SMALL LIPID NANOPARTICLES, AND CANCER VACCINE INCLUDING SAME
20220409708 · 2022-12-29
Inventors
Cpc classification
A61K39/3955
HUMAN NECESSITIES
A61K45/06
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
A61K2039/55555
HUMAN NECESSITIES
A61K2039/55561
HUMAN NECESSITIES
A61K2039/545
HUMAN NECESSITIES
International classification
A61K39/00
HUMAN NECESSITIES
A61K39/39
HUMAN NECESSITIES
A61K39/395
HUMAN NECESSITIES
Abstract
The present invention relates to small lipid nanoparticles, a small lipid nanoparticle (SLNP)-based nanovaccine platform including same, and a combination treatment regimen with an immune checkpoint inhibitor. Lipid nanoparticies according to the present invention can easily deliver antigens and anionic drugs into cells, and exhibit strong anti-tumor effects when loaded with tumor-associated antigens. Particularly, a cancer vaccine kit according to the present invention including lipid nanoparticles according to the present invention as a first vaccine composition and lipid nanoparticles and an immune checkpoint inhibitor as a second vaccine composition can be used to effectively suppress tumor regrowth and recurrence triggered by the occurrence of immunosuppression against a cancer nanovaccine.
Claims
1. A lipid nanoparticle comprising an antigen, a phospholipid, a cationic lipid, and an adjuvant.
2. The lipid nanoparticle of claim 1, wherein the antigen is a tumor-associated antigen.
3. The lipid nanoparticle of claim 1, wherein the phospholipid is a phospholipid having 14 to 22 aliphatic carbon atoms.
4. The lipid nanoparticle of claim 1, wherein the cationic lipid is at least one cationic lipid selected from the group consisting of Dimethyldioctadecyl-ammoniumbromide (DDAB), dimethyldioctadecylammonium (DDAB), (N,N-dimethyl-N-([2-sperminecarboxamido]ethyl)-2,3-bis(dioleyloxy)-1-propaniminium pentahydrochloride) (DOSPA), (N-[1-(2,3-dioleyloxy)propyl]-N, N, N-trimethylammonium) (DOTMA), (N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium) (DOTAP), 3β-[N-(N′,N′-dimethylaminoethane)-carbamoyl]cholesterol (DC-Chol), N4-cholesteryl-Spermine (GL67), 1,2-dioleyloxy-3-dimethylaminopropane (DODMA), O-alkyl phosphatidylcholines derivative, and dimethylammonium-propane (DAP) derivative.
5. The lipid nanoparticle of claim 1, wherein the cationic lipid is a cationic cholesterol derivative.
6. The lipid nanoparticle of claim 5, wherein the cationic cholesterol derivative is monoarginine-cholesterol (MA-Chol).
7. The lipid nanoparticle of claim 1, wherein the adjuvant is immunostimulatory single- or double-stranded oligonucleotide, immunostimulatory small-molecule compound, or a combination thereof.
8. The lipid nanoparticle of claim 7, wherein the single- or double-stranded oligonucleotide is a CpG oligonucleotide, a STING -active oligonucleotide, or a combination thereof.
9. A vaccine composition comprising the lipid nanoparticle claim 1 as an active ingredient.
10. The vaccine composition of claim 9, wherein the vaccine composition is for preventing or treating cancer.
11. A cancer vaccine kit which comprises a lipid nanoparticle including a tumor-associated antigen, a phospholipid, a cationic lipid, and an anionic drug as a first vaccine composition; and comprises the lipid nanoparticle and an immune checkpoint inhibitor as a second vaccine composition.
12. The cancer vaccine kit of cairn 11, wherein the immune checkpoint inhibitor is an anti-PD-1 antibody or an anti-PD-L1 antibody.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0116] Hereinafter, the present invention will be described more specifically with reference to examples. It will be apparent to those skilled in the art that these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.
EXAMPLE
[0117] Throughout this specification, “%” used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid and (volume/volume) % for liquid/liquid, unless otherwise stated.
Experimental Materials and Methods
Experimental Materials
[0118] 1,2-Distearoyl-sn-glycero-3-phosphoethanolamine-N-[carboxy(polyethylene glycol)-1000](DSPE-PEG.sub.1000), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[PDP(polyethylene glycol)-2000](DSPE-PEG.sub.2000-PDP), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (DPPE-Rhodamine) were purchased from Avanti Polar Lipids (Alabaster, Ala., USA). Boc-Arg(Pbf)-OH and cholesterol were purchased from Sigma Aldrich (St. Louis, Mo., USA). CpG oligodeoxynucleotide (CpG ODN; 5′-TCC ATG ACG TTC CTG ACG TT-3′) and control ODN (5′-TCC ATG AGC TTC CTG AGC TT-3′) were synthesized using a phosphorothioate backbone by Genotech (Daejeon, Korea). OVA.sub.257-264 SIINFEKL (OVAPEP) and SIINFEKL (C-OVA.sub.PEP) peptide with N-terminal cysteine were synthesized by Cosmo Genetec (Seoul, Korea). All other reagents were purchased from Sigma Aldrich unless otherwise indicated.
Animals and Cells
[0119] Female C57BL/6 mice were obtained from Orient Bio (Korea) and housed under pathogen free conditions. The animal care and experimental procedures have been approved by the Animal Care and Use Committee of the Korea Advanced Institute of Science and Technology (KAIST). The DC2.4 murine dendritic cell line was provided by Dr. K. L. Rock (University of Massachusetts Medical School, Worcester, Mass., USA). The B3Z murine CD8.sup.+ T hybridoma cell line was provided by Professor Yongtaek Lim (Sungkyunkwan University). DC2.4 and 83Z cells were maintained using RPMI-1640 medium (WELGENE, Geongsan-si, Korea) supplemented with 10% heat-inactivated fetal bovine serum (FBS; Welgene), 1% penicillin/streptomycin, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 1× non-essential amino acid and 50 μM 2-mercaptoethanol. EL4 murine lymphoma cell line, and E.G7-OVA murine EL4 lymphoma cell line transfected with Ovalbumin were purchased from ATCC (American Type Culture Collection; Manassas, Va., USA). EL4 cells were grown in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate and 50 μM 2-mercaptoethanol. E.G7-OVA cells were grown in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin/streptomycin, 2 mM L-glutamine, 4.5 g/L glucose, 10 mM HEPES, 1 mM sodium pyruvate, 50 μM 2-mercaptoethanol and 0.5 mg/mL G418 (Gibco, Grand Island, N.Y., USA). All cells were maintained at 37° C. in a humidified atmosphere containing 5% CO.sub.2.
Flow Cytometry
[0120] All antibodies were purchased from BioLegend (San Diego, Calif., USA), eBiosciences (San Diego, Calif., USA) and Tonbo Biosciences (San Diego, Calif., USA). Antibodies used included anti-CD16/CD32 (clone 2.4G2), anti-CD45 (clone 30-F11), anti-CD3 (clone 145-2C11), anti-CD8 (clone 53-6.7), anti-CD19 (clone 1D3), anti-CD169 (clone 3D6.112), anti-CD11b (clone M1/70), anti-CD11c (clone N418), anti-MHC II (clone M5/114.15.2), anti-CD40 (clone 3/23), anti-CD80 (clone 16-10A1), anti-CD86 (clone GL-1), anti-IFN-y (clone XMG1.2), anti-Granzyme B (clone 16G6), anti-PD-1 (clone 29F.1A12), and anti-PD-L1 (clone 10F.9G2). Cells were blocked with anti-CD16/CD32 antibody at 4° C. for 10 minutes, and immunostained with different antibodies at 4° C. for 20 to 30 minutes. Dead cells were excluded by staining with 7-AAD viability staining solution (BioLegend) or Ghost Dye™ Violet 450 (Tonbo Biosciences). Flow cytometry was performed using a LSRFortessa flow cytometer (BD Biosciences, San Jose, Calif., USA), and data were analyzed using FlowJo software (TreeStar),
Synthesis of OVA.SUB.PEP.-Phospholipid Conjugate
[0121] The C-OVAPEP peptide was conjugated to DSPE-PEG.sub.2000-PDP by a disulfide exchange reaction. Briefly, 2 mg of C-OVAPEP and 7.8 mg of DSPE-PEG.sub.2000PDP were dissolved in 200 μl DMSO and the solution was gently vortexed overnight at room temperature.
[0122] 200 μl of acetonitrile was added thereto, and the mixture was quenched, and purified by high performance liquid chromatography (HPLC, Agilent) using a C4 column (Nomura Chemical), The product-containing fraction was lyophilized to give a conjugate (DSPE-PEG2000-OVAPEP) as a white solid. The conjugate was further analyzed by matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) m as spectroscopy (Bruker).
Preparation and Characterization of OVA.SUB.PEP.-SLNP@CpG Nanovaccine
[0123] Monoarginine-cholesterol (MA-Chol) was synthesized as described above (Lee, J. et al. Theranostics 6, 192-203, 2016). A nanovaccine (OVA.sub.PEP-SLNP@CpG) based on small lipid nanoparticles (SLNP) was prepared by film formation and rehydration. Briefly, MA-Chol (3.89 μmol), DOPE (3.89 μmol), DSPE-PEG1000 (0.2 μmol) and DSPE-PEG2000-OVAPEP (0.02 μmol) were added to a glass vial and dried overnight under vacuum to completely remove the residual solvent. The resulting lipid film was rehydrated with 1 ml of HEPES-buffered glucose (HBG) containing 1.65 nmol of CpG ODN. The solution was sonicated for 10 minutes, then stirred with a magnetic bar at room temperature for at least 4 hours, and extruded at least 11 times using a small extruder (Avanti Polar Lipids). The morphology and size of OVA.sub.PEP-SLNP@CpG was evaluated by transmission electron microscopy (TEM) with 1% uranylacetate solution for negative staining. The average size of the nanoparticles was measured using ImageJ software (National Institutes of Health), and the hydrodynamic size and zeta potential thereof were measured at ambient temperature by dynamic light scattering (DLS) using a Zetasizer Nano range system (Malvern, Worcestershire, UK). The efficiency of CpG ODN loading was evaluated using a Sepharose CL-4B size exclusion column (Sigma Aldrich).
[0124] OVA.sub.PEP-SLNP@CpG was loaded onto the column washed with HEPES-buffered saline (HBS); 15 eluted fractions were collected, each CpG ODN was measured using Quant-iT OliGreen ssDNA reagent (Thermo Fisher). The loading efficiency of CpG ODN was determined by mixing 100 μl of each fraction with 20 μl of 5% Triton-X 100 and 100 μl of OliGreen, and measuring the fluorescence intensity at an excitation wavelength of 480 nm and an emission wavelength of 520 nm. Fractions 2-4 contained the nanovaccine, but fractions 6-10 contained free CpG ODN due to their size differences. The loading efficiency of 1.65 nmol of CpG ODN in 8 μmol of SLNP was almost 100%.
Cell Viability Assays
[0125] The cytotoxicity of the nanovaccine was assessed by analysis of water-soluble tetrazolium salt (WST-1) using the EZ-Cytox Cell Viability Assay kit (DoGenBio, Seoul, Korea) according to the manufacturer's instructions. Briefly, DC2.4 cells were seeded into 96-well plates at a density of 1×10.sup.4 cells per well in 100 μl medium, and incubated overnight at 37° C. The cells were treated with OVA.sub.PEP-SLNP@CpG and incubated at 37° C. for 24 hours. 10% volume of WST-1 reagent was added to each well and the plates were incubated at 37° C. for 4 hours, Absorbance was measured at 450 nm with a microplate reader (VERASmax™, Molecular Devices).
[0126] In Vitro Uptake of Dendritic Cells (DCs)
[0127] Intracellular uptake of the OVA.sub.PEP-SLNP@CpG nanovaccine was assessed by flow cytometry and confocal laser scanning microscopy. Briefly, DC2.4 cells were seeded into 6-well plates at a density of 5×10.sup.5 cells per well in 2 ml medium, and allowed to adhere overnight. To detect the intracellular uptake of the nanovaccine, 0.5 wt % of DPPE-rhodamine dye was added to the lipid nanoparticle formulation. Cells were incubated with 200 μM of rhodamine-labeled nanovaccine for 4 hours, and washed with PBS. Cell uptake was assessed by flow cytometry. To confirm cellular uptake by confocal microscopy, DC2.4 cells were seeded at a density of 4×10.sup.4 cells per well in 0.5 ml medium on coverslips of 24-well plates, grown and adhered overnight. Cells were incubated with 200 μM rhodamine-labeled nanovaccine for 4 hours, washed with PBS, and fixed with 10% formalin solution, and their nuclei were stained with DRAQ5 (Thermo Asher). All samples were imaged by a confocal laser scanning microscope (LSM 780; Carl Zeiss).
Generation, Uptake, Maturation and T Cell Cross-Priming of BMDCs
[0128] BMDC was produced as described in Kang, S. et al. (J Control Release 256, 56-67, 2017). To assess the intracellular uptake of nanovaccines, BMDCs were seeded into 12-well plates ata density of 3×10.sup.5 cells per well in 0.5 ml medium and allowed to adhere overnight. After incubation with 200 μM Rhodamine-labeled nanovaccine for 4 hours, cells were washed, harvested and stained with anti-CD11c-PE/Cy7 and anti-MHCII-APC antibodies,
[0129] Cell uptake was assessed by flow cytometry. To assess the ability of nanovaccines to enhance DC maturation, immature BMDCs were cultured in 12-well plates at a density of 5×10.sup.5 cells per well in 0.5 ml medium and allowed to adhere overnight. BMDCs were cultured in HBG buffer, soluble CpG, soluble OVA.sub.PEP, soluble OVA.sub.PEP+CpG, OVA.sub.PEP-SLNP@ODN or OVA.sub.PEP-SLNP@CpG (CpG: 0.1 μM; OVA.sub.PEP: 1.2 μM; SLNP: 0.48 mM) for 24 hours. Then, BMDC was washed, harvested, stained with anti-CD11c-PE/Cy7, anti-MHC±anti-CD80-FITC and anti-CD86-PE antibodies, and analyzed by flow cytometry.
[0130] To evaluate the cross-priming of T cells, BMDCs were seeded into 12-well plates at a density of 1×10.sup.6 cells per well in 1 ml medium and allowed to adhere overnight. BMDCs were incubated with HBG buffer, soluble CpG, soluble OVA.sub.PEP, soluble OVA.sub.PEP+CpG, OVA.sub.PEP-SLNP@ODN or OVA.sub.PEP-SLNP CpG (CpG: 0.1 μM; OVA.sub.PEP: 1.2 μM; SLNP: 0.48 mM) for 18 hours, harvested, and washed with citrate-phosphate buffer (pH 3.2) on ice for 3 minutes. Peptide/MHC class I complexes were removed from the surface,
[0131] These BMDCs were then co-cultured with B3Z CD8.sup.+ T hybridoma cells for 24 hours. Briefly, BMDCs were seeded into 96-well U-bottom plates at a density of 2×10.sup.4cells per well in 0.1 ml buffer, and then B3Z cells were added to each well at a density of 4×10.sup.4 cells per well in 0.1 ml medium and cultured for 24 hours. The suspension was centrifuged to isolate the cell pellet and supernatant.
[0132] β-galactosidase activity was assayed on cell pellets. Briefly, the harvested cell pellet was washed and resuspended in CPRG assay buffer (PBS containing 0.1% Triton X-100, 100 μM 2-mercaptoethanol, 10 mM MgCl.sub.2 and chlorophenol red-3-D-galactopyranoside (CPRG)). Each resuspended pellet was transferred to a well of a 96-well plate, and plates were incubated in the dark at 37° C. for 3 hours. The absorbance of each well at 570 nm was measured using a microplate reader, IL-2 concentration in the collected supernatant was assessed using an IL-2 ELISA kit (R&D Systems, Minneapolis, USA) according to the manufacturer's instructions.
In Vivo Lymphatic Drainage, Uptake and DCs Maturation
[0133] To assess the lymphatic drainage of OVA.sub.PEP-SLNP@CpG nanovaccine, 037 wt % of pegylated cypate dye was added to the nanoparticle formulation. The pegylated cypate-loaded nanovaccine was injected subcutaneously into the paw soles of C57BL/6 mice. After 2, 4, 8 and 12 hours, the fluorescence signal was assessed using an in vivo imaging system (IVIS). Lymphatic drainage was also assessed by confocal microscopy. Rhodamine dye-labeled nanovaccine was subcutaneously injected into the paw soles of mice, and popliteal LNs were removed after 8 hours. The removed LN was implanted in OCT compound (Leica, Germany) and frozen, and divided into 15 μm slices using a frozen microtome (CM1850; Leica), which was mounted on a glass slide. LN sections were fixed with 10% formalin solution and blocked with PBS containing 2% bovine serum albumin (BSA) for 1 hour at room temperature. Slides were mounted with a VectaMount™ AQ mounting medium (Vector Laboratories, Burlingame, Calif., USA) and imaged by confocal laser scanning microscopy. To confirm uptake by APC in LN, rhodamine-labeled nanovaccine was injected subcutaneously into the paw soles of mice. After 8 hours, the popliteal lymph node LN was removed. The removed LN was washed, excised and digested in collagenase type IV solution (1 mg/ml; Sigma Aldrich) at 37° C. for 30 minutes. The cells were washed again and passed through a 70 μm cell strainer (Falcon) to recover a single cell suspension. LN cells were cultured with anti-CD45-Pacific Blue, anti-CD3-PerCP/Cy5.5, anti-CD19-PerCP/Cy5.5, anti-CD169-F ITC, anti-CD11b-APC, anti-CD11c-PE/Cy7 antibody and with 7-AAD at 4° C. for 20 minutes. These cells were washed and analyzed by flow cytometry.
Immunization
[0134] Six-week-old female C57BU6 mice were immunized using a homologous prime-boost regimen. Mice were divided into four groups, which were injected subcutaneously with HBG buffer vehicle, soluble OVA.sub.PEP+CpG, OVA.sub.PEP-SLNP ODN, or OVA.sub.PEP-SLNP CpG (CpG: 0.4 nmol per mouse; OVA.sub.PEP: 5 nmol per mouse; SLNP: 2 μmol per mouse) into both paw soles at time points indicated, and immunized.
Evaluation of Antigen-Specific T Cell Responses
[0135] As mentioned above, mice divided into 4 groups were immunized 3 times at 10-day intervals and sacrificed 3 weeks after the last immunization. To evaluate antigen-specific T cell responses, splenocytes were restimulated ex vivo with OVA.sub.PEP (SIINFEKL peptide; 10 μg/ml). The amount of secreted IFN-γ was determined by enzyme-linked immunosorbent assay (ELISA) and the number of INF-γ producing cells was assessed by enzyme-linked immunospot (ELISpot) assay. INF-γ and granzyme B produced by CD8.sup.+ T cells were quantified by intracellular cytokine staining (ICS). To measure INF-γ levels by ELISA, splenocytes were seeded into 96-well U bottom plates at a density of 3×10.sup.5 cells per well and restimulated with OVA.sub.PEP for 72 hours. The culture supernatant was harvested and the IFN-γ concentration was measured using an IFN-γ ELISA kit (R&D Systems). To measure INF-γ producing cells by ELISpot, splenocytes were seeded into 96-well microplates coated with a monoclonal antibody specific for mouse IFN-γ at a density of 3×10.sup.5 cells per well, and the cells were restimulated with OVA.sub.PEP for 30 hours INF-γ producing spots were developed using a mouse IFN-γ ELISpot kit (R&D Systems) according to the manufacturer's protocol. After development, blue-black spots of cytokine localization sites were counted using an automated ELISpot reader (AID GmbH, Strassberg, Germany). For the ICS assay, splenocytes (3×10.sup.6 cells per round bottom test tube) were restimulated with OVAPEP for 1 hour. To suppress the intracellular transport of cytokines, GolgiStop™ or GolgiPlug™ (BD Biosciences) was added to each tube. The cells were incubated for 5 hours, and stained with Ghost Dye™ Violet 450 at 4° C. for 30 minutes, and the apoptotic cells were identified, and then stained with anti-CD3-PerCP/Cy5.5 and anti-CD8-APC/Cy7 antibodies at 4° C. for 20 minutes. For intracellular cytokine staining, the cells were permeabilized using Cytofix/Cytoperm™ solution (BD Biosciences) and incubated with PE-conjugated anti-IFN-γ and Alexa Fluor 647-conjugated anti-Granzyme B antibodies. Samples were washed and analyzed by flow cytometry.
In Vivo Cytotoxic T Lymphocyte Assay
[0136] As mentioned above, mice were divided into four treatment groups and immunized three times at 7-day intervals. Seven days after the last immunization, mice were injected with a mixture of cells prepared from splenocytes of non-immune C57BL/6 mice. Half of the splenocytes were pulsed with OVA.sub.PEP (1 μg/ml) at 37° C. for 1 hour, and the other half was not pulsed. Non-pulsed cells were labeled with 0.5 μM carboxyfluorescein succinimidyl ester (CFSE), and OVAPEP pulsed cells were labeled with 5 μM CFSE for 10 minutes. A 1:1 mixture of pulsed (CFSE.sup.high) and non-pulsed (CFSE.sup.low) cells was injected intravenously into immunized mice. 18 hours after injection, the splenocytes of recipient mice were harvested and analyzed by flow cytometry. The relative numbers of CFSE.sup.high and CFSE.sup.low cells were measured. Antigen-specific target targeted apoptosis was calculated using the following Equation:
Specific target apoptosis percentage=100−[100×{(% CFSEhigh immunized mouse/% CFSE.sup.low immunized mouse)/(% CFS.sup.high non-immunized mouse/%CFSE.sup.low N non-immunized mouse)}]
Antitumor Efficacy Test
[0137] To evaluate the therapeutic effect in tumor prevention, mice were immunized three times at 10-day intervals with each vaccine modality described above. 3 Weeks after the last immunization, 2×10.sup.5EL4 cells were inoculated subcutaneously in one flank of each mouse, and the other side was inoculated subcutaneously with 2×10.sup.5 E.G7-OVA cells. The tumor growth was monitored every two days using digital calipers, and the tumor volume was calculated as 0.5× length×width.sup.2, Mice were euthanized when the average tumor volume reached the ethical dead point (˜2000 mm.sup.3).
[0138] To analyze the effect of treatment in the tumor volume reduction. 2×10.sup.5 E.G7-OVA cells were subcutaneously inoculated into the right flank of each mouse. When the average tumor volume reached ˜50 mm.sup.3, mice were randomly divided into 4 treatment groups and immunized three times at 4-day intervals. To evaluate the efficacy of combination immunotherapy, mice underwent two immunization cycles with and without antibodies to mouse-PD-1 (alpha PD-1; BioXcell; done: RMP1-14). 2×10.sup.5 E.G7-OVA cancer cells were subcutaneously injected into the right flank and inoculated into mice. The first immunization cycle, consisting of three subcutaneous injections of OVA.sub.PEP-SLNP@CpG nanovaccine at 4-day intervals, started after 6 days. On the 20th day, mice were divided into mice with small-tumors (good responders) and mice with large-tumors (poor responders). Poor responders were sacrificed when the tumor volume reached ˜2000 mm.sup.3. Good responders started on the 26th day and started the second immunization cycle. The second immunization cycle consisted of two subcutaneous injections at 6-day intervals. Additionally, alpha PD-1 (200 μg per injection) was intraperitoneally administered to mice on days 1, 3 and 5 after each vaccination. When the dead point was reached, the tumor tissue was removed, weighed and photographed.
In Vitro Induction of PD-L1
[0139] E.G7-OVA cells were seeded into 24-well plates at a density of 1×10.sup.5 cells per well in 0.5 ml medium, Cells were treated with recombinant murine IFN-γ (100 ng/ml; Peprotech, Rocky Hill, N.J., USA) for 48 hours, washed, harvested and stained with PE-conjugated anti-PD-L1 antibody. In vitro induction of PD-L1 was confirmed by flow cytometry.
Histopathological Analysis of Tumor Tissues
[0140] The excised tumor tissue was implanted in OCT solution, immediately frozen, cut into 20 μm slices using a freezing slide, which was mounted on a glass slide. Tissue sections were fixed with 10% formalin solution for 10 minutes, and blocked with PBS containing 2% BSA for 1 hour at room temperature. The tissue sections were incubated with biotin-conjugated anti-mouse CD8a antibody (1:100 dilution; Tonbo Biosciences) overnight at 4° C., so that CD8+ T cell infiltration into tumor tissue was evaluated.
[0141] The tissue sections are then washed and incubated with PE-conjugated anti-streptavidin antibody (1:200 dilution; BD Biosciences) at room temperature for 1 hour. The tissue sections were incubated with rat monoclonal anti-PD-L1 antibody (1:100 dilution; Abcam) overnight at 4° C. so that PD-L1 induction in tumor tissue was evaluated. These sections were then washed and incubated with Alexa Fluor 488-conjugated goat anti-rat IgG antibody (1:100 dilution; Abcam) at room temperature for 1 hour.
[0142] Nuclei were stained with Hoechst 33342 (1:5000 dilution) and slides were mounted with VectaMount AQ mounting medium, All sections were imaged by confocal laser scanning microscopy. The excised tumor tissue was fixed with 10% formalin solution, and embedded in paraffin and cut into 4 μm slices. These sections were stained with H&E, and apoptotic cells were measured using the Dead End Colorimetric TUNEL system (Promega, Madison, Wis., USA) according to the manufacturer's instructions. AH slides were analyzed using a Nikon upright fluorescence microscope.
Tetramer Analysis
[0143] Mouse peripheral blood obtained by retroorbital bleed was collected in a serum separator tube (BD). Red blood cells (RBC) were removed by incubating with 1 ml of RBC lysis buffer (Biolegend) with gentle shaking at room temperature for 2 minutes. For tetramer staining, blood cells were incubated with iTAg H-2Kb OVA tetramer-PE (MBL, Japan) at 4° C. for 30 minutes. Then, the cells are washed, and incubated with Pacific Blue-conjugated anti-CD45, PE/Cy7 conjugated anti-CD3, Alexa Fluor 488 conjugated anti-CD8, and APC conjugated anti-PD-1 antibodies and 7-AAD at 4° C. for 20 minutes. Cells were washed again and analyzed by flow cytometry.
Statistical Analysis
[0144] Data were expressed as mean±S.E.M. Groups were compared by a one-way analysis of variance (ANOVA) with post hoc Tukey test using GraphPad Prism 5 (GraphPad Software). Statistical significance was defined as P<0.05.
Example 1: Synthesis and Characterization of Antigen- and Adjuvant-Carrying Nanovaccine (OVA.SUB.PEP.-SLNP@CpG) of the Present Invention
[0145] The small lipid nanoparticle (SLNP) used as antigen- and adjuvant-carrying nanovaccine in the present invention were prepared with two phospholipids: i) 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and ii) 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-[carboxy(polyethylene glycol)1000](DSPE-PEG1000); and a cholesterol derivative: monoarginine-cholesterol (MA-Chol).
[0146] The DOPE is a neutral lipid involved in endosome escape of lipid nanoparticles. Therefore, the incorporation of DOPE into SLNPs can enhance antigen migration from endosomes to the cytoplasm to promote antigen expression at the cell membrane. The DSPE-PEG.sub.1000 is a PEGylated phospholipid that increases colloidal stability of SLNP under physiological conditions to promote lymphatic drainage of SLNP.
[0147] The MA-Chol is a cationic molecule composed of arginine, cholesterol, and major components of SLNP, and enables complex formation between SLNP and oligonucleotides (see
[0148] The adjuvant used in the present invention is a toll-like receptor 9 agonistic CpG oligodeoxynucleotide (CpG ODN). The combination of CpG ODN and ICB immunotherapy has been reported to have potent synergistic antitumor efficacy, and several clinical trials of this combination are currently underway. The model tumor antigen was an MHC class I-restricted epitope of ovalbumin (SIINFEKL; named OVAPEP), which has been shown to stimulate CD8.sup.+ T cell responses. OVA.sub.PEP was chemically bound adhered to the end of PEGylated DSPE via a disulfide bond (see
[0149] When internalized into cells, this molecule was cleaved by glutathione in the cytoplasm, and the released free OVAPEP was presented to the cell membrane by MHC class I or II (see
[0150] An antigen-labeled, CpG adjuvant-containing SLNP, designated as OVAPEP-SLNP@CpG, was prepared by a one-pot sequential process of film formation and rehydration (see
[0151] The transmission electron microscopy (TEM) showed that OVA.sub.PEP-SLNP@CpG, prepared using 0.25 mol % of OVA.sub.PEP antigen, had a spherical morphology, and had an average diameter of ˜72 nm by analyzing 155 particles (see
[0152] These OVA.sub.PEP-SLNP@CpG particles had a hydrodynamic size of ˜104.5 nm and a zeta potential of +0.23 mV, indicating a neutral surface charge. As a control nanovaccine similar in size and zeta potential to OVAPEP-SLNP@CpG, a non-immunostimulant control ODN-conjugated SLNP (OVAPEP-SLNP@ODN) was prepared using the same procedure (see
Example 2: In Vitro DC Maturation and T Cell Cross-Priming Enhancing Effect of OVA.SUB.PEP.-SLNP@CpG Nanovaccine of the Present Invention
[0153] Since the in vivo nanovaccine is expected to be uptaken by dendritic cells (DC) or macrophages, the cytotoxicity of OVA.sub.PEP-SLNP@CpG to DCs was evaluated using the WST-1 assay. The nanovaccine of the present invention did not affect the viability of DC2.4 murine DC even at a high CpG concentration of 500 nM (see
[0154] The intracellular uptake of nanovaccines by DCs was evaluated using OVA.sub.PEP-SLNP@CpG labeled with rhodamine dye, and the flow cytometry showed a new band of rhodamine-positive cells, which was different from the bands of original DC2.4 cells and bone marrow-derived DCs (BMDCs) (see
[0155] Confocal laser scanning microscopy further confirmed the intracellular localization of the nanovaccine (see
[0156] Maturation of DCs and membrane presentation of delivered antigens via MHC class I molecules are essential for inducing an effective CD8+ T cell response. Flow cytometry was performed to evaluate whether OVA.sub.PEP-SLNP©CpG could enhance DC maturation. The frequency of mature DCs expressing the marker CD11c.sup.+ MHCIIhigh and the expression of costimulatory molecules (CD80 and CD86) were significantly higher than in other cell groups (see
[0157] A mixture of soluble antigen and adjuvant (OVA.sub.PEP+CpG CDN) or control OVA.sub.PEP-SLNP@ODN did not induce DC maturation, which indicates the importance of the CpG adjuvant. However, soluble CpG alone could not induce DC maturation, which suggests that DC needs an appropriate delivery system. These findings indicate that the OVA.sub.PEP-SLNP@CpG nanovaccine can be uptaken by DCs to induce maturation.
[0158] When the T cell receptor specifically recognizes the OVA.sub.PEP (SIINFEKL)-MHC complex, the cross-priming ability of nanovaccines in BMDC and CD8.sup.+ T cell hybridoma B3Z cells engineered to secrete β-galactosidase was evaluated. BMDCs were treated with each vaccine modality for 18 hours and washed thoroughly, whereby any antigenic peptides present on the MHC molecule were removed extracellularly so as to avoid intracellular processing and cross-presentation, and co-cultured with B3Z cells for 24 hours.
[0159] β-galactosidase assay and IL-2 enzyme-linked immunosorbent assays (ELISA) showed that OVA.sub.PEP-SLNP@CpG induced significantly higher levels of β-galactosidase and IL-2 secretion than other therapies (see
[0160] Taken together, the results of these in vitro studies suggest that the OVA.sub.PEP-SLNP@CpG nanovaccine is readily uptaken by DCs to induce maturation, and presents the antigen released to the surface via MHC, thereby an effectively cross-priming CD8.sup.+ T cells to antigen.
Example 3: Local Injection of OVAPEP-SLNP@CpG Results in Maturation of DCs in Lymph Nodes
[0161] Depending on size and surface function, locally injected nanovaccines have been shown to drain into regional lymph nodes (LNs), wherein the nanovaccine was uptaken by antigen presenting cells (APCs) such as DCs and macrophages. To evaluate the lymphatic drainage of the nanovaccine, the present inventors subcutaneously injected OVAPEP-SLNP@CpG labeled with a near-infrared dye into the paw soles of C57BL 6 mice. The in vivo imaging system (IVIS) showed a clear fluorescence signal intensity around the draining LN, and the fluorescence signal started after 2 hours and lasted for 12 hours (see
[0162] To investigate the distribution of nanovaccine in lymph nodes, OVA.sub.PEP-SLNP@CpG labeled with rhodamine dye was injected subcutaneously, and after 8 hours, draining popliteal LN was excised. Confocal microscopy showed that most of the nanovaccines were localized to the subcapsular sinus region of the lymph node (see
[0163] When OVA.sub.PEP-SLNP@CpG reaches the nearest lymph node within 2 hours, nanovaccines are more likely to be excreted directly into the LN via lymphatic vessels rather than being uptaken by DCs at the injection site and delivered to the lymph nodes. This process takes ˜24 hours. The ability of APCs to uptake dye-labeled nanovaccine in popliteal lymph nodes was assessed by flow cytometry-based gating (see
[0164] Approximately 19.7% of macrophages (CD169.sup.+ CD11b.sup.+) and 25% of DCs (CD169− CD11c.sup.+) were rhodamine fluorescence-positive, which suggests a high level of nanovaccine uptake by APCs in lymph nodes (see
[0165] After confirming the ability of OVA.sub.PEP-SLNP@CpG of the present invention to activate and promote DC maturation in vitro, the present inventors evaluated DC maturation in popliteal lymph nodes containing nanovaccines by a gating strategy using the maturation markers CD40 and CD86 (see
[0166] OVA.sub.PEP-SLNP@CpG significantly increased the expression of CD40 and CD86, but a mixture of soluble OVAPEP and CpG, or control OVA.sub.PEP-SLNP@ODN slightly increased the expression of these markers (see
[0167] Taken together, these in vivo results demonstrated that the OVA.sub.PEP-SLNP@CpG nanovaccine of the present invention can be delivered directly to regional lymph nodes with high efficiency, and uptaken by DCs and macrophages residing in lymph nodes, and can effectively induce DC maturation.
Example 4: Evaluation of Antigen-Specific Cytotoxic T Cell Responses In Vivo
[0168] To assess the antigen-specific CD8+ T cell response of i) vehicle, ii) soluble OVA.sub.PEP+CpG, iii) OVA.sub.PEP-SLNP@ODN or iv) OVA.sub.PEP-SLNP@CpG, each substance was immunized to mice three times (0 day, 10 days, 20 days) at 10-day intervals, and sacrificed on the third week (41 days) after the third immunization (see
[0169] After splenocytes were isolated from immunized mice and restimulated with OVA.sub.PEP (SIINFEKL peptide), the secretion of interferon-gamma (IFN-γ), which is a representative cytokine secreted by activated CD8.sup.+ T cells, was measured by ELISA and ELISpot assays.
[0170] OVA.sub.PEP-SLNP@CpG immunization induced greater secretion of INF-γ in ELISA (see
[0171] Intracellular cytokine staining (ICS) was performed to test the functionality of activated CD8.sup.+ T cells, and INF-γ and granzyme B were measured using the gating strategy shown in Supplementary
[0172] The in vivo antigen-specific killing activity of these CD8.sup.+ T cells was assessed by adoptively transferring of a mixture of half splenocytes obtained from nonpulsed mice and half splenocytes pulsed with OVAPEP to recipient mice immunized with the respective vaccine regimens. After 18 hours of adoptive transfer, the antigen-specific killing ability of CD8.sup.+ T cells was assessed by flow cytometry (see
[0173] Percentage of OVA.sub.PEP-specific killing of metastasized splenocytes in mice immunized with OVA.sub.PEP-SLNP©CpG (89%) than in mice immunized with soluble OVA.sub.PEP+CpG (60.7%) or OVA.sub.PEP-SLNP@ODN (82.9%). was higher (see
[0174] Taken together, these results suggest that OVA.sub.PEP-SLNP@CpG can induce much higher antigen-specific killing activity in CD8.sup.+ T cells than physical mixture of soluble antigen+adjuvant,
Example 5: Prophylactic Effect: Tumor Prevention by OVA.SUB.PEP.-SLNP@CpG Nanovaccine
[0175] The present inventors investigated the in vivo effect of each vaccine regimen to prevent tumor growth using two mouse lymphoma cell lines, EL4 and E.G7-OVA. E.G7-OVA was derived from EL4 cells by transfection of the OVA gene.
[0176] First, mice were immunized three times at 10-day intervals with each of the four vaccine modalities. Three weeks after the third immunization, EL4 and E.G7-OVA cells were injected into the contralateral flanks of these mice, respectively (see
[0177] Although OVA.sub.PEP-SLNP@CpG did not inhibit the growth of EL4-derived tumors, the growth of E.G7-OVA-derived tumors in the contralateral flank was completely prevented (
[0178] Soluble OVA.sub.PEP+CpG had no effect in preventing tumor growth in the two cell lines, but OVA.sub.PEP-SLNP@ODN was moderately effective against both, but not more effective than OVA.sub.PEP-SLNP@CpG against E.G7-OVA-derived tumors (
[0179] This finding means that the nanovaccine of the present invention prevented tumor growth in an antigen-specific manner.
Example 6: Therapeutic Efficacy of OVA.SUB.PEP.-SLNP@CpG in an Established Tumor Model
[0180] Next, the present inventors evaluated the therapeutic efficacy of the OVA.sub.PEP-SLNP@CpG nanovaccine of the present invention in E.G7-OVA tumor-bearing mice. When the average tumor volume reached ˜50 mm.sup.3, mice were randomly divided into 4 groups and immunized 3 times at 4-day intervals with each vaccine modality (see
[0181] Immunization induced with soluble OVA.sub.PEP+CpG or OVA.sub.PEP-SLNP@DN showed only moderate tumor growth inhibition compared to vehicle control, but the immunization induced with OVA.sub.PEP-SLNP@CpG of the present invention significantly inhibited tumor growth, and two of the seven mice lacked a tumor (see
[0182] Histopathological analysis of tumor tissues was performed to better understand the effects of nanovaccines at the cellular level, Immunohistochemistry (IHC) showed that CD8.sup.+ T cell infiltration was much higher in tumor tissues from mice immunized with our OVA.sub.PEP-SLNP@CpG of the present invention than in other groups (see
[0183] Although the tumor tissue was analyzed during the late rejection phase of the CTL response, the difference in the degree of T cell infiltration between groups was significant.
[0184] Hematoxylin and eosin (H&E) staining of tumor tissues showed that massive cell damage such as altered nuclei, enucleated necrotic cells and dead cell-derived debris occurred in the OVA.sub.PEP-SLNP@CpG-immunized group of the present invention, but this was not the case in the other groups (see
[0185] Terminal deoxynucleotidyl-transferase-mediated dUTP nick-end labeling (TUNEL) assay confirmed massive apoptosis in tumor tissues from the OVA.sub.PEP-SLNP@CpG-immunized group of the present invention (see
[0186] Taken together, these histopathological analysis indicate that the therapeutic efficacy of OVA.sub.PEP-SLNP@CpG immunization is caused by the mass death of cancer cells according to increased T cell infiltration into the tumor.
Example 7: Tumor Regrowth Inhibitory Effect of Sequential Combination of Immune Checkpoint Blocking and OVA PEP-SLNP tx. CpG Nanovaccine
[0187] Although the OVA.sub.PEP-SLNP@ CpG nanovaccine was highly effective in preventing and inhibiting tumor growth, the therapeutic response varied in individual mice. To evaluate the change in antitumor effect, tumors obtained from OVA.sub.PEP-SLNP@CpG-immunized mice were arbitrarily divided into two groups based on their relative size: large tumor group (>˜60 mm.sup.3, two of seven mice) and small tumor group (<˜60 mm.sup.3, three of seven mice).
[0188] IHC showed that PD-L1 expression was significantly higher in small tumor groups (classified as ‘good responders’) than in large tumor groups (classified as ‘poor responders’) or unvaccinated controls (see
[0189] The present inventors have also found that PD-L1 expression in E.G7-OVA cancer cells is markedly induced by treatment with IFN-γ, which is a typical antitumor cytokine secreted by activated CD8+ T cells (
[0190] Since PD-L1 expression in TME is increased by IFN-γ secreted from T cells, these findings suggest that greater antitumor efficacy in good responders may result from higher CD8.sup.+ T cell infiltration and cancer cell death by IFN-γ. However, since PD-L1 expression and co-localization of tumor-infiltrating T cells in tumor tissues are closely related to adaptive immune suppression and resistance, good responders with high PD-L1 induction in tumors can develop adaptive immune resistance through T cell depletion, leading to tumor recurrence. These findings suggest that new combinatorial strategies involving the order and timing of treatment of cancer nanovaccines and ICBs, need to be investigated.
[0191] To investigate the validity of sequentially combining nanovaccine with ICB therapy, 50 mice were vaccinated twice with or without antibody to mouse PD-1 (αPD-1), Specifically, after inoculation to the side with E.G7-OVA cancer cells, OVA.sub.PEP-SLNP@CpG nano-vaccine was subcutaneously injected 3 times after 6 days to perform a first immunization (
[0192] Tetramer assay showed that the percentage of OVA.sub.PEP (SIINFEKL)-specific CD8.sup.+ T cells was approximately 2-fold higher in good responders than in poor responders and unimmunized controls (see
[0193] This finding is in good agreement with reports showing that PD-1 expression is upregulated in antigen-specific CD8+ T cells induced by vaccination,
[0194] It is in good agreement with reports showing that it can be considered to reflect T cell depletion and activation, which can be considered to reflect T cell depletion and activation. The number of PD-1.sup.+ CD8+ T cells in TME was shown to be positively correlated with the number of these cells in peripheral blood. Further, infiltration of PD-1.sup.+ CD8.sup.+ T cells into the tumor was reported to be a positive marker for response to ICB therapy. The number of PD-1.sup.+ CD8.sup.+ T cells in peripheral blood was high in good responders (see
[0195] The 40 good responders were randomly divided into 4 groups of 10 mice each (see
[0196] i) one group was vehicle control, ii) another was treated with αPD-1 alone and iii) the other was reimmunized with OVA.sub.PEP-SLNP@CpG, and iv) the other was reimmunized with OVA.sub.PEP-SLNP@CpG and treated with αPD-1.
[0197] Starting on day 26, the last two groups of mice were immunized twice with the OVA.sub.PEP-SLNP@CpG of the invention at 6-day intervals, and the second and fourth groups of mice received 6 intraperitoneal injections of αPD-1 at 2-day intervals.
[0198] Only the vehicle control showed rapid regrowth of the tumor within a few days (see
[0199] Unlike the initial expectations by the present inventors, αPD-1 alone, which was expected to revitalize exhausted PD-1.sup.+ CD8.sup.+ T cells, could hardly inhibit tumor regrowth. Because antigen-specific T cells have only short-term activity, an additional nanovaccine was needed to boost the CTL response again. The second cycle of OVA.sub.PEP-SLNP@CpG alone immunization of the present invention partially inhibited tumor regrowth, but its efficacy was not significantly different compared to the vehicle control group or the αPD-1 group, which suggests that when PD-L1 expression is induced to high levels in tumors after the first immunization cycle, the treatment results of the second cycle immunization with the nanovaccine appear to be poor. In contrast, tumor regrowth was effectively inhibited by the combination of the OVA.sub.PEP-SLNP@CpG nanovaccine of the present invention+the second cycle of αPD-1 (see
[0200] These results suggest that the effect of combination therapy differs depending on the administration order and timing of nanovaccine and ICB therapy.
[0201] That is, initial immunization with nanovaccines can result in high tumor growth inhibition, but at the same time, it can induce tumor expression of PD-L1 and lead to antigen-specific T cell depletion. When treating a good responder for first-cycle immunization with a combination of second-cycle nanovaccine immunization+ICB it can lead to a strong therapeutic response (see
Discussion
[0202] Cancer nanovaccines using nanomaterials as antigen and/or adjuvant-delivery carriers can induce tumor antigen-specific T cell immunity, and have shown potential as a therapeutic method in in vivo animal models. Additionally, the combination of ICB immunotherapy and cancer nanovaccine can further enhance the anti-tumor efficacy of cancer nanovaccine.
[0203] The lack of an optimal vaccination regimen that can address not only the problems related to antigen-delivering nanomaterials themselves, such as toxicity and manufacturability, but also the adaptive resistance of tumors to cancer vaccines has hampered the clinical application of cancer nanovaccines.
[0204] To solve these problems, the present inventors have developed novel antigen/adjuvant-delivery nanoparticles made of biocompatible lipid components. These nanoparticles, in combination with ICB immunotherapy, showed very strong antitumor efficacy in both prophylactic and therapeutic tumor models, and have demonstrated the validity of a new treatment regimen based on the order and timing of modalities that effectively suppress tumor recurrence.
[0205] The lack of toxicity associated with antigen-delivery nanomaterials and the antitumor efficacy of nanovaccines are key factors for successful clinical application. The present invention has been disclosed the construction of a cancer nanovaccine using a biocompatible and non-toxic naturally occurring or synthetic components. Two biocompatible and non-toxic neutrally charged phospholipids, i.e., DOPE and DSPE-PEG1000, have been widely used in clinically available liposome-based therapies. The present inventors showed in previous studies that MA-Chol, which is a cationic cholesterol derivative, can form stable complexes with oligonucleotides such as siRNA and CpG ODN. MA-Chol is biodegradable and non-toxic because it is synthesized from endogenous arginine and cholesterol via an ester bond. Actually, all three biocompatible and non-toxic components were able to successfully form SLNPs with CpG ODN, and the antigen/adjuvant-carrying nanovaccine (OVA.sub.PEP-SLNP@CpG) was sufficiently stable in physiological media that it was released directly into the local LN according to local injection. These results shows that SLNP is clinically suitable for use in cancer nanovaccines. Further, the model tumor antigen (OVA.sub.PEP) was linked to the SLNP surface via a disulfide bond, so that the intact antigen was released in the cytoplasm and effectively displayed on the MHC of APC. In fact, the OVA.sup.PEP-SLNP@CpG nanovaccine presented antigens that were efficiently uptaken by DCs in vitro and in vivo and released on the DC surface via MHC, which made it possible to effectively cross-prime CD8.sup.+ T cells to antigen. Although the experimental conditions used in this example may be different from those of other nanovaccine systems, the antitumor efficacy of OVA.sub.PEP-SLNP@CpG was impressive because 4 of 6 mice in the prophylactic tumor model and 2 of 7 mice in the therapeutic tumor model had no tumor. The efficacy of these nanovaccines may be due to their ability to induce strong CTL responses against antigen-expressing E.G7 tumors. OVA.sub.PEP-SLNP©CpG is made of a biocompatible, non-toxic material and exhibits strong antitumor activity, so it has clinical potential for use as a therapeutic cancer nanovaccine.
[0206] The generation of adaptive resistance of tumors to cancer vaccines has interrupted clinical applications of cancer vaccines. PD-L1 expression and tumor-infiltrating CD8.sup.+ T cell colocalization are closely associated with adaptive immune resistance, This study focused on differences in therapeutic response to nanovaccines in individual E.G7 tumor-bearing mice. Tumor expression of PD-L1, infiltration of CD8.sup.+ T cells, and the ratio of circulating OVAPEP-specific/PD-1+CD8.sup.+ cells in peripheral blood were significantly higher in good responders than in poor responders and unvaccinated controls. This suggests that good responders can be regarded as ‘hot tumors’ that respond better to ICB therapy than ‘cold tumors’. On the other hand, these findings also indicated that good responders develop adaptive resistance to nanovaccines, leading to tumor recurrence if not properly treated. Therefore, only good responders were treated with the combination of nanovaccine and ICB therapy, Efforts were made to maximize treatment outcomes by varying the order and timing of each modality. The first cycle of vaccination was performed to systemically increase antigen-specific T cell immunity and induce PD-L1 expression in tumors. Good responders sensitive to ICB therapy were then treated with a second vaccination in combination with ICB antibody. However, treating good responders with αPD-1 alone was completely ineffective in inhibiting tumor regrowth, which suggests that booster vaccination is necessary to reactivate antigen-specific memory T cells derived from the first immunization cycle. Further, treating good responders with only the second cycle of OVA.sub.PEP-SLNP@CpG vaccination was ineffective. This indicates that T cell depletion by high PD-L1 induction in tumors is possible. The present inventors have found that only the combination of αPD-1 with the second cycle of OVA.sub.PEP-SLNP@CpG vaccination significantly improved the treatment outcome, amd resulted in effective inhibition of tumor regrowth or recurrence. This may be due to the re-boosting of antigen-specific T cell responses by the second vaccination, along with reversal of immune suppression by the ICB antibody. Taken together, these findings clearly indicate the importance of the treatment order and timing of each modality in combination therapies involving nanovaccines and ICB antibodies,
[0207] Despite their high therapeutic potential, cancer vaccines can stimulate cancer cells to produce immunosuppressive molecules and recruit immune regulatory cells to TME. For example, vaccine-induced CD8.sup.+ T cells upregulate PD-L1 and indoleamine-2,3-dioxygenase (IDO) expression and recruit T cells (Tregs) in a model of metastatic melanoma, thereby inducing immunosuppression. Further, cancer vaccines have been shown to upregulate the expression of NKG2A inhibitory receptors on tumor-infiltrating CD8.sup.+ T cells. Despite the presence of various inhibitory and immunosuppressive molecules, this study investigated the effect of nanovaccines on the expression of only one inhibitory molecule, PD-L1. Therefore, there is a need to investigate other immunosuppressive molecules induced by nanovaccines and their mechanisms of action. Furthermore, the reasons for the differences in therapeutic response to nanovaccines in individual mice with the same genetic background are unclear. Nevertheless, the results of the present invention indicate the importance of the order and timing of each modality in designing combination immunotherapy comprising nanovaccines. Although tumor size may not be a good marker for differentiating good and poor responders, the sequential combination strategy proposed in this study requires additional clinical evaluation of personalized therapy. Imaging modalities such as computer tomography to monitor tumor size and positron emission tomography to monitor tumor activity can be a criterion for distinguishing between good and bad responders.
[0208] In conclusion, the present inventors have developed a novel type of antigen/adjuvant-carrying nanovaccine composed of biocompatible and non-toxic lipid components. These nanovaccines showed very strong antitumor efficacy in both prophylactic and therapeutic tumor models. Further, a novel combination treatment regimen consisting of cancer nanovaccine and ICB immunotherapy was proposed according to the treatment order and timing. Such protocols can improve the persistence of anti-tumor immunity, including effective inhibition of tumor growth and recurrence. These findings further suggest the necessity for evaluating these new combination therapy regimens in other immunotherapy modalities.