| Targets |
The Ac-EEVC-OH linker does not have a biological target itself, but it is engineered to be cleaved by cathepsin B, a lysosomal cysteine protease overexpressed in many cancer cells. The linker contains a valine-citrulline (Val-Cit) dipeptide, which is the primary recognition site for cathepsin B. Upon internalization of the ADC into the target cell, the linker is proteolytically cleaved, releasing the attached cytotoxic payload (warhead). The acetylated N-terminus and the free carboxylic acid group provide handles for conjugation to both the antibody and the payload, respectively.
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|---|---|
| ln Vitro |
Ac-EEVC-OH is a chemical building block and is not directly evaluated for biological activity. It is used in the synthesis of ADCs. The specific recognition and cleavage of the Val-Cit motif by cathepsin B can be demonstrated in vitro using a cell-free cleavage assay. This ensures that the linker is stable in circulation and only releases the drug once it has entered the target cell, providing the targeted therapy effect.
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| ln Vivo |
ADCs synthesized using Ac-EEVC-OH can have potent in vivo activity. The linker is designed for high stability in circulation, minimizing premature payload release and off-target toxicity. After the ADC binds to the tumor antigen and is internalized, the linker is efficiently cleaved in the lysosome, leading to the specific release of the cytotoxic payload in the tumor microenvironment. This targeted delivery results in significant anti-tumor efficacy in xenograft models.
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| Enzyme Assay |
The enzymatic cleavage of the Ac-EEVC-OH linker can be assessed in a cell-free cathepsin B cleavage assay. The ADC (or a surrogate construct containing the linker and a fluorophore) is incubated with purified cathepsin B in cleavage buffer (50 mM sodium acetate, pH 5.0, containing 2 mM DTT) at 37degC for 1-4 hours. The release of the payload or the increase in fluorescence is measured over time to determine the cleavage kinetics. The stability of the linker in human plasma is also assessed by incubating the ADC in plasma at 37degC for up to 7 days and measuring the released payload by LC-MS/MS.
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| Cell Assay |
The specificity of the linker is evaluated by incubating the ADC with antigen-positive and antigen-negative cells. Only antigen-positive cells should internalize the ADC and release the payload, leading to cell death. The cytotoxicity of the ADC is measured using an MTT or CellTiter-Glo assay, and the selectivity index (IC50 on positive cells / IC50 on negative cells) is calculated. The mechanism of cell death (apoptosis) is confirmed by Annexin V/PI staining and caspase-3/7 activity assays.
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| Animal Protocol |
For in vivo efficacy, a xenograft mouse model is used. Female BALB/c nude mice (6-8 wk, n=8-10/group) bearing subcutaneous tumor xenografts are administered the ADC (1-10 mg/kg) intravenously once weekly for 2-3 weeks. Tumor volume is measured every 2-3 days with calipers. At the endpoint, tumors are excised and analyzed for payload concentration by LC-MS/MS to confirm tumor-specific drug release. Plasma samples are also analyzed to assess the stability of the linker in circulation.
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| ADME/Pharmacokinetics |
The Ac-EEVC-OH linker is a hydrophilic peptide designed to improve the solubility of the ADC and reduce aggregation. The PK of an ADC is largely dictated by the antibody. The linker is designed to be stable in circulation (with a half-life of days). The released payload's PK depends on its chemical properties. The glutathione (GSH) stability is not the primary mechanism for this non-reducible linker. For research use, the compound is stored as a powder at -20degC.
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| Toxicity/Toxicokinetics |
No specific toxicity data is available for Ac-EEVC-OH itself. The toxicity profile of an ADC is determined by the antibody, the payload (warhead), and the stability of the linker. A stable linker reduces systemic toxicity by preventing premature payload release. For research use, standard safety precautions for handling peptides should be followed: use PPE (gloves, lab coat, safety goggles), work in a fume hood.
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| References | |
| Additional Infomation |
Ac-EEVC-OH (CAS# 2921734-44-1) is a research-grade peptide ADC linker containing a cathepsin-cleavable Val-Cit motif. It is used to synthesize stable, site-specific antibody-drug conjugates (ADCs) for targeted cancer therapy. It is not an FDA-approved drug. Storage: Powder at -20degC for 3 years, 4degC for 2 years; In solvent at -80degC for 6 months, -20degC for 1 month.
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| Exact Mass |
686.385
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|---|---|
| CAS # |
2921734-44-1
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| PubChem CID |
172677434
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| Appearance |
Solid powder
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| Hydrogen Bond Donor Count |
7
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| Rotatable Bond Count |
23
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| Heavy Atom Count |
48
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| Complexity |
1160
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| Defined Atom Stereocenter Count |
4
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| InChi Key |
XYXTYHIOGGKKTC-CIEVZJJWSA-N
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| InChi Code |
InChI=1S/C31H54N6O11/c1-17(2)24(27(43)36-21(28(44)45)11-10-16-33-29(32)46)37-26(42)20(13-15-23(40)48-31(7,8)9)35-25(41)19(34-18(3)38)12-14-22(39)47-30(4,5)6/h17,19-21,24H,10-16H2,1-9H3,(H,34,38)(H,35,41)(H,36,43)(H,37,42)(H,44,45)(H3,32,33,46)/t19-,20-,21-,24-/m0/s1
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| Chemical Name |
(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-acetamido-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoyl]amino]-5-[(2-methylpropan-2-yl)oxy]-5-oxopentanoyl]amino]-3-methylbutanoyl]amino]-5-(carbamoylamino)pentanoic acid
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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 Note: Please store this product in a sealed and protected environment, 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) |
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.) |
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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