Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: Expression levels of ErbB2 and ErbB3 on cell lines

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Expressing

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: Affinity and binding kinetics of the anti-ErbB3 A5 scFv. k on and k off rates were determined by surface plasmon resonance and used to determine the binding affinity ( K D ) of the A5 scFv. ( A ) Sensorgram fit to 1 : 1 Langmuir binding model. ( B ) Analysis of data.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Binding Assay, SPR Assay

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: The anti-ErbB2/ErbB3 bs-scFv ALM. ( A ) Cartoon of ALM depicting scFv orientation, linker sequence and kinetic constants of ALM for each target antigen. ( B ) UV adsorption spectrum chromatograph of ALM over Superdex 75 size-exclusion column.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Sequencing, Adsorption

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: ALM selectively targets ErbB2/ErbB3 positive cells in vitro

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: In Vitro

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: The A5-linker-ML3.9 bs-scFv selectively binds BT-474 tumour cells in vitro . Non-labelled BT-474 (ErbB2‘+’/ErbB3‘+’) breast tumour cells were mixed with either an equal ( A and B ) or 18-fold excess ( C ) of fluorescently labelled MCF10a (ErbB2‘±’/ErbB3‘±’) normal breast epithelial cells. Cell mixtures were then incubated with buffer ( A ) or 100 n M ALM ( B and C ) and binding of ALM to each cell population was determined by flow cytometry with an anti-6XHis tag secondary antibody. MCF10a cells were sorted to the upper quadrants and the non-labelled BT-474 cells were sorted to the lower quadrants. Cells bound by the secondary antibody sorted to the respective right hand quadrants. Images on the left depict the raw flow cytometry data. Values on the right represent the absolute number and overall percentage of each cell type in the respective quadrants.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: In Vitro, Incubation, Binding Assay, Flow Cytometry

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: Bispecific binding is required for optimal tumour targeting of the ALM bs-scFv in vivo . The biodistributions of radioiodinated ALM, ALD and DLM bs-scFv were analysed 24 h post-injection into xenograft-bearing SCID mice ( n =5 per cohort). ( A ) Co-expression of ErbB2 and ErbB3 by the targeted tumour is required for optimal targeting of ALM in vivo . 125 I-ALM targeted ErbB2+/ErbB3+ tumour xenografts to ⩾3-fold higher levels than xenografts that express only one of the target antigens. ( B ) Radioiodinated ALM ( 125 I-ALM), which is capable of bivalent association with the surface of Sk-OV-3 tumour cells, exhibited increased targeting as compared with ALD and DLM that targeted the tumours monovalently. Error bars represent the standard error of the mean (s.e.m.).

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Binding Assay, In Vivo, Injection, Expressing

Journal: British Journal of Cancer

Article Title: Targeting ErbB2 and ErbB3 with a bispecific single-chain Fv enhances targeting selectivity and induces a therapeutic effect in vitro

doi: 10.1038/sj.bjc.6604700

Figure Lengend Snippet: The A5-linker-ML3.9 bs-scFv has intrinsic anti-tumour cell activity. ( A ) Treatment of BT-474 and MDA-361/DYT2 cells with ALM inhibits colony formation in clonogenicity assays. Treatment of ( B ) BT-474 or ( C ) MDA-361/DYT2 cells with A5 scFv, ML3.9 scFv or the combination of both indicates that the majority of the intrinsic anti-tumour cell activity of ALM is due to the anti-ErbB3 A5 scFv arm. Colonies larger than 0.35 m M were counted using an automatic colony counter. Error bars represent the standard deviation.

Article Snippet: Anti-ErbB2 (Becton Dickinson, cat no. 340552) and anti-ErbB3 (R&D, cat no. FAB3481P) conjugated to a 1 : 1 ratio with phycoerythrin (PE) by the manufacturers were used for monitoring expression levels with the mean of three independent experiments reported in .

Techniques: Activity Assay, Standard Deviation

Journal: Journal of Translational Medicine

Article Title: Membranous expression of Her3 is associated with a decreased survival in head and neck squamous cell carcinoma

doi: 10.1186/1479-5876-9-126

Figure Lengend Snippet: Characterization of anti-Her3 antibodies by western blotting . Three cell lines (lung; A549, Breast; MCF7, and Pancreatic; BxPC3) were tested with 30 μg of cell line lysates. 1, A549; 2, MCF7; 3, BxPC.

Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h, washed, and subsequently incubated overnight at 4°C in TBST with 5% BSA containing anti-Her3 antibodies (RTJ.2, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:1000).

Techniques: Western Blot

Journal: Journal of Translational Medicine

Article Title: Membranous expression of Her3 is associated with a decreased survival in head and neck squamous cell carcinoma

doi: 10.1186/1479-5876-9-126

Figure Lengend Snippet: Representative images of immunohistochemistry for Her2 ( a , membranous staining and Her3 ( b , membranous and cytoplasmic staining, and c , predominant cytoplasmic staining) 400 × magnification .

Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h, washed, and subsequently incubated overnight at 4°C in TBST with 5% BSA containing anti-Her3 antibodies (RTJ.2, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:1000).

Techniques: Immunohistochemistry, Staining

Journal: Journal of Translational Medicine

Article Title: Membranous expression of Her3 is associated with a decreased survival in head and neck squamous cell carcinoma

doi: 10.1186/1479-5876-9-126

Figure Lengend Snippet: Correlations between Her2/Her3 Expression and Clinicopathological Parameters

Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h, washed, and subsequently incubated overnight at 4°C in TBST with 5% BSA containing anti-Her3 antibodies (RTJ.2, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:1000).

Techniques: Expressing, Staining

Journal: Journal of Translational Medicine

Article Title: Membranous expression of Her3 is associated with a decreased survival in head and neck squamous cell carcinoma

doi: 10.1186/1479-5876-9-126

Figure Lengend Snippet: IHC Expression of Her2 and He3 in Primary Tumors, Paired Metastatic Carcinomas and Recurrent HNSCC

Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h, washed, and subsequently incubated overnight at 4°C in TBST with 5% BSA containing anti-Her3 antibodies (RTJ.2, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:1000).

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Membranous expression of Her3 is associated with a decreased survival in head and neck squamous cell carcinoma

doi: 10.1186/1479-5876-9-126

Figure Lengend Snippet: Kaplan-Meier survival analyses of HNSCC according to Her2 and Her3 expression . ( a, c ) The Log-rank test did not distinguish the patients with tumors that expressed high levels and low levels of Her2 membranous and Her3 cytoplasmic staining. ( b ) Patients with tumors displaying positive Her3 membranous expression (median survival, 22 months; n = 34) had a significantly worse survival time than those with tumors displaying negative membranous Her3 expression (median survival, 40 months; n = 344; log-rank test, p = 0.027).

Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h, washed, and subsequently incubated overnight at 4°C in TBST with 5% BSA containing anti-Her3 antibodies (RTJ.2, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:1000).

Techniques: Expressing, Staining

Journal: Journal of Translational Medicine

Article Title: Membranous expression of Her3 is associated with a decreased survival in head and neck squamous cell carcinoma

doi: 10.1186/1479-5876-9-126

Figure Lengend Snippet: Prognostic Factors in a Univariate and Multivariate Proportional Hazard Model of The Cox Regression

Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h, washed, and subsequently incubated overnight at 4°C in TBST with 5% BSA containing anti-Her3 antibodies (RTJ.2, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:1000).

Techniques: Staining

Journal: Journal of Translational Medicine

Article Title: Membranous expression of Her3 is associated with a decreased survival in head and neck squamous cell carcinoma

doi: 10.1186/1479-5876-9-126

Figure Lengend Snippet: Outcome of HNSCC Patients According to The Combined Status of Her3 and Her3 Membranous Staining

Article Snippet: The membranes were blocked with 5% nonfat dry milk in TBST (50 mM Tris, pH 7.5, 150 mM NaCl, 0.05% Tween-20) for 1 h, washed, and subsequently incubated overnight at 4°C in TBST with 5% BSA containing anti-Her3 antibodies (RTJ.2, mouse monoclonal; Santa Cruz Biotechnology, Santa Cruz, CA; dilution 1:1000).

Techniques:

Journal: Neural Regeneration Research

Article Title: Chitosan conduits enriched with fibrin-collagen hydrogel with or without adipose-derived mesenchymal stem cells for the repair of 15-mm-long sciatic nerve defect

doi: 10.4103/1673-5374.358605

Figure Lengend Snippet: Accession number and primer sequences of the analyzed genes

Article Snippet: HER3/ErbB3 , sc-285 , AB_2099723 , 1:1000 , Rabbit , Santa Cruz Biotechnology.

Techniques: Sequencing

Journal: Neural Regeneration Research

Article Title: Chitosan conduits enriched with fibrin-collagen hydrogel with or without adipose-derived mesenchymal stem cells for the repair of 15-mm-long sciatic nerve defect

doi: 10.4103/1673-5374.358605

Figure Lengend Snippet: Primary and secondary antibodies used in western blot analysis

Article Snippet: HER3/ErbB3 , sc-285 , AB_2099723 , 1:1000 , Rabbit , Santa Cruz Biotechnology.

Techniques: Western Blot

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: Journal of Translational Medicine

Article Title: HER3 receptor and its role in the therapeutic management of metastatic breast cancer

doi: 10.1186/s12967-024-05445-8

Figure Lengend Snippet: Structure of ErbB receptors, ligands, and conformational changes upon ligand binding. A The ErbB family (ErbB1-ErbB4; also referred to as EGFR, HER2, HER3, and HER4) comprises receptor tyrosine kinases (RTKs) with similar structures, each consisting of an extracellular domain (ECD), a single hydrophobic transmembrane region, and an intracellular domain. The intracellular domain includes a juxtamembrane region, a tyrosine kinase domain, and a tyrosine-rich carboxy-terminal tail. The extracellular domain is divided into four subdomains, designated as Subdomains I-IV. EGFR, HER3, and HER4 exist in a tethered ("closed") conformation in the absence of ligands, preventing the dimerization domain from interacting with the corresponding Erbb components. HER2, lacking a known ligand, exists in an active extended ("open") conformation and can readily dimerize permanently. Among EGFR ligands, EGF, transforming growth factor-alpha (TGFα), amphiregulin (AREG), and epigen (EPGN) interact exclusively with EGFR, whereas epiregulin (EREG), heparin-binding EGF-like growth factor (HB-EGF), and betacellulin (BTC) can also bind and activate HER4. A family of EGF-related ligands, the neuregulins (NRGs; composed of NRG1-NRG4), binds to HER3 and HER4. HER2 does not directly bind these EGF-related ligands. HER3 has an impaired tyrosine kinase domain and exhibits reduced kinase activity. Therefore, to activate and facilitate signaling through HER2 and HER3, heterodimerization with other ErbB family members is required. B Ligand binding to ErbB receptors induces a conformational change in the molecular fold of the dimerization domain, a step necessary for dimer formation and the functional activation of EGFR, ERBB3, and ERBB4. The interaction in the kinase domains is asymmetrical, where the amino-terminal lobe of one tyrosine kinase interacts with the carboxy-terminal lobe of another

Article Snippet: , ISU104 , HER3 mAb , ISU Abxis , Phase I , NCT03552406 , [ ] .

Techniques: Ligand Binding Assay, Binding Assay, Activity Assay, Functional Assay, Activation Assay

Journal: Journal of Translational Medicine

Article Title: HER3 receptor and its role in the therapeutic management of metastatic breast cancer

doi: 10.1186/s12967-024-05445-8

Figure Lengend Snippet: HER3 receptor and its signaling cascade. A The monomeric inactive form of HER3 comprises the extracellular, transmembrane, and intracellular regions. The extracellular region comprises subdomains I-IV, with subdomains I and III responsible for ligand binding and subdomain II containing a dimerization arm. The intracellular region is composed of the juxtamembrane domain, tyrosine kinase domain, and C-terminal tail with phosphorylation sites. B Upon NRG binding to subdomain I and III, a conformational change occurs in the extracellular region, exposing the dimerization arm. This leads to heterodimerization between HER3 and EGFR/HER2 receptors, subsequently resulting in the phosphorylation of HER3 C-terminal tail and the activation of downstream intracellular signaling cascades, including PI3K/AKT, MAPK, JAK/STAT, SRC, and PLCγ/PKC. These signaling pathways collectively promote cell survival, proliferation, migration, and growth. The abbreviations used are as follows: AKT (protein kinase B), GDP (guanosine diphosphate), GRB2 (growth factor receptor-bound protein 2), JAK (Janus kinase), MAPK (mitogen-activated protein kinase), MEK (mitogen-activated extracellular signal-regulated kinase), SOS (son of sevenless), STAT (signal transducer and activator of transcription)

Article Snippet: , ISU104 , HER3 mAb , ISU Abxis , Phase I , NCT03552406 , [ ] .

Techniques: Ligand Binding Assay, Phospho-proteomics, Binding Assay, Activation Assay, Protein-Protein interactions, Migration

Journal: Journal of Translational Medicine

Article Title: HER3 receptor and its role in the therapeutic management of metastatic breast cancer

doi: 10.1186/s12967-024-05445-8

Figure Lengend Snippet: A summary of HER3 overexpression and drug resistance mechanisms

Article Snippet: , ISU104 , HER3 mAb , ISU Abxis , Phase I , NCT03552406 , [ ] .

Techniques: Over Expression, Knockdown, Activation Assay, Blocking Assay, Phospho-proteomics, Activity Assay, Expressing

Journal: Journal of Translational Medicine

Article Title: HER3 receptor and its role in the therapeutic management of metastatic breast cancer

doi: 10.1186/s12967-024-05445-8

Figure Lengend Snippet: The current status quo of HER3-targeted therapies for MBC. Monoclonals antibodies (mAbs), bispecific antibodies (bAbs), antibody–drug conjugate (ADC) and other therapies such as antibody-derived molecules

Article Snippet: , ISU104 , HER3 mAb , ISU Abxis , Phase I , NCT03552406 , [ ] .

Techniques: Derivative Assay

Journal: Journal of Translational Medicine

Article Title: HER3 receptor and its role in the therapeutic management of metastatic breast cancer

doi: 10.1186/s12967-024-05445-8

Figure Lengend Snippet: A summary of the HER3-targeted therapies for MBC under evaluation in preclinical and clinical trials

Article Snippet: , ISU104 , HER3 mAb , ISU Abxis , Phase I , NCT03552406 , [ ] .

Techniques: Expressing