Well-defined Mannosylated Polymer for Peptide Vaccine Delivery with Enhanced Antitumor Immunity

Abstract

Peptide-based cancer vaccines offer production and safety advantages but have had limited clinical success due to their intrinsic instability, rapid clearance, and low cellular uptake. Nanoparticle-based delivery vehicles can improve the in vivo stability and cellular uptake of peptide antigens. Here, a well-defined, self-assembling mannosylated polymer is developed for anti-cancer peptide antigen delivery. The amphiphilic polymer is prepared by RAFT polymerization, and the peptide antigens are conjugated to the pH-sensitive hydrophobic block through the reversible disulfide linkage for selective release after cell entry. The polymer-peptide conjugates self-assemble into sub-100 nm micelles at physiological pH and dissociate at endosomal pH. The mannosylated micellar corona increases the accumulation of vaccine cargoes in the draining inguinal lymph nodes and facilitates nanoparticle uptake by professional antigen presenting cells. In vivo studies demonstrate that the mannosylated micelle formulation improved dendritic cell activation and enhanced antigen-specific T cell responses, resulting in higher antitumor immunity in tumor-bearing mice compared to free peptide antigen. The mannosylated polymer is therefore a simple and promising platform for the delivery of peptide cancer vaccines.

Graphical Abstract

A well-defined, self-assembling mannosylated polymer provides straightforward formulation of multiple peptide antigens for effective anti-tumor activity.

1. Introduction

The recent clinical translation of multiple immunotherapies is the largest paradigm shift in oncology since the use of chemo-drugs in the 1940s.^[1–5] The major types of cancer immunotherapy include immune checkpoint blocking (ICB) monoclonal antibodies, chimeric antigen receptor T cells (CAR T cells), and cancer vaccines.^[6–10] Cancer vaccines, which educate the immune system to recognize and remove cancer cells, are a powerful approach among various cancer immunotherapies.^[11–13] Therapeutic cancer vaccines that stimulate both CD8⁺ cytotoxic T lymphocytes (CTLs) and CD4⁺ T-helper (Th) cells by inducing antigen-presenting cells (APCs) like dendritic cells (DCs) to present antigens through MHC class I (MHC-I) and MHC class II (MHC-II) pathways can generate specific and effective antitumor responses. Cancer vaccines offer the possibility to eliminate cancer cells with sustained protection against recurrence. As a result, cancer vaccines have attracted great attention in recent years, and several therapeutic vaccines have been used in the treatment of different types of cancers.^{[14, 15]}

Antigen sources in cancer vaccines include (i) tumor cells and cell lysates, (ii) recombinant, purified or in situ-expressed proteins, and (iii) peptide epitopes.^{[1, 3]} Peptide antigens are particularly attractive given their direct and natural function as T-cell epitopes, well-defined structures, ease of manufacturing and modifications, low cost, and long-term storage stability.^[16–19] Despite these advantages, the application of peptide-based cancer vaccines in anticancer clinical trials only achieved modest benefit, which is mainly attributed to the intrinsic drawbacks from peptides themselves. Soluble peptide vaccines suffer from poor in vivo stability due to degradation, fast clearance, low DC uptake, and inefficient delivery to lymph nodes (LNs).^[20–22]

Nanoparticle (NP)-based delivery systems can overcome the aforementioned pharmacokinetic challenges of soluble peptides.^{[2, 23–26]} NPs can protect encapsulated peptide antigens against enzymatic degradation, enhance cellular uptake, and improve the accumulation in the lymphatic systems (e.g., LNs).^[27–29] In addition, peptide antigens can be co-formulated with immunoregulatory molecules such as active adjuvants in one nanocarrier for better immunostimulation.^{[16, 30–32]} Among various nanoparticulate systems, polymers are the most widely used carriers because they can be synthesized with a broad range of chemical and structural functionality.^{[23, 33–36]} The size, particle shape, surface properties of polymeric carriers can be tuned to optimize peptide vaccine efficacy. Polymers can also be functionalized with targeting ligands (such as mannose, and CD40) to enhance DC uptake and LN accumulation.^{[27, 37–39]} The most commonly used polymer carrier is poly(lactic-co-glycolic acid) (PLGA) which has limited capacity for functionalization with targeting ligands or other components that promote intracellular delivery.^[24] There remains a need for simple and scalable polymeric systems for peptide antigen delivery.

This work reports mannosylated polymer as an effective delivery platform for reversibly-conjugated peptide in cancer vaccine applications. The amphiphilic polymer is easily prepared by reversible addition-fragmentation chain transfer (RAFT) polymerization. The poly(mannose) block serves as the hydrophilic domain for micellization as well as the targeting ligand for improved LN accumulation. Peptides of both MHC-I and MHC-II epitopes were conjugated to the pH-sensitive block via disulfide bonds. In this design, polymers shield antigens at physiological pH and rapidly disassemble into unimers at endosomal pH after cellular uptake to facilitate antigen release (Scheme 1). In vivo LN accumulation studies demonstrate that the mannosylated micelles improve the DC uptake compared with free peptides and non-targeted NPs. The mannosylated micelles display improved DC activation, enhanced antigen-specific T cell response and higher antitumor immunity compared to free peptide, revealing a simple and effective platform for peptide cancer vaccine design.

Scheme 1.

Schematic illustration of a mannosylated block copolymer for the delivery of both MHC-I and MHC-II epitopes to dendritic cells.

2. Results and Discussion

2.1. Synthesis of Mannosylated Polymer Platform for Peptide Delivery

The mannosylated polymer (MAN-P) was synthesized by RAFT polymerization (Figure 1a). The mannose block was first prepared by polymerization of mannose methacrylate (MMA). The resulting polymer (PMMA) had a degree of polymerization (DP) of 38 and low dispersity (Ð = 1.08) determined by gel permeation chromatography (GPC) (Figure S1 and S2). The MAN-P diblock polymer was obtained by copolymerization of 2-diisopropylaminoethyl methacrylate (DIPAMA) and pyridyl disulfide ethyl methacrylate (PDSEMA) using PMMA as the macro chain transfer agent (mCTA) (Figure 1a). The DP of DIPAMA and PDSEMA were 47 and 3, respectively (Figure S3). GPC trace of MAN-P demonstrated a mono-model distribution and relatively low dispersity (Ð = 1.21, Figure S4). The DIPAMA block provides pH-sensitivity for the copolymer, and PDSEMA enables conjugation with thiol-containing peptide antigens. Therefore, peptides conjugated to MAN-P are protected inside the micellar core in extracellular environments but are exposed after cellular internalization.

Figure 1.

a) Synthetic pathway of the mannosylated block copolymer MAN-P. b) Conjugation of peptide antigens to MAN-P.

Peptide antigens were conjugated separately to MAN-P through the disulfide exchange reaction of thiolated peptides with PDSEMA (Figure 1b). CSSSIINFEKL, a variant of the chicken ovalbumin (OVA) MHC-I epitope, and CSSISQAVHAAHAEINEAGR, a variant of the OVA MHC-II epitope, were selected as the model antigens.^[40] Both the peptides were conjugated to MAN-P with at least 50% efficiency by simply mixing the peptides and polymer in the aqueous solution (Table 1). The obtained polymer-MHC-I and polymer-MHC-II conjugates are denoted as P-I and P-II, respectively.

Table 1.

Conjugation of MHC-I and MHC-II peptides to the MAN-P polymer.

Entry	Peptide per polymer	Peptide loading efficiency (%)	Peptide loading content (wt%)
P-I	2.0	67%	9.0
P-II	1.5	50%	11

P-I and P-II can self-assemble into micellar NPs in phosphate buffer saline (PBS) at physiological pH = 7.4. Both P-I and P-II micelles had diameters of 30–50 nm with narrow size distribution (Figure 2a, b, Table 2), which were smaller than the blank vehicle control (Figure S5). NPs encapsulating MHC-I and MHC-II peptides (P-I/II) are prepared by simply mixing P-I and P-II conjugates during the micelle fabrication process. The P-I/II mixed micelles demonstrated similar size distribution as compared to P-I and P-II micelles (Figure 2c). In addition, all of these micelles disassemble into free polymer unimers at acidic pH (Figure 3), exposing peptides after cellular internalization in endosomes.

Figure 2.

Size distribution of P-I, P-II and P-I/II micelles in PBS (pH = 7.4).

Table 2.

Diameter and dispersity of various micelle formulations.

Entry	Diameter (nm)	PDI
P-I micelles	33.7	0.26
P-II micelles	32.1	0.3
P-I/II mixed micelles	39.4	0.4

Figure 3.

pH transition study for Man-P, P-I, P-II and P-I/II micelles.

2.2. LN Accumulation

To test the effect of mannose functionalization on in vivo delivery of nanovaccines to DCs, the CSSSIINFEKL antigen was rhodamine-labeled and injected as free peptide or peptide-conjugated micelles subcutaneously at the right tail base of the mice. In addition to the free peptide control, two non-targeted micelles with the same micellar core but different coronas (PEGylated or cationic)^[41–43] were also utilized as control groups (Figure 4a). Fluorescent imaging of harvested lymph nodes 48 h after injection showed that the mannosylated micelles were retained more efficiently in draining inguinal lymph nodes (ILNs) than the other formulations, with preferential localization of micelles into the right ILN that corresponded with the injection site (Figure 4b). Flow cytometry analysis of lymph node-resident cells verified that mannosylated micelles and their antigen cargoes were primarily internalized by CD11c⁺MHCII⁺ DCs (Figure 4c). Mannosylated micelles generated an average of 16-, 79- and 36- fold increase in the number of DCs internalizing the rhodamine-labeled peptides in the lymph nodes compared with neutral micelles, cationic micelles and free peptide respectively. Taken together, these results confirm that display of mannose at micellar surface significantly improved the LN targeting and DC uptake in vivo.

Figure 4.

a) Cationic, neutral and mannose micelle structures. b) Fluorescence imaging of right and left draining ILNs (48 h post-treatment). Mannosylated micelles are retained more efficiently in lymph nodes. c) Flow cytometry analysis of lymph node-resident cells confirms that mannose-micelle formulations are internalized by CD11c⁺MHCII⁺ DCs. Data are presented as mean ± SD. N = 3 biological replicates. Statistical significance was calculated using one-way ANOVA with post-hoc Fisher’s LSD test (**P ≤ 0.01, ***P ≤ 0.001).

2.3. In Vivo DC Maturation

DC maturation is a critical step for T cell activation and immune responses.^[44] Hallmarks of DC maturation include increased expression of MHC II molecules and costimulatory molecules such as CD86.^{[45, 46]} To assess whether the mannosylated micelles enhance DC maturation in vivo, P-I/II micelles, free peptides and PBS were injected subcutaneously in mice at the tail base followed by ILN analysis 24 h post-immunization. All the formulations were confirmed to have low levels of endotoxin (Table S1). P-I/II micelles increased the percentage of CD86⁺MHCII⁺ cells in the lymph nodes to a greater extent than free peptides (Figure 5a). In addition, P-I/II micelles increased the expression of maturation marker CD86 in both the total DC population and the CD8⁺ DC subset (Figure 5b, 5c). CD8⁺ DCs are a special subset of DCs that can present antigens on MHC I molecules through cross-presentation,^[47] a process that is essential for the activation of CD8⁺ T cells that directly kill tumor cells.^[48] P-I/II micelles significantly increased the expression of costimulatory molecule CD86 in CD8⁺ DCs, whereas free peptides had no effect on CD86 expression (Figure 5c). The higher amount of activated CD8⁺ DCs could lead to enhanced activation of CD8⁺ T cells and robust antitumor responses. It is important to note that DC maturation in response to the formulations was not observed in vitro. Immature bone marrow-derived dendritic cells (BMDCs) were incubated with PBS, free peptides, MAN-P and P-I/II micelles for 24 h. Minimal change in expression of CD86, CD40 and MHCII was observed by flow cytometry after treatment with either peptides, control polymer or P-I/II micelles (Figure S6). Collectively, these data suggest that the micelles do not on their own have an adjuvant effect, but likely promote DC maturation in vivo by facilitating higher DC uptake through mannose targeting. The vaccine formulation can therefore be further improved in the future by combining with adjuvants like the stimulator of interferon genes (STING) agonists.^[49]

Figure 5.

P-I/II micelles enhance DC maturation in vivo. a) Percentage of CD86⁺ MHCII⁺ antigen-presenting cells in the ILNs 24 h after subcutaneous injection of PBS, free peptides and P-I/II micelles at the tail base. b) Median fluorescence intensity (MFI) of CD86 in CD11c⁺ MHCII⁺ DCs by flow cytometry 24 h after immunization. c) Median fluorescence intensity (MFI) of CD86 in CD8⁺ DC subset by flow cytometry 24 h after immunization. Data are presented as mean ± SD. N=4 biological replicates. Statistical significance was calculated using one-way ANOVA with post-hoc Tukey HSD test (**P ≤ 0.01, ***P ≤ 0.001, ****P ≤ 0.0001, ns: not significant).

2.4. In Vivo T Cell Activation

We next evaluated whether P-I/II micelles could generate antigen-specific T cell responses in vivo. Mice were immunized with PBS, free peptides and P-I/II micelles on day 0 and 14, and splenocytes were collected and analyzed on day 21 (Figure 6a). H-2K^b/SIINFEKL tetramer staining was used to analyze the population of antigen-specific T cells in the splenocytes. P-I/II micelles generated a significantly higher SIINFEKL-specific T cell response, with a 6.8-fold increase of SIINFEKL-specific CD8⁺ T cells in the splenocytes compared with that in the free peptides treatment group (Figure 6b, 6c). Intracellular cytokine staining (ICCS) was used to analyze cytokine production upon antigen restimulation.^[50] P-I/II micelles induced a significant increase in the number of IFN-γ- and TNF-α-producing CD8⁺ T cells in the splenocytes when stimulated with SIINFEKL, whereas free peptides only induced cytokine producing cells similar to the PBS control (Figure 6d, Figure S7). Similarly, P-I/II micelles induced a higher number of IFN-γ- and IL-2-producing CD4⁺ T cells when stimulated with OVA antigen, although the increase was not statistically significant (Figure 6e). Collectively, these results demonstrate that the P-I/II micelles generate a large amount of antigen-specific CD8⁺ T cells and robust cytokine production in both CD8⁺ and CD4⁺ T cells, a desirable trait as a peptide antigen carrier for cancer vaccines. The data also support the correlation between DC maturation and T cell activation, where more activated DCs lead to more robust T cell responses.

Figure 6.

a) Schematic of the study timeline. Female C57BL/6 mice were immunized on days 0 and 14 via subcutaneous injection at the tail base with PBS, free peptides or P-I/II micelles. Spleens were collected on day 21. b) Representative flow data of H-2K^b/SIINFEKL tetramer staining in CD8⁺ T cells. c) Percentage of SIINFEKL-specific CD8⁺ T cells in the spleens. d) Percentage of IFN-γ⁺ and TNF-α⁺ CD8⁺ T cells after restimulation with SIINFEKL peptide. e) Percentage of IFN-γ⁺ and IL-2⁺ CD4⁺ T cells after restimulation with OVA protein. Data are presented as mean ± SD. N=4 biological replicates. Statistical significance was calculated using one-way ANOVA with post-hoc Tukey HSD test (***P ≤ 0.001, ****P ≤ 0.0001).

2.5. Therapeutic Vaccination

Given the enhanced T cell activation generated by P-I/II micelles, we next evaluated the therapeutic effects of the vaccines. B16F10-OVA tumor-bearing mice were injected with PBS, free peptides, MAN-P micelles (vehicle control), and P-I/II micelles four days post tumor inoculation, followed by two boost injections at one week intervals (Figure 7a). Immune checkpoint blockade (ICB) anti-PD1 was given one day after vaccination through intraperitoneal injection to increase the efficacy of the treatments. P-I/II micelles delayed tumor growth compared with free peptides and MAN-P (Figure 7b). In addition, P-I/II micelles significantly prolonged the survival of tumor-bearing mice compared with free peptides (Figure 7c). These results align with the other in vivo data, further demonstrating the potential of P-I/II micelles as a therapeutic cancer vaccine. A similar trend was observed in the combination treatment with ICB. ICB alone was not able to achieve a therapeutic effect. However, the combination of ICB with free peptides or P-I/II micelles slowed tumor growth and prolonged survival (Figure 7d, 7e). The difference in tumor volume and survival between ICB + free peptides and ICB + P-I/II micelles was significant, substantiating the efficacy of the mannosylated-polymer delivery system in rodent tumor models. Both P-I/II micelles and ICB + P-I/II micelles inhibited tumor growth compared with other groups (Figure 7f). The inclusion of ICB slightly improved the efficacy of the P-I/II micelles (Figure S8). However, the difference was not significant as the B16F10 melanoma model is poorly immunogenic.^{[51, 52]} The treatment could be potentially improved by combining the anti-PD1 and anti-CTLA4 treatments to overcome the immune suppression in both the early and the late stages of an immune response.^[53] In addition, anti-PD1 was administered once between weekly vaccinations. Increasing the dosage frequency to two doses between weekly vaccinations could improve the efficacy of the vaccine.^[54]

Figure 7.

P-I/II micelles inhibit tumor growth and prolong survival in B16F10 tumor-bearing mice. a) Schematic of the study timeline. Female C57BL/6 mice were inoculated with B16F10-OVA cells on day −4 and immunized on day 0, 7 and 14 via subcutaneous injection at the tail base with PBS, free peptides, MAN-P or P-I/II micelles. Immune checkpoint blockade (ICB) anti-PD1 was given 1 day post immunization in some of the groups. b, c) Tumor growth curve and survival curve of mice treated with PBS, free peptides, polymer and P-I/II micelles. d, e) Tumor growth curve and survival of mice treated with PBS, ICB, ICB + free peptides and ICB + P-I/II micelles. f) Individual tumor growth curves. Data are presented as mean ± SD. N=6 biological replicates. Statistical significance of tumor volume was calculated using unpaired t-test. Survival analysis was performed using the log-rank test (*P ≤ 0.05, **P ≤ 0.01).

3. Conclusion

In summary, we report the development of mannosylated polymeric NPs for peptide cancer delivery. The polymer carrier design possesses several advantages including defined chemical structures, multiple functionalities, and ease of manufacturing. Various peptide antigens can be readily conjugated to the polymer under mild conditions. The obtained polymer-antigen peptide conjugates can self-assemble into sub-100 nm micelles at physiological environments and dissociate at endosomal acidic conditions. The mannose displayed on the surface of the micelles facilitates LN accumulation and DC uptake. The mannosylated micelles demonstrate significantly enhanced antitumor immunity. With a simple design and multi-functional capability for targeted delivery, peptide protection and triggered peptide release, this mannosylated polymer provides a useful and practical platform for the peptide cancer vaccine delivery. The combination of this mannosylated vaccine with immune-stimulating molecules will be investigated in the future studies.

4. Experimental Section

Materials, polymer synthesis, and characterization:

RAFT chain transfer agent 4-((((2-Carboxyethyl)thio)carbonothioyl)thio)-4-cyanopentanoic acid (CCC), azobisisobutyronitrile (AIBN), 4,4’-azobis(4-cyanovaleric acid) (ABCVA), and PDSEMA were purchased from Sigma-Aldrich and used as received. Mannose ethylmethacrylate (MMA) was purchased from Omm Scientific. DIPAMA was purchased from Sigma-Aldrich and was purified by passing through a column filled with basic alumina to remove the inhibitor prior to polymerization. Anhydrous N-Methyl-2-pyrrolidone (NMP, 99.5%), and dimethylsulfoxide (DMSO, > 99%) were purchased from Sigma-Aldrich and stored with activated molecular sieves. Endotoxin level was measured using the limulus amebocyte lysate (LAL) endosafe PTS assay from Charles River.

Characterization:

Polymer characterization.

All ¹H NMR spectra were recorded on a Bruker AV 300 (Bruker Corporation, Billerica, MA) nuclear magnetic resonance (NMR) instrument in various deuterated solvents as described below. Polydispersity index (Ɖ = M_w/M_n) of the polymers were determined by gel permeation chromatography (GPC). The mobile phase consisted of DMF containing 0.1 M LiBr at a flow rate of 1 mL min⁻¹. Samples were filtered through a 0.45 μm PTFE filter before analysis. Size and distribution of micelles were recorded on a dynamic light scattering (DLS) system (DynaPro NanoStar, Wyatt technology).

Polymer synthesis:

The mannose macro chain transfer agent PMMA was prepared through RAFT polymerization of MMA (40 mol equiv) using ABCVA (0.1 mol equiv) as initiator and CCC (1 mol equiv) as chain transfer agent in DMSO. All reaction components were dissolved in DMSO at 35% wt/wt final concentration in a round bottom flask. After purging with argon through a sealed septum for 15 minutes, the flask was vigorously stirred at 70 °C for 18 h before the reaction was terminated by perfusion of air. The reaction solution was diluted by N,N’-dimethylacetamide (DMAc) and then precipitated into acetone and ether mixture (1:1, v/v). PMMA was obtained as white powder after high vacuum and used without further purification. Yield: 70%. Then, PMMA (1 mol equiv), AIBN (0.25 mol equiv), DIPAMA (60 mol equiv), and PDSEMA (10 mol equiv) were dissolved in NMP at 20% wt/wt in a round bottom flask. After purging with argon through a sealed septum for 15 minutes, the flask was vigorously stirred at 70 °C for 18 h. The reaction was terminated by perfusion of air and the product purified through serial dialysis in NMP and deionized (DI) water before lyophilization. MAN-P was dissolved in DMSO-d₆ for ¹H NMR spectroscopy and block 2 degree of polymerization was confirmed relative to known block 1 MMA content. Yield: 90%.

Peptide synthesis and conjugation:

Antigen peptides (CSSSIINFEKL and CSSISQAVHAAHAEINEAGR) were synthesized using a Liberty Blue (CEM) microwave peptide synthesizer on Rink Amide resin (Millipore) before purification with reverse phase HPLC as in our previous work.^[43] Synthesized peptides were conjugated to polymers through disulfide exchange between cysteine and PDSEMA and conjugates were purified as in previous work.^[43] Briefly, both polymer and peptide were dissolved in DI water at > 40 mg/mL and incubated at room temperature until Abs343 nm due to pyridyl-2-thione displacement plateaued (~24 h), indicating completion of disulfide exchange. Then, the reaction solution was put into a MWCO 10000 dialysis bag and dialyzed against DI water for 3 days. P-I and P-II conjugates were obtained after lyophilization.

Micelle formation:

The vaccine micelles were prepared by a pH-transition method. Briefly, 10–20 mg of P-I and P-II was dissolved in 190 μL of 200 mM monobasic sodium phosphate (~pH 5.4), followed by addition of 810 μL of 200 mM dibasic sodium phosphate to bring the pH to 7.4. Micelles were allowed to form overnight before buffer exchange to nanopure distilled water on a 10 kDa cutoff Amicon centrifugal filter (Millipore) and stored at 4 °C before use. The pH transition point of the micelles was determined using a Nile Red assay as in previous work.^[43]

Cell lines and animals:

B16F10-OVA cells (gift of Prof. Amanda Lund) were cultured in DMEM supplemented with 10% FBS and 1% P/S at 37°C and 5% CO2. All studies with animals were carried out in accordance with protocols approved by the Institutional Animal Care and Use Committee at the University of Washington. Female C57BL/6 mice, aged between 6 and 8 weeks, were purchased from Charles River Laboratories.

In Vitro Dendritic Cell Activation:

BMDCs were generated by culturing bone marrow cells from the femur and tibia of female C57BL/6 mice. Cells were cultured in RPMI supplemented with 10% HI-FBS, 1% P/S, 2mM L-glutamine, 50uM β-mercaptoethanol and 20 ng/mL GM-CSF. Medium was half replaced on day 3 and day 6. Non-adherent and loosely adherent immature BMDCs were harvested on day 8. BMDCs were plated in 6-well non-TC treated plates at 3 * 10⁵ cells per well. PBS, free peptides containing 1μg/ml CSSSIINFEKL and 1μg/ml CSSISQAVHAAHAEINEAGR, P-I/II micelles containing equivalent antigen and MAN-P containing equivalent polymer were added to the cell culture and incubated for 24 h at 37°C and 5% CO2. After incubation, cells were stained with Zombie NIR viability kit, incubated with anti-CD16/32 and with anti-CD86-BV510, anti-MHC II-eFluor450, anti-CD11c-APC and anti-CD40-FITC. Flow data were acquired on the Attune NxT flow cytometer and analyzed using FlowJo software.

Vaccine localization to LN and DCs:

Mice were injected subcutaneously in the right tail base with various CSSSIINFEKL formulations (20 μg CSSSIINFEKL, 10% rhodamine-labeled) in 40 μL of 5% (w/v) glucose. The following formulations were used: (i) soluble CSSSIINFEKL, (ii) Cationic micelles, (iii) PEGylated micelles, and (iv) mannosylated micelles (n = 3 per treatment group). The cationic and PEGylated polymers were synthesized according to previous reports.^{[41, 42]} CSSSIINFEKL and rhodamine-labeled CSSSIINFEKL were conjugated to these polymers using the same reaction conditions as described above. Rhodamine fluorescence at the injection site was monitored by Xenogen (Caliper Life Sciences) over a period of 48 hours. After 48 hours, mice were sacrificed and ILNs were removed. Rhodamine fluorescence in the ILNs was visualized by Xenogen. Lymph node-resident cells were subsequently harvested as described above. Harvested cells were spun down, transferred to a 96-well U-bottom plate, and stained with Zombie Violet live/dead staining buffer for 15 minutes at room temperature After live/dead staining, cells were washed with mouse anti-CD16/CD32 for 10 minutes at 4 °C. After blocking, cells were stained for 30 minutes at 4 °C with the following antibodies: FITC-anti-CD45, CD11c-APC, and PerCP/Cy5.5-anti-MHCII. After antibody staining, cells were washed and resuspended in 250 μL of PBS + 1% bovine serum albumin. Flow cytometry was used to analyze DC populations. DCs from untreated mice were used as assay baselines.

In Vivo Dendritic Cell Activation:

Female C57BL/6 mice were injected subcutaneously at the tail base with PBS, free peptides containing 25 μg CSSSIINFEKL and 25 μg CSSISQAVHAAHAEINEAGR, or P-I/II micelles containing equivalent antigen. ILNs were harvested 24 h post injection. LNs were digested in RPMI 1640, 10% FBS, collagenase type IV (50 U/mL) and DNase I (100 U/mL). Tissues were mashed against a 70 μm cell strainer to obtain single-cell suspension. Cells were stained with Zombie NIR viability kit for 15 min at room temperature. Afterwards, cells were incubated with anti-CD16/32 for 30 min at 4°C. After blocking, cells were incubated with anti-CD86-BV510, anti-MHC II-PerCP/Cy5.5, anti-CD11c-APC, anti-CD8-AF488 for 20 min at 4°C. Flow data were acquired on the Attune NxT flow cytometer and analyzed using FlowJo software.

In Vivo Immunization and T Cell Responses:

Female C57BL/6 mice were immunized on day 0 and 14 via subcutaneous injection at the tail base with PBS, free peptides containing 25 μg CSSSIINFEKL and 25 μg CSSISQAVHAAHAEINEAGR, or P-I/II micelles containing equivalent antigen. On day 21, spleens were collected and passed through a 70 μm cell strainer to obtain single-cell suspension. The splenocytes were treated with ACK lysis buffer, washed and transferred to 96-well plates for tetramer staining and intracellular cytokine staining (ICCS). To analyze antigen-specific T cell responses, splenocytes were stained with Zombie NIR viability kit for 15 min at room temperature, followed by 15 min incubation with PE-labeled H-2K^b/SIINFEKL tetramer (NIH Tetramer Core). Cells were then incubated with anti-CD16/32 and stained with anti-CD3-eFluor450 and anti-CD8-FITC. Flow cytometry was used to determine the percentage of SIINFEKL-specific CD8⁺ T cells. Data were analyzed using FlowJo software. To analyze cytokine production, splenocytes were cultured in IMDM with 10% FBS and 1% P/S and restimulated with 5 μg/mL SIINFEKL peptide or 5 mg/mL OVA protein. After 3 h incubation at 37 °C and 5% CO₂, Protein Transport Inhibitor Cocktail (eBioscience) was added and cells were incubated for an additional 3 h. After restimulation, cells were incubated with Zombie NIR, anti-CD16/32, followed by anti-CD3-eFluor450, anti-CD8-FITC or anti-CD4-FITC. Fixation and permeabilization of cells were carried out using BD Cytofix/Cytoperm kit. ICCS was performed with anti-TNF-α-PE and anti-IFN-γ-APC for CD8⁺ T cells and anti-IL-2-PE and anti-IFN-γ-APC for CD4⁺ T cells. Flow data were acquired on the Attune NxT flow cytometer and analyzed using FlowJo software.

Tumor Studies:

Female C57BL/6 mice were inoculated with 10⁵ B16F10-OVA cells in the right hind flank on day −4. On day 0, 7 and 14, mice were immunized with PBS, free peptides containing 25 μg CSSSIINFEKL and 25 μg CSSISQAVHAAHAEINEAGR, P-I/II micelles containing equivalent antigen or MAN-P containing equivalent polymer at the tail base via subcutaneous injection. 100ug anti PD-1 was given via intraperitoneal injection on day 1, 8 and 15. Tumor volume was measured every other day using a caliper and the volume was calculated using the equation Volume = Width² * Length / 2. Mice were euthanized if tumor volume exceeded 1500 mm³, body weight loss exceeded 20%, or tumor discharge was observed.

Statistical Analysis:

All statistical analyses were carried out in GraphPad Prism software. Unpaired t-test was used for comparisons with two groups. One-way ANOVA with post-hoc Tukey HSD test or post-hoc Fisher’s LSD test was used for comparisons with more than two groups. Survival analysis was performed using the log-rank test.