Journal: Frontiers in Immunology

Article Title: Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer

doi: 10.3389/fimmu.2023.1168444

Figure Lengend Snippet: ELISA to assess the simultaneous binding of (A) 2Trastu-1Cetu-Fc to immobilized HER2 and EGFR-His (left) and immobilized EGFR and HER2-His (right); (B) 3Patri-2Trastu-Fc to immobilized HER3 and HER2-His (left) and immobilized HER2 and HER3-His (right); (C) 3Patri-1Cetu-Fc to immobilized HER3 and EGFR-His (left) and immobilized EGFR and HER3-His (right); and (D) 3Patri-1Matu-Fc to immobilized HER3 and EGFR-His (left) and immobilized EGFR and HER3-His (right). The EC50 values calculated from the ELISA binding curves are indicated.

Article Snippet: Tag-free recombinant human HER2 (SB #10004-HCCH) and HER3 (SB #10201-HCCH) ECD, and His-tagged recombinant human EGFR (SB #10001-H08H), HER2 (SB #10004-H08H) and HER3 (SB #10201-H08H) ECD were purchased from Sino Biological Inc (China).

Techniques: Enzyme-linked Immunosorbent Assay, Binding Assay

Journal: Frontiers in Immunology

Article Title: Design and selection of optimal ErbB-targeting bispecific antibodies in pancreatic cancer

doi: 10.3389/fimmu.2023.1168444

Figure Lengend Snippet: Comparison of the four lead BiXAb™ vs 2MAbs and single parental antibodies (1MAbs). (A) Effect on the phosphoproteome. Cells were pre-stimulated with BiXAb™, 2MAbs or 1MAbs for 20 min before adding a mixture of NRG1 and EGF for 10 min. Activation rate is relative to the maximal (100%) phosphorylation obtained in NRG1/EGFR-stimulated cells without antibodies. HuIgG1 IRR and HLA-DR-CD5-Fc were used as negative controls for 1MAbs and BiXAb™, respectively. The tyrosine kinase inhibitors iMEK and iPI3K were used for comparison. (B) Feedback loop strengths by modular response analysis in the indicated pancreatic cancer cell lines. (C) Effect on ErbB expression. The indicated cell lines were incubated with the indicated antibodies for 6 h. After cell lysis, EGFR, HER2 and HER3 levels were evaluated by sandwich ELISA (RD Systems) using receptor-specific antibodies. The percentage of expression was quantified relative to negative control (culture medium alone). The NRG1+EGF mixture was used as positive control. (D) Effect on ADCC assessed by monitoring CD107a degranulation and IFNγ secretion. The indicated cell lines were co-cultured with CD16-transfected NK92 cells and incubated with BiXAb™, 2MAbs or 1MAbs for 4 h. The percentage of effector NK cells positive for CD107a and/or IFNγ was measured by flow cytometry. Rituximab was used as irrelevant antibody. Data are the mean ± SEM from three replicates. The NRG1 + EGF mixture was used as negative control of ADCC. (E) Effect on cell viability. BxPC3 cells were incubated with BiXAb™, 2MAbs or 1MAbs for 5 days, and cell viability was measured using the MTS proliferation assay. (F) Effect on cell apoptosis. The indicated cells were incubated with BiXAb™ or 2MAbs for 48 h, and then labeled with fluorescein-conjugated Annexin V and 7-AminoActinomycin-D before apoptosis measurement (%) by flow cytometry. Medium and staurosporine (3 h incubation) were used as negative and positive controls of apoptosis, respectively. HLADR-CD5-Fc was used as irrelevant BiXAb™. Caspase 3/7 activation is reported in Supplementary Figure 5 .

Article Snippet: Tag-free recombinant human HER2 (SB #10004-HCCH) and HER3 (SB #10201-HCCH) ECD, and His-tagged recombinant human EGFR (SB #10001-H08H), HER2 (SB #10004-H08H) and HER3 (SB #10201-H08H) ECD were purchased from Sino Biological Inc (China).

Techniques: Activation Assay, Expressing, Incubation, Lysis, Sandwich ELISA, Negative Control, Positive Control, Cell Culture, Transfection, Flow Cytometry, Proliferation Assay, Labeling

Journal: British Journal of Cancer

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

doi: 10.1038/sj.bjc.6604700

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

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

Techniques: Expressing

Journal: British Journal of Cancer

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

doi: 10.1038/sj.bjc.6604700

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

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

Techniques: Binding Assay, SPR Assay

Journal: British Journal of Cancer

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

doi: 10.1038/sj.bjc.6604700

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

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

Techniques: Sequencing, Adsorption

Journal: British Journal of Cancer

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

doi: 10.1038/sj.bjc.6604700

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

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

Techniques: In Vitro

Journal: British Journal of Cancer

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

doi: 10.1038/sj.bjc.6604700

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

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

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

Journal: British Journal of Cancer

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

doi: 10.1038/sj.bjc.6604700

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

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

Techniques: Binding Assay, In Vivo, Injection, Expressing

Journal: British Journal of Cancer

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

doi: 10.1038/sj.bjc.6604700

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

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

Techniques: Activity Assay, Standard Deviation