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Currently, the only approved treatments for gastrointestinal stromal tumor (GIST) are tyrosine kinase inhibitors (TKI), which eventually lead to the development of secondary resistance mutations in KIT or PDGFRA and disease progression. Herein, we identified G protein–coupled receptor 20 (GPR20) as a novel non–tyrosine kinase target in GIST, developed new GPR20 IHC, and assessed GPR20 expression in cell lines, patient-derived xenografts, and clinical samples from two institutes (United States and Japan). We studied GPR20 expression stratified by treatment line, KIT expression, GIST molecular subtype, and primary tumor location. We produced DS-6157a, an anti-GPR20 antibody–drug conjugate with a novel tetrapeptide-based linker and DNA topoisomerase I inhibitor exatecan derivative (DXd). DS-6157a exhibited GPR20 expression–dependent antitumor activity in GIST xenograft models including a GIST model resistant to imatinib, sunitinib, and regorafenib. Preclinical pharmacokinetics and safety profile of DS-6157a support its clinical development as a potential novel GIST therapy in patients who are refractory or have resistance or intolerance to approved TKIs.Significance:GPR20 is selectively expressed in GIST across all treatment lines, regardless of KIT/PDGFRA genotypes. We generated DS-6157a, a DXd-based antibody–drug conjugate that exhibited antitumor activity in GIST models by a different mode of action than currently approved TKIs, showing favorable pharmacokinetics and safety profiles.This article is highlighted in the In This Issue feature, p. 1307

Characteristics for GIST patient samples, GPR20 expression in KIT/PDGFRA wild type GIST, GPR20 expression in normal, malignant and sarcoma tissue microarrays.

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

S1: Expression of GPR20 in clinical GIST specimens from NCCHE S2: Representative images of GPR20 expression in various sarcoma including GIST in University of Basel derived TMA. S3: GPR20 was not expressed in systemic mastocytosis. S4: Protein expression of human, cynomolgus monkey, rat, and mouse GPR20 on CHO-K1 cells. S5: Lack of DS-6157a efficacy in the GPR20-negative KPL-4 xenograft model. S6: Comparison of tumor growth curve of GIST-T1 and GIST-T1/GPR20 xenograft models.

Currently, the only approved treatments for gastrointestinal stromal tumor (GIST) are tyrosine kinase inhibitors (TKI), which eventually lead to the development of secondary resistance mutations in KIT or PDGFRA and disease progression. Herein, we identified G protein–coupled receptor 20 (GPR20) as a novel non–tyrosine kinase target in GIST, developed new GPR20 IHC, and assessed GPR20 expression in cell lines, patient-derived xenografts, and clinical samples from two institutes (United States and Japan). We studied GPR20 expression stratified by treatment line, KIT expression, GIST molecular subtype, and primary tumor location. We produced DS-6157a, an anti-GPR20 antibody–drug conjugate with a novel tetrapeptide-based linker and DNA topoisomerase I inhibitor exatecan derivative (DXd). DS-6157a exhibited GPR20 expression–dependent antitumor activity in GIST xenograft models including a GIST model resistant to imatinib, sunitinib, and regorafenib. Preclinical pharmacokinetics and safety profile of DS-6157a support its clinical development as a potential novel GIST therapy in patients who are refractory or have resistance or intolerance to approved TKIs. Significance: GPR20 is selectively expressed in GIST across all treatment lines, regardless of KIT/PDGFRA genotypes. We generated DS-6157a, a DXd-based antibody–drug conjugate that exhibited antitumor activity in GIST models by a different mode of action than currently approved TKIs, showing favorable pharmacokinetics and safety profiles. This article is highlighted in the In This Issue feature, p. 1307

Background: Trophoblast cell surface antigen 2 (TROP2) is highly expressed in various epithelial tumors, including non-small cell lung cancer (NSCLC) and triple negative breast cancer (TNBC), and correlates with poor prognosis. Datopotamab deruxtecan (Dato-DXd) is an antibody-drug conjugate (ADC) consisting of a humanized anti-TROP2 IgG1 monoclonal antibody covalently linked to a highly potent topoisomerase I (Topo I) inhibitor payload via a stable, tumor-selective, tetrapeptide-based cleavable linker. Dato-DXd has shown encouraging antitumor activity and a manageable safety profile in patients with NSCLC and TNBC as part of the first-in-human, Phase 1 study (NCT03401385). To enhance clinical responses, clinical trials evaluating combination therapy of Dato-DXd with PD-1/PD-L1 inhibitors in NSCLC and TNBC are currently ongoing (e.g. NCT04526691 and NCT03742102). Here we report preclinical evidence for supporting increased antitumor activity of Dato-DXd in combination with PD-1/PD-L1 inhibitors using a syngeneic mouse tumor model and its impact on tumor immunity. Methods: MC38 mouse colon adenocarcinoma cells stably transfected with human TROP2 (hTROP2_MC38) were inoculated subcutaneously in immunocompetent C57BL6 mice or immunodeficient nude mice and the antitumor activity of Dato-DXd with or without mouse PD-1/PD-L1 inhibitors was evaluated. The tumor infiltrating immune cells in hTROP2_MC38 tumors were analyzed by flow cytometry at 3 days and 10 days after dosing. In addition, the impact of CD8+ T cell depletion on the antitumor activity of Dato-DXd against hTROP2_MC38 tumors in C57BL6 mice was evaluated. Results: Dato-DXd showed stronger antitumor activity against hTROP2_MC38 tumors in immunocompetent C57BL6 mice than in immunodeficient nude mice, which suggests that immune cells play an important role in the mechanism of action (MoA) of Dato-DXd. The combination of Dato-DXd and mouse PD-1/PD-L1 inhibitors enhanced antitumor activity compared to monotherapy against hTROP2_MC38 tumors in C57BL6 mice. Activation of the dendritic cells in hTROP2_MC38 tumors in C57BL6 mice was observed after Dato-DXd monotherapy or combination therapy with mouse PD-1/PD-L1 inhibitors, which led to the increase of tumor infiltrating CD8+ T cells. Involvement of CD8+ T cells in the MoA of Dato-DXd was also supported by the result that CD8+ T cell depletion in C57BL6 mice decreased the antitumor activity of Dato-DXd against hTROP2_MC38 tumors. In addition, activation of tumor infiltrating NK cells and macrophages was observed after Dato-DXd monotherapy or combination therapy, suggesting the potential effect on innate immunity. Conclusion: These results suggest that Dato-DXd may stimulate tumor immunity and sensitize tumors to PD-1/PD-L1 inhibitors. The combination of Dato-DXd and PD-1/PD-L1 blockers could be a valuable therapy for patients with TROP2-positive tumors. Citation Format: Daisuke Okajima, Satoru Yasuda, Takami Suzuki, Michiko Kitamura, Junko Yamaguchi, Takanori Maejima, Tsuyoshi Karibe, Tadashi Toki, Penny Phillips, Toshinori Agatsuma. Datopotamab deruxtecan (Dato-DXd) enhances antitumor response to PD-1/PD-L1 inhibitors in TROP2-expressing tumors in mice [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 2932.

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.

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.

In the course of our screening, we discovered a novel compound, A-503451A, as a potent hypoxia-inducible factor (HIF) activator. In human hepatocarcinoma HepG2 cells, A-503451A induced HIF-mediated luciferase reporter gene expression and stabilized HIF-1α protein. A-503451A increased the mRNA expression levels and the protein secretion of HIF-dependent genes, vascular endothelial growth factor and erythropoietin. Addition of excess ferric chloride to the culture medium suppressed the HIF-induction activity of A-503451A. A-503451A did not have iron-chelating activity in vitro, but decreased the intracellular labile iron pool concentration. These data indicate that A-503451A is a unique HIF activator.

More than 85% of GISTs are driven by activating mutations in KIT proto-oncogene receptor tyrosine kinase (KIT) or platelet-derived growth factor receptor alpha (PDGFRA). Currently, the only approved treatments for GIST are KIT directed tyrosine kinase inhibitors (TKIs). However, treatment with approved TKIs eventually results in disease progression most often due to the development of secondary resistance mutations in KIT. In addition, these agents have limited activity in PDGFRA mutant GIST and KIT/PDGFRA wild type (WT) GIST as primary therapy. Therefore, it is essential to develop novel therapeutic strategies with different modes of action in advanced GIST. G protein-coupled receptor 20 (GPR20) is an orphan GPCR selectively and abundantly expressed in GIST. GPR20 expression is regulated by FOXF1 and ETV1, transcription factors that play critical roles in KIT-driven GIST initiation, proliferation, and survival. We hypothesize that GPR20 is a potential therapeutic target for ADC development for the treatment of GIST. In this study, 1) GPR20 and KIT protein expression was assessed by IHC staining on GIST samples from DFCI (n=144) and NCCHE (n=100) as well as on normal and malignant tissue microarrays obtained commercially, and 2) an anti-GPR20 ADC (DS-6157a) was generated to evaluate antitumor activity in GIST models and to assess safety. GPR20 was expressed in more than 88% of the GIST samples analyzed, with higher expression levels in samples that: (I) received multiple treatment lines compared to naïve/early treated samples, (II) expressed higher KIT levels, (III) were small intestinal GIST, and/or (IV) had no KIT mutation, including succinate dehydrogenase (SDH) deficient GIST and neurofibromatosis type 1 (NF1)-associated GIST. The interstitial cells of Cajal were the only normal cells positive for GPR20. Normal mast cells expressed KIT but not GPR20. DS-6157a is an ADC composed of a humanized anti-GPR20 antibody, a Gly-Gly-Phe-Gly tetra-peptide-based linker, and a DNA topoisomerase I (TOP1) inhibitor Dxd. DS-6157a exhibited GPR20 expression-dependent cell growth-inhibitory activity and induced tumor regression with dosing at 3 to 10 mg/kg in multiple GIST xenograft models. In addition, DS-6157a showed antitumor activity in a GIST patient-derived xenograft model that was resistant to imatinib, sunitinib, and regorafenib. In vitro, DS-6157a induced TOP1 inhibitor-associated markers of DNA damage (phosphorylation of Chk1) and apoptosis (cleaved PARP) in GPR20 expressing cells. In preclinical toxicology studies using rats and cynomolgus monkeys, the pharmacokinetics and safety profile of DS-6157a were favorable at up to 200 mg/kg and 30 mg/kg, respectively. These data support the clinical development of DS-6157a as a potential novel GIST therapy with activity in patients that are resistant, refractory, or intolerant to approved TKIs. Citation Format: Kenji Iida, Amr H. Abdelhamid, Akiko Kawano Nagatsuma, Tomoko Shibutani, Satoru Yasuda, Michiko Kitamura, Chiharu Hattori, Manabu Abe, Jun Hasegawa, Takuma Iguchi, Tsuyoshi Karibe, Takashi Nakada, Koichiro Inaki, Reiko Kamei, Yuki Abe, Jessica L. Andersen, Sandro Santagata, Matthew L. Hemming, Suzanne George, Toshihiko Doi, Atsushi Ochiai, George D. Demetri, Toshinori Agatsuma. Therapeutic targeting of GPR20, selectively expressed in gastrointestinal stromal tumor (GIST), with DS-6157a, an antibody-drug conjugate (ADC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5181.

Background: A trophoblast cell surface antigen 2 (TROP2) is a 36-kDa single-pass transmembrane protein overexpressed in various epithelial tumors including non-small cell lung cancer (NSCLC) with relatively low and restricted expression in normal tissues, and is associated with aggressive tumor behavior. Therefore, TROP2 could be an attractive target for cancer therapy. We created DS-1062a, TROP2-targeting antibody-drug conjugate (ADC) with Daiichi Sankyo DXd technology using a novel DNA topoisomerase I inhibitor DXd. In this study, the pharmacological activity and the mechanism of action of DS-1062a were evaluated in preclinical in vitro and in vivo models. Methods: In vitro cell growth inhibitory and in vivo antitumor activities of DS-1062a were evaluated using TROP2-high and -low tumor cell lines and xenograft mouse models. Internalization and intracellular trafficking of DS-1062a in tumor cells were analyzed by immunofluorescence microscopy. Induction of DNA damage and apoptosis to tumor cells by DXd payload released from DS-1062a were assessed by western blot and immunohistochemistry. Pharmacokinetic profiles of DS-1062a were also analyzed in xenograft mouse model. Results: DS-1062a showed in vitro cell growth inhibitory activity to TROP2-high tumor cells, but not to TROP2-low tumor cells. It was observed with confocal microscopy that DS-1062a was co-localized with a lysosomal marker LAMP-2 after internalizing into TROP2-high tumor cells. The amount of DXd payload released from TROP2-high tumor cells after the in vitro treatment with DS-1062a was higher than that of TROP2-low tumor cells. DNA damage and apoptosis were induced in TROP2-high tumor cells after the in vitro treatment with DXd and DS-1062a, but not with isotype control IgG ADC. Similarly, DS-1062a exhibited strong antitumor activity with tumor regression in several TROP2-high tumors including NSCLC and DXd accumulation and DNA damage in TROP2-high tumors were observed in DS-1062a-treated tumors, but not in isotype control IgG ADC-treated tumors. Pharmacokinetic profiles of DS-1062a in xenograft mouse model were preferable. Conclusion: Based on these preclinical results, DS-1062a could provide a valuable therapy with a potential benefit in TROP2-expressing cancers in the clinical setting. A first-in-human phase 1 study in patients with advanced solid tumors is in progress (NCT03401385). Citation Format: Daisuke Okajima, Junko Yamaguchi, Michiko Kitamura, Reiko Kamei, Takanori Maejima, Tomoko Shibutani, Satoru Yasuda, Tadashi Toki, Tsuyoshi Karibe, Tomomichi Fujitani, Takashi Nakada, Riki Goto, Yutaka Noguchi, Yuki Abe, Toshinori Agatsuma. DS-1062a, a novel TROP2-targeting antibody-drug conjugate with a novel DNA topoisomerase I inhibitor DXd, demonstrates potent antitumor activity in preclinical models [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C026. doi:10.1158/1535-7163.TARG-19-C026

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.

It has been pointed out that the motor evoked potential(MEP)with a subdural electrode is useful in the intraoperative monitoring for unruptured aneurysm surgery. However, in some cases, we experienced postoperative ischemic complications despite evaluating the motor function via MEP monitoring. Herein, we have reported the usefulness and problems of intraoperative monitoring with MEP to evaluate brain dysfunction caused by insufficiency of cerebral blood flow. Out of 279 aneurysm surgery procedures, we performed MEP monitoring in 142 cases and successfully recorded in 126 cases. We compared the ischemic complication rate of the group for which MEP was monitored with that of the group for which MEP was not monitored. The whole ischemic complication rate was decreased in the group that underwent MEP monitoring. Thus, it was suggested that MEP monitoring was useful for avoiding ischemic complications. In internal carotid artery aneurysms, the amplitude of MEP changed and recovered in 2 cases and disappeared in one case. In anterior cerebral artery aneurysms, the amplitude of MEP changed and recovered in 2 cases. In middle cerebral artery aneurysms, the amplitude of MEP changed and recovered in 5 cases. We could avoid ischemic complications by intraoperative MEP monitoring in many cases. However, in some cases, we found ischemic complications that were not detected by MEP monitoring with a subdural electrode. In these cases, transcranial stimulation in combination with subdural electrode might be effective in avoiding ischemic complications that might occur after dural closure.

e24206Background: DS-1062a is a trophoblast cell surface protein 2 (TROP2)-targeting antibody-drug conjugate (ADC) comprised of a humanized anti-TROP2 monoclonal antibody, enzymatically cleavable p...

In the course of our screening for activators of hypoxia-inducible factor (HIF), A-503451 A and virantmycin were isolated from the cultured broth of an actinomycete strain, Streptomyces sp. SANK 60101. From the same culture, the non-active homologs A-503451 B and D were also isolated. A-503451 A and virantmycin activated HIF-dependent reporter gene expression with EC