Fig. 3: Epitope on domain I of HER2 determines activity of fusion construct.

a Binders recognizing different epitopes on HER2 were tested as fusion partners in our format. Antibody 39s and A21 (shown as Fv fragments) recognize epitopes on top of HER2, but A21 is binding to D1 while 39s is binding to D2. b Crystal structures of three further antibodies complexed with HER2 (7C2, MF3958, and H2-18) were previously shown to partly overlap with the A21 epitope, but they bury less surface area upon binding (epitopes are colored). Structures were derived from PDB files 3H3B, 6ATT, 5O4G, and 3L3W and the 7C2 epitope³⁰. c Replacement of A21 in our scFv–IgG fusion protein by any binder in (b) led to a reduction in antiproliferative activity of the resulting fusions (n = 3 biological replicates, mean plus error bar SD), tested in BT474 and HCC1419 cells. The fusions with the TZB scFv are named by “4” followed by the A21 replacement name (left panels). Note that, in the case of 39s, the fusion even augmented the proliferation of BT474 cells. Single mAbs (middle panels) were only modestly active (n = 3, error bar SD). Equimolar combination treatments of the mAbs with TZB (right panels) were all indistinguishable (n = 3, error bar SD) from each other. 441 proved superior, especially in the TZB-insensitive cell line HCC1419 (lower row). Source data are available in the Source Data file.