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| Targets |
EX-A4328 is a cleavable ADC linker designed for the site-specific conjugation of cytotoxic payloads to antibodies. The linker contains a maleimide caproyl (MC) group that reacts with thiol groups on antibodies (typically generated by partial reduction of interchain disulfides) to form stable thioether bonds. The Gly-Gly-Phe-Gly peptide sequence is a substrate for lysosomal proteases, particularly cathepsin B, which is overexpressed in the lysosomes of cancer cells. Upon internalization of the ADC and trafficking to lysosomes, the peptide sequence is cleaved by cathepsin B, releasing the payload via the self-immolative glycolic acid spacer. This mechanism ensures selective payload release in target cells, minimizing off-target toxicity.
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| ln Vitro |
An ADC cytotoxin is connected to an antibody by use of an ADC linker to form an ADC.
EX-A4328 is a cleavable ADC linker that can be utilized to prepare active antibody-drug conjugates (ADC). The peptide sequence Gly-Gly-Phe-Gly serves as a substrate for cathepsin B, a lysosomal protease. The linker enables targeted delivery of cytotoxic payloads to cancer cells with selective release in the lysosomes. The maleimide caproyl (MC) group allows for conjugation to antibodies through thiol-maleimide chemistry. |
| ln Vivo |
In vivo, ADCs prepared using EX-A4328 linkers deliver cytotoxic payloads specifically to tumor cells expressing the target antigen. The linker's cleavable nature ensures that the payload is released inside target cells after ADC internalization and lysosomal trafficking, minimizing systemic toxicity. ADCs using similar peptide-based linkers have demonstrated potent antitumor activity in preclinical and clinical studies. The linker's design is optimized for stability in circulation and efficient payload release in the tumor microenvironment.
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| Enzyme Assay |
The activity of EX-A4328 as an ADC linker is assessed through conjugation efficiency and stability studies, payload release assays, and in vitro cytotoxicity assays of the resulting ADC. Conjugation efficiency is assessed by HPLC or mass spectrometry. The stability of the linker-payload conjugate is assessed in plasma or serum by LC-MS/MS. Payload release is assessed by incubating the ADC with cathepsin B or with cell lysates and measuring released payload by LC-MS/MS. The linker's self-immolative properties are confirmed by detecting the released payload and spacer fragments.
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| Cell Assay |
Cellular assays for ADCs prepared with EX-A4328 involve treating target antigen-positive and antigen-negative cell lines with the ADC. Cell viability is assessed using MTT or CellTiter-Glo assays after 72-120 hours of treatment. IC50 values are calculated from concentration-response curves. The specificity of the ADC is confirmed by comparing activity on antigen-positive versus antigen-negative cells. Payload internalization and release can be assessed by measuring intracellular payload levels by LC-MS/MS.
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| Animal Protocol |
In vivo efficacy of ADCs prepared with EX-A4328 is evaluated in mouse xenograft models using human cancer cell lines expressing the target antigen. Tumor-bearing mice are treated with the ADC (intravenous administration) at various doses. Tumor volumes are measured every 2-3 days. Pharmacokinetics of the ADC and released payload are assessed in plasma. Body weight and clinical signs are monitored for tolerability. The antitumor activity is compared with controls and with ADCs using other linkers.
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| ADME/Pharmacokinetics |
EX-A4328 has molecular formula C28H36N6O10 and molecular weight 616.62. It is a solid powder with purity ≥98%. The compound is soluble in DMSO (120 mg/mL). It should be stored at 4°C, away from moisture. The compound is a cleavable ADC linker used for the preparation of active antibody-drug conjugates.
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| Toxicity/Toxicokinetics |
Toxicological data for EX-A4328 are derived from studies of the resulting ADCs. As a linker component, its safety profile is assessed as part of the ADC toxicology package. The maleimide group may react with thiols in plasma, potentially leading to deconjugation. The peptide sequence is designed to be stable in circulation and cleaved only in lysosomes, minimizing systemic toxicity. Standard toxicology studies for ADCs would be conducted as part of drug development.
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| References | |
| Additional Infomation |
EX-A4328 is a cleavable ADC linker known as MC-Gly-Gly-Phe-Gly-NH-CH2-O-CH2COOH. It is used for the preparation of active antibody-drug conjugates (ADC). The linker contains a maleimide caproyl (MC) group for antibody conjugation, a Gly-Gly-Phe-Gly peptide sequence cleavable by cathepsin B, and a glycolic acid self-immolative spacer. It is a research-use only compound for ADC development.
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| Molecular Formula |
C28H36N6O10
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| Molecular Weight |
616.619647026062
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| Exact Mass |
616.249
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| CAS # |
1599440-25-1
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| Related CAS # |
MC-Gly-Gly-Phe-Gly-NH-CH2-O-CH2COOH;1599440-25-1
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| PubChem CID |
118305142
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| Appearance |
White to off-white solid powder
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| LogP |
-1.3
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| Hydrogen Bond Donor Count |
6
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| Hydrogen Bond Acceptor Count |
10
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| Rotatable Bond Count |
20
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| Heavy Atom Count |
44
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| Complexity |
1070
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| Defined Atom Stereocenter Count |
1
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| SMILES |
O=C([C@H](CC1C=CC=CC=1)NC(CNC(CNC(CCCCCN1C(C=CC1=O)=O)=O)=O)=O)N(COCC(=O)O)CC(N)=O
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| InChi Key |
SODPQQOBNODMSG-FQEVSTJZSA-N
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| InChi Code |
InChI=1S/C28H36N6O10/c35-21(9-5-2-6-12-34-25(39)10-11-26(34)40)29-14-22(36)30-16-24(38)33-20(13-19-7-3-1-4-8-19)28(43)31-15-23(37)32-18-44-17-27(41)42/h1,3-4,7-8,10-11,20H,2,5-6,9,12-18H2,(H,29,35)(H,30,36)(H,31,43)(H,32,37)(H,33,38)(H,41,42)/t20-/m0/s1
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| Chemical Name |
2-[[[2-[[(2S)-2-[[2-[[2-[6-(2,5-dioxopyrrol-1-yl)hexanoylamino]acetyl]amino]acetyl]amino]-3-phenylpropanoyl]amino]acetyl]amino]methoxy]acetic 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) |
DMSO : ~100 mg/mL (~162.17 mM)
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.86 mg/mL (4.64 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 28.6 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL. Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution. Solubility in Formulation 2: ≥ 2.86 mg/mL (4.64 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution. For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 28.6 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution. View More
Solubility in Formulation 3: ≥ 2.86 mg/mL (4.64 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution. |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 1.6217 mL | 8.1087 mL | 16.2174 mL | |
| 5 mM | 0.3243 mL | 1.6217 mL | 3.2435 mL | |
| 10 mM | 0.1622 mL | 0.8109 mL | 1.6217 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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