| Size | Price | Stock | Qty |
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| 1mg |
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| 5mg |
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| 10mg | |||
| Other Sizes |
| Targets |
Cathepsin B (for linker cleavage); MMAE binds to tubulin, inhibiting microtubule polymerization.
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| ln Vitro |
ADC cytotoxins are connected to antibodies through an ADC connector to form ADCs [1].
Mal-PEG8-Val-Cit-PAB-MMAE is a cleavable 8-unit PEG ADC linker conjugate that incorporates the potent tubulin inhibitor monomethyl auristatin E (MMAE). The Val-Cit dipeptide is a well-established protease-cleavable sequence that is selectively recognized and cleaved by lysosomal proteases, particularly cathepsin B, which is upregulated in many cancer cells. The PAB (p-aminobenzyl alcohol) spacer is a self-immolative group that, upon cleavage of the peptide bond, spontaneously rearranges to release the free MMAE payload. The maleimide group enables site-specific conjugation to reduced cysteine residues (e.g., in engineered cysteine residues or interchain disulfide bonds) on the antibody. The long, hydrophilic PEG8 spacer minimizes aggregation of the ADC, a common problem with highly potent and hydrophobic payloads like MMAE, and also improves the solubility and pharmacokinetic properties of the conjugate. MMAE is a synthetic antimitotic agent that is a derivative of the marine natural product dolastatin 10. It acts as a potent inhibitor of tubulin polymerization, blocking cell division and leading to G2/M phase arrest and apoptosis. The IC50 of MMAE against various cancer cell lines is in the low picomolar to nanomolar range. The complete agent-linker conjugate has no intrinsic activity until it is released from the antibody inside the target cell. Upon cleavage and release, free MMAE can also diffuse out of the cell and kill neighboring tumor cells (bystander effect). |
| ln Vivo |
The in vivo anti-tumor activity of an ADC incorporating Mal-PEG8-Val-Cit-PAB-MMAE is determined by the specificity of the antibody and the potency of the MMAE payload. ADCs built with this linker-payload have shown remarkable efficacy in preclinical xenograft models of various cancers, including breast cancer (HER2-positive, T-DM1 resistant), non-Hodgkin's lymphoma (CD30-positive), and multiple myeloma. For example, an anti-CD30 antibody conjugated to this linker-MMAE (brentuximab vedotin, Adcetris®) is clinically approved. The PEG8 spacer contributes to an improved therapeutic window compared to shorter, less hydrophilic linkers by reducing aggregation and non-specific uptake. The Val-Cit-PAB linker is highly stable in the circulation (half-life of days) but releases MMAE efficiently inside the lysosome of target cells (half-life of 1-4 hours). The in vivo data for the linker-payload itself is not generated separately; it is always generated as part of a specific ADC. The linker-payload is a reagent used to make the ADC.
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| Enzyme Assay |
The activity of Mal-PEG8-Val-Cit-PAB-MMAE is not measured in a standard in vitro biochemical or receptor binding assay because the entire conjugate is designed to be inactive until it is cleaved. However, the individual components can be assessed. The ability of the linker to be cleaved by cathepsin B is assessed in a biochemical assay. In this assay, the Mal-PEG8-Val-Cit-PAB-MMAE compound (or a surrogate model compound lacking the maleimide group) is incubated with recombinant cathepsin B in a lysosomal-like buffer (e.g., 50 mM sodium acetate, pH 5.0, 1 mM DTT, 1 mM EDTA) at 37degC. The release of free MMAE is then quantified over time by LC-MS. The half-life of the linker (time for 50% of MMAE to be released) is calculated. As a control, the reaction is also performed in the presence of the cathepsin B inhibitor CA-074, which should block cleavage. The stability of the linker in human plasma is also assessed. Mal-PEG8-Val-Cit-PAB-MMAE is spiked into fresh human plasma and incubated at 37degC. Aliquots are removed at various time points (e.g., 0, 1, 6, 24, 48, 96 hours). The amount of intact linker-MMAE, intermediate cleavage products, and free MMAE is quantified by LC-MS. The half-life in plasma is a measure of the linker's stability. Ideally, it should be long (>96 hours) to prevent premature release in the circulation. The binding of MMAE to tubulin can be measured in a separate assay. Purified tubulin is incubated with [3H]-MMAE in the presence or absence of excess unlabeled MMAE. The bound radioactivity is separated from free by filtration or by spin column, and the Kd for tubulin binding is determined.
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| Cell Assay |
The activity of an ADC built with Mal-PEG8-Val-Cit-PAB-MMAE is assessed in cell-based cytotoxicity assays. For this assay, the maleimide group on the linker-payload is used to conjugate it to an antibody that targets a specific tumor-associated antigen. The resulting ADC is then added to antigen-positive cancer cells (e.g., HER2-positive SK-BR-3 breast cancer cells) and antigen-negative cancer cells (e.g., HER2-negative MDA-MB-231 breast cancer cells). The ADC is tested at various concentrations (typically 0.001-100 nM based on the antibody concentration) and incubated with the cells for 96-120 hours. Cell viability is then measured using an MTT or CellTiter-Glo assay. The IC50 (the concentration of ADC required to kill 50% of the cells) is calculated. The specificity index is the ratio of the IC50 on antigen-negative cells to the IC50 on antigen-positive cells (a higher number indicates better specificity). To confirm the mechanism of action, the assay can be performed in the presence of a cathepsin B inhibitor (e.g., CA-074), which should block the ADC's cytotoxicity. The "bystander effect" is assessed in a co-culture assay. Antigen-positive and antigen-negative cells are mixed at a defined ratio and treated with the ADC. After 96 hours, the percentage of antigen-negative cells that are dead is measured by flow cytometry. If the ADC induces killing of antigen-negative cells, it is said to have a bystander effect, which is mediated by the diffusion of free MMAE from the antigen-positive cells.
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| Animal Protocol |
Mal-PEG8-Val-Cit-PAB-MMAE is not administered to animals as a standalone entity. It is first conjugated to an antibody to form an ADC, and then the ADC is administered to animals. A typical in vivo protocol for an ADC involves its administration to immunocompromised mice bearing subcutaneous xenografts of human cancer cells (e.g., NCI-N87 for HER2-targeted ADC). When the tumors reach a volume of approximately 150-200 mm3, the ADC is administered intravenously (i.v.) via the tail vein at doses of 1-10 mg/kg (based on the antibody content). A control group receives the vehicle (PBS) or a non-targeting ADC. Tumor volumes are measured with a caliper every 2-3 days. The animals are typically treated once weekly (QW) for 2-4 weeks. At the end of the study (typically after 4 weeks), the mice are euthanized, and the tumors are excised and weighed. Blood samples may be collected during the study for pharmacokinetic analysis. The linker-payload used in the ADC contributes to its efficacy, stability, and tolerability. The PEG8 spacer is known to improve the PK and reduce aggregation. The Val-Cit-PAB-MMAE linker-payload is a proven design that has been successful in several approved ADCs.
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| ADME/Pharmacokinetics |
The linker-payload Mal-PEG8-Val-Cit-PAB-MMAE itself is not typically used for pharmacokinetic analysis; rather, the PK of the complete ADC is measured. The ADC's PK is characterized by the antibody portion, with the linker-payload contributing to the overall exposure of the free payload. The PK of an ADC is typically biphasic. The elimination half-life of the ADC (conjugated antibody) can be 2-7 days, depending on the antibody. The linker-payload's stability is critical: premature deconjugation of the linker-payload from the antibody will lead to faster clearance of the payload. The concentration of total antibody (ADC + naked antibody), conjugated antibody (ADC only), and total MMAE (conjugated + unconjugated) are measured in plasma samples by ELISA and LC-MS. The half-life of MMAE release from the ADC is also calculated. The maleimide-thiol linkage is known to undergo a retro-Michael reaction over time, leading to deconjugation. The PEG8 spacer has been shown to reduce this deconjugation compared to shorter PEG linkers.
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| Toxicity/Toxicokinetics |
The toxicity of Mal-PEG8-Val-Cit-PAB-MMAE is assessed as part of the complete ADC. ADCs that use the Val-Cit-PAB-MMAE linker-payload have a well-characterized toxicity profile. The dose-limiting toxicities (DLTs) for this class of ADCs are typically neutropenia (low white blood cell count), peripheral neuropathy (due to the tubulin inhibitor payload), and sometimes liver toxicity (elevated liver enzymes). These toxicities are related to the on-target effects of MMAE on proliferating cells (bone marrow) and neurons. The PEG8 linker is non-toxic. The maleimide group can react with serum albumin, leading to off-target distribution of the payload and potential toxicity. However, newer conjugation strategies are being developed to avoid this. The Val-Cit linker is stable in the circulation, reducing premature release of MMAE. However, some release does occur, contributing to neutropenia. The overall safety of this linker-payload is considered acceptable, as evidenced by the approval of brentuximab vedotin (Adcetris®), which uses the Val-Cit-PAB-MMAE linker-payload.
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| References | |
| Additional Infomation |
Mal-PEG8-Val-Cit-PAB-MMAE is a commercially available, high-quality reagent used for the development of ADCs. ADCs represent a rapidly growing class of targeted cancer therapeutics that combine the specificity of monoclonal antibodies with the cytotoxic potency of small-molecule drugs. The Val-Cit-PAB linker is known as a "cleavable" linker, as it is designed to be stable in the bloodstream (pH 7.4) but rapidly cleaved by lysosomal proteases (cathepsin B) in the low pH (pH 4.5-5.0) environment of the lysosome. The PEG8 spacer is one of several lengths (e.g., PEG4, PEG6, PEG12) available; the optimal length is determined empirically for each ADC. This specific linker-payload is sometimes referred to as "vcMMAE." It is the same linker-payload used in the approved drugs brentuximab vedotin (Adcetris®) for CD30-positive lymphomas and polatuzumab vedotin (Polivy®) for CD79b-positive diffuse large B-cell lymphoma (DLBCL). The maleimide group (Mal) enables the conjugation to cysteine residues on the antibody. These can be native cysteines from reduced interchain disulfide bonds or engineered cysteines (THIOMAB technology). The product is supplied as a powder and is typically stored at -20degC.
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| Molecular Formula |
C81H131N11O23
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|---|---|
| Molecular Weight |
1626.97
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| Exact Mass |
1625.941
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| CAS # |
2353409-69-3
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| PubChem CID |
168012262
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| Appearance |
White to off-white solid powder
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| LogP |
2.6
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| Hydrogen Bond Donor Count |
8
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| Hydrogen Bond Acceptor Count |
23
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| Rotatable Bond Count |
60
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| Heavy Atom Count |
115
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| Complexity |
2920
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| Defined Atom Stereocenter Count |
11
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| SMILES |
[C@@H]([C@]1([H])CCCN1C(=O)CC(OC)[C@@]([H])([C@@H](C)CC)N(C)C(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)N(C)C(=O)OCC1C=CC(NC(=O)[C@H](CCCNC(=O)N)NC(=O)[C@H](C(C)C)NC(=O)CCOCCOCCOCCOCCOCCOCCOCCOCCN2C(C=CC2=O)=O)=CC=1)(OC)[C@@H](C)C(=O)N[C@H](C)[C@H](C1C=CC=CC=1)O
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| InChi Key |
XHWFOQLEVJXEET-PAALVUBQSA-N
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| InChi Code |
InChI=1S/C81H131N11O23/c1-15-56(8)72(64(105-13)51-68(96)91-33-20-24-63(91)74(106-14)57(9)75(98)84-58(10)73(97)60-21-17-16-18-22-60)89(11)79(102)70(54(4)5)88-78(101)71(55(6)7)90(12)81(104)115-52-59-25-27-61(28-26-59)85-76(99)62(23-19-32-83-80(82)103)86-77(100)69(53(2)3)87-65(93)31-35-107-37-39-109-41-43-111-45-47-113-49-50-114-48-46-112-44-42-110-40-38-108-36-34-92-66(94)29-30-67(92)95/h16-18,21-22,25-30,53-58,62-64,69-74,97H,15,19-20,23-24,31-52H2,1-14H3,(H,84,98)(H,85,99)(H,86,100)(H,87,93)(H,88,101)(H3,82,83,103)/t56-,57+,58+,62-,63-,64?,69-,70-,71-,72+,73+,74+/m0/s1
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| Chemical Name |
[4-[[(2S)-5-(carbamoylamino)-2-[[(2S)-2-[3-[2-[2-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrol-1-yl)ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]ethoxy]propanoylamino]-3-methylbutanoyl]amino]pentanoyl]amino]phenyl]methyl N-[(2S)-1-[[(2S)-1-[[(4R,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
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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: (1). This product requires protection from light (avoid light exposure) during transportation and storage. (2). This product is not stable in solution, please use freshly prepared working solution for optimal results. |
| 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 : ~110 mg/mL (~67.61 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 | 0.6146 mL | 3.0732 mL | 6.1464 mL | |
| 5 mM | 0.1229 mL | 0.6146 mL | 1.2293 mL | |
| 10 mM | 0.0615 mL | 0.3073 mL | 0.6146 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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