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Supplementary Data from Datopotamab Deruxtecan, a Novel TROP2-directed Antibody–drug Conjugate, Demonstrates Potent Antitumor Activity by Efficient Drug Delivery to Tumor Cells

Background: HER3 is broadly expressed in various solid tumor types, and its expression can be upregulated by treatment with receptor tyrosine kinase inhibitors (RTKi) such as EGFR TKIs used to treat EGFR-mutated NSCLC. HER3-DXd, a novel antibody-drug conjugate (ADC) composed of a human anti-HER3 IgG1 monoclonal antibody (patritumab) covalently linked to a topoisomerase I inhibitor payload (DXd), is currently being studied in clinical trials for breast cancer and NSCLC. As previously reported, HER3-DXd treatment transiently decreases HER3 expression levels in tumors and EGFR TKIs increase HER3 membrane expression. However, the impact of HER3 dynamics on payload delivery has not been clarified yet. In this study, we investigated HER3 dynamics including HER3 receptor turnover and payload delivery in cancer cells using HER3-DXd both as a single agent and in combination with RTKi including osimertinib, which is in clinical trials in combination with HER3-DXd. Methods: HER3/ADC internalization was evaluated by using confocal imaging in MDA-MB-453 cells treated with HER3-DXd. Internalization and payload release were quantitatively measured in 3 cancer cell lines treated with HER3-DXd. HER3 turnover on the cell surface was also evaluated upon wash-out of HER3-DXd. In xenograft models, mice were administered two doses of HER3-DXd at different doses and dosing intervals, and membrane HER3 expression and tumor payload concentration were examined over time. NSCLC cell lines harboring EGFR activating mutations, ROS1 fusions, or ALK fusions were used to evaluate the effect of osimertinib, lorlatinib, or ceritinib on cell surface HER3 expression and payload release (osimertinib only). Results: HER3-DXd was rapidly transferred to early endosomes after binding to HER3. HER3 dynamics varied among the cell lines tested in vitro, and payload release reflected cell surface HER3 expression levels, HER3 internalization speed and turnover rates. In xenograft models, a higher dosage of HER3-DXd resulted in a larger decrease in membrane HER3 expression. Dosing interval also affected membrane HER3 expression levels; the degree of tumor payload concentration increase after the second dose was dependent on the recovery of HER3 expression after the first dose. Furthermore, we confirmed that RTKi increased the cell surface HER3 expression in NSCLC cell lines with targetable driver genomic alterations and that osimertinib increased payload delivery in PC-9 cells through the upregulation of cell surface HER3 expression. Conclusion: HER3 expression was dynamically changed by HER3-DXd dosing regimen and by RTKi treatment, resulting in a substantial impact on payload release. These findings support our strategy of clinical studies using HER3-DXd after drugs that increase HER3 expression including EGFR TKI and indicate that HER3 dynamics may play a key role in achieving optimal efficacy of HER3-DXd. Citation Format: Nagiho Komatsu, Saori Sato, Sumie Muramatsu, Ryuichi Nakamura, Kumiko Koyama. The impact of HER3 dynamics on the efficacy of HER3-DXd, a novel HER3 directed antibody-drug conjugate. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3996.

Trophoblast cell surface antigen 2 (TROP2) is highly expressed on various epithelial tumors and correlates with poor prognosis. We developed the novel TROP2-directed antibody–drug conjugate (ADC), datopotamab deruxtecan (Dato-DXd, DS-1062a), with a potent DNA topoisomerase I inhibitor (DXd), and evaluated its antitumor activity and safety profiles in preclinical models. The pharmacologic activity and mechanism of action of Dato-DXd were investigated in several human cancer cell lines and xenograft mouse models including patient-derived xenograft (PDX) models. Safety profiles were also assessed in rats and cynomolgus monkeys. Dato-DXd bound specifically to TROP2 and was internalized into tumor cells followed by intracellular trafficking to lysosome and DXd release, which induced DNA damage and apoptosis in TROP2-expressing tumor cells in vitro. Dato-DXd exhibited in vivo antitumor activity with DNA damage induced by the accumulated DXd in TROP2-expressing xenograft tumors, but neither isotype control IgG-ADC nor anti-TROP2 antibody had this effect. Dato-DXd also showed potent antitumor activity with tumor regression in several TROP2-expressing xenograft tumors including NSCLC PDX models. Safety profiles of Dato-DXd in rats and cynomolgus monkeys were acceptable. Dato-DXd demonstrated potent antitumor activity against TROP2-expressing tumors by efficient payload delivery into tumors and acceptable safety profiles in preclinical models. These results suggest Dato-DXd could be a valuable treatment option for patients with TROP2-expressing tumors in the clinical setting.

Hepcidin has emerged as the central regulatory molecule in systemic iron homeostasis. The inhibition of hepcidin may be a favorable strategy for the treatment of anemia of chronic disease. Here, we have reported the design, synthesis, and structure-activity relationships (SAR) of a series of 4-aminopyrimidine compounds as inhibitors of hepcidin production. The optimization study of 1 led to the design of a potent and bioavailable inhibitor of hepcidin production, 34 (DS42450411), which showed serum hepcidin-lowering effects in a mouse model of interleukin-6-induced acute inflammation.

Hepcidin has emerged as the central regulatory molecule of systemic iron homeostasis. Inhibition of hepcidin could be a strategy favorable to treating anemia of chronic disease (ACD). We report herein the synthesis and structure-activity relationships (SARs) of a series of benzisoxazole compounds as orally active hepcidin production inhibitors. The optimization study of multi kinase inhibitor 1 led to a potent and bioavailable hepcidin production inhibitor 38 (DS79182026), which showed serum hepcidin lowering effects in a mouse IL-6 induced acute inflammatory model.

Hepcidin has emerged as the central regulatory molecule in systemic iron homeostasis, and its inhibition could be a favorable strategy for treating anemia of chronic disease (ACD). Here, we report the design, synthesis and structure-activity relationships (SAR) of a series of 4,6-disubstituted indazole compounds as hepcidin production inhibitors. The optimization study of multi-kinase inhibitor 1 led to the design of a potent and bioavailable hepcidin production inhibitor, 32 (DS28120313), which showed serum hepcidin-lowering effects in an interleukin-6-induced acute inflammatory mouse model.

Hepcidin has emerged as the central regulatory molecule of systemic iron homeostasis. Inhibition of hepcidin could be a strategy favorable to treating anemia of chronic disease (ACD). We report herein the synthesis and structure-activity relationships (SARs) of a series of indazole compounds as hepcidin production inhibitors. The optimization study of compound 1 led to a potent hepcidin production inhibitor 45, which showed serum hepcidin lowering effects in a mouse IL-6 induced acute inflammatory model.

We compared the antithrombotic properties of a factor Xa inhibitor (DX-9065a) with those of a thrombin inhibitor (melagatran) in a rat disseminated intravascular coagulation model and a rat venous thrombosis model. Rat disseminated intravascular coagulation and venous thrombosis models were produced by injection of tissue factor and platinum wire placement, respectively. DX-9065a exerted antithrombotic effects dose dependently in both models. Melagatran was also effective in the venous thrombosis model, whereas it showed an aggravation in the disseminated intravascular coagulation model at low but not high doses. In the in vitro study, DX-9065a decreased the C max of the thrombin generation curve in plasma irrespective of whether protein C was present or not. However, melagatran increased the C max at low concentrations when protein C was present. This increase was not detected in protein C-deficient plasma. These results suggest that, unlike DX-9065a, melagatran in low doses aggravates disseminated intravascular coagulation by increasing thrombin generation, which may be partly due to suppression of negative feedback by activated protein C.

Introduction: Bleeding risk accompanied with anti-platelet drugs is an ultimate dilemma in the treatment of thrombosis patient. Under high shear condition of blood flow, vWF- and collagen-induced signaling pathways are likely to trigger the platelet adhesion to the injured endothelium, which leads to the activation of platelets and arterial thrombus formation. Thus, the recent studies suggest that the selective inhibitor of these pathways is a new target of anti-platelet drugs with lower bleeding risk. We report here a pharmacological profile of DZ-697b, which selectively inhibits platelet aggregation evoked by ristocetin and collagen in vitro and ex vivo. Materials and methods: Human volunteers blood was processed platelet rich plasma (PRP) or washed platelets. PRP aggregation was induced by ristocetin and collagen. To reveal the selectivity, effect of DZ-697b on U46619 (TXA2 analogue), ADP, thrombin and TRAP induced aggregation in the washed platelets were examined. In guinea pigs and cynomolgus monkeys, effects of DZ-697b given orally were also examined on ex vivo PRP aggregation induced by collagen. To investigate the underlying mechanisms of DZ-697b, changes in phosphorylation of FcR γ chain, a common signaling pathway of both vWF- and collagen-induced platelet aggregation, were studied. Results: DZ-697b potently inhibited both ristocetin- and collagen-induced human PRP aggregation, the IC50 being 0.74 μM and 0.55 μM, respectively. In contrast, DZ-697b even at 50 μM did not show any influences on U46619, ADP, thrombin and TRAP induced platelet aggregation. DZ-697b did not affect ovine COX-1 and COX-2 activities at up to 300 μM. The bioavailability of this compound was more than 80% in monkeys. Oral administration of DZ-697b at 1–3 mg/kg significantly and persistently inhibited collagen induced PRP aggregation in monkeys and guinea pigs. Application of ristocetin, vWF, and collagen significantly increased the intensity of phosphorylation of FcR γ chain in washed platelets, which were inhibited by DZ-697b. Conclusion: DZ-697b is an orally active compound which selectively inhibits ristocetin- and collagen-induced platelet aggregation and seems to be promising as novel anti-platelet drug.

Introduction: DZ-697b, a novel anti-platelet compound, selectively inhibits ristocetin- and collagen-induced platelet aggregation, whose pharmacological profile is different from any current medicines. In order to clarify the benefit and risk balance as an anti-thrombotic agent, effects of DZ-697b and aspirin were compared on thrombus formation and gastric bleeding model in guinea pigs. Materials and Methods: Fasted male Hartly guinea pigs under anesthesia were used. DZ-697b (3 - 200 mg/kg) or aspirin (100–300 mg/kg) was orally administered 60 min prior to evaluation. PIT model: Left carotid artery was exposed. Photochemically induced thrombosis (PIT) was initiated by intravenous injection of rose-bengal (10 mg/kg) and exposure of green light (540 nm) to the carotid artery. The time to occlusion by thrombus formation was measured by pulse Doppler blood flow meter. Gastric bleeding model: The stomach was exposed, and esophagus and pylorus were ligated. A cannula was inserted into stomach and gastric hemorrhagic erosion was induced by intraluminal application of 0.7N HCl in a volume of 5mL. Gastric contents were collected every 10 min and measured hemoglobin (Hb) leakage as indices of bleeding. Results: DZ-697b at 3 mg/kg to 100 mg/kg dose-dependently prolonged the time to occlusion in the PIT model, and significant anti-thrombotic effects were observed from 10 mg/kg. However, gastric bleeding induced by 0.7N HCl in gastric lavage was not affected by DZ-697b up to 100 mg/kg. Statistically significant increase in gastric bleeding was observed when DZ-697b was administered at 200 mg/kg. Aspirin at doses of 100–300 mg/kg tended to lengthen the time to thrombotic occlusion in a dose-dependent manner, but there was no statistically significant difference. In contrast, aspirin dose-dependently increased gastric bleeding in the lavage and significant enhancement was found at doses of 200 and 300 mg/kg. Both DZ-697b and aspirin did not give any influences on the area of hemorrhagic erosion induced by gastric application of 0.7N HCl. Thus, the balance between anti-thrombotic effect and bleeding risk of DZ-697b seemed more favorable than that of aspirin. Conclusion: DZ-697b showed anti-thrombotic effects at doses much lower than those inducing gastric bleeding in comparison with aspirin. These results suggest that inhibitors of vWF and collagen related signaling pathway are promising target in the development of new anti-platelet drugs with lower bleeding risk.

Factor Xa (FXa) is a key serine protease in the coagulation cascade and is a promising target enzyme for developing a new antithrombotic agent. Our first clinical candidate for a small molecular direct FXa inhibitor DX-9065a potently inhibits FXa (Ki = 41 nM) and exerts antithrombotic effects in animal models. However, due to its poor bioavailability (10% in monkeys) the compound is used only as an injectable formulation in clinical studies. Here we report in vitro characteristics of serine proteases inhibition, anticoagulant effects and in vivo antithrombotic efficacy of DU-176b, a novel, potent and orally active direct FXa inhibitor. DU-176b competitively inhibited human FXa with a Ki value of 0.561 nM, indicating 70-fold increase in FXa inhibitory activity compared with DX-9065a. DU-176b demonstrated 10,000-fold selectivity relative to inhibition of thrombin (Ki = 6.00 μM), and had no effects on the enzymatic activities of factor VIIa, t-PA, plasmin, trypsin and chymotrypsin. In human plasma, DU-176b prolonged prothrombin time (PT) and activated partial thromboplastin time (APTT) in a concentration-dependent manner. Its concentrations for doubling these clotting times were 0.256 and 0.508 μM, respectively. After oral administration of DU-176b to rats, significant anti-Xa activity was observed in plasma over 4 h. The oral bioavailability of DU-176b (approximately 50%) was significantly higher than that of DX-9065a (10%) in monkeys. The antithrombotic efficacy of DU-176b was examined by oral administration to rats 30 minutes prior to thrombogenic stimuli. In a venous stasis thrombosis model, DU-176b (0.5 – 12.5 mg/kg, p.o.) dose-dependently inhibited thrombus formation, prolonged PT, and revealed plasma anti-Xa activity. DU-176b also exerted significant anticoagulant effect in a rat model of tissue factor-induced disseminated intravascular coagulation at doses of 0.1 – 2.5 mg/kg, p.o. These results demonstrate that DU-176b is a potent and selective factor Xa inhibitor that possesses antithrombotic effect after oral administration. DU-176b has the potential to be clinically useful for prophylaxis and treatment of several thrombotic diseases.