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CD147 is an immunoglobulin-like receptor that is highly expressed in various cancers and involved in the growth, metastasis, and activation of inflammatory pathways via interactions with various functional molecules, such as integrins, CD44, and monocarboxylate transporters. Through screening of CD147-targeting antibodies with antitumor efficacy, we discovered a novel rat monoclonal antibody #147D. This humanized IgG4-formatted antibody, h4#147D, showed potent antitumor efficacy in xenograft mouse models harboring the human PDAC cell line MIA PaCa-2, HCC cell line Hep G2, and CML cell line KU812, which featured low sensitivity to the corresponding standard-of-care drugs (gemcitabine, sorafenib, and imatinib, respectively). An analysis of tumor cells derived from MIA PaCa-2 xenograft mice treated with h4#147D revealed that cell surface expression of CD147 and its binding partners, including CD44 and integrin α3β1/α6β1, was significantly reduced by h4#147D. Inhibition of focal adhesion kinase (FAK), activation of multiple stress responsible signal proteins such as c-JunN-terminal kinase (JNK) and mitogen-activated protein kinase p38 (p38MAPK), and expression of SMAD4, as well as activation of caspase-3 were obviously observed in the tumor cells, suggesting that h4#147D induced tumor shrinkage by inducing multiple stress responsible signals. These results suggest that the anti-CD147 antibody h4#147D offers promise as a new antibody drug candidate.

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 DS-7300a, a DNA Topoisomerase I Inhibitor, DXd-Based Antibody–Drug Conjugate Targeting B7-H3, Exerts Potent Antitumor Activities in Preclinical Models

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.

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

B7-H3 is overexpressed in various solid tumors and has been considered as an attractive target for cancer therapy. Here, we report the development of DS-7300a, a novel B7-H3–targeting antibody–drug conjugate with a potent DNA topoisomerase I inhibitor, and its in vitro profile, pharmacokinetic profiles, safety profiles, and in vivo antitumor activities in nonclinical species.The target specificity and species cross-reactivity of DS-7300a were assessed. Its pharmacologic activities were evaluated in several human cancer cell lines in vitro and xenograft mouse models, including patient-derived xenograft (PDX) mouse models in vivo. Pharmacokinetics was investigated in cynomolgus monkeys. Safety profiles in rats and cynomolgus monkeys were also assessed.DS-7300a specifically bound to B7-H3 and inhibited the growth of B7-H3–expressing cancer cells, but not that of B7-H3–negative cancer cells, in vitro. Additionally, treatment with DS-7300a and DXd induced phosphorylated checkpoint kinase 1, a DNA damage marker, and cleaved PARP, an apoptosis marker, in cancer cells. Moreover, DS-7300a demonstrated potent in vivo antitumor activities in high–B7-H3 tumor xenograft models, including various tumor types of high–B7-H3 PDX models. Furthermore, DS-7300a was stable in circulation with acceptable pharmacokinetic profiles in monkeys, and well tolerated in rats and monkeys.DS-7300a exerted potent antitumor activities against B7-H3–expressing tumors in in vitro and in vivo models, including PDX mouse models, and showed acceptable pharmacokinetic and safety profiles in nonclinical species. Therefore, DS-7300a may be effective in treating patients with B7-H3–expressing solid tumors in a clinical setting.

Supplementary Figure S1. The chemical structure of DS-8201a. Supplementary Figure S2. Importance of targeted delivery by the ADC. Supplementary Figure S3. Reduced antitumor effect of DS-8201a in an immunocompromised mouse model. Supplementary Figure S4. Cell growth inhibition activity of DS-8201a and DXd. Supplementary Figure S5. Increased CD8+ cell infiltration into tumors. Supplementary Figure S6. Reduced antitumor effect of DS-8201a by CD8 depletion. Supplementary Figure S7. DXd, the payload of DS-8201a directly increased expression of MHC class I.

The FGFR4/FGF19 signaling axis is overactivated in 20% of liver tumors and currently represents a promising targetable signaling mechanism in this cancer type. However, blocking FGFR4 or FGF19 has proven challenging due to its physiological role in suppressing bile acid synthesis which leads to increased toxic bile acid plasma levels upon FGFR4 inhibition. An FGFR4-targeting antibody, U3-1784, was generated in order to investigate its suitability as a cancer treatment without major side effects.U3-1784 is a high-affinity fully human antibody that was obtained by phage display technology and specifically binds to FGFR4. The antibody inhibits cell signaling by competing with various FGFs for their FGFR4 binding site thereby inhibiting receptor activation and downstream signaling via FRS2 and Erk. The inhibitory effect on tumor growth was investigated in 10 different liver cancer models in vivo. The antibody specifically slowed tumor growth of models overexpressing FGF19 by up to 90% whereas tumor growth of models not expressing FGF19 was unaffected. In cynomolgus monkeys, intravenous injection of U3-1784 caused elevated serum bile acid and liver enzyme levels indicating potential liver damage. These effects could be completely prevented by the concomitant oral treatment with the bile acid sequestrant colestyramine, which binds and eliminates bile acids in the gut. These results offer a new biomarker-driven treatment modality in liver cancer without toxicity and they suggest a general strategy for avoiding adverse events with FGFR4 inhibitors.

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.

Growth inhibition activity in 8 cell lines with different HER3 expression levels

Cell surface expression of HER3 and HER2 on human cancer cell lines

Purpose:HER3 is a compelling target for cancer treatment; however, no HER3-targeted therapy is currently clinically available. Here, we produced U3-1402, an anti-HER3 antibody–drug conjugate with a topoisomerase I inhibitor exatecan derivative (DXd), and systematically investigated its targeted drug delivery potential and antitumor activity in preclinical models.Experimental Design:In vitro pharmacologic activities and the mechanisms of action of U3-1402 were assessed in several human cancer cell lines. Antitumor activity of U3-1402 was evaluated in xenograft mouse models, including patient-derived xenograft (PDX) models. Safety assessments were also conducted in rats and monkeys.Results:U3-1402 showed HER3-specific binding followed by highly efficient cancer cell internalization. Subsequently, U3-1402 was translocated to the lysosome and released its payload DXd. While U3-1402 was able to inhibit HER3-activated signaling similar to its naked antibody patritumab, the cytotoxic activity of U3-1402 in HER3-expressing cells was predominantly mediated by released DXd through DNA damage and apoptosis induction. In xenograft mouse models, U3-1402 exhibited dose-dependent and HER3-dependent antitumor activity. Furthermore, U3-1402 exerted potent antitumor activity against PDX tumors with HER3 expression. Acceptable toxicity was noted in both rats and monkeys.Conclusions:U3-1402 demonstrated promising antitumor activity against HER3-expressing tumors with tolerable safety profiles. The activity of U3-1402 was driven by HER3-mediated payload delivery via high internalization into tumor cells.

Summary of repeated dose toxicity studies in rats and monkeys

Supplementary materials and methods for xenograft studies and IHC

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.

Cell line and patient-derived xenograft studies

Histopathological findings in the intestines, bone marrow and lung in the repeated dose study in monkeys

Antitumor activity of U3-1402 in HER3-positive HCC1569 and MDA-MB-453 xenograft models

B7-H3 is overexpressed in various solid tumors and has been considered as an attractive target for cancer therapy. Here, we report the development of DS-7300a, a novel B7-H3–targeting antibody–drug conjugate with a potent DNA topoisomerase I inhibitor, and its in vitro profile, pharmacokinetic profiles, safety profiles, and in vivo antitumor activities in nonclinical species. The target specificity and species cross-reactivity of DS-7300a were assessed. Its pharmacologic activities were evaluated in several human cancer cell lines in vitro and xenograft mouse models, including patient-derived xenograft (PDX) mouse models in vivo. Pharmacokinetics was investigated in cynomolgus monkeys. Safety profiles in rats and cynomolgus monkeys were also assessed. DS-7300a specifically bound to B7-H3 and inhibited the growth of B7-H3–expressing cancer cells, but not that of B7-H3–negative cancer cells, in vitro. Additionally, treatment with DS-7300a and DXd induced phosphorylated checkpoint kinase 1, a DNA damage marker, and cleaved PARP, an apoptosis marker, in cancer cells. Moreover, DS-7300a demonstrated potent in vivo antitumor activities in high–B7-H3 tumor xenograft models, including various tumor types of high–B7-H3 PDX models. Furthermore, DS-7300a was stable in circulation with acceptable pharmacokinetic profiles in monkeys, and well tolerated in rats and monkeys. DS-7300a exerted potent antitumor activities against B7-H3–expressing tumors in in vitro and in vivo models, including PDX mouse models, and showed acceptable pharmacokinetic and safety profiles in nonclinical species. Therefore, DS-7300a may be effective in treating patients with B7-H3–expressing solid tumors in a clinical setting.

Regulatory T (Treg) cells, which are essential for maintaining self-tolerance, inhibit anti-tumor immunity, consequently hindering protective cancer immunosurveillance, and hampering effective anti-tumor immune responses in tumor-bearing hosts. Here, we show that depletion of Treg cells via targeting glycoprotein A repetitions predominant (GARP) induces effective anti-tumor immune responses. GARP was specifically expressed by highly suppressive Treg cells in the tumor microenvironment (TME) of multiple cancer types in humans. In the periphery, GARP was selectively induced in Treg cells, but not in effector T cells, by polyclonal stimulation. DS-1055a, a novel afucosylated anti-human GARP monoclonal antibody, efficiently depleted GARP+ Treg cells, leading to the activation of effector T cells. Moreover, DS-1055a decreased FoxP3+CD4+ T cells in the TME and exhibited remarkable anti-tumor activity in humanized mice bearing HT-29 tumors. We propose that DS-1055a is a new Treg-cell-targeted cancer immunotherapy agent with augmentation of anti-tumor immunity.

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.

Purpose: HER3 is a compelling target for cancer treatment; however, no HER3-targeted therapy is currently clinically available. Here, we produced U3-1402, an anti-HER3 antibody–drug conjugate with a topoisomerase I inhibitor exatecan derivative (DXd), and systematically investigated its targeted drug delivery potential and antitumor activity in preclinical models. Experimental Design: In vitro pharmacologic activities and the mechanisms of action of U3-1402 were assessed in several human cancer cell lines. Antitumor activity of U3-1402 was evaluated in xenograft mouse models, including patient-derived xenograft (PDX) models. Safety assessments were also conducted in rats and monkeys. Results: U3-1402 showed HER3-specific binding followed by highly efficient cancer cell internalization. Subsequently, U3-1402 was translocated to the lysosome and released its payload DXd. While U3-1402 was able to inhibit HER3-activated signaling similar to its naked antibody patritumab, the cytotoxic activity of U3-1402 in HER3-expressing cells was predominantly mediated by released DXd through DNA damage and apoptosis induction. In xenograft mouse models, U3-1402 exhibited dose-dependent and HER3-dependent antitumor activity. Furthermore, U3-1402 exerted potent antitumor activity against PDX tumors with HER3 expression. Acceptable toxicity was noted in both rats and monkeys. Conclusions: U3-1402 demonstrated promising antitumor activity against HER3-expressing tumors with tolerable safety profiles. The activity of U3-1402 was driven by HER3-mediated payload delivery via high internalization into tumor cells.

A novel DXd-ADC technology has been discovered with the study of ADC linker technology applied to a potent DNA topoisomerase I inhibitor. A high drug-to-antibody ratio with homogeneous conjugation, has strong potency on heterogeneous tumors by the bystander antitumor effect, and alleviates safety concerns in systemic circulation with its high linker-drug stability. Trastuzumab deruxtecan (T-DXd) could provide valuable therapy with great potential for providing effective treatment for breast cancer and other HER2-expressing cancers in clinical settings. Indeed, T-DXd was recently approved for the treatment of patients with HER2-positive unresectable or recurrent breast cancers in the United States, Japan, and EU and with HER2-positive unresectable or recurrent gastric cancers in the United States and Japan.

A major limitation of traditional chemotherapy for cancer is dose-limiting toxicity, caused by the exposure of non-tumor cells to cytotoxic agents. Use of molecular targeted drugs, such as specific kinase inhibitors and monoclonal antibodies, is a possible solution to overcome this limitation and has achieved clinical success so far. Use of an antibody-drug conjugate (ADC) is a rational strategy for improving efficacy and reducing systemic adverse events. ADCs use antibodies selectively to deliver a potent cytotoxic agent to tumor cells, thus drastically improving the therapeutic index of chemotherapeutic agents. Lessons learned from clinical failure of early ADCs during the 1980s to 90s have recently led to improvements in ADC technology, and resulted in the approval of four novel ADCs. Nonetheless, further advances in ADC technology are still required to streamline their clinical efficacy and reduce toxicity. [fam-] Trastuzumab deruxtecan (DS-8201a) is a next-generation ADC that satisfies these requirements based on currently available evidence. DS-8201a has several innovative features; a highly potent novel payload with a high drug-to-antibody ratio, good homogeneity, a tumor-selective cleavable linker, stable linker-payload in circulation, and a short systemic half-life cytotoxic agent in vivo; the released cytotoxic payload could exert a bystander effect. With respect to its preclinical profiles, DS-8201a could provide a valuable therapy with a great potential against HER2-expressing cancers in clinical settings. In a phase I trial, DS-8201a showed acceptable safety profiles with potential therapeutic efficacy, with the wide therapeutic index.

l‐Type amino acid transporter 1 (LAT1) disulfide linked to CD98 heavy chain (hc) is highly expressed in most cancer cells, but weakly expressed in normal cells. In the present study, we developed novel anti‐LAT1 mAbs and showed internalization activity, inhibitory effects of amino acid uptake and cell growth and antibody‐dependent cellular cytotoxicity, as well as in vivo antitumor effects in athymic mice. Furthermore, we examined the reactivity of mAbs with LAT1 of Macaca fascicularis to evaluate possible side‐effects of antihuman LAT1 mAbs in clinical trials. Antihuman LAT1 mAbs reacted with ACHN human and MK.P3 macaca kidney‐derived cells, and this reactivity was significantly decreased by siRNAs against LAT1. Macaca LAT1 cDNA was cloned from MK.P3, and only two amino acid differences between human and macaca LAT1 were seen. RH7777 rat hepatoma and HEK293 human embryonic kidney cells expressing macaca LAT1 were established as stable transfectants, and antihuman LAT1 mAbs were equivalently reactive against transfectants expressing human or macaca LAT1. Dual (high and low) avidity modes were detected in transfectants expressing macaca LAT1, MK.P3, ACHN and HCT116 human colon cancer cells, and KA values were increased by anti‐CD98hc mAb, suggesting anti‐LAT1 mAbs detect an epitope on LAT1‐CD98hc complexes on the cell surface. Based on these results, LAT1 may be a promising anticancer target and Macaca fascicularis can be used in preclinical studies with antihuman LAT1 mAbs.

Introduction: T-DXd (trastuzumab deruxtecan) is an antibody-drug conjugate (ADC) targeting human epidermal growth factor receptor 2 (HER2) composed of a humanized anti-HER2 monoclonal antibody conjugated to a DNA topoisomerase I inhibitor, DXd through a linker. Internalization of HER2-T-DXd followed by DXd release is a crucial process for T-DXd to exert its antitumor activity. T-DXd induced robust and durable responses in HER2 mutant metastatic non-small cell lung cancer (NSCLC) patients in DESTINY-Lung01 study, including those with low HER2 expression levels (Li BT, et al. NEJM 2021). One hypothesis of this T-DXd sensitivity of HER2 mutant NSCLC cells involves the potential of enhanced receptor and ADC internalization in these cells (Li BT, et al. Cancer Discov 2020). To examine this possibility further, we investigated the effect of HER2 mutations on T-DXd internalization and DXd release in transgenic cells expressing various HER2 mutants, as well as on T-DXd sensitivity in vivo. Methods: We established transgenic NCI-H322 cells expressing wild-type (WT) HER2 or various HER2 mutants and evaluated T-DXd internalization in these cells. Antitumor activity of T-DXd was examined in xenograft mouse models of transgenic NCI-H322 cells and compared between WT and each mutant. DXd concentration in tumor was also determined. In addition, in vitro internalization activity and antitumor activity of T-DXd were evaluated using NCI-H1781, the NSCLC cell line harboring an endogenous HER2 mutation (G776delinsVC). Results: Transgenic NSCLC cells expressing various HER2 mutants showed higher T-DXd internalization than WT HER2 expressing cells. There also was a negative correlation between the surface HER2 expression level and T-DXd internalization among various mutants and WT expressing cells. In xenograft models with transgenic cells, some HER2 mutant models showed higher response to T-DXd than WT HER2 model, with greater than 2-fold increase in G660D, YVMA, G776delinsVC, V777_G778insGSP, and V842I. Tumor DXd concentration was positively correlated with the antitumor activity. In an endogenous HER2 mutant NSCLC cell line (NCI-H1781), T-DXd also showed high internalization activity and potent antitumor activity. Conclusion: In vitro study with transgenic NSCLC cells suggested that T-DXd internalization in HER2 mutant tumors was more efficient than in WT HER2 tumor. Some of those HER2 mutant cell lines showed increased T-DXd sensitivity and higher payload release in the tumor in vivo, suggesting the enhanced internalization played a role in the mechanism of T-DXd sensitivity in these HER2 mutant transgenic NSCLC cells. Some mutants may be more or less clinically relevant. However, the relationship between the in vitro internalization efficiency and in vivo antitumor activity in each mutant was not consistent among different mutants, thus these results need to be interpreted cautiously. Citation Format: Atsushi Yamasaki, Misa Ikuta, Manabu Abe, Tsuneo Deguchi, Yasuki Kamai, Kenji Nakamaru, Toshinori Agatsuma. Pleclinical investigation of T-DXd internalization and payload release in HER2 mutant non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5376.

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.

ORAI1 constitutes the pore‐forming subunit of the calcium release‐activated calcium (CRAC) channel, which is responsible for store‐operated calcium entry into lymphocytes. It is known that ORAI1 is essential for the activation of T cells and mast cells and is considered to be a potent therapeutic target for autoimmune and allergic diseases. Here, we obtained a new humanized antibody, DS‐2741a, that inhibits ORAI1 function. DS‐2741a bound to human‐ORAI1 with high affinity and without cross‐reactivity to rodent Orai1. DS‐2741a demonstrated suppression of CRAC‐mediated human and mouse T‐cell activation and mast cell degranulation in human ORAI1 knock‐in mice. Furthermore, DS‐2741a ameliorated house dust mite antigen‐induced dermatitis in the human ORAI1 knock‐in mouse. Taken together, DS‐2741a inhibited T‐cell and mast cell functions, thus improving skin inflammation in animal models of atopic dermatitis and reinforcing the need for investigation of DS‐2741a for the treatment of allergic diseases in a clinical setting.

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.

Immune checkpoint blockers (ICBs) have drastically changed the clinical care of cancer; however, the population of patients who can benefit is relatively small because of intrinsic or acquired resistance to immune therapy. To evade immune destruction, tumors exploit several distinct strategies including immunosuppressive cells such as regulatory T (Treg) cells. Treg cells, an essential component for maintaining self-tolerance, inhibit antitumor immunity, consequently hindering protective cancer immunosurveillance and hampering effective antitumor immune responses in tumor-bearing hosts. It is often reported that a high ratio of Treg cells to effector CD8+ T cells is associated with poor prognosis in patients with cancer, and Treg cells represent one of the most important factors associated with resistance to ICBs, suggesting that Treg cells represent a new promising target for anti-cancer therapy. In this study, we performed a comprehensive screening to identify a tumor-infiltrating Treg cell-specific molecule among immune cell subsets in humans. Glycoprotein A repetitions predominant (GARP) was identified as a suitable target for selective Treg cell depletion in the tumor microenvironment (TME) of multiple caner types. In the periphery, GARP was selectively induced in Treg cells, but not in effector T cells, by polyclonal stimulation in humans. DS-1055a, a novel afucosylated anti-human GARP antibody, effectively depleted GARP+ Treg cells, leading to the activation of effector T cells. Moreover, DS-1055a decreased FOXP3+CD4+ T cell counts in the TME and exhibited remarkable antitumor activity in a human peripheral blood mononuclear cell-transplanted humanized mouse model bearing HT-29 tumors. From these results, we propose that DS-1055a can be a new Treg cell-targeted regent that augments antitumor T cell immunity by depleting GARP+ Treg cells. Currently, a Phase I study of DS-1055a is on-going (NCT04419532). Citation Format: Kazuki Satoh, Yoichi Kobayashi, Kaori Fujimaki, Masato Hata, Shinko Hayashi, Saori Ishida, Daisuke Sugiyama, Takahiko Sato, Lim Kyungtaek, Megumi Miyamoto, Shiho Kozuma, Michinori Kadokura, Kenichi Wakita, Kazuki Hirahara, Masato Amano, Ichiro Watanabe, Atsushi Okamoto, Andrea Tuettenberg, Helmut Jonuleit, Atsushi Tanemura, Shoichi Maruyama, Toshinori Agatsuma, Teiji Wada, Hiroyoshi Nishikawa. Anti-GARP antibody DS-1055a augments antitumor immunity by depleting highly suppressive GARP+ regulatory T cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1847.

Antibody–drug conjugates (ADCs) represent a promising class of drugs with a wider therapeutic index (TI) than conventional chemotherapeutic agents due to their efficient and specific drug delivery. ADC payload is attached to antibody by chemical linkage. Two classes of cytotoxins – DNA damaging agents and microtubule inhibitors – have been validated as ADC payloads in clinical trials and four ADCs, MYLOTARG®, ADCETRIS®, KADCYLA®, and BESPONSA®, have been approved so far. There are many different ADCs in clinical development stage. Novel linker-payload technologies are evaluated and a new class of payload DNA topoisomerase I inhibitor is emerging. This review will focus on introduction of the four approved ADC drugs and promising novel ADCs with DNA topoisomerase I inhibitors, Sacituzumab Govitecan and [fam-] Trastuzumab Deruxtecan, in late stages of clinical development.

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.

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.

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.

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.

Purpose Neovascular age-related macular degeneration (nAMD) results from choroidal neovascularization (CNV) and causes severe vision loss. Intravitreal anti-vascular endothelial growth factor (VEGF) therapies have significantly improved therapeutic outcomes; however, a substantial number of patients experience disease progression. Roundabout 4 (ROBO4) has been reported to be a vascular-specific protein that stabilizes vasculature in ocular pathological angiogenesis. To explore ROBO4 targeting as a novel treatment against neovascularization, we generated a humanized anti-human ROBO4 antibody, DS-7080a, and evaluated its efficacy. Methods ROBO4 mRNA in human whole eye cross-sections was examined by in situ hybridization. Human umbilical vein endothelial cell (HUVEC) migration was measured in the presence of VEGF, basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), or conditioned medium of primary human retinal pigment epithelial (HRPE) cells. CNV was induced in cynomolgus monkeys by laser irradiation. Vascular leakage was measured by fluorescein angiography, and pathological changes were determined by histology. Results ROBO4 mRNA was detected in choroidal vessels of nAMD patients. DS-7080a suppressed HGF- or bFGF-induced HUVEC migration in addition to that induced by VEGF. Further, HUVEC migration induced by HRPE-conditioned medium was inhibited by either DS-7080a or ranibizumab in a similar manner, and the combination of these showed further inhibition. In a laser-induced CNV monkey model, single intravitreous administration of 1.1 mg/eye of DS-7080a reduced the incidence of grade 4 leakage from 44.45% in control eyes to 1.85% (P < 0.05 by Dunnett's test). Conclusions Anti-ROBO4 antibody DS-7080a suppressed HUVEC migration in a distinctly different fashion from anti-VEGF agents and improved laser-induced CNV in non-human primates. Translational Relevance DS-7080a may be a novel treatment option for nAMD.

CD147, a type I membrane protein, is highly expressed in various cancers and is involved in the growth, metastasis, and activation of inflammatory signals in cancer cells via interaction with various molecules, such as integrins, CD44, and monocarboxylate transporters. Additionally, the expression of CD147 in tumors positively correlates with poor prognosis in various cancers. Therefore, CD147 is an attractive target for cancer therapy. However, there have been no successful drugs targeting CD147 so far, possibly because it is a multifunctional protein interacting with multiple molecules, making it challenging to discover a relevant CD147-targeting drug candidate by general in vitro screening methodologies. In this study, we developed novel anti-CD147 antibodies, which showed significant antitumor efficacy in immune-deficient mice bearing human pancreatic cancer cell lines, PANC-1 or MIA PaCa-2. Six anti-CD147 antibodies showing in vivo anti-tumor efficacy were obtained; one mouse IgG3, two rat IgG2b, two rat IgG1, and one rat IgG2a antibodies. Analysis of antibody dependent cellular toxicity, antibody-dependent cellular phagocytosis, and complement-dependent cytotoxicity of chimeric and humanized antibodies with human IgG2 or IgG4P derived from the obtained 6 anti-CD147 antibodies, #147A/B/C/D/E/F, suggested no significant immune-effector function for these antibodies. Despite lacking effector function, these antibodies exhibited significant in vivo antitumor activity, suggesting that the mechanism underlying their anti-tumor efficacy did not involve their effector function. We also found that the anti-tumor efficacy of antibodies #147A and #147B positively correlated with SMAD4 protein expression rather than CD147 protein expression in 10 pancreatic cancer xenograft models (correlation coefficient of #147B: anti-tumor efficacy vs. CD147, 0.245; vs. SMAD4, 0.9328). Moreover, the administration of anti-CD147 antibodies upregulated rhoB expression in an anti-CD147 antibody-sensitive SMAD4-positive MIA PaCa-2 graft mouse model. The stable expression of SMAD4 increased the sensitivity to the anti-CD147 antibody, ch4#147A (chimeric IgG4P derived from #147A) in xenograft mice inoculated with the BxPC-3 pancreatic cancer cell line, which is originally negative for SMAD4 and has low sensitivity to anti-CD147 antibodies. These data suggest that the expression of SMAD4 and activation of SMAD signaling are functionally important for susceptibility to anti-CD147 antibody treatment. The humanized anti-CD147 IgG4P antibody, h4#147D, derived from #147D with cross reactivity to monkey CD147, showed superior anti-tumor efficacy with complete tumor reduction, compared to standard anticancer drugs, including gemcitabine, imatinib, and sorafenib, in xenograft mice bearing corresponding cancer cell lines, MIA PaCa-2, KU812 chronic myeloid leukemia, and HepG2 liver cancer cell lines, respectively. No toxicity of h4#147D was observed in cynomolgus monkeys at a single dose of 100 mg/kg. These data suggest that h4#147D might be a novel anti-CD147 antibody, which could induce SMAD activation and tumor shrinkage in multiple xenograft models. Therefore, it has potential as a promising antitumor therapeutic antibody with superior anti-tumor efficacy to existing therapy. Citation Format: Keisuke Fukuchi, Kayoko Nanai, Shoji Yamamoto, Jun Tsukada, Yusuke Totoki, Koichiro Inaki, Masato Ishigami, Naoyuki Makita, Yoko Nakano, Chigusa Yoshimura, Kozo Yoneda, Masato Amano, Kensuke Nakamura, Yoshiyuki Kanari, Yoko Oda, Haruyuki Nishigohri, Rika Nakano, Atsuko Nishida, Kenji Murakami, Yumi Matsui, Naomi Kasanuki, Shoji Midori, Satoko Funo, Sayako Takahashi, Hironobu Komori, Toshiaki Ohtsuka, Toshinori Agatsuma. Novel anti-CD147 antibodies inducing activation of SMAD signaling and tumor shrinkage in intractable cancer 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 B095. doi:10.1158/1535-7163.TARG-19-B095

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

The FGFR4/FGF19 signaling axis is overactivated in 20% of liver tumors and currently represents a promising targetable signaling mechanism in this cancer type. However, blocking FGFR4 or FGF19 has proven challenging due to its physiological role in suppressing bile acid synthesis which leads to increased toxic bile acid plasma levels upon FGFR4 inhibition. An FGFR4-targeting antibody, U3-1784, was generated in order to investigate its suitability as a cancer treatment without major side effects. U3-1784 is a high-affinity fully human antibody that was obtained by phage display technology and specifically binds to FGFR4. The antibody inhibits cell signaling by competing with various FGFs for their FGFR4 binding site thereby inhibiting receptor activation and downstream signaling via FRS2 and Erk. The inhibitory effect on tumor growth was investigated in 10 different liver cancer models in vivo. The antibody specifically slowed tumor growth of models overexpressing FGF19 by up to 90% whereas tumor growth of models not expressing FGF19 was unaffected. In cynomolgus monkeys, intravenous injection of U3-1784 caused elevated serum bile acid and liver enzyme levels indicating potential liver damage. These effects could be completely prevented by the concomitant oral treatment with the bile acid sequestrant colestyramine, which binds and eliminates bile acids in the gut. These results offer a new biomarker-driven treatment modality in liver cancer without toxicity and they suggest a general strategy for avoiding adverse events with FGFR4 inhibitors.

Antibody-drug conjugates (ADCs) selectively deliver large amounts of antitumor drugs to tumor tissue and show significant antitumor effects with a wide therapeutic window. We developed a new linker-drug technology platform with an exatecan derivative, which is a highly potent topoisomerase I inhibitor. The major advantages of the technology are: 1) high and homogeneous drug-to-antibody ratio (DAR) availability; 2) potent antitumor activity in conjunction with bystander killing; 3) few safety concerns because of the stable linker limiting release of free drug; and 4) a wide application to therapeutic antibodies. Using this linker-drug technology, we generated an anti-HER2 ADC, namely DS-8201a. DS-8201a, in which almost all eight cysteine residues of the antibody are bound to drug, was effective against trastuzumab DM1 (T-DM1)-insensitive patient-derived xenograft (PDX) models with high HER2 expression and also demonstrated antitumor efficacy against several breast cancer PDX models with low HER2 expression. DS-8201a was well tolerated in rats and monkeys following repeated administration. These results suggest that DS-8201a may be efficacious in a broader population of HER2-positive cancer patients and also confirm the importance of this new class of novel topoisomerase I inhibitor-based ADC technology.

[Fam-] trastuzumab deruxtecan (DS-8201a) is a HER2 (ERBB2)-targeting antibody-drug conjugate, composed of a HER2-targeting antibody and a topoisomerase I inhibitor, exatecan derivative, that has antitumor effects in preclinical xenograft models and clinical trials. Recently, [fam-] trastuzumab deruxtecan was reported to enhance antitumor immunity and was beneficial in combination with an anti-PD-1 antibody in a mouse model. In this study, the antitumor effect of [fam-] trastuzumab deruxtecan in combination with an anti-CTLA-4 antibody was evaluated. [Fam-] trastuzumab deruxtecan monotherapy had antitumor activity in an immunocompetent mouse model with EMT6 human HER2-expressing mouse breast cancer cells (EMT6-hHER2). [Fam-] trastuzumab deruxtecan in combination with the anti-CTLA-4 antibody induced more potent antitumor activity than that by monotherapy with either agent. The combination therapy increased tumor-infiltrating CD4+ and CD8+ T cells in vivo. Mechanistically, cured mice with treatment of [fam-] trastuzumab deruxtecan and an anti-CTLA-4 antibody completely rejected EMT6-mock cells similar to EMT6-hHER2 cells, and splenocytes from the cured mice responded to both EMT6-hHER2 and EMT6-mock cells as measured by interferon-gamma release. Taken together, these results indicate that antitumor immunity is induced by [fam-] trastuzumab deruxtecan and is facilitated in combination with anti-CTLA-4 antibody.

Background: Human epidermal growth factor receptor 3 (HER3), a member of the ErbB receptor tyrosine kinase family, is overexpressed in a variety of human cancers and plays an important role in cell proliferation and survival. U3-1402 is a novel HER3-targeting antibody-drug conjugate (ADC) consisting of a fully human anti-HER3 antibody (patritumab), a tetrapeptide-based linker, and a topoisomerase I inhibitor payload. It is currently being investigated in clinical trials for HER3-positive breast cancer (phase 1/2) and NSCLC (phase 1). In principle, effective action of ADCs depends on multiple dynamic factors including antigen-specific binding, internalization, trafficking to lysosomes, and payload release, as well as other inherent characteristics such as payload potency and linker stability. Methods: To assess the targeted delivery potential of U3-1402, the HER3-mediated molecular dynamics of U3-1402 (cell surface binding, internalization, trafficking, and payload release) were investigated in 8 cancer cell lines with various HER3 expression levels, along with cell growth inhibition activity. In vitro molecular dynamics were compared between U3-1402 and trastuzumab (anti-HER2 antibody) using the MDA-MB-453 cell line, which expresses both HER2 and HER3. The activity of patritumab (naked antibody of U3-1402) was tested. In vivo efficacy of U3-1402 was also evaluated and immunostaining of HER3 and γH2AX (marker of DNA double-stranded breaks) as well as drug concentration assessment were performed. Results: U3-1402 bound to the cell surface of HER3-positive cell lines in HER3 expression level-dependent manner, and was then internalized into cells. Interestingly, the internalization rate of U3-1402 in MDA-MB-453 cells at 1 h was 64%, which was higher than that observed with trastuzumab (2%). Subsequently, U3-1402 was translocated to the lysosome, followed by payload release, leading to an improved cell growth inhibition compared to that noted with patritumab. In vivo antitumor efficacy was also demonstrated in a HER3-dependent manner in MDA-MB-453 xenograft model. In the study, internalization of HER3 from cell membrane into the intracellular compartment by U3-1402 treatment was observed, followed by increased free payload and γH2AX levels in tumor tissue. Conclusion: U3-1402 has a high internalization property for effective payload delivery to HER3-expressing cancer cells, resulting in a favorable ADC-driven efficacy. Citation Format: Kumiko Koyama, Yuuri Hashimoto, Yasuki Kamai, Yoshinobu Shiose, Manabu Abe, Yuki Kaneda, Naoyuki Maeda, Kenji Hirotani, Yusuke Ogitani, Tsuyoshi Karibe, Takashi Kagari, Kenichi Wakita, Suguru Ueno, Toshinori Agatsuma, Masato Murakami. U3-1402, a novel HER3-targeting antibody-drug conjugate, exhibits its antitumor activity through increased payload intracellular delivery via highly efficient drug internalization [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-275.