CL264
目录号 : GC39361
CL264 是一种用于先天免疫信号研究的 TLR7 特异性激动剂。
Cas No.:1510712-69-2
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
- Purity: >98.00%
- COA (Certificate Of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
| Cell experiment [1]: | |
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Cell lines |
Lewis lung carcinoma (LLC),Bone marrow derived macrophages(BMSMs) |
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Preparation Method |
Macrophages were cultivated in the presence or absence of CL264 for 24 h, before tumor cells were added and co-cultured for 42 h. Radiolabeled thymidine was used to detect tumor cell growth and was added to the co-cultures 18 h before cell harvest. |
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Reaction Conditions |
Macrophages,CL264(40ng/ml),LLC co-cultured for 42 h |
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Applications |
CL264 stimulating the BMDMs and it resulted in inhibition of LLC cells, but only when it was used together with IFN-γ. |
| Animal experiment [2]: | |
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Animal models |
ild-type C57Bl/6J mice,TLR7 Knock out C57Bl/6J mice |
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Preparation Method |
Cancer cells - Murine lung adenocarcinoma LLC-luc cells (1E6 in 100μl PBS/injection) were injected into the flank of recipient mice. Mice then received intra-tumor injections of CL264 at day 0, 3 and 6 after tumor grafting. |
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Dosage form |
40μg/injection, intra-tumor injection |
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Applications |
Intratumoral injection of the TLR7 agonist CL264 induced a pro-tumorigenic effect.CL264 in TLR7 KO mice, similar to that observed in WT mice.These results clearly demonstrate that the pro-tumorigenic effect of CL264 effect is mediated by an effect on TLR7 expressed by malignant (rather than immune) cells. |
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References: [1]. Müller E, Christopoulos PF, et al. Toll-Like Receptor Ligands and Interferon-γ Synergize for Induction of Antitumor M1 Macrophages. Front Immunol. 2017 Oct 26;8:1383. [2]. Dajon M, Iribarren K, et al. Toll like receptor 7 expressed by malignant cells promotes tumor progression and metastasis through the recruitment of myeloid derived suppressor cells. Oncoimmunology. 2018 Oct 11;8(1):e1505174. |
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CL264 is a a potent and highly specific toll-like receptor 7 (TLR7) ligand. Immune cells utilize toll-like receptors (TLRs) to sense pathogen associated molecular patterns (PAMPs), which represents the starting point of the innate immune response s[1].
Using concentrations from 0.5 to 10?μg/mL, stimulation with CL264 resulted in a dose-dependent secretion of TNF-α?after 6 hours. CAL-1 cells were stimulated with CL264 (5?μg/mL) for up to 12 hours. ?CAL-1 cells were seeded into 96-well plates. After overnight resting, cells were stimulated with CL264 or 9.2s RNA or the combination of both. After incubation as indicated, cell supernatant was analyzed for TNF-α, IL-6, and IFN-β?by ELISA. CL264 led to a robust, time-dependent release of TNF-α?reaching absolute levels of 1347?pg/mL TNF-α?into the supernatant[1].Peripheral blood mononuclear cells (PBMCs) stimulated with CL264, IL-6, IL-8 and IL-12 were significantly stimulated[2].
The TLR7 agonist CL264 ,40 μg/injectionat day 0, 3 and 6 after tumor grafting, stimulated tumor growth, and this pro-tumorigenic effect was completely lost in the absence of MDSCs, both in WT mice and in TLR7 KO mice. The tumor growth exacerbating effect of TLR7 stimulation is mediated by the increased number of MDSCs within the tumor microenvironment.The secretion of CCL2 and GM-CSF by tumor cells was confirmed in vitro, which was found increased in supernatants of CL264-stimulated cells,these results suggest that CL264 may stimulate the recruitment of MDSCs via the induction of CCL2 and GM-CSF.[3].
References:
[1].Hilbert T, Steinhagen F, et al. Synergistic Stimulation with Different TLR7 Ligands Modulates Gene Expression Patterns in the Human Plasmacytoid Dendritic Cell Line CAL-1. Mediators Inflamm. 2015;2015:948540.
[2].Dajon M, Iribarren K, et al. Toll like receptor 7 expressed by malignant cells promotes tumor progression and metastasis through the recruitment of myeloid derived suppressor cells. Oncoimmunology. 2018 Oct 11;8(1):e1505174.
CL264 是一种有效且高度特异性的 Toll 样受体 7 (TLR7) 配体。免疫细胞利用 Toll 样受体 (TLR) 感知病原体相关分子模式 (PAMP)，这是先天免疫反应的起点 s[1]。
使用 0.5 至 10μg/mL 的浓度，用 CL264 刺激会在 6 小时后导致 TNF-α 的剂量依赖性分泌。用 CL264 (5μg/mL) 刺激 CAL-1 细胞长达 12 小时。将 CAL-1 细胞接种到 96 孔板中。过夜休息后，用 CL264 或 9.2s RNA 或两者的组合刺激细胞。如所示孵育后，通过 ELISA 分析细胞上清液中的 TNF-α、IL-6 和 IFN-β。 CL264 导致上清液中 TNF-α 的稳健、时间依赖性释放达到 1347pg/mL TNF-α 的绝对水平[1]。用 CL264 刺激的外周血单核细胞 (PBMC)， IL-6、IL-8和IL-12均得到显着刺激[2]。
TLR7 激动剂 CL264，40 μg/ 次注射，在肿瘤移植后的第 0、3 和 6 天，刺激肿瘤生长，并且在没有 MDSCs 的情况下，这种促肿瘤发生作用在 WT 小鼠和 TLR7 KO 小鼠中完全消失. TLR7 刺激的肿瘤生长加剧作用是由肿瘤微环境中 MDSC 数量的增加介导的。体外证实了肿瘤细胞分泌 CCL2 和 GM-CSF，这在 CL264 刺激细胞的上清液中发现增加，这些结果表明，CL264 可能通过诱导 CCL2 和 GM-CSF 刺激 MDSCs 的募集。[3]。
| Cas No. | 1510712-69-2 | SDF | |
| Canonical SMILES | O=C(O)CNC(C1=CC=C(CN2C(NC3=C(N)N=C(NCCCC)N=C23)=O)C=C1)=O | ||
| 分子式 | C19H23N7O4 | 分子量 | 413.43 |
| 溶解度 | Soluble in DMSO | 储存条件 | Store at -20°C |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 2.4188 mL | 12.0939 mL | 24.1879 mL |
| 5 mM | 0.4838 mL | 2.4188 mL | 4.8376 mL |
| 10 mM | 0.2419 mL | 1.2094 mL | 2.4188 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。
[The study of increased immunogenicity of UCMSC Stimulated by TLR7 agonist CL264]
Sichuan Da Xue Xue Bao Yi Xue Ban 2015 Mar;46(2):191-6.PMID:25924427doi
Objective: To study the change of immune status of umbilical cord mesenchymal stem cells (UCMSC) stimulated by toll like receptor 7 (TLR7) agonist CL264. Methods: TLR7 specific ligand CL264 was used to stimulate the UCMSC. Flow cytometry was conducted to assay the expression of co-stimulators [human leukocyte antigen (HLA)-E, CD80 and CD86] and surface markers of stem cells (CD29, CD59 and CD90). Quantitative PCR was applied to measure the expression variation of immune-related molecules and stem cell markers. Cell differentiation experiment was used to study the change of differentiation ability of UCMSC upon CL264 stimulation. Peripheral blood mononuclear cells (PBMC) were isolated from healthy human and then cocultured with UCMSC in the presence of CL264. Cytotoxicity assay was used to measure the attack of PBMC to UCMSC. Results: Expression of cotimulatory molecules CD86 and HLA-E were enhanced in UCMSC upon CL264 stimulation. Real-time PCR indicated that many pro-inflammatory molecules [interleukin (IL)-1beta, IL-6, IL-8, IL-10, interferon (IFN)-beta, IFN-gamma, nuclear factor-KB (NF-kappaB), transforming growth factor-beta (TGF-beta)] were induced in the presence of CL264 while the expression of stem cells markers were inhibited [Kruppel-like factor-4 (Klf4), Nestin, SRY-related high-mobility-group-box protein-2 (Sox2), Lin28]. Activation of TLR7 also increased the immune attack of PBMC on UCMSC. Our study also indicated that the treatment of CL264 did not influence the differentiation ability of UCMSC. Conclusion: TLR7 agonist CL264 could increase the immunogenicity of UCMSC.
Synthetic Polymeric Mixed Micelles Targeting Lymph Nodes Trigger Enhanced Cellular and Humoral Immune Responses
ACS Appl Mater Interfaces 2018 Jan 24;10(3):2874-2889.PMID:29285934DOI:10.1021/acsami.7b14004.
It has been widely accepted that lymph nodes (LNs) are critical targets of cancer vaccines because antigen presentation and initiation of T-cell-mediated immune responses occur primarily at these locations. In this study, amphiphilic diblock copolymer poly(2-ethyl-2-oxazoline)-poly(d,l-lactide) (PEOz-PLA) combined with carboxylterminated-Pluronic F127 was used to construct mixed micelles [carboxylated-nanoparticles (NPs)] for codelivery of antigen ovalbumin (OVA) and Toll-like receptor-7 agonist CL264 (carboxylated-NPs/OVA/CL264) to the LN-resident dendritic cells (DCs). The results showed that the small, sub-60 nm size of the self-assembled mixed micelles enables them to rapidly penetrate into lymphatic vessels and reach draining lymph nodes after subcutaneous injection. Furthermore, the surface modification with carboxylic groups imparted the carboxylated-NPs with endocytic receptor-targeting ability, allowing for DC internalization of carboxylated-NPs/OVA/CL264 via the scavenger receptor-mediated pathway. Because stimulation of CL264 in early endosomes will lead to a more effective immune response than that in late endo/lysosomes, the mass ratio of PEOz-PLA to carboxylated-Pluronic F127 in the mixed micelles was adjusted to release the encapsulated CL264 to the early endosome, resulting in increased expression of costimulatory molecules and secretion of stimulated cytokines by DCs. Moreover, the incorporation of PEOz outside the micellar shell effectively augmented MHC I antigen presentation through facilitating endosome escape and cytosolic release of antigens. This in turn evoked potent immune responses in vivo, including activation of antigen-specific T-cell responses, production of antigen-specific IgG antibodies, and generation of cytotoxic T-lymphocyte responses. Finally, immunization with the codelivery system in E.G7-OVA tumor-bearing mice could not only significantly inhibit tumor growth but also markedly prolong the survival of tumor-bearing mice. Taken together, carboxylated-NPs/OVA/CL264 have demonstrated great potential for clinical applications as an effective antitumor vaccine for further immunotherapy.
TLR7 agonist loaded airway epithelial targeting nanoparticles stimulate innate immunity and suppress viral replication in human bronchial epithelial cells
Int J Pharm 2022 Apr 5;617:121586.PMID:35181464DOI:10.1016/j.ijpharm.2022.121586.
Nanoparticle-based delivery is a strategy for increasing the therapeutic window of inhaled immunomodulatory drugs that have inflammatory activity. TLR7 agonists are a class of immunomodulators that have been considered for the treatment of virus-induced respiratory diseases. However, due to high immune-stimulatory activity, TLR7 agonists, delivered via direct exposure, generally have a narrow therapeutic window. To address this, we have developed lipid/polymer hybrid nanoparticles (NPs) conjugated with anti-EpCAM monoclonal antibody for targeted delivery of TLR7 agonist (CL264) to airway epithelial cells (AECs)2 - the primary site of respiratory virus infection. These airway epithelial targeting nanoparticles (AEC-NPs)3 showed safety and biocompatibility, and approximately two-fold increased cellular uptake compared to non-targeting NPs. Upon cell entry, AEC-NPs were able to deliver CL264 to cytoplasm and endosomes where TLR7 is located. CL264 delivered by AEC-NPs significantly increased innate immune response through expression of IFN-β, IFN-λ 2/3 and IFN-stimulated genes and suppressed more than 92% of viral load at 48 h post-infection compared to the drug alone and non-targeting NPs. In conclusion, AEC-NPs exhibited increased cellular uptake leading to enhanced innate immune activation and suppression of viral replication. These findings support the use of AEC-targeting approach for delivering drugs with a narrow therapeutic window.
Level of Decoy Receptor 3 for Monitoring Clinical Progression of Severe Burn Patients
J Burn Care Res 2021 Sep 30;42(5):925-933.PMID:34213565DOI:10.1093/jbcr/irz170.
The clinical value of Decoy receptor 3 (DcR3) in severe burn is investigated. Ten patients with severe burns were monitored for DcR3, PCT, CRP, IL6, SOFA score, white blood cell (WBC), and platelet. The correlations were analyzed. DcR3 increased on day 1. The nonsurvivors had a steady high level of DcR3 while the survivors had a relatively low level of DcR3. The peak magnitude of DcR3 was high in five nonsurvivors and low in five survivors without overlap. Three patients had a continuously increasing DcR3 level and then died. In the other two nonsurvivors, DcR3 reached the peak and then decreased before death. DcR3 correlated well with PCT (ρ = 0.4469, P < .0001), less with CRP, platelet, IL6, SOFA score and WBC (ρ = 0.4369, 0.4078, 0.3995, 0.2631, 0.1504, respectively, all P < .001). To explore the mechanisms, the HaCaT or THP-1 cells were stimulated by the plasma of burn patients, 45°C, LPS or stimulators of TLRs or NOD2 (PGN, CL264, MDP, iE-DAP, Gardiquimod), and their DcR3 was increased, which could be reduced by GDC-0941 or BEZ235 (inhibitors of PI3K and mTOR). The levels of DcR3 appeared to be a useful biomarker for monitoring the clinical severity and a predictor of mortality of severe burns.
Targeting of Immune Cells by Dual TLR2/7 Ligands Suppresses Features of Allergic Th2 Immune Responses in Mice
J Immunol Res 2017;2017:7983217.PMID:29204451DOI:10.1155/2017/7983217.
Background: TLR ligands can promote Th1-biased immune responses, mimicking potent stimuli of viruses and bacteria. Aim: To investigate the adjuvant properties of dual TLR2/7 ligands compared to those of the mixture of both single ligands. Methods: Dual TLR2/7 ligands: CL401, CL413, and CL531, including CL264 (TLR7-ligand) and Pam2CysK4 (TLR2-ligand), were used. Immune-modulatory capacity of the dual ligands with the individual ligands alone or as a mixture in mouse BMmDCs, BMmDC:TC cocultures, or BMCMCs was compared and assessed in naïve mice and in a mouse model of OVA-induced intestinal allergy. Results: CL413 and CL531 induced BMmDC-derived IL-10 secretion, suppressed rOVA-induced IL-5 secretion from OVA-specific DO11.10 CD4+ TCs, and induced proinflammatory cytokine secretion in vivo. In contrast, CL401 induced considerably less IL-10 secretion and led to IL-17A production in BMmDC:TC cocultures, but not BMCMC IL-6 secretion, or IL-6 or TNF-α production in vivo. No immune-modulating effects were observed with single ligands. All dual TLR2/7 ligands suppressed DNP-induced IgE-and-Ag-specific mast cell degranulation. Compared to vaccination with OVA, vaccination with the mixture CL531 and OVA, significantly suppressed OVA-specific IgE production in the intestinal allergy model. Conclusions: Based on beneficial immune-modulating properties, CL413 and CL531 may have utility as potential adjuvants for allergy treatment.