Fig 2: RNF43 and ZNRF3 bivalent antibody campaigns facilitate rational design of ligase-based bispecific PROTABs that tether endogenous RNF43 or ZNRF3 to IGF1R.a) SPR analysis of RNF43 or ZNRF3 bivalent antibodies from rabbit and rat antibody campaigns binding to purified human RNF43 or ZNRF3 extracellular domain. Graph depicts KD values of individual campaign antibodies from one high throughput screen and line as the mean value per campaign. b) Schematic representation of HiBiT-LgBiT Nano-luciferase technology. Endogenous IGF1R was N-terminally tagged with HiBiT. Incubation of live cells with LgBiT and substrate allows HiBiT-LgBiT Nano-luciferase reconstitution and substrate catalysis. This can be quantified using luminescence as a proxy for IGF1R cell-surface levels. c) IGF1R cell-surface clearance in HT29 HiBiT-IGF1R knock-in (KI) cells subjected to LgBiT + substrate incubation following 24 h treatment with RNF43*IGF1R bispecific PROTABs. Graph depicts IGF1R clearance (%) from one screening campaign with values from technical repeats overlaid. Assay controls and PROTABs used subsequently are highlighted. d, e) IGF1R cell-surface clearance in HT29 HiBiT-IGF1R KI cells subjected to LgBiT + substrate incubation following treatment with indicated antibodies. Graphs depict IGF1R clearance (%) time kinetics (top) or dose response (bottom) (d) and impact of dosing, recovery and redosing (e) from two independent experiments. Data are presented as non-linear curves (d and e) and mean (e) with values from biological repeats overlaid. f, g) Western blot lysate analysis from SW1417 (f) or RKO (g) cells left untreated (-) or subjected to indicated antibodies for 48 h. Endogenous IGF1R-ß was detected. a and ß-TUBULIN were used as loading controls. Data are representative of three independent experiments. h) Flow cytometry histograms of PE signal in HT29 wildtype (WT), RNF43 Knock Out (KO) or ZNRF3 KO cells stained with an antibody against RNF43, ZNRF3 or the matched isotype control from two independent experiments. i) Western blot lysate analysis from HT29 WT, RNF43 KO or ZNRF3 KO cells that were left untreated (-) or subjected to indicated antibodies for 24 h. Endogenous IGF1R-ß protein was detected. ß-TUBULIN was used as a loading control. Data are representative of two independent experiments. j) Western blot lysate analysis from SW48 xenograft in vivo tumours derived from mice left untreated (-) or subjected to indicated antibodies for 72 h. Endogenous IGF1R-ß was detected. GAPDH was used as a loading control. N = 4 animals per group. k) Mice bodyweight following treatment with indicated antibodies for 7 days. Graph depicts changes of bodyweight (% of day 0) over time from four animals per group. Data are presented as mean ± s.e.m. For gel source data, see Supplementary Fig. 2. Source data

Fig 3: Dimerization of IGF1R to the Wnt-responsive E3 ubiquitin ligases RNF43 and ZNRF3 leads to IGF1R degradation.a) Comparative gene set enrichment analysis of wild type (WT) and APC truncation mutant (Apc-/-) colon organoids highlighting MSigDB C2 Wnt pathway gene sets from three independent experiments. b) RNA expression profile of indicated E3 ubiquitin ligases with predicted transmembrane domains in healthy control and colon adenomas. Box plots represent data distribution (minimum, first quartile, median, third quartile and maximum) with individual gene probes overlayed from 8 independent healthy control and 15 colon adenoma samples. c) Heatmap depicting RNF43 and ZNRF3 TCGA RNA expression profile in human tumors compared to their respective normal tissues. d) Schematic representation of iDimerize technology utilized to chemically induce the interaction of C-terminally DmrA-HA-tagged RNF43 or ZNRF3 with C-terminally DmrC-FLAG-tagged IGF1R following treatment with the A/C heterodimerizer. e) Western blot lysate analysis from HEK293T cells co-expressing C-terminally DmrA-HA-tagged ZNRF3 and C-terminally DmrC-FLAG-tagged IGF1R subjected to HA immunoprecipitation (IP) following no treatment (-) or 24 h treatment with indicated A/C heterodimerizer concentrations. Precipitated and total DmrA-HA-tagged ZNRF3 and DmrC-FLAG-tagged IGF1R were detected. a-TUBULIN was used as a loading control. Data are representative of three independent experiments. f) Summary of Surface Plasmon Resonance (SPR) analysis of IGF1R bivalent antibodies binding to purified human IGF1R. Graph depicts bivalent antibody affinity to human IGF1R detected using Biacore T200 at 37 °C in HBSEP buffer with representative sensorgrams from three independent experiments. Data are presented as mean ± s.e.m. with values from biological repeats overlaid. For Gani one outlier data point was excluded. g) Epitope binning analysis of IGF1R bivalent antibodies binding to purified human IGF1R. For (f and g) IGF1R bivalent antibodies were tested (Cixu = Cixutumumab; Figi = Figitumumab; Gani = Ganitumab; h10H5.V1; Istira = Istiratumab; Roba = Robatumumab; Tepro = Teprotumumab). For gel source data, see Supplementary Fig. 2. Source data

Fig 4: Multiple cell-surface E3 ubiquitin ligases can degrade plasma membrane targets.a,b, Levels of HER2 (a) and PD-L1 (b) in SW48 xenografts 48 h after intraperitoneal administration with indicated antibodies. GAPDH was used as a loading control. n = 4 mice per group. c, Frequency of detectable cell-surface gD epitopes in HT29 cells expressing indicated gD–ligase–Flag constructs versus parental cells. Graph depicts gD-positive cells from three independent experiments. Data are mean ± s.e.m. with values from biological repeats overlaid. d, Levels of IGF1Rß in HT29 cells expressing indicated gD–ligase–Flag constructs following treatment with specified antibodies for 24 h. Data are representative of three independent experiments. e, Levels of cell-surface FZD5 in ASPC1 cells expressing indicated gD–ligase–Flag constructs following antibody treatment at 1 or 10 µg ml-1 for 24 h. Data are presented as lines connecting mean values from two independent experiments with values from biological repeats overlaid. f, Schematic representation of PROTAB antibody engineering and the effect on downstream degradation. g–i, Levels of cell-surface IGF1R and RNF43 assessed by flow cytometry in HT29 cells expressing gD–RNF43–Flag following treatment with various antibody formats for 24 h: 2 (target) + 1 (ligase) (g), 2 (ligase) + 1 (target) (h), or one-arm Fv–IgG (i). The 2 (target) + 1 (ligase) and 2 (ligase) + 1 (target) antibodies are referred to as ‘2+1 Fab–IgGs’. Graphs depict IGF1R or RNF43 clearance from three independent experiments. Data are mean ± s.e.m. For g–i, samples were processed simultaneously using Cixu*NIST and Cixu*RNF43-37.39 as common controls for two (g,i) or three (g,h) biological repeats. j, Levels of IGF1R in DLD1 cells treated with indicated antibodies for 24 h. a-Tubulin was used as a loading control. Data are representative of two independent experiments. For gel source data and molecular masses of gD–ligase–Flag proteins in d, see Supplementary Fig. 1.Source data

Fig 5: The Wnt-responsive E3 ubiquitin ligases RNF43 and ZNRF3 can degrade IGF1R.a, Left, CRISPR–Cas9 strategy used to generate APC mutant organoids. Right, phase-contrast images of wild-type (WT) and APC truncation mutant (Apc-/-) colon organoids. Data are representative of two independent experiments. Scale bars, 250 µm. b, Gene expression analysis of wild-type and Apc-/- colon organoids. Genes encoding proteins with predicted transmembrane domains (TM) (grey), E3 ubiquitin (Ub) ligases (light blue) or both (dark blue) are shown. Data are the mean expression from three independent experiments. FC, fold change; FDR, false discovery rate. c, In situ hybridization in AKPS colon organoids probing for Rnf43 and Znrf3. Data are representative of two independent experiments. Scale bars, 50 µm. d, Schematic representation of the iDimerize construct. HA, haemagglutinin tag. e, Levels of total and immunoprecipitated IGF1R and RNF43 following HA tag immunoprecipitation (IP) following treatment of HEK293T cells harbouring the RNF43–IGF1R iDimerize construct with the A/C heterodimerizer. f, Schematic representation of gD-based PROTAB-mediated IGF1R degradation. g, Levels of cell-surface IGF1R assessed by flow cytometry following treatment of HT29 cells expressing gD–RNF43–Flag or gD–ZNRF3–Flag with 10 µg ml-1 of the indicated gD-based PROTABs for 48 h. Graphs depict IGF1R clearance from four independent experiments. Data are mean ± s.e.m., with values from individual biological repeats overlaid. h,i, Levels of IGF1Rß following treatment of parental HT29 cells or cells expressing gD–RNF43–Flag (h) or gD–ZNRF3–Flag (i) with 0.5 µg ml-1 of the indicated PROTABs for 24 h. In e,h,i, a-tubulin was used as a loading control. Data are representative of three independent experiments. IGF1R-binding antibodies: Cixu, cixutumumab; Figi, figitumumab; Gani, ganitumab; h10H5.V1; Istira, istiratumab; Roba, robatumumab; Tepro, teprotumumab. For gel source data, see Supplementary Fig. 1.Source data