Topoisomerase

Biochemical and Structural Basis of Exatecan as a Higher Potency TOP1 inhibitor [1]
DXd was derived from exatecan by the chemical modification of NH2 on the F-ring by a 2-hydroxyacetyl (COCH2OH) group (Supplementary Fig. S1A). Exatecan showed an average of 10 to 20 times more in vitro cytotoxic potency than DXd with subnanomolar IC50 (Supplementary Fig. S1B; Supplementary Table S1) in a panel of cancer cell lines. Cell-based TOP1 inhibition assays and structural modeling provided a mechanism of higher cytotoxic potency of exatecan. The level of DNA-trapped TOP1 (TOP1ccs) of exatecan in comparison with DXd and SN-38 was examined by a modified rapid approach to DNA adduct recovery (RADAR) assay (21). Exatecan was the most potent drug and induced TOP1ccs at a lower concentration than DXd and SN-38 (Fig. 1A; Supplementary Fig. S1C). DNA-trapped TOP1 is typically removed from DNA and degraded. To measure the rate of TOP1 degradation, cells were treated with exatecan or DXd/SN-38 for 2 hours and then allowed to grow without drugs for 30 minutes for the reversal of TOP1ccs and TOP1 degradation. Exatecan induced TOP1 degradation in a dose-dependent manner and was more effective than DXd and SN-38 (Fig. 1B).

---

Exatecan Shows Less Sensitivity to MDR Mechanisms than DXd and SN38 [1]
The MDR transporter ABCG2- and P-gp–mediated efflux of exatecan, DXd, and SN-38 was determined in Caco-2 cells (Supplementary Fig. S1E). DXd showed an order of magnitude higher efflux ratios than exatecan without or with the ABCG2 inhibitor novobiocin, the P-gp inhibitor verapamil, or the dual inhibitor GF120918 (Fig. 1D). Interestingly, SN-38 efflux ratios were higher than exatecan but lower than DXd. Immunofluorescence (IF) examination of the intracellular accumulation of exatecan and DXd showed that DXd accumulation was lower than exatecan in the ABCG2 high expression cell line NCI-H460 (Fig. 1E; Supplementary Fig. S1F–1J). Consistent with MDR substrate experiment results, the cytotoxic potency difference between DXd and exatecan showed a correlation with endogenous ABCG2/P-gp expression (mRNA and protein; Supplementary Fig. S2A–S2C). In general, the IC50 ratio of DXd/exatecan was higher in cells with a higher ABCG2 or P-gp expression (Fig. 1F; Supplementary Fig. S2D and S2E). The inhibition of ABCG2 or P-gp improved the cytotoxicity of DXd/SN-38 but had a much smaller effect on exatecan (Fig. 1G). Even with an improved IC50 with inhibitor for DXd/SN-38, exatecan remained more potent (Supplementary Fig. S2F and S2G). Collectively, exatecan demonstrated less sensitivity to the MDR genes than DXd/SN38, potentially conferring higher cytotoxicity in cancer cells along with a more prevalent resistance mechanism.

Antibody-drug conjugates (ADC) using DNA topoisomerase I inhibitor DXd/SN-38 have transformed cancer treatment, yet more effective ADCs are needed for overcoming resistance. We have designed an ADC class using a novel self-immolative T moiety for traceless conjugation and release of exatecan, a more potent topoisomerase I inhibitor with less sensitivity to multidrug resistance (MDR). Characterized by enhanced therapeutic indices, higher stability, and improved intratumoral pharmacodynamic response, antibody-T moiety-exatecan conjugates targeting HER2, HER3, and TROP2 overcome the intrinsic or treatment resistance of equivalent DXd/SN-38 ADCs in low-target-expression, large, and MDR+ tumors. T moiety-exatecan ADCs display durable antitumor activity in patient-derived xenograft and organoid models representative of unmet clinical needs, including EGFR ex19del/T790M/C797S triple-mutation lung cancer and BRAF/KRAS-TP53 double-mutant colon cancer, and show synergy with PARP/ATR inhibitor and anti-PD-1 treatment. High tolerability of the T moiety-exatecan ADC class in nonhuman primates supports its potential to expand the responding patient population and tumor types beyond current ADCs. Significance: ADCs combining a novel self-immolative moiety and topoisomerase I inhibitor exatecan as payload show deep and durable response in low-target-expressing and MDR+ tumors resistant to DXd/SN-38 ADCs without increasing toxicity. This new class of ADCs has the potential to benefit an additional patient population beyond current options. See related commentary by Gupta et al., p. 817. This article is highlighted in the In This Issue feature, p. 799 [1].

In Vitro Stability of MTX-1000/DS-8201a in Plasma [1]
The release rate of exatecan/DXd from MTX-1000/DS-8201a at the concentration of 10 mg/mL at 37°C up to 21 days was evaluated in mouse, rat, monkey, and human plasma.

---

ELISA [1]
For a binding assay, immune plates were coated with 2.5 mg/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 using a PathScan Phospho-Akt1 (Ser473) Sandwich ELISA Kit and PathScan Total-Akt1 Sandwich ELISA Kit according to the manufacturer's instructions. The relative pAkt of each sample well was calculated by dividing treated normalized pAkt values by untreated normalized pAkt values.

---

Immunoblotting [1]
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 and 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 (Abcam) 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.

Cell Line and PDX Studies [1]
\nAll tumor-bearing models were established in female BALB/c nude mice that were purchased from the Shanghai Family Planning Research Institute. All in vivo studies were performed in accordance with the local guidelines of the Institutional Animal Care and Use Committee of Multitude Therapeutics and with the approval of the committee. Briefly, 4- to 6-week-old mice were housed together in sterilized cages and maintained under required pathogen-free conditions. Each cell suspension or tumor fragment was inoculated subcutaneously into 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 started. Antibodies and ADCs were administered intravenously to the mice. Small-molecule drugs were administered orally. Tumor volume defined as 1/2 × length × width2 was measured twice (3–4 days) a week. TGI (%) was calculated as follows: TGI (%) = [1 – (mean of treatment group tumor volume on evaluation day)/(mean of control group tumor volume on evaluation day)] × 100. The mice were euthanized with CO2 gas when they reached endpoints (tumor volume exceeding 3,000 mm3, >10% reduction of body weight, or clinical signs indicating that mice should be euthanized for ethical reasons).\n

---

\n\nSyngeneic Model [1]
\nFemale BALB/c mice, weighing approximately 18 to 20 g, were maintained with 5 mice per cage in an individually ventilated cage in a specific pathogen–free animal facility with autoclaved cages. After 9 days of acclimation, animals were subcutaneously inoculated with CT26-hHER2 cells (3 × 105 cells/0.1 mL/mouse) on the right flank for tumor development. Ten days after inoculation, 36 mice with tumor sizes ranging from 100 to 200 mm3 (average tumor size 178 mm3) were enrolled into the efficacy study and randomized into six therapy groups, and the treatments were initiated on the randomization day (defined as D0). MTX-1000, DS-8201a (10 mg/kg), and anti–PD-1 antibody (5 mg/kg) were administered intravenously at a volume of 10 mL/kg to mice. As a control, ABS buffer (10 mmol/L acetate buffer, 5% sorbitol, pH 5.5) was administered at the same volume as the drugs. MTX-1000 and DS-8201a were administered on days 0 and 7. Anti–PD-1 antibody was administered on days 0, 3, 7, and 10.\n\nFor flow-cytometric analysis of T cells, dendritic cells, and tumor cells, when the average volume of tumors reached approximately 250 to 400 mm3 (8 days after tumor inoculation), the mice were treated with vehicle or DS-8201a (10 mg/kg, once, intravenously; day 0). The mice were euthanized with CO2 asphyxiation on day 8, and tumors were cut into small pieces and dissociated with the Tumor Dissociation Kit by the gentleMACS Octo Dissociator with Heaters. The resultant single cells were blocked with Mouse BD Fc Block reagent and stained with antibodies against mouse CD3, CD4, CD8, CD11c, CD45, CD86, granzyme B, MHC class I, MHC class II, and PD-L1 and human HER2.\n

---

\n\nTissue Multiplex IHC [1]
\nSections from formalin-fixed, paraffin-embedded tumor tissue blocks were stained using Opal multiplex according to the manufacturer's protocol for DAPI, CD45, CD4, and CD8. Slide scanning was performed on the Vectra 3.0 instrument. All the images were analyzed with the HALOTM Image Analysis platform. The whole section was analyzed, and the big necrosis area and stroma area were excluded. Single-positive cells were counted separately. Images were spectrally unmixed, evaluated for staining intensities and morphology, tissue segmented based on tissue markers, cell segmented based on nuclear and membrane markers, and phenotypically scored.\n

---

\n\nPK of MTX-1000/DS-8201a in Rat/Mouse [1]
\nConcentrations of ADC and the total antibody in plasma were determined with a validated ligand-binding assay; the lower limit of quantitation was 0.02 μg/mL. Briefly, immunoplates were coated with 1 μg/mL Human HER2 Protein, His Tag in coating buffer and kept overnight at 4°C. After washing, the plates were blocked, and each serially diluted sample was added to the wells. After incubation for 1 to 2 hours at 37°C, the plates were washed and incubated with HRP-conjugated anti-human IgG Fc secondary antibody for total antibody measurement or a biotin-labeled anti-exatecan/DXd antibody for ADC measurement. After reaction at 37°C for 1 to 2 hours, TMB solution was added directly or after incubation with Streptavidin Protein, HRP for 40 to 60 minutes at 37°C. A450 in each well was measured with a microplate reader. Concentrations of payload exatecan/DXd in plasma were determined with a validated LC/MS-MS method; the lower limit of quantitation was 0.05 ng/mL. MTX-1000/DS-8201a was intravenously administered at 4.0 mg/kg to rats. Plasma concentrations of ADC, total antibody, and DXd were measured up to 21 days after dose.

Partial conjugation transforms ethatecan into a superior antibody-drug conjugate: physicochemical and pharmacological properties, stability and toxicity [1]
T-part design: hydrophilic modification of the PABC spacer for seamless conjugation [1]
To achieve seamless conjugation and release of ethatecan, we used a dipeptide VA linker and a modified self-cleaving spacer p-aminobenzyl (pAB), referred to as the T-part (Fig. 2A; S1 in Supplementary Fig. S4A). VA was chosen because it produces a more hydrophilic antibody-drug conjugate with higher DAR compared to other peptide linkers VC or Gly–Gly–Phe–Gly (GGFG) (S0 in Supplementary Fig. S4A; References 29–30). Direct conjugation of the HER2-targeting drug Tras and ethatecan using the unmodified peptide linker VA/VC/GGFG resulted in high aggregation (Supplementary Fig. S4B). Integrating the T moiety with a PEG group (T900; S3 in Supplementary Figure S4A) significantly reduced the aggregation to an acceptable 2%. However, modifying the MC with the same PEG (referred to as the M moiety, M900; S2 in Supplementary Figure S4A) resulted in high aggregation of >50% (Supplementary Figure S4B), highlighting the importance of careful selection of modification sites. Considering the potential for fatal adverse reactions from PEG-based biopharmaceuticals (31), we decided to use polysarcosine (pSAR) due to its higher solubility and biocompatibility (Supplementary Figure S4C; Reference 32). pAB (T1000) modified with 10 units of polysarcosine (pSAR10) yielded a homogeneous antibody-drug conjugate (ADC) with negligible aggregation (Supplementary Figure S4B). Notably, the intermediate structure T800, by linking pSAR10 to pAB with m-methylaminomethyl modification, yielded a homogeneous ADC with a high drug-to-antibody ratio (DAR) and no aggregation. In contrast, attaching the same m-methylaminomethyl group to the VA linker (VK-pABC, M800, CLogP = 4.98) resulted in an 10% aggregation rate of ethanotecan, even though T800 (CLogP = 4.73) and M800 were considered to be chemically identical, supporting the selection of pAB as the optimal modification site. The coupling yield of the T-part ADC was >90%. Replacing the VA linker with the GGFG linker in T1000 also yielded a homogeneous ADC, and no aggregation was observed (T1001; Supplementary Figure S4B).

---

Tras–T1000–Exatecan (MTX-1000) exhibits superior physicochemical properties[1]
To compare the physicochemical parameters of Tras–T1000–Exatecan (MTX-1000) with DS-8201a, we used commercially available DS-8201a or internally produced Tras–GGFG–DXd. The physicochemical properties (Supplementary Figures S5A and S5B), in vivo potency (Supplementary Figures S5C–S5E), and mouse pharmacokinetics (PK) (Supplementary Figure S5F) of the two were highly matched. Similar to Tras–GGFG–DXd, the binding of ethanotecan to the HER2 antibody did not affect the binding of the HER2 antibody to the target (as detected by flow cytometry or ELISA) (Supplementary Figures S6A and S6B). Tras–ethanotecan conjugates using T800 or T900 also exhibit stronger hydrophilicity than Tras–GGFG–DXd (Supplementary Figures S6C and S6D). Both DAR8 and DAR4 ADCs can be readily prepared (Supplementary Figure S6E). The stability of Tras–T-part–ethanotecan ADCs (Tras–T800–ethanotecan/MTX-800 and Tras–T1000–ethanotecan/MTX-1000, both DAR8) is similar to or better than that of Tras–GGFG–DXd. Stability was determined by the aggregates formed after prolonged incubation at 37°C and repeated freeze-thaw cycles (Supplementary Figure S6F). The thermal and photostable stability of the T-part–ethanotecan antibody-drug conjugates (ADCs) is also superior to that of DS-8201a (Supplementary Figures S6G and S6H). Notably, the stability of VA and pSAR ADCs (T1000) is superior to other linkers (GGFG) and modifications (PEG; T900 or T1001; Supplementary Figures S6C, S6D, S6F and S6G), and they exhibit lower aggregation, highlighting the influence of linker and coupling chemical selection on the physicochemical properties of ADCs.

Exatecan mesylate was well tolerated in rats [1]
We conducted an intermittent intravenous toxicity study of esaxetcan mesylate in rats (6 animals per group) for 4 weeks (days 1, 8, 15, 22, and 29), with a 4-week recovery period (3 out of 6 animals). At doses up to 10 mg/kg (based on free esaxetcan base), esaxetcan mesylate was well tolerated in rats. At doses up to 30 mg/kg, esaxetcan mesylate caused weight loss, increased morbidity (reduced food intake, abnormal clinical symptoms), and/or death (occurring 5 or 6 days after administration). In the 3 mg/kg and 10 mg/kg groups, dose-dependent transient suppression of weight gain (Supplementary Figure S3A), decreased blood cell count (Supplementary Figures S3B–S3E), and elevated levels of key serum enzymes (Supplementary Figures S3F and S3G) were observed, but were largely reversible at the end of the 4-week recovery period (Supplementary Table S2), indicating that essanotecan has similar tolerability to DXd or SN-38.

[1]. Antibody-Exatecan Conjugates with a Novel Self-immolative Moiety Overcome Resistance in Colon and Lung Cancer. Cancer Discov. 2023 Apr 3;13(4):950-973.

[2]. Intermediate for synthesizing camptothecin derivatives using exatecan mesylate and its preparation method and application. China, CN111470998. 2020-07-31.

In non-human primate studies, the T-group-exatecan antibody-drug conjugate (ADC) maintained high efficacy without increasing HNSTD toxicity, achieving efficacy levels comparable to or higher than other TOP1 inhibitor ADCs. Notably, side effects following T-group-exatecan ADC administration were typically more pronounced in the gastrointestinal tract (diarrhea) and hematologic system (reticulopenia), but these symptoms were completely reversible. The hematologic and gastrointestinal toxicities of the T-group-exatecan ADC were similar to those of ethatecan monotherapy in human trials. However, compared to ethatecan, the T-group-exatecan ADC demonstrated superior gastrointestinal safety and extremely low myelotoxicity, potentially reflecting the superior physicochemical properties of this ADC design. The higher stability, longer half-life, lower free payload release, and lower myelotoxicity of the T-group-exatecan ADC compared to the free payload likely contributed to its favorable toxicity profile. In clinical trials, the incidence of interstitial lung disease was slightly higher than 10% in patients treated with DXd-based ADCs. Although we did not observe any signs of structural damage or inflammation in the lungs of animals treated with T-group-exatecan ADCs, it remains uncertain whether long-term treatment will lead to additional pulmonary toxicity. However, it is conceivable that T-group-exatecan ADCs may have a different target-independent tissue distribution than DXd-based ADCs, resulting in different symptom presentations. For tumors that respond poorly to or are resistant to TOP1 inhibitors, payload classes with different mechanisms of action may be required. The T-group is a modular structure that is compatible with a variety of linker chemistry and is suitable for constructing ADCs with dual payloads. In summary, T-part-exatecan ADCs have the potential to meet patient needs that current ADCs cannot meet, and the versatility and scalability of the T-part can facilitate the introduction of payloads with completely different mechanisms of action to address the ongoing challenge of drug resistance. [1]

C13H13NO5

263.25

263.079

102978-40-5

Exatecan Intermediate 1;110351-94-5;(R)-Exatecan Intermediate 1;110351-91-2

359849

White to light yellow solid powder

0.089

1

5

1

19

574

0

IGKWOGMVAOYVSJ-UHFFFAOYSA-N

InChI=1S/C13H13NO5/c1-2-13(18)8-5-9-10(15)3-4-14(9)11(16)7(8)6-19-12(13)17/h5,18H,2-4,6H2,1H3

4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10-trione

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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