Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: Design of FR-engineered e23sFv derivatives. (A) Amino acid sequence alignment of the VL and VH domains of mouse anti-HER2 single-chain variable fragment, e23sFv, and their five most homologous counterparts identified in the National Centre for Biotechnology Information protein database. L1-L5 represent VL homologous sequences and H1-H5 represent VH homologous sequences. CDRs and FRs are indicated in columns. The residues that are identical to those of e23sFv are indicated with dashed lines, and missing residues in the CDRs are indicated with asterisks. Non-identical FR residues in e23sFv and all their five homologs in the VL or VH collection are in red. Introduced site-directed mutations are indicated by blue triangles, above which are the corresponding substituted residues. (B) The schematic structure of three e23sFv derivatives. EMEY includes 11 mutated residues in the FRs of e23sFv, as indicated by triangles. EX1 and EX2 represent CDR grafts of e23sFv in the L1-H1 and L2-H2 FR scaffolds, respectively. FR, framework region; VL, light-chain variable region; VH, heavy-chain variable region; CDR, complementarity-determining region.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: Sequencing

Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: In vitro binding of the e23sFv-derived scFvs to recombinant HER2. (A) Affinity measurement by ELISA. HER2-coated microplates were incubated with the e23sFv derivatives at various concentrations, and the bound scFvs were detected using an anti-His antibody. scFv15 served as the negative control. (B) One-shot kinetics of SPR. Five sensorgrams indicated the response of HER2-immobilized sensor chips with five diluted concentrations of the e23sFv derivatives. (C) Comparison of K on , K off and K D of the e23sFv derivatives calculated from SPR sensing. scFv, single-chain variable fragment; SPR, surface plasmon resonance; RU, resonance unit; K on , association rate constant; K off , dissociation rate constant, K D , equilibrium constant; Chi 2 , goodness-of-fit between the binding model and theoretical affinity; OD, optical density.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: In Vitro, Binding Assay, Derivative Assay, Recombinant, Enzyme-linked Immunosorbent Assay, Incubation, Negative Control, SPR Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: Binding of the e23sFv-derived single-chain variable fragments to HER2 on the cell surface. HER2-positive cells (BT-474 and SKOV-3 cells) and HER2-negative cells (MCF-7 cells) were incubated with FITC-labelled e23sFv derivatives and subjected to flow cytometry analysis. (A) Representative dataset of one-parameter histograms. For the isotype control, FITC-labelled scFv15 against HBsAg was used. For the positive control, a commercial FITC-conjugated anti-HER2 antibody was used. (B) Statistical analysis from three independent parallel experiments. * P<0.05; ** P<0.01 and *** P<0.001. ns, non-significant.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: Binding Assay, Derivative Assay, Incubation, Flow Cytometry, Positive Control

Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: Internalization of the e23sFv-derived single-chain variable fragments by HER2-positive cells. Following incubation with FITC-labelled e23sFv derivatives, BT-474, SKOV-3 and MCF-7 cells were observed under fluorescence microscopy. MCF-7 cells served as the HER2-negative cell controls and scFv15 was the non-specific binding control. Scale bar, 100 µm. The data are representative of at least three independent experiments.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: Derivative Assay, Incubation, Fluorescence, Microscopy, Binding Assay

Journal: Experimental and Therapeutic Medicine

Article Title: Syngeneic homograft of framework regions enhances the affinity of the mouse anti-human epidermal receptor 2 single-chain antibody e23sFv

doi: 10.3892/etm.2020.9568

Figure Lengend Snippet: Docking mechanism of the enhanced EX1-HER2 interaction. (A-D) In silico docking of e23sFv, EMEY, EX1 and EX2 and their interactions with the predicted surface models of the HER2 ECD. All the scFv fragments form distinct but overlapping interfaces with domain IV of the HER2 ECD. The 3D structures of (A) e23sFv, (B) EMEY, (C) EX1 and (D) EX2 are presented as coloured ribbons. (E) Binding energy with HER2 and the predicted binding epitopes of all the scFv fragments. ECD, extracellular domain; scFv, single-chain variable fragment.

Article Snippet: In addition, a FITC-labelled anti-HER2 antibody (1:2,000; cat. no. 10004-R511-F; Sino Biological) was used as a positive control.

Techniques: In Silico, Binding Assay

Journal: Journal of traditional Chinese medicine = Chung i tsa chih ying wen pan

Article Title: Modulation of expression of p16 and her2 in rat breast tissues of mammary hyperplasia model by external use of rupifang extract.

doi: 10.1016/s0254-6272(13)60087-4

Figure Lengend Snippet: Figure 2 her2 expression in rat breast tis- sues in each group (SP×200) A: bank control group (-); B: model group (+); C: the low dose group of Rupifang(+); D: the middle dose group of Rupifang (+); E: the high dose group of Rupifang (-).

Article Snippet: Rabbit-Anti-Human her2 and Rabbit-Anti-Human p16 SP immunohistochemical kit (Fuzhou Maixin Biological Technology Development Co., Ltd.); DAB chromogenic reagents (Beijing Zhongshan Gold Bridge Biotechnology Co., Ltd.); Estradiol Benzoate Injection: 1ml:1mg (Tianjin Jinyao Amino Acid Co., Ltd.); Progesterone injection: 1 ml∶20 mg (Tianjin Jinyao Amino Acid Co., Ltd.).

Techniques: Expressing, Control

Journal: Oncotarget

Article Title: ERRF sensitizes ERBB2-positive breast cancer cells to lapatinib treatment likely by attenuating MCL1 and ERBB2 expression.

doi: 10.18632/oncotarget.16425

Figure Lengend Snippet: Figure 1: Lapatinib upregulates ERRF expression in SK-BR-3 and BT-474 breast cancer cell lines, and higher levels of ERRF correlate with lapatinib sensitivities and better patient survival. (A) Illustration of ERRF expression after lapatinib treatment (100 nM) for 12 and 24 hours in SK-BR-3 cells based on the data from the Array Express database [43]. (B, C) Lapatinib upregulates ERRF expression in BT-474 and SK-BR-3 breast cancer cell lines, both express ERRF and respond to ERBB2 inhibition. Lapatinib treatment was at the indicated concentrations (µM) for 48 hours, and ERRF expression was measured by real-time RT-PCR. (D) Row signal of ERRF in SK-BR3 and BT-474 breast cancer cell lines and lapatinib resistant clones derived from them, as detected in a microarray study in the GEO database [44]. (E) Correlation of ERRF mRNA levels with sensitivities to ERBB2 inhibition in breast cancer cell lines, according to published information [45] and the CCLE database [58]. (F) Higher levels of ERRF expression are associated with better prognosis in ERBB2-positive breast cancer, as determined in the BreastMark database (HR = 0.5442, p = 0.028, n = 107).

Article Snippet: Antibodies used in this study included: ERRF (1:1000 dilution, Sigma-Aldrich, St Louis, MO), ERBB2 (1:1000 dilution, OriGene, Rockville, MD), β-actin (1:5000 dilution, Sigma-Aldrich), and MCL1 (1:1000 dilution, Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Inhibition, Quantitative RT-PCR, Clone Assay, Derivative Assay, Microarray

Journal: Oncotarget

Article Title: ERRF sensitizes ERBB2-positive breast cancer cells to lapatinib treatment likely by attenuating MCL1 and ERBB2 expression.

doi: 10.18632/oncotarget.16425

Figure Lengend Snippet: Figure 2: Ectopic expression of ERRF enhances lapatinib sensitivity in ERBB2-positive and lapatinib-resistant JIMT- 1 and MDA-MB-453 breast cancer cell lines. Lentivirus-mediated stable expression of ERRF was confirmed by western blotting (A, D) and cell viability was determined by the CCK-8 assay (B, E) and the sphere formation assay was in matrigel or in ultralow attachment plates (C, F). Lapatinib treatment was for 6 days at indicated concentrations in the CCK-8 assay and for 6 days at 2 µM in sphere formation assays. *p < 0.05; **p < 0.01; ***p < 0.001; pLHCX, vector control.

Article Snippet: Antibodies used in this study included: ERRF (1:1000 dilution, Sigma-Aldrich, St Louis, MO), ERBB2 (1:1000 dilution, OriGene, Rockville, MD), β-actin (1:5000 dilution, Sigma-Aldrich), and MCL1 (1:1000 dilution, Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Western Blot, CCK-8 Assay, Tube Formation Assay, Plasmid Preparation, Control

Journal: Oncotarget

Article Title: ERRF sensitizes ERBB2-positive breast cancer cells to lapatinib treatment likely by attenuating MCL1 and ERBB2 expression.

doi: 10.18632/oncotarget.16425

Figure Lengend Snippet: Figure 3: Knockdown of ERRF decreases lapatinib sensitivities in ERBB2-positive breast cancer cells. SK-BR-3 (A–C) and BT-474 (D–I) cell lines were used, and the knockdown effect for siRNAs (#1 and #2) was confirmed by real-time PCR (A, F, H). The CCK-8 assay was used to detect cell viability (B, C, D, G, I), and sphere formation assay was also performed for BT-474 cells (E). Lapatinib treatments were at indicated times and doses (B, C, D). For BT-474 cells, different concentrations of siRNA were also applied (G, I) **p < 0.01; ***p < 0.001; C, negative control.

Article Snippet: Antibodies used in this study included: ERRF (1:1000 dilution, Sigma-Aldrich, St Louis, MO), ERBB2 (1:1000 dilution, OriGene, Rockville, MD), β-actin (1:5000 dilution, Sigma-Aldrich), and MCL1 (1:1000 dilution, Cell Signaling Technology, Danvers, MA).

Techniques: Knockdown, Real-time Polymerase Chain Reaction, CCK-8 Assay, Tube Formation Assay, Negative Control

Journal: Oncotarget

Article Title: ERRF sensitizes ERBB2-positive breast cancer cells to lapatinib treatment likely by attenuating MCL1 and ERBB2 expression.

doi: 10.18632/oncotarget.16425

Figure Lengend Snippet: Figure 4: ERRF expression sensitizes xenograft breast cancer to the therapeutic effect of lapatinib and predicts response to lapatinib in breast cancer patients. Lapatinib resistant and ERBB2-positive JIMT-1 cells expressing ERRF or vector control (pLHCX) were injected subcutaneously into nude mice, lapatinib treatment was started at day 21, and tumor volumes (A) weights at excision (B) and images (C) are shown. (D). Detection of Ki67 in tumor sections by IHC staining. (E) Correlation of higher levels of ERRF expression with pathologic complete response (pCR) in patients treated with chemotherapy combined with lapatinib, as determined using a publically available data in the GEO database. NS, not significant; *P < 0.05; **P < 0.01.

Article Snippet: Antibodies used in this study included: ERRF (1:1000 dilution, Sigma-Aldrich, St Louis, MO), ERBB2 (1:1000 dilution, OriGene, Rockville, MD), β-actin (1:5000 dilution, Sigma-Aldrich), and MCL1 (1:1000 dilution, Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Plasmid Preparation, Control, Injection, Immunohistochemistry

Journal: Oncotarget

Article Title: ERRF sensitizes ERBB2-positive breast cancer cells to lapatinib treatment likely by attenuating MCL1 and ERBB2 expression.

doi: 10.18632/oncotarget.16425

Figure Lengend Snippet: Figure 6: ERBB2 and MCL1 are involved in ERRF mediated sensitization to lapatinib treatment. (A) Expression of ERRF inversely correlates with that of MCL1 in breast cancer samples, as determined by the Pearson correlation analysis using data from the TCGA database (Supplementary Table 1). (B) Overexpression of ERRF inhibits ERBB2 and MCL1 expression in the JIMT-1 ERBB2 positive breast cancer cell line, as determined by western blotting. (C, D). Knockdown of ERRF in the BT-474 breast cancer cell line, as confirmed by real-time PCR (C), increases ERBB2 and MCL1 expression, as determined by western blotting (D). (E, F) Knockdown of MCL1 by siRNA transfection (50 nM) rescued decreased sensitivity of BT-474 cells to lapatinib mediated by ERRF knockdown (12.5 nM and 25 nM siRNA). ERRF and MCL1 expression levels were examined by real-time PCR or western blotting. Con, control; #1 and #2, siRNAs against ERRF.

Article Snippet: Antibodies used in this study included: ERRF (1:1000 dilution, Sigma-Aldrich, St Louis, MO), ERBB2 (1:1000 dilution, OriGene, Rockville, MD), β-actin (1:5000 dilution, Sigma-Aldrich), and MCL1 (1:1000 dilution, Cell Signaling Technology, Danvers, MA).

Techniques: Expressing, Over Expression, Western Blot, Knockdown, Real-time Polymerase Chain Reaction, Transfection, Control

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: The expression of human HER2 in murine liver by recombinant adenovirus delivery. ( A ) Design of the adenovirus vector carrying a truncated human HER2/ERBB2 ( tHER2 ) gene and a firefly luciferase gene, whose expression are driven by the CMV and SV40 promoter, respectively. ( B ) In vitro testing luciferase and HER2/ERBB2 expression in B16 cell (the murine melanoma cell line). A two-tailed unpaired t -test of luciferase and HER2/ERBB2 level was used for statistical analysis. ( C ) In vivo imaging of luciferase expression in mice injected with 0, 2.5E + 8, 5E + 8 and 1E + 9 PFU of Ad5-HER2 8 days post-infection (8dpi). ( D ) IHC staining of liver section from mice that received different dosage of Ad5-HER2 for human HER2 (brown). ( E ) Quantification of HER2 in the indicated liver tissue. Data presented as mean ± SEM, each group contained 4 mice. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance of different groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, *** P ＜0.001, **** P ＜0.0001, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques: Expressing, Recombinant, Plasmid Preparation, Luciferase, In Vitro, Two Tailed Test, In Vivo Imaging, Injection, Infection, Immunohistochemistry

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: CAR-T cells are activated by human antigen and accumulated in murine liver. Human HER2 was expressed in murine liver following 0, 2.5E + 8, 5E + 8 and 1E + 9 PFU of Ad5-HER2 adenovirus delivery. Mice were then received either 5E + 6 HER2 CAR-T cells or UTD. ( A ) Systemic cytokine secretion by T cells was determined in murine serum (n = 3–4) by cytometric beads array 5 days after T cells transfer. ( B ) Frequency of HER2 CAR-T cells (up panel) and HER2 CAR-T cell counts (down panel) among live nucleated cells in the indicated tissues 7 days after T cells infusion. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance of different groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques:

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: HER2 CAR-T cells cause acute on-target liver toxicity in mice. ( A ) Flow chart of the experimental design for evaluating on-target, liver injury of CAR-T cells. NSG mice received 0, 2.5E + 8, 5E + 8 and 1E + 9 PFU of Ad5-HER2 and then infused with either 5E + 6 HER2 CAR-T cells or untransduced T cells (UTD) 8dpi. An n = 3–4 mice per group is shown in each panel unless stated otherwise. ( B ) Weight change shown by percent change from initial weight ± SEM in mice that received different dosage of Ad5-HER2 and then either HER2 CAR-T cells or UTD. ( C ) Survival curves of NSG mice that received different dosage of Ad5-HER2 and then T cells transfer. Statistical analysis was performed using a log-rank Mantel-Cox test. ( D ) Liver function profile as determined by serum ALT and AST levels collected days as indicated after T cells infusion. Mean ALT/AST ± SEM in mice received different dosage of Ad5-HER2 and then T cells injection. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance of different groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, *** P ＜0.001, **** P ＜0.0001, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques: Injection

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: Hypoxia-response CAR-T cells don’t cause acute on-target liver toxicity in mice expressing high levels of human antigen. ( A ) Flow chart of the experimental design for evaluating on-target, liver injury of CAR-T cells. NSG mice received 1E + 9 PFU of Ad5-HER2 and then infused with either 5E + 6 HER2 CAR-T cells or hypoxia-response CAR-T cells. A human antigen negative control group of NSG mice received 5E + 6 HER2 CAR-T cells but no Ad5-HER2 (i.e. Ad-). An n = 3–4 mice per group is shown in each panel unless stated otherwise. ( B ) Weight change shown by percent change from initial weight ± SEM in mice that received 1E + 9 PFU of Ad5-HER2 and then either CAR-T cells or hypoxia-response CAR-T cells. ( C ) Survival curves of NSG mice that received 1E + 9 PFU of Ad5-HER2 and then T cells transfer. Statistical analysis was performed using a log-rank Mantel-Cox test. ( D ) Liver function profile as determined by serum ALT levels collected days as indicated after T cells infusion. Mean ALT ± SEM in mice (n = 3–4) received 1E + 9 PFU of Ad5-HER2 and then T cells injection. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance between UTD group and other groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, *** P ＜0.001, **** P ＜0.0001, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques: Expressing, Negative Control, Injection

Journal: Journal of Advanced Research

Article Title: Rapid generation of a mouse model for evaluating on-target normal tissue toxicity of human CAR-T cells using replication-defective recombinant adenovirus

doi: 10.1016/j.jare.2022.08.008

Figure Lengend Snippet: Hypoxia-response CAR-T cells are not activated and accumulated by human antigen in murine liver. Human HER2 was expressed in murine liver following 1E + 9 PFU of Ad5-HER2 delivery. Mice were then received 5E + 6 HER2 CAR-T cells or hypoxia-response HER2 CAR-T cells. Control groups included mice that had hepatic HER2 expression but untransduced T cells (UTD) and mice that received HER2 CAR-T cells but lacked hepatic HER2 expression (i.e. Ad-). ( A ) Systemic cytokine secretion by T cells was determined in murine serum (n = 4–13) by cytometric beads array 7 days after T cells transfer. ( B ) Quantitation of inflammatory infiltration lesions in the liver 7 days after T cells transfer. ( C ) Frequency of CAR-T cells (up panel) and CAR-T cell counts (down panel) among live nucleated cells in the indicated tissues 7 days after T cells infusion. A one-way ANOVA with Dunnett's multiple comparisons test was used to test statistical significance of different groups. Statistical significance is denoted as * P ＜0.05, ** P ＜0.01, ns not significant.

Article Snippet: Briefly, the slides were stained with anti-human HER2/ERBB2 (Cat #: 10004-T56, SinoBiological) or anti-human CD47 (ab218810, Abcam) for immunohistochemistry (IHC), and immunostained with anti-human CD3 (Cat #: ab11089, Abcam), anti-FLAG tag (Cat #: 637301, Biolegend) antibodies and DAPI for immunofluorescence, which performed by the immunoassay platform at the Shanghai Institute for Emerging and Re-emerging Infectious Diseases of SPHCC.

Techniques: Expressing, Quantitation Assay

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Rapid internalization of anti-HER2-Bs compared with trastuzumab or 39S in BT-474 cells. Internalization time courses of trastuzumab-AF647 ( A – C ), 39S-AF647 ( D – F ), and anti-HER2-Bs-AF647 ( G – I ) in cells stained with cytoplasmic dye, CFSE) (green), are shown. Before the initiation of internalization, fluorescence antibodies are localized on the cell surface ( A , D , E ). At 30 min, both trastuzumab-AF647 (red) and 39S-AF647 (red) are slowly internalized into the cells ( B , E ), while a majority of anti-HER2-Bs-AF647 (red) are already internalized ( H ). At 2 h, the surface-bound anti-HER2-Bs-AF647 is completely internalized ( I ), while both trastuzumab-AF647 and 39S-AF647 remain on the cell surface ( C , F ). ( J ) Antibody internalization time course was quantified by an image analysis algorithm. Data are plotted as mean ± standard deviation from 6 wells. Scale bar is 10 μm.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques: Staining, Fluorescence, Standard Deviation

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Effect of dynamin and clathrin inhibitors on anti-HER2-Bs and trastuzumab internalization. Overlays of anti-HER2-Bs-AF647 ( A – F ) and trastuzumab-AF647 ( G – L ) with the cytoplasm dye (green) in BT-474 cells treated with either DMSO ( A , D , G , J ), dynamin inhibitor Dyngo 4a ( B , E , H , K ), or clathrin light chain inhibitor Pitstop 2 ( C , F , I , L ) are shown. At T = 0, anti-HER2-Bs-AF647 ( A , B , C ) or trastuzumab-AF647 ( G , H , I ) was bound on the surface of the treated cells. At 2 h, the internalization of both anti-HER2-Bs-AF647 (red) and trastuzumab-AF647 (red) was inhibited in the presence of Dyngo 4a (compare B , E to A , D for anti-HER2-Bs; compare H , K to G , J for trastuzumab) or Pitstop 2 (compare C , F to A , D for anti-HER2-Bs; compare I , L to G , J for trastuzumab). Scale bar is 10 μm.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques:

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: The combination of antibodies did not accelerate internalization. However, the addition of a secondary antibody accelerated trastuzumab internalization. Combined treatment with trastuzumab-AF647 (red) and unlabeled 39S ( C ) did not accelerate internalization of trastuzumab compared to trastuzumab-AF647 alone at 1 h ( A ). The internalization rate of trastuzumab-AF647 in the presence of 39S ( C ) is similar to the single antibody treatment with trastuzumab-AF647 ( A ) or 39S-AF647 (red) ( B ). By contrast, and similar to the previous figures, anti-HER2-Bs-AF647 (red) internalized rapidly into the cells ( E ). ( D ) Addition of anti-human IgG accelerated the internalization of trastuzumab compared to the untreated cells (compare D to A ). However, the internalization rate of anti-HER2-Bs is still much more pronounced compared to the trastuzumab-AF647/anti-human IgG-treated cells (compare E to D ). Cytoplasm dye, CFSE, is shown in green. Scale bar is 10 μm.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques:

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Blocking one epitope on HER2 receptors slowed down the internalization of anti-HER2-Bs-AF647 significantly. Cytoplasm dye, CFSE, is shown in green. Similar to the previous figures shown, anti-HER2-Bs-AF647 (red) internalized rapidly ( C ) compared to either trastuzumab-AF647 (red) ( A ) or 39S-AF647 (red) ( B ) at 2 h. ( D ) By contrast, blocking of trastuzumab-binding epitopes significantly slowed down the internalization of anti-HER2-Bs-AF647 at 2 h (compare D to C ) and resembled the internalization of 39S alone (compare D to B ). ( E ) Similarly, blocking of the other epitopes (39S-binding epitopes) also slowed down the anti-HER2-Bs-AF647 internalization (compare E to C ). Note that at the start of internalization (T = 0), similar fluorescent signals (AF647) were observed for all tested conditions ( A – E ) (see ). Scale bar is 10 μm.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques: Blocking Assay, Binding Assay

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Anti-HER2-Bs, but not trastuzumab, 39S, or both in combination, induced efficient HER2 degradation. ( A ) The level of total HER2 was not changed in the cells treated with trastuzumab, 39S, or both in combination. However, treatment with anti-HER2-Bs resulted in a decrease in total HER2 expression by 6 h. ( B ) Anti-HER2-Bs induces HER2 receptor ubiquitination, and this coincides with HER2 degradation. Treatment with anti-HER2-Bs resulted in an increase in total HER2 ubiquitination at 1–2 h and K63-specific ubiquitination at 1 h. ( C ) Quantification of HER2 total ubiquitination level normalized to total HER2 receptor level. The normalized level of HER2 total ubiquitination increased from 30 min post treatment and was sustained during 2–6 h post treatment.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques: Expressing

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: Anti-HER2-Bs and trastuzumab mediated distinct intracellular trafficking of internalized HER2 receptors. Internalization time courses of anti-HER2-Bs-AF647 ( A – C ) and trastuzumab-AF647 ( D – F ) are shown. ( A , D ) Before the initiation of internalization, both anti-HER2-Bs-AF647 and trastuzumab-AF647 (red) were colocalized with HER2 receptors on the cell surface (green) and appeared yellow. ( B , E ) At 30 min, the antibody-HER2 complexes (appearing as yellow puncta) were internalized into the cells. Consistent with the previous figures, the internalization of anti-HER2-Bs-AF647 is more rapid compared to trastuzumab-AF647 (compare D to G ). ( C , F ) Anti-HER2-Bs-AF647 and trastuzumab-AF647 mediated strikingly different trafficking of HER2 receptors at 2 h. ( C ) Internalized anti-HER2-Bs-AF647 (red) remained colocalized with HER2 receptors (green) and appeared as yellow puncta. However, trastuzumab-AF647 was dissociated from HER2 receptors and appeared as red puncta in the cells ( F ). ( G , H ) Treatment with anti-HER2-Bs, but not trastuzumab, resulted in complete surface clearance of HER2 receptors at 8 h. ( I , J ) Internalized anti-HER2-Bs-AF647 and trastuzumab-AF647 are colocalized with a lysosomal marker, LAMP1-AF488, at 8 h. The puncta of internalized anti-HER2-Bs-AF647 ( I ) or trastuzumab-AF647 ( J ) (red), colocalized with the LAMP1-AF488-positive compartments (green) merging in yellow. Treatment with anti-HER2-Bs resulted in the complete clearance of the antibody from the cell surface by 8 h ( I ). By contrast, there was a significant amount of trastuzumab remaining on the cell surface ( J ). Scale bars are 10 μm. ( K ) Treatment with anti-HER2-Bs resulted in a ~93% reduction of HER2 ectodomain (ECD) shedding in the conditioned culture media compared to the untreated cells. Treatment with trastuzumab also reduced HER2 ECD shedding by ~76%. ( L , M ) In addition, treatment with anti-HER2-Bs also reduced the number of exosome particles and the level of HER2 expression in the normalized exosomal fraction. CD63 is the protein marker for exosomes purified from conditioned culture media.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques: Marker, Expressing, Purification

Journal: Antibodies

Article Title: Molecular Mechanism of HER2 Rapid Internalization and Redirected Trafficking Induced by Anti-HER2 Biparatopic Antibody

doi: 10.3390/antib9030049

Figure Lengend Snippet: A proposed model of anti-HER2-Bs- and trastuzumab-mediated internalization, trafficking, and the fate of HER2 receptors. Please note that the receptors are drawn unproportionally to highlight the crosslinking on the cell surface and the intracellular trafficking. ( A ) In the untreated cells, the HER2 receptors are predominately localized on the cell surface. As part of the normal turnover process, a basal level of HER2 continuously undergoes internalization and catabolism. The pool of cell surface HER2 is replenished by the de novo synthesis of HER2 receptors. The internalized HER2 is then trafficked to multivesicular bodies (MVBs) for subsequent sorting. The HER2-loaded MVBs can fuse either with lysosomes for degradation, or with plasma membrane to release intra-luminal vesicles (ILVs) as exosomes. In addition, HER2-ECDs are constantly shedding from the receptors on the cell surface. ( B ) Treatment with trastuzumab resulted in a slow internalization of HER2 receptors via a clathrin-mediated pathway. The internalized trastuzumab-HER2 complexes are dissociated in the endosomes. The dissociated HER2 receptors are then recycled back to the cell surface. Similar to the untreated cells, a basal level of HER2 is internalized and trafficked to MVBs as a process of normal turnover, leading to either degradation of HER2 or release of HER2-loaded exosomes. On the other hand, the dissociated trastuzumab is trafficked to MVBs and lysosomes for degradation. Treatment with trastuzumab did not affect the level of exosome formation. However, binding of trastuzumab to the domain IV of the HER2 receptors on the cell surface inhibits the cleavage of HER2-ECD. ( C ) Concurrent engagement of HER2 epitopes by the bivalent bispecific anti-HER2-Bs antibody may induce crosslinking of HER2 receptors to form high molecular weight complexes on the cell surface. The anti-HER2-Bs-HER2 complexes are internalized rapidly via a clathrin-mediated pathway. The anti-HER2-Bs-HER2 complexes stay associated and traffic through the endocytic pathway. Instead of being recycled back to the cell surface, anti-HER2-Bs induces redirected trafficking of HER2 receptors to lysosomes for degradation. The degradation of HER2 receptors coincides with an increase in HER2 ubiquitination (total and K63). As a result of the lysosomal targeting, the anti-HER2-Bs-HER2-loaded multivesicular bodies are not able to fuse with the cell membrane, and thus this results in reduced exosomal export. In addition, rapid internalization of surface anti-HER2-Bs-HER2 complexes results in a fast clearance of surface HER2 receptors, thus leading to a reduction of HER2 ECD shedding on the cell surface.

Article Snippet: A standard-bind Meso Scale Discovery (MSD) 96-well assay plate (Meso Scale Discovery, Rockville, MD, USA) was coated with capture anti-human HER2 ECD antibody (Clone R002, Cat# 10004-R002, Sino Biological Inc, Chesterbrook, PA, USA) overnight at 4 °C.

Techniques: Binding Assay, Molecular Weight