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

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.


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 "peptidebinding 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 costimulatory 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-Ipeptide 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).
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 tumorbearing mice with tumor epitope-loaded NPs combined with poly-IC and anti-PD1 mAb is a powerful strategy to treat tumors.

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-2 b ), 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).

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-2K b mAb-conjugated NPs and H-2K b -attached NPs (NP-K b )
Anti-mouse H-2K b 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-2K b 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-2K b 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 tumorderived membrane proteins for 24 h at 4°C. The resulting H-2K b -attached NPs (NP-K b ) were washed twice with PBS and resuspended in 1 ml PBS. GL for 18 h at 37°C. OVA 257-264 -pulsed NP-K b was stained with mAbs recognizing H-2K b -OVA 257-264 complexes (clone 25-D1.16) and analyzed by flow cytometry. In some experiments, native peptides associated with H-2K b molecules of NP-K b were exchanged with TRP2 180-188 by incubating 5 mg of NP-K b with 100 μM TRP2 180-188 with or without 20 mM dipeptide GM for 18 h at 37°C, washed twice with PBS, and resuspended in PBS. In vivo generation of peptide-specific CD8 + T cells C57BL/6 mice were immunized intravenously with PBS or the indicated NPs (1×10 9 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-2K b -OVA 257-264 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 (1×10 9 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, OVA 257-264 -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 OVA 257-264 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 OVA 257-264 peptide pulsing. A 1:1 mixture of each target cell population was injected into immunized mice via the tail vein (1×10 7 cells/mouse). After 18 h, the specific killing of OVA 257-264 -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 OVA 257-264 -unpulsed cells (CFSE Low ) versus pulsed cells (CFSE High ) was calculated to represent cytotoxicity.

Anti-tumor activity
The right flanks of C57BL/6 mice were shaved and subcutaneously inoculated with EG7-OVA cells (5×10 5 cells/mouse). The mice were intravenously administered with PBS or the indicated NPs (1×10 9 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 mm 3 . Tumor volume was calculated using the following formula: Tumor Volume [mm 3 ]=0.52×(Long Diameter [mm])×(Short Diameter [mm]) 2 . Spleen or lymph node cells were isolated from tumor-bearing mice, stained with H-2K b -OVA 257-264 tetramers and mAbs Epitope-Coated NPs and Poly-IC/Anti-PD1 mAbs for Immunotherapy 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 (1×10 5 cells/mouse). The mice were intravenously administered with PBS or the indicated NPs (1×10 9 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-2K b -TRP2 180-188 tetramers and mAbs against mouse CD8α, and analyzed using flow cytometry according to the manufacturer's instructions.

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.

Fabrication and characterization of NP-K b pulsed with OVA 257-264
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-2K b mAbs (isotype mIgG2b) via carbodiimide conjugation and then attached to H-2K b molecules by incubating the mAb-conjugated NPs with solubilized membrane proteins isolated from the EG7-OVA tumor mass (H-2 b ) to generate H-2K b -attached NPs (NP-K b ). Native peptides associated with the H-2K b molecules of NP-K b were exchanged with a model tumor-specific peptide, OVA 257-264 , 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 Epitope-Coated NPs and Poly-IC/Anti-PD1 mAbs for Immunotherapy concentrations of OVA 257-264 . The peptide exchange efficiency was maximal when NP-K b was treated with 100 μM OVA 257-264 in the presence of 20 mM GL, whereas GM and GCha were ineffective (Supplementary Fig. 1). The relative degree of H-2K b -OVA 257-264 complexes on NP-K b was 2.8-fold higher when NP-K b was treated with 100 μM OVA 257-264 in the presence of 20 mM GL (NP-SIIN Hi ) than when NP-K b was treated with 100 μM OVA 257-264 only (NP-SIIN Lo ) (Fig. 1B). The H-2K b -OVA 257-264 complexes on NPs remained stable for up to 5 days (Fig. 1C). The average NP-SIIN Hi size was 1,032.1±44.8 nm, and the average ζ-potential was −19.48±1.61 mV.

NPs loaded with higher amounts of OVA 257-264 induce OVA 257-264 -specific CTLs more efficiently in normal mice
To compare the OVA 257-264 -specific CTL-inducing activity of NP-SIIN Lo and NP-SIIN Hi , mice were intravenously injected with the NPs (1×10 9 particles/mouse) three times at 7-day intervals. Control mice were intravenously injected with PBS or NP-K b . Five days after the first and last immunizations, OVA 257-264 -specific CTLs in peripheral blood were analyzed using H-2K b -OVA 257-264 tetramers. After priming, the percentage of OVA 257-264 -specific CTLs among CD8 + T cells reached 1.28% and 1.71% in mice treated with NP-SIIN Lo and NP-SIIN Hi , respectively ( Fig. 2A and B). After boosting, the percentage of OVA 257-264 -specific CTLs among CD8 + T cells increased to 3.36% and 5.63% in mice treated with NP-SIIN Lo and NP-SIIN Hi , respectively ( Fig. 2C and D). Seven days after the last immunization, OVA 257-264 -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-SIIN Hi induced OVA 257-264 -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-SIIN Lo (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 OVA 257-264 efficiently induced potent CTL responses.

Combining NP-SIIN Hi with poly-IC further increases OVA 257-264 -specific CTL induction in normal mice
The OVA 257-264 -specific CTL-inducing activity of NP-SIIN Hi , in combination with poly-IC, was examined in normal mice. In this experiment, mice were intravenously injected three times with NP-SIIN Hi (1×10 9 particles/mouse) alone or with poly-IC at 7-day intervals. Five days after the first and last immunizations, OVA 257-264 -specific CTLs in peripheral blood were analyzed using H-2K b -OVA 257-264 tetramers. In primed mice, the combination of NP-SIIN Hi with poly-IC did not induce discernible increases in OVA 257-264 -specific CTLs among CD8 + T cells compared to that with NP-SIIN Hi alone (1.98% vs. 1.61%) ( Fig. 2I and J). However, after boosting, the combination of NP-SIIN Hi with poly-IC significantly increased the percentage of OVA 257-264specific CTLs among CD8 + T cells compared to that with NP-SIIN Hi 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-SIIN Hi , but not during priming.
Seven days after the last immunization, OVA 257-264 -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-SIIN Hi combined with poly-IC potently induced OVA 257-264 -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-SIIN Hi alone (specific target cell killing of 56.2% in the spleen and 53.4% in the lymph node) ( Fig. 2N and P).

NP-SIIN Hi and poly-IC combination exerts potent anti-tumor effects in EG7-OVA tumor-bearing mice with increased production of OVA 257-264 -specific CTLs
The anti-tumor therapeutic potential of NP-SIIN Hi 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-SIIN Hi , poly-IC, or NP-SIIN Hi plus poly-IC at 7-day intervals. The combination of NP-SIIN Hi and poly-IC exerted much greater anti-tumor effects than NP-SIIN Hi alone (Fig. 3A). Poly-IC alone did not show anti-tumor activity. The anti-tumor therapeutic potential of NP-SIIN Hi plus poly-IC was also confirmed by the survival rate (Fig. 3B).
Upon completion of the anti-tumor experiment (day 25 of Fig. 3A), the induction degree of OVA 257-264 -specific CTLs was measured in the spleen and lymph nodes isolated from EG7-OVA tumor-bearing mice. The percentage of OVA 257-264 -specific CTLs in the spleen was increased 2.1-fold in mice immunized with NP-SIIN Hi plus poly-IC (12.9%) compared to that in mice immunized with NP-SIIN Hi alone (6.18%) (Fig. 3C and D). The percentage of OVA 257-264 -specific CTLs in the lymph node was also increased 1.8-fold in mice immunized with NP-SIIN Hi plus poly-IC (2.04%) compared to that in mice immunized with NP-SIIN Hi 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

Combination of NP-SIIN Hi 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-SIIN Hi 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-SIIN Hi , or NP-SIIN Hi 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-SIIN Hi , or NP-SIIN Hi plus poly-IC (Fig. 4A). Immunization with NP-SIIN Hi plus poly-IC significantly inhibited tumor growth, and its combination with anti-PD1 mAbs increased the anti-tumor activity of NP-SIIN Hi plus poly-IC (Fig. 4B).
On the last day of the experiment, OVA 257-264 -specific CTLs were measured in the spleen and lymph nodes. NP-SIIN Hi and poly-IC combination significantly increased the percentage of OVA 257-264 -specific CTLs among CD8 + T cells compared to that with NP-SIIN Hi 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-SIIN Hi or NP-SIIN Hi plus poly-IC did not further increase the percentage of OVA 257-264 -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 OVA 257-264 -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 TRP2 180-188 peptide (NP-SVYD) in combination with poly-ICLC potently induces TRP2 180-188 -specific CTLs in normal mice
The therapeutic efficacy of the tumor epitope-loaded NPs was extended to a melanoma mouse model. NP-K b was first prepared using the methods described, and then, native peptides associated with H-2K b molecules were exchanged with a melanoma-associated peptide, TRP2 180-188 . Because a mAb recognizing the H-2K b -TRP2 180-188 complex was not available, FITC-labeled TRP2 180-188 (FITC-TRP2 180-188 ) was used to optimize reactions, and the degree of FITC-TRP2 180-188 exchange was analyzed using flow cytometry. The peptide exchange efficiency was maximal when NP-K b was treated with 100 μM FITC-TRP2 180-188 in the presence of 20 mM GM (Supplementary Fig. 3). The binding of unlabeled TRP2 180-188 to NP-K b was confirmed by competitively blocking the binding of FITC-TRP2 180-188 to NP-K b with unlabeled TRP2 180-188 (Fig. 5A).
The TRP2 180-188 -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-K b or NP-SVYD (1×10 9 particles/mouse) together with poly-ICLC at 5-day intervals. Seven days after the last immunization, TRP2 180-188 -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 TRP2 180-188specific CTL activity (specific target cell killing of 39.5%) compared to that with NP-K b 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   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-2K b -OVA 257-264 tetramers and anti-mouse CD8α mAbs and analyzed using flow cytometry. (G) Spleen cells of each experimental group were also co-cultured with OVA 257-264 -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. inoculated with B16-F10 tumor cells (1×10 5 cells/mouse) on day 0. PBS, NP-SVYD or NP-SVYD plus poly-ICLC (1×10 9 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-2K b -TRP2 180-188 tetramers and anti-mouse CD8α mAbs and analyzed using flow cytometry. (E) Spleen cells of each experimental group were also co-cultured with TRP2 180-188 -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. 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 antitumor activity of NP-SVYD was observed when used in combination with both poly-ICLC and anti-PD1 mAb ( Fig. 6B and C).

Epitope-Coated
On the last day of the experiment, the degree of TRP2 180-188 -specific CTL induction was measured in the spleen. The combination of NP-SVYD with poly-ICLC significantly increased the percentage of TRP2 180-188 -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 TRP2 180-188specific 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 TRP2 180-188 -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.

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-2K b .
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 Epitope-Coated NPs and Poly-IC/Anti-PD1 mAbs for Immunotherapy 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-2K b in the NP-K b 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 Abconjugated 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 TRP2 180-188 . 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 1.56×10 4 / 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 1.56×10 4 molecules/NP (29).

Epitope-Coated NPs and Poly-IC/Anti-PD1 mAbs for Immunotherapy
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.

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Epitope-Coated NPs and Poly-IC/Anti-PD1 mAbs for Immunotherapy