| Size | Price | Stock | Qty |
|---|---|---|---|
| 100mg |
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| Other Sizes |
| Targets |
Protease Cleavable Linker Cleavable Linker
Fmoc-Gly3-Val-Cit-PAB itself has no biological target; it is a structural component of an ADC. Its target, when incorporated into an ADC, is the lysosomal cysteine protease, cathepsin B. The Val-Cit dipeptide is a specific substrate for this enzyme, which is highly active in the endolysosomal compartments of cells, including cancer cells. The ultimate target is the intracellular machinery affected by the payload drug that is released. The tri-glycine spacer is introduced to reduce steric hindrance and improve enzyme accessibility to the cleavage site, thereby enhancing the efficiency of payload release from the ADC. |
|---|---|
| ln Vitro |
ADCs are comprised of an antibody to which is attached an ADC cytotoxin through an ADC linker[1].
No direct in vitro activity is reported for Fmoc-Gly3-Val-Cit-PAB alone. The "in vitro activity" of an ADC containing this linker is measured in cytotoxicity assays on a panel of cancer cell lines. The IC₅0 values typically range from nanomolar to picomolar levels. The cleavability of the linker is confirmed in an in vitro lysosomal extract assay. The ADC is incubated with purified cathepsin B or a lysosomal extract, and the release of the payload is monitored by LC-MS. A significant decrease in cytotoxicity upon co-incubation with a cathepsin B inhibitor (e.g., CA-074) confirms the mechanism of activation. |
| ln Vivo |
No in vivo activity is reported for the linker alone. For an ADC incorporating a Gly3-Val-Cit-PAB linker, in vivo efficacy is demonstrated in mouse xenograft models. A typical study involves dosing mice bearing established human tumor xenografts intravenously with the ADC. The Val-Cit linker is known for its stability in circulation and efficient intratumoral release, leading to robust tumor growth inhibition. The efficacy is often superior to ADCs with non-cleavable linkers. The pharmacodynamic effect is confirmed by immunohistochemical analysis of tumor sections for cleaved caspase-3, a marker of apoptosis, and by measuring the concentration of the released payload in the tumor tissue by LC-MS.
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| Enzyme Assay |
A typical non-cellular cleavage assay for this linker involves incubating the ADC (10-100 ug/mL) with 10-50 nM of purified human cathepsin B in 100 uL of activation buffer (20 mM sodium acetate, pH 5.0, containing 2 mM DTT and 1 mM EDTA) at 37degC. The reaction is carried out for 1-24 hours. At various time points, the reaction is quenched with E-64, a cysteine protease inhibitor. The samples are then analyzed by HPLC or LC-MS to separate and quantify the released payload versus the intact linker-payload. The half-life (t1/2) of cleavage can be determined from the kinetic data. A control without the enzyme is run to assess stability.
|
| Cell Assay |
A typical in vitro cell-based assay for an ADC containing this linker uses flow cytometry and immunofluorescence to track the linker's cleavage. Cells are seeded in a chamber slide or 6-well plate and incubated with the ADC (10 ug/mL) for 6-24 hours. The cells are then fixed, permeabilized, and stained with an anti-payload antibody. Colocalization with LysoTracker (a marker for acidic organelles) is performed to confirm that the ADC reaches the lysosomes, where cleavage occurs. The appearance of the payload in the cytosol and nucleus is a strong indicator of efficient linker cleavage. The effect on the cell cycle is assessed by staining with propidium iodide.
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| Animal Protocol |
Animal studies for this linker are part of ADC evaluation. A standard in vivo efficacy study uses 5-6-week-old female BALB/c nude mice implanted with antigen-positive human cancer cells. When tumors reach approximately 100-200 mm3, mice (n=8-10/group) are randomized and treated with a single intravenous dose of the ADC at 3, 10, and 30 mg/kg (n=8-10 per group). Tumor volume is measured twice weekly. For a PK-PD study, additional groups are euthanized at 6, 24, 48, 72, and 168 hours post-dose. The major organs are collected and homogenized for LC-MS/MS analysis to quantify the intact ADC and the released payload. An ex vivo imaging study can be performed using an ADC labeled with a near-infrared dye.
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| ADME/Pharmacokinetics |
The pharmacokinetic properties are not reported for the linker alone. For an ADC containing a Val-Cit linker, the antibody component drives the overall PK. The PK profile is typically biphasic: a rapid distribution phase (alpha phase) followed by a slower elimination phase (beta phase). The half-life (t1/2) of the ADC is often in the range of 1-2 weeks in humans and several days in mice. The Val-Cit linker is designed to be stable, and minimal payload release is observed in circulation. The payload, once released, is often rapidly cleared from the plasma. The major elimination pathway for the ADC is through proteolytic degradation and subsequent excretion of the payload metabolites.
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| Toxicity/Toxicokinetics |
No toxicity data is available for Fmoc-Gly3-Val-Cit-PAB alone. The toxicity profile of an ADC using this linker is largely determined by the conjugated payload. In preclinical studies, the MTD is determined in rodents. Dose-limiting toxicities (DLTs) often include bone marrow suppression (resulting in neutropenia and thrombocytopenia) and hepatotoxicity. The use of the Val-Cit linker is a strategy to mitigate systemic toxicity by ensuring that payload release is largely restricted to the acidic, protease-rich environment of the target cell's lysosomes, thus widening the therapeutic window compared to traditional chemotherapy.
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| References |
[1]. Beck A, et al. Strategies and challenges for the next generation of antibody-drug conjugates. Nat Rev Drug Discov. 2017 May;16(5):315-337.
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| Additional Infomation |
Fmoc-Gly3-Val-Cit-PAB is not a drug and is not approved for clinical use. It is an advanced, research-grade linker for the development of ADCs. Its mechanism involves being stable in the bloodstream but being efficiently cleaved by cathepsin B after ADC internalization into target cancer cells, leading to targeted payload release. The Gly3 spacer improves cleavage efficiency. It is a critical component of many modern ADCs in development. No clinical trials are registered for this linker alone. For research use only; not for human therapeutic or diagnostic applications.
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| Molecular Formula |
C39H48N8O9
|
|---|---|
| Molecular Weight |
772.85
|
| Exact Mass |
772.354
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| CAS # |
2647914-09-6
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| PubChem CID |
125373407
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| Appearance |
White to off-white solid powder
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| LogP |
1.3
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| Hydrogen Bond Donor Count |
9
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| Hydrogen Bond Acceptor Count |
9
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| Rotatable Bond Count |
20
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| Heavy Atom Count |
56
|
| Complexity |
1340
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| Defined Atom Stereocenter Count |
2
|
| SMILES |
CC(C)[C@@H](C(=O)N[C@@H](CCCNC(=O)N)C(=O)NC1=CC=C(C=C1)CO)NC(=O)CNC(=O)CNC(=O)CNC(=O)OCC2C3=CC=CC=C3C4=CC=CC=C24
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| InChi Key |
ANTAWRUQHZQAGZ-ZJJOJAIXSA-N
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| InChi Code |
InChI=1S/C39H48N8O9/c1-23(2)35(37(53)46-31(12-7-17-41-38(40)54)36(52)45-25-15-13-24(21-48)14-16-25)47-34(51)20-43-32(49)18-42-33(50)19-44-39(55)56-22-30-28-10-5-3-8-26(28)27-9-4-6-11-29(27)30/h3-6,8-11,13-16,23,30-31,35,48H,7,12,17-22H2,1-2H3,(H,42,50)(H,43,49)(H,44,55)(H,45,52)(H,46,53)(H,47,51)(H3,40,41,54)/t31-,35-/m0/s1
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| Chemical Name |
9H-fluoren-9-ylmethyl N-[2-[[2-[[2-[[(2S)-1-[[(2S)-5-(carbamoylamino)-1-[4-(hydroxymethyl)anilino]-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-2-oxoethyl]amino]-2-oxoethyl]amino]-2-oxoethyl]carbamate
|
| 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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture. |
| 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) |
DMSO: 100 mg/mL (129.39 mM)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: 2.5 mg/mL (3.23 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 25.0 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. Solubility in Formulation 2: 2.5 mg/mL (3.23 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 25.0 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. View More
Solubility in Formulation 3: ≥ 2.5 mg/mL (3.23 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.2939 mL | 6.4696 mL | 12.9391 mL | |
| 5 mM | 0.2588 mL | 1.2939 mL | 2.5878 mL | |
| 10 mM | 0.1294 mL | 0.6470 mL | 1.2939 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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