Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Identification of variants binding HER3. ( A ) Binding activity of TK-hu A3 and TK-hu A4 antibody variants to human HER3, as measured by ELISA. Six combinations of TK-hu A3 heavy (H) and light (L) chains and nine combinations of TK-hu A4 variants were transiently expressed in HEK293 cells. Supernatants were harvested 72 h post-transfection and assessed for HER3 binding. Bars represent mean absorbance ± SD of triplicate measurements. H/L ratio: DNA heavy chain to DNA light chain ratio. ( B ) Expression and integrity of recombinant mAbs H1L1 (lane 1), H2L1 (lane 2) from TK-hu A3, and H3L1 (lane 3) from TK-hu A4 were analyzed by SDS-PAGE under reducing conditions (1 mM DTT) followed by Coomassie blue staining. Molecular weight markers corresponding to the heavy chain (~50 kDa) and light chain (~25 kDa) are indicated.

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Binding Assay, Activity Assay, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Recombinant, SDS Page, Staining, Molecular Weight

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Antibodies species cross-reactivity. Binding of TK-murine A3, TK-hu A3, and TK-hu A4 purified antibody variants to recombinant mouse, rat, human, and rhesus HER3 was determined in a dose–response experiment by ELISA. Data represent mean absorbance ± SD from triplicate wells.

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Binding Assay, Purification, Recombinant, Enzyme-linked Immunosorbent Assay

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Binding activity of TK-hu A3 and TK-hu A4 antibody variants. ( A ) TK-murine A3 and TK-murine A4 antibodies, as well as TK-hu A3 and TK-hu A4 purified antibody variants, were analyzed for their binding to members of the ErbB tyrosine kinase receptor family: EGFR, ErbB2, HER3, and ErbB4. Antibodies were used at a concentration of 10 µg/mL, and binding was analyzed by ELISA. Absorbance values are plotted on the y-axis with respect to the antibody tested on the different recombinant proteins. ( B ) Serial dilutions of TK-murine A3, TK-hu A3, and TK-hu A4 purified antibody variants were analyzed by ELISA to assess their binding to human HER3 in a dose-dependent manner. Absorbance values are plotted on the y-axis with respect to the antibody tested at different dilutions.

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Binding Assay, Activity Assay, Purification, Concentration Assay, Enzyme-linked Immunosorbent Assay, Recombinant

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Epitope mapping of murine and humanized anti-HER3 antibodies. ( A ) Peptide array binding assays for TK-hu A3-H1L1, TK-hu A3-H2L1, and the murine antibody TK-A3. Reactivity was measured by ELISA using overlapping 15-mer peptides covering the HER3 extracellular domain (ECD). Each colored bar represents the optical density (OD) at 405 nm for an individual peptide. Strong binding was observed for peptides #54 and #55 in TK-hu A3-H1L1 and TK-A3, indicating conserved epitope recognition. In contrast, TK-hu A3-H2L1 showed loss of binding to these peptides and gained reactivity for peptide #66, suggesting a shift in epitope specificity. C+ = positive control; C− = negative control. ( B ) Structural mapping of the identified epitopes onto the HER3 ECD (PDB entry: 1M6B). Left: location of peptide #54 and #55 (orange) shared by TK-A3 and TK-hu A3-H1L1. Right: location of peptide #66 (purple), recognized exclusively by TK-hu A3-H2L1. Protein domains are indicated with Roman numerals. Only HER3 domain II is rendered as surface. ( C ) Amino acid sequences of the relevant peptides with conserved regions highlighted in bold. Peptides #54 and #55 share the core motif HCFGPNPNQCC, while peptide #66 contains a distinct sequence (QPLVYNKLTFQLEPN), reflecting altered antigen recognition in TK-hu A3-H2L1.

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Peptide Microarray, Binding Assay, Enzyme-linked Immunosorbent Assay, Positive Control, Negative Control, Sequencing

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: Structure of HER3::TK-hu A3 Fab complex. ( A ) Front view (left) and side view (right) of HER3 (cyan) in complex with TK-hu A3 Fab (light chain, V L , in light green and heavy chain, V H , in coral). Only HER3 domain II is rendered as surface; domains I–IV are indicated with Roman numerals. The domain II dimerization arm (residues 242–257, chain B) is shown in magenta. HER3 domain II surface colored by Fab contacts (atom–atom cutoff 4.0 Å). ( B ) Residues contacting V L only are light green, residues contacting V H only are coral, and residues contacting both chains are yellow. The HER3 cartoon is shown underneath (cyan); the Fab is omitted for clarity. (Contact patches computed with PDBePISA). Atomic details of HER3::TK-hu A3 Fab interface region. ( C , D ) TK-hu A3 Fab binds to the N-terminus region of domain II of HER3 mostly via hydrogen bonds, except for R84, which is involved in a π-cation interaction with residue Y33/F. ( C ): interactions between V L (variable light chain, light green) and HER3 domain II (cyan). ( D ): interactions between V H (variable heavy chain, coral) and HER3 domain II (cyan) (PDB entry: 9I1Q).

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Residue

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: TK-hu A3 and TK-hu A4 antibodies variants bind HER3 receptor in its native conformation. MCF7 cells were incubated on ice for 1 h in the presence or absence of TK-murine A3 ( A ), TK-hu A3-H1L1 ( B ), TK-hu A3-H2L1 ( C ), and TK-hu A4-H3L1 ( D ) antibody variants at concentrations of 10 µg/mL and 100 µg/mL. After incubation, cells were washed, and antibody binding was detected using anti-mouse or anti-human IgG antibody conjugated to Alexa Fluor 488 (gray bars). Cells were analyzed using a CytoFLEX flow cytometer platform. Histograms display the percentage of HER3-positive cells. CTRL antibody: The APC anti-human HER3/HER-3 Antibody was used as a positive control (white bars).

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

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

Journal: Antibodies

Article Title: Novel Humanized Anti-HER3 Antibodies: Structural Characterization and Therapeutic Activity

doi: 10.3390/antib14040084

Figure Lengend Snippet: TK-hu A3 and TK-hu A4 antibodies inhibit NRG-driven HER3 signaling and reduce cancer cell viability. ( A ) Neuregulin competition assay. SK-BR-3 cells were incubated for 1 h at 4 °C with increasing concentrations of TK-hu A3 or TK-hu A4, either in the presence or absence of recombinant neuregulin (NRG). Binding to cell-surface HER3 was assessed by flow cytometry. The plot shows representative binding curves: TK-hu A3 ± NRG (purple and yellow), and TK-hu A4 ± NRG (light green and dark green). The percentage of HER3-positive cells is plotted on the y-axis versus antibody concentration (log10 [µg/mL], x-axis). ( B ) Western blot analysis of HER3 signaling pathway activation in MCF7, FaDu, and BxPC-3 cells. Cells were pretreated for 6 h with increasing concentrations of TK-hu A3 or TK-hu A4 antibodies, then stimulated with NRG. The Control (Ctrl) lane represents untreated, unstimulated cells, while the NRG lane represents NRG-stimulated cells in the absence of antibody. Blots were probed for phosphorylated HER3 (pHER3, Tyr1289), total HER3, phosphorylated Akt (pAkt, Ser473), and phosphorylated p42/44 MAPK (Thr202/Tyr204). β-tubulin served as the loading control. ( C ) Colony-formation assay. BxPC-3 cells were seeded in 6-well plates, treated or not with increasing concentrations of TK-hu A3-H1L1 or TK-hu A4-H3L1 and cultured to allow colony formation; colonies were then fixed, stained, and counted. Only colonies comprising >50 cells were scored as survival colonies. The percentage of inhibition of colony formation (% inh) relative to untreated controls is plotted against the logarithmic antibody concentration (log 10 [µg/mL]), and data are fitted with a four-parametric non-linear regression curve (GraphPad v8.0).

Article Snippet: The interactions of HER3 Fc chimera protein (R&D Systems, Minneapolis, MN, USA) with TK-murine A3, TK-murine A4, TK-hu A3, and TK-hu A4 antibodies were studied by surface plasmon resonance detection using a Biacore X100 instrument (Cytiva, Marlborough, MA, US).

Techniques: Competitive Binding Assay, Incubation, Recombinant, Binding Assay, Flow Cytometry, Concentration Assay, Western Blot, Activation Assay, Control, Colony Assay, Cell Culture, Staining, Inhibition