Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: (A) Cells expressing either ERBB4 (upper panel), ERBB4 and ERBB3 (middle panel) or ERBB3 receptors (lower panel) were cultured in medium without ligand, in the presence of 10 ng/ml IL3, or in the presence of 10 or 100 ng/ml NRG1. Cell numbers were counted at the indicated time points. (B) Parental Ba/F3 or Ba/F3 cells expressing ERBB3, ERBB4 or both receptors were incubated in the absence of IL3 and treated with 100 ng/mL NRG1 where indicated. Total cell lysate were then prepared and immunoblotted with phosphoepitope- and protein-specific antibodies for ERBB3, ERBB4, Akt and MEK.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques: Expressing, Cell Culture, Incubation

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: Ba/F3 ERBB3/ERBB4 or ERBB4 cells were treated in the three replicate experiments as indicated. Upon lysis and proteolytic digestion, peptides were differentially labeled with the three isotopic variant of mTRAQ and then pooled prior to peptide separation by high pH reversed phase chromatography and IMAC phosphopeptide enrichment. Phosphopeptide fractions were then analyzed by quantitative LC-MS on a LTQ Orbitrap Velos instrument. Lower panel: Characteristic mTRAQ patterns shown for a peptide harboring a NRG1-induced phosphosite in ERBB3/ERBB4 cells, which less strongly up-regulated in the absence of ERBB3 in ERBB4-expressing Ba/F3 cells.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques: Lysis, Labeling, Variant Assay, Reversed-phase Chromatography, Phospho-proteomics, Liquid Chromatography with Mass Spectroscopy, Expressing

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: (A) Volcano plot of NRG1-regulated phosphorylation in Ba/F3 cells expressing ERBB3 and ERBB4. (B) Volcano plot comparison of phosphorylation sites in NRG1-treated ERBB3/ERBB4 versus NRG1-treated ERBB4 expressing Ba/F3 cells. In both comparisons, log 10 -transformed, average phosphosite ratios are plotted against their standard deviations determined from mTRAQ replicate quantifications. Significantly regulated class I sites according to the Global Mean Rank test are depicted in red, all other sites in blue. The dashed grey lines indicate two-fold regulation.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques: Phospho-proteomics, Expressing, Comparison, Transformation Assay

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: Scatter plot of the mean ERBB3/ERBB4 ± NRG1 ratios with mean ERBB3/ERBB4 versus ERBB4 ratios from NRG1-treated cells. Reproducibly quantified ERBB3 phosphosites are encircled.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques:

Journal: PLoS ONE

Article Title: Quantitative Phosphoproteomics Analysis of ERBB3/ERBB4 Signaling

doi: 10.1371/journal.pone.0146100

Figure Lengend Snippet: Selected phosphorylation sites induced by NRG1 treatment.

Article Snippet: To measure ERBB3 cell surface expression, Ba/F3 cells expressing ERBB3 and ERBB4 and, as a control, cells expressing ERBB4 alone were analyzed on a FACSCanto flow cytometer (BD Biosciences) with an ERBB3 antibody conjugated to phycoerythrin (FAB3481P, R&D Systems).

Techniques: Phospho-proteomics, Sequencing

Journal: Cancer letters

Article Title: Inhibition of the endosomal recycling pathway downregulates HER2 activation and overcomes resistance to tyrosine kinase inhibitors in HER2-positive breast cancer.

doi: 10.1016/j.canlet.2022.01.003

Figure Lengend Snippet: Fig. 2. RCP expression correlates with HER2 and HER3 expression. a Western blot analysis of RCP, HER2, HER3 and EGFR protein expression levels in lysates of the indicated breast cancer cell lines. NM non-malignant, HER2+ HER2 amplified, HR+ hor mone receptor positive, TNBC triple-negative breast cancer, ILC invasive lobular carcinoma. b RCP expression positively correlates with HER2 expres sion in breast cancer cell lines from the Cancer Cell Line Encyclopedia (N = 57). Data equals mean ± std dev. A Mann-Whitney U statistical analysis test was performed (**p < 0.01). c RCP expression positively correlates with ERBB3 (HER3) expression in breast cancer cell lines from the Cancer Cell Line Encyclopedia (N = 57). Data equals mean ± std dev. A Mann-Whitney U statistical analysis was per formed (****p < 0.0001). d RCP expression posi tively correlates with ERBB3 expression in breast tumours analysed by the METABRIC study and accessible in cBioPortal (N = 1923). The violin plot depicts the median and 25th and 75th percentiles and the whiskers extend to the maximum and minimum values. Data equals mean ± std dev. A Mann-Whitney U statistical analysis test was per formed (****p < 0.0001). e Proteomic analysis of RCP and HER3 protein levels of breast cancer cell lines available in the DepMap Portal database (N = 29). f Proteomic analysis of RCP and HER2 protein levels of breast cancer cell lines available in the DepMap Portal database (N = 29). g BT474 cells were stimulated with 20 ng/ml HRGβ1 for the indicated times. Lysates were made and immuno blotted with the indicated antibodies. The histo gram represents the fold difference in RCP protein levels at each time point relative to unstimulated cells.

Article Snippet: Antibodies specific for HER2 (Rabbit, #4290), HER3 (Rabbit, #12708), p-Y1221/1222 HER2 (Rabbit, #2243), p-Y1248 HER2 (Rabbit, #2247), p-Y1289 HER3 (Rabbit, #2842), pan-Akt (Mouse, #2920) p-S473 Akt (Rabbit, #9271), Erk 1/2 (Mouse, #4696), p-T202/Y204 Erk 1/2 (Rabbit, #4370), mTor (Rabbit, #2972), p-S2448 mTor (Rabbit, #2971), S6 Ribosomal Protein (Mouse, #2971), and p-S235/236 S6 Ribosomal Protein (Rabbit, #2211) were from Cell Signalling Technology, Danvers, MA.

Techniques: Expressing, Western Blot, Amplification, MANN-WHITNEY

Journal: Cancer letters

Article Title: Inhibition of the endosomal recycling pathway downregulates HER2 activation and overcomes resistance to tyrosine kinase inhibitors in HER2-positive breast cancer.

doi: 10.1016/j.canlet.2022.01.003

Figure Lengend Snippet: Fig. 4. Depletion of RCP downregulates HER3. a Lysates of BT474 cells depleted of RCP were immunoblotted with the indicated antibodies. The histogram rep resents the fold difference in protein levels relative to cells transfected with a non-targeting siRNA duplex. b SK-BR-3 cells were transfected with control non-targeting or RCP-targeting siRNA duplexes and cell proliferation was monitored 5 days post-transfection.

Article Snippet: Antibodies specific for HER2 (Rabbit, #4290), HER3 (Rabbit, #12708), p-Y1221/1222 HER2 (Rabbit, #2243), p-Y1248 HER2 (Rabbit, #2247), p-Y1289 HER3 (Rabbit, #2842), pan-Akt (Mouse, #2920) p-S473 Akt (Rabbit, #9271), Erk 1/2 (Mouse, #4696), p-T202/Y204 Erk 1/2 (Rabbit, #4370), mTor (Rabbit, #2972), p-S2448 mTor (Rabbit, #2971), S6 Ribosomal Protein (Mouse, #2971), and p-S235/236 S6 Ribosomal Protein (Rabbit, #2211) were from Cell Signalling Technology, Danvers, MA.

Techniques: Transfection, Control

Journal: Cancer letters

Article Title: Inhibition of the endosomal recycling pathway downregulates HER2 activation and overcomes resistance to tyrosine kinase inhibitors in HER2-positive breast cancer.

doi: 10.1016/j.canlet.2022.01.003

Figure Lengend Snippet: Fig. 3. HRGβ1 induces internalisation and colocal ization of HER2 and HER3 with RCP. a Represen tative images of serum-starved JIMT-1 HER2+ cells stimulated with 20 ng/ml HRGβ1 for the indicated times prior to fixation and labelling with anti-RCP (green) and anti-HER2 (red). b Representative im ages of serum-starved JIMT-1 HER2+ cells stimu lated with 20 ng/ml HRGβ1 for the indicated times prior to fixation and labelling with anti-RCP (green) and anti-HER3 (red). The colocalization between RCP and HER2 or HER3 was quantified by deter mining their Pearson’s correlation coefficient (PCC) displayed in the box and whisker plots to the right, which depict the median, 25th and 75th percentiles with the whiskers extending to the maximum and minimum values. Between 15 and 27 cells were analysed per condition (*p < 0.05, **p < 0.01) from two biological replicates. Bar, 10 μm.

Article Snippet: Antibodies specific for HER2 (Rabbit, #4290), HER3 (Rabbit, #12708), p-Y1221/1222 HER2 (Rabbit, #2243), p-Y1248 HER2 (Rabbit, #2247), p-Y1289 HER3 (Rabbit, #2842), pan-Akt (Mouse, #2920) p-S473 Akt (Rabbit, #9271), Erk 1/2 (Mouse, #4696), p-T202/Y204 Erk 1/2 (Rabbit, #4370), mTor (Rabbit, #2972), p-S2448 mTor (Rabbit, #2971), S6 Ribosomal Protein (Mouse, #2971), and p-S235/236 S6 Ribosomal Protein (Rabbit, #2211) were from Cell Signalling Technology, Danvers, MA.

Techniques: Whisker Assay

Journal: Cancer letters

Article Title: Inhibition of the endosomal recycling pathway downregulates HER2 activation and overcomes resistance to tyrosine kinase inhibitors in HER2-positive breast cancer.

doi: 10.1016/j.canlet.2022.01.003

Figure Lengend Snippet: Fig. 5. Inhibition of the endosomal recycling pathway diverts HER2 and HER3 into lysosomes for degradation. a HER2 recycling assay in control or PQ-treated BT474 cells. Antibody-bound HER2 was allowed to internalise overnight. Shown are confocal images of cells fixed 0 and 60 min after trastuzumab was stripped from HER2 on the cell surface. The ratio of surface/internalised HER2 is displayed in the box plots (n = 34 Control and 35 PQ-treated cells from two independent experiments; statistical analysis was a two-way ANOVA). b BT474 cells were treated with or without 10 μM PQ for 24 h in serum-free medium. Lysates were immunoblotted with the indicated antibodies and the histogram indicates the fold difference in pro tein levels relative to untreated cells (n = 3). c BT474 cells were treated with or without 10 μM PQ for 24 h in serum-free medium and analysed for HER2 and HER3 mRNA expression by quantitative real-time, reverse transcriptase PCR (qRT-PCR). The results (mean ± s.e.m. of three biological replicates, each performed in triplicate) are expressed as mRNA expression levels relative to that for control cells. d BT474 cells were treated with 10 μM PQ plus or minus 20 mM NH4Cl or 1 μM MG132 for 24 h in serum-free medium. Lysates were immuno blotted with the indicated antibodies and the his togram indicates the fold difference in protein levels relative to untreated cells (n = 4).

Article Snippet: Antibodies specific for HER2 (Rabbit, #4290), HER3 (Rabbit, #12708), p-Y1221/1222 HER2 (Rabbit, #2243), p-Y1248 HER2 (Rabbit, #2247), p-Y1289 HER3 (Rabbit, #2842), pan-Akt (Mouse, #2920) p-S473 Akt (Rabbit, #9271), Erk 1/2 (Mouse, #4696), p-T202/Y204 Erk 1/2 (Rabbit, #4370), mTor (Rabbit, #2972), p-S2448 mTor (Rabbit, #2971), S6 Ribosomal Protein (Mouse, #2971), and p-S235/236 S6 Ribosomal Protein (Rabbit, #2211) were from Cell Signalling Technology, Danvers, MA.

Techniques: Inhibition, Control, Expressing, Reverse Transcription, Quantitative RT-PCR

Journal: Cancer letters

Article Title: Inhibition of the endosomal recycling pathway downregulates HER2 activation and overcomes resistance to tyrosine kinase inhibitors in HER2-positive breast cancer.

doi: 10.1016/j.canlet.2022.01.003

Figure Lengend Snippet: Fig. 7. Primaquine synergises with the HER2- targeting tyrosine kinase inhibitor lapatinib. a Clo nogenic assays of BT474 cells individually or dual treated with 20 nM lapatinib and 5 μM PQ for 10 days. Histogram indicates the surviving cells expressed as a percentage of the untreated control (n = 5). b BT474 spheroids treated with 10 nM lapatinib, 5 μM PQ or the combination for 5 days were labelled with Calcein AM (green) and ethidium bromide (red) to label the live and dead cells, respectively. Fluorescence microscopy images of the live spheroids were recorded. The line plots depict the fluorescence intensity profiles along a line through the equator of the spheroids for both channels under each condition. c Immunoblots of lysates prepared from BT474 cells treated with lapatinib and PQ for 24 h. The histogram indicates the fold difference in p-Y1221/1222 HER2 phos phorylation levels in the treated cells relative to control cells (n = 6). d Immunoblots of lysates prepared from BT474 cells treated with lapatinib and PQ for 24 h. The histogram indicates the fold difference in p-Y1289 HER3 phosphorylation levels in the treated cells relative to control cells (n = 6).

Article Snippet: Antibodies specific for HER2 (Rabbit, #4290), HER3 (Rabbit, #12708), p-Y1221/1222 HER2 (Rabbit, #2243), p-Y1248 HER2 (Rabbit, #2247), p-Y1289 HER3 (Rabbit, #2842), pan-Akt (Mouse, #2920) p-S473 Akt (Rabbit, #9271), Erk 1/2 (Mouse, #4696), p-T202/Y204 Erk 1/2 (Rabbit, #4370), mTor (Rabbit, #2972), p-S2448 mTor (Rabbit, #2971), S6 Ribosomal Protein (Mouse, #2971), and p-S235/236 S6 Ribosomal Protein (Rabbit, #2211) were from Cell Signalling Technology, Danvers, MA.

Techniques: Control, Fluorescence, Microscopy, Western Blot, Phospho-proteomics

Journal: Cancer letters

Article Title: Inhibition of the endosomal recycling pathway downregulates HER2 activation and overcomes resistance to tyrosine kinase inhibitors in HER2-positive breast cancer.

doi: 10.1016/j.canlet.2022.01.003

Figure Lengend Snippet: Fig. 10. Schematic depicting the effect of endo somal recycling inhibitors on HER2-amplified breast cancer cells. a HER2/HER3 heterodimers are continuously internalised from the plasma membrane and transported to early endosomes. From here they are recycled back to the cell surface, directly or indirectly via the recycling compart ment. b Small molecule or genetic inhibition of the endosomal recycling pathway simultaneously in hibits autophagic flux and prevents internalised HER2 and HER3 from returning to the plasma membrane, resulting in their diversion to lysosomes for degradation.

Article Snippet: Antibodies specific for HER2 (Rabbit, #4290), HER3 (Rabbit, #12708), p-Y1221/1222 HER2 (Rabbit, #2243), p-Y1248 HER2 (Rabbit, #2247), p-Y1289 HER3 (Rabbit, #2842), pan-Akt (Mouse, #2920) p-S473 Akt (Rabbit, #9271), Erk 1/2 (Mouse, #4696), p-T202/Y204 Erk 1/2 (Rabbit, #4370), mTor (Rabbit, #2972), p-S2448 mTor (Rabbit, #2971), S6 Ribosomal Protein (Mouse, #2971), and p-S235/236 S6 Ribosomal Protein (Rabbit, #2211) were from Cell Signalling Technology, Danvers, MA.

Techniques: Amplification, Clinical Proteomics, Membrane, Inhibition

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: PTEN loss and ERBB2/ERBB3-mediated AKT reactivation drive resistance to MET inhibition in MET-amplified hepatocellular carcinoma

doi: 10.1007/s13402-025-01097-y

Figure Lengend Snippet: MET inhibition triggers feedback activation of the ERBB2/ERBB3-dependent AKT pathway in HCC. ( A ) Immunoblots were performed with the indicated antibodies in PTEN -knockout cells (MHCC97H and HCCLM3) as well as their corresponding parental cells treated with capmatinib (0.5 µM) for the indicated time points. HSP90 served as a loading control. ( B ) Heatmap of transcriptional changes (fold-change) in MHCC97H cells treated with vehicle (control) or capmatinib for 24 h. ( C ) The mRNA levels of ERBB2 and ERBB3 were measured in PTEN -knockout cells (MHCC97H and HCCLM3) as well as their corresponding parental cells treated with capmatinib (0.5 µM) for 24 h. ( D - E ) Immunoblots were performed with the indicated antibodies in PTEN -knockout and parental cells (MHCC97H and HCCLM3), under the treatment with capmatinib (0.5 µM) for 24 to 120 h

Article Snippet: Primary antibodies targeting PTEN (9188), MET (8198), phospho-MET (3077), neurofibromin 1 (14623), phospho-AKT (4060), AKT (4685), ERBB2 (2165), ERBB3 (12708), phospho-ERBB2 (2243), and phospho-ERBB3 (4561) were obtained from Cell Signaling Technology.

Techniques: Inhibition, Activation Assay, Western Blot, Knock-Out, Control

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: PTEN loss and ERBB2/ERBB3-mediated AKT reactivation drive resistance to MET inhibition in MET-amplified hepatocellular carcinoma

doi: 10.1007/s13402-025-01097-y

Figure Lengend Snippet: Reactivation of the ERBB2/ERBB3-AKT signaling axis in HCC xenografts following MET inhibitor treatment. ( A ) Tumour growth curves of MHCC97H xenografts in BALB/c nude mice treated with vehicle (control) or capmatinib (5 mg/kg, oral gavage [i.g.], daily) for 35 days. ( n = 5). Statistical significance was determined by two-way ANOVA with Geisser-Greenhouse correction. ( B ) Body weights of mice in the MHCC97H xenograft models. Data are presented as mean ± SEM. ( C ) GSEA analysis of the PI3K-AKT signaling pathway in RNA-seq data from MHCC97H capmatinib-resistant and parental tumours. ( D ) Heatmap of transcriptional changes (fold-change) in MHCC97H capmatinib-resistant and parental tumours.( E ) Representative images of hematoxylin and eosin (H&E), p-AKT, p-ERBB2, ERBB2, and PCNA staining in MHCC97H xenografts with the indicated treatments. ( F ) Quantification of p-AKT, p-ERBB2, ERBB2 and PCNA staining in MHCC97H xenografts with the indicated treatments. Data are presented as mean ± SEM. Statistical significance was determined by unpaired two-tailed Student’s t-test. ** P < 0.01

Article Snippet: Primary antibodies targeting PTEN (9188), MET (8198), phospho-MET (3077), neurofibromin 1 (14623), phospho-AKT (4060), AKT (4685), ERBB2 (2165), ERBB3 (12708), phospho-ERBB2 (2243), and phospho-ERBB3 (4561) were obtained from Cell Signaling Technology.

Techniques: Control, RNA Sequencing, Staining, Two Tailed Test

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: PTEN loss and ERBB2/ERBB3-mediated AKT reactivation drive resistance to MET inhibition in MET-amplified hepatocellular carcinoma

doi: 10.1007/s13402-025-01097-y

Figure Lengend Snippet: Dual inhibition of MET and ERBB2/ERBB3 overcomes MET inhibitor resistance in HCC cells. ( A-B ) Immunoblots for p-AKT and AKT were performed in PTEN -knockout MHCC97H and HCCLM3 cells as well as their corresponding parental cells treated with capmatinib (0.5 µM), EGFR/ERBB2 dual inhibitor afatinib (0.5 µM) or their combination for 96 h. HSP90 served as a loading control. ( C-D ) Long-term cell proliferation assays were performed with combinations of capmatinib (0.5 µM) or tepotinib (0.5 µM) and afatinib or dacomitinib at the indicated concentrations for 10–14 days in PTEN -knockout cells (MHCC97H and HCCLM3). ( E-F ) Cell viability assays were performed on PTEN -knockout MHCC97H and HCCLM3 cells under combinations of capmatinib (0.5 µM) and afatinib (1 µM) or dacomitinib (1 µM) for 5 days. ( G-H ) Flow cytometry analysis of EdU (5-ethynyl-2´-deoxyuridine) incorporation into PTEN -knockout MHCC97H and HCCLM3 cells treated with capmatinib (0.5 µM), afatinib (1 µM) and their combination for 72 h. ( I-J ) Quantitative analysis of the percentage of EdU-positive cells ( n = 3 biological replicates). Data are presented as mean ± SD. Statistical significance was calculated by one-way ANOVA with Tukey’s multiple comparisons test (E, F, I and J). ** P < 0.01; *** P < 0.001; ns, not significant

Article Snippet: Primary antibodies targeting PTEN (9188), MET (8198), phospho-MET (3077), neurofibromin 1 (14623), phospho-AKT (4060), AKT (4685), ERBB2 (2165), ERBB3 (12708), phospho-ERBB2 (2243), and phospho-ERBB3 (4561) were obtained from Cell Signaling Technology.

Techniques: Inhibition, Western Blot, Knock-Out, Control, Flow Cytometry

Journal: Cellular Oncology (Dordrecht, Netherlands)

Article Title: PTEN loss and ERBB2/ERBB3-mediated AKT reactivation drive resistance to MET inhibition in MET-amplified hepatocellular carcinoma

doi: 10.1007/s13402-025-01097-y

Figure Lengend Snippet: Schematic model depicting the mechanisms of MET inhibitor resistance and potential therapeutic strategies in HCC. (Left panel) PTEN loss induces MET inhibitor resistance through ERBB2/ERBB3-mediated reactivation of the AKT signaling pathway. (Right panel) Proposed therapeutic interventions: (1) Co-targeting MET and AKT by using capmatinib with MK-2206, or (2) Dual MET/ERBBs inhibition by combining capmatinib with afatinib/dacomitinib

Article Snippet: Primary antibodies targeting PTEN (9188), MET (8198), phospho-MET (3077), neurofibromin 1 (14623), phospho-AKT (4060), AKT (4685), ERBB2 (2165), ERBB3 (12708), phospho-ERBB2 (2243), and phospho-ERBB3 (4561) were obtained from Cell Signaling Technology.

Techniques: Inhibition

Journal: The Journal of Neuroscience

Article Title: Regulation of Neuregulin-Mediated Acetylcholine Receptor Synthesis by Protein Tyrosine Phosphatase SHP2

doi: 10.1523/JNEUROSCI.19-21-09426.1999

Figure Lengend Snippet: SHP2 interaction with ErbB proteins in neuregulin (NRG)-stimulated muscle cells.A, C2 myotubes were stimulated for the indicated times with 2 nm neuregulin. Cell lysates were subjected to immunoprecipitation with a SHP2 monoclonal antibody followed by SDS-PAGE and immunoblot using anti-phosphotyrosine antibody. A doublet was observed in the 180 kDa range, and subsequent reprobing demonstrated that the higher molecular weight band comigrated with erbB2, whereas the lower one comigrated with ErbB3. The 70 kDa protein that showed a stimulation-dependent increase in phosphotyrosine content was SHP2. Approximately equal amounts of SHP2 were immunoprecipitated for each time point, as indicated in the bottom panel.B, Interaction of SHP2 SH2 domains with 180 kDa proteins. C2 myotubes were stimulated with 2 nm neuregulin for 5 min and then incubated with agarose-bound GST or GST fusion proteins containing both SHP2 SH2 domains (2SH2), the N-terminal (NSH2), or the more C-terminal (CSH2) SH2 domain. Bound proteins were eluted in SDS-sample buffer followed by SDS-PAGE and immunoblot analysis using an anti-phosphotyrosine antibody. The 180 kDa proteins were determined to contain ErbB proteins based on parallel experiments with specific antibodies. No association occurred with GST alone.

Article Snippet: Polyclonal antibodies to ErbB2 (C-18), ErbB3 (C-17) EGF receptor (EGFR) (SC-O3), and monoclonal anti-SHP2 antibody (B-1) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Immunoprecipitation, SDS Page, Western Blot, Molecular Weight, Incubation

Journal: The Journal of Neuroscience

Article Title: Regulation of Neuregulin-Mediated Acetylcholine Receptor Synthesis by Protein Tyrosine Phosphatase SHP2

doi: 10.1523/JNEUROSCI.19-21-09426.1999

Figure Lengend Snippet: ERK activation in response to NRG and EGF in CT and M2 fibroblasts. A, Immunoblot analysis demonstrating the relative expression of ErbB2, ErbB3, and SHP2 in SHP2 mutant M2 cells, and wild-type CT cells. ErbB4 expression was not detected in either cell line. Notice the lower apparent molecular weight of the mutant SHP2, resulting from deletion of amino acids 46–110 of the N-terminal SH2 domain. B, ERK activation assayed using MBP as a substrate. Serum-starved CT (open circles) and M2 (closed circles) fibroblasts were stimulated with EGF or neuregulin for the indicated times. ERK1 was immunoprecipitated from cell lysates and subjected to an in vitro kinase assay using MBP as a substrate. Shown are the averages of ERK1 activity from four to six independent experiments. C, ERK activation assayed by anti-phospho-ERK antibodies. Lysates from cells treated as in B were subjected to SDS-PAGE and Western blot analysis with an antibody that specifically recognizes activated ERK1 and ERK2. The same membrane was stripped and reprobed with a different antibody that recognizes both phosphorylated and nonphosphorylated ERK1 and ERK2. Shown is a Western blot from a representative experiment that was repeated three times with similar results.

Article Snippet: Polyclonal antibodies to ErbB2 (C-18), ErbB3 (C-17) EGF receptor (EGFR) (SC-O3), and monoclonal anti-SHP2 antibody (B-1) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Activation Assay, Western Blot, Expressing, Mutagenesis, Molecular Weight, Immunoprecipitation, In Vitro, Kinase Assay, Activity Assay, SDS Page, Membrane

Journal: The Journal of Neuroscience

Article Title: Regulation of Neuregulin-Mediated Acetylcholine Receptor Synthesis by Protein Tyrosine Phosphatase SHP2

doi: 10.1523/JNEUROSCI.19-21-09426.1999

Figure Lengend Snippet: Neuregulin (NRG)- and EGF-stimulated receptor tyrosine phosphorylation and association with SHP2. CT and M2 cells were stimulated with neuregulin or EGF for the indicated times. Cell lysates were subjected to immunoprecipitation with anti-ErbB2, anti-ErbB3, or anti-EGFR polyclonal antibodies. Immunocomplexes were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with anti-phosphotyrosine and anti-SHP2 antibodies. Half of each immunocomplex was run on a parallel gel and probed with same antibody used for immunoprecipitation to demonstrate equal amount of immunoprecipitated protein.

Article Snippet: Polyclonal antibodies to ErbB2 (C-18), ErbB3 (C-17) EGF receptor (EGFR) (SC-O3), and monoclonal anti-SHP2 antibody (B-1) were from Santa Cruz Biotechnology (Santa Cruz, CA).

Techniques: Phospho-proteomics, Immunoprecipitation, SDS Page

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

Journal: Clinical Cancer Research

Article Title: PI3Kα Inhibitor and Degrader Inavolisib Can Co-opt FGFR2 to Enhance Responses in Patients with PIK3CA -Mutated Solid Tumors and in Preclinical Models

doi: 10.1158/1078-0432.CCR-25-1459

Figure Lengend Snippet: Inavolisib sensitivity depends on high FGFR2 expression. A, Legend related to panels in B–I . B, Cell lines were treated with 0.03 μmol/L of the FGFR2i or 2-μmol/L lapatinib for 1 hour followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). C, Cell lines were treated with inavolisib or the FGFR2i at various concentrations for 1 hour. Cell lysates were immunoprecipitated with an antibody against p85β, followed by immunoblotting with the antibodies indicated at left. Representative results from experiments ( n = 2). IB, immunoblotting. D, Following RAS-GTP pulldown, cell lines were treated with the FGFR2i or lapatinib for different durations and immunoblotted with the antibodies indicated at left. Representative results from experiments ( n = 2). E, Cell lysates from cells treated with inavolisib alone or in combination with the FGFR2i or lapatinib for 4 hours were immunoprecipitated with RAS antibody and blotted with p110α antibody. F, Mechanistic model of the effects of FGFR2 and HER2 inhibition on HER3 and RAS activity. FGFR2-high–expressing cell lines induced PI3K signaling through both HER3 and WT RAS activity (top) compared with HER2-induced PI3K signaling through HER3 but not RAS activity (bottom). G, FGFR2-high–expressing cell lines, MFM223 and SUM52PE, were treated with inavolisib, FGFR2i, or lapatinib for 1 hour. Membrane fractions were analyzed by reciprocal co-IP with one another using HER3 or FGFR2 antibody and Western blotting with FGFR2, HER3, RAS, and p85β antibody. Representative results from experiments ( n = 2). H, SUM52PE, MFM223, and MFE280 cells were treated with inavolisib single-agent or in combination with the FGFR2i or lapatinib for 6 hours. Ubiquitinated proteins were pulled down from the membrane fraction with TUBE1 reagent and blotted with p110α antibody. Representative results from experiments ( n = 2). I, Western blots of the inhibitor response in PI3K signaling (pHER3 and pAKT) in PIK3CA mutant MFM223 and PIK3CA WT SUM52PE; cell lines were treated with 0.5-μmol/L inavolisib or 1-μmol/L alpelisib for different durations. Representative results from experiments ( n = 2). J, Ratio of inavolisib and alpelisib GR 50 values in FGFR2-high ( n = 12) vs. FGFR2-low ( n = 9) expressing cell lines harboring PIK3CA mutations, as assessed in a 5-day viability assay. Data are represented as median (center line) ± IQR (25th to 75th percentile, box) and ± full range (minimum to maximum, whiskers). P value was calculated using Wilcoxon rank-sum test. Representative results from experiments ( n = 2). WB, Western blotting.

Article Snippet: Antibodies to p110α (cat. No. 4249, RRID: AB_2165248), pAKT Ser473 (cat. No. 4060, RRID: AB_2315049), pS6 S235/236 (cat. No. 2211, RRID: AB_331679), HER3 (cat. No. 12708, RRID: AB_2721919), HER2 (cat. No. 2242, RRID: AB_331015), pHER2 Y1221/Y1222 (cat. No. 2243, RRID: AB_490899), pHER3 Y128 (cat. No. 4791, RRID: AB_2099709), pHER3 Y1328 (cat. No. 14525, RRID: AB_2798501), pPLCγ Y783 (cat. No. 14008, RRID: AB_2728690), pERK T202/T204 (cat. No. 9101, RRID: AB_331646), p4EBP T37/46 (cat. No. 9459, RRID: AB_330985), FGFR1 (cat. No. 9740, RRID: AB_11178519), FGFR2 (cat. No. 11835, RRID: AB_2797742), FGFR3 (cat. No. 4574, RRID: AB_2246903), FGFR4 (cat. No. 8562, RRID: AB_10891199), pFGFR Y653/654 (cat. No. 3476, RRID: AB_331369), and pFRS2A Y196 (cat. No. 3864, RRID: AB_2106222) were obtained from Cell Signaling Technology.

Techniques: Expressing, Western Blot, Immunoprecipitation, Inhibition, Activity Assay, Membrane, Co-Immunoprecipitation Assay, Mutagenesis, Viability 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: 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: For a binding assay, immune plates were coated with 1 μg/mL Human His tag EGFR/HER2/HER3/HER4 protein, Cynomolgus His tag HER3 protein, and Mouse His tag HER3 protein that all purchased from ACROBiosystems in coating buffer and kept overnight 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: For a binding assay, immune plates were coated with 1 μg/mL Human His tag EGFR/HER2/HER3/HER4 protein, Cynomolgus His tag HER3 protein, and Mouse His tag HER3 protein that all purchased from ACROBiosystems in coating buffer and kept overnight 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: For a binding assay, immune plates were coated with 1 μg/mL Human His tag EGFR/HER2/HER3/HER4 protein, Cynomolgus His tag HER3 protein, and Mouse His tag HER3 protein that all purchased from ACROBiosystems in coating buffer and kept overnight 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: For a binding assay, immune plates were coated with 1 μg/mL Human His tag EGFR/HER2/HER3/HER4 protein, Cynomolgus His tag HER3 protein, and Mouse His tag HER3 protein that all purchased from ACROBiosystems in coating buffer and kept overnight 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: For a binding assay, immune plates were coated with 1 μg/mL Human His tag EGFR/HER2/HER3/HER4 protein, Cynomolgus His tag HER3 protein, and Mouse His tag HER3 protein that all purchased from ACROBiosystems in coating buffer and kept overnight 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: For a binding assay, immune plates were coated with 1 μg/mL Human His tag EGFR/HER2/HER3/HER4 protein, Cynomolgus His tag HER3 protein, and Mouse His tag HER3 protein that all purchased from ACROBiosystems in coating buffer and kept overnight at 4°C.

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