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| Targets |
Traditional Cytotoxic Agents; DGN549-C (1b) itself does not have a specific biological target. As part of an antibody-drug conjugate, it targets the antigen recognized by the antibody (e.g., FRα or CD123). Its mechanism of action is DNA alkylation after intracellular release. The catabolite identified from both lysine-linked and CYSAMB DGN549 ADCs is the monoimine indolinobenzodiazepine aniline species (4). [1]
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| ln Vitro |
DGN549-C (1b) is not tested as a free drug; its activity is assessed as part of ADCs (e.g., ADC 2b and 3b). [1]
In FRα-positive cell lines (KB, T47D, NCI-H2110), the site-specific ADC 2b (DGN549-C conjugated to mAb1 at HC-C442) shows specific cytotoxicity with IC50 values (based on ADC concentration) of 5 × 10⁻¹² M (KB), 1 × 10⁻¹¹ M (T47D), and 4 × 10⁻¹¹ M (NCI-H2110). The corresponding IC50 values based on DGN549 payload concentration are 7 × 10⁻¹² M (KB), 3 × 10⁻¹¹ M (T47D), and 9 × 10⁻¹¹ M (NCI-H2110). Cytotoxicity is blocked by excess unconjugated targeting antibody, confirming target specificity. [1] In CD123-positive cell lines (EOL-1, HNT-34, MV4-11), the site-specific ADC 3b (DGN549-C conjugated to mAb2 at HC-C442) shows specific cytotoxicity with IC50 values (based on ADC concentration) of 2 × 10⁻¹² M (EOL-1), 1 × 10⁻¹² M (HNT-34), and 8 × 10⁻¹³ M (MV4-11). The corresponding IC50 values based on DGN549 payload concentration are 4 × 10⁻¹² M (EOL-1), 2 × 10⁻¹² M (HNT-34), and 2 × 10⁻¹² M (MV4-11). Non-targeting control ADCs are substantially less potent (IC50 2 × 10⁻⁹ to 4 × 10⁻⁹ M). [1] Bystander activity: both ADC 2a (lysine-linked) and ADC 2b (site-specific) kill FRα-negative cells in mixed population assays at subnanomolar concentrations, consistent with release of a cell-permeable catabolite. [1] |
| ln Vivo |
DGN549-C (1b)-based ADCs show efficacy in mouse xenograft models. [1]
In NCI-H2110 (FRα-positive NSCLC) xenografts, ADC 2b (3 μg DGN549/kg) is minimally active (T/C = 39%); at 9 μg DGN549/kg, it induces durable complete regressions (6/6 complete). Activity is similar to lysine-linked ADC 2a. [1] In Molm-13 (CD123-positive AML) disseminated xenografts, ADC 3b at 0.3 μg DGN549/kg produces 262% increased life span; at 1 μg DGN549/kg, median survival exceeds 152 days (>443% ILS). The site-specific ADC 3b shows improved activity compared to lysine-linked ADC 3a at the 0.3 μg DGN549/kg dose (262% vs 59% ILS). [1] |
| Cell Assay |
Cell binding assay (flow cytometry) : Cells (0.5–1 × 10⁵) are incubated with serial dilutions of test antibodies for 1 hour at 4°C, washed, and incubated with FITC-conjugated goat anti-human IgG secondary antibody for 1 hour at 4°C. After washing, cells are fixed and analyzed by flow cytometry. EC50 values for ADCs are comparable to parent antibodies. [1]
Cytotoxicity assay (CellTiter-Glo) : Cells (1500–3000/well) are seeded in 96-well plates and incubated overnight. Serial dilutions of ADCs are added with or without blocking antibody (20 μg/mL). After 4 days at 37°C, CellTiter-Glo reagent is added, and luminescence is measured. IC50 values are calculated. [1] Bystander activity assay : A mixed population of FRα-negative parental 300.19 cells and FRα-positive transfectants is treated with ADC. Cell viability is determined after 4 days using CellTiter-Glo. [1] Catabolite identification assay (LC-MS) : KB cells are treated with saturating concentration of ADC for 24 h. Catabolites in media are concentrated by binding to an anti-IGN antibody, extracted into acetone, dried, redissolved, and analyzed by UPLC coupled to high-resolution mass spectrometry. [1] |
| Animal Protocol |
Efficacy (NCI-H2110 xenograft) : Female CB-17 SCID mice (n=6/group) are inoculated subcutaneously with 1 × 10⁷ NCI-H2110 cells. When tumors reach ~100 mm³ (day 8), a single intravenous dose of ADC (3 or 9 μg DGN549/kg) is administered. Tumor size is measured twice weekly. [1]
Efficacy (Molm-13 disseminated xenograft) : Female thymic nude mice (n=10/group) are inoculated intravenously with 1 × 10⁷ Molm-13 cells. On day 7, a single intravenous dose of ADC (0.1, 0.3, or 1 μg DGN549/kg) is administered. To block Fc receptors, mice receive 400 mg/kg of a non-targeting IgG1 antibody intraperitoneally 24 h before ADC, followed by 100 mg/kg on days 4 and 9. [1] Tolerability : Non-tumor-bearing CD-1 mice (n=8/group) receive a single intravenous dose of ADC. Body weight is monitored daily for 14 days. Maximum tolerated dose (MTD) is defined as the maximum dose with no deaths or euthanasia due to >20% body weight loss. For ADC 2b (site-specific), MTD is ~100–125 μg DGN549/kg (approx. 6–8 mg antibody/kg). [1] Pharmacokinetics : Non-tumor-bearing CD-1 mice receive a single intravenous dose of ADC (2.5 mg antibody/kg). Plasma samples are collected at various time points (2 min to 28 days). ADC concentrations are measured by two ELISA methods: one for total antibody (anti-human IgG capture) and one for conjugated antibody (anti-IGN capture). [1] |
| ADME/Pharmacokinetics |
For site-specific ADC 2b (DGN549-C conjugated to mAb1 at HC-C442) in CD-1 mice at 2.5 mg antibody/kg: total antibody Cmax = 52.1 μg/mL, AUC0-∞ = 10,795 h•μg/mL, clearance = 0.23 mL/h/kg, t½ = 5.7 days, Vss = 31.5 mL/kg. Conjugated antibody Cmax = 65.4 μg/mL, AUC0-∞ = 9,159 h•μg/mL, clearance = 0.27 mL/h/kg, t½ = 6.0 days, Vss = 23.4 mL/kg. Compared to lysine-linked ADC 2a, site-specific ADC 2b shows 1.6-fold greater exposure and 1.7-fold slower clearance. [1]
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| Toxicity/Toxicokinetics |
In mouse tolerability studies, the maximum tolerated dose (MTD) of ADC 2b (site-specific, DAR ~2) is ~100–125 μg DGN549/kg (approximately 6–8 mg antibody/kg), which is approximately 2-fold higher than the MTD of lysine-linked ADC 2a (DAR ~2.5), indicating improved tolerability with site-specific conjugation. For ADC 3b (site-specific, DAR ~2), MTD is ~100 μg DGN549/kg, formally the same as lysine-linked ADC 3a, but at the 125 μg DGN549/kg dose, the majority of animals in the 3b group recover and gain weight following acute toxicity, while those in the 3a group require euthanasia, suggesting slightly improved tolerability. A site-specific ADC with higher DAR (ADC 2c, DAR ~3.5) has an intermediate MTD of 125 μg/kg, suggesting that both site-specificity and lower DAR contribute to improved tolerability. [1]
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| References | |
| Additional Infomation |
Synthesis: DGN549-C (1b) is generated from DGN549-L (1a) via acylation of aminoethylmaleimide hydrochloride. [1]
Conjugation method (CYSAMB) : Engineered cysteine mutant antibodies (e.g., HC-S442C) are globally reduced and selectively reoxidized. The resulting partially reduced antibodies are conjugated with 1b in an aqueous buffer containing propylene glycol as a nondenaturing cosolvent. The conjugation yields ADCs with DAR of approximately 2.0 for single-mutant antibodies. [1] Catabolite: The only major catabolite species identified from both lysine-linked and CYSAMB DGN549 ADCs is the monoimine indolinobenzodiazepine aniline species (4), confirming that the conjugation chemistry does not alter the mechanism of action. [1] Hydrophobicity: By HIC analysis, CYSAMB DGNS49 ADCs appear more hydrophobic than their lysine-linked counterparts, eluting at the very end of the HIC gradient, yet they show improved tolerability and slower clearance, suggesting that HIC retention does not correlate with poor PK properties for this payload class. [1] |
| Molecular Formula |
C60H61N9O12
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|---|---|
| Molecular Weight |
1100.18
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| Exact Mass |
1099.443
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| CAS # |
2058075-34-4
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| PubChem CID |
118912740
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| Appearance |
Typically exists as solid at room temperature
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| LogP |
3.8
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| Hydrogen Bond Donor Count |
5
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| Hydrogen Bond Acceptor Count |
14
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| Rotatable Bond Count |
21
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| Heavy Atom Count |
81
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| Complexity |
2360
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| Defined Atom Stereocenter Count |
4
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| SMILES |
O=C1C2=CC(OC)=C(OCC3C=C(NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)CCCCC(=O)NCCN4C(C=CC4=O)=O)C=C(COC4C=C5N=C[C@]6([H])CC7=CC=CC=C7N6C(=O)C5=CC=4OC)C=3)C=C2NC[C@]2([H])CC3=CC=CC=C3N12
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| InChi Key |
ARZPUSPNNAYGKH-RCAQRAEBSA-N
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| InChi Code |
InChI=1S/C60H61N9O12/c1-34(64-54(71)16-10-9-15-53(70)61-19-20-67-55(72)17-18-56(67)73)57(74)65-35(2)58(75)66-40-22-36(32-80-51-28-45-43(26-49(51)78-3)59(76)68-41(30-62-45)24-38-11-5-7-13-47(38)68)21-37(23-40)33-81-52-29-46-44(27-50(52)79-4)60(77)69-42(31-63-46)25-39-12-6-8-14-48(39)69/h5-8,11-14,17-18,21-23,26-30,34-35,41-42,63H,9-10,15-16,19-20,24-25,31-33H2,1-4H3,(H,61,70)(H,64,71)(H,65,74)(H,66,75)/t34-,35-,41-,42-/m0/s1
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| Chemical Name |
N'-[(2S)-1-[[(2S)-1-[3-[[(12aS)-8-methoxy-6-oxo-12a,13-dihydroindolo[2,1-c][1,4]benzodiazepin-9-yl]oxymethyl]-5-[[(12aS)-8-methoxy-6-oxo-11,12,12a,13-tetrahydroindolo[2,1-c][1,4]benzodiazepin-9-yl]oxymethyl]anilino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]-N-[2-(2,5-dioxopyrrol-1-yl)ethyl]hexanediamide
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| Synonyms |
2058075-34-4; orb1691297; SCHEMBL17553282; SCHEMBL30018815;
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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
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| Solubility (In Vivo) |
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 Formulations
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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)] Oral Formulations
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 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.9089 mL | 4.5447 mL | 9.0894 mL | |
| 5 mM | 0.1818 mL | 0.9089 mL | 1.8179 mL | |
| 10 mM | 0.0909 mL | 0.4545 mL | 0.9089 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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