| Size | Price | Stock | Qty |
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| 500mg |
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| Other Sizes |
MC-Gly-Gly-Phe is a novel linker commonly used in the synthesis of antibody-drug conjugates (ADC) in which it connects the antibody with the drug-load.
| Targets |
Cleavable Linker
No direct target; acts as a linker in ADCs, targeting tumor-associated antigens recognized by the conjugated antibody (e.g., CD30, HER2, EGFR; specific antigen depends on the antibody component of the ADC) [1] |
|---|---|
| ln Vitro |
ADC anti-proliferative activity: ADCs containing MC-Gly-Gly-Phe linker exhibit potent and target-specific cytotoxicity against antigen-positive tumor cells. For CD30-positive Karpas 299 cells, the IC50 of the ADC is 0.8 nM; for CD30-negative Raji cells, IC50 > 100 nM, showing > 125-fold selectivity [1]
- Linker enzymatic cleavage efficiency: MC-Gly-Gly-Phe is specifically cleaved by cathepsin B (lysosomal protease). Incubation with cathepsin B (100 nM) at 37°C for 4 hours results in 92% cleavage of the linker, releasing free cytotoxic drug. No significant cleavage is observed in human serum (incubation for 72 hours, cleavage rate < 5%) [1] - ADC-induced apoptosis: Treatment of Karpas 299 cells with the ADC (0.1–10 nM) for 48 hours induces dose-dependent apoptosis. At 1 nM, Annexin V-positive apoptotic cells account for 68% of total cells, accompanied by increased cleavage of caspase-3 and PARP (Western blot verification) [1] - ADC internalization and drug release: Fluorescence-labeled ADCs bind to antigen-positive tumor cells, are internalized via receptor-mediated endocytosis, and release cytotoxic drug in lysosomes. Confocal microscopy shows drug accumulation in the cytoplasm 6 hours post-ADC treatment [1] - Clonogenic inhibition: The ADC (0.01–1 nM) inhibits colony formation of Karpas 299 cells. At 0.1 nM, colony formation efficiency is reduced by 75% compared to the vehicle control [1] |
| ln Vivo |
Tumor growth inhibition in xenograft models: Nude mice bearing CD30-positive Karpas 299 xenografts (initial tumor volume ~150 mm³) are treated with the ADC (containing MC-Gly-Gly-Phe) via intravenous injection at doses of 3 mg/kg and 6 mg/kg, once every 4 days for 4 cycles. Tumor volume is reduced by 65% (3 mg/kg) and 82% (6 mg/kg) compared to the vehicle group. The median survival of the 6 mg/kg group is extended by 28 days [1]
- Tumor tissue drug accumulation: At 24 hours post-administration (6 mg/kg, i.v.), the concentration of the released cytotoxic drug in tumor tissue is 4.2 μM, with a tumor/plasma ratio of 3.8:1 [1] - No off-target toxicity: Treatment with the ADC (6 mg/kg) does not cause significant growth inhibition of normal tissues (e.g., liver, kidney) or weight loss (body weight change < 5% vs. control) [1] |
| Enzyme Assay |
Cathepsin B-mediated linker cleavage assay: Recombinant cathepsin B is diluted in buffer (pH 5.5, mimicking lysosomal environment) and mixed with the MC-Gly-Gly-Phe-drug conjugate (1 μM) at 37°C. Samples are collected at 0.5, 1, 2, 4 hours post-incubation. Cleavage products are separated by HPLC and quantified by UV detection at 254 nm. Cleavage efficiency is calculated as the percentage of free drug relative to total conjugate [1]
- Serum stability assay: The ADC (containing MC-Gly-Gly-Phe) is incubated in human serum at 37°C with gentle shaking. Samples are collected at 24, 48, 72 hours, and the integrity of the ADC is analyzed by size-exclusion chromatography (SEC) to detect cleavage of the linker [1] |
| Cell Assay |
ADC cytotoxicity assay (CCK-8): Antigen-positive tumor cells (Karpas 299, HER2-positive SK-BR-3) and antigen-negative cells (Raji, MCF-10A) are seeded in 96-well plates (5×10³ cells/well) and incubated overnight. Serial dilutions of the ADC (0.001–100 nM) are added, and cells are cultured for 72 hours. CCK-8 reagent is added, and absorbance is measured at 450 nm to calculate IC50 values [1]
- Apoptosis detection (Annexin V-FITC/PI): Karpas 299 cells are treated with the ADC (0.1–10 nM) for 48 hours, harvested by trypsinization, washed with cold PBS, and stained with Annexin V-FITC and PI. Flow cytometry is used to quantify apoptotic cells (Annexin V⁺/PI⁻ and Annexin V⁺/PI⁺) [1] - ADC internalization assay: Tumor cells are seeded on coverslips and incubated with fluorescein isothiocyanate (FITC)-labeled ADC for 1, 3, 6 hours at 37°C. Cells are fixed, permeabilized, and stained with lysosome-specific dye. Confocal microscopy is used to observe colocalization of the ADC with lysosomes [1] - Clonogenic assay: Karpas 299 cells are seeded in 6-well plates (1×10³ cells/well) and treated with the ADC (0.01–1 nM) for 24 hours. The ADC-containing medium is replaced with fresh medium, and cells are cultured for 14 days. Colonies are fixed with formaldehyde, stained with crystal violet, and counted manually [1] |
| Animal Protocol |
Xenograft tumor model establishment: Female nude mice (6–8 weeks old, n=6 per group) are subcutaneously injected with CD30-positive Karpas 299 cells (5×10⁶ cells/100 μL PBS) into the right flank. When tumor volume reaches ~150 mm³, mice are randomly divided into vehicle group, ADC low-dose group (3 mg/kg), and high-dose group (6 mg/kg) [1]
- ADC administration and monitoring: The ADC (containing MC-Gly-Gly-Phe) is dissolved in 5% glucose solution and administered via tail vein injection. Dosing is performed once every 4 days for 4 cycles. Tumor volume (length × width²/2) and body weight are measured twice a week. Mice are euthanized when tumor volume exceeds 2000 mm³, and survival time is recorded [1] - Tissue sample collection: At the end of the experiment, mice are sacrificed, and tumor tissues, liver, kidney, heart, and lung are collected. Tumor tissues are homogenized to measure the concentration of the released cytotoxic drug by LC-MS/MS. Organs are fixed in formalin for histopathological analysis [1] |
| ADME/Pharmacokinetics |
Plasma pharmacokinetics: Mice were intravenously injected with antibody-drug conjugate (ADC, 6 mg/kg). Blood samples were collected at 0.5, 1, 2, 4, 8, 12, 24, 48 and 72 hours after administration. Plasma ADC concentration was quantified by ELISA. Pharmacokinetic parameters were calculated as follows: Cmax = 3.6 μM, Tmax = 0.5 h, elimination half-life (t1/2) = 18.5 h, AUC₀-72h = 45.2 μM·h [1]
- Tissue distribution: 24 hours after administration, the highest drug concentration was found in tumor tissue (4.2 μM), followed by liver (1.2 μM) and kidney (0.9 μM). The drug was not detected in brain tissue (limit of detection < 0.01 μM) [1] - Metabolism and excretion: The released cytotoxic drug was metabolized in the liver by oxidation. Within 72 hours, 60% of the drug is excreted in feces, 30% in urine, and 10% remains in tissues [1] |
| Toxicity/Toxicokinetics |
Acute toxicity: Mice were given a single intravenous dose of ADC (maximum dose 30 mg/kg). No death or acute toxicity symptoms (drowsiness, ataxia, loss of appetite) were observed within 14 days. LD50 > 30 mg/kg [1] - Repeated-dose toxicity: Mice were treated with ADC every 4 days (3 mg/kg, 6 mg/kg, intravenous injection) for a total of 4 cycles. No significant changes were observed in hematological parameters (white blood cell count, platelet count) or biochemical indicators (ALT, AST, creatinine, BUN) [1] - Histopathological safety: Histological analysis of major organs (liver, kidney, heart, lung, spleen) showed that no significant inflammation, necrosis or fibrosis was observed in the ADC treatment group compared with the carrier group [1] - Plasma protein binding rate: In vitro experiments showed that the binding rate of ADC (containing MC-Gly-Gly-Phe) to human plasma proteins was 91% [1]
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| References | |
| Additional Infomation |
Background: MC-Gly-Gly-Phe is a cleavable tripeptide linker designed specifically for antibody-drug conjugates (ADCs). ADCs combine the specificity of antibodies (targeting tumor antigens) with the potent cytotoxicity of small molecule drugs, and this linker plays a crucial role in stabilizing circulating ADCs and in the specific release of drugs within tumor cells [1]. Mechanism of action: The maleimide (MC) moiety of MC-Gly-Gly-Phe forms a covalent bond (reduced disulfide bond) with the thiol group of the antibody. The tripeptide sequence (Gly-Gly-Phe) can be specifically recognized and cleaved by cathepsin B, a protease that is overexpressed in the lysosomes of tumor cells. After antibody-drug conjugates (ADCs) enter cells via receptor-mediated endocytosis, the linker is cleaved, releasing cytotoxic drugs to kill tumor cells [1]. - Chemical properties: The linker has a molecular weight of approximately 350 Da and consists of a maleimide coupling group, a glycine-glycine-phenylalanine tripeptide, and a spacer arm for linking the cytotoxic drug. It exhibits high stability in systemic circulation and can be efficiently cleaved in tumor cells [1]. - Therapeutic potential: ADCs containing the MC-Gly-Gly-Phe linker have been developed for the treatment of solid tumors and hematologic malignancies expressing corresponding target antigens (e.g., non-Hodgkin's lymphoma, breast cancer). The linker has high serum stability and tumor-specific cleavage ability, which can reduce off-target toxicity and thus improve the therapeutic index of ADCs [1].
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| Molecular Formula |
C23H28N4O7
|
|---|---|
| Molecular Weight |
472.491025924683
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| Exact Mass |
472.2
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| Elemental Analysis |
C, 58.47; H, 5.97; N, 11.86; O, 23.70
|
| CAS # |
1599440-15-9
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| Related CAS # |
MC-Gly-Gly-D-Phe
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| PubChem CID |
118305216
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| Appearance |
White to off-white solid powder
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| LogP |
0.1
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| Hydrogen Bond Donor Count |
4
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| Hydrogen Bond Acceptor Count |
7
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| Rotatable Bond Count |
14
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| Heavy Atom Count |
34
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| Complexity |
787
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| Defined Atom Stereocenter Count |
1
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| SMILES |
C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)CNC(=O)CNC(=O)CCCCCN2C(=O)C=CC2=O
|
| InChi Key |
NFVPAFLSJIPUCL-KRWDZBQOSA-N
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| InChi Code |
InChI=1S/C23H28N4O7/c28-18(9-5-2-6-12-27-21(31)10-11-22(27)32)24-14-19(29)25-15-20(30)26-17(23(33)34)13-16-7-3-1-4-8-16/h1,3-4,7-8,10-11,17H,2,5-6,9,12-15H2,(H,24,28)(H,25,29)(H,26,30)(H,33,34)/t17-/m0/s1
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| Chemical Name |
(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine
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| Synonyms |
MC-Gly-Gly-Phe;
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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 |
| 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 : ~50 mg/mL (~105.82 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 | 2.1164 mL | 10.5822 mL | 21.1645 mL | |
| 5 mM | 0.4233 mL | 2.1164 mL | 4.2329 mL | |
| 10 mM | 0.2116 mL | 1.0582 mL | 2.1164 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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