Orn06 lyovec

Antibody targeting EGFR (Cetuximab) was purchased from Merck Serono. Gardiquimod, R848 (Resiquimod), ssRNA40/LyoVec and ORN06/LyoVec were purchased from InvivoGen. The 257–264 OVA peptide (SIINFEKL) was provided by Dr. S. Amigorena (Institut Curie, Paris, France). TLR3/dsRNA Complex Inhibitor was purchased from Calbiochem.

In total, 50,000 cells/well were plated in a tissue-culture treated 96-well plate (Costar, No. 3596) in unsupplemented DMEM. Cells were then treated with 100 μM R848 (InvivoGen, No. tlrl-r848), 5 μg/mL ssRNA40/LyoVec (InvivoGen, No. tlrl-lrna40), and/or 5 μg/mL ORN06/LyoVec (InvivoGen, No. tlrl-orn6) and the indicated concentrations of the compounds. Cells were incubated overnight at 37 °C in a humidified atmosphere of 5% CO₂ and assayed for NF-κB signaling. Cell media was assayed with Quanti-Blue (InvivoGen, No. rep-qbs3) per the manufacturer’s recommendations. Specificity test in HEK-Blue hTLRs cells for CU-CPD107 using TLR-specific ligands to selectively activate the corresponding TLRs: 100 ng/mL of Pam₃CSK₄, 100 ng/mL of Pam₂CSK₄, 5 μg/mL of polyriboinosinic: polyribocytidylic acid [poly(I:C)], 20 ng/mL of LPS (lipopolysaccharide), 50 ng/mL of Flagellin, 2.9 µM of R848, 22.8 µM of R848, and 0.5 μM of ODN2006 were used to selectively activate hTLR1/2, hTLR2/6, hTLR3, hTLR4, hTLR5, hTLR7, hTLR8, and hTLR9 cells, respectively. The SEAP reporter is constructed as a IFN-β promoter fused to five NF-κB and AP-1-binding sites. SEAP was quantified by measuring absorbance at 620 nm. Data were normalized with ligand + DMSO as 100% activity and untreated cells as 0. Each data point represents the average and standard deviation of at least three biological replicates.

This study was carried out in strict accordance with the recommendations in the Guide for the Care and use of Laboratory Animals of the National Institutes of Health. The protocol was approved by Texas A&M University Animal Use and Care Committee approved the protocol (Protocol Number: IACUC 2017–0414). 5-week old 20 g male and female C57BL/6 mice (Jackson Laboratories, Bar Harbor, ME) were given an oropharyngeal aspiration of 1 mg/kg (or less where indicated) ORN06/LyoVec (a ssRNA oligonucleotide with 6 UGU repeats complexed with a lipid which protects the ssRNA from degradation; henceforth referred to as ORN06) (InvivoGen, San Diego, CA) in 50 μl phosphate-buffered saline (PBS), or an oropharyngeal aspiration of an equal volume of PBS as a control, following [51 (link),52 (link)]. At 24 and 48 hours after ORN06 insult, the control mice and some of the ORN06-treated mice were given intraperitoneal injections of 100 μl PBS, or SAP diluted to a dose of 2.5 mg/kg in 100 μl PBS. All the mice were monitored twice daily to observe any sign of distress. At day 3, mice were euthanized by CO₂ inhalation, and bronchoalveolar lavage fluid (BALF) was obtained as previously described [34 (link),51 (link)]. All mice survived until euthanasia at day 3, and 20 mice were used in the study.