Biolayer interferometry (BLI) profiles of antigen CRDs to antibodies.

(A) Binding of hASGR1 to 8M24-IgG1; (B) Non-binding of mASGR1, hASGR2, and mASGR2 to 8M24-IgG1; (C) Binding of hASGR1 to 8G8-IgG1; (D) Binding of hASGR2 to 8G8-IgG1; (E) Binding of mASGR1 to 8G8-IgG1; and (F) Binding of mASGR2 to 8G8-IgG1. Binding profiles for hASGR1, hASGR2, mASGR1, and mASGR2 are shown in brown, magenta, orange, and red traces, respectively.

Kinetics parameters of binding of ASGR1 and ASGR2 to 8M24 and 8G8 antibodies.

Structure of hASGR1:8M24 complex.

(A) A cartoon representation of overall structure of hASGR1CRD:8M24-Fab complex. The heavy- and light- chains of 8M24 are colored orange and olive, respectively, with their CDR loops marked as H1, H2, H3, L1, L2, and L3. hASGR1CRD traced in blue to red from the N- to C-terminus. Three calcium ions are represented as magenta spheres. A glycerol molecule bound to the calcium ion at the ASGR substrate binding site is highlighted in the stick representation. (B) Epitope of 8M24 highlighted on the surface of hASGR1. Antigen residues that are within 4.5 Å from 8M24 heavy- and light- chains are shown in orange and olive colors, respectively. (C) Residues of hASGR1 involved in polar interactions with the 8M24 antibody of shown as sticks on the secondary structure elements (cyan traces) with most of the residues situated on the helix ∝1 of hASGR1. (D). A close-up view of polar interactions between 8M24 and hASGR1 near the N-terminus of helix ∝1. (E) A close-up view of polar interactions between 8M24 and hASGR1 near the C-terminus of helix ∝1.

Crystallography structure determination statistics.

Comparison of hASGR1:8M24 and hASGR2:8G8 complex structures.

(A). An alignment, made with MultAlin (Corpet, 1988) and ESPript (Gouet et al., 2003) of human, mouse, and rat ASGR1 and ASGR2 sequences with the secondary structure elements of hASGR1CRD (PDB Code: 1DV8, Meier et al., 2000) depicted at the top and labeled according to the color scheme in Fig. 1A. Paratope residues from the heavy- and light- chains of 8M24 are marked with orange and olive circles, respectively. Paratope residues from the heavy- and light- chains of 8G8 are marked with purple and teal circles, respectively. Important residues are highlighted with a magenta downward arrow. hASGR2 residue Ser172 is marked for reference to the sequence numbering. (B). A dot representation showing snug-fit of hASGR1 Asn180 of the cavity formed by Phe91-Trp92-Gly93 (LCDR3 loop) and Asn32 (LCDR1 loop) of 8M24. hASGR1 Asn180 is replaced by either Lys or Gln h/m/rASGR2 and m/rASGR1, respectively. (C). Superimposed structures of hASGR1:8M24 and hASGR2:8G8 complexes illustrating that the interaction surfaces of antibodies are situated on the opposite surfaces of the antigens are non-overlapping. The overall folds of hASGR1 and hASGR2 are shown in cyan and green, respectively, with their N- and C- terminus highlighted as blue and red spheres. Three calcium ions are represented as magenta spheres. A glycerol molecule bound to the calcium ion at the ASGR substrate binding site is highlighted in the stick representation.

Structure of hASGR2:8G8 complex.

(A) A cartoon representation of overall structure of hASGR2CRD:8G8-Fab complex. The heavy- and light- chains of 8G8 are colored in purple and teal, respectively, with their CDR loops marked as H1, H2, H3, L1, L2, and L3. hASGR2CRD traced in blue to red from the N- to C-terminus. Three calcium ions are represented as magenta spheres. A glycerol molecule bound to the calcium ion at the ASGR substrate binding site is highlighted in the stick representation. (B) Epitope of 8G8 highlighted on the surface of hASGR2. Antigen residues that are within 4.5 Å from 8G8 heavy- and light- chains are shown in purple and teal colors, respectively. (C) Residues of hASGR2 involved in polar interactions with the 8G8 antibody of shown as sticks on the secondary structure elements (green traces) with most of the residues situated on the helix ∝2 of hASGR2. (D). A close-up view of polar interactions between 8M24 and hASGR2 near the N-terminus of helix ∝2. (E) A close-up view of polar interactions between 8M24 and hASGR2 near the C-terminus of helix ∝2.

Both 8M24 and 8G8 RSPO2RA SWEETS molecules enhance Wnt signal:

(A) Diagrams of the SWEETS molecules. RSPO2RA is fused at the N-terminus of the heavy chain of IgG. (B) Both 8M24 and 8G8 RSPO2RA SWEETS molecules enhance WNT signaling in HuH-7 STF cells which has the WNT response reporter. (C, D) Both 8M24 and 8G8 RSPO2RA SWEETS molecules also enhance WNT signaling in ASGR1 overexpressed HEK293 STF cells which has the WNT response reporter, but not in parental HEK293 cells without ASGR1 overexpression. Data are representative of three independent experiments performed in triplicates and are shown as mean ± standard deviation (SD).

SWEETS induce degradation of ASGR1 in HuH-7 cells.

(A) Dose-dependent ASGR1 degradation promoted by different concentrations of 4F3-, 8M24-, and 8G8-RSPO2RA SWEETS. (B) Time course of ASGR1 degradation treated with 10nM of 4F3-, 8M24-, and 8G8-RSPO2RA for 24h. (C) Western blot analysis demonstrating the efficacy of ASGR1 degradation in cells treated with 4F3-, 8M24-, and 8G8-RSPO2RA SWEETS compared to ASGR1 antibodies lacking the RSPO2RA domain. (D) Western blot data showing total protein levels of ASGR1, Ubiquitin, and LC3B in HuH-7 cells pre-treated with DMSO, lysosomal pathway inhibitor bafilomycin A1 (Baf.A1), proteasome inhibitor MG132, and E1 ubiquitin ligase inhibitor TAK-243 to test which degradation pathways govern ASGR1 degradation by SWEETS. Data in (A-C) are representatives of three independent experiments, while data in (D) are representatives of two independent experiments. For (A-B), total ASGR1 levels were normalized to generate graphs representing the mean of those three experiments. In (C), data are represented as mean ± s.e.m. of normalized total ASGR1 levels and one-way ANOVA with Tukey’s post hoc test was used for statistical analysis. *p<0.05, **p<0.01.

8M24 is specific to human ASGR1 and 8G8 binds to both ASGR1 and ASGR2 with mouse cross-reactivity:

(A) Interaction of 8M24-Fab with human and mouse ASGR1 and ASGR2 CRDs. Left-shifted peak, corresponding to complex formation, is observed only with hASGR1-CRD, revealing complex formation. (B) Interaction of 8G8-Fab with human and mouse ASGR1 and ASGR2 CRDs. Left-shifted peak, corresponding to complex formation, is observed for four proteins human and mouse ASGR1 and ASGR2 CBDs. Elution time is shown in the x-axis and the absorption unit in the y-axis are not to the actual scale. Samples were analyzed on an Agilent AdvanceBio SEC 300Å, 2.7 μm, 7.8 x 300 mm column, equilibrated with 20 mM HEPES pH 7.5, 150 mM sodium chloride, mounted on a Vanquish (Thermoscientific, USA) UHPLC system.

ASGR1 degradation promoted by SWEETS was determined in an additional hepatocyte cell line, HepG2 cells.

(A) ASGR1 degradation induced by different concentrations of 4F3-, 8M24-, and 8G8-RSPO2RA showing dose-response curve of SWEETS. (B) Time course of ASGR1 degradation treated with 10nM of 4F3-, 8M24-, and 8G8-RSPO2RA for 24h. (C) Western blot analysis showing the comparisons of ASGR1 degradation induced by SWEETS and ASGR1 antibody. (D) Total protein levels of ASGR1, Ubiquitin, and LC3B in HepG2 cells pre-incubated with DMSO, lysosomal pathway inhibitor bafilomycin A1 (Baf.A1), proteasome inhibitor MG132, and E1 ubiquitin ligase inhibitor TAK-243 to determine the degradation pathway for SWEETS-mediated ASGR1 degradation. The Western blot data in (A-C) are representatives of three independent experiments, whereas data in (D) are representatives of two independent experiments. For (A-B), total ASGR1 levels were normalized by total loading control protein levels (Vinculin) to generate graphs representing the mean of those three experiments. For (C), data are represented as mean ± s.e.m. of normalized total ASGR1 levels and one-way ANOVA with Tukey’s post hoc test was used for statistical analysis. **p<0.01, ***p<0.001.