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| 100mg |
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| Targets |
This is an ADC linker; the maleimide group reacts with antibody thiols, and the Val-Cit-PABC is cleaved by cathepsin B in the lysosome, releasing the payload.
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| ln Vitro |
ADC cytotoxins are connected to antibodies through an ADC connector to form ADCs [1].
Mal-PEG1-Val-Cit-PABC-OH is a maleimide-based cleavable ADC linker with a PEG spacer and Val-Cit-PABC for targeted drug release. The maleimide group facilitates thiol conjugation in ADC synthesis for cancer therapy. The Val-Cit-PABC segment is a cathepsin B-sensitive dipeptide linker commonly used in ADCs, where enzymatic cleavage triggers self-immolative release of the attached payload. The PEG1 spacer reduces aggregation and improves solubility. The linker is designed to be stable in the bloodstream (neutral pH) but cleaved inside the lysosomes of target cells (acidic pH and presence of cathepsin B). Upon internalization of the ADC into the target cancer cell and trafficking to the lysosome, the Val-Cit dipeptide is selectively cleaved by cathepsin B, a protease that is overexpressed in many cancer cells. Cleavage of the amide bond between the valine and citrulline residues initiates the self-immolative elimination of the PABC spacer, releasing the free cytotoxic payload (e.g., MMAE, MMAF) into the cytoplasm. The released payload then exerts its cytotoxic effect (typically by binding to tubulin, inhibiting microtubule polymerization, causing G2/M arrest and apoptosis). The maleimide group is reactive with free thiols on the antibody, which are typically generated by mild reduction of interchain disulfide bonds. The linker has no inherent biological activity but serves as the bridge between the targeting antibody and the potent payload. |
| ln Vivo |
In vivo, ADCs built with the Val-Cit-PABC linker (including this PEG1 variant) have demonstrated potent anti-tumor activity in various xenograft models. The Val-Cit-PABC linker is the same linker used in the clinically approved ADC brentuximab vedotin (Adcetris®). The PEG1 spacer is shorter than the PEG8 spacer used in some commercial linkers, which may affect solubility and aggregation but is still effective. The PEG1 spacer provides some flexibility and reduces steric hindrance during conjugation. The in vivo efficacy of an ADC built with this linker depends on the specificity of the antibody, the potency of the payload (typically MMAE or MMAF), and the stability of the linker in circulation. The linker is stable in human plasma (t1/2 > 96 hours) but is efficiently cleaved in the lysosome (t1/2 of 1-4 hours). This design minimizes premature payload release in the bloodstream, reducing off-target toxicity. ADCs using this linker have shown objective responses in clinical trials for CD30-positive lymphomas (brentuximab vedotin) and other cancers.
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| Enzyme Assay |
The cleavability of the Val-Cit-PABC linker by cathepsin B is assessed in a biochemical assay. The linker (or a model conjugate with a fluorophore attached via the PABC spacer) is incubated with recombinant human cathepsin B in a lysosomal-like buffer (e.g., 50 mM sodium acetate, pH 5.0, 1 mM DTT, 1 mM EDTA) at 37degC. Samples are taken at various time points (0, 0.5, 1, 2, 4, 8, 24 hours), and the release of the fluorophore is monitored by HPLC or by measuring the increase in fluorescence (if the fluorophore is quenched when attached). The half-life of the linker (time for 50% of the payload to be released) is calculated. A control reaction without cathepsin B or with the cathepsin B inhibitor CA-074 is run to confirm specificity. The plasma stability of the linker (or the complete ADC) is assessed by spiking the compound into fresh human plasma at 37degC and measuring the amount of intact linker-payload (or ADC) and free payload over time by LC-MS. The desired profile is high stability in plasma (t1/2 > 96 hours) and rapid cleavage in the presence of cathepsin B (t1/2 < 4 hours). The maleimide-thiol conjugation efficiency is assessed by reacting the linker with a model thiol (e.g., cysteine or glutathione) in PBS at pH 6.5-7.5 at room temperature, monitoring the reaction by HPLC or LC-MS. The maleimide group reacts specifically with thiols to form a stable thioether bond.
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| Cell Assay |
As a linker, Mal-PEG1-Val-Cit-PABC-OH is not used directly in cell-based assays. The complete ADC (antibody conjugated to a cytotoxic payload via this linker) is tested. A typical cell-based assay for an ADC built with this linker involves testing the ADC on antigen-positive and antigen-negative tumor cells. Target cells (e.g., CD30-positive Karpas 299 or L540 cells for brentuximab vedotin) are seeded in 96-well plates at 5×103 cells per well in RPMI-1640 medium containing 10% FBS. After overnight attachment, the ADC is added at varying concentrations (typically 0.0001-100 nM based on antibody concentration) and incubated for 72-120 hours. Cell viability is measured by CellTiter-Glo or MTT assays. The IC50 for antigen-positive cells is calculated. Antigen-negative cells (e.g., CD30-negative Raji cells) serve as a control for target specificity; they should have minimal cytotoxicity (IC50 > 100 nM). To confirm the mechanism of action, the assay can be performed in the presence of a cathepsin B inhibitor (e.g., CA-074, 10 microM) or an endocytosis inhibitor (e.g., dynasore, 100 microM), which should block the ADC's cytotoxicity. The free payload (e.g., MMAE) is used as a positive control for cell killing; it should be highly potent (IC50 in the sub-nanomolar to low nanomolar range) regardless of antigen expression. The linker itself should have no cytotoxicity (CC50 > 100 microM).
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| Animal Protocol |
The ADC built with Mal-PEG1-Val-Cit-PABC-OH is administered to animals. A typical in vivo protocol for an ADC using this linker (e.g., brentuximab vedotin) involves a murine xenograft model. Female SCID or athymic nude mice (6-8 weeks old, 18-22 g) are injected subcutaneously with 5-10×10⁶ antigen-positive tumor cells (e.g., CD30-positive L540 or Karpas 299 cells) in 100 microL of PBS mixed 1:1 with Matrigel. When tumors reach a volume of 100-200 mm3 (approximately 7-14 days), mice are randomized into treatment groups (n=8-10). The ADC is formulated in PBS or 0.9% saline and administered intravenously (i.v.) via the tail vein at doses of 1-10 mg/kg (based on antibody content). Control groups receive vehicle alone, non-targeting ADC (isotype control), unconjugated antibody, or free payload. Treatment is typically administered once weekly (QW) or once every 3 weeks (Q3W) for 2-4 cycles. Tumor volumes are measured with a caliper every 2-3 days, and body weights are recorded. At the end of the study (when control tumors reach ~2000 mm3), mice are euthanized, and tumors are excised and weighed. Tumors are processed for histology (H&E staining, Ki-67 IHC for proliferation, cleaved caspase-3 IHC for apoptosis) and for analysis of payload concentrations by LC-MS. Blood is collected for PK analysis.
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| ADME/Pharmacokinetics |
The pharmacokinetics of the ADC are studied, not the linker alone. For an ADC with a Val-Cit-PABC linker, the PK is characterized by the antibody portion. The linker is stable in circulation, with the maleimide-thiol bond (thioether) being irreversible under physiological conditions, and the Val-Cit dipeptide resistant to plasma proteases (plasma stability t1/2 > 48-96 hours). Therefore, the conjugated antibody (ADC) has a PK profile similar to the unconjugated antibody. The terminal elimination half-life for an IgG1-based ADC is typically 4-7 days in mice and 10-21 days in humans. The volume of distribution (Vd) is low, similar to plasma volume. Clearance (CL) is low, primarily via catabolism of the antibody. The PEG1 spacer has minimal effect on PK due to its short length. The released payload (e.g., MMAE) has a short half-life (hours) and is rapidly cleared.
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| Toxicity/Toxicokinetics |
The toxicity profile is determined for the complete ADC, not the linker alone. ADCs built with the Val-Cit-PABC-MMAE linker (e.g., brentuximab vedotin) have a well-characterized safety profile. The dose-limiting toxicities are neutropenia (low white blood cell count) and peripheral neuropathy (due to the tubulin inhibitor payload). These toxicities are reversible upon dose reduction or treatment interruption. Other common adverse events include fatigue, nausea, diarrhea, and rash. The linker itself contributes to the safety profile by being stable in circulation, reducing off-target toxicity. The PEG1 spacer, being short, has minimal impact on safety. For laboratory handling, Mal-PEG1-Val-Cit-PABC-OH should be handled with standard chemical safety precautions: use gloves, lab coat, eye protection. The maleimide group is reactive with thiols and can cause skin sensitization. Store the compound at -20degC in a dry, dark environment under inert atmosphere (argon or nitrogen) to prevent hydrolysis and oxidation. Solutions should be prepared fresh and used immediately.
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| References | |
| Additional Infomation |
Mal-PEG1-Val-Cit-PABC-OH is a cleavable, one-unit polyethylene glycol (PEG) ADC linker that contains a maleimide for cysteine conjugation and a Val-Cit-PABC protease-cleavable spacer. The Val-Cit-PABC segment is a cathepsin B-sensitive dipeptide linker used in many ADCs. This linker is designed for conjugation to free thiols on the antibody (interchain disulfides reduced to generate cysteine residues) and to a cytotoxic payload (typically a tubulin inhibitor like MMAE or MMAF). The hydroxyl group (-OH) is typically activated to a carbonate or other leaving group for conjugation to the payload. The PEG1 spacer is minimal but still provides some flexibility; longer PEG spacers (PEG4, PEG8, PEG12) are also available. This product is for research use only and is not for therapeutic use. It is supplied as a solid and should be stored at -20degC, protected from light and moisture.
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| Molecular Formula |
C27H38N6O8
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| Molecular Weight |
574.626026630402
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| CAS # |
2055041-37-5
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| Appearance |
White to off-white solid powder
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| SMILES |
C([C@@H](N(C1C=CC(CO)=CC=1)C([C@@H](NC(CCOCCN1C(=O)C=CC1=O)=O)C(C)C)=O)CCCNC(=O)N)(=O)N
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| Synonyms |
MalPEG1ValCitPABCOH; Mal PEG1 Val Cit PABC OH
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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.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.7403 mL | 8.7013 mL | 17.4025 mL | |
| 5 mM | 0.3481 mL | 1.7403 mL | 3.4805 mL | |
| 10 mM | 0.1740 mL | 0.8701 mL | 1.7403 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
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