A multivalent CD44 glycoconjugate vaccine candidate for cancer immunotherapy

Targeted and Self-Adjuvated Nanoglycovaccine Candidate for Cancer Immunotherapy

“…Serum samples from each animal were utilized to measure the concentration of Immunoglobulin G (IgG) or Immunoglobulin M (IgM) antibodies using the Mouse IgG or IgM ELISA Antibody Pair Kit assay from STEMCELL Technologies, following the manufacturer's guidelines and previously described by us. 49 Serum was appropriately diluted to 1:50000 for IgG and 1:100000 for IgM analysis. Briefly, ELISA plates were coated overnight at 4 °C with the Mouse IgG or IgM capture antibody.…”

“…IgG specificity against glycans and glycoconjugates on cancer cells was evaluated by flow cytometry using T24 wild type (T24 WT; MUC16 positive ; Tn negative ) and T24 C1GALT1 KO (T24 C1GALT1 KO; MUC16 positive , Tn high ) as previously described us. 49 Briefly cells were stained with the murine IgG isolated from mice primed with the 3 μg of MUC16-Tn glyconanovaccine for 1 h at RT (1:50), followed by the secondary antibody Alexa Fluor 488 antimouse IgG (A11029, Thermo Fisher Scientific) for 30 min at a 1:300 dilution in FACS buffer under mild agitation at RT. A secondary antimouse antibody was used as control.…”

“…The metabolic activity of mo-DCs stimulated with of Control NPs, MUC16 functionalized nanoparticles (MUC16 NPs), glyconanovaccine (MUC16-Tn NPs), monophosphoryl lipid A (MPLA) adjuvated glycovaccine (MUC16-Tn NPs plus MPLA), and free MUC16-Tn was evaluated using the MTT assay as previously described by us. 49 Accordingly, mo-DCs were seeded into 96 well plates at a cell density of 1 × 10 4 and exposed to increasing concentrations of nanoformulations (0.1, 1, 10, 50 μg/mL). After 24 h exposure, conditioned medium was replaced by 100 μL of fresh culture medium and 10 μL of a 12 mM 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Thermo Fisher Scientific) stock solution was added to each well, following a 4 h of incubation at 37 °C in a humidified chamber.…”

“…Mo-DCs profile evaluation was performed as previously described by us. 49 The mo-DCs were seeded into six-well plates at a cell density of 1 × 10 6 cells and cultured during 24 h with the following formulations (1 μg/mL): Control NPs, MUC16 NPs, glyconanovaccine, MPLA (2 μg/mL) adjuvated glyconanovaccine (MUC16-Tn NPs plus MPLA), free MUC16-Tn, and KLH-MUC16-Tn. Unstimulated DCs were used as negative control and 50 ng/mL LPS (Sigma-Aldrich) was used as mo-DCs activation positive control.…”

“…Mice liver, spleen, kidneys, heart, brain, and pancreas were gathered to evaluate the toxic effects resulting from the administration of various nanovaccine formulations and processed as previously described by us. 49 Accordingly, freshly collected tissue samples were fixed and preserved in a 10% aqueous formaldehyde solution overnight. Following fixation, the tissue specimens underwent manual dehydration using a series of ethanol concentrations and xylene.…”