| Size | Price | Stock | Qty |
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| 1mg |
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| Other Sizes |
| Targets |
The compound is designed to target Toll-like receptors 7 and 8 (TLR7 and TLR8), which are key components of the innate immune system and are primarily expressed in endosomes of immune cells like dendritic cells and macrophages [13L10-L11]. Upon internalization and release of the payload in target cells (e.g., cancer cells), the agonist moiety activates TLR7 and TLR8, initiating a signaling cascade that leads to the production of pro-inflammatory cytokines and type I interferons. This "immunogenic cell death" can potentially convert a non-immunogenic "cold" tumor into an immunogenic "hot" tumor, stimulating a systemic anti-tumor immune response.
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| ln Vitro |
The in vitro activity of this compound is typically validated after it has been conjugated to a targeting antibody to form a complete ADC. The ADC's activity is measured using in vitro cell-based assays. For example, target-positive cancer cells are treated with the ADC, which binds to a cell surface antigen and is internalized. After processing, the released TLR7/8 agonist activates TLR7/8 in the endosomes of the cancer cell or in co-cultured immune cells. Activation is measured by quantifying the secretion of cytokines (e.g., IFN-alpha, TNF-alpha, IL-6) in the cell culture supernatant via ELISA. Additionally, the activation of NF-kappaB and IRF pathways can be measured using luciferase reporter cell lines.
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| ln Vivo |
The ultimate in vivo activity of this compound is realized as part of a complete ADC in animal models of cancer. The TLR7/8 agonist 4 hydroxy-PEG6-acid is not active as a standalone small-molecule drug; its activity is highly dependent on being targeted to the tumor microenvironment by a conjugated antibody. The ADC is designed to be non-cleavable, ensuring the payload remains attached to the antibody until it is internalized and degraded within the target cell. This targeted approach aims to concentrate the potent immune activation at the tumor site while minimizing systemic toxicity.
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| Enzyme Assay |
The binding of the TLR7/8 agonist component to its target receptors can be studied using cell-free binding assays. A standard protocol is a competitive binding assay using isolated endosomes or purified recombinant TLR7 and TLR8 proteins. The protein is immobilized on a 96-well plate. A known fluorescently labeled TLR7/8 ligand is added to the plate, along with varying concentrations of the free TLR7/8 agonist 4 (the payload part of the linker-conjugate). After incubation and washing, the remaining fluorescence signal is measured. The ability of the free TLR7/8 agonist to displace the labeled ligand is directly proportional to its binding affinity (IC50) for the TLR7/8 receptor.
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| Cell Assay |
To evaluate the activity of the complete ADC in cellulo, the target cells (e.g., a cancer cell line expressing the target antigen) are seeded in 96-well plates. Serial dilutions of the ADC are added to the cells and incubated for 3-5 days. Subsequently, the cells are lysed, and the activation of TLR7/8 signaling is measured by quantifying the levels of downstream chemokines like IP-10 (CXCL10) and pro-inflammatory cytokines using a multiplex assay or ELISA. Alternatively, a co-culture system can be used where target cancer cells are incubated with peripheral blood mononuclear cells (PBMCs), and the ADC is added. Activation of the immune cells is then measured by flow cytometry (e.g., upregulation of co-stimulatory markers CD80/CD86 on dendritic cells).
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| Animal Protocol |
The complete ADC is typically tested in murine xenograft models to assess its in vivo antitumor efficacy. Immunodeficient mice (e.g., NSG mice) are humanized by engrafting human immune cells (e.g., PBMCs) or are used as a syngeneic model with a murine TLR7/8 agonist. The mice are subcutaneously implanted with a target-positive tumor cell line. Once tumors reach a certain size (e.g., 100-200 mm3), the ADC is administered intravenously (i.v.) at a specific dose (e.g., 1-10 mg/kg) once or twice a week for 2-3 weeks. Tumor volumes and body weights are measured bi-weekly. Blood is collected to measure cytokine levels, and tumors are harvested at the end of the study for flow cytometric analysis of immune cell infiltration (e.g., CD8+ T cells, dendritic cells).
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| ADME/Pharmacokinetics |
The TLR7/8 agonist 4 hydroxy-PEG6-acid hydrochloride is a building block for ADCs; therefore, its pharmacokinetic (PK) properties are not intended for standalone use. Once conjugated to an antibody, the PK profile is largely determined by the antibody's characteristics. However, the hydrophilic PEG6 (polyethylene glycol) linker is specifically chosen to improve the overall water solubility of the ADC and prevent aggregation, which can affect its stability and clearance. The hydrochloride salt form (HCl) is used to enhance the solubility of the compound for easier handling and formulation during the ADC synthesis process.
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| Toxicity/Toxicokinetics |
Specific toxicity data for the standalone TLR7/8 agonist 4 hydroxy-PEG6-acid hydrochloride is not publicly available. However, systemic activation of TLR7/8 can be highly toxic, leading to severe cytokine release syndrome (CRS), also known as a "cytokine storm," which can be fatal. This is the primary reason this potent agonist is conjugated to an antibody-to restrict its activity to the target cells and avoid systemic toxicity. Standard laboratory safety precautions should be taken when handling the powder, as it is a potent immune activator. The product is strictly for research use and is not intended for human or clinical use.
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| References | |
| Additional Infomation |
TLR7/8 agonist 4 hydroxy-PEG6-acid hydrochloride is a research-grade drug-linker conjugate used in the synthesis of antibody-drug conjugates (ADCs) [36L20-L21]. The compound combines a potent TLR7/8 agonist with a PEG6-based non-cleavable linker, and the hydrochloride salt enhances its solubility. By attaching this conjugate to a targeting antibody, researchers can create ADCs that deliver a potent immune agonist specifically to the tumor microenvironment, aiming to trigger a targeted anti-tumor immune response. This compound is strictly for research applications, not for clinical or therapeutic use. It should be stored at -20degC, protected from light and under nitrogen, to maintain its stability [36L6-L8].
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| Molecular Formula |
C33H53CLN6O8
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|---|---|
| Molecular Weight |
697.26
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| Related CAS # |
TLR7/8 agonist 4 hydroxy-PEG6-acid;2388520-23-6
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| Appearance |
White to off-white solid powder
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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). Please store this product in a sealed and protected environment (e.g. under nitrogen), 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) |
H2O :~50 mg/mL (~71.71 mM)
DMSO :< 1 mg/mL |
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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 | 1.4342 mL | 7.1709 mL | 14.3419 mL | |
| 5 mM | 0.2868 mL | 1.4342 mL | 2.8684 mL | |
| 10 mM | 0.1434 mL | 0.7171 mL | 1.4342 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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