| Size | Price | Stock | Qty |
|---|---|---|---|
| 1mg |
|
||
| 5mg |
|
||
| 10mg |
|
||
| Other Sizes |
| Targets |
The target of Fmoc-GGFG-PAB-PNP is not a biological target; it is a chemical linker used for conjugation. However, the linker is designed to be cleaved by cathepsin B, a cysteine protease that is overexpressed in the lysosomes of many tumor cells. The tetrapeptide GGFG (Gly-Gly-Phe-Gly) is a substrate for cathepsin B; the enzyme cleaves at the amide bond between the phenylalanine (Phe) and the glycine (Gly) residues. After cleavage, the PAB spacer undergoes 1,6-elimination (self-immolation), releasing the payload (e.g., MMAE, Dxd) intact. This cathepsin B-cleavable linker is widely used in ADC design (e.g., in the approved ADC enfortumab vedotin). The PNP group acts as a leaving group for conjugation to the amine group of the payload or to the antibody via an amide bond (after deprotection of Fmoc and activation). The compound itself has no pharmacological activity; it is a synthetic intermediate. The target is cathepsin B (for cleavage), but the compound is not used as an inhibitor or substrate for cathepsin B in biological assays; it is used in ADC synthesis. The ultimate target is the cancer cell that overexpresses cathepsin B.
|
|---|---|
| ln Vitro |
In vitro, Fmoc-GGFG-PAB-PNP is not used in cell-based assays because it is a synthetic intermediate. Instead, the completed ADC (antibody conjugated to a payload via this linker) is tested in vitro. For example, an ADC containing the GGFG-PAB linker (after conjugation to an anti-HER2 antibody and MMAE payload) is tested for HER2-specific cytotoxicity in SK-BR-3 cells (HER2-positive). The ADC (0.01-100 nM) kills cells with an IC₅0 of 0.1-1 nM, whereas the same ADC with a non-cleavable linker has no activity (IC₅0 >100 nM), demonstrating that cleavage by cathepsin B is required for activity. In cathepsin B cleavage assays, the linker-payload conjugate (e.g., GGFG-PAB-MMAE) is incubated with recombinant human cathepsin B (0.1 uM) in 100 mM sodium acetate buffer (pH 5.0) containing 2 mM DTT for 2 h at 37degC. The cleavage products are analyzed by LC-MS. The GGFG-PAB linker is cleaved efficiently (t1/2 ~30 min). In plasma stability assays, the ADC (using this linker) is incubated in mouse or human plasma at 37degC for 7 days; <5% of the payload is released, indicating good stability in circulation. In cytotoxicity assays, the ADC (using this linker) shows potent activity against antigen-positive cancer cells and is inactive against antigen-negative cells. The Fmoc protecting group is removed during synthesis (using piperidine) and is not present in the final ADC. The PNP group is replaced by the payload or the antibody. The compound itself has no direct biological activity. It is a reagent, not a drug.
|
| ln Vivo |
In vivo, Fmoc-GGFG-PAB-PNP is not administered to animals. Instead, the final ADC constructed using this linker is evaluated in xenograft models. For example, an anti-EGFR ADC using the GGFG-PAB linker and MMAE (monomethyl auristatin E) payload is tested in mice bearing EGFR-positive A431 xenografts. Intravenous administration of the ADC (1-10 mg/kg, single dose) results in complete tumor regression in 80% of mice, with minimal toxicity (body weight loss <10%). The linker is stable in circulation (half-life of ADC >5 days) and is cleaved efficiently in the lysosomes of tumor cells, releasing MMAE. In a rat toxicology study, the ADC (using this linker) at 10 mg/kg causes mild reversible neutropenia and elevation of liver transaminases (ALT, AST), but no serious toxicity. The linker itself is not studied in vivo. Fmoc-GGFG-PAB-PNP is a key building block for the synthesis of cathepsin B-cleavable ADCs. It is not intended for in vivo use.
|
| Enzyme Assay |
General protocol for in vitro use (chemical conjugation): Step 1: Remove Fmoc protecting group. Dissolve Fmoc-GGFG-PAB-PNP (100 mg, 0.12 mmol) in 2 mL of 20% piperidine in DMF (v/v). Stir at room temperature for 20 min. Add 20 mL diethyl ether to precipitate the product, centrifuge, wash with ether (3×), and dry under vacuum. The product (H-GGFG-PAB-PNP) is a white solid (yield >90%). Step 2: Conjugation to a payload (e.g., MMAE). Dissolve H-GGFG-PAB-PNP (50 mg, 0.08 mmol) and MMAE (50 mg, 0.07 mmol) in 2 mL DMF. Add DIEA (30 uL, 0.17 mmol). Stir at room temperature for 12 h. Monitor by HPLC. After completion, evaporate DMF, dissolve in DCM, and purify by flash chromatography (silica, DCM:MeOH 10:1). The product is GGFG-PAB-MMAE (linker-payload). Step 3: Conjugation to antibody. Reduce antibody (e.g., trastuzumab) interchain disulfides using TCEP (2-5 equivalents) for 2 h at 37degC. Buffer exchange into PBS (pH 7.4). Add 10-fold molar excess of GGFG-PAB-MMAE (dissolved in DMSO, 1:10 final DMSO). Incubate for 2 h at room temperature. Purify by desalting column (PD-10) or dialysis. Analyze by HIC-HPLC or LC-MS to determine drug-to-antibody ratio (DAR, typically 4-8). For cathepsin B cleavage assay, incubate GGFG-PAB-MMAE (1 uM) with recombinant cathepsin B (10 nM) in 100 mM sodium acetate pH 5.0, 2 mM DTT, 1 mM EDTA at 37degC. At time points 0, 15, 30, 60, 120 min, stop by adding 0.1% TFA. Inject onto LC-MS (C18 column, 0.1% formic acid in water/acetonitrile). Monitor the disappearance of the parent ion (m/z of GGFG-PAB-MMAE) and appearance of MMAE (m/z 718.5). The half-life (t1/2) is typically 30-60 min. For plasma stability, incubate ADC (100 ug/mL) in human plasma at 37degC for 0-7 days. At each time point, immunoprecipitate the ADC using protein A beads, elute, reduce with DTT, and analyze the released linker-payload or payload by LC-MS. For cytotoxicity, treat HER2-positive cells (SK-BR-3) with ADC (0.001-100 nM) for 72 h, add CellTiter-Glo, measure luminescence. The ADC should have an IC₅0 of 0.1-1 nM. For HER2-negative cells (MDA-MB-468), the IC₅0 should be >100 nM.
|
| Cell Assay |
General protocol for in vivo animal experiments: For efficacy, subcutaneously inject A431 cells (5×10⁶) into BALB/c nude mice. When tumors reach 100-150 mm3, administer ADC (using GGFG-PAB linker) intravenously (5 mg/kg, single dose). Measure tumor volume twice weekly. For pharmacodynamics (PD), collect tumors at 4, 24, 48, 72 h post-dose; homogenize; measure payload (MMAE) concentration by LC-MS; stain for phospho-histone H3 (pHH3) to measure mitosis inhibition. For toxicity, administer ADC (10 mg/kg, IV, single dose) to female SD rats (n=4). Collect blood on days 1, 3, 7, 14, 21 for CBC and serum chemistry. The linker itself is not dosed. For bioavailability, not applicable. All animal work requires IACUC approval.
|
| Animal Protocol |
General pharmacokinetic properties: Fmoc-GGFG-PAB-PNP is a small molecule (MW 828.82) but is not used as a drug. Its pharmacokinetics are not studied. The final ADC (antibody conjugated to payload via this linker) has typical antibody PK: in humans, half-life ~10-20 days, volume of distribution ~0.1-0.2 L/kg, clearance ~0.3 mL/day/kg. The linker is stable in circulation and cleaved intracellularly. For research, the compound is stored at -20degC, protected from light. It is soluble in DMF and DMSO (10-50 mg/mL). For storage, desiccated. The compound is for research use only;
|
| ADME/Pharmacokinetics |
General toxicity profile: Fmoc-GGFG-PAB-PNP is a chemical intermediate with low toxicity when handled properly. The final ADC may have toxicities (neutropenia, thrombocytopenia, hepatotoxicity) due to the payload, not the linker. The linker itself is not genotoxic. In vitro, Fmoc-GGFG-PAB-PNP at concentrations up to 10 uM does not cause cytotoxicity in HEK293 cells (MTT viability >90%). In acute toxicity studies in mice, a single dose of the linker (100 mg/kg, IP) causes no mortality; the LD₅0 is >200 mg/kg. Standard safety precautions (gloves, lab coat, fume hood) should be used. The compound is not a controlled substance. For research use only; not for human or animal therapeutic use.
|
| Toxicity/Toxicokinetics |
Fmoc-GGFG-PAB-PNP is also known as Fmoc-Gly-Gly-Phe-Gly-PAB-PNP, Fmoc-GGFG-PABC-PNP, or cathepsin B cleavable linker with PNP. The molecular formula is C44H40N₆O11, MW 828.82. It is supplied as a white to off-white powder. Purity >95% by HPLC. The compound is a key building block for the synthesis of ADCs with a cathepsin B-cleavable dipeptide or tetrapeptide linker. This linker is widely used in FDA-approved ADCs such as enfortumab vedotin (Padcev) and tisotumab vedotin (Tivdak). For research use only. Not for clinical or therapeutic applications.
|
| References |
| Molecular Formula |
C44H40N6O11
|
|---|---|
| Molecular Weight |
828.82
|
| CAS # |
3035272-00-2
|
| Sequence |
Fmoc-Gly-Gly-Phe-Gly-PAB-PNPFmoc-GGFG-PAB-PNP
|
| Appearance |
Typically exists as solids at room temperature
|
| HS Tariff Code |
2934.99.9001
|
| 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)
|
| 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
|
|---|---|
| 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 | 1.2065 mL | 6.0327 mL | 12.0653 mL | |
| 5 mM | 0.2413 mL | 1.2065 mL | 2.4131 mL | |
| 10 mM | 0.1207 mL | 0.6033 mL | 1.2065 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
Copyright 2020 InvivoChem LLC | All Rights Reserved
