MUC1 Specific Immune Responses Enhanced by Coadministration of Liposomal DDA/MPLA and Lipoglycopeptide

Mucin 1 (MUC1), a well-known tumor-associated antigen and attractive target for tumor immunotherapy, is overexpressed in most human epithelial adenomas with aberrant glycosylation. However, its low immunogenicity impedes the development of MUC1-targeted antitumor vaccines. In this study, we investigated three liposomal adjuvant systems containing toll-like receptor 4 (TLR4) agonist monophosphoryl lipid A (MPLA) and auxiliary lipids of different charges: cationic lipid dimethyldioctadecylammonium (DDA), neutral lipid distearoylglycerophosphocholine (DSPC) or anionic lipid dioleoylphosphatidylglycerol (DOPG), respectively. ELISA assay evidenced that the positively charged DDA/MPLA liposomes are potent immune activators, which induced remarkable levels of anti-MUC1 antibodies and exhibited robust Th1-biased immune responses. Importantly, the antibodies induced by DDA/MPLA liposomes efficiently recognized and killed MUC1-positive tumor cells through complement-mediated cytotoxicity. In addition, antibody titers in mice immunized with P2-MUC1 vaccine were significantly higher than those from mice immunized with P1-MUC1 or MUC1 vaccine, which indicated that the lipid conjugated on MUC1 antigen also played important role for immunomodulation. This study suggested that the liposomal DDA/MPLA with lipid-MUC1 is a promising antitumor vaccine, which can be used for the immunotherapy of various epithelial carcinomas represented by breast cancer.


Vaccine formulation and immunization
The composition of each vaccine candidate is shown in Table S1. Vaccines A-D: the liposomes of mixture of adjuvant (each mouse MPLA 17 g), antigen (MUC1 for A, P1-MUC1 for B, P2-MUC1 for C, and P3-MUC1 for D), and auxiliary lipid DDA in a molar ratio of 10:10:80 were dissolved in a mixture of DCM/MeOH (1:1, v/v, 2 mL); Vaccines E-G: the liposomes of mixture of adjuvant (MPLA 17 g), antigen (P2-MUC1 28 µg), and auxiliary lipid (DSPC for E, DOPG for F, no auxiliary lipid for G) in a molar ratio of 10:10:80 were dissolved in a mixture of DCM/MeOH (1:1, v/v, 2 mL); Vaccines H-K: the liposomes of mixture of antigen (P2-MUC1 28 µg) and auxiliary lipid (DDA for H, DSPC for I, DOPG for J, no auxiliary lipid for K) in a molar ratio of 10:80 were dissolved in a mixture of DCM/MeOH (1:1, v/v, 2 mL), and the solvents were removed under reduced pressure through rotary evaporation, which generated a thin lipid film on the flask wall. This film was hydrated and subjected to freeze/thaw cycles to produce multilamellar vesicles, then the Tris buffer (20 mM, pH 7.4) was added and shaking the mixture under an argon atmosphere at 25 °C for 1 h. The milky suspension was finally sonicated for 20 min to obtain the optimal liposomes.

Vaccines
Auxiliary lipid (80 nmol Scheme S6. Study design for MUC1 vaccine candidates in mice 2 . Mice were immunized on the 1st, 15th, and 29th days, and the blood was taken on day 0 before initial immunization, and 14th, 28th, and 42nd days before the boost immunizations.

Biological testing methods
Mouse immunization schedule BALB/c mice (five per group) were vaccinated via intraperitoneal injection of vaccines (A-I) on days 1, 15 and 29 of a 2-week cycle by intraperitoneal injection, sera were collected on 2 h, 24 h and days 14, 28, 42 after the first immunization. All the animal experiments were conducted strictly in accordance with the principles of welfare and ethics of laboratory animals.

Protocol for ELISA
The antibody titers and antibody isotypes generated by the vaccine candidates were measured by ELISA. The biotinylated MUC1 (0.125 μg/mL) and avidin (1.16 μg/mL) were directly dissolved in the prepared NaHCO3/Na2CO3 buffer (50 mM, pH 9.5) with a final concentration of 0.125 μg/mL and 1.16 μg/mL, then 96-well ELISA plates (costar 3590) were coated with avidin-biotin-MUC1 complex solution and incubated at 4°C overnight. Then the plates were washed three times with 0.05% Tween PBS and blocked with PBS containing 3% BSA for 1 hour at 37 ℃. After the plates were washed three times with 0.05% PBST, the diluted antisera were added to each well (100 µL/well) and incubated for 1 h at 37 ℃. After washing, the horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG (-chain specific), IgM (-chain specific), IgG1, IgG2a, IgG2b and IgG3 (1:2000 dilution) in 0.1% BSA/PBS were added to each well (100 µL/well) respectively, and incubated for 1 h at 37 ℃. After washing, the freshly prepared substrate solutions [9.5 mL critic buffer at pH 5.0, 0.5 mL 1.6 mg/mL tetramathyl benzidine (TMB), and 32 µL 3% (w/v) urea hydrogen peroxide] were added to each well (100 µL/well) in the dark for 5 minutes, then 2 M H2SO4 (50 µL/well) was added to quench the reaction. Optical density was then measured at a wavelength of 450 nm with a microplate reader (Synergy H1). The titer of antibody was defined as the highest dilution, indicating that the absorbance was 0.1, minus the background. Data were analyzed with the software GraphPad (San Diego, CA).

Cell cytotoxicity
To assess whether the induced antibodies of the antisera from vaccinated mice can mediate complement lysis via activation of the complement dependent cytotoxicity (CDC). For this purpose, MCF-7 cells were incubated with these antisera (at a antisera dilution of 1:50) (50 μL per well) from vaccinated mice, after washing with PBS solution, the prepared rabbit sera (at a rabbit sera dilution of 1:50) in 1% BSA/PBS were added as complement supplier (RC: rabbit complement; RC-inactive: inactivated rabbit complement). MCF-7 cells were incubated with antisera diluted to 1:50 in 1% BSA/PBS (50 μL per well). After washing, the rabbit sera diluted to 1:5 in 1% BSA/PBS, and 50 μL/well of the solution were added. After incubation for 4 hours, 0.5% MTT solution in PBS (MTT: 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) was added (20 μl/well) and incubated for 2 h. After removing the medium, DMSO was added (150 μL/well) and the absorption was analyzed at the wavelength of 490 nm. The survival rate of cells was measured with the following formula. All assays were conducted in five independent experiments.