MOLECULAR CANCER THERAPEUTICS | LARGE MOLECULE THERAPEUTICS A Novel Therapeutic Anti-ErbB3, ISU104 Exhibits Potent Antitumorigenic Activity by Inhibiting Ligand Binding and ErbB3 Heterodimerization Mirim Hong 1 , Youngki Yoo 2 , Miyoung Kim 1 , Ju Yeon Kim 1 , Jeong Seok Cha 2 , Myung Kyung Choi 2 , Uijin Kim 2 , Kyungyong Kim 1 , Youngsoo Sohn 1 , Donggoo Bae 1 , Hyun-Soo Cho 2 , and Seung-Beom Hong 1 ABSTRACT ◥ 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 antican- cer 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. Biochem- ical studies confirmed these predictions. ISU104 inhibited ligand binding, ligand-dependent heterodimerization and phosphoryla- tion, 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. Introduction 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 signal- ing 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 ben- efits 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 acti- vation 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. Materials and Methods 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% CO 2 . 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 mg/mL in PBS, pH 7.4, with the selection criterion being positive binding to human ErbB3. The variable light 1 Research Institute, ISU ABXIS Co., Ltd., Sungnam-si, Republic of Korea (South). 2 Department of Systems Biology, Yonsei University, Seoul, Republic of Korea (South). Note: Supplementary data for this article are available at Molecular Cancer Therapeutics Online (http://mct.aacrjournals.org/). M. Hong and Y. Yoo contributed equally to this article. Corresponding Authors: Seung-Beom Hong, Research Center, ISU ABXIS, 22 Daewangpangyo-ro, 712-beon-gil, Seongnam-si, Gyeonggi-do 13488, Republic of Korea (South). Phone: 82-31-696-4723; Fax: 82-31-696-4693; E-mail: hongsb@isu.co.kr; and Hyun-Soo Cho, Yonsei-ro 50, Seodaemun-gu, Seoul 03722, Republic of Korea (South). Phone: 82-2-2123-5651; E-mail: hscho8@yonsei.ac.kr Mol Cancer Ther 2021;20:1142–52 doi: 10.1158/1535-7163.MCT-20-0907 Ó2021 American Association for Cancer Research. AACRJournals.org | 1142 Downloaded from http://aacrjournals.org/mct/article-pdf/20/6/1142/3104036/1142.pdf by guest on 11 June 2022