Journal: Molecular Cancer Therapeutics

Article Title: AMT-562, a Novel HER3-targeting Antibody–Drug Conjugate, Demonstrates a Potential to Broaden Therapeutic Opportunities for HER3-expressing Tumors

doi: 10.1158/1535-7163.MCT-23-0198

Figure Lengend Snippet: Generation and characterization of Ab562, a novel HER3-targeting antibody. A, Ab562 binds to an epitope located in ECD1 determined by non–cross-reactive mouse HER3 domain swaps. Binding sites of other HER3-targeting mAbs: MM-121 or lumretuzumab (domain I) prevents NRG1 binding, LJM716 binds an epitope created by ECD domains II and IV in the HER3 closed conformation and patritumab binds the extracellular domain II. Modified from Kinisha Gala; Sarat Chandarlapaty. Molecular Pathways: HER3 Targeted Therapy. Clin Cancer Res 20, 1410–1416 (2014). B, Ab562 binding to HER3 in the absence (top) or presence (bottom) of NRG1 measured by BLI analysis. C, Target overexpression (colocalization of red mAb staining and green GFP-tagged target in 293T). Negative staining on the untransfected cells (blue only) is indicated by two red arrows. Scale bars, 10 μm. D, Ab562 caused less cell surface HER3 expression decrease in PC9 measured by flow cytometry. Change of HER3-positive cell percentage is tabulated on the right. Data shown are mean of triplicate measurements and error bars are SEM. Unpaired two-sided t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. E, Effect of Ab562 on HER3 signaling. Western blot analysis for the phosphorylation of HER3, AKT, ERK1/2. Cells were incubated with 30 nmol/L patritumab or Ab562 with or without 1 ng/mL neuregulin-1 (NRG-1) for 1 hour. Western blot results from two ADCs, AMT-562 and P-GGFG-DXd, were also shown here (described in the section “ In vivo functional validation of P-GGFG-DXd/U3-1402 and comparison with AMT-562”). F, In vitro proliferation experiments of Ab562 and patritumab. Cancer cell lines with HER3 and NRG1 expression data (labeled) treated with 100 μg/mL anti-HER3 antibodies for 5 days with cell viability determined by CTG assay. Cell proliferation values are relative to untreated cells and represent average of three replicates ± SEM. Unpaired two-sided t test. *, P < 0.05; **, P < 0.01. G, In vivo efficacy of Ab562 (blue) and patritumab in a breast cancer cell line xenograft model. High HER3 expression in BT-474 was shown as flow cytometry data on the left. Mice were intraperitoneally administered with antibodies (20 mg/kg) and on day 0 (tumor size reached an average of 150–200 mm 3 ) and subsequent dates indicated by red arrows. Each value represents the mean and SEM ( n = 5). Unpaired two-sided t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: The membranes were blocked and probed overnight with anti-phospho-HER3 antibody (#4791, Cell Signaling Technology), anti-HER3 antibody (#12708, Cell Signaling Technology), anti-phospho-AKT antibody (T40067, Abmart), anti-AKT antibody (T55561, Abmart), anti-phospho-ERK1/2 antibody (#4370, Cell Signaling Technology), anti-ERK1/2 antibody (#4695, Cell Signaling Technology), anti-β-tubulin antibody (R20005, Abmart) at 4°C.

Techniques: Binding Assay, Modification, Over Expression, Staining, Negative Staining, Expressing, Flow Cytometry, Western Blot, Phospho-proteomics, Incubation, In Vivo, Functional Assay, Biomarker Discovery, Comparison, In Vitro, Labeling, CTG Assay

Journal: Molecular Cancer Therapeutics

Article Title: AMT-562, a Novel HER3-targeting Antibody–Drug Conjugate, Demonstrates a Potential to Broaden Therapeutic Opportunities for HER3-expressing Tumors

doi: 10.1158/1535-7163.MCT-23-0198

Figure Lengend Snippet: Structure, stability, and cellular dynamics of AMT-562. A, HER3 and HER2 expression level on cancer cell surface measured by mAb562 and anti-HER2 mouse mAb (ab264541, Abcam). Expression was defined by number of HER3 and HER2 molecules per cell measured by flow cytometry (QIFIKIT). HER3 and HER2 expression on the same cell line was shown for comparison (connected by dotted lines). Expression level was classified as low, medium, and high by the number of target molecules per cell: 0–10,000 as low, 10,000–100,000 as medium, and 100,000–1 million as high. The average copy number of HER3 was 2,900 versus 120,000 for HER2. B, Structure of AMT-562. Exatecan was attached to T moiety, a hydrophilically modified self-immolative (indicated by a red scissor) pAB spacer. T moiety–exatecan was then linked to Ab562 through MC-VA linker. Exatecan is released as payload. T1000 with a pSAR or T800 with a methylaminomethyl group modification (rectangle box) was shown. C, In vivo efficacy of AMT-562, Ab562-T1000-exatecan, and P-GGFG-DXd in PC9 xenograft model. D, Representative hematoxylin and eosin staining of toxicity organs of AMT-562 and Ab562-T1000-exatecan. Scale bar, 200 μm. E, ADC half-life of AMT-562 and Ab562-T1000-exatecan from repeated dose toxicity studies in cynomolgus monkeys. F, Exatecan release of AMT-562 and Ab562-T1000-exatecan from repeated dose toxicity studies in cynomolgus monkeys. G, In vivo efficacy of orthogonal combinations of antibody and linker-payload pairs. ADCs were tested in an in vivo xenograft model of SW620. Four doses of 10 mg/kg ADCs were intravenously administered on day 0 (tumor size reached an average of 150–200 mm 3 ) and days indicated by red arrows. Each value represents the mean and SEM ( n = 5). Unpaired two-sided t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. H, Internalization rate of AMT-562, P-GGFG-DXd, and P-T800-exatecan on cancer cell lines at 8 hours after ADC binding measured by flow cytometry. I, Correlation of exatecan release and target expression. HER3 expression on each cell line is determined by flow cytometry and exatecan concentration in the culture media at 24 hours after treatment with 100 nmol/L AMT-562 was determined by LC/MS-MS ( n = 3).

Article Snippet: The membranes were blocked and probed overnight with anti-phospho-HER3 antibody (#4791, Cell Signaling Technology), anti-HER3 antibody (#12708, Cell Signaling Technology), anti-phospho-AKT antibody (T40067, Abmart), anti-AKT antibody (T55561, Abmart), anti-phospho-ERK1/2 antibody (#4370, Cell Signaling Technology), anti-ERK1/2 antibody (#4695, Cell Signaling Technology), anti-β-tubulin antibody (R20005, Abmart) at 4°C.

Techniques: Expressing, Flow Cytometry, Comparison, Modification, In Vivo, Staining, Binding Assay, Concentration Assay, Liquid Chromatography with Mass Spectroscopy

Journal: Molecular Cancer Therapeutics

Article Title: AMT-562, a Novel HER3-targeting Antibody–Drug Conjugate, Demonstrates a Potential to Broaden Therapeutic Opportunities for HER3-expressing Tumors

doi: 10.1158/1535-7163.MCT-23-0198

Figure Lengend Snippet: Pharmacodynamics of AMT-562 in vivo and in organoids. A, Bystander killing effect of AMT-562 and P-GGFG-DXd in coculture conditions in vitro . PC9GR and COLO320DM cells were cocultured and treated with 50 nmol/L ADCs for 5 days. After collecting adherent cells, cell number and ratio of HER3‐positive and HER3-negative cells were determined by a cell counter and a flow cytometer, respectively. Each bar represents the mean and SD ( n = 3). B, In vivo efficacy of AMT-562 and P-GGFG-DXd in COLO205 xenograft model. CDX mice were intravenously administered with indicated ADCs and vehicle control (10 mg/kg) on day 0 (tumor size reached an average of 150–200 mm 3 ) indicated by red arrows. Each value represents the mean and SEM ( n = 2). C and D, γH2AX foci induction in the HER3+ COLO205 tumor model by IHC analysis. Tumors were collected at the indicated timepoints and FFPE for IHC analysis. γH2AX IHC antibody (#9718, Cell Signaling Technology) was used for detection. Representative images were shown for indicated days. Positive γH2AX foci (black spots) were indicated by red arrows. Scale bar, 20 μm ( C ). Time course of γH2AX H-score. Each value represented the mean (2 mice, two measurements each; D ). E and F, Cell death induction in COLO205 xenograft model by IHC analysis of Cleaved Caspase-3. Cleaved Caspase-3 IHC antibody (#9579, Cell Signaling Technology) was used for detection. Representative images were shown for indicated days. Positive signals (black spots) were indicated by red arrows. Scale bar, 20 μm ( E ). Time course of Cleaved Caspase-3 H-score. Each value represented the mean (2 mice, two measurements each; F ). G, Colon cancer PDOs (CO117&CO15) response to AMT-562 and P-GGFG-DXd. Representative images of brightfield (left) and live/dead cells (right) for CO117 was shown. Colon cancer PDOs were treated with AMT-562 or P-GGFG-DXd at a serial concentration for 6 days. Live cells were stained by Calcein AM (green) and dead cells by PI (red). Scale bar, 200 μm. H and I, Organoid size at day 0 and day 6 of ADCs treatment. Surviving organoid size was measured. Data shown are means ± SEM from two independent measurements.

Article Snippet: The membranes were blocked and probed overnight with anti-phospho-HER3 antibody (#4791, Cell Signaling Technology), anti-HER3 antibody (#12708, Cell Signaling Technology), anti-phospho-AKT antibody (T40067, Abmart), anti-AKT antibody (T55561, Abmart), anti-phospho-ERK1/2 antibody (#4370, Cell Signaling Technology), anti-ERK1/2 antibody (#4695, Cell Signaling Technology), anti-β-tubulin antibody (R20005, Abmart) at 4°C.

Techniques: Drug discovery, In Vivo, In Vitro, Flow Cytometry, Control, Concentration Assay, Staining

Journal: Molecular Cancer Therapeutics

Article Title: AMT-562, a Novel HER3-targeting Antibody–Drug Conjugate, Demonstrates a Potential to Broaden Therapeutic Opportunities for HER3-expressing Tumors

doi: 10.1158/1535-7163.MCT-23-0198

Figure Lengend Snippet: In vivo antitumor efficacy of AMT-562 in pancreatic, esophagus and gastric cancer. A–E, Information of CDX/PDX models of pancreatic ( A ) or esophagus ( E ) cancer. Shown is mutation status of TP53/KRAS/BRAF . HER3 expression level is indicated on the basis of IHC score or flow cytometry. Subtype of esophagus cancer was indicated. B–D, In vivo efficacy of AMT-562 and P-GGFG-DXd and control ADCs in pancreatic cancer models. F–H, In vivo efficacy of AMT-562 and P-GGFG-DXd and control ADCs in esophagus cancer models. For all in vivo models, target and cell line/PDX tumor type is labeled. Target expression in each model is shown as flow cytometry (rMFI) and/or IHC image on untreated mouse tumor tissue and H-score. Scale bars, 20 μm. CDX/PDX mice were intravenously administered with indicated ADCs (10 mg/kg unless otherwise labeled) and on day 0 (tumor size reached an average of 150–200 mm 3 ) and subsequent dates indicated by red arrows. Each value represents the mean and SEM ( n = 4 or 5). Unpaired two-sided t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. I, A summary of tumor volume change of AMT-562 versus P-GGFG-DXd in pancreatic, esophagus, and gastric cancer models.

Article Snippet: The membranes were blocked and probed overnight with anti-phospho-HER3 antibody (#4791, Cell Signaling Technology), anti-HER3 antibody (#12708, Cell Signaling Technology), anti-phospho-AKT antibody (T40067, Abmart), anti-AKT antibody (T55561, Abmart), anti-phospho-ERK1/2 antibody (#4370, Cell Signaling Technology), anti-ERK1/2 antibody (#4695, Cell Signaling Technology), anti-β-tubulin antibody (R20005, Abmart) at 4°C.

Techniques: In Vivo, Mutagenesis, Expressing, Flow Cytometry, Control, Labeling

Journal: Molecular Cancer Therapeutics

Article Title: AMT-562, a Novel HER3-targeting Antibody–Drug Conjugate, Demonstrates a Potential to Broaden Therapeutic Opportunities for HER3-expressing Tumors

doi: 10.1158/1535-7163.MCT-23-0198

Figure Lengend Snippet: AMT-562 is more effective as single agent or in combination therapy for colorectal cancer. A–D, Single ADC agent efficacy in PDX models. A PDX with liver metastasis of a relatively high HER3 expression ( A ). A low HER3 expression PDX ( B ). A tumor resistant to Rabusertib (CHEK1 inhibitor; C ). A high HER3 expression PDX ( D ). A summary of tumor volume change of AMT-562 versus P-GGFG-DXd in colorectal cancer models ( E ) and information of CDX/PDX models of colorectal cancer ( F ). Shown is mutation status of TP53/KRAS/BRAF and other amplifications. HER3 expression level is indicated on the basis of IHC score. G–I, Combination therapy of ADC with VEGF/EGFR/CHEK1 inhibitor, respectively. In vivo efficacy of single-agent AMT-562, P-GGFG-DXd and control ADCs or in combination treatment of colon cancer models. Target and cell line/PDX tumor type is labeled. Target expression in each model is shown as flow cytometry (rMFI) and/or IHC image on untreated mouse tumor tissue and H-score. Scale bars, 20 μm. CDX/PDX mice were intravenously administered with indicated ADCs (10 mg/kg unless otherwise labeled) and on day 0 (tumor size reached an average of 150–200 mm 3 ) and subsequent dates indicated by red arrows. Each value represents the mean and SEM ( n = 4 or 5). Unpaired two-sided t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. For combination treatments, bevacizumab was dosed at 5 mg/kg, i.p., once a week for 3 weeks. Cetuximab was dosed at 1 mg/animal, i.p., once a week for 3 weeks. Rabusertib was dosed at 100 mg/kg, orally, every day for 21 days.

Article Snippet: The membranes were blocked and probed overnight with anti-phospho-HER3 antibody (#4791, Cell Signaling Technology), anti-HER3 antibody (#12708, Cell Signaling Technology), anti-phospho-AKT antibody (T40067, Abmart), anti-AKT antibody (T55561, Abmart), anti-phospho-ERK1/2 antibody (#4370, Cell Signaling Technology), anti-ERK1/2 antibody (#4695, Cell Signaling Technology), anti-β-tubulin antibody (R20005, Abmart) at 4°C.

Techniques: Expressing, Mutagenesis, In Vivo, Control, Labeling, Flow Cytometry

Journal: Molecular Cancer Therapeutics

Article Title: AMT-562, a Novel HER3-targeting Antibody–Drug Conjugate, Demonstrates a Potential to Broaden Therapeutic Opportunities for HER3-expressing Tumors

doi: 10.1158/1535-7163.MCT-23-0198

Figure Lengend Snippet: AMT-562 targets lung cancer resistance alone or in combination therapy. A–E, Single ADC agent efficacy in PDX models. PDXs resistant to third-generation EGFR TKI AZD9291( A – D ). PDX with EGFR WT genotype ( E ). F, Information of CDX/PDX models of NSCLC and tumor volume change of AMT-562 versus P-GGFG-DXd in NSCLC models. Shown is mutation status of EGFR, other mutations/amplifications. HER3 expression level is indicated on the basis of IHC score or flow cytometry. G, Combination treatment of lowered dose of ADCs and EGFR TKI AZD9291. The efficacy of 10 mg/kg of ADCs in this model was shown in C . H, Combination treatment of ADCs and AZD9291 in an AMT-562–sensitive but P-GGFG-DXd–resistant PDX model. See B . I, In vivo efficacy of HER3-targeting ADCs alone or in combination with KRAS G12C inhibitor AMG510 (Sotorasib) in a NSCLC PDX with KRAS G12C mutation. Target and cell line/PDX tumor type is labeled. Target expression in each model is shown as flow cytometry (rMFI), gene expression copy number and/or IHC image on untreated mouse tumor tissue and H-score. Scale bars, 20 μm. CDX/PDX mice were intravenously administered with indicated ADCs (10 mg/kg unless otherwise labeled) or other drugs (dose indicated) on day 0 (tumor size reached to an average of 150–200 mm 3 ) and subsequent dates indicated by red arrows. Each value represents the mean and SEM ( n = 4 or 5). Unpaired two-sided t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001. For combination treatments, AZD9291 was dosed at 10 mg/kg, orally, every day for 21 days. AMG510 was dosed at 30 mg/kg, orally, every day for 21 days.

Article Snippet: The membranes were blocked and probed overnight with anti-phospho-HER3 antibody (#4791, Cell Signaling Technology), anti-HER3 antibody (#12708, Cell Signaling Technology), anti-phospho-AKT antibody (T40067, Abmart), anti-AKT antibody (T55561, Abmart), anti-phospho-ERK1/2 antibody (#4370, Cell Signaling Technology), anti-ERK1/2 antibody (#4695, Cell Signaling Technology), anti-β-tubulin antibody (R20005, Abmart) at 4°C.

Techniques: Mutagenesis, Expressing, Flow Cytometry, In Vivo, Labeling, Gene Expression

Journal: Scientific Reports

Article Title: Neuregulin 1 improves complex 2-mediated mitochondrial respiration in skeletal muscle of healthy and diabetic mice

doi: 10.1038/s41598-017-02029-z

Figure Lengend Snippet: NRG1 treatment increases ERBB4 activation in gastrocnemius muscle. C57BL/6JRJ (C57) control and db/db (Db) male mice were treated with vehicle (VHL; 0.9% NaCl solution; n = 8/each condition), or with NRG1 (50 μg . kg −1 ; n = 8/each condition), three days per week for eight weeks. Western blot analysis ( A ) cropped images) was used to quantify in gastrocnemius muscle samples the abundance of full length (115 kDa) ( B ) and cleaved (42 kDa) ( C ) NRG1 and the NRG1 cleavage index (the ratio between cleaved and full length NRG1) ( D ) as well as the abundance ( E ) and phosphorylation ratios ( F ) of ERBB2, ERBB3 and ERBB4. Results are the mean ± SEM (n = 8 per group) relative to the level in untreated healthy mice (C57-VHL, white bars). Diabetes (healthy vs db/db mice) and NRG1 (saline vs NRG1) effects were investigated with a 2 × 2 ANOVA. When a significant interaction was found, the Tuckey’s test was used for post-hoc multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, NS: not significant.

Article Snippet: The anti-porin (1/1000), -AKT (1/1000), -p-AKT Ser473 (1/1000), -p-AKT Thr308 (1/1000), -ERK (1/1000), -p-ERK, (1/1000), -AMPK (1/1000), -p-AMPK (1/1000), ACC (1/1000), -p-ACC (1/1000), -ACL (1/1000), -p-ACL (1/1000), -GLUT4 (1/1000), -ERBB3 (1/200) and -p-ERBB3 (1/200) antibodies were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Activation Assay, Western Blot

Journal: Scientific Reports

Article Title: Neuregulin 1 improves complex 2-mediated mitochondrial respiration in skeletal muscle of healthy and diabetic mice

doi: 10.1038/s41598-017-02029-z

Figure Lengend Snippet: Summary of the main effects of diabetes and NRG1 treatment in gastrocnemius muscle. The abundance of ERBB receptors is increased in diabetic db/db mice. This is associated with increased phosphorylation ratio of ERBB2 and decreased phosphorylation ratios of ERBB3 and ERBB4. Similarly, phosphorylation (activation) of the metabolic regulators AMPK, ACC and ACL is reduced in diabetic mice as well as Pparb and Tfam mRNA expression level. However, mitochondrial respiration in permeabilised fibres is similar in diabetic and control healthy mice. Chronic NRG1 treatment increases ERBB4 phosphorylation and tends to decrease ERBB3 phosphorylation. Among the regulators of the mitochondrial biogenesis pathway, only Pparb mRNA expression is slightly increased by NRG1. However, in NRG1-treated mice, complex 2-mediated mitochondrial respiration and complex 2 subunit abundance are increased. Effect of diabetes: red arrows. Effect of NRG1: green arrows. Figure was produced using Servier Medical Art image bank ( http://www.servier.com/Powerpoint-image-bank ), which is licensed under a Creative Commons Attribution 3.0 Unported License (http://creativecommons.org/licenses/by/3.0/).

Article Snippet: The anti-porin (1/1000), -AKT (1/1000), -p-AKT Ser473 (1/1000), -p-AKT Thr308 (1/1000), -ERK (1/1000), -p-ERK, (1/1000), -AMPK (1/1000), -p-AMPK (1/1000), ACC (1/1000), -p-ACC (1/1000), -ACL (1/1000), -p-ACL (1/1000), -GLUT4 (1/1000), -ERBB3 (1/200) and -p-ERBB3 (1/200) antibodies were purchased from Cell Signaling (Beverly, MA, USA).

Techniques: Activation Assay, Expressing, Produced

Journal: Journal of Biological Chemistry

Article Title: Epidermal Growth Factor as a Biologic Switch in Hair Growth Cycle

doi: 10.1074/jbc.m212082200

Figure Lengend Snippet: FIG. 4. Immunoblots of crude skin samples from specific time points. a, on both Day 7 and Day 14, expression and phosphorylation of EGFR were detected in both wild type and transgenic mice. On Day 21 and Day 35, normally the telogen phase, both the expression level and phosphorylation level of EGFR were intensively down-regulated. How- ever, samples from transgenic animals had comparatively higher levels of EGFR expression and phosphorylation than samples from wild type animals. Complete down-regulation of ErbB2 and ErbB3 expression was observed on Day 35 in wild type but not in transgenic skin. Com- parable amounts of the mature form of EGF were detected at each stage in transgenic and wild type samples. b, immunoprecipitation of ErbB2 and ErbB3. On Day 14, no observable difference between wild type and transgenic skin was detected with respect to phosphorylation of ErbB2 and EGFR/ErbB2 heterodimerization. Similar results were obtained for ErbB3. On Day 35 (telogen), only transgenic skin showed EGFR/ErbB2 heterodimerization and phosphorylation.

Article Snippet: The primary antibodies and working concentrations are as follows: rabbit anti-mouse EGF (Upstate Biotechnology, Inc., 2 g/ml); mouse anti-human EGFR (Transduction Laboratories, 1 g/ml); rabbit anti-mouse ErbB2 (Santa Cruz Biotechnology, 0.2 g/ml); rabbit anti-mouse ErbB3 (Santa Cruz Biotechnology, 0.2 g/ml); and mouse anti-phosphotyrosine PY20 (Transduction Laboratories, 1 g/ml).

Techniques: Western Blot, Expressing, Phospho-proteomics, Transgenic Assay, Immunoprecipitation