Improving the Adjuvanticity of Small Molecule Immune Potentiators Using Covalently Linked NF-κB Modulators

Small molecule immune potentiators (SMIPs) such as imidazoquinolinone derivatives that activate Toll-like receptor (TLR) 7/8 have immense potential as vaccine adjuvants and as antitumor agents. However, these molecules have high bioavailability that results in unacceptable levels of systemic inflammation due to adjuvant toxicity, thereby greatly limiting their use. To address this challenge, here we report the design and synthesis of novel imidazoquinolinone-NF-κB immunomodulator dimers. Employing in vitro assays, we screened a select library of synthesized dimers and selected viable candidates for further in vivo experiments. With ovalbumin as a model antigen, we vaccinated mice and demonstrated that these dimers reduce the systemic toxicity associated with SMIPs to baseline levels while simultaneously maintaining the adjuvanticity in a vaccine formulation. Additionally, we showed that select dimers improved efficacy in a CT26 mouse colon carcinoma tumor model while eliciting minimal adjuvant toxicity.


Biological Procedures 1. RAW264.7 Macrophage (RAW-Blue) NF-κB assay
RAW-Blue cells, (InvivoGen) were cultured as described by the manufacturer. Cells were grown in complete culture media composed of Dulbecco's Modified Eagle's Medium (DMEM) with 4.5 g/L glucose (Life Technologies), 2 mM L-glutamine, 10,000 U/mL penicillin, 10 mg/mL streptomycin, 25μg/mL amphotericin B, supplemented with 10% fetal bovine serum (FBS, Thermo Fisher Scientific). RAW-Blue cells (passage 5-15) were plated in a 96 well plate at a density of 100,000 cells/well in 180 μL DMEM containing 10% heat-inactivated FBS (HI-FBS) and selective antibiotics. The cells were treated with agonist and agonist dimers and LPS control (50 ng/mL) for 20 h at 37 °C and 5% CO2. NF-κB activity was measured by a QUANTI-Blue (InvivoGen) assay and the absorbance was measured at 620 nm using a Multiskan FC plate reader (Thermo Scientific).

Bone Marrow-Derived Dendritic Cell Harvest and Culture.
Bone marrow-derived dendritic cells (BMDCs) were harvested from female C57Bl/6 mice as previously described. 1 Femur bones were aseptically removed from mice and the bone marrow was extracted into PBS buffer and the cell suspension centrifuged at 300 RCF for 10 min at RT to pellet the cells. ACK Lysing Buffer (3 mL, Lonza) was added to the cell pellet and incubated for 2 min at RT. PBS buffer (13 mL) was then added to the cell suspension, and the cell solution was centrifuged at 300 RCF for 10 min at RT. Next, the cell pellet was resuspended in BMDC complete media (RPMI 1640, 10% heat-inactivated FBS, 20 ng/mL granulocyte-macrophage colony-stimulating factor (GM-CSF), 2 mM L-glutamine (Life Technologies), 10,000 U/mL penicillin, 10 mg/mL streptomycin, 25μg/mL amphotericin B, and 50 μM beta-mercaptoethanol). The cells were then plated at 1x10 6 cells/mL in 100 mm petri dishes in 10 mL complete media and incubated at 37 °C in a CO2 incubator. On day 3, 10 mL of fresh BMDC media was added to each petri dish. On day 6, BMDCs were released and plated in untreated 12-well plates at 1x10 6 cells/mL for cell surface marker activation and cytokine secretion experiments.

Western blot analysis of pathway proteins
RAW264.7 Macrophage cells (InvivoGen) cultured in complete media were plated in 6 well plates at 1 x10 6 cells/mL and allowed to adhere for 12 h at 37 °C in a CO2 incubator. The cells were then treated with agonist, agonist dimers, and LPS control for 16 h. The treated cells were washed and scraped into cold phosphate-buffered saline (PBS) and centrifuged at 400 × g at 4°C for 5 min. The cell pellets were resuspended in triple detergent lysis buffer (10 mL) containing one protease inhibitor cocktail (cOmplete™ ULTRA Tablets, Sigma) and centrifuged to yield whole cell lysate. The lysate was quantified using a Pierce™ BCA Protein Assay Kit. 50 μg of total protein was separated using 4-15% SDS-PAGE and blotted onto PVDF membranes (Bio-Rad). The membranes were probed using monoclonal antibodies for COX-2 at a dilution of 1:1000 (Cayman Chemicals, MI) GAPDH (14C10) at a dilution of 1:1000 and Rabbit mAb and at a dilution of 1:500 iNOS (D6B6S) Rabbit mAb Visualization was achieved using IRDye® 800CW (Abcam) at a dilution of 1:10000 and imaged on Azure biosystems imager. Densitometric analysis was done using Image J.

Flow Cytometry for Cell Surface Marker Upregulation and Cytokine secretion analysis.
BMDCs were plated in untreated 12-well plates at 1x10 6 cells/mL and incubated with agonist and agonist dimers in culture media for 8 h at 37 °C with 5% CO2. The cells were released from the plate by pipetting vigorously and centrifuged at 2500 RPM at 4 °C for 10 min. The cell culture media was saved for IL-6 cytokine quantification using ELISA (BioLegend). The cell pellet was resuspended in cold FACS buffer (PBS, 10% FBS, and 0.1% sodium azide) buffer (300 μL) and incubated with CD16/32 FcR blocking antibodies (1.0 μg/1x106 cells) on ice for 15 min. The cell suspension was pelleted, and the supernatant was removed. Next, the cell pellet was resuspended in cold FACS buffer (100 μL) and incubated with PE-CD11c (1.0μg/1x106 cells) and APC CD40 (1.0 μg/1x106 cells), on ice and in the dark for 30 min. The samples were then washed twice with 300 μL FACS buffer. The pelleted cells were resuspended in cold FACS buffer (200 μL) and kept on ice until being loaded onto the flow cytometer for analysis.

In vivo vaccination of mice
Female C57/BL6 mice were briefly anesthetized with isoflurane and injected intramuscularly in the right hind leg with 50 μL containing ovalbumin (100 μg), adjuvant, adjuvant dimers, vanillin, and dopamine (0.07 µmoles) and a PBS vehicle control group.

Plasma cytokine analysis and Antibody quantification
Mouse blood was collected via the submandibular vein in 0.2 mL heparin-coated collection tubes (VWR Scientific) 1 h after vaccination. Serum was isolated by allowing blood to clot for 30 min RT and centrifugation at 2000 x g for 10 min. Supernatant was collected and stored at -80 °C until use. Serum was analyzed using BD Cytometric Bead Array Mouse Inflammation cytokine kit or LEGENDplex™ Mouse Inflammation Panel (Biolegend) according to manufacturer's protocol. For antibody quantification, mouse blood was collected via cardiac puncture 28 days after vaccination in 0.2 mL heparin-coated collection tubes (VWR Scientific). Serum was isolated by allowing blood to clot for 30 min RT and centrifuging at 2000 x g for 10 min. Serum was analyzed using a quantitative anti-ovalbumin total Ig's, IgA, and IgG ELISA kits (Alpha Diagnostic International) according to the manufacture's protocol.

Tumor studies
0.2 × 10 6 CT-26 cells were injected subcutaneously into the flank of 6-week-old Balb/c mice (n = 10 per group) in 100 µL of PBS. The tumor size was monitored on alternating days. Tumor volumes were measured using the equation V =1/2×L×W×W. When the tumors reached a size of approximately 75 mm 3 (day 11), treatment was started. Various formulations (20 nmols of each agonist or PBS) were injected peritumorally every 4 days (day 15, 19, and 23). Mice were euthanized when the tumors reached 20 mm in any linear dimension. Five mice for each group were used for blood analysis. Blood was collected two days post the first injection for hematological toxicity analysis and two hours post the second injection for systemic cytokine analysis.

Nitric oxide assay
RAW264.7 Macrophage (InvivoGen) cultured in complete media were plated in 12-well plates at 1 x10 6 cells/mL and treated with agonist and agonist dimers for 16 h at 37 °C in a CO2 incubator. The cell supernatant was collected and the quantity of nitrite in the culture medium was measured as an indicator of NO production. Amounts of nitrite, a stable metabolite of NO, were measured using Griess reagent (1% sulfanilamide and 0.1% naphthyl ethylenediamine dihydrochloride in 2.5% phosphoric acid). 100 µL of cell culture medium was mixed with 100 µL of Griess reagent. After incubation at room temperature for 10 min, the absorbance at 540 nm was measured in a microplate reader. The quantity of nitrite was determined from a sodium nitrite standard curve.

Proliferation Assay
Proliferation assay was performed as previously reported. 2 Splenocytes were isolated from C57BL/6 mice and plated at 5×10 4 in 96-well plates. The splenocytes were then incubated with 3 µM of agonist and agonist dimers for 48 h at 37 °C in a CO2 incubator. 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) (20 µl, 5 mg/ml in PBS) was added 4 h before the end of the incubation period. Purple crystals were dissolved in sterile DMSO and incubated for 5 minutes to ensure complete dissolution. The absorbance was measured at 590 nm using a Multiskan FC plate reader (Thermo Scientific). The proliferation rate was determined as follows: Abs(sample)/Abs(PBS)×100%.

Cell viability (MTT) assay
RAW264.7 Macrophage cells were incubated with agonist and agonist dimers for 16 h and subjected to cell viability assays. 3-(4,5-dimethylthiazol-2-yl)-2,5-diiphenyltetrazolium bromide (MTT) was dissolved in PBS to final concentration of 5 mg/mL and sterile-filtered. Treated cells were resuspended in fresh RPMI medium with 10 % FBS at concentration and plated in a 96-well plate at a concentration of 1 x 10 5 cell/mL. To these cells, 10 L of MTT solution was added, then incubated at 37 °C with 5 % CO2 for 3 h. When purple crystals were visible, 75 L of supernatant was removed. Purple crystals were dissolved in sterile DMSO and incubated for 5 minutes to ensure complete dissolution. The absorbance was measured at 590 nm using a Multiskan FC plate reader (Thermo Scientific). %viability was calculated as follows: Abs(sample)-Abs(blank)/Abs(Rest cells)-Abs(blank) x 100%.