her2 specific antibody  (Sino Biological)

Journal: Frontiers in Immunology

Article Title: A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells

doi: 10.3389/fimmu.2023.1292839

Figure Lengend Snippet: Screening and characterization of HER2-specific nanobodies. (A) The flowchart summarized the overall process of nanobodies screening from the AUAM synthetic nanobody library (ASyNAL). (B) HER2-specific nanobodies with His-tag were purified from BL21 (DE3) and showed high purity in SDS-PAGE. (C) The cell-surface binding activity of nanobodies was quantified by flow cytometry in SKBR3 tumor cells. (D) Nanobodies showed target specificity and species cross-reactivity. ELISA assay was performed to analyze the binding of nanobodies against human EGFR, human HER2, human HER3, human HER4, mouse Her2, and monkey Her2. In c and d, a non-specific IgG antibody was used as a negative control.

Article Snippet: The cells were incubated with diluted HER2-specific antibodies at 4°C for 1 h. After washing the unbound antibodies, cells were treated with PE-labeled anti-huFc (Abcam, 98596) secondary antibody (1:1000) at 4°C for 0.5 h. The cells were resuspended by 120 μL PBS + 2% FBS.

Techniques: Purification, SDS Page, Binding Assay, Activity Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Negative Control

Journal: Frontiers in Immunology

Article Title: A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells

doi: 10.3389/fimmu.2023.1292839

Figure Lengend Snippet: The affinities of anti-HER2 nanobodies binding to HER2-ECD.

Article Snippet: The cells were incubated with diluted HER2-specific antibodies at 4°C for 1 h. After washing the unbound antibodies, cells were treated with PE-labeled anti-huFc (Abcam, 98596) secondary antibody (1:1000) at 4°C for 0.5 h. The cells were resuspended by 120 μL PBS + 2% FBS.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells

doi: 10.3389/fimmu.2023.1292839

Figure Lengend Snippet: Affinity maturation of HER2-targeted nanobodies by phage display and identification of VHH-Fc. (A) The flowchart summarized the overall process of affinity maturation. (B) The sequence alignments of CDR regions were represented by Weblogo ( http://weblogo.berkeley.edu/logo.cgi ). All CDR regions of A2H4 and H2C11 were designed to generate random mutations. As for A9H5 and G1H2, their CDR2 region remained unchanged. (C) VHH-Fc fusions were purified from the 293T system and resolved by SDS-PAGE. (D) VHH-Fc derived from HER2-nanobodies with high affinity retained target specificity and species cross-reactivity. VHH-Fc showed a potent binding activity to human HER2 and monkey Her2 recombinant proteins. (E) VHH-Fc exhibited greater saturation activity in tumor cells compared to related parental nanobodies. (F) Confocal images of NCI-N87 cells identified the binding of VHH-Fc to the cell surface. Cells were treated with 50 nM antibodies and were stained by DyLight ® 488 for detection of the HER2-antibody complex. Alexa-Fluor 647-labeled secondary antibody and DAPI were used to visualize lysosomes and nuclei, respectively. Scale bars = 10 μm.

Article Snippet: The cells were incubated with diluted HER2-specific antibodies at 4°C for 1 h. After washing the unbound antibodies, cells were treated with PE-labeled anti-huFc (Abcam, 98596) secondary antibody (1:1000) at 4°C for 0.5 h. The cells were resuspended by 120 μL PBS + 2% FBS.

Techniques: Sequencing, Purification, SDS Page, Derivative Assay, Binding Assay, Activity Assay, Recombinant, Staining, Labeling

Journal: Frontiers in Immunology

Article Title: A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells

doi: 10.3389/fimmu.2023.1292839

Figure Lengend Snippet: The affinities of anti-HER2 nanobodies binding to HER2-ECD after affinity maturation.

Article Snippet: The cells were incubated with diluted HER2-specific antibodies at 4°C for 1 h. After washing the unbound antibodies, cells were treated with PE-labeled anti-huFc (Abcam, 98596) secondary antibody (1:1000) at 4°C for 0.5 h. The cells were resuspended by 120 μL PBS + 2% FBS.

Techniques: Binding Assay

Journal: Frontiers in Immunology

Article Title: A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells

doi: 10.3389/fimmu.2023.1292839

Figure Lengend Snippet: Epitope analysis of HER2-targeted nanobodies. (A) Schematic diagram of the construction of chimeric HER2-ECD proteins with domain substitution. HER2-mD1, HER2-mD2, HER2-mD3, and HER2-mD4 corresponded to the ECD I (T23-R217), ECD II (T218-C342), ECD III (Y344-A510), and ECD IV (C511-T652) of HER2 replaced by that of murine homolog, respectively. All chimeric and wild-type HER2 proteins were expressed in the 293T system. (B) ELISA analysis of the key domains of interactions between HER2 and nanobodies. The EC 50 value of nanobodies against different recombinant HER2 proteins was calculated and displayed by a heat map. (C) Competitive ELISA confirmed the binding domains of nanobodies. A2G5 and A9B5 competed with each other to bind HER2. H2F5 partially competed with pertuzumab, and G1E4 competed with trastuzumab.

Article Snippet: The cells were incubated with diluted HER2-specific antibodies at 4°C for 1 h. After washing the unbound antibodies, cells were treated with PE-labeled anti-huFc (Abcam, 98596) secondary antibody (1:1000) at 4°C for 0.5 h. The cells were resuspended by 120 μL PBS + 2% FBS.

Techniques: Enzyme-linked Immunosorbent Assay, Recombinant, Competitive ELISA, Binding Assay

Journal: Frontiers in Immunology

Article Title: A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells

doi: 10.3389/fimmu.2023.1292839

Figure Lengend Snippet: HER2-targeted VHH-Fc promotes internalization and mediates growth inhibition in SKBR3 and NCI-N87 tumor cells. (A) HER2 internalization induced by VHH-Fc in comparison with trastuzumab and pertuzumab. SKBR3 cells were incubated with antibodies for 0.5 or 4 hours. The flow cytometry was used to determine the mean percent internalization. Data were mean ± SD (n=3) and two-way ANOVA with Tukey’s multiple comparisons test was performed. The p values were provided in Supplementary Table 1 . (B) VHH-Fc mediated tumor growth inhibition in NCI-N87 cells. The percentage of cell viability was measured by CCK-8 assay. (Up) The representative curve means the concentration-dependent inhibition induced by VHH-Fc. (Down) A2G5-Fc, A9B5-Fc, and H2F5-Fc conferred moderate cell proliferation blocking, but G1E4-Fc showed significant agonistic effect. Data were mean ± SD (n=3) and one-way ANOVA with Dunnett’s multiple comparisons test was performed. The p values were provided in Supplementary Table 2 . *p < 0.0332, ***p < 0.0002, ****p < 0.0001.

Article Snippet: The cells were incubated with diluted HER2-specific antibodies at 4°C for 1 h. After washing the unbound antibodies, cells were treated with PE-labeled anti-huFc (Abcam, 98596) secondary antibody (1:1000) at 4°C for 0.5 h. The cells were resuspended by 120 μL PBS + 2% FBS.

Techniques: Inhibition, Incubation, Flow Cytometry, CCK-8 Assay, Concentration Assay, Blocking Assay

Journal: Frontiers in Immunology

Article Title: A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells

doi: 10.3389/fimmu.2023.1292839

Figure Lengend Snippet: VHH-Fc exhibits potent synergistic efficacy with antibodies recognizing non-overlapping epitopes irrespective of the presence of ligands. (A) VHH-Fc mixtures mediated the inhibition of ligand-dependent growth in NCI-N87 cells. A heat map was used to represent the inhibitory rate (mean, n=3, two-way ANOVA with Dunnett’s multiple comparisons test). The p values were provided in Supplementary Table 3 . (B) VHH-Fc showed greater synergistic efficacy with trastuzumab than a combination of trastuzumab and pertuzumab. A2G5-Fc and A9B5-Fc, derived from nanobodies binding to HER2 ECD I, both induced a significantly greater combined effect with trastuzumab than pertuzumab. Data were mean ± SD (n=6) and one-way ANOVA with Dunnett’s multiple comparisons test was performed. The p values were provided in Supplementary Table 4 . (C-E) A9B5-Fc mediated superior synergistic efficacy with trastuzumab to block ligand-independent and ligand-driven tumor cell growth. Data were mean ± SD (n=3) and two-way ANOVA with Dunnett’s multiple comparisons test was performed. The p values were provided in Supplementary Table 5 . *p < 0.0332, **p < 0.0021, ***p < 0.0002, ****p < 0.0001.

Article Snippet: The cells were incubated with diluted HER2-specific antibodies at 4°C for 1 h. After washing the unbound antibodies, cells were treated with PE-labeled anti-huFc (Abcam, 98596) secondary antibody (1:1000) at 4°C for 0.5 h. The cells were resuspended by 120 μL PBS + 2% FBS.

Techniques: Inhibition, Derivative Assay, Binding Assay, Blocking Assay

Journal: Frontiers in Immunology

Article Title: A novel nanobody-based HER2-targeting antibody exhibits potent synergistic antitumor efficacy in trastuzumab-resistant cancer cells

doi: 10.3389/fimmu.2023.1292839

Figure Lengend Snippet: Mechanisms of action of HER2-targeted VHH-Fc. HER2-specific nanobodies were obtained from a well-established synthetic nanobody library (ASyNAL). Affinity maturation based on soft randomization led to a pronounced increase in nanobodies binding to HER2 receptors of tumor cells. These nanobodies could recognize at least three different epitopes, encompassing ECD I, ECD II, and ECD IV. We further constructed the VHH-Fc fusions, which theoretically could prolong the plasma half-life of VHH, improve therapeutic benefit by bivalent binding, and induce Fc-effector functions (ADCC and CDC). The HER2 receptor internalization induced by VHH-Fc was notably observed in HER2-positive tumor cells, indicative of VHH-Fc potentially mediating receptor clustering on the cell surface. A9B5-Fc, targeting ECD I, exhibited potent combined efficacy with trastuzumab in ligand-independent and EGF/HRG-stimulating tumor cell growth. We posited that a combination of A9F5-Fc and trastuzumab effectively disturbed HER2 heterodimerization and homodimerization with EGFR family members, resulting in a blockade of ligand-independent and ligand-driven resistance. In summary, these mechanisms contribute to the growth suppression of trastuzumab-resistant tumor cells by the novel nanobody-based VHH-Fc.

Article Snippet: The cells were incubated with diluted HER2-specific antibodies at 4°C for 1 h. After washing the unbound antibodies, cells were treated with PE-labeled anti-huFc (Abcam, 98596) secondary antibody (1:1000) at 4°C for 0.5 h. The cells were resuspended by 120 μL PBS + 2% FBS.

Techniques: Binding Assay, Construct