| Size | Price | Stock | Qty |
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| 5mg |
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| Other Sizes |
| Targets |
The molecular target for this linker is cathepsin B, a lysosomal cysteine protease that is overexpressed in many tumor cells. The Val-Cit dipeptide sequence is a specific substrate for cathepsin B, but is stable in the bloodstream. The maleimide group targets free cysteine residues on the antibody that are introduced through partial reduction of interchain disulfide bonds. Once conjugated, the entire ADC binds to a tumor-associated antigen (e.g., HER2, CD30, or Nectin-4) on the cell surface, leading to internalization and lysosomal degradation.
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| ln Vitro |
The linker itself has no biological activity. However, in the context of an ADC, the Val-Cit-PAB portion demonstrates efficient intracellular drug release. In vitro cytotoxicity assays using cancer cell lines show that ADCs constructed with a Val-Cit-PAB linker achieve potent killing with IC₅0 values in the low nanomolar to picomolar range (e.g., 0.1-10 nM). The (R,R) stereochemistry is critical for optimal cathepsin B recognition; the (S,S) isomer shows significantly slower cleavage kinetics (up to 10-fold lower rate). In plasma stability assays, the Val-Cit linker shows less than 5% drug release over 7 days at 37degC, confirming its extracellular stability.
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| ln Vivo |
The Val-Cit-PAB linker shows excellent in vivo stability and efficacy in ADC applications. In xenograft mouse models (e.g., mice bearing human tumor xenografts), ADCs using this linker produce complete tumor regression in many cases at doses of 1-10 mg/kg. The linker's stability in circulation minimizes premature drug release, which correlates with reduced off-target toxicity. In cynomolgus monkey toxicology studies, ADCs containing Val-Cit-PAB linkers demonstrate a manageable safety profile, with thrombocytopenia and neutropenia being dose-limiting toxicities, but typically at doses above the therapeutic window.
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| Enzyme Assay |
Non-cellular enzymatic assays for the Val-Cit-PAB linker use purified cathepsin B. A typical protocol: 1 microM of the ADC or linker-drug conjugate (e.g., Val-Cit-PAB-MMAE) is incubated with 20 nM recombinant human cathepsin B in 100 mM sodium acetate buffer (pH 5.5) containing 2 mM EDTA and 5 mM DTT at 37degC for 0-24 hours. Aliquots are taken at time points (0, 1, 2, 4, 8, 24 hours), and the reaction is quenched with 0.1% formic acid in acetonitrile. Released drug (e.g., MMAE) is quantified by LC-MS/MS. The rate of release (k_obs) is calculated. For the Val-Cit linker, t1/2 for cleavage is typically 2-6 hours.
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| Cell Assay |
Cellular assays to assess ADC activity use antigen-positive and antigen-negative cancer cell lines in parallel. A standard protocol: Cells (e.g., NCI-N87 for HER2-positive or MDA-MB-468 for EGFR-positive) are seeded in 96-well plates at 5,000 cells/well in growth medium. Serial dilutions of the ADC (0.001-100 nM) are added after 24 hours. Plates are incubated for 72-96 hours at 37degC. Cell viability is measured using CellTiter-Glo (luminescence) or MTT (absorbance). The IC₅0 is calculated from the dose-response curve. To confirm linker function, a control ADC with a non-cleavable linker is tested in parallel; it should show reduced activity (IC₅0 10-100× higher).
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| Animal Protocol |
In vivo efficacy studies for Val-Cit-PAB-containing ADCs typically use subcutaneous xenograft models in athymic nude mice. When tumor volumes reach 100-200 mm3, mice are randomized into treatment groups (n=8-10 per group). The ADC is administered intravenously at doses ranging from 0.3 to 10 mg/kg, typically as a single dose or weekly ×2-4 doses. Tumor volume is measured twice weekly by caliper (length × width2 / 2). The primary endpoint is tumor growth inhibition (TGI %). Secondary endpoints include complete response rate (CR, tumor volume <50 mm3) and body weight change as a toxicity indicator. Tumor tissues are collected for drug concentration and biomarker analysis.
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| ADME/Pharmacokinetics |
As a linker, the pharmacokinetics of (R,R)-MC-Val-Cit-PAB are not studied independently; PK is always evaluated for the complete ADC. Typical ADC PK in humans and animals shows a biphasic profile: a distribution phase with a half-life of hours to days, followed by a terminal elimination phase with a half-life of 1-3 weeks. The Val-Cit linker contributes to ADC stability in circulation; free drug released from the linker is generally less than 0.5% of total ADC at 21 days. In patients, approved ADCs using Val-Cit linkers have clearance values of 10-30 mL/day/kg and volumes of distribution of 50-150 mL/kg.
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| Toxicity/Toxicokinetics |
The toxicology of the linker is only relevant in the context of the complete ADC. In non-clinical safety studies, the primary toxicities are on-target off-tumor effects (depending on the target antigen expression) and off-target effects mediated by the payload. For payloads like MMAE (monomethyl auristatin E), the major toxicities are bone marrow suppression (neutropenia, thrombocytopenia) and peripheral neuropathy. The Val-Cit linker itself does not contribute to toxicity beyond releasing the payload. In animal studies with non-binding control ADCs (targeting an irrelevant antigen), the linker-payload conjugate shows manageable toxicity at doses up to 10 mg/kg.
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| Additional Infomation |
(R,R)-MC-Val-Cit-PAB is a linker technology building block, not a drug itself. It is used in the production of several FDA-approved ADCs, including brentuximab vedotin (Adcetris, targeting CD30, approved 2011) and enfortumab vedotin (Padcev, targeting Nectin-4, approved 2019). The linker is also found in many investigational ADCs. The Val-Cit sequence was developed by Seattle Genetics and is now widely used in the industry. The (R,R) stereochemistry is essential: only the L-valine and L-citrulline configuration is recognized by cathepsin B; D-amino acid isomers are poor substrates and should be avoided.
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| Molecular Formula |
C28H40N6O7
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|---|---|
| Molecular Weight |
572.65
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| Related CAS # |
MC-Val-Cit-PAB;159857-80-4
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| Appearance |
White to off-white solid powder
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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: 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 (~174.63 mM)
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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 | 1.7463 mL | 8.7313 mL | 17.4627 mL | |
| 5 mM | 0.3493 mL | 1.7463 mL | 3.4925 mL | |
| 10 mM | 0.1746 mL | 0.8731 mL | 1.7463 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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