Fig 1.
Graphical representation of the domain-exchanged antibody.
(A) Superimposition of cartoon diagrams of structures of the 4D5 fragment (PDB: 1FVE) and CH3 domains (PDB: 1OQO). The figure was prepared using PyMol Molecular Graphics System. (B) Schematic of domain-exchanged antibody and the analogous Fab-like fragment. VH: dark blue, CH1: light blue, Vκ: lemon, Cκ: teal, CH2: gray, CH3: black. Heterodimerization motif is indicated with white symbols dot and crescent for “Knob and Hole”. Cysteine bond connecting the novel CH3 domains is in orange.
Fig 2.
The HPLC profiles of the domain-exchanged antibody and the corresponding Fab-like fragments.
(A) From top to bottom: trace for TRA-CH3KiH, trastuzumab and the gel filtration standard. (B) From top to bottom: trace for TRA-Fab-CH3ZW1, TRA-Fab-CH3KiH, 4D5 Fab and the gel filtration standard.
Fig 3.
Thermal stability of the domain-exchanged antibody and the Fab-like fragments.
(A) Deconvolution of the thermogram for TRA-CH3KiH (upper trace) and trastuzumab (lower trace), (B) from top to bottom: deconvolution of the thermogram of TRA-Fab-CH3ZW1, TRA-Fab-CH3KiH and 4D5 Fab.
Fig 4.
Cell surface binding of domain-exchanged antibody and the Fab-like fragments to SK-BR3 cells.
(A) Binding to HER2/neu positive SK-BR3 cells of trastuzumab (full line—full triangles), TRA-CH3KiH (dotted line—full squares) and TRA-CH3KiH H:Phe404Tyr//L:Phe404Tyr (dashed line—full circles). No binding to HER2/neu negative MB-MDA468 could be detected with either trastuzumab (empty triangle), TRA-CH3KiH (empty circle) or TRA-CH3KiH H:Phe404Tyr//L:Phe404Tyr (empty square). (B) Binding to HER2/neu positive SK-BR3 cells of trastuzumab Fab (full line—full triangles), TRA-Fab-CH3KiH (dotted line—full squares) and TRA-Fab-CH3KiH H:Phe404Tyr//L:Phe404Tyr (dashed line—full circles), (C) Binding to HER2/neu positive SK-BR3 cells of trastuzumab Fab (full line—full triangles), TRA-Fab-CH3ZW1 (dotted line—full squares) and TRA-Fab-CH3ZW1 H:Phe404Tyr//L:Phe404Tyr (dashed line—full circles). No binding to HER2/neu negative MB-MDA468 could be detected with any of the constructs (corresponding empty markers in each panel).
Fig 5.
Domain-exchanged antibody elicits a more potent activation of NFAT pathway in FcγRIIIa-overexpressing T-cells than the parental antibody trastuzumab due to stronger binding to FcγRIIIa.
(A) T-cell activation was tested at an E:T ratio of 5:1 (upper pannel) and 15:1 (lower panel). The activation of T-cells in response to HER2/neu positive SK-BR3 cells coated with trastuzumab (full line–full triangles) or TRA-CH3KiH H:Phe404Tyr//L:Phe404Tyr (dashed line—full circles) and HER2/neu—negative MB-MDA468 cells incubated with trastuzumab (full line–empty triangles) or TRA-CH3KiH H:Phe404Tyr//L:Phe404Tyr (dashed line—empty circles). (B) Results of BLI assay for trastuzumab (upper panel) and TRA-CH3KiH H:Phe404Tyr//L:Phe404Tyr (lower panel) binding to FcγRIIIa. The curves correspond to the response of 2500, 1250, 625, 312.5 and 156.3 nM antibody. Dotted lines indicate the start of dissociation. (C) ADCC assay with NK cells from two donors (upper and lower panel) with trastuzumab (full line–full triangles) or TRA-CH3KiH H:Phe404Tyr//L:Phe404Tyr (dashed line—full circles) and HER2/neu—negative MB-MDA468 cells incubated with trastuzumab (full line–empty triangles) or TRA-CH3KiH H:Phe404Tyr//L:Phe404Tyr (dashed line—empty circles). Incubation of target cells with isotype control is indicated with an empty square and the lysis of the control MB-MDA468 cells, induced with Cetuximab, with a full square.