Fig. 1

Characterization of anti-HER2 llama V_HHs. a Size exclusion chromatography profiles of anti-HER2 V_HHs. Approximately 0.5 mg of each V_HH was injected over a Superdex™ 75 GL column (GE Healthcare) connected to an ÄKTA FPLC protein purification system (GE Healthcare) in a mobile phase consisting of HBS-EP + (10 mM HEPES, pH 7.4, containing 150 mM NaCl, 3 mM EDTA and 0.05% surfactant P20). Maximum A₂₈₀ values were normalized to 100 for each V_HH. b Single-cycle kinetic analysis of V_HHs binding to human HER2 by SPR. All V_HHs were purified by preparative size exclusion chromatography prior to analysis. Approximately 1323 response units (RUs) of human HER2 were immobilized on adjacent flow cells of a CM5 Series S sensor chip in 10 mM acetate, pH 4.0, using an amine coupling kit (GE Healthcare). An ethanolamine-blocked flow cell served as the reference. Monomeric V_HHs at concentrations ranging from 1–400 nM were injected over the surfaces in HBS-EP+ buffer at a flow rate of 40 µL min⁻¹. The contact time was 120 s and the dissociation time was 600 s. The surfaces were regenerated using 10 mM glycine, pH 1.5. Data were analyzed using Biacore T200 Software v3.0 (GE Healthcare) and fitted to a 1:1 binding model (black lines show data and red lines show fits). Affinity and kinetic parameters (25 °C) are shown in Table 1, and sensorgrams showing binding to cynomolgus and murine HER2 are shown in Additional file 2. c Binding of V_HHs to HER2⁺ SKOV3 cells by flow cytometry. SKOV3 cells were grown to 70–80% confluency at 37 °C in a humidified 5% CO₂ atmosphere in RPMI-1640 medium supplemented with 10% fetal bovine serum, 100 U mL⁻¹ penicillin, 100 µg mL⁻¹ streptomycin and 250 ng mL⁻¹ amphotericin B. Cells were dissociated from flasks using Accutase^® solution, washed in PBS and then resuspended in PBS containing 1% bovine serum albumin. Approximately 1 × 10⁵ cells were stained sequentially on ice for 30 min with: (i) 10 µg mL⁻¹ of each V_HH, (ii) 5 µg mL⁻¹ of mouse anti-c-Myc IgG (clone 9E10), and (iii) 5 µg mL⁻¹ of APC-conjugated goat anti-mouse IgG (Thermo-Fisher). The cells were washed with PBS in between each staining step, and after the final wash, data (10,000 events) were acquired on a BD FACSCanto™ instrument (BD Biosciences, San Jose, CA). d Summary of epitope binning of anti-HER2 V_HHs by SPR. HER2 was immobilized as described in B. In the first injection, each V_HH at a concentration equivalent to 20 × K_D or trastuzumab (20 nM) was injected at a flow rate of 20 µL s⁻¹ for 300 s contact time to saturate the HER2 surface. The second injection consisted of the same V_HH along with a second V_HH (both at 20 × their respective K_Ds). All co injection experiments were performed in both orientations. Blue circles represent distinct epitopes conserved between human and cynomolgus HER2, and green circle represents a distinct epitope conserved across human, cynomolgus and mouse HER2