Antibodies used in the present study were as follows: phosphorylated (p)-ErbB-3 (Tyr1289; clone 21D3; product no. 4791), ErbB-3 (clone D22C5; product no. 12708), p-EGFR (Tyr1068; clone D7A5; product no. 3777), EGFR (clone D38B1; product no. 4267), GAPDH (clone D16H11; product no. 5174), p-Akt (Ser473; clone D9E; product no. 4060), Akt (product no. 9272), p-Erk1/2 (Thr202/Tyr204; clone D.13.14.4E; product no. 4370), Erk1/2 (clone 137F5; product no. 4695), all from Cell Signaling Technology, Inc.; and anti-β-actin (product no. A5441) was purchased from Sigma-Aldrich; Merck KGaA. Neuregulin-1β (NRG-1β; product no. 5218SC) was purchased Cell Signaling Technology, Inc. Recombinant human EGF (cat. no. AF-100-15) was purchased from ProSpec-Tany TechnoGene Ltd. EV20 antibody was produced as previously described (22,23). Free MMAF (cat. no. HY-15579A) and sorafenib (cat. no. HY-10201) were purchased from MedChemExpress. The multidrug resistance-associated protein inhibitors PSC833 (CAS no. 121584-18-7; product no. SML0572), Reversan (CAS no. 313397-13-6; product no. SML0173) and MK571 (CAS no. 115104-28-4; product no. MK-571), used in combination with free MMAF in the MTT assay, were purchased from Sigma-Aldrich; Merck KGaA. T-DM1 was kindly provided by Professor Atanasio Pandiella from Centro De Investigaciòn del Càncer (Barcelona, Spain).

A375m human melanoma cell line (CRL3223) and SJSA-1 human osteosarcoma cell line (CRL2098) were purchased from ATCC. Liver cancer cell lines (HepG2, Hep3B, HuH7, SNU449, and PLC/PRF/5) were kindly provided by Dr Dituri Francesco from the National Institute of Gastroenterology ‘S. de Bellis’ Research Hospital (Castellana Grotte, Bari, Italy). HepG2 cells were authenticated by ATCC using Short Tandem Repeat (STR) DNA analysis. HepG2SR cells were obtained from culturing HepG2 parental cells in the presence of increasing doses of sorafenib up to a final concentration of 2 µM. All cell lines were cultured less than 3 months after resuscitation. The cells were cultured according to manufacturer's instructions, using EMEM for HepG2, Hep3B and PLC/PRF/5 cells, DMEM for HuH7 and A375m cells, and RPMI-1640 medium (all from Thermo Fisher Scientific, Inc.) for SNU449 and SJSA-1 cells, supplemented with 10% heat-inactivated fetal bovine serum (FBS; Invitrogen; Thermo Fisher Scientific, Inc.), L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich; Merck KGaA), and incubated at 37°C in humidified air with 5% CO₂.

EV20/MMAF was generated by Levena Biopharma (http://www.levenabiopharma.com/) as previously described (24,25). For site-specific conjugation, EV20 was engineered to EV20-sss as previously reported (26). Briefly, the cysteine residues of the heavy chain in positions 220, 226 and 229 were mutated into serine. EV20-sss-vc/MMAF was obtained as follows: EV20-sss was reduced using 60 M excess of Tris(2-carboxyethyl)phosphine (TCEP; Thermo Fisher Scientific, Inc.) in phosphate-buffered saline (PBS; Sigma-Aldrich; Meck KGaA), pH 7.4. The reaction was carried out overnight at room temperature. The reaction was stopped by passing the EV20-sss/TCEP mixture through a PD10 column (Cytiva) equilibrated in PBS, pH 7.4. The reduced antibody was then reacted with 10 M excess of MMAFvc in PBS overnight at room temperature. The reaction was stopped by adding 500-fold molar excess iodoacetamide (Sigma-Aldrich; Merck KGaA). To eliminate unreacted free auristatin, the reaction mixture was passed through a G25 Sephadex column equilibrated in PBS/5% sucrose/10% DMA in an isocratic way with a flow rate of 1 ml/min. The final concentration of the ADCs was estimated by UV–VIS spectrophotometry, using an extinction coefficient ε280 = 1.5 M⁻¹ cm⁻¹. The auristatin MMAFvc was provided by Levena Biopharma (http://www.levenabiopharma.com/). To evaluate the drug antibody ratio (DAR), hydrophobic chromatography (HIC)-HPLC was performed on conjugated and unconjugated antibody sample, dialyzed in solution containing 1.5 ammonium sulphate, 50 mM sodium phosphate, isopropanol 5%, pH 7. Subsequently, both samples (0.3 mg/ml) were analyzed in HIC-HPLC (Sol.A: 1.5 ammonium sulphate, 50 mM sodium phosphate, isopropanol 5%, pH 7; Sol.B: 50 mM sodium phosphate, isopropanol 20%, pH 7) with gradient 0-100% Sol.B in 20 min, flow rate 1 ml/min.

For HER receptors surface expression analysis, flow cytometry was performed as follows. Approximately one million growing cells were harvested and labeled with 1 µg/ml of primary antibody for 30 min on ice. For EGFR, HER-2 and HER-3 staining, primary antibodies used were chimeric anti-EGFR cetuximab (cat. no. A2000), humanized anti-HER-2 trastuzumab (cat. no. A2007), both purchased from Selleck Chemicals, and EV20 (humanized anti-HER-3, developed in our laboratory (Mediapharma srl, University of Chieti-Pescara, Chieti, Italy) (22,23). All primary antibodies were used at the dilution of 1 µg/1×10⁶ cells. After washing, the cells were labelled with PE-conjugated goat anti-Human Fc as secondary antibody at a dilution of 1:300 (cat. no. H10104; Molecular Probes; Life Technologies; Thermo Fisher Scientific, Inc.) for 30 min on ice. Regarding HER-4 analysis, cells were fixed with 1% paraformaldehyde for 15 min at room temperature and permeabilized with 0,1% Triton X-100 for 5 min at room temperature, then stained for 30 min on ice with 1 µg/ml of anti-HER4 antibody (cat. no. MA1-861; Invitrogen; Thermo Fisher Scientific, Inc.) as a primary antibody followed by staining for 30 min on ice with goat anti-mouse IgG Alexa-Fluor 488-conjugated at a dilution of 1:300 (cat. no. A11001; Molecular Probes, Life Technologies; Thermo Fisher Scientific, Inc.). For the in vivo binding assay of EV20 and EV20-based ADCs, A375m cells were detached and labelled with 10 µg/ml of EV20 and EV20-sss-vc/MMAF for 30 min on ice, followed by staining for 30 min on ice with PE-conjugate goat anti-Human Fc as a secondary antibody at a dilution of 1:300 (cat. no. H10104). Analysis was performed using FACSCantoII cytometer (BD Biosciences). Data were analyzed with FlowJo software V10.7 (FlowJo, LLC).

Cell proliferation was assessed by 3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma-Aldrich; Merck KGaA). Cell lines were seeded into 24-well plates at a density ranging between 4×103 cells/well and 7×10³ cells/well in 500 µl of complete culture medium. Then, cells were treated with drugs at indicated concentrations in triplicates and further incubated for 120 h. At the end of the incubation period, cells were incubated with 200 µl of MTT solution (serum-free medium with 0.5 mg/ml of MTT) for a further 2 h. After removal of MTT solution, 200 µl of dimethyl sulfoxide (DMSO) was added to the wells for 10 min and the absorption value at 570 nm was measured using a multi-plate reader. All experiments were performed in triplicate and the IC₅₀ values were calculated using GraphPad Prism 5.0 software (GraphPad Software, Inc.).

Lysates from cells in culture were prepared by washing cells twice in cold PBS followed by lysis with RIPA Buffer (50 mM Tris-HCl, 1% NP-40, 0.1% SDS, 150 mM NaCl) supplemented with protease and phosphatase inhibitors (Sigma-Aldrich: Merck KGaA) for 10 min at 4°C. Insoluble materials were removed by centrifugation (16,000 × g for 10 min at 4°C) and protein concentration was assessed using a Bradford assay. Equal amounts of protein (30 µg) were separated by SDS/PAGE on 10% polyacrylamide gel and transferred to nitrocellulose membranes. Membranes were blocked with 5% non-fat dry milk in PBS containing 0.1% Tween-20 for 1 h at room temperature and incubated with following primary antibodies: p-ErbB-3, ErbB-3, p-EGFR, EGFR, p-Akt, Akt, p-Erk1/2, Erk1/2, all from Cell Signaling Technology, Inc. and anti-β-actin from Sigma-Aldrich; Merck KGaA, as aforementioned. All the antibodies were used at a dilution of 1:1,000 in PBS containing 0.1% Tween-20 overnight at 4°C, except for anti-β-actin, which was used at a dilution of 1:40,000 in PBS containing 0.1% Tween-20. After washing, the membranes were hybridized for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibodies at a dilution of 1:20,000 [(HRP-conjugated goat anti-mouse IgG; product code STAR207P) and (HRP-conjugated goat anti-rabbit IgG; product code STAR208P; both purchased from Bio-Rad Laboratories, Inc.]. Detection was performed with Plus-ECL chemiluminescence kit (Bio-Rad Laboratories, Inc.). Densitometric analysis of bands was performed using ImageJ software V1.53 (National Institutes of Health).

For the evaluation of HER-3 protein expression in human specimens, tumor and peritumor samples, as well as normal liver samples, were collected, snap-frozen, and analyzed by western blotting. Male (n=9) and female (n=2) patients aged between 48 and 80 years were included. Samples were collected at the Department of Emergencies and Organ Transplant of the Policlinic Hospital of Bari (Bari, Italy), between September 2016 and September 2017. All patients provided written consent for the use of their specimens for research purposes; none were identifiable. Frozen specimens of tissues were homogenized with a Polytron homogenizer in a lysis buffer T-PER Tissue Protein Extraction (cat. no. 78510; Thermo Fisher Scientific, Inc.) supplemented with proteinases/phosphatases inhibitor cocktail (cat. no. 1861280; Thermo Fisher Scientific, Inc.). The homogenates were then centrifuged at 16,000 × g for 10 min at 4°C, and the protein concentration was determined using a Bio-Rad assay kit according to the manufacturer's instructions (cat. no. 131947; Bio-Rad Laboratories, Inc.). Equal amounts of proteins (20 µg) were separated by SDS/PAGE on 10% polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was probed overnight at 4°C with the HER-3 (product no. 12708) and GAPDH (product no. 5174; both 1:1000; both from Cell Signaling Technology, Inc.) antibodies, washed with TBS-T (TBS 1X + 0.05% Tween-20), and then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (HRP-conjugated goat anti-rabbit IgG; cat. no. STAR208P; Bio-Rad Laboratories, Inc.) at the dilution of 1:20,000 in TBB buffer (TBS-T + 5% nonfat milk). Detection was performed with Clarity Max Western ECL Substrate (Bio-Rad Laboratories, Inc.). This study was approved by the Local Ethics Committee, Azienda Ospedaliero Universitaria Consorziale Policlinico di Bari (Bari, Italy); protocol no. 254; date of release, February 2012.

For flow cytometric quantification, A375m and HepG2 cells were plated in 60 mm plates and grown in DMEM containing 10% FBS, for 24 h. Cells were then incubated with 10 µg/ml of EV20 in complete medium on ice for 30 min before returning the plates in the incubator at 37°C for 1 h, maintaining control cells on ice in the presence of the antibody. Finally, the cells were detached and stained with PE-conjugate goat anti-Human Fc at a dilution of 1:300 (cat. no. H10104) for 30 min on ice. Analysis was performed using FACSCantoII cytometer (BD Biosciences). Data were analyzed with FlowJo software V10.7 (FlowJo, LLC).

For confocal microscopy, A375m and HepG2 cells were seeded on round cover slips in 12-well plates to 70% confluence in complete medium for 24 h. Cells were then incubated with 10 µg/ml of EV20 in complete medium on ice for 30 min, after which they were then incubated again at 37°C for 2 h. The antibody was washed away and the cells were fixed for 15 min at room temperature with 4% paraformaldehyde (pH 7.4). Cells were then permeabilized for 5 min at room temperature with 0.5% Triton X-100 and labeled with goat anti-human IgG Alexa-Fluor 488-conjugated at a dilution of 1:200 (cat. no. A11013; Molecular Probes, Life Technologies; Thermo Fisher Scientific, Inc.) and Draq5 (product no. 4084; Cell Signaling Technologies, Inc.) to visualize nuclei. Images were acquired at a magnification of ×63 with a Zeiss LSM 510 meta-confocal microscope (Zeiss AG) using 488- and 633-nm lasers.

Recombinant HER-3 extracellular domain (ECD) (cat. no. ER3-H5223; AcroBiosystems) (1 µg/ml) was pre-coated overnight at 4°C on 96 well-plates NUNC Maxisorp modules. After blocking with 1% BSA in PBS for 1 h at room temperature, increasing concentrations (ranging between 0.05 nM and 6.6 nM) of EV20-sss or EV20-sss-vc/MMAF were incubated for 1 h at room temperature. After several washes with PBS + 0,05% Tween-20, a goat anti-human IgG-HRP solution at a dilution of 1:5,000 (product no. A0170; Sigma-Aldrich; Merck KGaA) was added to each well and incubated for 1 h at room temperature. After washing, stabilized chromogen was added to each well for at least 10 min in the dark, then the reaction was stopped with the addition of H₂SO₄ 1N and the absorbance was read at 450 nm with an ELISA reader.

Homozygous Balb/c nu/nu athymic female mice (4-6-weeks old) were purchased from Charles River Laboratories and maintained at 22-24°C and relative humidity (40-60%) under pathogen-limiting conditions as required. Cages, bedding, and food were autoclaved before use. Mice were provided with a standard diet and water ad libitum and acclimatized for 2 weeks before the start of the experiments. Housing and all procedures involving the mice were performed according to the protocol approved by the Institutional Animal Care and Use Committee of the Italian Ministry of Health (authorization no. 292/2017-PR).

Five million of exponentially growing HepG2 cells were implanted subcutaneously (s.c.) into the right flank of the mice in a ratio of 1:6 with Matrigel (Cultrex Basement Membrane Matrix; cat. no. 3432-001-01; Trevigen, Inc.). When tumors became palpable (approximately 150 mm³), animals were randomly divided into two groups and treated intravenously via tail vein with vehicle (PBS, once a week for a total of 4 injections), or EV20-sss-vc/MMAF (10 mg/kg, once a week for a total of 4 injections), respectively. The tumor volume was monitored weekly by a caliper and calculated using the following formula: tumor volume (mm³) = (length × width²)/2. A tumor volume of 1.5 cm³ was selected as the endpoint for all experiments after which mice were sacrificed using CO₂ inhalation (20-70%).

For in vivo xenograft curves and HER-3 expression level in tumor tissues, P-values were determined by a paired Student's t-test and considered significant for P<0.05. Statistical analysis of PI3K/AKT activation was performed using one-way ANOVA, followed by Tukey's multiple comparisons test. Experimental sample numbers (n) are indicated in the figure legends. All statistical analysis was performed with GraphPad Prism 5.0 software (GraphPad Software, Inc.). Survival curves were evaluated by Kaplan-Meier and analyzed by the log-rank test with GraphPad Prism 5.0 software (GraphPad Software, Inc.).

A panel of human LC cell lines for surface expression of HER-3 and its preferred partner HER-2 were screened by flow cytometry. The HER-3 and HER-2 receptors were expressed in all the LC cell lines assessed except for SNU449 cells, where HER-3 expression was found to be markedly low (Fig. 1A and B). Next, whether the downstream signalling of HER-3 was activated in these cells was evaluated. To this end, western blot analysis was performed of lysates prepared from this panel of LC cells stimulated with either HER-3 ligand neuregulin1b (NRG-1b) or epidermal growth factor (EGF), the ligand for the other member of the receptor family, EGFR. Notably, despite the fact that EGFR was highly expressed in this panel of cell lines (Fig. 1A), NRG-1b activated the PI3K/AKT the survival signaling pathway more potently than EGF (Fig. 1B). ERK activation was more pronounced upon NRG-1b stimulation in HepG2 cells. By contrast, a stronger activation upon EGF stimulation was observed in HuH7 and Hep3B cells, while PLC/PRF/5 and SNU449 exhibited high basal, but no inducible ERK phosphorylation (Fig. 1B).

Next, HER-3 receptor expression levels were evaluated by western blotting in 11 tumoral and corresponding peritumoral LC tissues. HER-3 expression was revealed to be significantly higher in tumoral than corresponding peritumoral tissues. Notably, HER-3 expression was barely detectable in normal liver tissues (Fig. 1C). All together these data indicated that HER-3 receptor was expressed in LC and that the NRG-1b/HER-3/Akt signalling axis was activated in these cancers, thus reinforcing the hypothesis that HER-3 may represent a suitable target for an ADC-based therapy.

The antitumor activity of EV20/MMAF, an anti-HER-3 ADC, which we have recently revealed to possess a potent and specific therapeutic activity in melanoma and breast cancer models (24,25) was evaluated. Surprisingly, the activity of EV20/MMAF in LC cell lines was revealed to be significantly lower in comparison to the activity observed in non-LC cell lines. In fact, IC₅₀ values ranging between ~25 and ~70 nM were observed for HuH7 and PLC/PRF/5 cells, respectively, whereas an IC₅₀ >100 nM was observed for the other three cell lines assessed (Fig. 2A).

To rule out the possibility that low efficacy of EV20/MMAF in LC cells was due to the impairment of an internalization process of the antibody/receptor complex, the EV20 internalization rate in HepG2 (LC, low responders) vs. A375m cells, which in our previous work were revealed to be markedly sensitive to this ADC (24), were compared. As revealed in Fig. 2B, no significant differences were observed between the two cell lines, indicating that the internalization process was functional in LC cells. Notably, HepG2 sensitivity to free MMAF was not significantly different from that of high-responder A375m melanoma cells (Fig. 2C, left panel) neither was it increased by multidrug resistance-associated protein inhibitors (such as Reversan, PSC833 and MK571 as reported in other systems (27,28) (Fig. 2C, right panel).

To improve the ADC activity, we generated a novel EV20-based ADC (named EV20-sss-vc/MMAF) maintaining the same cytotoxic payload coupled to an engineered variant of EV20 (named EV20-sss) generated by means of a cleavable linker, as described in the method section and in our previous work (26). The results obtained are presented in HIC in Fig. 2D, by detecting at ε280 nm: with naked antibody (DAR =0) as a negative control, eluting with a retention time of 5 min, and present only in the sample of the unconjugated but not in the ADC chromatogram (0%); the antibody conjugated with DAR =1 is present in a small percentage (18%) (retention time 6 min); the antibody conjugated with DAR =2 is present at 7 min (82%). Percentages were obtained by integration of the peak area. Due to the site-specific conjugation process, this ADC had a fixed DAR of 2 or 1 (Fig. 2D). Moreover, EV20-sss-vc/MMAF was revealed to possess the same in vitro and cell binding ability of that observed with the naked EV20-sss (Fig. 2D) as well as receptor downregulatory capacity, while inhibition of ligand-induced HER-3 phosphorylation appeared slightly reduced in conjugated vs. naked EV20 antibody (Fig. 2E).

Notably, EV20-sss-vc/MMAF displayed higher cell killing activity than EV20/MMAF (Fig. 2A), although the two ADCs exhibited superimposable cell killing activity in A375m melanoma cells (Fig. 3A).

Additionally, EV20-sss-vc/MMAF cell killing activity was revealed to be to be strictly target-dependent. This was demonstrated by using HER-3 negative osteosarcoma SjSa-1cells, in which no cell killing could be observed (Fig. 3B) and by a competition assay with a 500-fold molar excess of naked EV20 antibody (Fig. 3C). Finally, the HER-3 low-expressing SNU 449 cells were revealed to be nearly insensitive to the ADC (Fig. 2A).

As HER-2 was revealed to be highly expressed in LC cells, the activity of EV20-sss-vc/MMAF was compared to the activity of the clinically approved anti-HER-2 ADC (T-DM1) in HepG2 cells. Despite the cells expressing similar levels of HER-2 and HER-3 (Fig. 1A), EV20-sss-vc/MMAF exhibited a significantly increased cell killing activity, suggesting this ADC was more effective in comparison to T-DM1 (Fig. 3D).

As HER-3 expression/activation is upregulated in response to several drugs, including sorafenib (11,20,29,30), it was investigated whether the activity of EV20-sss-vc/MMAF could be potentiated by combination with this agent. In line with a previous study (9), treatment of LC cells with sorafenib induced a potent cell killing activity with an IC₅₀ ranging between ~7 and 1 µM (Fig. 4A, left panels). However, cell killing activity was not associated with the upregulation of either HER-3 or HER-2 receptors (Fig. 4A, right panels). Similarly, no upregulation of HER-3 expression was detected in HepG2 cells grown under chronic exposure (up to 5 months) of 2 µM of sorafenib, (Fig. 4B, upper panel) and no increase of EV20-sss-vc/MMAF cell killing activity was observed in these cells (Fig. 4B, lower panel).

The therapeutic activity of EV20-sss-vc/MMAF ADC as a single agent in LC-derived xenografts was evaluated in xenografts assays. Although different methodological approaches were assessed, only mice harbouring tumours derived from subcutaneous injection of HepG2 were available for therapeutic study. As revealed in Fig. 5A, within 6 weeks after the start of administration, the tumor volumes in the EV20-sss-vc/MMAF-treated group were significantly smaller compared with the vehicle-treated group. The growth suppression effect was accompanied by increased survival (Fig. 5B).