Agatolimod targets Toll-like receptor 9 (TLR9), which recognizes unmethylated CpG DNA motifs and activates innate immune responses. It is the optimal CpG sequence for humans and strongly activates B cells and plasmacytoid dendritic cells (pDCs).

In HD11 chicken macrophage cells, Agatolimod stimulates strong production of NO2 and IL-6. It strongly activates human B cells, induces IL-6 secretion, activates NF-kappaB signaling pathways, but weakly stimulates IFN-alpha secretion in pDCs. It exhibits potent immunostimulatory activity.

Agatolimod has been investigated in clinical trials (e.g., NCT00471471) for cancer immunotherapy, including melanoma and breast cancer. As a vaccine adjuvant, it enhances antigen-specific immune responses and shows potential antitumor activity by activating TLR9-mediated innate immunity.

Not applicable, as Agatolimod is an oligonucleotide agonist that acts on the intracellular TLR9 receptor. Binding is assessed in cell-based assays measuring TLR9 activation and downstream signaling, rather than classical cell-free receptor-ligand binding assays.

Human PBMCs or specific immune cell subsets (B cells, pDCs) are seeded in 96-well plates. Agatolimod (0.1-10 ug/mL) is added to the culture medium for 6-48 hours. B cell activation (CD69, CD86 expression) is assessed by flow cytometry. Cytokine secretion (IL-6, IL-12, IFN-alpha, TNF-alpha) in supernatants is measured by ELISA. NF-kappaB activation is evaluated by Western blot or reporter assays.

Agatolimod has been studied in mouse tumor models. Sequence: 5‘-tcgtcgttttgtcgttttgtcgtt-3'. Administration routes include subcutaneous or intratumoral injection, often in combination with vaccines or other immunotherapies. Doses vary by study (e.g., 0.1-1 mg/mouse). Anti-tumor efficacy is assessed by tumor growth inhibition and survival prolongation. Immunomonitoring is performed on blood and tumor tissues.

Following subcutaneous or intravenous administration in humans, Agatolimod has a half-life of approximately 30-60 minutes and is rapidly cleared but induces a sustained pharmacodynamic response due to TLR9 activation. It accumulates in lymphoid tissues. The compound is formulated in saline for clinical and preclinical use.

In clinical studies, Agatolimod has shown a manageable safety profile, with most common adverse events being injection site reactions, flu-like symptoms, and mild to moderate cytokine release syndrome. Dose-limiting toxicities have not been observed at the doses tested in clinical trials.

[1]. CpG-induced immunomodulation and intracellular bacterial killing in a chicken macrophage cell line. Dev Comp Immunol. 2003 Oct;27(9):823-34.

[2]. Oligodeoxynucleotides ODN 2006 and M362 Exert Potent Adjuvant Effect through TLR-9/-6 Synergy to Exaggerate Mammaglobin-A Peptide Specific Cytotoxic CD8+T Lymphocyte Responses against Breast Cancer Cells. Cancers (Basel). 2019 May 14;11(5):672.

Agatolimod is being investigated in the clinical trial NCT00471471 (Vaccine therapy for patients with unresectable recurrent stage III or IV melanoma). Agatolimod is a synthetic 24-meric oligonucleotide containing three CpG motifs and possesses potential antitumor and immunostimulatory activities. After administration, atoratolimod selectively targets Toll-like receptor 9 (TLR9), thereby activating dendritic cells and B cells, and stimulating cytotoxic T cell and antibody responses against tumor cells expressing tumor antigens.

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Agatolimod (also known as PF-3512676, CpG 7909, ODN 2006) has been evaluated in clinical trials for cancer immunotherapy and vaccine adjuvants. It is not yet approved for clinical use but has shown promise in combination with other agents. Molecular formula: C236H326N70O133P23S23; MW ~7704.

7705.798

525625-52-9

Biotin-labeled Agatolimod sodium;FITC-labeled Agatolimod sodium;Agatolimod sodium;541547-35-7

56841790

White to off-white solid powder

-6.6

55

162

142

486

23800

0

CC1=CN(C(=O)NC1=O)C2CC(C(O2)COP(=S)(O)OC3CC(OC3COP(=S)(O)OC4CC(OC4COP(=S)(O)OC5CC(OC5COP(=S)(O)OC6CC(OC6COP(=S)(O)OC7CC(OC7COP(=S)(O)OC8CC(OC8COP(=S)(O)OC9CC(OC9COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1COP(=S)(O)OC1CC(OC1CO)N1C=C(C(=O)NC1=O)C)N1C=CC(=NC1=O)N)N1C=NC2=C1N=C(NC2=O)N)N1C=C(C(=O)NC1=O)C)N1C=CC(=NC1=O)N)N1C=NC(=C1C)C(=O)NC(=C)N)N1C=C(C(=NC1=O)N)C)N1C=C(C(=NC1=O)N)C)N1C=C(C(=O)NC1=O)C)N1C=C(C(=O)NC1=O)C)N1C=NC2=C1N=C(NC2=O)N)N1C=C(C(=O)NC1=O)C)N1C=CC(=NC1=O)N)N1C=NC2=C1N=C(NC2=O)N)N1C=C(C(=NC1=O)N)C)N1C=C(C(=NC1=O)N)C)N1C=C(C(=NC1=O)N)C)N1C=C(C(=NC1=O)N)C)N1C=NC2=C1N=C(NC2=O)N)N1C=C(C(=O)NC1=O)C)N1C=CC(=NC1=O)N)N1C=NC2=C1N=C(NC2=O)N)N1C=C(C(=O)NC1=O)C)O

GUVMFDICMFQHSZ-UHFFFAOYSA-N

InChI=1S/C238H314N75O127P23S23/c1-93-49-294(225(334)266-191(93)244)167-31-115(424-450(357,473)376-69-139-116(32-168(400-139)295-50-94(2)192(245)267-226(295)335)425-451(358,474)379-72-142-120(36-172(403-142)299-54-98(6)196(249)271-230(299)339)429-456(363,479)392-85-155-131(47-183(416-155)312-91-259-189-200(312)275-219(253)280-214(189)328)439-462(369,485)386-79-149-123(39-175(410-149)304-59-103(11)206(320)286-235(304)344)431-448(355,471)375-68-138-114(30-165(399-138)293-24-20-160(243)265-224(293)333)422-446(353,469)391-84-154-129(45-181(415-154)310-89-257-187-198(310)273-217(251)278-212(187)326)437-460(367,483)384-75-145-121(37-173(406-145)302-57-101(9)204(318)284-233(302)342)423-441(348,464)371-64-134-109(315)25-161(395-134)300-55-99(7)202(316)282-231(300)340)140(401-167)70-378-453(360,476)427-119-35-171(298-53-97(5)195(248)270-229(298)338)405-144(119)74-383-459(366,482)436-128-44-180(309-88-256-186-197(309)272-216(250)277-211(186)325)413-152(128)83-390-445(352,468)421-113-29-164(292-23-19-159(242)264-223(292)332)398-137(113)67-374-449(356,472)432-124-40-176(305-60-104(12)207(321)287-236(305)345)411-150(124)80-387-463(370,486)440-132-48-184(313-92-260-190-201(313)276-220(254)281-215(190)329)417-156(132)86-393-457(364,480)434-126-42-178(307-62-106(14)209(323)289-238(307)347)408-147(126)77-381-455(362,478)433-125-41-177(306-61-105(13)208(322)288-237(306)346)407-146(125)76-380-454(361,477)428-117-33-169(296-51-95(3)193(246)268-227(296)336)402-141(117)71-377-452(359,475)426-118-34-170(297-52-96(4)194(247)269-228(297)337)404-143(118)73-382-458(365,481)435-127-43-179(308-87-255-185(107(308)15)210(324)261-108(16)239)412-151(127)81-388-443(350,466)419-112-28-163(291-22-18-158(241)263-222(291)331)397-136(112)66-373-447(354,470)430-122-38-174(303-58-102(10)205(319)285-234(303)343)409-148(122)78-385-461(368,484)438-130-46-182(311-90-258-188-199(311)274-218(252)279-213(188)327)414-153(130)82-389-444(351,467)420-111-27-162(290-21-17-157(240)262-221(290)330)396-135(111)65-372-442(349,465)418-110-26-166(394-133(110)63-314)301-56-100(8)203(317)283-232(301)341/h17-24,49-62,87-92,109-156,161-184,314-315H,16,25-48,63-86,239H2,1-15H3,(H,261,324)(H,348,464)(H,349,465)(H,350,466)(H,351,467)(H,352,468)(H,353,469)(H,354,470)(H,355,471)(H,356,472)(H,357,473)(H,358,474)(H,359,475)(H,360,476)(H,361,477)(H,362,478)(H,363,479)(H,364,480)(H,365,481)(H,366,482)(H,367,483)(H,368,484)(H,369,485)(H,370,486)(H2,240,262,330)(H2,241,263,331)(H2,242,264,332)(H2,243,265,333)(H2,244,266,334)(H2,245,267,335)(H2,246,268,336)(H2,247,269,337)(H2,248,270,338)(H2,249,271,339)(H,282,316,340)(H,283,317,341)(H,284,318,342)(H,285,319,343)(H,286,320,344)(H,287,321,345)(H,288,322,346)(H,289,323,347)(H3,250,272,277,325)(H3,251,273,278,326)(H3,252,274,279,327)(H3,253,275,280,328)(H3,254,276,281,329)

N-(1-aminoethenyl)-1-[4-[[5-(4-amino-5-methyl-2-oxopyrimidin-1-yl)-3-[[5-(4-amino-5-methyl-2-oxopyrimidin-1-yl)-3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(4-amino-5-methyl-2-oxopyrimidin-1-yl)-3-[[5-(4-amino-5-methyl-2-oxopyrimidin-1-yl)-3-[[5-(4-amino-5-methyl-2-oxopyrimidin-1-yl)-3-[[5-(4-amino-5-methyl-2-oxopyrimidin-1-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[hydroxy-[[3-[hydroxy-[[3-hydroxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy]phosphinothioyl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy]phosphinothioyl]oxyoxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxyoxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxyoxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxyoxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxyoxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxyoxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxy-5-(2-amino-6-oxo-1H-purin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxy-5-(2-amino-6-oxo-1H-purin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxyoxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxyoxolan-2-yl]methoxy-hydroxyphosphinothioyl]oxy-5-[[[2-[[[2-[[[5-(2-amino-6-oxo-1H-purin-9-yl)-2-[[[5-(4-amino-2-oxopyrimidin-1-yl)-2-[[hydroxy-[2-(hydroxymethyl)-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-3-yl]oxyphosphinothioyl]oxymethyl]oxolan-3-yl]oxy-hydroxyphosphinothioyl]oxymethyl]oxolan-3-yl]oxy-hydroxyphosphinothioyl]oxymethyl]-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-3-yl]oxy-hydroxyphosphinothioyl]oxymethyl]-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-3-yl]oxy-hydroxyphosphinothioyl]oxymethyl]oxolan-2-yl]-5-methylimidazole-4-carboxamide

2934.99.9001

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.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ≥ 125 mg/mL (~16.23 mM)

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.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*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).

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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)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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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).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)