Resiquimod r 848

Tissue culture media and media supplements, mannan (Saccharomyces cerevisiae), laminarin (Laminaria digitata), Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), f-MLF (N-Formyl-methionyl-leucyl-phenylalanine), epicatechin, polyinosinic:polycytidylic acid, sodium salt (poly (I:C)), lipopolysaccharide (LPS) from Escherichia coli, TNF-alpha, and GM-CSF were obtained from Sigma-Aldrich (St. Louis, USA). 4-(N-Maleimidomethyl) cyclo-hexanecarboxylic-acid N-hydroxysuccinimide ester (SMCC) was provided by Thermo Scientific (Erembodegem, Belgium). Biocompatible Anchor for cell Membrane (BAM, Mw 4000) was obtained from NOF EUROPE (Grobbendonk, Belgium). N-Formyl-methionyl-leucyl-phenylalanine with two lysine molecules (f-MLFKK) was synthesized by Schafer-N (Copenhagen, Denmark). Imiquimod (R-837) was delivered by Merck Millipore (Billerica, USA), monophosphoryl lipid A (MPLA) by Avanti Polar Lipids (Alabaster, USA), and resiquimod (R-848) by Tocris Bioscience (Bristol, UK).

Mannan from Saccharomyces cerevisiae, lipoteichoic acid (LTA) from Bacillus subtilis, and polyinosinic:polycytidylic acid (poly(I:C)) was obtained from Sigma-Aldrich (Saint Lous, MO, USA). BAM was obtained from NOF Corporation (White Plains, NY, USA). Resiquimod (R-848) was obtained from Tocris Bioscience (Minneapolis, MN, USA). Monoclonal anti-CD40 (clone FGK4.5/FGK45) was obtained from BioXCell (West Lebanon, NH, USA). Mannan-BAM synthesis was performed as previous reported (25 (link)). After development of tumors (average tumor volume 55 mm³), mice were treated intratumorally in days 0, 1, 2, 8, 9, 10, 16, 17, 18, 24, 25, and 26 with 50 μL of the therapeutic mixture consisting of 0.5 mg R-848 (HCl form), 0.5 mg poly(I:C), 0.5 mg LTA, and 0.4 mg anti-CD40 per mL of 0.2 mM mannan-BAM in PBS (MBTA therapy).

The Biocompatibility Anchors for Cell Membranes (BAM) were acquired from NOF America (White Plains, NY). Mannan from Saccharomyces cerevisiae, polyinosinic-polycytidylic acid sodium salt, and lipoteichoic acid from Bacillus subtilis were obtained from Sigma-Aldrich (St. Louis, MO). Resiquimod (R-848) was purchased from Tocris Bioscience (Minneapolis, MN). Anti-mouse CD40 (clone: FGK4.5/GFK45) was procured from BioXCell (West Lebanon, NH). Flow cytometry antibodies, including anti-CD4, anti-CD8, anti-CD11c, anti-IFN-γ, anti-CD44, anti-CD62L, anti-Ly6G, anti-Ly6C, anti-CD11b were obtained from BD biosciences (Franklin Lakes, NJ); anti-CD45, anti-CD107a, anti-CD80, anti-CD86, anti-CD137L, anti-MHCII, anti-TNF-α, anti-CD19, anti-MHCII, anti-TCRβ and anti-GranB were obtained from Bioligand (San Diego, CA). The ELISA kits for detecting IFN-γ, TNF-α, and IL-6 were obtained from Bioligand (San Diego, CA).