ErbB3, a member of the ErbB receptor family, is a potent mediator in the development and progression of cancer, and its activation plays pivotal roles in acquired resistance against anti-EGFR therapies and other standard-of-care therapies. Upon ligand (NRG1) binding, ErbB3 forms heterodimers with other ErbB proteins (i.e., EGFR and ErbB2), which allows activation of downstream PI3K/Akt signaling. In this study, we developed a fully human anti-ErbB3 antibody, named ISU104, as an anticancer agent. ISU104 binds potently and specifically to the domain 3 of ErbB3. The complex structure of ErbB3-domain 3::ISU104-Fab revealed that ISU104 binds to the NRG1 binding region of domain 3. The elucidated structure suggested that the binding of ISU104 to ErbB3 would hinder not only ligand binding but also the structural changes required for heterodimerization. Biochemical studies confirmed these predictions. ISU104 inhibited ligand binding, ligand-dependent heterodimerization and phosphorylation, and induced the internalization of ErbB3. As a result, downstream Akt phosphorylation and cell proliferation were inhibited. The anticancer efficacy of ISU104 was demonstrated in xenograft models of various cancers. In summary, a highly potent ErbB3 targeting antibody, ISU104, is suitable for clinical development.

The ErbB family (EGFR, ErbB2, ErbB3, and ErbB4) belongs to the plasma membrane–embedded receptor family and plays important roles in the development and progression of cancers (1–3). These receptors form active homo- and/or heterodimers upon ligand binding to their ectodomains and induce MEK/MAPK and PI3K/AKT signaling through tyrosine kinase activities (4, 5). Unlike other receptors, ErbB3 does not have kinase activity and forms only heterodimers with EGFR or ErbB2 (6–10).

ERBITUX (cetuximab) and HERCEPTIN (trastuzumab), targeting EGFR and ErbB2, respectively, have been used as anticancer therapeutics (11–14). Although these drugs can provide clinical benefits to some patients, a large proportion of patients are not responsive or soon become resistant to these drugs (15–17). To overcome these limitations, combination therapies targeting multiple ErbB family proteins are being attempted.

As ErbB3 is known for its relevance to acquired resistance to standard-of-care therapeutics, it has become a new treatment target in patients with cancer. However, currently, there is no marketed treatment that targets ErbB3. Multiple studies have reported that continuous exposure to EGFR- or ErbB2-targeting treatments, or chemotherapeutics (e.g., paclitaxel and tamoxifen), leads to the activation of ErbB3/PI3K/Akt bypass signals through an increase in NRG1 (ligand of ErbB3), and thereby induces resistance to these therapeutics (18–21). Moreover, overexpression of ErbB3 in the head and neck (HNSCC), breast, lung, gastric, ovarian, colon, prostate, and bladder cancers (22, 23) has been shown to be related to low survival rate and cancer recurrence (24–26). In other words, if an ErbB3-targeting mAb drug is used as a combination partner with existing standard treatments or as a secondary treatment, it is expected to provide clinical benefits to the nonresponsive/tolerant patient group by blocking the alternative survival signal through ErbB3.

Here, we developed a human monoclonal anti-ErbB3 (ISU104) with high affinity, selectivity, and potency for clinical use. The mechanism of action and the preclinical efficacy of ISU104 were explored in this study.

Cell lines

All cell lines used in this study were sourced from the ATCC. Cells were cultured in RPMI1640 Medium (Invitrogen) or Eagle Minimum Essential Medium (Invitrogen) containing 10% FBS (Invitrogen) at 37°C in a water-saturated atmosphere containing 5% CO2.

Anti-human ErbB3 antibody generation

A synthetic human single-chain variable fragment (scFv) phage library (27) was screened with Magnetic Beads (Dynabeads, M-270) coated with recombinant human ErbB3 extracellular domain (ECD). Phages bound to the beads were eluted with 100 mmol/L citrate and amplified in Escherichia coli for the next round of panning. Five rounds of panning were performed to enrich ErbB3-binding phages. ErbB3-specific phage clones were identified by ELISA using plates coated with human ErbB3 ECD at 1 μg/mL in PBS, pH 7.4, with the selection criterion being positive binding to human ErbB3. The variable light (VL) chain and variable heavy (VH) chain from positive phage clones were generated by PCR and inserted into a human IgG1 expression vector (Selexis SA). These IgG1-formated antibody leads were further screened in cell-based functional assays to select the most potent lead suppressing ligand-induced ErbB3 activity. On the basis of efficacy data, multiple functional antibodies were selected and subjected to affinity maturation, production, and characterization. Among them, a clone (clone No. 442S1) was selected as a final candidate and named ISU104 (patent No. US10413607, seq. ID No. 2 and No. 32; see Supplementary Table S1 for sequential and Kabat numbering).

FACS

Cells were treated with 10 μg/mL ISU104. Unbound antibodies were removed, and the cells were labeled with anti-human IgG-FITC (1:100). ISU104 binding was determined by FACS analysis using FITC fluorescence intensity.

Binding ELISA with ErbB3 from other species

Human ErbB3 ECD (R&D Systems), mouse ErbB3 ECD (R&D Systems), rat ErbB3 ECD (Sino Biologicals), and monkey ErbB3 (Sino Biologicals) were coated on 96-well plates (Nunc) and blocked with 5% BSA in PBS. ISU104 was titrated and incubated on plates containing each immobilized protein in the presence of 5% BSA in PBS. Plates were washed with PBST, incubated with a horseradish peroxidase (HRP)-linked secondary goat anti-human antibody, washed again, and developed with TMB Substrate (Thermo Fisher Scientific).

Binding ELISA with other ErbB families

Protein purification of human EGFR ECD was performed as described previously (28). Human ErbB2 ECD was engineered to contain a His-tag at the C-terminal and was expressed in HEK293 cells. Human ErbB2 ECD was purified over Ni-NTA Resin (GE Healthcare) and eluted with an imidazole step gradient in PBS.

Human EGFR ECD and human ErbB2 ECD were coated on 96-well plates (Nunc) and blocked with 5% BSA in PBS. ISU104, and commercially available cetuximab and trastuzumab, were serially diluted and added to the plate. Plates were washed with PBST, incubated with an HRP-linked secondary goat anti-human antibody, washed again, and developed with TMB Substrate (Thermo Fisher Scientific).

Surface plasmon resonance

ISU104 and ISU104-Fab were diluted to six concentration levels (ISU104 ranged from 6.25 to 0.19 nmol/L and ISU104-Fab ranged from 1,000 to 62.5 nmol/L). Recombinant human ErbB3 ECD-Fc proteins (R&D Systems) were immobilized on the surface of a CM5 chip and ISU104 or ISU104-Fab was injected over the surface at a constant flow rate (Osong Medical Innovation Foundation). Changes in mass concentrations as a consequence of association and dissociation between the molecules (Kon and Koff, respectively) were measured in real time using Biacore T200 (GE Healthcare). To analyze KD value, curve fitting was performed using a model assuming 1:1 binding ratio between molecules. In addition, a bivalent analyte model was applied to determine whether the χ2 value (reliability of the fitting) met the maximum response (Rmax) of ≤10% in a 1:1 model analysis. The binding avidity and affinity (KD value) of ISU104 and ISU104-Fab to recombinant human ErbB3-Fc were measured in this manner.

Epitope mapping by the interaction with EGFR/ErbB3 hybrid domain mutants

Twelve EGFR/ErbB3 hybrid domain mutants and two wild-type ECD proteins (ErbB3 and EGFR) were engineered to contain a human Fc-tag at their C-termini and were expressed in 293F cells (Invitrogen) and subsequently purified using MabSelect Resin (GE Healthcare). Bio-layer interferometry (BLI) experiments were performed using OCTET QK384 (ForteBio). ISU104 was immobilized on an amin reactive second-generation sensor chip using an amine-coupling method. ECD hybrid mutants and two wild-type ECD proteins (0, 0.4, 2, and 10 nmol/L) were loaded onto a 384-well plate.

Epitope mapping by hydrogen/deuterium exchange mass spectrometry

Hydrogen/deuterium exchange mass spectrometry (MS) was performed to determine a specific epitope of ISU104 on the ErbB3 (Osong Medical Innovation Foundation). A complex of ErbB3 ECD (16 μmol/L) and ISU104 (16 μmol/L) was prepared and preincubated at 4°C for 3 hours. ErbB3 ECD and the ErbB3 ECD-ISU104 complex were diluted with D2O buffer (1 μmol/L), respectively. Both of them were deuterated for 0.33, 1, 10, 60, and 240 minutes, and then the reaction was terminated using a quenching buffer. After the reaction, the proteins were digested with pepsin and loaded onto a chromatographic system to isolate individual peaks before MS analysis. The mean differences in deuterium levels were calculated between the respective peptides from the ErbB3 ECD and the ErbB3 ECD complexed with ISU104. A difference of ≥0.5 Da between them was considered significant. As the exchange rate depends on protein conformation, the regions with significant differences in deuterium levels were considered to be involved in protein–protein interactions.

Production of ErbB3-domain 3

Human ErbB3-domain 3, corresponding to amino acid residues 309–500, was amplified using PCR and subcloned into a baculovirus expression vector consisting of an N-terminal gp67 signal sequence, a deca-histidine tag, maltose binding protein, and tobacco etch virus protease cleavage site. ErbB3-domain 3 was expressed by insect cells using a Bac-to-Bac Baculovirus Expression System (Invitrogen). Three days post-infection, the medium was collected by centrifugation and filtered. The medium was loaded into a HisTrap Excel Column (GE Healthcare) and washed with a buffer containing 20 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, and 20 mmol/L imidazole. Bound ErbB3-domain 3 was eluted with 20 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, and 300 mmol/L imidazole. The tobacco etch virus recognition site was cleaved using tobacco etch virus protease for 16 hours at 4°C. After desalting using 20 mmol/L Tris-HCl (pH 7.5) and 50 mmol/L NaCl, the ErbB3-domain 3 protein was loaded into a Cation Exchange Chromatography Column (Hitrap SP HP, GE Healthcare) and eluted with 20 mmol/L Tris-HCl (pH 7.5) and 150 mmol/L NaCl. The protein was deglycosylated using Endo H (Promega) for 16 hours at 4°C.

Production of ISU104-Fab

ISU104 antibody was cleaved into Fc and Fab domains using papain protease for 1 hour at 37°C. Next, to deactivate papain protease activity, iodoacetamide was added to a final concentration of 10 mmol/L and incubated for 20 minutes at 20°C. Using Protein A Column (GE Healthcare), ISU104-Fab was collected from the flow through.

Crystallography

Equal amounts of purified ErbB3-domain 3 and ISU104-Fab dissolved in HEPES (pH 7.5) and 100 mmol/L NaCl were subjected to gel filtration (HiLoad 16/600 Superdex 200pg, GE Healthcare). ErbB3-domain 3 and ISU104-Fab complex proteins found at the same elution fraction were concentrated to 10 mg/mL. Crystallization screening was performed with a Crystallization Kit (Hampton Research) and the Mosquito Machine (TTP Labtech) using a sitting-drop vapor-diffusion method. Complex protein crystals with a trigonal pyramidal shape appeared in the solution containing 0.2 mol/L sodium acetate and 20% polyethylene glycol 3350 within 3 months. The complex crystals were cryoprotected by the mother liquor with 20% ethylene glycol. X-ray diffraction of the complex crystals was performed at the Pohang Accelerator Laboratory using BL-11C beamline.

The diffraction datasets were integrated and scaled using XDS (29). The complex crystal belonged to the space group P1 21 1, with unit cell parameters of a = 92.3 Å, b = 77.8 Å, and c = 93.9 Å, and α = 90°, β = 113.4°, and γ = 90°. The complex structure was solved by molecular replacement with PHASER using the ErbB3 structure (PDB ID code: 1M6B) and the Fab structure (PDB ID code: 3P11; refs. 30–32). For structure analysis, sequential numbering was employed for the heavy and light chains of ISU104. PHENIX (v1.18.2) and COOT (v0.8.9.2) were used for structural refinement (PMID: 31588918 and 15572765). All figures were prepared using PyMOL (The PyMOL Molecular Graphics System, version 2.0 Schrödinger, LLC; refs. 33–35).

NRG1-ErbB3 binding assay

The Octet System (Octet QK384, Fortébio) was used to detect ErbB3/NRG1 binding and its inhibition by ISU104. With NRG1 immobilized on the surface of Amine Reactive Second-generation Biosensors (Fortébio), human ErbB3 ECD-Fc protein (50 nmol/L) and ISU104 (0, 3, 30, and 300 nmol/L) were loaded onto a 384-well plate. NRG1 and human ErbB3 were purchased from R&D Systems. An ISU104-free reactant was used as a negative control. ErbB3/NRG1 binding (%) was calculated in the presence of ISU104 drug substance versus a negative control.

ErbB2/ErbB3 heterodimerization

The inhibitory effect of ISU104 at the cell level was identified by establishing a method of detecting ErbB3/ErbB2 dimer levels in MDA-MB-453 breast cancer cells using immunoprecipitation. MDA-MB-453 cells were inoculated onto a 6-well plate and incubated under serum starvation for 24 hours, followed by treatment with ISU104 (1 μg/mL) and NRG1 (100 ng/mL). Lysis buffer (containing protease and phosphatase inhibitor) was added to each well and the proteins collected from the cell lysate were incubated with anti-ErbB3 (Cell Signaling Technology; clone 1B2) and protein-A agarose (Cell Signaling Technology). The amount of ErbB2 and ErbB3 isolated with anti-ErbB3 antibody was detected by Western blotting using anti-ErbB2 (Cell Signaling Technology; clone D8F12) and anti-ErbB3 antibody (Merck; clone 2F12), respectively.

Cell survival signaling

FaDu (HNSCC) cells were seeded in a 12-well plate and incubated for 24 hours. Following this step, the medium was replaced with serum-free medium, followed by a further 24-hour incubation. The cells were treated with ISU104 (10 μg/mL) and NRG1 (100 ng/mL), and incubated again for 24 hours. After incubation, cells were collected and lysed using lysis buffer and total protein was quantitated and subjected to SDS-PAGE analysis using the same amount of each protein sample. Western blotting was performed using phosphorylated-S6 and S6 antibodies (Cell Signaling Technology) to compare the intensity of phosphorylated-S6 bands between antibody-free and antibody-treated samples. Human IgG1 (10 μg/mL) was used as a negative control.

ErbB3 and Akt phosphorylation

Cancer cells were inoculated onto a 48-well plate and incubated under serum starvation for 24 hours. Thereafter, these cells were treated with ISU104 at various concentrations (0.00025, 0.001, 0.004, 0.016, 0.065, 0.26, 1.04, 4.17, 16.7, and 66.7 nmol/L) for 2 hours at 37°C, followed by treatment with NRG1 (100 ng/mL) for 15 minutes at 37°C. After NRG1 stimulation, cells were washed twice with cold PBS and lysed with Cell Lysis Buffer (Cell Signaling Technology) containing protease inhibitors on ice for 1 hour. ErbB3 or Akt phosphorylation levels in the collected total protein lysates were measured using PathScan Phospho-HER3/ErbB3 (panTyr) Sandwich ELISA Kit (Cell Signaling Technology) and PathScan Phospho-Akt1 (Ser473) Sandwich ELISA Kit (Cell Signaling Technology). In each cancer cell line, the IC50 values of ISU104 against pErbB3/pAkt1 were calculated.

In vitro inhibition of cell proliferation

Cancer cell lines, including BxPC3 (pancreas) and SKBR3 (breast), were seeded in 96-well plates and incubated. The next day, to induce NRG1-dependent cell proliferation (BxPC3), the plating medium was removed and ISU104 and NRG1 (100 ng/mL) mixture in a medium containing 0.1% FBS was added. Plates were then incubated in 5% CO2 at 37°C for 3 days. SKBR3 cells were used as ligand-independent models. To induce NRG1-independent cell proliferation (SKBR3), the plating medium was removed and an antibody in complete medium was added. Plates were then incubated in 5% CO2 at 37°C for 5 days. Equal volumes of CellTiter-Glo Reagent (Promega) were added to each well at the end of each timepoint. Plates were rocked on a plate shaker for 10 minutes at 20°C to ensure complete cell lysis. Luminescence was measured using Victor 3 (PerkinElmer).

ErbB3 internalization assay

MDA-MB-468 breast cancer cells were treated with ISU104 (10 μg/mL) for 6 hours. After treatment, the extracellular membrane proteins were biotin labeled and the biotinylated proteins were separated using avidin-conjugated agarose resin. The content of ErbB3 protein in the presence of ISU104 was determined via Western blotting using an ErbB3-specific antibody.

MDA-MB-453 cells were resuspended in serum-free medium for 5 minutes. Cells were then incubated with 10 μg/mL ISU104 on ice. Following a 60-minute incubation on ice, cells were washed with a FACS buffer (PBS, pH 7.4, containing 0.05% BSA and 0.02% NaN3). The samples were then incubated at 37°C for 60 minutes. Goat anti-human ErbB3/HER3 antibody (1 μg/mL, R&D Systems; catalog No. AF234) was added at 1 μg/mL and incubated for 60 minutes on ice. ISU104 was confirmed to have no negative impact (epitope masking) on the binding of the goat anti-ErbB3 to ErbB3 (Supplementary Fig. S1). The detection antibody was confirmed to have no negative impact (epitope masking) on the binding of ISU104 to ErbB3. FITC-conjugated rabbit anti-goat IgG antibody was added at 1:100 and incubated for 30 minutes on ice for analysis using flow cytometry. The cells were washed with FACS buffer and analyzed using a Beckman Coulter Gallios Flow Cytometer (Beckman Coulter). Data collected from the experiments were analyzed using Kaluza (analysis software from Beckman Coulter).

Mouse xenograft models

Female Balb/c nude or NOD/SCID mice 5 to 8 weeks of age were implanted with either A431, A549, BxPC3, CAL27, LoVo, MDA-MB-468, BT474, FaDu, or ZR-75-1 cells. All cells were suspended in 50% Matrigel/PBS, except for A549, BxPC3 and FaDu, which were suspended in PBS alone. Xenografts were established by subcutaneously injecting 5 × 106 (A431, MDA-MB-468, A549, BxPC3, FaDu, and CAL27) or 1 × 107 cells (LoVo) per mouse. Breast carcinoma xenografts were established orthotopically by injecting 0.5 × 107 and 1 × 107 of ZR-75-1 and BT474 cells, respectively, per mouse into the fat pad. For BT474 and ZR-75-1, estrogen pellets were placed under the skin of the left flank 1 to 5 days prior to cell implantation. Mice were staged and randomized into 10 mice per group when tumors reached 200 to 300 mm3. ISU104 was prepared by dilution with PBS. All mice treated with antibody therapeutics were dosed intravenously twice a week according to body weight (10 mg/kg). Both tumor size and body weight were measured twice weekly and tumor volume was calculated using the equation (L × W2)/2, where L and W refer to the length and width dimensions, respectively. All animal studies were reviewed and approved by the Institutional Animal Care and Use Committees of contracted research organizations.

Development of anti-ErbB3 antibody, ISU104

ISU104 is a human anti-ErbB3 antibody that shows anticancer effects through selective binding and inhibition of ErbB3. To obtain a human anti-ErbB3 antibody that inhibits ErbB3 activity, a synthetic human scFv phage library was screened. The scFv fragments that selectively bound to ErbB3 were acquired. On the basis of the scFv sequence, lead antibodies were produced in the form of IgG1. Through an optimization process, ISU104, a final candidate with improved affinity, physical stability, and potency was selected. CHO-K1 cells were transfected with the ISU104 expression vector and selected by antibiotic resistance. Multiple rounds of single-cell cloning using ClonPix were performed to ensure the clonality of ISU104-expressing cells. Master cell banks and working cell banks are generated in a GMP facility. ISU104 expressed by CHO-K1 cells was purified using multiple chromatographic procedures.

Specificity, selectivity, and cross-reactivity

To examine the specific binding of ISU104 to cellular ErbB3, FACS analysis was performed. ISU104 bound to the four different ErbB3(+) cells (ZR-75-30, ZR-75-1, MDA-MB-468, and FaDu), but not to ErbB3(−) (A498) cells (Fig. 1A). The binding affinity of ISU104-Fab and the avidity of ISU104 to recombinant human ErbB3 protein, measured by surface plasmon resonance assay, were very high (KD, <75 and 23 pmol/L, respectively; Fig. 1B). ISU104 showed interspecies cross-reactivity; similar avidities to recombinant human, mouse, rat, and monkey ErbB3 were represented by the dose-dependent binding profiles and similar EC50 values (∼20 pmol/L) as measured by ELISA (Fig. 1C). The cross-reactivity analysis using BLI showed similar results (Supplementary Table S2). While ISU104 bound to ErbB3, it did not bind to other members of the ErbB family (i.e., EGFR or ErbB2), demonstrating its selectivity (Fig. 1D).

Figure 1.

ISU104 binds to domain 3 of ErbB3 with high affinity/avidity, specificity, and selectivity. A, Binding of fluorescently conjugated ISU104 to ErbB3-expressing cancer cells. B, Kinetic value of ISU104 and ISU104-Fab binding to human ErbB3-ECD measured by surface plasmon resonance analysis. C, Cross-reactivity of ISU104. D, No binding of ISU104 to EGFR or ErbB2. E, Domain 3 of ErbB3 is required for ISU104 binding. F, The two peptides (374-ITGYLNIQ-381 and 405-YNRGFSLL-412) in domain 3 were hindered by ISU104 from hydrogen-deuterium exchange.

Figure 1.

ISU104 binds to domain 3 of ErbB3 with high affinity/avidity, specificity, and selectivity. A, Binding of fluorescently conjugated ISU104 to ErbB3-expressing cancer cells. B, Kinetic value of ISU104 and ISU104-Fab binding to human ErbB3-ECD measured by surface plasmon resonance analysis. C, Cross-reactivity of ISU104. D, No binding of ISU104 to EGFR or ErbB2. E, Domain 3 of ErbB3 is required for ISU104 binding. F, The two peptides (374-ITGYLNIQ-381 and 405-YNRGFSLL-412) in domain 3 were hindered by ISU104 from hydrogen-deuterium exchange.

Close modal

ISU104 interaction with the domain 3 of ErbB3

To map the ISU104 binding region in ErbB3, we generated various ErbB3 and EGFR ECD hybrid mutants by swapping single or multiple equivalent domains. A total of 12 ECD hybrid mutants and two wild-type ECD proteins (ErbB3-ECD and EGFR-ECD) were produced and analyzed for their binding to ISU104 using BLI analysis (Supplementary Table S3). As expected, ISU104 bound to wild-type ErbB3-ECD, but not to EGFR-ECD. Among the 12 ErbB3-EGFR hybrid domain mutants, all the six mutants with the domain 3 of ErbB3, but not the other six mutants without the domain 3, were bound by ISU104 (Fig. 1E; Supplementary Fig. S2). This result indicates that the epitope of ISU104 resides mainly in domain 3 of ErbB3.

Hydrogen/deuterium exchange MS was applied to further determine the epitope of ISU104 in ErbB3. ErbB3-ECD and ErbB3-ECD::ISU104 protein complex were deuterated by incubating in D2O solution. Deuterium uptake into the polypeptides of ErbB3 was measured by MS after pepsin digestion. Compared with the corresponding peptides from ErbB3-ECD, there were notable (>1 Da) decreases in deuterium uptake levels in the two peptides from the ErbB3-ECD::ISU104 complex, which corresponds to amino acid residues 374–381 and 405–412 located in the domain 3. This result demonstrated that ISU104 binds the domain 3 of ErbB3 and masks deuterium uptake (Fig. 1F).

ErbB3-domain 3::ISU104-Fab complex structure

To figure out the molecular mechanism of ISU104, we elucidated the complex structure of ErbB3-domain 3 and ISU104-Fab at a resolution of 2.5 Å (PDB ID: 7D85; Fig. 2A). Trigonal pyramidal crystals were obtained through crystallization under the conditions of 0.2 mol/L sodium acetate and 20% (w/v) PEG3350 within 3 months (Supplementary Fig. S3A). X-ray diffraction experiments for the crystal were conducted at the Pohang Accelerator Laboratory (Supplementary Fig. S3B). The ErbB3-domain 3 structure was determined using molecular replacement methods using the domain 3 of the previously solved ErbB3 structure (32). The crystal belonged to the P21 space group, and two ErbB3-domain 3 molecules and two ISU104-Fab molecules can be found in the asymmetric unit. The ErbB3-domain 3 showed a difference in root-mean-square deviation of approximately 1.4 Å compared with that of the previously solved ErbB3 structure (PDB ID: 1M6B). When the complex structure was superimposed on the ErbB3 tethered structure, ISU104-Fab did not collide or interact with other domains (Fig. 2B), which is consistent with the domain swapping data (Fig. 1E). The buried surface area of ErbB3 was 919 Å2 by ISU104-Fab, where the VH and VL chain regions of Fab contributed 498 Å2 and 476 Å2, respectively (Fig. 2C and D; ref. 36).

Figure 2.

Structural analysis of the interaction between ErbB3 and ISU104. A, Crystal structure of the ErbB3-domain 3::ISU104-Fab complex (PDB ID: 7D85). B, Superimposition results of previously reported ErbB3 ECD and ISU104-Fab complex on tethered ErbB3 surface (PDB ID: 1M6B). C, Open-book representation of ErbB3-domain 3::ISU104-Fab complex. The epitope of ISU104-Fab is highlighted either as a blue surface [recognized by Fab heavy chain (HC)] or a pink surface [recognized by Fab light chain (LC)] within the ErbB3-domain 3 surface. Similarly, the paratope of ISU104-Fab is denoted as a blue surface (Fab HC) and a pink surface (Fab LC) on the surface of ISU104-Fab. D, Detailed epitope residues in ErbB3 that interact with ISU104-Fab. The epitope recognized by Fab HC is colored blue, and that recognized by Fab LC is shown in red. E, Multiple interactions of ErbB3 Arg407 residue with ISU104-Fab. The guanidinium group of ErbB3 Arg407 interacts with the CDR (HC, Asp33H, Asp99H, Thr50H, and Ser35H and LC, Trp92L and His99L) of ISU104-Fab through a hydrogen bond or salt bridge (red dashed line). F, Hydrophobic interactions on the buried surface area between ErbB3-domain 3 and ISU104 Fab. Met102H of the Fab HC is buried on the hydrophobic core (arc line). ErbB3 Tyr436 forms a hydrogen bond with His101H of the Fab HC (red dashed line). All H superscripts represent HC residues of ISU104-Fab; all L superscripts represent LC residues of ISU104-Fab.

Figure 2.

Structural analysis of the interaction between ErbB3 and ISU104. A, Crystal structure of the ErbB3-domain 3::ISU104-Fab complex (PDB ID: 7D85). B, Superimposition results of previously reported ErbB3 ECD and ISU104-Fab complex on tethered ErbB3 surface (PDB ID: 1M6B). C, Open-book representation of ErbB3-domain 3::ISU104-Fab complex. The epitope of ISU104-Fab is highlighted either as a blue surface [recognized by Fab heavy chain (HC)] or a pink surface [recognized by Fab light chain (LC)] within the ErbB3-domain 3 surface. Similarly, the paratope of ISU104-Fab is denoted as a blue surface (Fab HC) and a pink surface (Fab LC) on the surface of ISU104-Fab. D, Detailed epitope residues in ErbB3 that interact with ISU104-Fab. The epitope recognized by Fab HC is colored blue, and that recognized by Fab LC is shown in red. E, Multiple interactions of ErbB3 Arg407 residue with ISU104-Fab. The guanidinium group of ErbB3 Arg407 interacts with the CDR (HC, Asp33H, Asp99H, Thr50H, and Ser35H and LC, Trp92L and His99L) of ISU104-Fab through a hydrogen bond or salt bridge (red dashed line). F, Hydrophobic interactions on the buried surface area between ErbB3-domain 3 and ISU104 Fab. Met102H of the Fab HC is buried on the hydrophobic core (arc line). ErbB3 Tyr436 forms a hydrogen bond with His101H of the Fab HC (red dashed line). All H superscripts represent HC residues of ISU104-Fab; all L superscripts represent LC residues of ISU104-Fab.

Close modal

The epitope of ISU104 was widely distributed in the domain 3 of ErbB3. The most significant interaction was Arg407 of ErbB3 deeply inserted into the space between the heavy and light chains of ISU104-Fab with Asp33H, Ser35H, Thr50H, Asp99H, Trp92L, and His99L (superscripts H and L represent the heavy and light chain of ISU104-Fab, respectively; Fig. 2E and F; Supplementary Table S5). Interestingly, a cleft exists between the VH and VL chains of the ISU104-Fab (Supplementary Fig. S4C and S4D). The ErbB3 Arg407 residue is buried deep in the cleft, forming strong interactions (Fig. 2E; Supplementary Fig. S4C and S4D). First, the guanidinium group of Arg407 interacts by ionic interactions with Asp33H and Asp99H. In addition, Arg407 forms weak hydrogen bonds with Ser35H and Thr50H. Trp92L of the VL is located alongside the backbone to β, γ carbon of Arg407, and the side chain of Arg407 positioned as guidance. The ϵ-nitrogen of Trp92L forms a hydrogen bond with Arg407 carbonyl oxygen. Finally, VL His99L forms hydrogen bonds with the ϵ-nitrogen of Arg407.

In contrast, Met102H deeply penetrates the domain 3, forming hydrophobic interactions with Phe409, Leu412, Met414, and Tyr436 of ErbB3 (Fig. 2F). These residues are located in the center of the whole complex structure. As hydrophobic interactions exclude water molecules, the stability of interactions is enhanced. In addition, Tyr436 of ErbB3 forms a hydrogen bond with His101H.

Mechanism of action of ISU104

ErbB3 is activated by ligand binding to the domains 1 and 3, which breaks interdomain tethering between the domain 2 and domain 4, allowing a 130° rotation of domains 1 and 2 about a pivot point between the domains 2 and 3. The dimerization loop in the domain 2 of ErbB3 is then exposed to induce heterodimerization through interaction with the domain 2 of other ErbB family proteins (EGFR, ErbB2, and ErbB4). Heterodimerization allows asymmetric dimerization of cytosolic kinase domains, inducing trans-phosphorylation.

Although no crystal structure of ErbB3 in complex with NRG1 has been reported till date, the ligand-binding sites can be predicted (Fig. 3A; Supplementary Movie S1), which is supported by the previous small-angle X-ray scattering experiment of NRG1 and ErbB3 (37). The predicted ligand-binding regions within the domain 3 overlapped with the regions recognized by ISU104-Fab (Fig. 3B). Accordingly, ISU104 directly prevented ligand binding to ErbB3 (Supplementary Movie S2). In addition, binding of ISU104 with the domain 3 would occupy the space between the domain 1 and domain 3 and, thereby interfere with the rotation of the domains 1 and 2 by steric hindrance, resulting in inhibition of heterodimerization. Consequently, this would block the ErbB3-mediated growth signaling cascade in cancer cells (Fig. 3C).

Figure 3.

ISU104 blocks NRG1 binding to ErbB3 directly and inhibits conformational changes for dimerization. A, Predicted NRG binding site (yellow surface) on the ErbB3-domain 3 surface (white surface) based on modeling of interaction between NRG (orange cartoon) and ErbB3. Modeling was performed on the basis of the crystal structure of ErbB4 in complex with NRG1 (PDB ID: 3U7U). B, Overlap between ISU104 epitope and NRG1 binding site. The buried surface area of ISU104-Fab is colored blue, and the NRG1 binding site is colored yellow. The overlapping site is colored red. C, Schematic representation of the mechanism of action of ISU104 inhibiting both ligand binding to ErbB3 and heterodimerization with other ErbB family proteins. Tethered ErbB3 ECD is stretched by binding to NRG1 (yellow; the NRG1 binding site is indicated as a red star). Fully activated ErbB3 interacts with EGFR or ErbB2 to form a heterodimer complex (black arrow). As a result, cellular signal transduction is induced by these complexes. However, ISU104-Fab (brown) is a competitive inhibitor that binds to the NRG binding site within ErbB3-domain 3, and, thus, prevents the binding of NRG (red arrow). Conclusively, the tethered conformation of ErbB3 is forced by ISU104-Fab, which blocks the formation of ErbB3 heterodimeric complex with other members of the EGFR family.

Figure 3.

ISU104 blocks NRG1 binding to ErbB3 directly and inhibits conformational changes for dimerization. A, Predicted NRG binding site (yellow surface) on the ErbB3-domain 3 surface (white surface) based on modeling of interaction between NRG (orange cartoon) and ErbB3. Modeling was performed on the basis of the crystal structure of ErbB4 in complex with NRG1 (PDB ID: 3U7U). B, Overlap between ISU104 epitope and NRG1 binding site. The buried surface area of ISU104-Fab is colored blue, and the NRG1 binding site is colored yellow. The overlapping site is colored red. C, Schematic representation of the mechanism of action of ISU104 inhibiting both ligand binding to ErbB3 and heterodimerization with other ErbB family proteins. Tethered ErbB3 ECD is stretched by binding to NRG1 (yellow; the NRG1 binding site is indicated as a red star). Fully activated ErbB3 interacts with EGFR or ErbB2 to form a heterodimer complex (black arrow). As a result, cellular signal transduction is induced by these complexes. However, ISU104-Fab (brown) is a competitive inhibitor that binds to the NRG binding site within ErbB3-domain 3, and, thus, prevents the binding of NRG (red arrow). Conclusively, the tethered conformation of ErbB3 is forced by ISU104-Fab, which blocks the formation of ErbB3 heterodimeric complex with other members of the EGFR family.

Close modal

ISU104 inhibits ligand binding to ErbB3 and dimerization of ErbB2-ErbB3, and induces ErbB3 internalization

As the crystallographic results indicated the potential inhibition of ligand binding and heterodimerization by ISU104, we performed biochemical assays to confirm these predictions. We first established a BLI-based assay that measures the interaction between NRG1 and ErbB3. NRG1 was immobilized on sensor chips and a recombinant ErbB3-ECD protein solution was loaded. The real-time interactions between them were monitored and reported as sensorgrams. When ErbB3-ECD (50 nmol/L) was preincubated with ISU104 (0, 3, 30, and 300 nmol/L), ErbB3-ECD interactions with NRG1 decreased in an ISU104 concentration–dependent manner (Fig. 4A).

Figure 4.

ISU104 prevents ErbB3 signaling and cancer cell proliferation by inhibiting ligand binding, heterodimerization between ErbB2 and ErbB3, and ErbB3 internalization. ISU104 prevents binding of NRG1 to ErbB3 (A) and NRG1-induced heterodimerization between ErbB3 and ErbB2 in cells (B). ISU104 binding induced internalization of ErbB3 as demonstrated by the reduction in ErbB3 levels in plasma membrane: FACS (C) and Western blotting (biotin-labeled plasma membrane cell extract; D). E and F, Inhibition of NRG1-induced activation of PI3K–AKT signaling. NRG1-dependent (G) and -independent cell proliferation (H). IB, immunoblot; IP, immunoprecipitation.

Figure 4.

ISU104 prevents ErbB3 signaling and cancer cell proliferation by inhibiting ligand binding, heterodimerization between ErbB2 and ErbB3, and ErbB3 internalization. ISU104 prevents binding of NRG1 to ErbB3 (A) and NRG1-induced heterodimerization between ErbB3 and ErbB2 in cells (B). ISU104 binding induced internalization of ErbB3 as demonstrated by the reduction in ErbB3 levels in plasma membrane: FACS (C) and Western blotting (biotin-labeled plasma membrane cell extract; D). E and F, Inhibition of NRG1-induced activation of PI3K–AKT signaling. NRG1-dependent (G) and -independent cell proliferation (H). IB, immunoblot; IP, immunoprecipitation.

Close modal

Next, we examined whether ISU104 can inhibit heterodimerization between ErbB2 and ErbB3 in the breast cancer cell line, MDA-MB-453. Increased interactions between cellular ErbB2 and ErbB3 were observed upon NRG1 treatment (100 ng/mL) in a coimmunoprecipitation assay, but cotreatment of NRG1 with ISU104 (1 μg/mL) inhibited the interactions (Fig. 4B).

ErbB family receptors, including ErbB3, expressed on cell surfaces can be internalized upon binding by ligands or targeting antibodies. The internalization of receptors was determined by measuring changes in receptor expression on cell surfaces following antibody binding. ErbB3-expressing MDA-MB-453 cells were exposed to ISU104 at a concentration of 10 μg/mL and ErbB3 levels on the cell surface were measured by FACS analysis. Contrary to the control (hIgG1), ISU104 treatment reduced ErbB3 signal intensities on the cell surface by 47% (Fig. 4C). To directly measure ErbB3 levels associated with the plasma membrane, plasma membrane fractions were isolated and subjected to Western blot analysis. ErbB3 levels were decreased by 50% compared with those in the two negative controls (no treatment and hIgG1; Fig. 4D).

ISU104 inhibits ligand-dependent and -independent cell signaling and proliferation

NRG1 binding to ErbB3 induced phosphorylation of ErbB3 and its downstream signaling molecules, such as Akt1 and S6 (Fig. 4E). As expected, ISU104 effectively suppressed the activation of NRG1-dependent signaling, as shown by the decrease in pErbB3, pAkt1, and pS6 in FaDu cells. To investigate the efficacy of ISU104 in various cancer cell lines, inhibition of ErbB3 and Akt1 phosphorylation by ISU104 was measured using sandwich ELISA, and IC50 value was calculated (Fig. 4F; Supplementary Table S6). The IC50 values for pErbB3 and pAkt1 ranged from 0.06 to 0.46 and 0.21 to 0.96 nmol/L, respectively. Maximal inhibition was observed at concentrations of 1 to 10 nmol/L of ISU104.

ErbB3 signaling is important for cancer cell proliferation and survival. The inhibitory effect of ISU104 on cell proliferation was examined using BxPC3 and SKBR3 cell lines, which are models for NRG1-dependent and -independent cell proliferation, respectively (Fig. 4G and H). BxPC3 cell proliferation (measured by cell viability) induced by NRG1 was inhibited by ISU104 in a dose-dependent manner. Up to 50% inhibition was observed at a maximum concentration of 333 nmol/L. SKBR3 cells cultured in media containing 10% FBS were treated with 0.0067, 0.067, 0.67, and 6.7 nmol/L ISU104. Up to 40% inhibition of cell proliferation was observed at a maximum concentration of 6.7 nmol/L.

Potent tumor growth inhibition by ISU104 in various xenograft models

In vivo efficacy of ISU104 was investigated using nine different cancer cell–derived xenograft models, including HNSCC, pancreatic, lung, skin, colon, and breast cancers, which are known to be related to ErbB3 oncogenic signaling. From the tumor models, the most superior tumor growth inhibitory effect was observed in the HNSCC models (FaDu model, tumor regression and CAL27 model, 93% inhibition; Fig. 5). In the BxPC3 pancreatic cancer model, 84% inhibition was observed, followed by 81% in the MDA-MB-468 breast cancer model, 73% and 70% each in the A549 lung cancer and BT474 breast cancer models, respectively. In contrast, lower inhibition effects of 59%, 33%, and 1% were seen in the A431 skin cancer, LoVo colon cancer, and the ZR-75-1 breast cancer models, respectively. These in vivo efficacy data suggest that ISU104 is applicable to treat patients with various cancer types, including HNSCC.

Figure 5.

Potent tumor growth inhibition (TGI) by ISU104 in various xenograft models. ISU104 treatment was started when the tumor size reached 200–300 mm3. Animals were dosed intravenously twice weekly at 10 mg/kg ISU104. TGI% is calculated using the following equation: TGI% = 100 − (average tumor volume in drug treatment group)/(average tumor volume in vehicle treatment group) × 100.

Figure 5.

Potent tumor growth inhibition (TGI) by ISU104 in various xenograft models. ISU104 treatment was started when the tumor size reached 200–300 mm3. Animals were dosed intravenously twice weekly at 10 mg/kg ISU104. TGI% is calculated using the following equation: TGI% = 100 − (average tumor volume in drug treatment group)/(average tumor volume in vehicle treatment group) × 100.

Close modal

In this study, we discovered and developed a novel therapeutic, fully human monoclonal anti-ErbB3, ISU104. ISU104 inhibited ligand binding to ErbB3, heterodimerization between ErbB2 and ErbB3, and induced ErbB3 internalization through selective binding to the domain 3 of ErbB3. Structural analysis confirmed the epitope of ISU104, which partly overlapped with the potential ligand-binding region within the domain 3 of ErbB3. Pharmacologic activities of ISU104, such as inhibition of ErbB3/Akt signaling, cell proliferation, and tumor growth, were demonstrated using various cancer cell models.

As mentioned previously, Met102H of the heavy chain and Arg407 of ErbB3 are considered as characteristic residues in this complex structure, which are located in the center of the antibody-antigen interface. The long side chains of arginine and methionine are involved in strong ionic and hydrophobic interactions, respectively, within the deep clefts in their counterparts. These interactions are distinctly different features from those of other anti-ErbB3 mAbs, including CDX-3379 and RG7116 (38, 39). Although the domain 3s of the ErbB family are structurally similar to each other overall, the structure around the Arg407 residue in ErbB3 is different from those of the superimposed corresponding residues in other ErbB proteins (EGFR and ErbB2; Supplementary Fig. S5). Only three of the 24 residues within the ErbB3-domain 3 that showed interaction with ISU104 were conserved among the four ErbB families (Supplementary Fig. S5C). This explains the selectivity of ISU104 only toward ErbB3, but not toward other family members.

ISU104 bound to cellular ErbB3 expressed on the plasma membrane of various cancer cell lines, including three breast cancer–derived ZR-75-30, ZR-75-1, and MDA-MB-468, and HNSCC-derived FaDu cell line. ISU104 showed specific and selective binding to ErbB3 with very high affinity (sub-nanomolar KD), but not to EGFR or ErbB2. Considering these observations, the pharmacologic activity of ISU104 is likely to be target specific, and unwanted off-target effects and toxicities would be avoided. ISU104 showed cross-reactivity to the species (mouse, rat, and monkey) that are generally used for preclinical studies, such as pharmacokinetics, pharmacodynamics, and toxicology. Physiologically relevant information can be obtained from preclinical studies using these species.

The epitope of ISU104 overlapped with the NRG1-binding region within the domain 3 of ErbB3. In addition, it is predicted that binding of ISU104 to ErbB3 would sterically prevent conformational changes required for heterodimerization. An EGFR-targeting antibody, cetuximab, shows a similar mechanism of action. Cetuximab binds exclusively to the domain 3 of EGFR, partially masking the ligand-binding region and sterically preventing structural changes for dimerization (12). Therapeutic anti-ErbB3 antibodies under clinical development bind to various ECDs (domains 1–4) of ErbB3 (DL11 and MF3178 bind to domain 3, CDX-3379 to domains 2 and 3, RG7116 to domain 1, and LJM-716 to domains 2 and 4); thus, their mechanisms of action would be different from that of each other (30, 38–41). Similarly to ISU104, DL11, MF3178, and RG7116 not only inhibit ligand binding directly, but also restrict conformational changes required for heterodimerization. In contrast, CDX-3379 and LJM-716 bind to the region distinct from the ligand-binding site and rather lock the ECDs in an inactive configuration. Overall, the mechanism of action of ISU104 is quite similar to the previously developed antibodies in terms of ligand blocking and inhibition of dimerization. Some residues in the epitope of ISU104 are commonly recognized by CDX-3379, DL11, and MF3178, but many are uniquely recognized by ISU104, which would render its distinctive binding characteristics (i.e., binding affinity/avidity and conformation of interaction, which may contribute to ISU104's biological activity). The epitopes of various anti-ErbB3 antibodies are listed and compared (Supplementary Table S7).

Toxicity could be an issue in the development of anti-ErbB3 therapy as in the case of RG7116, a glycoengineered antibody with enhanced antibody-dependent cellular cytotoxicity (ADCC) activity. About 50% of patients treated with RG7166 experienced diarrhea, which may potentially limit its clinical use (42). Contrary to RG7166, ISU104 only showed limited toxicity (13% or 2/15 patients experienced diarrhea) in the ongoing clinical study (43). This limited toxicity might be due to low or no ADCC activity of ISU104. ISU104 did not show any ADCC activity in an in vitro ADCC report bioassay (Supplementary Fig. S6). Although further work would be required to clarify the mechanism, it is less likely that ADCC is a major part of ISU104's mechanism of action.

Overall, a novel anti-ErbB3, ISU104, showing unique binding characteristics, exhibited antitumorigenic potencies in various preclinical models. The clinical application of ISU104 is expected to provide benefits to patients with various ErbB3-dependent cancers.

M. Hong reports grants from The Korea Health Industry Development Institute during the conduct of the study, has a patent for US 10413607 issued, and a patent for KR 10-1927732 issued. M. Kim reports grants from The Korea Health Industry Development Institute during the conduct of the study, has a patent for US 10413607 issued, and a patent for KR 10-1927732 issued. J.Y. Kim reports grants from the Korea Health Industry Development Institute during the conduct of the study. K. Kim reports grants from The Korea Health Industry Development Institute during the conduct of the study. Y. Sohn reports grants from the Korea Health Industry Development Institute during the conduct of the study and outside the submitted work. S.-B. Hong reports grants from the Korea Health Industry Development Institute during the conduct of the study and personal fees from National Institute for Mathematical Sciences, Human Asia, and Korea Basic Science Institute outside the submitted work. No disclosures were reported by the other authors.

M. Hong: Investigation, visualization, methodology, writing–original draft. Y. Yoo: Resources, formal analysis, investigation, writing–original draft. M. Kim: Conceptualization, supervision, funding acquisition, investigation, writing–original draft. J.Y. Kim: Data curation, investigation, writing–review and editing. J.S. Cha: Resources, writing–review and editing. M.K. Choi: Resources, writing–review and editing. U. Kim: Resources, writing–review and editing. K. Kim: Resources, supervision. Y. Sohn: Resources, supervision. D. Bae: Conceptualization, supervision, funding acquisition, writing–review and editing. H.-S. Cho: Conceptualization, supervision, funding acquisition, writing–original draft. S.-B. Hong: Conceptualization, supervision, writing–original draft.

H.-S. Cho was supported by the National Research Foundation of Korea (NRF) grant funded by the Ministry of Science and ICT (NRF-2016R1A5A1010764, NRF-2017M3A9F6029755, and NRF-2019M3E5D6063903), the Strategic Initiative for Microbiomes in Agriculture and Food grant funded by the Ministry of Agriculture, Food and Rural Affairs (918012-4), and a grant funded by ISU ABXIS Co. Ltd. M. Hong, M. Kim, K. Kim, Y. Sohn, and D. Bae were supported by a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute, funded by the Ministry of Health & Welfare, Republic of Korea (HI16C0528). We thank the staff scientists for assistance with the Beamline 11C at the Pohang Light Source, Pohang Accelerator Laboratory.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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Supplementary data