Open Access, Peer-reviewed
pISSN 1598-2629 eISSN 2092-6685
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    Immune Netw. 2022 Oct;22(5):e42. English.
    Published online Oct 25, 2022.
    https://doi.org/10.4110/in.2022.22.e42
    Copyright © 2022. The Korean Association of Immunologists
    Original Article

    Efficient Anti-Tumor Immunotherapy Using Tumor Epitope-Coated Biodegradable Nanoparticles Combined With Polyinosinic-Polycytidylic Acid and an Anti-PD1 Monoclonal Antibody

    Sang-Hyun Kim,1 Ji-Hyun Park,1 Sun-Jae Lee,1 Hee-Sung Lee,1 Jae-Kyung Jung,1 Young-Ran Lee,2 Hyun-Il Cho,3 Jeong-Ki Kim,4 Kyungjae Kim,5 Chan-Su Park,1 and Chong-Kil Lee1
      • 1Department of Pharmaceutics, College of Pharmacy, Chungbuk National University, Cheongju 28644, Korea.
      • 2Center for Convergence Bioceramic Materials, Korea Institute of Ceramic Engineering and Technology, Cheongju 28160, Korea.
      • 3Research and Development Division, ViGenCell Inc., Seoul 06591, Korea.
      • 4Department of Pharmacy, Korea University College of Pharmacy, Sejong 30019, Korea.
      • 5Department of Pharmacy, College of Pharmacy, Sahmyook University, Seoul 01795, Korea.
    • Correspondence to Chong-Kil Lee. Department of Pharmaceutics, College of Pharmacy, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju 28644, Korea. Email: cklee@chungbuk.ac.kr
      Correspondence to Chan-Su Park. Department of Pharmaceutics, College of Pharmacy, Chungbuk National University, 1 Chungdae-ro, Seowon-gu, Cheongju 28644, Korea. Email: cpark@chungbuk.ac.kr
    Received October 05, 2022; Accepted October 16, 2022.

    This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Abstract

    Vaccination with tumor peptide epitopes associated with MHC class I molecules is an attractive approach directed at inducing tumor-specific CTLs. However, challenges remain in improving the therapeutic efficacy of peptide epitope vaccines, including the low immunogenicity of peptide epitopes and insufficient stimulation of innate immune components in vivo. To overcome this, we aimed to develop and test an innovative strategy that elicits potent CTL responses against tumor epitopes. The essential feature of this strategy is vaccination using tumor epitope-loaded nanoparticles (NPs) in combination with polyinosinic-polycytidylic acid (poly-IC) and anti-PD1 mAb. Carboxylated NPs were prepared using poly(lactic-co-glycolic acid) and poly(ethylene/maleic anhydride), covalently conjugated with anti-H-2Kb mAbs, and then attached to H-2Kb molecules isolated from the tumor mass (H-2b). Native peptides associated with the H-2Kb molecules of H-2Kb-attached NPs were exchanged with tumor peptide epitopes. Tumor peptide epitope-loaded NPs efficiently induced tumor-specific CTLs when used to immunize tumor-bearing mice as well as normal mice. This activity of the NPs significantly was increased when co-administered with poly-IC. Accordingly, the NPs exerted significant anti-tumor effects in mice implanted with EG7-OVA thymoma or B16-F10 melanoma, and the anti-tumor activity of the NPs was significantly increased when applied in combination with poly-IC. The most potent anti-tumor activity was observed when the NPs were co-administered with both poly-IC and anti-PD1 mAb. Immunization with tumor epitope-loaded NPs in combination with poly-IC and anti-PD1 mAb in tumor-bearing mice can be a powerful means to induce tumor-specific CTLs with therapeutic anti-tumor activity.

    Keywords
    Anti-PD1 monoclonal antibody; Cytotoxic T lymphocytes; Immunotherapy; Polyinosinic-polycytidylic acid; Tumor epitope nanoparticles; Tumor peptide epitopes

    INTRODUCTION

    In anti-tumor immunotherapy, tumor-specific CTLs are central to eliminating tumor cells (1, 2, 3, 4). These cells must first be primed with and expanded in response to tumor epitopes, comprised of short peptides (~8–10 amino acids in length) and associated with the “peptide-binding groove” of MHC class I (MHC-I) molecules, before exerting tumor Ag-specific cytotoxic effects (5). Tumor Ags are classified into two general categories, tumor-associated Ags (TAAs) and tumor-specific Ags (TSAs) (6). TAAs are self-Ags that are either preferentially or abnormally overexpressed in tumors, whereas TSAs are expressed only in tumors, such as oncoviral Ags and neoantigens generated by somatic mutations (6, 7). Peptide epitopes presented by MHC-I molecules (first signal) on Ag-presenting cells (APCs) are recognized by TCRs on naïve CD8+ T cells (1). Following recognition of peptide epitope-MHC-I complexes, co-stimulatory molecules (second signal), such as CD80 and CD86, synergize with the first signal to promote T cell activation and expansion (8, 9, 10).

    Vaccination with tumor epitopes associated with the peptide-binding groove of MHC-I molecules is an attractive approach to inducing tumor-specific CTLs (11, 12, 13). In most studies, the peptide epitopes were synthetic peptides derived from TAAs or TSAs. Peptide epitope vaccines have several advantages over other vaccine types, including tumor-specific proteins, nucleic acids, and viral vectors. Peptide epitopes are easy to synthesize and purify, safe because they lack hypersensitivity-inducing or carcinogenic potential, and are highly specific in eliciting peptide-specific CTLs in vivo (14, 15, 16, 17). However, challenges remain in improving the therapeutic efficacy of peptide epitope vaccines. One major limitation is that peptide epitopes have low immunogenicity and inefficiently induce peptide-specific CTLs. For efficient naïve CD8+ T cell priming, peptide epitopes must bind to MHC-I molecules on professional APCs expressing co-stimulatory molecules because efficient T cell activation requires at least two distinct APC-derived signals, MHC-I-peptide complexes and co-stimulatory molecules (18, 19). However, peptide epitopes can also bind to MHC-I molecules expressed on all nucleated cells that cannot perform professional APC functions. MHC-I-peptide complex presentation in the absence of co-stimulation induces peptide-specific immune tolerance (14, 20). Thus, the binding of in vivo-administered peptide epitopes to MHC-I molecules expressed in non-APCs would critically limit the peptide epitope vaccine effectiveness. Other limitations include the short half-life of the peptides and insufficient stimulation of innate immune components in vivo (18).

    Numerous strategies have been developed to overcome the limitations of peptide epitope vaccines. These include the combination of peptide epitopes with TLR agonists, such as polyinosinic-polycytidylic acid (poly-IC) and CpG oligomers, which induce the upregulation of co-stimulatory molecules and pro-inflammatory cytokines through APC activation, and peptide epitopes nano-encapsulation with a biodegradable polymer (21, 22). Peptide epitopes Encapsulation with a biodegradable polymer would not only protect peptides from degradation mediated by serum or tissue peptidases but also could deliver the peptide epitopes preferentially to phagocytic cells, including dendritic cells (DCs), via phagocytosis (21, 23). The combination of peptide epitope vaccines with an immune checkpoint inhibitor (ICI), such as anti-PD1 mAb, anti-PD-L1 mAb, and anti-CTLA-4 mAb, has received intense research recently (17, 24, 25, 26). By interrupting co-inhibitory signaling pathways, ICIs reinvigorate exhausted CTLs to regain their function and eradicate tumor cells (27, 28). However, ICI immunotherapy is only effective for cancer patients producing tumor-specific CTLs that recognize tumor Ags because ICI would not work in a microenvironment with no tumor-specific CTLs. Thus, for “immune desert cancers” with no tumor-specific CTLs, a strategy to induce tumor-specific CTLs would be the first objective before ICI use.

    Here, the aim was to develop and test an efficient anti-tumor immunotherapy strategy using tumor epitope-loaded nanoparticles (NPs) in combination with poly-IC and anti-PD1 mAb. NPs attached to MHC-I molecules were first fabricated with a biodegradable polymer, poly(lactic-co-glycolic acid) (PLGA); then, the native peptides associated with MHC-I molecules were exchanged with tumor epitopes using a peptide exchange reaction. The tumor epitope-loaded NPs were examined for their capacity to induce tumor-specific CTLs in tumor-bearing mice as well as normal mice. Furthermore, the anti-tumor therapeutic potential of the tumor epitope-loaded NPs alone and in combination with poly-IC, anti-PD1 mAb, or both was examined in tumor-bearing mice. Results showed that immunizing tumor-bearing mice with tumor epitope-loaded NPs combined with poly-IC and anti-PD1 mAb is a powerful strategy to treat tumors.

    MATERIALS AND METHODS

    Mice and cell lines

    Female C57BL/6 mice (8–12 weeks old) were purchased from Kosa Bio, Inc. (Seongnam, Korea). All experimental animal procedures were performed in accordance with guidelines and regulations approved by the Institutional Animal Care and Use Committee of Chungbuk National University (CBNUA-1490-21-02). EG7-OVA cells, a mouse EL4 lymphoma cell line expressing OVA (H-2b), were purchased from the American Type Culture Collection (Manassas, VA, USA). B16-F10 mouse melanoma cells were purchased from the Korean Cell Line Bank (Seoul, Korea).

    Peptides, Abs, and reagents

    OVA257–264 peptide (SIINFEKL), TRP2180–188 peptide (SVYDFFVWL), FITC-labeled TRP2180–188 peptide (FITC-TRP2180–188), and dipeptide glycyl-cyclohexylalanine (GCha) were purchased from Peptron (Daejeon, Korea). Dipeptide glycyl-leucine (GL) and glycyl-methionine (GM) were from Tokyo Chemical Industry (Tokyo, Japan) and BLD Pharmatech (Shanghai, China), respectively. mAbs specific to mouse H-2Kb (clone Y-3) and mouse PD1 (clone RMP1-14) were from BioXCell (West Lebanon, NH, USA). Fluorescence-labeled mAbs specific to mouse H-2Kb-OVA257–264 complex (clone 25-D1.16) and mouse CD8α (clone KT15) were from BioLegend (San Diego, CA, USA) and Invitrogen (San Diego, CA, USA), respectively. Fluorescence-labeled H-2Kb-OVA257–264 tetramers and H-2Kb-TRP2180–188 tetramers were from MBL (Tokyo, Japan). Poly-IC was from InvivoGen. Poly-IC stabilized with poly-L-lysine and carboxy-methylcellulose (poly-ICLC) was provided by Dr. Hyun-Il Cho (ViGenCell Inc., Seoul, Korea).

    Preparation of carboxylated NPs

    Carboxylated NPs were prepared using PLGA (lactide:glycolide 50:50, Mw 24,000–38,000, acid terminated; Evonik Industries, Essen, Germany) and poly(ethylene/maleic anhydride) (PEMA; Polysciences Inc., Warrington, PA, USA), as described previously (29). Briefly, 200 mg of PLGA was dissolved in 4 ml of ethyl acetate and combined with 8 ml of 1% PEMA. The mixture was emulsified using a homogenizer (HG-15D Homogenizer, DAIHAN Scientific, Seoul, Korea) at 14,000 rpm for 4 min to form a water-in-oil (w/o) emulsion, which was transferred to a beaker containing a 0.2% PEMA and 0.1% polyvinyl alcohol (Sigma-Aldrich, St. Louis, MS, USA) mixture and stirred at room temperature for 18 h. The resulting NPs were centrifuged at 3,500×g for 20 min at 4°C and resuspended in 10 mL of sterile distilled water. The mean size and ζ-potential of the carboxylated NPs were assessed using a particle size analyzer (ELS-Z, Otsuka, Japan). The NPs were counted using qNano Gold (IZON Science, Christchurch, New Zealand).

    Isolation of tumor-derived membrane protein

    Tumor-derived membrane proteins were isolated from the EG7-OVA tumor mass as described previously (29). Briefly, 1 g of the tissue was dissociated with 10 ml of extraction buffer containing 1X protease inhibitor cocktail (Sigma-Aldrich) using a gentleMACS dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany). Cell debris and the lipid layer were removed by centrifugation for 5 min at 350×g, and the supernatant was ultracentrifuged for 45 min at 80,000×g at 4°C (Beckman Coulter, Brea, CA, USA). After ultracentrifugation, the pellet was resuspended in extraction buffer (0.5 ml) and homogenized using a tissue grinder (SHS-30E, SciLab, Seoul, Korea). The homogenate was mixed with solubilization buffer (0.5 ml) and solubilized for 90 min at 4°C. The homogenate containing solubilized membrane proteins was centrifuged at 25,000×g for 30 min at 4°C, and the supernatant containing the membrane proteins was transferred to a new conical tube. The amount of isolated membrane protein was determined using a microbicinchoninic acid assay kit (Thermo Fisher Scientific, Waltham, MA, USA).

    Preparation of anti-mouse H-2Kb mAb-conjugated NPs and H-2Kb-attached NPs (NP-Kb)

    Anti-mouse H-2Kb mAbs were conjugated to the surface of carboxylated NPs using a modified carbodiimide coupling method, and the NPs conjugated with mAbs were attached with H-2Kb molecules, as described previously (29). Briefly, 10 mg of carboxylated NPs was resuspended in 1.2 ml of 50 mM activation buffer, added to 4 μl of 300 mM 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC; Thermo Fisher Scientific) and 8 μl of 300 mM N-hydroxysuccinimide (Sigma-Aldrich), and incubated for 1 h at room temperature. EDC-activated NPs were washed twice with 50 mM activation buffer, resuspended in 850 μl of 50 mM activation buffer, added to 150 μl of 4 mg/ml anti-mouse H-2Kb mAbs, and incubated for 18 h at room temperature. To block non-specific conjugation, mAb-conjugated NPs were treated with 30 μl of ethanolamine (Sigma-Aldrich) for 30 min and washed twice with PBS. The NPs conjugated with mAbs were incubated with 5 mg of tumor-derived membrane proteins for 24 h at 4°C. The resulting H-2Kb-attached NPs (NP-Kb) were washed twice with PBS and resuspended in 1 ml PBS.

    Generation of bone marrow-derived DCs (BMDCs) from bone marrow cells

    BMDCs were generated from mouse bone marrow cells using GM-CSF and IL-4, as described previously (29). Briefly, bone marrow cells from mouse femurs were cultured in 6-well plates (5×106 cells/well) in a culture medium with 40 ng/ml GM-CSF and 20 ng/ml IL-4 (both from Creagene, Seongnam, Korea). After 3 days, non-adherent cells were removed by gently shaking the plate and replacing the medium. On day 4, non-adherent cells were removed using the same method. On day 6, half of the culture medium was replaced with a fresh medium. To induce maturation, BMDCs were exposed to 100 ng/ml LPS (Sigma-Aldrich) for 24 h. BMDCs were harvested by gentle pipetting on day 7.

    Peptide exchange reaction on H-2Kb-attached NPs

    Native peptides associated with the H-2Kb molecules of NP-Kb were exchanged with OVA257–264 by incubating 5 mg of NP-Kb with 100 μM OVA257–264 peptide with or without 20 mM dipeptide GL for 18 h at 37°C. OVA257–264-pulsed NP-Kb was stained with mAbs recognizing H-2Kb-OVA257–264 complexes (clone 25-D1.16) and analyzed by flow cytometry. In some experiments, native peptides associated with H-2Kb molecules of NP-Kb were exchanged with TRP2180–188 by incubating 5 mg of NP-Kb with 100 μM TRP2180–188 with or without 20 mM dipeptide GM for 18 h at 37°C, washed twice with PBS, and resuspended in PBS.

    Competitive blocking assay for peptide epitope bindings

    NP-Kb (1 mg) was incubated with a fixed concentration of FITC-TRP2180–188 (0.1 μM) and various concentrations of unlabeled TRP2180–188 (0–100 μM) with 20 mM dipeptide GM for 18 h at 37°C, washed twice with PBS, and resuspended in PBS. Levels of FITC-TRP2180–188 binding to NP-Kb were analyzed using flow cytometry.

    In vivo generation of peptide-specific CD8+ T cells

    C57BL/6 mice were immunized intravenously with PBS or the indicated NPs (1×109 particles/mouse) on days 0, 7, and 14. In poly-IC-treated mice, the NPs were mixed with poly-IC (50 μg/mouse) before intravenous injection. On days 5 and 19, whole blood cells were collected from the facial vein, stained with the H-2Kb-OVA257–264 tetramers and mAbs against mouse CD8α, and analyzed using flow cytometry.

    Measurement of peptide-specific CTL activity

    C57BL/6 mice were immunized intravenously with PBS or the indicated NPs (1×109 particles/mouse) on days 0, 7, and 14. In poly-IC-treated mice, NPs were mixed with poly-IC (50 μg/mouse) before intravenous injection. On day 21, OVA257–264-specific CTL activity was assessed by an in vivo CTL assay, as described previously (29). In brief, splenocytes from naïve syngeneic mice were pulsed with 1 μM OVA257–264 for 1 h at 37°C and then labeled with a high concentration of CFSE (25 μM). The other control target population was syngeneic splenocytes labeled with a low concentration of CFSE (5 μM) without OVA257–264 peptide pulsing. A 1:1 mixture of each target cell population was injected into immunized mice via the tail vein (1×107 cells/mouse). After 18 h, the specific killing of OVA257–264-pulsed target cells was determined using spleen or lymph node cells isolated from each recipient mouse by flow cytometry. The ratio between the percentage of OVA257–264-unpulsed cells (CFSELow) versus pulsed cells (CFSEHigh) was calculated to represent cytotoxicity.

    To administer the NPs exchanged with the TRP2180–188 peptide, C57BL/6 mice were intravenously immunized with PBS or the indicated NPs (1×109 particles/mouse) on days 0 and 5. In poly-ICLC-treated mice, the NPs were mixed with poly-ICLC (50 μg/mouse) before intravenous injection. On day 12, TRP2180–188-specific CTL activity was assessed using an in vivo CTL assay.

    Anti-tumor activity

    The right flanks of C57BL/6 mice were shaved and subcutaneously inoculated with EG7-OVA cells (5×105 cells/mouse). The mice were intravenously administered with PBS or the indicated NPs (1×109 particles/mouse) on days 2, 9, and 16. In poly-IC-treated mice, the NPs were mixed with poly-IC (50 μg/mouse) before intravenous injection. For combination therapy, anti-PD1 mAbs (100 μg/mouse) were intraperitoneally administered six times before or 2 days after each NP immunization. The mice were euthanized when the tumor volume reached 2,500 mm3. Tumor volume was calculated using the following formula: Tumor Volume [mm3]=0.52×(Long Diameter [mm])×(Short Diameter [mm])2. Spleen or lymph node cells were isolated from tumor-bearing mice, stained with H-2Kb-OVA257–264 tetramers and mAbs against mouse CD8α, and analyzed using flow cytometry according to the manufacturer’s instructions.

    For the B16-F10 melanoma model, the right flanks of C57BL/6 mice were shaved and subcutaneously inoculated with B16-F10 cells (1×105 cells/mouse). The mice were intravenously administered with PBS or the indicated NPs (1×109 NPs/mouse) on days 2, 7, and 12. In poly-ICLC-treated mice, the NPs were mixed with poly-ICLC (50 μg/mouse) before intravenous injection. For combination therapy, anti-PD1 mAbs (100 μg/mouse) were intraperitoneally administered six times before or 1 day after NP immunization. Spleen cells were isolated from tumor-bearing mice, stained with H-2Kb-TRP2180–188 tetramers and mAbs against mouse CD8α, and analyzed using flow cytometry according to the manufacturer’s instructions.

    Restimulation of splenocytes isolated from tumor-bearing mice

    Splenocytes (2×106 cells/well) isolated from tumor-bearing mice were co-cultured with peptide epitope-pulsed BMDCs (2×105 cells/well) in 24-well plates for 3 days. Peptide epitope-pulsed BMDCs were prepared by pulsing LPS-stimulated BMDCs with 10 μM peptide epitopes for 1 h at 37°C. IFN-γ levels in the culture supernatant were measured using commercial ELISA kits (BD Biosciences, San Jose, CA, USA).

    Anti-metastatic activity

    C57BL/6 mice were intravenously inoculated with B16-F10 cells (1×105 cells/mouse) into the tail vein. Mice were intravenously administered with PBS or the indicated NPs (1×109 particles/mouse) on days 1, 6, and 11. In poly-ICLC-treated mice, the NPs were mixed with poly-ICLC (50 μg/mouse) before intravenous injection. For combination therapy, anti-PD1 mAbs (100 μg/mouse) were intraperitoneally administered six times before or 1 day after NP immunization. Mice were euthanized on day 17 after tumor cell injection, and lungs were harvested and fixed in Bouin’s solution (BioGnost, Zagreb, Croatia). Metastatic nodules on the lung surface were counted.

    Statistical analysis

    A two-tailed paired Student’s t-test was performed for a single comparison of two groups after a normality evaluation. One-way or two-way ANOVA with post-hoc Tukey’s test was performed to compare the significance of multiple groups; p≤0.05 was considered statistically significant.

    RESULTS

    Fabrication and characterization of NP-Kb pulsed with OVA257–264

    Carboxylated NPs were prepared using PLGA and PEMA using the w/o emulsion solvent evaporation method. The carboxylated NPs were covalently conjugated with anti-H-2Kb mAbs (isotype mIgG2b) via carbodiimide conjugation and then attached to H-2Kb molecules by incubating the mAb-conjugated NPs with solubilized membrane proteins isolated from the EG7-OVA tumor mass (H-2b) to generate H-2Kb-attached NPs (NP-Kb). Native peptides associated with the H-2Kb molecules of NP-Kb were exchanged with a model tumor-specific peptide, OVA257–264, via a peptide exchange reaction (Fig. 1A). The peptide exchange reaction was maximized by optimizing the reaction conditions, such as the use of dipeptides, GL, GM, and GCha, which are known to enhance the peptide exchange reaction (30), and the concentrations of OVA257–264. The peptide exchange efficiency was maximal when NP-Kb was treated with 100 μM OVA257–264 in the presence of 20 mM GL, whereas GM and GCha were ineffective (Supplementary Fig. 1). The relative degree of H-2Kb-OVA257–264 complexes on NP-Kb was 2.8-fold higher when NP-Kb was treated with 100 μM OVA257–264 in the presence of 20 mM GL (NP-SIINHi) than when NP-Kb was treated with 100 μM OVA257–264 only (NP-SIINLo) (Fig. 1B). The H-2Kb-OVA257–264 complexes on NPs remained stable for up to 5 days (Fig. 1C). The average NP-SIINHi size was 1,032.1±44.8 nm, and the average ζ-potential was −19.48±1.61 mV.

    Figure 1
    Comparison of the OVA257–264-loaded degree between NP-SIINLo and NP-SIINHi and the stability of H-2Kb-OVA257–264 complexes on the NP-SIINHi. (A) NP-Kb was incubated with 100 μM OVA257–264 in the presence or absence of 20 mM GL over 18 h at 37°C. (B) H-2Kb-OVA257–264 complexes on the NPs were stained using a mAb (Clone 25-D1.16) and analyzed by flow cytometry. NP-Kb loaded with OVA257–264 in the absence or presence of GL was denoted as NP-SIINLo or NP-SIINHi, respectively. (C) NP-SIINHi was stored in PBS at 4°C. At indicated time points, H-2Kb-OVA257–264 complexes on the NP were stained using a mAb (Clone 25-D1.16) and analyzed by flow cytometry. The data are presented as the mean ± SD of at least three independent experiments.
    *p<0.05, ***p<0.001.

    NPs loaded with higher amounts of OVA257–264 induce OVA257–264-specific CTLs more efficiently in normal mice

    To compare the OVA257–264-specific CTL-inducing activity of NP-SIINLo and NP-SIINHi, mice were intravenously injected with the NPs (1×109 particles/mouse) three times at 7-day intervals. Control mice were intravenously injected with PBS or NP-Kb. Five days after the first and last immunizations, OVA257–264-specific CTLs in peripheral blood were analyzed using H-2Kb-OVA257–264 tetramers. After priming, the percentage of OVA257–264-specific CTLs among CD8+ T cells reached 1.28% and 1.71% in mice treated with NP-SIINLo and NP-SIINHi, respectively (Fig. 2A and B). After boosting, the percentage of OVA257–264-specific CTLs among CD8+ T cells increased to 3.36% and 5.63% in mice treated with NP-SIINLo and NP-SIINHi, respectively (Fig. 2C and D). Seven days after the last immunization, OVA257–264-specific CTL activity in the spleen and lymph nodes was analyzed using an in vivo CTL assay. Representative histograms are shown in Fig. 2E and G, respectively. Immunization with NP-SIINHi induced OVA257–264-specific CTL activity more efficiently (specific target cell killing of 57.2% in the spleen and 53.6% in the lymph node) than immunization with NP-SIINLo (specific target cell killing of 34.7% in the spleen and 36.8% in the lymph node) (Fig. 2F and H). These results showed that immunization with NPs loaded with high amounts of OVA257–264 efficiently induced potent CTL responses.

    Figure 2
    NP-SIINHi efficiently induces OVA257–264-specific CTLs in normal mice. (A–H) PBS, NP-Kb, NP-SIINLo or NP-SIINHi (1×109 particles/mouse) was i.v. injected into C57BL/6 mice on days 0, 7, and 14. (A, B) Five days after priming, peripheral blood cells were stained with H-2Kb-OVA257–264 tetramers and anti-mouse CD8α mAbs (Clone KT15) and analyzed using flow cytometry. (C, D) Five days after boosting, peripheral blood cells were stained with H-2Kb-OVA257–264 tetramers and anti-mouse CD8α mAbs. On day 21, OVA257–264-specific CTL activity was assessed using an in vivo CTL assay. Target cells comprised a 1:1 mixture of syngeneic cells pulsed with OVA257–264 and then labeled with a high concentration of CFSE and syngeneic cells unpulsed and labeled with a low concentration of CFSE. Specific killing of the target cells was analyzed for the spleen (E, F) and lymph nodes (G, H). (I–P) NP-SIINHi (1×109 particles/mouse) with or without poly-IC was i.v. injected into C57BL/6 mice on days 0, 7, and 14. Five days after priming (I, J) and boosting (K, L), peripheral blood cells were stained with H-2Kb-OVA257–264 tetramer. On day 21, OVA257–264-specific CTL activity was assessed using an in vivo CTL assay. Specific killing of the target cells is shown for the spleen (M, N) and lymph node (O, P). Histograms are representatives of each experiment, and the bar data are presented as the mean ± SD of at least three independent experiments (n=4–5 mice/group in each experiment).
    *p<0.05, **p<0.01, ***p<0.001.

    Combining NP-SIINHi with poly-IC further increases OVA257–264-specific CTL induction in normal mice

    The OVA257–264-specific CTL-inducing activity of NP-SIINHi, in combination with poly-IC, was examined in normal mice. In this experiment, mice were intravenously injected three times with NP-SIINHi (1×109 particles/mouse) alone or with poly-IC at 7-day intervals. Five days after the first and last immunizations, OVA257–264-specific CTLs in peripheral blood were analyzed using H-2Kb-OVA257–264 tetramers. In primed mice, the combination of NP-SIINHi with poly-IC did not induce discernible increases in OVA257–264-specific CTLs among CD8+ T cells compared to that with NP-SIINHi alone (1.98% vs. 1.61%) (Fig. 2I and J). However, after boosting, the combination of NP-SIINHi with poly-IC significantly increased the percentage of OVA257–264-specific CTLs among CD8+ T cells compared to that with NP-SIINHi alone (8.79% vs. 5.66%) (Fig. 2K and L). These results suggest that the adjuvant activity of poly-IC is prominent during boosting with NP-SIINHi, but not during priming.

    Seven days after the last immunization, OVA257–264-specific CTL activity in the spleen and lymph nodes was analyzed using an in vivo CTL assay. Representative histograms are shown in Fig. 2M and 2O, respectively. In accordance with tetramer analysis results, immunization with NP-SIINHi combined with poly-IC potently induced OVA257–264-specific CTL activity (specific target cell killing of 84.7% in the spleen and 75.3% in the lymph node), compared to that upon immunization with NP-SIINHi alone (specific target cell killing of 56.2% in the spleen and 53.4% in the lymph node) (Fig. 2N and P).

    NP-SIINHi and poly-IC combination exerts potent anti-tumor effects in EG7-OVA tumor-bearing mice with increased production of OVA257–264-specific CTLs

    The anti-tumor therapeutic potential of NP-SIINHi combined with poly-IC was examined with EG7-OVA tumor-bearing mice. Mice were subcutaneously inoculated with EG7-OVA tumor cells, and 2 days later, they were intravenously injected three times with PBS, NP-SIINHi, poly-IC, or NP-SIINHi plus poly-IC at 7-day intervals. The combination of NP-SIINHi and poly-IC exerted much greater anti-tumor effects than NP-SIINHi alone (Fig. 3A). Poly-IC alone did not show anti-tumor activity. The anti-tumor therapeutic potential of NP-SIINHi plus poly-IC was also confirmed by the survival rate (Fig. 3B).

    Figure 3
    Combination of NP-SIINHi and poly-IC exerts potent anti-tumor effects in EG7-OVA tumor-bearing mice with increased production of OVA257–264-specific CTLs. C57BL/6 mice were s.c. inoculated with EG7-OVA tumor cells (5×105 cells/mouse) on day 0. PBS, poly-IC, NP-SIINHi, or NP-SIINHi plus poly-IC (1×109 particles/mouse) was i.v. injected on days 2, 9, and 16. (A) Tumor volume was measured every 2 days. (B) Mice of each experimental group were sacrificed when the tumor volume reached 2,500 mm3, and survival data were analyzed. The data are presented as the mean ± SD of at least three independent experiments (n=6 mice/group in each experiment). Spleen cells (C, D) and lymph node cells (E, F) were stained with H-2Kb-OVA257–264 tetramers and anti-mouse CD8α mAbs and analyzed using flow cytometry. (G) Spleen cells of each experimental group were also co-cultured with OVA257–264-pulsed mature BMDCs. After 3 days, IFN-γ production was analyzed using an ELISA. The data are presented as the mean ± SD of at least three independent experiments (n=6 mice/group in each experiment).
    **p<0.01, ***p<0.001.

    Upon completion of the anti-tumor experiment (day 25 of Fig. 3A), the induction degree of OVA257–264-specific CTLs was measured in the spleen and lymph nodes isolated from EG7-OVA tumor-bearing mice. The percentage of OVA257–264-specific CTLs in the spleen was increased 2.1-fold in mice immunized with NP-SIINHi plus poly-IC (12.9%) compared to that in mice immunized with NP-SIINHi alone (6.18%) (Fig. 3C and D). The percentage of OVA257–264-specific CTLs in the lymph node was also increased 1.8-fold in mice immunized with NP-SIINHi plus poly-IC (2.04%) compared to that in mice immunized with NP-SIINHi alone (1.13%) (Fig. 3E and F). IFN-γ production by CTLs is a major characteristic of activated CTLs (31, 32). To confirm tumor-specific CTL functional activation, spleen cells isolated from EG7-OVA tumor-bearing mice were co-cultured with OVA257–264-pulsed mature BMDCs, and IFN-γ levels in the culture supernatants were measured using ELISA. Spleen cells isolated from mice immunized with NP-SIINHi plus poly-IC produced more IFN-γ (2.8-fold) than those immunized with NP-SIINHi alone (Fig. 3G). Mice immunized with PBS or poly-IC did not show OVA257–264-specific CTL induction (Fig. 3D and F) or IFN-γ production from re-stimulated spleen cells (Fig. 3G).

    Combination of NP-SIINHi with poly-IC and anti-PD1 mAb further increases anti-tumor activity in EG7-OVA tumor-bearing mice

    The anti-tumor therapeutic potential of NP-SIINHi in combination with both poly-IC and anti-PD1 mAbs was examined in EG7-OVA tumor-bearing mice. Mice were subcutaneously inoculated with EG7-OVA tumor cells, and 2 days later, the mice were intravenously injected three times with PBS, NP-SIINHi, or NP-SIINHi plus poly-IC at 7-day intervals with or without anti-PD1 mAbs. In groups treated with anti-PD1 mAbs, anti-PD1 mAbs were intraperitoneally administered six times before or 2 days after intravenous injection with PBS, NP-SIINHi, or NP-SIINHi plus poly-IC (Fig. 4A). Immunization with NP-SIINHi plus poly-IC significantly inhibited tumor growth, and its combination with anti-PD1 mAbs increased the anti-tumor activity of NP-SIINHi plus poly-IC (Fig. 4B).

    Figure 4
    Combination of NP-SIINHi with poly-IC and anti-PD1 mAb further increases anti-tumor activity in EG7-OVA tumor-bearing mice. (A) C57BL/6 mice were s.c. inoculated with EG7-OVA tumor cells (5×105 cells/mouse) on day 0. PBS, NP-SIINHi or NP-SIINHi plus poly-IC (1×109 particles/mouse) was i.v. injected on days 2, 9, and 16. In groups treated together with anti-PD1 mAbs, anti-PD1 mAbs were i.p. administered six times before or 2 days after each NP immunization. (B) Tumor volume was measured every 2 days. Spleen cells (C, D) and lymph node cells (E, F) were stained with H-2Kb-OVA257–264 tetramers and anti-mouse CD8α mAbs and analyzed using flow cytometry. (G) Spleen cells of each experimental group were also co-cultured with OVA257–264-pulsed mature BMDCs. After 3 days, IFN-γ production was analyzed using an ELISA. The data are presented as the mean ± SD of at least three independent experiments (n=6 mice/group in each experiment).
    *p<0.05, **p<0.01, ***p<0.001.

    On the last day of the experiment, OVA257–264-specific CTLs were measured in the spleen and lymph nodes. NP-SIINHi and poly-IC combination significantly increased the percentage of OVA257–264-specific CTLs among CD8+ T cells compared to that with NP-SIINHi alone in the spleens (10.3% vs. 1.57%) (Fig. 4C and D) and lymph nodes (3.17% vs. 1.44%) (Fig. 4E and F). However, the combination of anti-PD1 mAbs with NP-SIINHi or NP-SIINHi plus poly-IC did not further increase the percentage of OVA257–264-specific CTLs, although the addition of anti-PD1 mAbs significantly increased the anti-tumor activity (Fig. 4C-F). The combination with anti-PD1 mAbs did not affect the number of CTLs or the functional CTL activity. The IFN-γ-producing capacity of spleen cells re-stimulated with OVA257–264-pulsed mature BMDCs was not increased by combination therapy with anti-PD1 mAbs, compared to that without anti-PD1 mAbs (Fig. 4G). These results suggest that the increased anti-tumor activity with the anti-PD1 mAb combination was due to the blockade of inhibitory signals. In fact, we found that EG7-OVA tumor cells expressed PD-L1, and the expression of PD-L1 was further increased by IFN-γ treatment (Supplementary Fig. 2A).

    NPs loaded with melanoma TRP2180–188 peptide (NP-SVYD) in combination with poly-ICLC potently induces TRP2180–188-specific CTLs in normal mice

    The therapeutic efficacy of the tumor epitope-loaded NPs was extended to a melanoma mouse model. NP-Kb was first prepared using the methods described, and then, native peptides associated with H-2Kb molecules were exchanged with a melanoma-associated peptide, TRP2180–188. Because a mAb recognizing the H-2Kb-TRP2180–188 complex was not available, FITC-labeled TRP2180–188 (FITC-TRP2180–188) was used to optimize reactions, and the degree of FITC-TRP2180–188 exchange was analyzed using flow cytometry. The peptide exchange efficiency was maximal when NP-Kb was treated with 100 μM FITC-TRP2180–188 in the presence of 20 mM GM (Supplementary Fig. 3). The binding of unlabeled TRP2180–188 to NP-Kb was confirmed by competitively blocking the binding of FITC-TRP2180–188 to NP-Kb with unlabeled TRP2180–188 (Fig. 5A).

    Figure 5
    NP-SVYD in combination with poly-ICLC potently induces TRP2180–188-specific CTLs in normal mice. (A) Confirmation of the binding of TRP2180–188 to NP-Kb. NP-Kb was incubated with 0.1 μM FITC-TRP2180–188 plus 0, 0.1, 1, 10, or 100 μM unlabeled TRP2180–188 in the presence of 20 mM GM for 18 h at 37°C. The relative amounts of FITC-TRP2180–188 bound to NP-Kb were analyzed using flow cytometry. (B, C) PBS, NP-Kb or NP-SVYD plus poly-ICLC (1×109 particles/mouse) was i.v. injected into C57BL/6 mice on days 0, 5, and 10. On day 17, TRP2180–188-specific CTL activity was assessed using an in vivo CTL assay. The target cells comprised a 1:1 mixture of syngeneic cells pulsed with TRP2180-188 and then labeled with a high concentration of CFSE and syngeneic cells unpulsed and labeled with a low concentration of CFSE. The data are presented as the mean ± SD of at least three independent experiments (n=6 mice/group in each experiment).
    *p<0.05, **p<0.01, ***p<0.001.

    The TRP2180–188-specific CTL-inducing activity of NP-SVYD in combination with poly-ICLC, a synthetic derivative that stabilizes poly-IC through the addition of poly-L-lysine and carboxymethyl cellulose (33), was examined in mice. In this experiment, mice were intravenously injected twice with NP-Kb or NP-SVYD (1×109 particles/mouse) together with poly-ICLC at 5-day intervals. Seven days after the last immunization, TRP2180–188-specific CTL activity in the spleen was analyzed using an in vivo CTL assay. Representative histograms are shown in Fig. 5B. Immunization with NP-SVYD plus poly-ICLC efficiently induced TRP2180–188-specific CTL activity (specific target cell killing of 39.5%) compared to that with NP-Kb plus poly-ICLC immunization (specific target cell killing of 8.2%) (Fig. 5B and C).

    NP-SVYD in combination with poly-ICLC and anti-PD1 mAb exerts potent anti-tumor activity in B16-F10 tumor-bearing mice

    The anti-tumor therapeutic potential of NP-SVYD in combination with poly-ICLC, anti-PD1 mAb, or both was examined in B16-F10 tumor-bearing mice. Mice were subcutaneously inoculated with B16-F10 tumor cells, and 2 days later, the mice were intravenously injected with NP-SVYD plus poly-ICLC three times at 5-day intervals. Control mice were intravenously injected with PBS or NP-SVYD. In groups treated with anti-PD1 mAbs, anti-PD1 mAbs were intraperitoneally administered six times before or 1 day after intravenous injection with NP-SVYD or NP-SVYD plus poly-ICLC (Fig. 6A). Immunization with NP-SVYD significantly inhibited melanoma growth, and the anti-tumor activity of NP-SVYD was potently increased upon immunization in combination with poly-ICLC or anti-PD1 mAb. The most potent anti-tumor activity of NP-SVYD was observed when used in combination with both poly-ICLC and anti-PD1 mAb (Fig. 6B and C).

    Figure 6
    NP-SVYD in combination with poly-ICLC and anti-PD1 mAb exerts potent anti-tumor effects in B16-F10 tumor-bearing mice. (A) C57BL/6 mice were s.c. inoculated with B16-F10 tumor cells (1×105 cells/mouse) on day 0. PBS, NP-SVYD or NP-SVYD plus poly-ICLC (1×109 particles/mouse) was i.v. injected on days 2, 7, and 12. In groups treated together with anti-PD1 mAbs, anti-PD1 mAbs were i.p. administered six times before or 2 days after each NP immunization. (B, C) Tumor volume was measured every 2 days. (D) Spleen cells were stained with H-2Kb-TRP2180–188 tetramers and anti-mouse CD8α mAbs and analyzed using flow cytometry. (E) Spleen cells of each experimental group were also co-cultured with TRP2180–188-pulsed mature BMDCs. After 3 days, IFN-γ production was analyzed using an ELISA. The data are presented as the mean ± SD of at least three independent experiments (n=6 mice/group in each experiment).
    *p<0.05, **p<0.01, ***p<0.001.

    On the last day of the experiment, the degree of TRP2180–188-specific CTL induction was measured in the spleen. The combination of NP-SVYD with poly-ICLC significantly increased the percentage of TRP2180–188-specific CTLs among CD8+ T cells compared to that with NP-SVYD alone (2.68% vs. 1.53%) (Fig. 6D). However, the combination of anti-PD1 mAbs with NP-SVYD or NP-SVYD plus poly-ICLC did not further increase the percentage of TRP2180-188-specific CTLs, although the addition of anti-PD1 mAbs significantly increased anti-tumor activity (Fig. 6B and C). The combination of anti-PD1 mAbs did not affect the number of CTLs or the functional CTL activity. The IFN-γ-producing capacity of spleen cells re-stimulated with TRP2180–188-pulsed mature BMDCs was not increased by the combination therapy with anti-PD1 mAbs, compared to that without anti-PD1 mAbs (Fig. 6E). These results suggest that the increased anti-tumor activity mediated by the anti-PD1 mAb combination is due to the blockade of inhibitory signals. In fact, we found that B16-F10 tumor cells expressed PD-L1, which was further increased by IFN-γ treatment (Supplementary Fig. 2B).

    Combined NP-SVYD with poly-ICLC and anti-PD1 mAb potently reduces B16-F10 melanoma lung metastasis

    The anti-metastatic activity of NP-SVYD combined with poly-ICLC and anti-PD1 mAb was examined in a mouse B16-F10 melanoma model. Mice were intravenously injected with B16-F10 tumor cells, and 1 day later the mice were intravenously injected with NP-SVYD alone or in combination with poly-ICLC three times at 5-day intervals. In mice treated with NP-SVYD plus poly-ICLC together with anti-PD1 mAbs, anti-PD1 mAbs were intraperitoneally administered six times before or 2 days after intravenous injection with NP-SVYD plus poly-ICLC (Fig. 7A). NP-SVYD Immunization significantly reduced the number of lung metastatic nodules, and the combination of NP-SVYD with poly-ICLC further reduced this number (Fig. 7B and C). The most potent anti-metastatic activity was observed in mice treated with NP-SVYD combined with poly-ICLC and anti-PD1 mAb.

    Figure 7
    Combination of NP-SVYD with poly-ICLC and anti-PD1 mAb potently reduces B16-F10 melanoma lung metastasis. (A) C57BL/6 mice were i.v. injected with B16-F10 tumor cells (1×105 cells/mouse) on day 0. NP-SVYD or NP-SVYD plus poly-ICLC (1×109 particles/mouse) was i.v. injected on days 1, 6, and 11. Anti-PD1 mAbs were i.p. administered six times before or 2 days after each NP immunization. (B) Representative images of the lungs from each group. (C) The number of metastatic nodules on the surface of the lung was counted on day 16. The data are presented as the mean ± SD of at least three independent experiments (n=8 mice/group in each experiment).
    ***p<0.001.

    DISCUSSION

    One of the major findings of the present study is that tumor peptide epitope-loaded NPs can efficiently induce tumor-specific CTLs when used to immunize tumor-bearing mice as well as normal mice. The CTL-mediated anti-tumor activity of the tumor epitope-loaded NPs was confirmed with two mouse tumor models, EG7-OVA lymphoma and B16-F10 melanoma. Moreover, the combination of tumor epitope-loaded NPs with poly-IC, a TLR3 agonist, dramatically increased tumor-specific CTL-mediated anti-tumor activity. Furthermore, we showed that the combination of tumor epitope-loaded NPs with both poly-IC and anti-PD1 mAb elicited the most potent anti-tumor activity, although anti-PD1 mAb alone did not increase the generation of tumor-specific CTLs. The tumor epitopes used in this study were synthetic peptides that bind to the Ag-binding cleft of the MHC-I molecule, H-2Kb.

    Tumor epitope-based vaccination strategies, aiming to elicit tumor-specific CTLs using synthetic tumor peptides binding to MHC-I molecules, could be an attractive means of tumor immunotherapy (17, 34), because short peptides have several advantages such as their low toxicity, high specificity, straightforward chemical synthesis, and cost-effectiveness, as compared to other immunotherapies (35). However, most peptide epitope-based vaccines have demonstrated only a modest capacity to elicit tumor-specific CTLs in patients with limited therapeutic efficacy and disappointing clinical outcomes (36, 37). The most critical drawback of MHC-I-binding epitope vaccines is that the epitopes could bind to a variety of cells expressing MHC-I molecules (14, 38). For efficient CTL generation, peptide epitopes must be presented to CD8 T cells by professional APCs expressing co-stimulatory molecules, such as CD80 and CD86 (1, 9). Further, the presentation of peptide epitopes in the absence of co-stimulatory signals can even induce T cell anergy or apoptosis (14). Accordingly, the strategy developed in the present study would provide a novel approach that circumvents this issue (i.e., prompting the uptake of peptide epitope-loaded NPs by professional APCs is advantageous because peptide epitopes can be delivered specifically to professional APCs). In addition, peptide epitope pre-binding to H-2Kb in the NP-Kb formulation prevents rapid degradation of the peptide epitopes mediated by tissue and serum peptidases.

    Immunization using short peptide epitopes typically generates unsatisfactory immune responses owing to the insufficient stimulation of innate immune components (18). Hence, suitable delivery systems and additional stimuli, such as immune adjuvants, are necessary to efficiently induce innate immune responses against peptide epitopes (18, 23). In this regard, our vaccination strategy using NPs coated with peptide epitopes has distinctive merits in that they can stimulate innate immune components. Our previous studies showed that NP phagocytosis itself induces the activation of innate immune cells, including DCs, as demonstrated by the increased expression of co-stimulatory molecules and cytokine production (39). Furthermore, the NPs fabricated here can be phagocytosed specifically by cells expressing Fc receptors, because some of the Abs on NPs might be conjugated via the Ag-binding site, leaving the Fc region outside the NPs. Indeed, we have shown that Ab-conjugated NPs are much more efficient in upregulating co-stimulatory molecule expression and inducing pro-inflammatory cytokines in DCs than NPs unconjugated with Abs (29).

    Immune adjuvants, such as poly-IC, activate innate immune components, including the optimization of Ag presentation, recruitment of DCs, and creation of a cytokine environment that supports the generation of CTLs (14). Poly-IC induces DC activation leading to the upregulation of co-stimulatory signals required for the generation of tumor-specific CTLs (40). We examined the CTL-inducing activity of the tumor epitope-loaded NPs in combination with poly-IC and showed that this combination could further inhibit tumor growth by efficiently enhancing tumor-specific CTLs, compared to that with the NPs alone. Notably, poly-ICLC, instead of poly-IC, was used in the melanoma model which used NPs loaded with TRP2180–188. Poly-ICLC is a synthetic derivative of poly-IC that prevents poly-IC degradation by RNases, thereby sustaining and enhancing its activity (33, 41).

    ICI immunotherapy, which uses mAbs specific to co-inhibitory molecules, has emerged as one of the most promising approaches to invigorate anti-tumor immune responses (28). However, anti-tumor immunotherapy using ICIs has some clinical limitations. Some patients exhibit complete and durable responses that prolong survival, whereas others do not respond to ICI immunotherapy at all (42). Because ICIs block the co-inhibitory signaling induced by tumors, ICI immunotherapy will not work in patients without sufficient levels of pre-existing tumor-specific CTLs. For such patients, strategies to generate tumor-specific CTLs must be applied before ICI use (27). Our vaccination strategy using tumor epitope-loaded NPs could overcome the shortcomings of anti-tumor immunotherapy using ICIs. Our results showed that the anti-tumor activity of tumor epitope-loaded NPs could be further enhanced when combined with anti-PD1 mAbs. However, the combination with anti-PD1 mAbs did not increase the generation of tumor-specific CTLs in tumor-bearing mice. This suggests that the anti-PD1 mAb used in combination with the NPs exerted its effect by blocking the inhibitory signals delivered by PD-L1 in tumors. We confirmed that tumor cells indeed expressed PD-L1.

    PLGA is an extensively investigated biocompatible and biodegradable polymer approved by the US Food and Drug Administration for clinical applications in humans (43). It is degraded via hydrolysis of the ester bonds into lactic and glycolic acid and is commonly used in the production of NPs that are investigated for the delivery of drugs and/or vaccine Ags (44, 45). NPs prepared using PLGA, for which the particle size ranges from 500 nm to 2 μm, are efficiently internalized by the phagocytosis of professional APCs (22, 46). PLGA NPs with a size range below 200 nm are inappropriate for the delivery of Ags to APCs because they are minimally taken up through phagocytic recognition (47). The average size of tumor epitope-loaded NPs fabricated in the present study was approximately 1,032 nm, which can be taken up by professional APCs. The average number of tumor epitopes loaded onto the NPs was not measured exactly; however, it could be estimated as a maximum of 1.56×104/NP. In our previous study, we measured the average number of MHC-I molecules attached to NPs fabricated using the same methods described in this study and found that the number of MHC-I molecules immobilized per NP was 1.56×104 molecules/NP (29).

    In conclusion, the vaccination strategy described here would provide a novel approach that circumvents most of the drawbacks of peptide epitope-based vaccines such as difficulties in delivering the peptides specifically to professional APCs, short peptide half-life in vivo, and limited peptide immunogenicity. This study comprises the development and testing of a novel strategy to elicit potent CTL responses against tumor epitopes, based on vaccination using tumor epitope-loaded NPs combined with poly-IC and an anti-PD1 mAb. The present study demonstrates that vaccination with tumor epitope-loaded NPs combined with poly-IC and an anti-PD1 mAb is a novel strategy for efficient induction of tumor-specific CTLs for tumor immunotherapy.

    SUPPLEMENTARY MATERIALS

    Supplementary Figure 1

    Effect of dipeptides on the OVA257–264 exchange reaction with NP-Kb. (A, C, E) NP-Kb was incubated with 20 μM OVA257–264 in the presence or absence of 10 mM of each dipeptide (GL, GM, or GCha) for 18 h at 37°C. OVA257–264-pulsed NP-Kb was stained using a mAb (clone 25-D1.16) and analyzed by flow cytometry. (B, D, F) Mean fluorescence intensities of OVA257–264-pulsed NP-Kb. Data are presented as the mean ± SD of at least five independent experiments.

    Click here to view.(1M, ppt)

    Supplementary Figure 2

    Expression levels of PD-L1 in EG7-OVA and B16-F10 tumor cells. EG7-OVA (A) and B16-F10 (B) tumor cells were cultured in the presence or absence of 100 μg/mL IFN-γ. After 24 h, PD-L1 expression was analyzed using flow cytometry. Data are presented as the mean ± SD of at least three independent experiments.

    Click here to view.(588K, ppt)

    Supplementary Figure 3

    Effect of dipeptides on FITC-TRP2180–188 exchange reaction with NP-Kb. (A) NP-Kb was incubated with 100 μM FITC-TRP2180–188 in the presence or absence of 20 mM of each dipeptide (GL, GM, or GCha) for 18 h at 37°C. FITC-TRP2180–188-pulsed NP-Kb was analyzed using flow cytometry. (B) Mean fluorescence intensity of FITC-TRP2180–188-pulsed NP-Kb. Data are presented as the mean ± SD of at least five independent experiments.

    Click here to view.(666K, ppt)

    Notes

    Conflict of Interest:The authors declare no potential conflicts of interest.

    Author Contributions:

    • Conceptualization: Kim SH, Lee CK.

    • Data curation: Kim SH, Park JH.

    • Formal analysis: Kim SH, Park JH.

    • Funding acquisition: Lee CK.

    • Investigation: Kim SH, Park JH, Lee SJ, Lee HS.

    • Methodology: Kim SH, Jung JK, Lee CK.

    • Supervision: Lee CK.

    • Writing - original draft: Kim SH, Lee CK.

    • Writing - review & editing: Kim SH, Lee YR, Cho HI, Kim JK, Kim K, Park CS, Lee CK.

    Abbreviations

    APC Ag-presenting cell
    BMDC bone marrow-derived dendritic cell
    DC dendritic cell
    EDC 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride
    GCha glycyl-cyclohexylalanine
    GL glycyl-leucine
    GM glycyl-methionine
    ICI immune checkpoint inhibitor
    NP nanoparticle
    PEMA poly(ethylene/maleic anhydride)
    PLGA poly(lactic-co-glycolic acid)
    poly-IC polyinosinic-polycytidylic acid
    poly-ICLC poly-IC stabilized with poly-L-lysine and carboxy-methylcellulose
    TAA tumor-associated Ag
    TSA tumor-specific Ag

    ACKNOWLEDGEMENTS

    This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (NRF-2020R1A2C1009484, MRC-2017R1A5A2015541). The authors would like to thank Jonathan Yewdell, M.D., Ph.D., Cellular Biology Section Laboratory of Viral Diseases NIAID, NIH, MD, USA, for his critical and productive comments.

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    MeSH Terms
    Animals
    Antibodies, Monoclonal*
    Epitopes
    Histocompatibility Antigens Class I
    Immunization
    Immunotherapy*
    Melanoma
    Mice
    Nanoparticles*
    Peptides
    Poly I-C*
    Polylactic Acid-Polyglycolic Acid Copolymer
    T-Lymphocytes, Cytotoxic
    Thymoma
    Vaccination
    Vaccines
    Metrics
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    ORCID IDs
    Funding Information
    PERMALINK