Yusuke Ogitani

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.

Novel antibody drug conjugates containing exatecan derivative-based cytotoxic payloads

Trastuzumab conjugates consisting of exatecan derivatives were prepared and their biological activities and physicochemical properties were evaluated. The ADCs showed strong efficacy and a low aggregation rate. The exatecan derivatives were covalently connected via a peptidyl spacer (Gly-Gly-Phe-Gly), which is assumed to be stable in circulation, and were cleaved by lysosomal enzymes following ADC internalization into tumor tissue. These anti-HER2 ADCs exhibited a high potency, specifically against HER2-positive cancer cell lines in vitro. The ADCs, bearing exatecan derivatives which have more than two methylene chains, exhibited superior cytotoxicity. It was speculated that steric hindrance of the cleavable amide moiety could be involved in the drug release. The adequate alkyl lengths of exatecan derivatives (13, 14, 15) were from two to four in terms of aggregation rate. The ADC having a hydrophilic moiety showed good efficacy in a HER2-positive and Trastuzumab-resistant breast carcinoma cell model in mice.

DS-8201a, a new HER2-targeting antibody–drug conjugate incorporating a novel DNA topoisomerase I inhibitor, overcomes HER2-positive gastric cancer T-DM1 resistance

Anti‐HER2 therapies are beneficial for patients with HER2‐positive breast or gastric cancer. T‐DM1 is a HER2‐targeting antibody–drug conjugate (ADC) comprising the antibody trastuzumab, a linker, and the tubulin inhibitor DM1. Although effective in treating advanced breast cancer, all patients eventually develop T‐DM1 resistance. DS‐8201a is a new ADC incorporating an anti‐HER2 antibody, a newly developed, enzymatically cleavable peptide linker, and a novel, potent, exatecan‐derivative topoisomerase I inhibitor (DXd). DS‐8201a has a drug‐to‐antibody‐ratio (DAR) of 8, which is higher than that of T‐DM1 (3.5). Owing to these unique characteristics and unlike T‐DM1, DS‐8201a is effective against cancers with low‐HER2 expression. In the present work, T‐DM1‐resistant cells (N87‐TDMR), established using the HER2‐positive gastric cancer line NCI‐N87 and continuous T‐DM1 exposure, were shown to be susceptible to DS‐8201a. The ATP‐binding cassette (ABC) transporters ABCC2 and ABCG2 were upregulated in N87‐TDMR cells, but HER2 overexpression was retained. Furthermore, inhibition of ABCC2 and ABCG2 by MK571 restored T‐DM1 sensitivity. Therefore, resistance to T‐DM1 is caused by efflux of its payload DM1, due to aberrant expression of ABC transporters. In contrast to DM1, DXd payload of DS‐8201a inhibited the growth of N87‐TDMR cells in vitro. This suggests that either DXd may be a poor substrate of ABCC2 and ABCG2 in comparison to DM1, or the high DAR of DS‐8201a relative to T‐DM1 compensates for increased efflux. Notably, N87‐TDMR xenograft tumor growth was prevented by DS‐8201a. In conclusion, the efficacy of DS‐8201a as a treatment for patients with T‐DM1‐resistant breast or gastric cancer merits investigation.

Safety, pharmacokinetics, and antitumour activity of trastuzumab deruxtecan (DS-8201), a HER2-targeting antibody–drug conjugate, in patients with advanced breast and gastric or gastro-oesophageal tumours: a phase 1 dose-escalation study

BACKGROUND Antibody-drug conjugates have emerged as a powerful strategy in cancer therapy and combine the ability of monoclonal antibodies to specifically target tumour cells with the highly potent killing activity of drugs with payloads too toxic for systemic administration. Trastuzumab deruxtecan (also known as DS-8201) is an antibody-drug conjugate comprised of a humanised antibody against HER2, a novel enzyme-cleavable linker, and a topoisomerase I inhibitor payload. We assessed its safety and tolerability in patients with advanced breast and gastric or gastro-oesophageal tumours. METHODS This was an open-label, dose-escalation phase 1 trial done at two study sites in Japan. Eligible patients were at least 20 years old with breast or gastric or gastro-oesophageal carcinomas refractory to standard therapy regardless of HER2 status. Participants received initial intravenous doses of trastuzumab deruxtecan from 0·8 to 8·0 mg/kg and dose-limiting toxicities were assessed over a 21-day cycle; thereafter, dose reductions were implemented as needed and patients were treated once every 3 weeks until they had unacceptable toxic effects or their disease progressed. Primary endpoints included identification of safety and the maximum tolerated dose or recommended phase 2 dosing and were analysed in all participants who received at least one dose of study drug. The dose-escalation study is the first part of a two-part study with the second dose-expansion part ongoing and enrolling patients as of July 8, 2017, in Japan and the USA. This trial is registered at ClinicalTrials.gov, number NCT02564900. FINDINGS Between Aug 28, 2015, and Aug 26, 2016, 24 patients were enrolled and received trastuzumab deruxtecan (n=3 for each of 0·8, 1·6, 3·2, and 8·0 mg/kg doses; n=6 for each of 5·4 and 6·4 mg/kg). Up to the study cutoff date of Feb 1, 2017, no dose-limiting toxic effects, substantial cardiovascular toxic effects, or deaths occurred. One patient was removed from the activity analysis because they had insufficient target lesions for analysis. The most common grade 3 adverse events were decreased lymphocyte (n=3) and decreased neutrophil count (n=2); and grade 4 anaemia was reported by one patient. Three serious adverse events-febrile neutropenia, intestinal perforation, and cholangitis-were reported by one patient each. Overall, in 23 evaluable patients, including six patients with low HER2-expressing tumours, ten patients achieved an objective response (43%, 95% CI 23·2-65·5). Disease control was achieved in 21 (91%; 95% CI 72·0-98·9) of 23 patients. Median follow-up time was 6·7 months (IQR 4·4-10·2), with nine (90%) of ten responses seen at doses of 5·4 mg/kg or greater. INTERPRETATION The maximum tolerated dose of trastuzumab deruxtecan was not reached. In this small, heavily pretreated study population, trastuzumab deruxtecan showed antitumour activity, even in low HER2-expressing tumours. Based on safety and activity, the most likely recommended phase 2 dosing is 5·4 or 6·4 mg/kg. FUNDING Daiichi Sankyo Co, Ltd.

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

Abstract A145: DS-8201a, a novel HER2-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a potent anti-tumor efficacy with differentiation from T-DM1 in preclinical studies

Antibody-drug conjugates (ADCs) represent a promising drug class which expresses a wider therapeutic window than conventional chemotherapeutic agents by effecting efficient and specific drug delivery to antigen-expressing tumor cells. DS-8201a is a HER2-targeting ADC structurally composed of a humanized anti-HER2 antibody, enzymatically cleavable peptide-linker, and a novel topoisomerase I inhibitor (DXd), which is cell-membrane permeable and more potent than SN-38 the active metabolite of irinotecan. This ADC achieves a high drug-to-antibody-ratio (DAR, 7 to 8) with homogeneous conjugation with DXd. DS-8201a is cleaved by lysosomal enzymes and releases DXd in the cytoplasm after it binds to HER2 receptors and is internalized in tumor cells. In order to evaluate the pharmacological potential of DS-8201a, in vitro and in vivo studies were performed in comparison with T-DM1. T-DM1 is a commercialized HER2-targeting ADC with a tubulin polymerization inhibitor that is approved for treatment for HER2-positive breast cancer. In vitro studies indicated that DS-8201a exhibited a HER2 expression-dependent cell growth inhibitory activity. In vivo studies using a HER2-positive gastric cancer NCI-N87 cell line-derived xenograft (CDX) model suggested that DS-8201a induced a dose-dependent tumor growth inhibition and tumor regression with a single dosing at more than 1 mg/kg. In comparison with T-DM1, 1) DS-8201a was effective against T-DM1-insensitive breast and gastric patient-derived xenograft (PDX) models with high HER2 expression. 2) DS-8201a, but not T-DM1, demonstrated anti-tumor efficacy against several breast cancer PDX models with low HER2 expression. 3) DS-8201a showed a complete response in mice inoculated with a mixture of HER2-positive and -negative cells while T-DM1 did not. These differentiations may be due to the different mechanisms of action of each conjugated drug (topoisomerase I inhibition vs. tubulin polymerization inhibition), higher DAR of DS-8201a (7 to 8 vs. 3.5), and DS-8201a9s more potent bystander killing due to higher cell membrane permeability of conjugated toxin. When taken together with over 30 CDX and PDX models from 8 tumor types (breast, gastric, cholangiocarcinoma, esophagus, colon, NSCLC, pancreas, and ovary) conducted so far, DS-8201a induced tumor regression in over 20 models with various HER2 expression levels though T-DM1 induced regression in only 8 models with high HER2 expression. In conclusion, these studies suggest that DS-8201a, a novel HER2-targeting ADC, may be more efficacious in a broader patient population than T-DM1, and in tumors which current anti-HER2 therapies are ineffective, such as T-DM1 insensitive tumors and low HER2-expressing tumors, and in tumors with high HER2 heterogeneity. The first in human study will be initiated in 2015 3Q. Citation Format: Yusuke Ogitani, Junko Yamaguchi, Chiaki Ishii, Takehiro Hirai, Ryo Atsumi, Koji Morita, Ichiro Hayakawa, Hiroyuki Naito, Takeshi Masuda, Takashi Nakada, Takahiro Jikoh. DS-8201a, a novel HER2-targeting ADC with a novel DNA topoisomerase I inhibitor, demonstrates a potent anti-tumor efficacy with differentiation from T-DM1 in preclinical studies. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A145.

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.

Abstract 1193: DS-8201a, a novel HER2-targeting ADC with a novel DNA topoisomerase I inhibitor, abrogates the resistance to T-DM1 in HER2-positive gastric cancer: a preclinical study

Background Anti-HER2 therapies are beneficial for patients with HER2-positive breast and gastric cancer. T-DM1 is an HER2-targeting antibody-drug conjugate (ADC), which is structurally composed of the anti-HER2 antibody trastuzumab and the tubulin inhibitor DM1. T-DM1 has shown efficacy in patients with advanced breast cancer, but all patients eventually develop resistance to T-DM1. DS-8201a is a novel ADC composed of an anti-HER2 antibody and a novel potent topoisomerase I inhibitor DX-8951 derivative. DS-8201a achieved a high drug-to-antibody-ratio (DAR: 7-8) and homogeneous drug conjugation. The aim of this study was to elucidate the mechanisms of T-DM1 resistance, and evaluate the efficacy of DS-8201a in a T-DM1-resistant HER2-positive gastric cancer cell line. Materials and methods The T-DM1-resistant NCI-N87 cell line (N87-TDMR) was established by a step-wise method—the parent HER2-positive N87 cell line was exposed to up to 4 µg/ml of T-DM1. The profile of N87-TDMR was assessed by immunoblotting, DNA microarray, and quantitative reverse transcription-PCR (qRT-PCR). The sensitivity of parent N87 and N87-TDMR cells to T-DM1 or DS8201a was assessed by an in vitro growth inhibition assay as well as through the mouse xenograft model study. Results N87-TDMR cells were found to be resistant to T-DM1 in the in vitro growth inhibition assay (50% growth inhibitory concentration; >10 µg/ml in N87-TDMR, 0.055 µg/ml in N87) as well as in the in vivo xenograft model study, but HER2 expression was maintained in the N87-TDMR cells. A comprehensive expression analysis revealed that N87-TDMR cells showed increased ATP-binding cassette (ABC) transporter expression such as ABCC2 and ABCG2 compared with N87 cells (50.2 times; 8.0 times higher than that measured by qRT-PCR relatively). Inhibition of ABCC2 and ABCG2 by MK571 recovered the sensitivity to T-DM1 in N87-TDMR cells. Since DS-8201a has higher DAR than T-DM1 and the unique topoisomerase I inhibitor, this resistance was expected to be abrogated. DS-8201a showed anti-tumor efficacy against N87-TDMR xenograft cells in the mouse model (relative tumor growth rate: 244.2% with T-DM1, -39.2% with DS-8201a; n=10 for each treatment). Conclusions The HER2-positive gastric cancer cell line used in this preclinical study acquired resistance to T-DM1 depending on the up-regulation of ABCC2 and ABCG2 expression. However, DS-8201a could abrogate this T-DM1 resistance. These results provide a rationale for DS-8201a to be investigated for its efficacy in patients with T-DM1-resistant breast or gastric cancer. Citation Format: Yoshikane Nonagase, Naoki Takegawa, Kimio Yonesaka, Kazuko Sakai, Yusuke Ogitani, Junji Tsurutani, Kazuto Nishio, Kazuhiko Nakagawa. DS-8201a, a novel HER2-targeting ADC with a novel DNA topoisomerase I inhibitor, abrogates the resistance to T-DM1 in HER2-positive gastric cancer: a preclinical study [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 1193. doi:10.1158/1538-7445.AM2017-1193