Toshinori Agatsuma

DS-8201a, A Novel HER2-Targeting ADC with a Novel DNA Topoisomerase I Inhibitor, Demonstrates a Promising Antitumor Efficacy with Differentiation from T-DM1

Purpose: An anti-HER2 antibody–drug conjugate with a novel topoisomerase I inhibitor, DS-8201a, was generated as a new antitumor drug candidate, and its preclinical pharmacologic profile was assessed. Experimental Design: In vitro and in vivo pharmacologic activities of DS-8201a were evaluated and compared with T-DM1 in several HER2-positive cell lines and patient-derived xenograft (PDX) models. The mechanism of action for the efficacy was also evaluated. Pharmacokinetics in cynomolgus monkeys and the safety profiles in rats and cynomolgus monkeys were assessed. Results: DS-8201a exhibited a HER2 expression-dependent cell growth–inhibitory activity and induced tumor regression with a single dosing at more than 1 mg/kg in a HER2-positive gastric cancer NCI-N87 model. Binding activity to HER2 and ADCC activity of DS-8201a were comparable with unconjugated anti-HER2 antibody. DS-8201a also showed an inhibitory activity to Akt phosphorylation. DS-8201a induced phosphorylation of Chk1 and Histone H2A.X, the markers of DNA damage. Pharmacokinetics and safety profiles of DS-8201a were favorable and the highest non-severely toxic dose was 30 mg/kg in cynomolgus monkeys, supporting DS-8201a as being well tolerated in humans. DS-8201a was effective in a T-DM1–insensitive PDX model with high HER2 expression. DS-8201a, but not T-DM1, demonstrated antitumor efficacy against several breast cancer PDX models with low HER2 expression. Conclusions: DS-8201a exhibited a potent antitumor activity in a broad selection of HER2-positive models and favorable pharmacokinetics and safety profiles. The results demonstrate that DS-8201a will be a valuable therapy with a great potential to respond to T-DM1–insensitive HER2-positive cancers and low HER2–expressing cancers. Clin Cancer Res; 22(20); 5097–108. ©2016 AACR.

Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity.

Antibody–drug conjugates deliver anticancer agents selectively and efficiently to tumor tissue and have significant antitumor efficacy with a wide therapeutic window. DS‐8201a is a human epidermal growth factor receptor 2 (HER2)‐targeting antibody–drug conjugate prepared using a novel linker‐payload system with a potent topoisomerase I inhibitor, exatecan derivative (DX‐8951 derivative, DXd). It was effective against trastuzumab emtansine (T‐DM1)‐insensitive patient‐derived xenograft models with both high and low HER2 expression. In this study, the bystander killing effect of DS‐8201a was evaluated and compared with that of T‐DM1. We confirmed that the payload of DS‐8201a, DXd (1), was highly membrane‐permeable whereas that of T‐DM1, Lys‐SMCC‐DM1, had a low level of permeability. Under a coculture condition of HER2‐positive KPL‐4 cells and negative MDA‐MB‐468 cells in vitro, DS‐8201a killed both cells, whereas T‐DM1 and an antibody–drug conjugate with a low permeable payload, anti‐HER2‐DXd (2), did not. In vivo evaluation was carried out using mice inoculated with a mixture of HER2‐positive NCI‐N87 cells and HER2‐negative MDA‐MB‐468‐Luc cells by using an in vivo imaging system. In vivo, DS‐8201a reduced the luciferase signal of the mice, indicating suppression of the MDA‐MB‐468‐Luc population; however, T‐DM1 and anti‐HER2‐DXd (2) did not. Furthermore, it was confirmed that DS‐8201a was not effective against MDA‐MB‐468‐Luc tumors inoculated at the opposite side of the NCI‐N87 tumor, suggesting that the bystander killing effect of DS‐8201a is observed only in cells neighboring HER2‐positive cells, indicating low concern in terms of systemic toxicity. These results indicated that DS‐8201a has a potent bystander effect due to a highly membrane‐permeable payload and is beneficial in treating tumors with HER2 heterogeneity that are unresponsive to T‐DM1.

MRGD, a MAS-related G-protein Coupled Receptor, Promotes Tumorigenisis and Is Highly Expressed in Lung Cancer

To elucidate the function of MAS-related GPCR, member D (MRGD) in cancers, we investigated the in vitro and in vivo oncogenic function of MRGD using murine fibroblast cell line NIH3T3 in which MRGD is stably expressed. The expression pattern of MRGD in clinical samples was also analyzed. We found that overexpression of MRGD in NIH3T3 induced focus formation and multi-cellular spheroid formation, and promoted tumors in nude mice. In other words, overexpression of MRGD in NIH3T3 induced the loss of contact inhibition, anchorage-independent growth and in vivo tumorigenesis. Furthermore, it was found that the ligand of MRGD, beta-alanine, enhanced spheroid formation in MRGD-expressing NIH3T3 cells. From investigation of clinical cancer tissues, we found high expression of MRGD in several lung cancers by immunohistochemistry as well as real time PCR. Based on these results, MRGD could be involved in tumorigenesis and could also be a novel anticancer drug target.

Identification of Physiologically Active Substances as Novel Ligands for MRGPRD

Mas-related G-protein coupled receptor member D (MRGPRD) is a G protein-coupled receptor (GPCR) which belongs to the Mas-related GPCRs expressed in the dorsal root ganglia (DRG). In this study, we investigated two novel ligands in addition to beta-alanine: (1) beta-aminoisobutyric acid, a physiologically active substance, with which possible relation to tumors has been seen together with beta-alanine; (2) diethylstilbestrol, a synthetic estrogen hormone. In addition to the novel ligands, we found that transfection of MRGPRD leads fibroblast cells to form spheroids, which would be related to oncogenicity. To understand the MRGPRD novel character, oncogenicity, a large chemical library was screened in order to obtain MRGPRD antagonists to utilize in exploring the character. The antagonist in turn inhibited the spheroid proliferation that is dependent on MRGPRD signaling as well as MRGPRD signals activated by beta-alanine. The antagonist, a small-molecule compound we found in this study, is a potential anticancer agent.

Novel anti-EPHA2 antibody, DS-8895a for cancer treatment

ABSTRACT Overexpression of EPHA2 has been observed in multiple cancers and reported to be associated with poor prognosis. Here, we produced an afucosylated humanized anti-EPHA2 monoclonal antibody (mAb), DS-8895a for cancer treatment. The antibody recognizes the extracellular juxtamembrane region of EPHA2 and therefore can bind to both full-length and truncated forms of EPHA2, which are anchored to cell membranes and recently reported to be produced by post-translational cleavage in tumors. DS-8895a exhibited markedly increased antibody dependent cellular cytotoxicity (ADCC) in vitro and also inhibited tumor growth in EPHA2-positive human breast cancer MDA-MB-231 and human gastric cancer SNU-16 xenograft mouse models. Moreover, DS-8895a in combination with cisplatin (CDDP) showed better efficacy than each of the monotherapies did in the human gastric cancer model. These results suggest that a novel antibody, DS-8895a has therapeutic potential against EPHA2-expressing tumors.

Wide application of a novel topoisomerase I inhibitor-based drug conjugation technology

To establish a novel and widely applicable payload-linker technology for antibody-drug conjugates (ADCs), we have focused our research on applying exatecan mesylate (DX-8951f), a potent topoisomerase I inhibitor, which exhibits extensive antitumor activity as well as significant myelotoxicity, as the payload part. Through this study, we discovered a promising exatecan derivative (DX-8951 derivative, DXd), that has the characteristics of low membrane permeability and shows considerably less myelotoxicity than that shown by exatecan mesylate in an in vitro human colony forming unit-granulocyte macrophage assay. DXd was further used for drug conjugation by using commercially or clinically useful monoclonal antibodies to evaluate the potency of the ADC. The result revealed that the DXd-ADCs targeting CD30, CD33, and CD70 were effective against each of their respective target-expressing tumor cell lines. Moreover, a novel DXd-ADC targeting B7-H3, which is a new target for ADCs, also showed potent antitumor efficacy both in vitro and in vivo. In conclusion, this study showed that this novel topoisomerase I inhibitor-based ADC technology is widely applicable to a diverse number of antibodies and is expected to mitigate myelotoxicity, thereby possibly resulting in better safety profiles than that of existing ADC technologies.

Abstract 3092: U3-1402a, a novel HER3-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a potent antitumor efficacy

Background HER3 (human epidermal growth factor receptor 3) is a member of HER family, and overexpressed in breast cancer, NSCLC, melanoma, gastric cancer and pancreatic cancer patients` tissues. U3-1402a is an antibody-drug conjugate (ADC) comprised of a fully human anti-HER3 monoclonal immunoglobulin G1 (IgG1) antibody (U3-1287) covalently conjugated via a cleavable peptide linker to exatecan derivative (DXd). The DXd is released after internalization of U3-1402a and leads to apoptosis of the target tumor cells by the inhibition of topoisomerase I. This ADC achieves a high drug-to-antibody-ratio (DAR 7 to 8) with homogeneous conjugation with the topoisomerase I inhibitor. The aim of this study was to preclinically evaluate the efficacy of U3-1402a in breast cancer models. Materials and methods In order to evaluate the pharmacological potential of U3-1402a, in vitro and in vivo studies were performed. In vitro growth inhibition assay evaluated the sensitivity of U3-1402a in HER3-positive human breast cancer cell line (HCC1569) and HER3-negative human cervical carcinoma cell line (C33A). Cells were treated with U3-1402a or MAAA-1181 (payload of U3-1402a) depending on its concentration (U3-1402a: 0.153 to 10 000 ng/mL, MAAA-1181: 2.44 to 160,000 pg/mL). In vivo growth inhibition study evaluated the dose-dependent sensitivity of U3-1402a in HER3-positive breast cancer xenograft model, MDA-MB-453. In addition, several xenograft models with different HER3 expression were tested with its sensitivity to U3-1402a. These models were HCC1569 (human breast cancer cell line, HER3 IHC score 3+), MDA-MB-453 (human breast cancer cell line, HER3 IHC score 2+), NIBIO-G016 (human gastric cancer patient-derived xenograft, HER3 IHC score 1+) and MDA-MB-231 (human breast cancer cell line, HER3 IHC score 0). R esults In vitro study, U3-1402a exhibited anti-tumor killing activity in HER3-positive human breast cancer cell line, HCC1569. C-33A human cervical carcinoma cell line was not sensitive to U3-1402a even MAAA-1181 itself exhibited anti-tumor killing activity to this cell line. In vivo study, U3-1402a showed dose-dependent anti-tumor killing activity in a HER3-positive breast cancer MDA-MB-453 xenograft model. Finally, in vivo tumor regression was only observed in HER3 2+ and 3+ models. Conclusions U3-1402a preclinically exhibited its efficacy in breast cancer model in vitro and in vivo. In vivo efficacy was correlated with HER3 expression. These studies suggest that U3-1402a, a novel HER3-targeting ADC, would be efficacious in a broader patient population with HER3 expression like breast cancer, melanoma, NSCLC, gastric cancer and pancreatic cancer. Citation Format: Suguru Ueno, Kenji Hirotani, Reimar Abraham, Sabine Blum, Birgit Frankenberger, Mauricio Redondo-Muller, Johannes Bange, Yusuke Ogitani, Akiko Zembutsu, Koji Morita, Takashi Nakada, Shuji Majima, Yuki Abe, Toshinori Agatsuma. U3-1402a, a novel HER3-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a potent antitumor efficacy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3092. doi:10.1158/1538-7445.AM2017-3092

Development of DS-5573a: A novel afucosylated mAb directed at B7-H3 with potent antitumor activity

B7‐H3 is highly overexpressed in a variety of human clinical tumors, and its expression is significantly associated with poor outcomes. In our study, we aimed to develop new antitumor mAbs by employing cancer cell immunization, and succeeded in generating a mouse anti‐human B7‐H3 antibody (M30) that shows antitumor activity. M30 was humanized (Hu‐M30), and an afucosylated Hu‐M30 (DS‐5573a) was also generated. To assess the potency of DS‐5573a as a therapeutic mAb, we characterized this mAb and evaluated its antitumor activity in vitro and in vivo. Flow cytometry analysis showed that B7‐H3 proteins were expressed on various types of cancer cell lines broadly, and DS‐5573a binds to IgC1 and IgC2 domains of human B7‐H3. Antibody‐dependent cellular cytotoxicity activity of DS‐5573a was drastically enhanced against medium to high B7‐H3‐expressing cancer cell lines MDA‐MB‐231 and NCI‐H322. DS‐5573a also induced high antibody‐dependent cellular cytotoxicity activity against low B7‐H3‐expressing cancer cell line COLO205, whereas Hu‐M30 induced little activity against it. In addition, DS‐5573a was found to be a novel anti‐B7‐H3 antibody which showed antibody‐dependent cellular phagocytosis activity. Furthermore, DS‐5573a showed dose‐dependent and significant antitumor efficacy (0.03–3 mg/kg) in MDA‐MB‐231‐bearing SCID mice (which have functional natural killer cells and macrophages), but little antitumor efficacy in NOG mice (which lack natural killer cells and have reduced macrophage function). These results suggest that antitumor activity of DS‐5573a is mediated by effector cells, and this mAb could be a promising antitumor therapy for patients with a wide range of B7‐H3‐expressing tumors.

Abstract 3852: U3-1784, a human anti-FGFR4 antibody for the treatment of cancer

Fibroblast Growth Factor Receptor 4 (FGFR4) is the fourth member of the Fibroblast Growth Factor Receptor (FGFR) family of receptor tyrosine kinases. All of the FGFR9s have been implicated in cancer development due to increased activation of their enzymatic activity either by gene mutation, over-expression or inadvertent ligand-mediated stimulation. One important alteration that may lead to FGFR4 activation in cancer is the overexpression of its ligand FGF19 in 20-40% of primary liver cancer. Here, we report the development of U3-1784, a phage display-derived fully human antibody that specifically binds to FGFR4 but not to isoforms of FGFR1-3. The antibody binds to an epitope in the putative ligand binding domain of the receptor and consequently inhibits ligand binding and downstream signaling. In a panel of 10 tumor models derived from hepatocellular carcinoma, U3-1784 significantly inhibits the growth of FGF19-expressing models up to 90% whereas models without FGF19 expression are insensitive. These results strongly suggest that the FGFR4/FGF19 axis is an oncogenic driver in hepatocellular carcinoma. U3-1784 is currently in phase I clinical trials. Citation Format: Rene Bartz, Keisuke Fukuchi, Tanja Lange, Katrin Gruner, Toshiaki Ohtsuka, Ichiro Watanabe, Shinko Hayashi, Mauricio Redondo-Muller, Mizuki Takahashi, Toshinori Agatsuma, Johannes Bange, Reimar Abraham. U3-1784, a human anti-FGFR4 antibody for the treatment of cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3852.

A HER2-Targeting Antibody–Drug Conjugate, Trastuzumab Deruxtecan (DS-8201a), Enhances Antitumor Immunity in a Mouse Model

Trastuzumab deruxtecan (DS-8201a), a HER2-targeting antibody–drug conjugate with a topoisomerase I inhibitor exatecan derivative (DX-8951 derivative, DXd), has been reported to exert potent antitumor effects in xenograft mouse models and clinical trials. In this study, the immune system–activating ability of DS-8201a was assessed. DS-8201a significantly suppressed tumor growth in an immunocompetent mouse model with human HER2-expressing CT26.WT (CT26.WT-hHER2) cells. Cured immunocompetent mice rejected not only rechallenged CT26.WT-hHER2 cells, but also CT26.WT-mock cells. Splenocytes from the cured mice responded to both CT26.WT-hHER2 and CT26.WT-mock cells. Further analyses revealed that DXd upregulated CD86 expression on bone marrow–derived dendritic cells (DC) in vitro and that DS-8201a increased tumor-infiltrating DCs and upregulated their CD86 expression in vivo. DS-8201a also increased tumor-infiltrating CD8+ T cells and enhanced PD-L1 and MHC class I expression on tumor cells. Furthermore, combination therapy with DS-8201a and anti–PD-1 antibody was more effective than either monotherapy. In conclusion, DS-8201a enhanced antitumor immunity, as evidenced by the increased expression of DC markers, augmented expression of MHC class I in tumor cells, and rejection of rechallenged tumor cells by adaptive immune cells, suggesting that DS-8201a enhanced tumor recognition by T cells. Furthermore, DS-8201a treatment benefited from combination with anti–PD-1 antibody, possibly due to increased T-cell activity and upregulated PD-L1 expression induced by DS-8201a. Mol Cancer Ther; 17(7); 1494–503. ©2018 AACR.