U3-1402, a Novel HER3-Targeting Antibody-Drug Conjugate, for the Treatment of Colorectal Cancer

Home , Duligotuzumab, Panitumumab, Patritumab, Trastuzumab deruxtecan

Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/1535-7163.MCT-19-0452 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

U3-1402, a novel HER3-targeting antibody–drug conjugate, for the treatment of Colorectal Cancer

Shigehiro Koganemaru1, Yasutoshi Kuboki1, Yoshikatsu Koga2, Takashi Kojima3,

Mayumi Yamauchi3, Naoyuki Maeda4, Takashi Kagari5, Kenji Hirotani6,

Masahiro Yasunaga2, Yasuhiro Matsumura2, Toshihiko Doi1

1 Department of Experimental Therapeutics, National Cancer Center Hospital East,

Kashiwa, Japan

2 Division of Developmental Therapeutics, Research Center for Innovative Oncology,

National Cancer Center Hospital East, Kashiwa, Japan

3 Department of Gastrointestinal Oncology, National Cancer Center Hospital East,

Kashiwa, Japan

4 Biomarker Department, Daiichi Sankyo Co., Ltd., Tokyo, Japan

5 Oncology Research Laboratories I, Daiichi Sankyo Co., Ltd., Tokyo, Japan

6 Oncology Clinical Development Department, Daiichi Sankyo Co., Ltd., Tokyo, Japan

Running title: Preclinical Activity of novel HER3-ADC in Colorectal Cancer

Keywords: HER3, antibody drug conjugate, colorectal cancer, armed antibody, monoclonal antibody

Financial support: This research was financially supported by Daiichi Sankyo Co.,

Ltd.

Corresponding author: Yasutoshi Kuboki, Department of Experimental Therapeutics,

National Cancer Center Hospital East, Kashiwa, Chiba, 277-8577, Japan

Tel: +81-4-7133-1111; Fax: +81-4-7131-9960; E-mail: [email protected]

Downloaded from mct.aacrjournals.org on September 26, 2021. © 2019 American Association for Cancer Research. Author Manuscript Published OnlineFirst on August 8, 2019; DOI: 10.1158/1535-7163.MCT-19-0452 Author manuscripts have been peer reviewed and accepted for publication but have not yet been edited.

Disclosure of Potential Conflicts of Interest

Y.K. reports receiving grants and research funding from Daiichi Sankyo. N.M, T.K. and

K.H are all employees of Daiichi Sankyo. T.D. is a consultant/advisory board member for Daiichi Sankyo and reports receiving research funding from Daiichi Sankyo. No potential conflicts of interest were disclosed by the other authors.

Contains: 3,085 words (main text); 2 tables, 4 figures, 6 supplementary figures

Abstract

HER3 (human epidermal growth factor receptor 3) is overexpressed in several cancers including colorectal cancer (CRC). Although therapies with anti-HER3 antibodies have been investigated, significant clinical benefits have not been reported. U3-1402 is a novel HER3-antibody-drug conjugate (ADC), composed of the HER3 antibody patritumab and a novel topoisomerase I inhibitor, DX-8951 derivative (DXd). The sensitivity of DXd was evaluated by a growth inhibition assay. The antitumor activity of

U3-1402 was evaluated in a murine xenograft model in which its effects on cells, with a range of HER3 expression levels, were compared with those of patritumab alone, irinotecan, control-ADC and saline. In the growth inhibition assay, all CRC cell lines were sensitive to DXd. In the tumor xenograft model, significant tumor regression with

U3-1402 was observed both in the DiFi cell line (high HER3 expression; KRAS wild type) and in SW620 (high HER3 expression; KRAS mutation), but no treatment effect was observed in Colo320DM (low HER3 expression). Notably, SW620 tumor growth was significantly suppressed with U3-1402 compared with the saline-treated group (P <

0.001) and showed greater activity compared with the irinotecan group. By contrast, patritumab alone, control-ADC and saline did not significantly differ in tumor growth inhibition. The antitumor activity of U3-1402 was dependent on HER3 expression level, but not on KRAS mutation status. These results support further investigation of development strategies for U3-1402 in patients with HER3-expressing CRC.

Introduction

Colorectal cancer (CRC) is a major cause of cancer death worldwide (1). Multimodality therapies that include surgery, chemotherapy, and radiotherapy are required to treat operable CRC (2-4), whereas chemotherapy is the main treatment for inoperable cancers.

Despite recent advances in cytotoxic chemotherapy, targeted therapies and immune therapy (5-9), the long-term prognosis remains poor, especially for patients with recurrent or inoperable CRC (10).

The human epidermal growth factor receptor (EGFR/ErbB) family is composed of four known members (HER1 or EGFR, HER2, HER3 and HER4), which have important functions in the pathogenesis of solid tumors (11). Some anti-EGFR or anti-HER2 antibodies are widely used to treat several cancer types. For instance, anti-EGFR antibodies such as cetuximab and panitumumab are already used as a standard treatment in patients with RAS wild-type metastatic colorectal cancer (mCRC); and dual-targeted therapy with anti-HER2 antibody (trastuzumab) and lapatinib have shown activity in patients with KRAS codon 12/13 wild-type/HER2 positive mCRC in clinical trials

(12-14).

Among these ErbB family members, HER3 is unique, as it lacks significant kinase activity and requires heterodimerization with other members of ErbB family to activate

downstream signaling pathways (11,15). HER3 is reported to be overexpressed in several cancer types, using immunohistochemistry (IHC), although the definition or antibody and its cutoff value for high HER3 expression are different in each article (16).

In general, HER3 overexpression was found in about 20–75% of CRC (17), which was much higher than HER2 expression (18,19). High HER3 expression has been suggested to predict poor prognosis (20). HER3 signaling is also associated with the resistance mechanism of anti-EGFR/HER2 therapies in several malignancies (21-23). Therefore,

HER3-targeted therapies have been developed (24,25); however, monoclonal antibody against HER3, such as patritumab, or dual-target monoclonal antibodies against

EGFR/HER3 such as duligotuzumab, have not shown any significant clinical benefit in mCRC patients in early clinical trials (26,27).

Antibody–drug conjugates (ADCs) consist of monoclonal antibodies conjugated to cytotoxic agents (known as their payloads) via synthetic linkers. In addition to the antibodies’ antitumor activity, highly potent agents can induce cancer cell death; therefore, the activity of ADCs is not necessarily dependent on downstream signaling, but rather on target expression. Although ADC development has been driven by hematological cancers (28-30), a new approach with HER2-targeted ADCs, such as trastuzumab emtansine or trastuzumab deruxtecan, has led to breakthrough results in

solid tumors (31,32), or with other targeted ADCs has shown promising results in preclinical study (33-37), and is one of the fastest growing classes of oncology therapies.

U3-1402, a potential first-in-class HER3-targeting ADC, is composed of a humanized anti-HER3 IgG1 antibody (patritumab), an enzymatically cleavable peptide-linker, and a novel topoisomerase-I inhibitor. To explore new strategies that target HER3 with ADC, we investigated the antitumor activity of U3-1402, using 9 CRC cell lines.

Materials and Methods

Cell culture

Eight human CRC cell lines (Colo320DM, DLD-1, HCT-15, HCT-116, LoVo, SW480,

SW620 and WiDr) were purchased from the American Type Culture Collection (ATCC)

(Manassas, VA). The DiFi human CRC cell line was kindly provided by Dr. Kimio

Yonesaka, of the Kindai University Faculty of Medicine. These cell lines were authenticated by short tandem repeat DNA profiling in 2018 by the JCRB Cell Bank

(Osaka, Japan) or purchased from ATCC in 2017. The Colo320DM, DLD-1, HCT-15,

HCT-116, LoVo, SW480, SW620, and WiDr cells were kept in Roswell Park Memorial

Institute-1640 medium, supplemented with 10% fetal bovine serum (FBS); and DiFi

cells in Dulbecco's Modified Eagle's Medium with high glucose, supplemented with

10% FBS. All media were supplemented with 1% penicillin/streptomycin (Wako, Osaka,

o Japan). Cells were maintained under a humidified atmosphere with 5% CO2 at 37 C.

The cells’ genetic background status, such as RAS, BRAF, PIK3CA, were reported in the public database, COSMIC (https://cancer.sanger.ac.uk/cosmic). We also analyzed HER3 expression levels in these cell lines by flow cytometry (FCM), as described below.

Compounds and antibodies

Irinotecan was purchased from Towa Pharmaceutical Co., Ltd. (Tokyo, Japan). U3-1402

(HER3-ADC), U3-1287 (anti-human HER3 antibody, patritumab), control-ADC and

DX-8951 derivative (DXd) were provided by Daiichi Sankyo Co., Ltd (Tokyo, Japan).

U3-1402 or control-ADC were composed of antibody (patritumab or control antibody) and DXd that were conjugated by a tetra-peptide (Gly-Gly-Phe-Gly)-based cleavable linker at a drug/antibody ratio of 7 to 8 (Supplementary Fig. S1) (38).

FCM analysis and determination of antigen receptor density

HER3 expression by the cell lines were analyzed by FCM analysis. We incubated 105 harvested cells with 0.2 µg of each antibody (anti-HER3-VioBright FITC and

REA-control-FITC, Miltenyi Biotec) for 30 min at 4oC. After washing with PBS that

contained 0.5% bovine serum albumin and 2 mM EDTA, cells were analyzed by FCM using Guava easyCyte (Millipore, Billerica, MA). Quantitative analyses of cell surface

HER3 antigen used QIFIKIT (Dako, Grostrup, Denmark) according to manufacturer’s instruction. Briefly, the mean fluorescence intensity of the beads’ known antigen levels were compared against a calibration curve to determine cell-surface antigen levels.

Western blotting

Cells were lysed in a radio immunoprecipitation assay buffer containing 50 mM

Tris-HCL (pH 8.0), 150 mM sodium chloride, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, and 1% ND-40 (Wako, Osaka, Japan) with protease inhibitor and phosphatase inhibitor (Wako). Total protein lysates were loaded on SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Bio-Rad, Hercules, CA) using a

Trans-Blot Turbo transfer machine (Bio-Rad). Immunostaining of the membranes was performed using the iBind Western System (Life Technology, Carlsbad, CA) according to the manufacturer's protocol. Primary antibodies were purchased from Cell Signaling

Technology (Beverly, MA) and the following antibodies were used: HER3, phosphorylated-HER3 (Tyr1289), ERK1/2, phosphorylated-ERK1/2 (Thr202/Tyr204), AKT, phosphorylated AKT (Ser473) and β-actin. Secondary antibody (donkey-anti-mouse IgG) was purchased from Jackson Immuno Research (West Grove, PA). Primary antibodies

and secondary antibody were diluted to appropriate concentrations in iBind buffer.

Subsequently, membranes were washed with a tris-buffered saline containing 0.1%

Tween 20, and proteins were visualized with using ECL prime substrate (GE Healthcare,

Piscataway, NJ). Western blots were imaged using a ChemiDoc XRS Plus System

(Bio-Rad).

In vitro growth inhibition assay

Cells were seeded into 96-well plates at a density of 1.0 ×103 cells/100 μL (for

Colo320DM and LoVo) and 3.0 ×103 cells/100 μL (for DiFi, DLD-1, HCT15, HCT116,

SW480, SW620 and WiDr), and incubated for 24 hours at 37oC. Cells were then treated with various concentrations of DXd for 144 hours under the same conditions. After the removal of medium, cell viability was analyzed using the Cell Counting Kit-8 (WST-8 assay; Dojindo, Kumamoto, Japan) according to manufacturer’s instruction. The 50%

inhibitory concentration (IC50) was determined from dose–response curves. The experiment was repeated three times.

Experimental mice model

Six- or eight-week-old female BALB/c nude mice (Charles River Laboratories Japan,

Yokohama, Japan) were used in this study. The mice were maintained in cages under

specific pathogen-free conditions, provided with standard food, and given free access to sterilized water. Mice were monitored daily, and tumor volume (for the tumor xenograft model) and their body weights were measured twice a week. All animal procedures were carried out in compliance with the Guidelines for the Care and Use of

Experimental Animals established by the Committee for Animal Experimentation from the National Cancer Center, Japan. These guidelines meet the ethical standards required by law and also comply with the guidelines for the use of experimental animals in Japan.

Their tumor volume was calculated as: L× W2 (L: length, W: width)/2 of the subcutaneous tumor. Mice were euthanized when tumor volume reached a maximum

3,000 mm3 or they developed other morbidities, whichever occurred first. If a mouse reached a humane endpoint and was excluded from the study, the remaining mice were carefully monitored for tumor growth until the next evaluating day.

In vivo safety study

To determine the maximum tolerated dose (MTD), various doses of U3-1402 and irinotecan were administered weekly, for four times (Days 0, 7, 14 and 21), to 8-week female BALB/c nude mice without tumors, by peritoneal injection. The treatment schedule was determined based on the previous preclinical study using U3-1402 or trastuzumab deruxtecan that used the same linker-payload system as U3-1402 (39,40).

MTD was defined as the highest dose at which mice lost less than 10% of their initial body weight during the treatment (41).

In vivo screening assay

To evaluate the antitumor effect of U3-1402, viable cells (Colo320DM: 1.0 × 107, DiFi:

1.0 × 107, DLD-1: 5.0 × 106, HCT-15: 5.0 × 106, HCT-116: 2.0 × 106, LoVo: 5.0× 106,

SW480: 1.0 × 107, SW620: 1.0 × 107, and WiDr: 2.0 × 106) suspended with 0.1 mL of

PBS were injected subcutaneously into the right trunk of 6-week female BALB/c nude mice. When tumor volumes reached 200–250 mm3, U3-1402 was administered to the mice, at 10 mg/kg weekly for three times (Days 0, 7 and 14) using peritoneal injections.

In vivo tumor growth inhibition assay

To evaluate tumor growth inhibition, the mice were randomly divided into several treatment groups (n=5 or 6 for each group) when tumor volume reached 200–250 mm3

(Day 0). Each drug was administered intraperitoneally on Days 0, 7, 14, and 21.

U3-1402 was administered at 5 or 10 mg/kg in the first experiment. U3-1402, control-ADC and patritumab at 10 mg/kg, and irinotecan at 100 mg/kg, were administered in the second experiment. Saline was administered as an untreated control.

Complete regression was defined by tumor volume ≤ 25 mm3 for at least three consecutive measurements (42).

Statistical analyses

All results were expressed as mean ± standard deviation (SD) and statistical significance was analyzed using ANOVA with Dunnett multiple comparisons. Statistical analyses were performed using SPSS Statistics version 25 (IBM Corporation, Armonk, NY). All statistical tests were two-sided. P < 0.05 was considered significant.

Results

HER3 expression of each cell line

The number of HER3 molecules expressed on the surface of individual cells in each cell line was calculated by a QIFIKIT. HER3 expression levels were defined as high (>5,000 molecules/cell), intermediate (2,000–5,000 molecules/cell), and low (<2,000 molecules/cell; Table 1; Supplementary Fig. S2). The DiFi, SW480, SW620 and WiDr cell lines had high HER3 expression, the Colo320DM cell line had low HER3 expression and other cell lines had intermediate HER3 expression.

In vitro cytotoxicity of DXd against tumor cell lines

To explore the impact of payload on U3-1402 activity, we analyzed in vivo cytotoxicity

of DXd against tumor cell lines. The IC50 values of DXd for CRC cell lines are

described in Table 1. The IC50 value of DXd for the cell lines ranged from 2.05 ± 0.11 nM to 17.88 ± 2.63 nM. All cell lines were sensitive to DXd.

Maximum tolerated doses of U3-1402 and Irinotecan

The MTDs for weekly injections of U3-1402 and irinotecan was determined using non-tumor-bearing nude mice (Table 2). The drugs were injected intraperitoneally because high concentrations of the drugs injected intravenously were more likely to cause acute death. We used dose ranges of 3–10 mg/kg for U3-1402, and 50–200 mg/kg for irinotecan. In the U3-1402-treated mice, weight loss >10% was not observed for any of the treatment doses, but was observed in the irinotecan-treated mice at 150 mg/kg and 200 mg/kg. We were concerned about raising off-target effects of U3-1402 by using more than 10 mg/kg; therefore, 10 mg/kg U3-1402 and 100 mg/kg irinotecan were considered recommended dose in vivo study.

In vivo screening study

To evaluate antitumor activity in a tumor xenograft model, we administered U3-1402 at

10 mg/kg weekly, for three times (Days 0, 7 and 14). Activity of U3-1402 was assessed by the maximum tumor regression rate during the treatment (Fig. 1). Tumor regression was observed in xenografted tumors derived from five of the nine cell lines (DiFi,

DLD-1, LoVo, SW480, and SW620). The highest regression rates were seen in tumors from SW620 cells (high HER3 with KRAS mutation), and DiFi (high HER3 expression with KRAS wild type). These results showed that U3-1402 was more effective against xenograft models with high HER3 expression, regardless the KRAS mutation status.

U3-1402 antitumor activity in xenograft models

To confirm that antitumor activity of U3-1402 depended on HER3 expression levels, we next compared the antitumor activity of U3-1402 at 5 mg/kg and 10 mg/kg with the saline-treated group, using xenografts from cells with a range of HER3 expression levels, including Colo320DM (low HER3 expression, Fig. 2A, B), HCT15 (intermediate expression; Fig. 2C, D) and SW620 (high expression; Fig. 2E, F). Each drug was administered on Days 0, 7, 14 and 21. Tumor growth was significantly suppressed in the

U3-1402 groups compared with the saline groups for the SW620 group (P < 0.001), and the HCT15 group (P < 0.005). U3-1402 also showed dose-dependent antitumor activity.

In contrast, tumor growth between the U3-1402 groups and the saline group did not significantly differ as for the Colo320DM group. These results reveal that the antitumor activity of U3-1402 was dependent on HER3 expression level. Body weight loss was not observed in any of the treatment groups (Supplementary Fig. S3).

U3-1402 antitumor activity compared with other drugs

We hypothesized that the antitumor activity in xenograft models with HER3 expression was dependent on both patritumab and DXd. We tested this hypothesis by immunoblot assays and tumor xenografts, using high HER3-expressing DiFi and SW620 cell lines.

Although western blotting revealed the presence of HER3 in both DiFi and SW620 cells, only DiFi cells expressed phosphorylated HER3 (Supplementary Fig. S4). As expected,

HER3 phosphorylation was slightly decreased only in DiFi cells in the presence of patritumab at 10 µg/ml (Fig. 3A). In the DiFi xenograft tumors, patritumab alone showed only slight treatment effects, whereas the U3-1402-treated group showed tumor regression (Fig. 3B). None of these treatment groups showed body weight loss

(Supplementary Fig. S5).

To compare the antitumor activity of U3-1402 with standard chemotherapy, we chose irinotecan as control chemotherapy because of the payload of U3-1402 is a novel topoisomerase I inhibitor. In the SW620 xenograft tumors, growth was also

significantly suppressed with U3-1402 compared with the saline group (P < 0.001), whereas tumor growth inhibition did not significantly differ among the groups treated with same-dose patritumab alone or control-ADC and saline (Fig. 4A, B). Notably,

U3-1402 had greater activity than irinotecan for SW620 tumors. Tumor regression was only observed in U3-1402-treated group; 4 of the 6 mice in the U3-1402-treated SW620 group achieved complete regression. Moreover, 3 of the 4 U3-1402-treated mice in the

SW620 that achieved complete regression showed no evidence of regrowth at their last follow-up. These results indicate that U3-1402 is most effective against tumors with high HER3 expression. None of the treatment groups showed any body weight loss

(Supplementary Fig. S6).

Discussion

Therapeutic strategies that target HER3 molecules have not yet shown clinically meaningful success. Although the reasons for this are unclear, a possible explanation is the difficulty in identifying patients whose tumor cells rely on HER3 signaling. Patient selection in early clinical trials was based on HER3 expression of tumor tissue samples, as determined by conventional immunohistochemistry (26,43). However, the oncogenic activity of HER3 expression is reported to depend on kinase activity of other ErbB members (44). In this study, we showed that DiFi cells expressed HER3 and

phosphorylated HER3. Patritumab-alone treatment slightly inhibited HER3 phosphorylation (Fig. 3A); only a marginal effect was observed in the patritumab-alone-treated group (Fig. 3B). These results suggest that the strategy of inhibiting HER3 signaling by anti-HER3 antibody may not be enough to inhibit tumor growth, which is in line with previous clinical studies that found no objective response in CRC patients who received anti-HER3 antibody monotherapy (26,43). In addition, we showed that SW620 tumor growth was significantly suppressed with U3-1402 compared with the saline-treated group, while control-ADC and saline did not significantly differ in tumor growth inhibition. These results showed that the antitumor activity of U3-1402 was specific to HER3 expression. The mode of action of U3-1402 is distinct from conventional anti-HER3 antibodies, i.e., HER3 is used as a carrier for cytotoxic agents. Therefore, U3-1402 might overcome the limitations of earlier anti-HER3 antibodies.

Activation of downstream signal pathways is a common mechanism of primary or acquired resistant to monoclonal antibodies. Mutations in RAS, BRAF or PIK3CA were reported to cause primary or acquired resistance to anti-EGFR antibody therapies in

CRC (45-48). Furthermore, recent clinical trial of HER2-targeting antibodies showed promising activity in patients with KRAS wild-type CRC, but no objective response

observed in those with KRAS mutations (49). Our study showed significant xenograft tumor eradication for both DiFi (KRAS wild type) and SW620 (KRAS mutation) cells treated with U3-1402. These results suggest that the activity of U3-1402 was regardless of KRAS mutation status, possibly because the mode of action of U3-1402 might be achieved by both, HER3 signal inhibition and cytotoxic agents. Indeed, in a phase III clinical trial that used anti-HER2 ADC T-DM1, standard HER2-directed therapies were less effective in tumors with PI3KCA mutations, whereas T-DM1 was apparently effective in both PI3KCA-mutated and wild-type tumors (50).

The payload of U3-1402 is DXd (DX-8951 derivative), which is a novel, highly membrane-permeable topoisomerase I inhibitor, which has a different mechanism of action from commonly used tubulin polymerization inhibitors. Topoisomerase-I inhibitors such as irinotecan are widely used in cancer therapy, including CRC.

DX-8951 was reportedly more efficacious than irinotecan in various tumor xenograft models, including an irinotecan-resistant model (51). Indeed, in our xenograft model,

U3-1402 was more efficacious than irinotecan, and this effect was long-lasting; after 50 days of treatment, no mouse in the U3-1402 group reached a humane endpoint (Fig. 4A).

These results suggest that U3-1402 might be useful against CRCs that are refractory to irinotecan. A recent phase I study of trastuzumab deruxtecan, which is a novel

anti-HER2 ADC that uses the same linker-payload system as U3-1402, showed promising results in patients with gastric cancer with HER2 positive who were previously treated with irinotecan (32).

In this study, we showed no antitumor activity in HER3 low-expressing tumor xenografts; however, it is well known that there is heterogeneity of target antigen in solid tumor, which is far different from tumor xenograft models. U3-1402 is expected to have a bystander effect due to its highly membrane-permeable payload (52); therefore, it might be expected that U3-1402 show the antitumor activity in patients with both

HER3 high and low expression. In fact, trastuzumab deruxtecan showed activity in patients who expressed both low and high antigen levels (32,53,54).

In summary, we have shown that the effect of U3-1402 is dependent on HER3 expression, but not on KRAS mutation status. A phase I study of U3-1402 has demonstrated durable tumor shrinkage and was well tolerated in patients with HER3 positive breast cancer (55). These results provide a strong rationale for investigating the activity of U3-1402 in patients with HER3 positive colorectal cancer.

Authors’ contributions

Conception and design: S. Koganemaru, Y. Kuboki, Y. Koga, T. Kojima, T. Doi

Development of methodology: S. Koganemaru, Y. Koga

Acquisition of data (provided animals, provided facilities, etc.): S. Koganemaru, Y.

Koga, M. Yamauchi

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): S. Koganemaru, Y. Kuboki, Y. Koga, N. Maeda, T. Kagari, K. Hirotani, M.

Yasunaga, Y. Matsumura, Y. Kuboki, T. Kojima, T. Doi

Writing, review, and/or revision of the support (i.e., reporting or organizing data, constructing databases): S. Koganemaru, Y. Kuboki, Y. Koga, T. Kojima, M. Yamauchi,

N. Maeda, T. Kagari, K. Hirotani, M. Yasunaga, Y. Matsumura, T. Doi

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): N. Maeda, T. Kagari, K. Hirotani

Study supervision: Y. Matsumura, T. Doi

Acknowledgment

We thank Marla Brunker, from Edanz Group (www.edanzediting.com/ac) for editing a draft of this manuscript with financial support from Daiichi Sankyo Co., Ltd.

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Tables

Table 1. Colorectal cancer cell line characteristics

HER3 a HER3 Genetical background status c DXd Cell line b (molecules/cell) Expression KRAS BRAF PIK3CA IC50 (nM) Colo320DM 900 Low WT WT WT 5.67±0.80 LoVo 2500 Intermediate G13D WT WT 6.51±1.04 HCT116 4000 Intermediate G13D WT H1047R 3.47±0.57 DLD-1 4200 Intermediate G13D WT E545K 14.38±1.53 HCT15 4500 Intermediate G13D WT E545K; D549N 5.23±3.50 SW480 5300 High G12V WT WT 2.44±0.72 SW620 6200 High G12V WT WT 2.05±0.11 WiDr 10000 High WT V600E WT 17.88±2.63 DiFi 18200 High WT WT WT 11.38±2.02 aQuantitative analysis of cell-surface HER3 antigen was determined by using QIFIKIT. bHER3 expression levels were defined by the number of HER3 molecules per cell, as high: > 5,000, intermediate: 2,000–5,000, and low: < 2,000. cThe genetic background status was obtained from the public database, COSMIC.

Table 2. Maximum tolerated dose studies for U3-1402 and Irinotecan

Dose (mg/kg) Route Days Maximum weight loss (%)a U3-1402 3 i.p. 0, 7, 14, 21 +4.0 5 i.p. 0, 7, 14, 21 −1.8 10 i.p. 0, 7, 14, 21 +0.5 Irinotecan 50 i.p. 0, 7, 14, 21 −2.8 100 i.p. 0, 7, 14, 21 −2.9 150 i.p. 0, 7, 14, 21 −10.5 200 i.p. 0, 7, 14, 21 −15.1 i.p.; intraperitoneally. aMaximum tolerated dose (MTD) was defined as the highest dose that caused less than 10% weight loss compared with the initial pre-treatment weight during the treatment.

Figure legends

Figure 1. Waterfall plot for tumor xenograft models. U3-1402 at 10 mg/kg was administered weekly for three times (Days 0, 7 and 14), by peritoneal injection.

Figure 2. Antitumor activity of U3-1402 against tumor xenograft models that exhibited a range of HER3 expression levels. The activity of U3-1402 was evaluated in a tumor xenograft model in Colo320DM (A, B), with low HER3 expression, HCT15

(C, D), with intermediate HER3 expression and SW620 (E, F), with high HER3 expression. In each model, U3-1402 and saline were administered weekly for four times

(Days 0, 7, 14 and 21); arrows indicate days of administration.

Figure 3. HER3 expression and activation of signal proteins and antitumor activity for U3-1402 used against DiFi cells (high HER3 expression; KRAS wild-type) in a tumor xenograft model. DiFi and SW620 cells were treated with or without patritumab

(10 µg/ml) for 24 hours. Cells were then lysed and cell extracts were analyzed by western blotting for total or phosphorylated HER3, AKT and ERK (and β-actin used as a loading control) (A). The antitumor activity of U3-1402 at 10 mg/kg was compared with saline and same-dose patritumab in the DiFi tumor xenograft model (B). Each drug was administered weekly for four times (Days 0, 7, 14 and 21); arrows: days of administration.

Figure 4. The antitumor activity of U3-1402 compared with other drugs against the

SW620 harboring KRAS mutation in a tumor xenograft model. The antitumor activity of U3-1402 at 10 mg/kg compared with saline, same dose control-ADC, same dose patritumab and irinotecan at 100 mg/kg in the SW620 tumor xenograft model (A,

B). All agents were administered weekly for four times (Days 0, 7, 14 and 21); arrows: days of administration.

U3-1402, a novel HER3-targeting antibody-drug conjugate, for the treatment of Colorectal Cancer

Shigehiro Koganemaru, Yasutoshi Kuboki, Yoshikatsu Koga, et al.

Mol Cancer Ther Published OnlineFirst August 8, 2019.

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