Journal: Protein Engineering, Design and Selection

Article Title: Identification of high affinity HER2 binding antibodies using CHO Fab surface display

doi: 10.1093/protein/gzy004

Figure Lengend Snippet: The hu4D5 Fab was effectively displayed on the surface of CHO cells. (A) Schematic of the Fab CHO expression construct. The open reading frame inserted into pPyEBV consisted of a murine IgΚ secretion signal (SS) fused to the hu4D5 IgΚ chain (VL–CL) DNA sequence, followed by DNA encoding a furin cleavage site and F2A peptide (2 A). The hu4D5 VH and CH1 coding sequence (VH–CH) fused to a short glycine–serine linker and the PDGFR transmembrane domain (TM) immediately followed the 2A peptide. (B) Schematic of the Fab CHO display and staining system. The displayed Fab was stained with HER2-Fc, then anti-human Fc-Alexa Fluor 647, and anti-human IgK-FITC was added for flow cytometric detection. For microscopy visual confirmation of the Fab CHO expression and staining procedure, CHO cells were transfected with either (C) blank pPyEBV or (D) pPyhu4D5disp and stained for HER2 binding, then imaged by confocal microscopy.

Article Snippet: The geometric mean of the goat–anti-human Fc-Alexa Fluor 647 fluorescence (MFI) of the forward/side scatter gated cells was fit to a one site specific binding equation using GraphPad Prism 7.03 (Fig. B).

Techniques: Expressing, Construct, Sequencing, Staining, Microscopy, Transfection, Binding Assay, Confocal Microscopy

Journal: Protein Engineering, Design and Selection

Article Title: Identification of high affinity HER2 binding antibodies using CHO Fab surface display

doi: 10.1093/protein/gzy004

Figure Lengend Snippet: Fab display was optimized to limit the number of plasmids per cell and modulate expression level. Epi-CHO cells were transfected with: (A) no plasmid or pPy4D5disp and pPyEGFP at a ratio of (B) 0:1, (C) 1:1, (D) 4:1, (E) 9:1 or (F) 19:1. The percent in quadrant I, positive for both GFP and HER2-Fc/anti-human Fc-AF 647, were 0%, 0.6%, 19.5%, 12.5%, 8.2% and 3.8%, respectively. Plasmids encoding expression level Kozak variants pPy4D5disp0 (0), pPy4D5disp1 (1), pPy4D5disp2 (2), pPy4D5disp3 (3), pPy4D5disp4 (4) and unmodified pPy4D5disp (5) were transfected to CHO-T cells, stained with HER2-Fc/anti-human Fc- Alexa Fluor 647, and scanned by flow cytometry. Data was collected for (G) mean fluorescence intensity (MFI) of Alexa Fluor 647 in cells displaying Fab and H, the percentage of total cells expressing Fab.

Article Snippet: The geometric mean of the goat–anti-human Fc-Alexa Fluor 647 fluorescence (MFI) of the forward/side scatter gated cells was fit to a one site specific binding equation using GraphPad Prism 7.03 (Fig. B).

Techniques: Expressing, Transfection, Plasmid Preparation, Staining, Flow Cytometry, Fluorescence

Journal: Protein Engineering, Design and Selection

Article Title: Identification of high affinity HER2 binding antibodies using CHO Fab surface display

doi: 10.1093/protein/gzy004

Figure Lengend Snippet: Full-length variants of bD1 have improved binding to both the soluble and membrane-bound form of HER2. (A) Each purified antibody was analyzed by ELISA to determine relative binding to soluble HER2-Fc or milk-coated (control) wells. (B) SK-OV-3 cells were incubated with purified antibody and stained with goat-anti-Fc-Alexa Fluor 647, then analyzed by flow cytometry. Control samples were antibody incubated with CHO-K1 cells and SK-OV-3 cells incubated with an irrelevant antibody isotype control.

Article Snippet: The geometric mean of the goat–anti-human Fc-Alexa Fluor 647 fluorescence (MFI) of the forward/side scatter gated cells was fit to a one site specific binding equation using GraphPad Prism 7.03 (Fig. B).

Techniques: Binding Assay, Purification, Enzyme-linked Immunosorbent Assay, Incubation, Staining, Flow Cytometry