| Size | Price | Stock | Qty |
|---|---|---|---|
| 5mg |
|
||
| 10mg |
|
||
| Other Sizes |
| Targets |
The molecular target of the conjugate is tubulin, via the MMAE payload. Upon internalization of the ADC into target cells and delivery to the lysosome, the Val‑Cit dipeptide is cleaved by cathepsin B, a protease that is upregulated in many cancer cells. The PAB spacer undergoes spontaneous elimination, releasing free MMAE. MMAE then binds to tubulin at the vinca binding site, inhibiting microtubule polymerization and causing G2/M cell cycle arrest and apoptosis. The Boc group protects the N‑terminus during chemical synthesis; it is removed before conjugation. The overall target is defined by the antibody portion of the ADC; this linker‑payload confers anti‑tumor activity on the ADC.
|
|---|---|
| ln Vitro |
In vitro, the activity of Boc‑Val‑Cit‑PAB‑MMAE is assessed after conjugation to a targeting antibody. The free linker‑payload is not used in cell assays because it cannot enter cells efficiently. However, the release mechanism can be validated in a cell‑free cathepsin B cleavage assay. In a standard cathepsin B assay, Boc‑Val‑Cit‑PAB‑MMAE (10 uM) is incubated with recombinant human cathepsin B (10 nM) at pH 5.0 for 24 h, and the release of MMAE is monitored by LC‑MS. The Val‑Cit dipeptide is efficiently cleaved, with >90% MMAE released by 24 h. In the absence of cathepsin B, the conjugate is stable. This confirms the linker is cleavable and functional.
|
| ln Vivo |
In vivo, a complete ADC constructed with the Val‑Cit‑PAB‑MMAE linker‑payload (de‑protected, conjugated to an antibody) exhibits potent anti‑tumor activity in xenograft models. Typically, an ADC with this linker (e.g., ADC targeting CD30, HER2, or other antigens) at 1‑3 mg/kg, i.v., single dose, can induce complete tumor regressions. The Val‑Cit linker is stable in circulation (plasma stability >48 h) but cleaved in lysosomes, ensuring targeted delivery of MMAE. Boc‑Val‑Cit‑PAB‑MMAE is the protected building block used to prepare the functional ADC.
|
| Enzyme Assay |
General in vitro cathepsin B cleavage assay: Prepare Boc‑Val‑Cit‑PAB‑MMAE (10 uM) in 50 mM sodium acetate buffer (pH 5.0) containing 2 mM DTT and 0.5 mM EDTA. Add recombinant human cathepsin B (10 nM). Incubate at 37degC. At time points 0, 1, 4, 8, 24 h, remove 50 uL aliquots and quench with 100 uL of cold acetonitrile. Centrifuge at 14,000 rpm for 10 min. Analyze the supernatant by LC‑MS to monitor the disappearance of the parent compound and the appearance of free MMAE. The expected result: >90% release by 24 h. For control, incubate without cathepsin B; parent compound remains stable. For plasma stability, incubate the conjugate (10 uM) in mouse or human plasma at 37degC for up to 72 h and analyze by LC‑MS; the linker‑payload should be stable (t½ >48 h).
|
| Cell Assay |
General in vitro cytotoxicity assay (for the final ADC): After conjugating the de‑protected linker‑payload to an antibody, test the ADC on antigen‑positive cells. Seed cells in 96‑well plates at 5×103 cells/well. Treat with ADC (0.0001‑10 ug/mL) for 96 h. Add CellTiter‑Glo reagent and measure luminescence. The ADC should have an IC₅0 in the low ng/mL range (e.g., 0.01‑0.1 ug/mL). For the free Boc‑Val‑Cit‑PAB‑MMAE alone, treat cells with the same concentrations (0.0001‑10 uM) as a control; it should have little or no activity (IC₅0 >1 uM) because it cannot enter cells without an antibody.
|
| Animal Protocol |
General in vivo protocol for ADC efficacy: Female NCr nu/nu mice (n=8 per group) are inoculated subcutaneously with antigen‑positive tumor cells. When tumors reach 150‑200 mm3, administer the ADC (constructed using the de‑protected Val‑Cit‑PAB‑MMAE linker) intravenously (i.v.) at 1, 3, 6 mg/kg, single dose. Control groups receive vehicle (PBS) and a non‑targeting isotype control ADC (same linker‑payload). Measure tumor volume twice weekly for 28 days. The ADC should cause dose‑dependent tumor growth inhibition (TGI) and regressions at higher doses. The conjugate Boc‑Val‑Cit‑PAB‑MMAE is the building block; it is not administered alone.
|
| ADME/Pharmacokinetics |
Boc‑Val‑Cit‑PAB‑MMAE is a large, lipophilic molecule that is not intended for in vivo administration as a free compound. It is a synthetic intermediate. Its ADME properties are not characterized; rather, the final ADC has the PK profile of an antibody (long half‑life, days). The Boc protecting group is removed prior to conjugation. The Val‑Cit‑PAB‑MMAE portion is stable in plasma but cleaved in lysosomes. For research use, the compound is stored as a solid powder at ‑20degC, soluble in DMSO.
|
| Toxicity/Toxicokinetics |
The compound is not intended for direct in vivo administration, so its toxicity profile is not a primary consideration. When conjugated to an antibody, the ADC may cause on‑target and off‑target toxicity, including neutropenia (due to MMAE). The free building block is not genotoxic. For use as a reference standard, standard laboratory safety precautions are sufficient.
|
| Additional Infomation |
Background: The Val‑Cit‑PAB linker is one of the most widely used cleavable linkers in FDA‑approved ADCs (e.g., Adcetris® [brentuximab vedotin] and Polivy® [polatuzumab vedotin]). The Boc protecting group is used during chemical synthesis to protect the N‑terminus of the Val residue. Boc removal (with TFA) yields the free amine, which can be conjugated to an antibody or other carrier. This linker‑payload is a fundamental building block for the production of many ADCs. It is stored at ‑20degC and is for research use only.
|
| Molecular Formula |
C63H102N10O14
|
|---|---|
| Molecular Weight |
1223.54
|
| Exact Mass |
1222.758
|
| CAS # |
2226611-21-6
|
| PubChem CID |
135219644
|
| Appearance |
White to off-white solid powder
|
| Hydrogen Bond Donor Count |
8
|
| Rotatable Bond Count |
35
|
| Heavy Atom Count |
87
|
| Complexity |
2220
|
| Defined Atom Stereocenter Count |
12
|
| SMILES |
CC[C@H](C)[C@@H]([C@@H](CC(=O)N1CCC[C@H]1[C@@H]([C@@H](C)C(=O)N[C@H](C)[C@H](C2=CC=CC=C2)O)OC)OC)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)N(C)C(=O)OCC3=CC=C(C=C3)NC(=O)[C@H](CCCNC(=O)N)NC(=O)[C@H](C(C)C)NC(=O)OC(C)(C)C
|
| InChi Key |
KRHSITWMWRCCDT-VGVQYDFXSA-N
|
| InChi Code |
InChI=1S/C63H102N10O14/c1-18-39(8)52(47(84-16)34-48(74)73-33-23-27-46(73)54(85-17)40(9)55(76)66-41(10)53(75)43-24-20-19-21-25-43)71(14)59(80)50(37(4)5)69-58(79)51(38(6)7)72(15)62(83)86-35-42-28-30-44(31-29-42)67-56(77)45(26-22-32-65-60(64)81)68-57(78)49(36(2)3)70-61(82)87-63(11,12)13/h19-21,24-25,28-31,36-41,45-47,49-54,75H,18,22-23,26-27,32-35H2,1-17H3,(H,66,76)(H,67,77)(H,68,78)(H,69,79)(H,70,82)(H3,64,65,81)/t39-,40+,41+,45-,46-,47+,49-,50-,51-,52-,53+,54+/m0/s1
|
| Chemical Name |
[4-[[(2S)-5-(carbamoylamino)-2-[[(2S)-3-methyl-2-[(2-methylpropan-2-yl)oxycarbonylamino]butanoyl]amino]pentanoyl]amino]phenyl]methyl N-[(2S)-1-[[(2S)-1-[[(3R,4S,5S)-1-[(2S)-2-[(1R,2R)-3-[[(1S,2R)-1-hydroxy-1-phenylpropan-2-yl]amino]-1-methoxy-2-methyl-3-oxopropyl]pyrrolidin-1-yl]-3-methoxy-5-methyl-1-oxoheptan-4-yl]-methylamino]-3-methyl-1-oxobutan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]-N-methylcarbamate
|
| HS Tariff Code |
2934.99.9001
|
| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). 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)
|
| Solubility (In Vitro) |
DMSO : ~200 mg/mL (~163.46 mM; with sonication)
|
|---|---|
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 5 mg/mL (4.09 mM)(saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween-80 + 45% Saline (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 50.0 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix thoroughly. Then add 50 μL of Tween-80 to the above system and mix thoroughly. Finally, add 450 μL of physiological saline to bring the volume to 1 mL. Preparation of physiological saline: Dissolve 0.9 g of sodium chloride in ddH₂O and bring the volume to 100 mL to obtain a clear and transparent physiological saline solution. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 0.8173 mL | 4.0865 mL | 8.1730 mL | |
| 5 mM | 0.1635 mL | 0.8173 mL | 1.6346 mL | |
| 10 mM | 0.0817 mL | 0.4087 mL | 0.8173 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.
Products are for research use only; We do not sell to patients
Copyright 2020 InvivoChem LLC | All Rights Reserved
