures of the generated anti-ASGR1 antibodies were analyzed in transgenic mice expressing human FcRn. Contrary to published reports of increased serum exposure of pH/Ca²⁺-dependent antibodies, the pH/Ca²⁺-dependent anti-ASGR1 antibody had rapid serum clearance in comparison to a conventional anti-ASGR1 antibody. We conducted sub-cellular trafficking studies of the anti-ASGR1 antibodies along with receptor quantification analysis for mechanistic understanding of the rapid serum clearance of pH/Ca²⁺-dependent anti-ASGR1 antibody. The findings from our study provide valuable insights in identifying the antigens, especially membrane bound, that may benefit from targeting with pH/Ca²⁺-dependent antibodies to obtain increased serum exposure.

Keywords: Antigen-antibody trafficking; TMDD; fluorescence microscopy; pH/Ca2+-dependent antibodies; pharmacokinetics.

Figure 1.

Differential clearance of non-CAR and CAR anti-ASGR1 antibodies in human FcRn Tg mice. (a) mice were intravenously administered with antibodies and bled at indicated time points to measure antibody concentrations over time. Individual measurements were marked by (▼) for 0.3 mg/kg, (▲) 3 mg/kg, (■) 10 mg/kg, and (●) 30 mg/kg. (b) the inset presents a zoom in of the non-CAR and CAR antibody data from 0 to 24 hours.

Figure 2.

Moderate increase in serum exposure of anti-ASGR1 antibodies incorporating YTE mutations in comparison to WT variant antibodies in human FcRn Tg mice. (a) Mice were intravenously administered with antibodies and bled at indicated time points to measure antibody concentrations over time. Individual measurements for CAR and non-CAR antibodies were marked by (●) and (■) for WT and YTE, respectively. Non-CAR-IHH antibody concentrations over time are marked by (▲). (b) Serum exposure of antibodies AUC_last was plotted against the administered dose. (c) Fold increase in serum exposure [mean AUC_{(CAR-antibody)} /mean AUC_{(non-CAR antibody)}] for WT and YTE antibodies by introducing catch-and-release binding to ASGR1 (left). Fold increase in serum exposure [mean AUC_{(antibody-YTE)} /mean AUC_{(antibody-WT)}] for non-CAR antibody and CAR antibody by introducing YTE mutations (right).

Figure 3.

Sub-cellular localization of human ASGR1 and its colocalization with early and late endosomal markers. HepG2 cells were fixed with paraformaldehyde and permeabilized prior to staining with anti-ASGR1 antibody labeled with alexa-647, anti-Rab11 antibody labeled with alexa-568, and anti-LAMP1 antibody labeled with alexa-488. Stained cells were imaged and pseudo-colored red (alexa-647) or green (alexa-488 or alexa-568). An image of a representative field of view of cells is shown. Fluorescence intensities along the lines in the overlays are shown in the fluorescence intensity plot. The scale bars represent 20 µm.

Figure 4.

Internalized anti-ASGR1 antibodies colocalize with intracellular FcRn. AML12 cells were co-pulsed with 10 µg/mL of alexa-568 labeled FcRn marker (Abdeg) and alexa-647 labeled CAR and non-CAR anti-ASGR1 antibodies at 37°C, followed by wash and fix with paraformaldehyde. Fixed cells were imaged, and a representative field of view is presented. Alexa fluorophores 568, 647 and hoescht were pseudo-colored green, red, and blue, respectively. Fluorescence intensity along the dotted line over an endosomal compartment is presented in the fluorescence intensity plot. The scale bar for the panel of cells is 20 µm.

Figure 5.

Anti-ASGR1 antibodies localize to lysosomal compartments at the end of the chase phase. AML12 cells were pre-pulsed with alexa-568 labeled dextran for four hours and chased overnight at 37°C. Following the dextran pulse-chase, alexa-647 labeled CAR and non-CAR anti-ASGR1 antibodies were pulsed for 30 minutes and chased for four hours at 37°C. Chased cells were washed and fixed with paraformaldehyde and imaged. Alexa-647 labeled antibodies and alexa-568 labeled dextran are presented in pseudo-colored red and green, respectively. Fluorescence intensity along the dotted line is presented in the fluorescence intensity plot. The scale bars for the panel of cells is 20 µm.

Figure 6.

Treatment of HepG2 cells with CAR and non-CAR’ anti-ASGR1 antibodies decreases ASGR1 levels. HepG2 cells were pulsed with varying concentrations (1, 10 and 100 µg/mL) of CAR antibody and non-CAR’ antibody (an antibody binding to human ASGR1 with no pH/Ca-sensitivity) for 24, 48 and 72 hours. Treated cells were lysed and analyzed for ASGR1 levels through western blot.