Camptothecins; TOP I; topoisomerase I

Antibody-drug conjugates deliver anticancer agents selectively and efficiently to tumor tissue and have significant antitumor efficacy with a wide therapeutic window[2]. The antitumor activity of [fam-] trastuzumab deruxtecan for CRC with five CRC cell lines that possess different biological characteristics was investigated. The expression of HER2 at both mRNA and protein levels in these various cell lines was first examined. Immunoblot analysis and RT and real-time polymerase chain reaction (PCR) analysis revealed that the amounts of HER2 protein and HER2 mRNA were much smaller in all the CRC cell lines than in NCI-N87 cells. [fam-] trastuzumab deruxtecan attenuated the viability of NCI-N87 cells, consistent with previous results, whereas all five CRC cell lines showed resistance to this agent. These findings suggested that the expression level of HER2 protein might determine sensitivity to [fam-] trastuzumab deruxtecan.

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Dxd (Exatecan derivative for ADC) is a potent inhibitor of DNA topoisomerase I that is used as a conjugated drug with HER2-targeting ADC (DS-8201a), having an IC50 of 0.31 μM. With IC50s ranging from 1.43 nM to 4.07 nM, Dxd is cytotoxic to human cancer cell lines of KPL-4, NCI-N87, SK-BR-3, and MDA-MB-468; however, control IgG-ADC, of which Dxd is the payload, exhibits no inhibition on the four cell lines (expressing HER2). With IC50 values of 26.8, 25.4, and 6.7 ng/mL, respectively, DS-8201a (the payload is Dxd) exhibits significant suppression on the HER2-positive KPL-4, NCI-N87, and SK-BR-3 cell lines, but no such inhibition is seen on MDA-MB-468 (IC50, >10,000 ng/mL)[3].

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The structure of DS-8201a is shown in Fig. 1A. DS-8201a is a HER2-targeting ADC and is composed of an anti-HER2 antibody and a derivative of DX-8951 (Dxd), a topoisomerase I inhibitor, which are bound together by a maleimide glycyn-glycyn-phenylalanyn-glycyn (GGFG) peptide linker. The linker-payload is conjugated with the antibody via the cysteine residues after the interchain disulfide bounds are reduced with a reducing agent, tris (2-carboxyethyl) phosphine hydrochloride (TCEP HCl). As the tetrapeptide is decomposed by lysosomal enzymes such as cathepsins B and L which are highly expressed in tumor cells, it is supposed that DS-8201a is cleaved by lysosomal enzymes and releases Dxd, which attacks target molecules specifically in tumor cells after it binds to HER2 receptors and is internalized in tumor cells. By using RPC, the DAR of DS-8201a was determined as approximately 8, which is the theoretical maximum drug loading number for conventional interchain cysteine conjugation. Therefore, homogeneous drug distribution was observed in the HIC chart (Fig. 1B). We confirmed that Dxd is more potent in inhibitory activity in topoisomerase I than SN-38 as well as DX-8951f, as measured by a topoisomerase I–mediated DNA relaxation assay (Fig. 1C)[3].

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Inhibition of cancer cell growth by DS-8201a [3]
The inhibitory activity of DS-8201a against cancer cell growth was compared with an anti-HER2 Ab and control IgG–ADC–conjugated with Dxd against various human cancer cell lines in vitro. HER2 expression on the cell surface of the cell lines KPL-4, NCI-N87, SK-BR-3, and MDA-MB-468 was firstly evaluated by flow cytometric analysis (Fig. 2A). The relative MFIs of KPL-4, NCI-N87, and SK-BR-3 were 95.7, 101.6, and 56.2, respectively, suggesting that HER2 is clearly expressed on the cell surfaces, whereas the relative MFI was 1.0 for MDA-MB-468, indicating no expression in the MDA-MB-468. Remarkable inhibitory activity to the cell growth was observed for DS-8201a against HER2-positive KPL-4, NCI-N87, and SK-BR-3, with the IC50 values of 26.8, 25.4, and 6.7 ng/mL, respectively, whereas no such inhibition was seen against MDA-MB-468 with the IC50 value of >10,000 ng/mL (Fig. 2B). Although the anti-HER2 Ab showed cell growth–inhibitory activity against NCI-N87 and SK-BR-3, these activities were rather weaker than that of the DS-8201a; the IC50 values of NCI-N87 and SK-BR-3 were 204.2 and 65.9 ng/mL, respectively (Fig. 2B). Also, control IgG-ADC did not show cell growth–inhibitory activities in any of the four cell lines (Fig. 2B), although all four cell lines were sensitive to the payload, Dxd (IC50: 1.43 nmol/L–4.07 nmol/L). These results indicate that the cell growth–inhibitory activity of DS-8201a was remarkably enhanced by drug conjugation to the anti-HER2 Ab, and also that DS-8201a shows target-specific growth inhibition against HER2-positive cell lines.

The efficacy of [fam-] trastuzumab deruxtecan in HER2-expressing xenograft tumor models was tested. It was first confirmed that HER2 protein expression levels by immunohistochemistry (IHC) in subcutaneous tumors formed in nude mice by HCT116-Mock, HCT116-H2L or HCT116-H2H cells. Administration of [fam-] trastuzumab deruxtecan at a dose of 3.0 mg/kg markedly inhibited the growth of tumors formed by HCT116-H2L or HCT116-H2H cells but not that of those formed by HCT116-Mock cells. The extents of the inhibition by [fam-] trastuzumab deruxtecan were 60 and 93% compared to PBS vehicle for HCT116-H2L and HCT116-H2H cells, respectively, on day 24. Treatment with [fam-] trastuzumab deruxtecan had no effect on body weight in any of the three groups of mice. These findings thus indicated that the sensitivity of tumors to [fam-] trastuzumab deruxtecan in xenograft models is dependent on HER2 expression level and that such treatment is not associated with overt toxicity.

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DS-8201a (DXdis the payload, 10 mg/kg, i.v.) demonstrates potent ntitumor activity in HER2-low-expressing ST565 and ST313 models with HER2 IHC 1+/FISH-negative expression as well as in HER2-positive models with KPL4, JIMT-1, and Capan-1[3].

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Antitumor activity in vivo [3]
The in vivo antitumor activity of DS-8201a was evaluated in a HER2-positive NCI-N87 xenograft model. DS-8201a induced tumor growth inhibition in a dose-dependent manner and tumor regression with a single dosing at more than 1 mg/kg without inducing any abnormalities in the general condition or body weight changes of the mice (Fig. 2C). In the same model, 4 mg/kg administration of anti-HER2 Ab partially inhibited the tumor growth, indicating 31% of tumor growth inhibition (TGI) compared with the control group on day 21 (Fig. 2D). On the other hand, DS-8201a clearly showed more potent antitumor efficacy, indicating 99% TGI at the same dose of 4 mg/kg, so that the enhancement of efficacy by drug conjugation was observed in vivo as well as in the in vitro models (Fig. 2D). Moreover, it is suggested that the in vivo efficacy of DS-8201a depends on its HER2 binding, as no inhibition of tumor growth was seen for the control-IgG ADC (Fig. 2D).

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Antitumor activity of DS-8201a in low HER2–expressing tumors [3]
T-DM1 has been approved for HER2-positive metastatic breast cancer patients, defined as being HER2 IHC 3+ or IHC 2+/FISH–positive according to the current guidelines, and there are still clinical unmet needs in FISH-negative, HER2 1+ and 2+ populations for HER2-targeting therapies. Therefore, the antitumor activity of DS-8201a was evaluated in various mice xenograft models with different HER2 expression levels; KPL-4 (strong positive), JIMT-1 (moderate positive), Capan-1 (weak positive), and GCIY (negative) (Fig. 4A and B). Anti-HER2 ADC with the same drug-linker as DS-8201a and about half the DAR (DAR 3.4) was also evaluated to investigate the effect of DAR on antitumor activity. While T-DM1 was effective against only the KPL4 model, DS-8201a was effective against all HER2-positive models with KPL4, JIMT-1, and Capan-1. Both ADCs were not effective in the GCIY model. Anti-HER2 ADC (DAR 3.4) inhibited tumor growth against all HER2-positive models, and the efficacy was HER2 expression–dependent. A stronger efficacy was apparently observed for DS-8201a than anti-HER2 ADC (DAR 3.4) in the HER2 weak–positive Capan-1 model. These results suggest that the high DAR ADC, DS-8201a, enables the delivery of sufficient payload amounts into cancer cells, indicating cytotoxicity even with low HER2 levels. In case of HER2 strong –positive models, even a low DAR ADC is able to deliver a sufficient amount of payload for cell death. DS-8201a was effective in tumors with broader HER2 levels due to its high DAR, approximately 8. To confirm HER2-specificity of DS-8201a in a HER2 low–expressing model, a competitive inhibition study was performed in a HER2 low CFPAC-1 model (Fig. 4C). The efficacy of DS-8201a was cancelled by the prior treatment of the anti-HER2 Ab, and the control IgG-ADC did not inhibit tumor growth at a 3-fold higher dose than DS-8201a. From these results, the HER2 specificity of DS-8201a in a HER2 low–expressing model was confirmed.

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Comparison with T-DM1 in PDX models [3]
In addition to the cell line–based xenograft models, several PDX evaluations were performed to assess clinical benefits more precisely. In a gastric cancer PDX model, NIBIO G016, DS-8201a demonstrated potent antitumor activity with tumor regression, but T-DM1 did not (Fig. 5A). As the HER2 status in this model was IHC 3+/FISH+, it is supposed that this difference in antitumor efficacy between DS-8201a and T-DM1 is based on the different sensitivity of payload due to dissimilar mechanism of action of each payload. In breast cancer PDX models, although both DS-8201a and T-DM1 were effective in the HER2 IHC 2+/FISH–positive ST225 model, complete tumor regression was observed on day 21 in 3 of 5 mice treated with DS-8201a, not T-DM1 (Fig. 5B). Furthermore, DS-8201a showed an antitumor activity in HER2 low–expressing ST565 and ST313 models with HER2 IHC 1+/FISH–negative expression (Fig. 5C and D), but T-DM1 did not. This result indicated a similar tendency to the cell line–based xenograft models such as Capan-1 and CFPAC-1 (Fig. 4B and C). Consequently, DS-8201a showed more potent antitumor activity than T-DM1 in all 4 of these models with several HER2 expression levels. These results suggest that DS-8201a has a differentiable potential from T-DM1, which shows effectiveness in T-DM1–insensitive and HER2 low–expressing tumors, resulting from the different mechanisms of action of the conjugated drug and the high DAR of DS-8201a.

Parallel artificial membrane permeability assay (PAMPA) was carried out using a Freedom EVO200 system. The filter membrane of the acceptor plate was coated with GIT‐0 lipid solution. Each compound solution in DMSO (10 mM) was added to Prisma HT buffer (Pion) to obtain 5‐μM donor solutions (containing 0.05% DMSO, pH 5.0 and pH 7.4), and then placed on a donor plate. The acceptor plate was filled with an acceptor sink buffer. The donor plate was stacked onto the acceptor plate and incubated for 4 h at 25°C. After incubation, the concentrations of compounds in both plates were measured by an LC‐MS/MS system (API 4000). The permeability coefficient (Peff; 10−6 cm/s) was calculated using PAMPA Evolution DP software (Pion).[1]

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Topoisomerase I inhibitory assay [3]
SN-38, DX-8951f and DX-8951 derivative (DXd) were synthesized in-house. The inhibitory activities of SN-38, DX-8951f, and DXd against human topoisomerase I were evaluated by a topoisomerase I–mediated DNA relaxation assay according to a previous report. Briefly, recombinant human topoisomerase I was incubated with each drug for 5 minutes. Then, supercoiled DNA pBR322 was added and incubated at 25°C for 60 minutes. After the electrophoresis of the mixture on an agarose gel, the amount of the supercoiled DNA was measured with a CCD imager.

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In vitro stability ofDS-8201a in plasma [3]
The release rate of DXd from DS-8201a at the concentration of 10 μg/mL at 37°C up to 21 days was evaluated in mouse, rat, monkey, and human plasma.

Cells were seeded in a 96‐well plate at 1000 cells/well for KPL‐4 and 2000 cells/well for MDA‐MB‐468. After overnight incubation, a serially diluted solution of each ADC was added. Cell viability was evaluated after 5 days using a CellTiter‐Glo luminescent cell viability assay from Promega according to the manufacturer's instructions. For coculture study, KPL‐4 and MDA‐MB‐468 cells were seeded in a 6‐well plate at 1 × 105 cells and 3 × 105 cells, respectively, in 2 mL/well culture medium. After overnight incubation, the supernatant was removed from the plate and each ADC diluent (10 nM) was added at 6 mL/well. Viable cells were detached from the plate after 5 days of culture, and the cell number in each well was determined using a cell counter. In order to determine the ratio of KPL‐4 and MDA‐MD‐468 cells of the total viable cells, the cells were stained with anti‐HER2/nue FITC and incubated on ice for 20 min. After washing, fluorescent signals of 2 × 104 stained cells were measured using a flow cytometer. Based on the number and ratio of HER2‐positive and HER2‐negative cells in each treatment well, the number of KPL‐4 or MDA‐MB‐468 cells was calculated.[1]

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A 96-well plate is seeded with 1,000 cells per well. DXd is added following a night of incubation. A CellTiter-Glo Luminescent Cell Viability Assay is used to assess cell viability six days later. To identify HER2 expression in each cell line, FITC Mouse IgG1, κ Isotype Control, or anti-HER2/neu FITC are incubated on ice for 30 minutes. Following washing, FACSCalibur is used to analyze the labeled cells. The calculation of relative mean fluorescence intensity (rMFI) is done[3].

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Cytotoxic assay [3]
Cells were seeded to a 96-well plate at 1,000 cells per well. After overnight incubation, each diluted substance was added. Cell viability was evaluated after 6 days using a CellTiter-Glo Luminescent Cell Viability Assay according to the manufacturer's instructions. For the detection of HER2 expression in each cell line, cells were incubated on ice for 30 minutes with FITC Mouse IgG1, κ Isotype Control, or anti-HER2/neu FITC, the labeled cells were analyzed by FACSCalibur. Relative mean fluorescence intensity (rMFI) was calculated by the following equation:

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ELISA [3]
For a binding assay, immunoplates were coated with 2.5 μg/mL His-tagged HER2-ECD protein in coating buffer and kept overnight at 4°C. After washing, the plates were blocked and each serially diluted substance was added to the wells. After incubation for 1.5 hours at 37°C, the plates were washed and incubated with HRP-conjugated anti-human IgG secondary antibody for 1 hour at 37°C. After washing, TMB solution was added and A450 in each well was measured with a microplate reader. For the detection of phosphorylated Akt (pAkt), SK-BR-3 cells were preincubated in a 96-well plate for 4 days and then incubated with each substance for 24 hours. After incubation, the cells were lysed and intercellular pAkt and total Akt were detected by using a PathScan Phospho-Akt1 (Ser473) Sandwich ELISA Kit and PathScan Total-Akt1 Sandwich ELISA Kit according to the manufacturer's instructions. Relative pAkt of each sample well was calculated by dividing treated normalized pAkt values by untreated normalized pAkt values.

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ADCC evaluation [3]
Antibody-dependent cell-mediated cytotoxicity (ADCC) activities were evaluated using human peripheral blood mononuclear cells (PBMC) derived from a donor as effector cells and the SK-BR-3 cells as target cells. The effector cells (2 × 105 cells) and the 51Cr-labeled target cells (1 × 104 cells) were incubated with each substance, and the indicating effector:target (E:T) ratio was 20:1. After 4 hours of incubation, ADCC activity was measured by radioactivity in the culture supernatant.

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Immunoblotting [3]
KPL-4 cells were treated with each substance. After 24, 48, or 72 hours, the cells were harvested and lysed with M-PER lysis buffer containing Halt Protease & Phosphatase Inhibitor Cocktail. The samples were loaded and separated by SDS-PAGE and blotted onto polyvinylidene difluoride membranes. The membranes were blocked, and probed overnight with anti-phospho-Chk1 (Ser345; 133D3) rabbit mAb, anti-Chk1 (2G1D5) mouse mAb, anti-cleaved PARP (Asp214) antibody, anti-β-actin (8H10D10) Mouse mAb, anti-phospho-Histone H2A.X (Ser139) antibody, and anti-Histone H2A.X antibody at 4°C. Then, the membranes were washed and incubated with fluorescence-labeled secondary antibodies for 10 minutes using SNAP intradermally. The fluorescence signal was detected using an Odyssey imaging system.

In vivo xenograft studies
All in vivo studies were carried out in accordance with the local guidelines of the Institutional Animal Care and Use Committee. Specific pathogen‐free female CAnN.Cg‐Foxn1nu/CrlCrlj mice (BALB/c nude mice) aged 5 weeks were used. All models were established by s.c. inoculation in the flanks of the mice. NCI‐N87 and MDA‐MB‐468‐Luc models were established by injecting 5 × 106 and 1 × 107 cells suspended in a Matrigel matrix, respectively. After 6 days for NCI‐N87, and 9 days for MDA‐MB‐468‐Luc models, the tumor‐bearing mice were randomized into treatment and control groups based on the tumor volume, and dosing initiated (day 0). Each ADC was given i.v. to the mice at a dose of 3 or 10 mg/kg, and a volume of 10 mL/kg. As a vehicle, ABS buffer (10 mM acetate buffer, 5% sorbitol, pH 5.5) was given at the same volume as the ADCs. The tumor volume was defined as 1/2 × length × width2.[1]

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In vivo luciferase imaging
Seven days after inoculating the mixture of 5 × 10~6 NCI‐N87 cells and 1 × 107 MDA‐MB‐468‐Luc cells suspended in the Matrigel matrix into the right flank of the mice at a total volume of 100 μL, the tumor‐bearing mice were randomized into treatment and control groups based on the tumor volume, and dosing initiated (day 0). Each ADC or the vehicle was given i.v. to the mice. Luciferase activity of each mouse was measured using an in vivo imaging system twice a week in parallel with the measurement of tumor length 10 min after administering 150 mg/kg luciferin i.v. The amount of luminescence was analyzed using analysis software as average radiance (p/s/cm2/sr). For another study, MDA‐MB‐468‐Luc cells at a density of 1.5 × 107 cells were inoculated into the left flank of the mice in addition to the inoculation of the mixture into the right flank. Seven days after inoculation, dosing and evaluation were undertaken in a similar manner as described above.[1] Mice: In brief, specific pathogen-free female nude mice are subcutaneously injected with each cell suspension or tumor fragment. Dosing begins on day 0 and the tumor-bearing mice are randomized into treatment and control groups based on the tumor volumes once the tumor has grown to an appropriate size. The mice receive intravenous injections of DS-8201a (1 or 10 mg/kg, i.v.; Dxd is the payload). One calculates tumor growth inhibition (TGI, %)[1].

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Cell line and patient-derived xenograft studies [3]
Detailed study procedures are written in the supplement. Briefly, each cell suspension or tumor fragment was inoculated subcutaneously into specific pathogen-free female nude mice. When the tumor had grown to an appropriate volume, the tumor-bearing mice were randomized into treatment and control groups based on the tumor volumes, and dosing was initiated on day 0. Each substance was administered intravenously to the mice. Tumor growth inhibition (TGI, %) was calculated according to the following equation:

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Pharmacokinetics of DS-8201a in cynomolgus monkeys [3]
Concentrations of DS-8201a and the total antibody in plasma were determined with a validated ligand-binding assay; the lower limit of quantitation was 0.100 μg/mL. Concentrations of DXd in plasma were determined with a validated liquid chromatography-tandem mass spectrometry (LC/MS-MS) method; the lower limit of quantitation was 0.100 ng/mL. DS-8201a was intravenously administered at 3.0 mg/kg to male cynomolgus monkeys. Plasma concentrations of DS-8201a, total antibody, and DXd were measured up to 672 hours postdose.

Pharmacokinetics in cynomolgus monkeys [3]
The plasma DS-8201a concentrations decreased exponentially after a single intravenous administration of DS-8201a. The volume of distribution at steady state (Vss) of DS-8201a and total antibody was close to the plasma volume (data not shown). No clear difference was observed in the pharmacokinetic profile between DS-8201a and the total antibody, indicating that the peptide-linker of DS-8201a is stable in plasma even at DAR 8 (Fig. 2E). A low level of DXd was detected only at the limited time points (Fig. 2E).

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In vitro stability in plasma [3]
The release rates of DXd from DS-8201a ranged from 1.2% to 3.9% on day 21 in mouse, rat, monkey, and human plasma (Fig. 2F), and these were comparable or rather lower than those of other ADCs, such as T-DM1, SGN-35 (Brentuximab vedotin), and inotuzumab ozogamicin (35–37). These results indicate that DS-8201a is stable in plasma.

Safety profile of DS-8201a [3]
A repeated intravenous dosing (every 3 weeks for 3 doses) study was conducted in cynomolgus monkeys, the cross-reactive species for DS-8201a, and in rats (antigen–non-binding species; Table 1). In the rat study, no deaths or life-threatening toxicities were found at dose levels up to 197 mg/kg, the maximum dose. Therefore, the severely toxic dose of 10% in animals (STD10) was considered to be >197 mg/kg. In the monkey study, one female at the highest dose of 78.8 mg/kg was euthanized due to moribundity on day 26. The cause of the moribundity appeared to be the deteriorated condition of the animal, which included decreased body weight and food consumption, as well as bone marrow toxicity and intestinal toxicity. Microscopic findings in the intestines, bone marrow and lungs in the surviving monkeys are shown in Supplementary Table S1. Gastrointestinal toxicity and bone marrow toxicity are typical dose-limiting factors in the clinical use of topoisomerase I inhibitors. The effects of DS-8201a on the intestines were very slight, and severe changes were not pronounced in any animal at up to 78.8 mg/kg. The bone marrow toxicity was produced only at 78.8 mg/kg, and was accompanied by decreases in reticulocyte ratios. No abnormalities in leukocyte and erythrocyte counts were observed in monkeys at 10 and 30 mg/kg. The repeated dose of DS-8201a caused moderate pulmonary toxicity in monkeys at 78.8 mg/kg, and findings graded as slight or very slight after the 6-week recovery period at ≥30 mg/kg. On the basis of the mortality and severity of the findings above, the highest non-severely toxic dose (HNSTD) for monkeys was considered to be 30 mg/kg. DS-8201a was well tolerated at the doses up to 197 mg/kg in rats and 30 mg/kg in monkeys following the repeated administration corresponding to the clinical regimen, and the nonclinical safety profile was acceptable for entry into human trials.

[1]. Bystander killing effect of DS-8201a, a novel anti-human epidermal growth factor receptor 2 antibody-drug conjugate, in tumors with human epidermal growth factor receptor 2 heterogeneity. Cancer Sci. 2016 Jul;107(7):1039-46.

[2]. METHOD FOR SELECTIVELY MANUFACTURING ANTIBODY-DRUG CONJUGATE. WO2017002776A1.

[3]. DS-8201a, A Novel HER2-Targeting ADC with a Novel DNA Topoisomerase I Inhibitor, Demonstrates a Promising Antitumor Efficacy with Differentiation from T-DM1. Clin Cancer Res. 2016 Oct 15;22(20):5097-5108.

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.[1]

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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. [3]

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Most of the ADCs currently in the market and in clinical development carry tubulin polymerization inhibitors such as T-DM1 and SGN-35 (Brentuximab vedotin; ref. 13). We synthesized a novel ADC with a topoisomerase I inhibitor, which has a different mechanism of action from tubulin polymerization inhibitors, and a novel self-immolative linker system using an aminomethylene (AM) moiety. Although other cleavable linker systems applied to SGN-35 (Brentuximab vedotin) and several ADCs release amino group–containing payloads, this AM self-immolative linker system is able to release DXd containing the hydroxyl group from DS-8201a. Moreover, this novel linker-payload system enables a reduction in the hydrophobicity of the ADC and helps increase its DAR. In the case of T-DM1, lysine conjugation and noncleavable systems are used, and it is quite a different system from DS-8201a. DS-8201a showed potent HER2-specific efficacy both in vitro and in vivo, and by drug conjugation maintained the functional effects of trastuzumab equal to those of T-DM1. Furthermore, the safety profiles of DS-8201a in rats and cynomolgus monkeys showed DS-8201a as being well tolerated.[3]

C52H56FN9O13

1034.05195617676

1,033.40

C, 60.40; H, 5.46; F, 1.84; N, 12.19; O, 20.11

1599440-13-7

Exatecan mesylate;169869-90-3;Deruxtecan-d6;2760715-89-5;Deruxtecan-d5;Exatecan mesylate dihydrate;197720-53-9; 171335-80-1; 144008-87-7 (HCl)

118305111

White to yellow solid powder

1.48±0.1 g/cm3

1491.1±65.0 °C

-0.4

7

15

22

75

2360

3

FC1=CC2=C3C(=C1C)CC[C@@H](C3=C1C(C3=CC4[C@](C(=O)OCC=4C(N3C1)=O)(CC)O)=N2)NC(COCNC(CNC([C@H](CC1C=CC=CC=1)NC(CNC(CNC(CCCCCN1C(C=CC1=O)=O)=O)=O)=O)=O)=O)=O

WXNSCLIZKHLNSG-MCZRLCSDSA-N

InChI=1S/C52H56FN9O13/c1-3-52(73)33-19-38-48-31(24-62(38)50(71)32(33)25-75-51(52)72)47-35(14-13-30-28(2)34(53)20-36(60-48)46(30)47)58-43(67)26-74-27-57-41(65)22-56-49(70)37(18-29-10-6-4-7-11-29)59-42(66)23-55-40(64)21-54-39(63)12-8-5-9-17-61-44(68)15-16-45(61)69/h4,6-7,10-11,15-16,19-20,35,37,73H,3,5,8-9,12-14,17-18,21-27H2,1-2H3,(H,54,63)(H,55,64)(H,56,70)(H,57,65)(H,58,67)(H,59,66)/t35-,37-,52-/m0/s1

Glycinamide, N-[6-(2,5-dihydro-2,5-dioxo-1H-pyrrol-1-yl)-1-oxohexyl]glycylglycyl-L-phenylalanyl-N-[[2-[[(1S,9S)-9-ethyl-5-fluoro-2,3,9,10,13,15-hexahydro-9-hydroxy-4-methyl-10,13-dioxo-1H,12Hbenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl]amino]-2-oxoethoxy]methyl]-

Deruxtecan; DS-8201a; DS8201a; DX-8951 derivative; Trastuzumab deruxtecan; DS 8201a; exatecan derivative; DX 8951; DX8951; Deruxtecan; 1599440-13-7; Mc-ggfg-dxd(1); 5SEB972CO4; Deruxtecan [USAN]; UNII-5SEB972CO4;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

Note: (1) This product is not stable in solution, please use freshly prepared working solution for optimal results. (2) This product requires protection from light (avoid light exposure) during transportation and storage.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~35 mg/mL (~33.85 mM)
H2O : Insoluble (< 1 mg/mL)

Solubility in Formulation 1: 1.75 mg/mL (1.69 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 17.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 1.75 mg/mL (1.69 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 17.5 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 1.75 mg/mL (1.69 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 17.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 10% DMSO+ 40% PEG300+ 5% Tween-80+ 45% saline: 1.75 mg/mL (1.69 mM)

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 (Please use freshly prepared in vivo formulations for optimal results.)