Oxygen-generating cryogel vaccines help overcome tumor antigen tolerance and induce durable antitumor immunity in prostate cancer

Summary

Therapeutic cancer vaccines represent a promising approach to boost patients’ own immune system to fight cancer; however, they have shown limited success in clinical trials largely due to insufficient antigen delivery to overcome tolerance and hypoxia-mediated immunosuppression. Cryogel-based delivery scaffolds offer a promising platform for cancer vaccines due to their biocompatibility and macroporous structure, enabling effective delivery to infiltrating antigen-presenting cells. However, these systems are limited by rapid, diffusion-mediated burst release of encapsulated proteins and local hypoxia-driven immunosuppression. Click conjugation of a tumor-associated protein within cryogel-based vaccines, combined with an O₂-generating platform (Click O₂-Cryogel_VAX), overcomes immunosuppression and weak antigenicity, and primes effective antitumor immunity. Sustained antigen delivery promotes cellular memory and Th1-driven responses, while O₂ release enhances humoral immunity. Together, Click O₂-Cryogel_VAX supports a robust antitumor response and prolongs survival in a therapeutic prostate cancer model, supporting further investigation as an effective delivery platform for cancer vaccines.

Graphical Abstract

eTOC Blurb

Therapeutic cancer vaccines often fail due to poor antigen delivery and tumor-driven immunosuppression. This study introduces Click O₂-Cryogel_VAX, an engineered cryogel that couples sustained antigen release with local oxygen generation to overcome hypoxia-mediated immunosuppression, elicit robust antitumor immunity, and significantly prolong survival in a prostate cancer model.

Introduction

Renewed interest in therapeutic cancer vaccines has followed major developments in vaccine technology and breakthroughs in cancer immunotherapy, including immune checkpoint inhibitors (ICIs) and adoptive cell therapy (ACT).^1,2 Prostate cancer is one of the leading causes of cancer-associated death in men, with a 5-year survival rate of 30 % in patients with metastatic castration-resistant prostate cancer (mCRPC).^3–5 In response, several therapeutic vaccines targeting prostate cancer have been developed to help prime an immune response to reject recurrent disease or delay progression.^4,6 Sipuleucel-T (PROVENGE^®), an antigen-loaded dendritic cell (DC)-based vaccine, was the first official cancer vaccine/cell-based immunotherapy approved by the Food and Drug Administration (FDA) in 2010 but has had limited success.^7–10 ProstVac-VF, a viral-based recombinant vaccine, showed improved median overall survival and was easier to administer as an “off-the-shelf” therapy compared to Sipuleucel-T; however, it showed no improvement as a monotherapy in Phase III trials.^8,9,11,12 Recent advances in mRNA vaccine technology have also led to the exploration of neoadjuvant and personalized therapeutic cancer mRNA vaccines.^13,14 BioNTech recently completed a Phase I/IIa trial investigating BNT112, an mRNA lipoplex vaccine as a monotherapy and in combination with anti-PD-1 (NCT04382898).¹⁵ While this trial was promising, Phase III trials will be required to reveal the clinical efficacy of the platform. Despite their ability to robustly boost B-cell and T-cell immunity against tumor cells, therapeutic cancer vaccines are limited by inadequate delivery methods, tumor heterogeneity, and the immunosuppressive tumor microenvironment (TME).^2,6,13 It is now clear that success will require not only improved formulations of antigen delivery vehicles but also innovative strategies to overcome tolerance and immunosuppression.^16–19

To overcome immunosuppression and immune tolerance, and maintain effective humoral and cellular immunity against tumor-associated antigens (TAAs) or tumor-specific antigens (TSAs), strategies are required to improve delivery vehicles, optimize routes and timing of administration, and identify effective adjuvants to activate immune cells.² To this end, biomaterial platforms, such as cryogels (a subclass of hydrogels), have been employed in preclinical models targeting melanoma, breast cancer, and other cancer subtypes.^20–30 These scaffold-based vaccine platforms offer a promising solution through the protection of biomolecules from rapid clearance and degradation.^31–33 The macroporous structure of cryogel scaffolds provides several benefits for the delivery of therapeutic cancer vaccines, including biocompatibility, mechanical strength, and injectability. The porous structure also facilitates nutrient diffusion and cell infiltration, providing an avenue for recruiting and activating immune cells in situ.^25,34–36 Cell-based therapies, TAA/TSA proteins or peptides, various adjuvants, and cytokines can be physically encapsulated or conjugated within the scaffold, along with granulocyte-macrophage colony-stimulating factor (GM-CSF), to recruit and stimulate antigen-presenting cells (APCs), such as DCs, at the injection site.^22,24,25,36 Scaffold-based delivery of therapeutic cancer vaccines enhances the ability of the antitumor immune response to recognize and eliminate tumor cells by extending antigen release from the scaffold and allowing for effective antigen capture and activation of DCs to prime cellular (e.g., cytotoxic T cells) and humoral (e.g., antibody-dependent cellular cytotoxicity (ADCC)) responses.^36–39

Loading efficiency and release kinetics of vaccine antigens and adjuvants have been a focus of improvement in scaffold-based delivery vehicles. Recombinant proteins typically exhibit rapid, diffusion-mediated burst release when physically encapsulated in hydrogels, including cryogel scaffolds, and protein encapsulation and release can be highly variable due to difficult-to-control cargo distribution during fabrication.^40,41 Extended vaccine exposure has been established as necessary to achieve the potency and breadth of immune responses required for adaptive immunity against challenging diseases, such as cancer.^42–44 Strategies to enhance protein antigen loading and release include implementing pre-absorbed protein cargo on Laponite nanoparticles to increase the loading of protein and provide sustained release from an alginate hydrogel.⁴⁰ Alternatively, tuning the viscoelastic properties of a poly(vinyl alcohol)-based hydrogel to promote sustained release of protein cargo has been recently explored.⁴⁵ However, the ability of such strategies to enhance immunological outcomes has not been tested. Moreover, biorthogonal crosslinked hyaluronic acid (HA)-based cryogels supported sustained release of vaccine components, resulting in increased intensity of the adaptive immune response over bolus injection, but were only evaluated with a highly immunogenic model antigen, ovalbumin (OVA), in a melanoma (B16-OVA) model.²³ Sustained antigen exposure was also recently demonstrated by carbodiimide-mediated crosslinking of OVA peptides to create porous antigen-conjugated scaffolds, which were shown to support sustained peptide release via polymer hydrolysis and successfully activate antigen-specific immune cells.⁴⁶

Despite improvements in antigen delivery, these platforms remain limited by physiological immunosuppression and tolerance mechanisms. Subcutaneous scaffolds experience local hypoxia (0–3 % O₂ in tissues) due to poor vascularization and high cellular activity during infiltration and the innate immune response.^47–49 In inflamed and cancerous tissues, hypoxia-driven signaling promotes the accumulation of extracellular adenosine, which potently inhibits effector functions of antitumor immune cells.^50–53 Our previous work has shown that oxygenation of the TME (via respiratory hyperoxia and/or oxygenation agents) reduces intratumoral hypoxia and immunosuppressive extracellular adenosine while reprogramming the cytokine/chemokine/metabolome profile away from immunosuppression and toward immune reactivity.^17,18,54,55 Here, we apply the discovery that oxygenation potently reverses immunosuppression in the TME to our cryogel-based vaccine development. We previously engineered an HA-glycidyl methacrylate (HAGM) cryogel-based O₂ delivery platform (O₂-Cryogel) that generates O₂ and restores the function of DCs and T cells under hypoxic conditions.^56–58 We have also shown the benefit of localized O₂ availability and cryogel-based delivery in improving the efficacy of a protein subunit vaccine against COVID-19 (O₂-Cryogel_VAX).⁵⁹ Local O₂ generation within the scaffold can mitigate hypoxia and oxygenate infiltrating cells, thereby functioning as a powerful co-adjuvant that reinforces DC-mediated responses by preventing vaccine-associated hypoxia at the vaccination site.^56,59 O₂-Cryogel_VAX promoted a stronger humoral immune response, showing an increase in long-lasting anti-SARS-CoV-2 IgG antibodies compared to Cryogel_VAX and Bolus_VAX. Additionally, O₂ generation promoted the production of Th1-biased and proinflammatory cytokines.

Herein, we explored how enhanced antigen loading and release, in combination with local O₂ generation, could enhance our Cryogel_VAX and O₂-Cryogel_VAX platforms as effective delivery vehicles for a subunit protein cancer vaccine targeting prostate cancer. First, we explored the effectiveness of click chemistry (Click Cryogel_VAX) via copper-free, strain-promoted azide-alkyne cycloaddition (SPAAC) for delivering a TAA protein, prostatic acid phosphatase (PAP). SPAAC-mediated conjugation has been shown to be a safe approach for biomolecule conjugation in HAGM-based cryogel scaffolds.⁶⁰ Click Cryogel_VAX was evaluated against Cryogel_VAX to determine how extended exposure of TAA to infiltrating APCs might enhance B- and T-cell responses post-immunization. Subsequently, local oxygenation was further evaluated as a potent co-adjuvant (O₂-Cryogel_VAX, Click O₂-Cryogel_VAX). Finally, we evaluated the efficacy of Click O₂-Cryogel_VAX to elicit a strong, balanced immune response against the loaded TAA, thereby supporting an effective antitumor response in a TRAMP-C2 prostate cancer model.

Results

Fabrication of prostate cancer-associated antigen cryogel-based vaccines

To engineer a prostate cancer vaccine that elicits immunologic memory against murine prostate cancer cells (TRAMP-C2), the overexpressed protein PAP was used in addition to the immunomodulatory factors: granulocyte-macrophage colony-stimulating factor (GM-CSF) and polyinosinic:polycytidylic acid (Poly(I:C)). Previous cryogel-based vaccine formulations relied on physical entrapment of antigens and adjuvants during cryogelation to deliver the vaccine. Here, we found that PAP exhibits poor loading through physical encapsulation, therefore we evaluated if chemical conjugation of PAP through click bioconjugation would influence vaccine efficacy. First, the polymer HAGM was modified with Azido-PEG3-amine via carbodiimide (EDC/NHS) coupling, and the resulting polymer, HAGM-PEG3-Azido, was purified. Conjugation of the Azido-PEG3 group to the HAGM backbone was confirmed with Proton Nuclear Magnetic Resonance (¹H NMR) and Attenuated Total Reflectance Fourier-Transform Infrared (ATR-FTIR) analysis (Figs. S1, S2). Additionally, PAP was reacted with DBCO-PEG4-NHS ester to yield DBCO-PEG4-PAP, which was then purified. HAGM or HAGM-PEG3-Azido were combined with PAP or DBCO-PEG4-PAP, respectively, and GM-CSF and Poly(I:C) in solution overnight at 4 °C before cryopolymerization at −20 °C for 18 h and then thawed to form cryogel-based vaccines, Cryogel_VAX (HAGM) or Click Cryogel_VAX (HAGM-PEG3-Azido) (Fig. 1A). To form O₂-generating cryogel-based vaccines, ball-milled calcium peroxide (CaO₂) particles (1% w/v) and acrylate-PEG-catalase (CAT) (1% w/v) were incorporated into the polymer solution before freezing (Fig. 1A). CaO₂ hydrolyzes with water (H₂O) to form hydrogen peroxide (H₂O₂) and O₂. CAT is added to support the decomposition of H₂O₂ into H₂O and O₂. Additionally, formation of cryogels with HAGM-PEG3-Azido compared to HAGM did not affect the pore connectivity, swelling ratio, or mechanical strength of Cryogels or O₂-Cryogels (Fig. S3).

Figure 1. Characterization of PAP-containing cryogel-based vaccines.

(A) Schematic depicting the fabrication of Cryogel_VAX and O₂-Cryogel_VAX using HAGM or HAGM-PEG3-Azido, prostate cancer antigenic proteins, and O₂ generation. In this process, the polymer is dissolved with biomolecules and CaO₂ before being mixed with the initiator system (APS/TEMED), followed by incubation at −20 °C for 18 h to allow cryopolymerization. (B) Loading efficiency (%) of PAP inside cryogel-based vaccines. Values are presented as mean ± SEM of three independent experiments. (C) Representative confocal microscopy images of FITC-conjugated PAP and DBCO-PEG4-PAP inside Cryogel_VAX and Click Cryogel_VAX. (D) In vitro release curves for PAP and GM-CSF from cryogel vaccines over time. Values are presented as mean ± SD (n = 3–4). (E) O₂ release from O₂-Cryogel_VAX and Click O₂-Cryogel_VAX over time. Values are presented as mean (n = 3). Statistical analysis was performed using one-way ANOVA and a Bonferroni post hoc test (α = 0.05/6 = 0.0083): ****p < 0.0001.

Encapsulation of PAP through physical entrapment resulted in poor loading efficiency: 10 ± 1.7% in Cryogel_VAX and 18 ± 2.4% in O₂-Cryogel_VAX. As expected, click conjugation ensured nearly complete loading of the DBCO-modified protein with 93 ± 2.0% and 95 ± 6.6% loading efficiency in Click Cryogel_VAX and Click O₂-Cryogel_VAX, respectively (Fig. 1B). Confocal microscopy of FITC-labeled protein confirmed protein within the polymer walls of macroporous cryogel scaffolds (Fig. 1C). PAP was released over 96 h, with full recovery of the encapsulated protein from Cryogel_VAX and O₂-Cryogel_VAX. As expected, chemical conjugation prevented release of PAP from Click Cryogel_VAX; however, almost all the encapsulated PAP (~90%) was released from Click O₂-Cryogel_VAX within 96 h (Fig. 1D). Similar to previously reported findings, GM-CSF and Poly(I:C) were physically entrapped within Cryogel_VAX, 99 ± 23% and 60 ± 5.4% respectively, and most of the encapsulated cargo was released within 12–24 h (Figs. 1D, S4). No significant differences were found with the addition of click moieties and O₂ generation.

O₂-generating cryogel vaccines released O₂ for 48 h, with the O₂ level peaking at ~200–225 Torr (corresponding to ~26–30% O₂) after 3 h. Both O₂-Cryogel_VAX and Click O₂-Cryogel_VAX exhibited very similar release profiles for O₂ (Fig. 1E). To assess any potential effects associated with the production and CAT-mediated decomposition of H₂O₂, O₂-Cryogel_VAX and Click O₂-Cryogel_VAX were incubated in PBS with and without covalently incorporated CAT. Decomposition of H₂O₂ by CAT accounted for the majority of O₂ production, and the released H₂O₂ in the supernatant was measured. As expected, inclusion of CAT (1% w/v) effectively depleted H₂O₂, maintaining concentrations below 10 μM, a range previously shown to be safe and non-toxic to cells, comparable to Cryogel controls, whereas CAT-free O₂-Cryogels produced ~30 μM H₂O₂ (Fig. S5). While we have confirmed that the inclusion of CAT maintains safe levels of H₂O₂ and have previously shown no negative effects of O₂-Cryogels on immune cell viability and functionality, residual H₂O₂ could have redox-mediated signaling effects on sensitive immune cells.^58,61,62 Additionally, the low levels of H₂O₂ likely contributed to the oxidation-mediated degradation of HAGM and PEG linkages, thereby releasing conjugated PAP in Click O₂-CryogelVAX.^57,63–65

Immunization with Click O₂-Cryogel_VAX improves TAA delivery and APC response in draining lymph nodes (LNs)

We hypothesized that APCs would traffic to the injection site, pick up antigens, become activated and migrate to the draining LNs to prime effective B- and T-cell responses against the delivered antigen (Fig. 2A). Previously, we have shown that O₂-Cryogel_VAX prevents infiltrating immune cells from becoming hypoxic; herein, we confirmed this aspect by subcutaneous injection of the Cryogel_VAX formulations. Cryogels were explanted after 72 h, and the fraction of hypoxic infiltrating immune cells was quantified. As expected, O₂ generation inside the scaffold significantly reduced cellular hypoxia in vivo. Immune cells (CD45⁺) collected from Cryogel_VAX and Click Cryogel_VAX were 75 ± 5.7% and 63 ± 5.6%, respectively, compared with 41 ± 2.9% and 42 ± 3.4% in O₂-Cryogel_VAX and Click O₂-Cryogel_VAX, respectively (Fig. 2B).

Figure 2. Click O₂-Cryogel_VAX reduces cellular hypoxia, enhances antigen uptake, and promotes downstream APC and B-cell responses.

(A) Schematic illustrating cryogel-based vaccine-supported immunity through recruitment of APCs that will migrate to the LNs and prime antitumor T and B cell responses. Created in BioRender. Noonan, B. (2026) https://BioRender.com/rjetc4n. (B) Fraction of hypoxic cryogel-infiltrating leukocytes at 72 h and representative histogram of Hypoxyprobe⁺ expression. (C) Percentage of pHrodo^™ Green (FITC)⁺ expressing leukocytes (CD45⁺) and conventional DCs (CD11c⁺, MHC-II⁺) from cryogels 3 and 7 d after injection. Values are indicative of the average from each animal and are presented as mean ± SEM (n = 5). Statistical analysis was performed using one-way ANOVA and Bonferroni post hoc test (α = 0.05/6 = 0.0083): *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. (D) Counts of conventional DCs (CD11c⁺, MHC-II⁺), macrophages (F4/80⁺), antigen-presenting B cells (CD19⁺ MHC-II⁺), and mature B cells (CD19⁺ IgD⁺) in inguinal LNs 28 d after vaccination. Two cryogels or draining LNs were analyzed per animal. Values are indicative of the average from each animal and are presented as mean ± SEM (n = 6). Statistical analysis was performed using one-way ANOVA and Bonferroni post hoc test (α = 0.05/15 = 0.0033): *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

To investigate the phagocytosis of protein antigen by infiltrating immune cells, PAP and DBCO-PEG4-PAP were labeled with pHrodo^™ Green, incorporated into Cryogel_VAX formulations, and cryogels were subcutaneously injected. After 3 and 7 d, cryogels were explanted, and the percentages of immune cells (CD45⁺) and conventional dendritic cells (cDCs) (CD11c⁺, MHC-II⁺) that were positive for the protein antigen were quantified. Strikingly, Click O₂-Cryogel_VAX significantly increased the fraction of immune cells and cDCs that were positive for phagocytosed protein 3 d after injection (p < 0.0001). Additionally, Click O₂-Cryogel_VAX maintained higher fractions of cells positive for phagocytosed protein 7 d after injection (Figs. 2C, S6A–B). To confirm whether click-modified PAP could have influenced phagocytosis, bone marrow-derived DCs (BMDCs) were co-cultured with pHrodo^™ Green-labeled PAP and DBCO-PEG4-PAP, and no differences in phagocytosis were observed (Fig. S6C).

To test whether the Cryogel_VAX formulations were effective at promoting immune-cell responses in the draining LNs, C57BL/6 mice were immunized and, after 14 d, boosted with subcutaneous injections on both flanks. Mice were euthanized 28 d after initial immunization, and the inguinal LNs were dissected for evaluation of total APC counts through flow cytometry (Fig. S7A). Vaccination with Click O₂-Cryogel_VAX created the strongest inflammatory response with the highest number of CD45⁺ leukocytes (Fig. S7B). Click O₂-Cryogel_VAX significantly increased the total count of CD11c⁺ MHC-II⁺ cDCs compared to Bolus_VAX injection (p = 0.0064) and Cryogel_VAX (p = 0.016) (Fig. 2D). Additionally, Click O₂-Cryogel_VAX significantly increased the total count of F4/80⁺ macrophages compared to Bolus_VAX (p = 0.0005), O₂-Cryogel (p = 0.0035), and Cryogel_VAX (p = 0.0033) (Fig. 2D). Next, the vaccine’s ability to enhance B-cell activity in the LNs was evaluated. Click O₂-Cryogel_VAX significantly increased the number of antigen-presenting CD19⁺, MHC-II⁺ B cells over Bolus_VAX (p = 0.0008), O₂-Cryogel (p = 0.013), and Cryogel_VAX (p = 0.032) (Fig. 2D). Additionally, Click O₂-Cryogel_VAX increased total counts of mature CD19⁺, IgD⁺ B cells compared to Bolus_VAX (p = 0.0009), O₂-Cryogel (p = 0.014), and Cryogel_VAX (p = 0.030) (Fig. 2D). These results suggest that Click O₂-Cryogel_VAX exhibits a higher APC prevalence in the draining LNs than previous cryogel vaccine formulations and Bolus vaccination.

O₂ generation increases Th1 antibody response and improves TAA-specific antibody production

To determine whether increased antigen phagocytosis and B-cell presence after vaccination was effective in eliciting a robust antibody response, blood was collected from the submandibular vein of the mice 2 weeks after the prime and boost, and the serum was isolated (Fig. 3A). Antibody titers were evaluated through serial dilution of mouse sera for each treatment group (Fig. S8, Table S1). As expected, inclusion of O₂-generation improved the overall humoral immune response. Click O₂-Cryogel_VAX significantly increased PAP-specific immunoglobulin production by day 28 over Bolus_VAX (p = 0.0078), O₂-Cryogel alone (p = 0.0093), Cryogel_VAX (p = 0.0083), and Click Cryogel_VAX (p = 0.0090), additionally, while not statistically significant, O₂-Cryogel_VAX also showed a similar increase in PAP-specific immunoglobulin (Fig. 3B). Immunization with Click O₂-Cryogel_VAX also significantly increased the levels of primary antibody IgM compared to Bolus_VAX (p = 0.028), Cryogel_VAX (p = 0.015), and Click Cryogel_VAX (p = 0.0063) (Fig. 3C).

Figure 3. O₂-generating cryogel-based vaccines promote production of Th1-associated antibodies.

(A) C57BL/6 mice were immunized subcutaneously on day 0 and day 14. Mouse sera was collected 14 d after each immunization and analyzed for anti-PAP antibodies and IgG subtypes. Schematic created in BioRender. Noonan, B. (2026) https://BioRender.com/ryf92i4. (B) Anti-PAP IgG, (C) IgM, (D) IgG1, (E) IgG2b, (F) IgG2c, (G) IgG3 EC50 titers 14 and 28 d after prime and boost with Bolus_VAX, O₂-Cryogel alone or Cryogel_VAX formulations. Values are presented as mean ± SEM (n = 4–6). Statistical analysis was performed using one-way ANOVA and Bonferroni post hoc test (α = 0.05/15 = 0.0033): *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

To better understand the balance between Th1/Th2 immune responses after vaccination, the overall production of several IgG subclasses was evaluated. Mice that received O₂-Cryogel_VAX or Click O₂-Cryogel_VAX had elevated IgG1 titers (Fig. 3D). While there were no significant differences after 28 d, O₂-Cryogel_VAX significantly increased the levels of IgG2b and IgG2c on day 14, and a similar tendency is observed on day 28 (Fig. 3E–F). However, vaccination with Cryogel_VAX and Click Cryogel_VAX displayed a similar increase in IgG2c as with oxygenation (Fig. 3F). Interestingly, we found that Click O₂-Cryogel_VAX had significantly lower levels of IgG3 on day 14, which were overcome by day 28 with significantly higher levels compared to the other treatments (Fig. 3G). These findings suggest that cryogel delivery of the vaccine components supports Th1-associated immune responses, as indicated by increased IgG2c levels, while O₂ generation increased IgG1 and IgG3 levels, suggesting a potential role in promoting antibody-dependent cellular cytotoxicity (ADCC). Indeed, IgG1 and IgG3 can engage Fc receptor-mediated effector functions that contribute to NK cell-mediated targeting of cancer cells.⁶⁶

Improved loading and delivery of TAA support memory cell generation and cytotoxic responses

To evaluate the cellular immune response after vaccination, the spleens were dissected after 28 d and memory T cell populations were characterized with flow cytometry (Fig. S9). Strikingly, Cryogel_VAX and Click Cryogel_VAX resulted in a significant shift in the proportion of central memory (CD44⁺ CD62L⁺) T cells over effector memory (CD44⁺ CD62L⁻) in both CD4⁺ and CD8⁺ T cells, suggesting that cryogel vaccines supported the creation of long-lasting immunity compared to Bolus_VAX (Fig. 4A–F). Cryogel_VAX and Click Cryogel_VAX increased CD4⁺ central memory T cells over Bolus_VAX (p = 0.0081, 0.0016) and Click O₂-Cryogel_VAX maintained a significant increase compared to Bolus_VAX (p = 0.018) (Fig. 4B). Similarly, in the CD8⁺ central memory T cells, Cryogel_VAX and Click Cryogel_VAX increased over Bolus_VAX (p = 0.0099, 0.0018), and Click O₂-Cryogel_VAX maintained a significant increase compared to Bolus_VAX (p = 0.0044) (Fig. 4E). Interestingly, the increase in central memory population was maintained, or potentially slightly attenuated, by the inclusion of O₂ generation, suggesting that cryogel-supported vaccine delivery plays a central role in the generation of memory T cells. Additionally, Cryogel_VAX, Click Cryogel_VAX, and Click O₂-Cryogel_VAX significantly increased the population of Naïve T cells, indicating an altered proportion of T-cell population after vaccination (Fig. 4C, 4F).

Figure 4. Vaccination with Click Cryogel_VAX and Click O₂-Cryogel_VAX promote the generation of robust cellular memory to tumor-associated antigens.

(A–F) Flow cytometric analysis of memory T cell populations in spleen after receiving vaccine regimen including percentages of CD4⁺ (A) effector memory (CD44⁺, CD62L⁻), (B) central memory (CD44⁺, CD62L⁺), and (C) naïve (CD44⁻, CD62L⁺), and percentages of CD8⁺ (D) effector memory (CD44⁺, CD62L⁻), (E) central memory (CD44⁺, CD62L⁺), and (F) naïve (CD44⁻, CD62L⁺). (G) Percentages of TNF-α-producing (CD44⁺, TNF-α⁺) and IL-2-producing (CD44⁺, IL-2⁺) activated CD8⁺ T cells in response to splenocyte restimulation with TAAs. (H–J) TAA specific IFN-γ producing splenocytes, as a proxy for T cells after culture with peptides and tumor cells. Values are presented as mean ± SEM (n = 5–6). Statistical analysis was performed using one-way ANOVA and Bonferroni post hoc test (α = 0.05/15 = 0.0033): *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

To evaluate TAA-specific memory, the collected splenocytes were co-cultured with TAA-peptides (mPAP_114–122, mPAP_128–136), recombinant PAP, TRAMP-C2 cells, PMA/ionomycin (positive-control), or culture media alone (negative-control) for 6 h, and the cellular immune response was evaluated by measuring the generation of Th1 cytokines: TNF-α, IL-2, and IFN-γ. Vaccination with Click O₂-Cryogel_VAX significantly increased the percentage of CD44⁺ TNF- α⁺ CD8⁺ T cells in response to the tumor cells (Figs. 4G, S10) and the percentage of CD44⁺ IL-2⁺ CD8⁺ T cells in response to the tumor cells and TAA peptide mPAP_128–136 (Figs. 4G, S10). Splenocytes collected from mice that received Click Cryogel_VAX and Click O₂-Cryogel_VAX also produced more IFN-γ when exposed to TAA peptides mPAP_114–122 and mPAP_128–136, as well as the tumor cells (Fig. 4H–J). These results suggest enhanced anti-cancer outcomes with click-loaded protein by increased production of Th1 cytokines, which support the differentiation and activation of cytotoxic T cells, macrophages, and natural killer cells.

O₂-Cryogel_VAX and Click O₂-Cryogel_VAX significantly reduce tumor growth in a therapeutic TRAMP-C2 model

Based on our results above, demonstrating that Click O₂-Cryogel_VAX improved delivery of PAP to APCs, enhanced APC and B-cell responses in draining lymph nodes, promoted humoral immunity, and improved cellular memory and Th1 responses after vaccination, we evaluated the therapeutic efficacy of Click O₂-Cryogel_VAX. Because Click O₂-Cryogel_VAX consistently demonstrated superior overall immunological performance and because the primary objective of the therapeutic study was to determine the contribution of O₂ generation to antitumor efficacy, subsequent therapeutic studies focused on O₂-generating formulations. TRAMP-C2 cells were derived from a mouse prostate adenocarcinoma and are considered an immunologically “cold” tumor model.^67,68 TRAMP-C2 tumors were subcutaneously developed and when the mean tumor size exceeded 50 mm³, mice received a prime (0 d) and boost (14 d) immunization of Bolus_VAX, O₂-Cryogel, O₂-Cryogel_VAX, or Click O₂-Cryogel_VAX in both flanks, and tumor size and mouse survival were monitored over time (Fig. 5A). As compared to our control, Bolus_VAX, the use of O₂-cryogels, as described before, slowed tumor progression and slightly increased survival by 2 d. Moreover, when combined with TAA, O₂-Cryogel_VAX significantly hindered tumor growth compared to Bolus_VAX (p = 0.0005) and O₂-Cryogel alone (p = 0.0003) (Fig. 5B–C). Notably, improved loading of the TAA in Click O₂-Cryogel_VAX led to an enhanced antitumor response with a significant decrease in tumor growth rate. Treatment with Click O₂-Cryogel_VAX significantly slowed tumor growth compared to Bolus_VAX (p = 0.0005) and O₂-Cryogel alone (p < 0.0001) (Fig. 5B–C).

Figure 5. Vaccination with Click O₂-Cryogel_VAX significantly delays TRAMP-C2 tumor growth.

(A) C57BL/6 mice were subcutaneously injected with TRAMP-C2 tumor cells. After tumors reached a mean volume of 50 mm³, mice were immunized and boosted 2 weeks apart, and tumors were measured with calipers until tumor burden exceeded euthanasia criteria. Schematic created in BioRender. Noonan, B. (2026) https://BioRender.com/lyv0b3n. (B) Average tumor volume over time post-treatment and (C) individual subject tumor volume over time post-treatment with Bolus_VAX, O₂-Cryogel, O₂-Cryogel_VAX, and Click O₂-Cryogel_VAX. Data are presented as mean ± SEM (n = 10–11). Statistical analysis was performed using Mixed effects analysis and Bonferroni post hoc test (α = 0.05/6 = 0.0083): *p < 0.05, ***p < 0.001, and ****p < 0.0001. (D) Overall Kaplan–Meier survival curves of treated mice (n = 9–11). Statistical analysis was performed using log-rank (Mantel-Cox) test and Bonferroni post hoc test (α = 0.05/6 = 0.0083): *p < 0.05, and ***p < 0.001. (E) Flow cytometric characterization of tumor-infiltrated T cells. Data are presented as mean ± SEM (n = 6–8). Statistical analysis was performed using one-way ANOVA and Bonferroni post hoc test (α = 0.05/6 = 0.0083): *p < 0.05, and ***p < 0.001. Black colored * indicates comparison to Bolus_VAX, and purple colored * indicates comparison to O₂-Cryogel.

Strikingly, treatment with O₂-Cryogel_VAX and Click O₂-Cryogel_VAX increased the median survival to 64 and 76 d, respectively, compared to 47 d with Bolus_VAX (p = 0.026, p = 0.0004) (Fig. 5D, Table S2). The median lifespan of mice receiving Click O₂-Cryogel_VAX was extended by 62%. In addition, Click O₂-Cryogel_VAX induced durable tumor growth control in 2 mice until study endpoint. When characterizing the tumor-infiltrating T cells (Fig. S11), O₂-Cryogel_VAX and Click O₂-Cryogel_VAX incrementally increased the presence of CD8⁺ tumor-infiltrating T cells, resulting in an increased ratio of CD8⁺/CD4⁺ T cells (Fig. 5E). While not statistically significant, both O₂-Cryogel_VAX (23 ± 3.3%) and Click O₂-Cryogel_VAX (24 ± 3.6%) similarly decreased the percentage of CD25⁺ FoxP3⁺ regulatory T cells compared to less effective treatments Bolus_VAX (32 ± 0.95%) and O₂-Cryogel (29 ± 2.5%) (Fig. 5E). Furthermore, O₂-Cryogel_VAX (17 ± 2.0%) and Click O₂-Cryogel_VAX (18 ± 1.6%) significantly decreased the percentage of PD-1⁺ CD4⁺ cells over Bolus (34 ± 1.6%) and O₂-Cryogel (27 ± 2.9%) (p < 0.001, p < 0.05) (Fig. 5E). A similar trend was also obtained in PD-1⁺ CD8⁺ cells. Taken together, these results suggest that vaccination with O₂-Cryogel_VAX and Click O₂-Cryogel_VAX promote antitumor T-cell responses resulting in significant prostate tumor regression.

Discussion

Recent advancements in vaccine development provide promising opportunities to enhance the design and delivery of effective therapeutic cancer vaccines.^69,70 However, creating an effective adaptive immune response capable of overcoming barriers to cancer therapy, such as the immunosuppressive TME, tolerance to self-antigens, and tumor heterogeneity, has proven challenging.^69,70 Harnessing biomaterial scaffolds, such as cryogels, as a delivery platform may be key to enhancing vaccine efficacy.^{22,24,27,36,38} By delaying the release of vaccine components, scaffold-based delivery enables improved adaptive memory responses after immunization.^29,37,40,42 Additionally, we demonstrated that O₂ delivery can be utilized as a co-adjuvant to boost antitumor responses, overcome physiological immunosuppression, and have incorporated this effective strategy into our vaccine design.^56,59,71,72 The enhanced immune responses observed may be explained by the restoration of DC function under hypoxic conditions, as previously shown.^56,58 O₂-Cryogels prevent tolerogenic DC phenotypes and restore antigen presentation and T-cell priming. We leveraged click-mediated covalent bioconjugation to improve the retention and sustained delivery of an antigenic subunit protein within a cryogel-based vaccine platform, thereby enhancing adaptive memory, while utilizing O₂ as a potent co-adjuvant to boost Th1-associated proinflammatory responses. Together, these two unique features of our vaccine platform promoted a strong antitumor response.

To formulate a Cryogel_VAX platform for murine prostate cancer, we incorporated TAA protein PAP, along with recombinant murine GM-CSF to recruit APCs to the cryogel and the adjuvant Poly(I:C) to activate APCs against the self-antigen. The biopolymer HAGM was selected for its demonstrated performance in formulating biocompatible, injectable cryogels and was previously used in Cryogel_VAX and O₂-Cryogel_VAX.^59,60,73 Additionally, HA-based polymers are biodegradable compared to alginate or synthetic polymers.^63,74,75 However, the loading efficiency of PAP in the HAGM-based cryogel was low, despite the sufficient loading efficiency of GM-CSF. GM-CSF also exhibited more of a “burst” release profile. These discrepancies are likely to be due to differences in molecular weight, with PAP being three times larger, and both proteins have a net negative charge in physiological buffers.⁷⁶ To address the challenge of PAP delivery, we investigated how covalent bioconjugation of PAP via click chemistry would influence the immune response after immunization. By integrating an azido-group into the HAGM backbone and adding a DBCO-group to the protein antigen, we were able to encapsulate 100% of the dose within the cryogel scaffold. This is paramount for increasing the clinical translatability of the delivery vehicle, as it prevents the undesirable loss of biomolecules during formulation. SPAAC chemistry was chosen because it has already been shown to be a biocompatible and effective strategy for conjugating proteins to HAGM cryogels via click chemistry.⁶⁰ However, we altered the strategy to conjugate the protein prior to cryopolymerization to ensure compatibility with O₂-release, and incorporated PEG linkers to encourage specific receptor-ligand interactions.⁷⁷ Future studies should be conducted to investigate the influence of linker length on vaccine efficacy. Additionally, PEG has been suggested to be immunogenic, therefore it might influence the activation of infiltrating APCs.⁷⁸ Furthermore, O₂ generation likely induces oxidative degradation of the cryogel matrix, enabling controlled release of covalently bound antigens, as supported by prior work.⁵⁷ This dual mechanism combines sustained antigen retention with environment-triggered release.

Notably, Click O₂-Cryogel_VAX improved the delivery of TAA to cryogel-infiltrating cells in vivo, consistent with the improved TAA release observed in vitro. O₂-Cryogel_VAX and Click O₂-Cryogel_VAX displayed the highest percentage of TAA-positive DCs after 7 d. The enhanced delivery of TAA likely accounts for the enhanced adaptive immune responses observed including increased production of PAP-specific antibodies and increased TNF-α and IL-2 T-cell responses found in mice that received O₂-Cryogel_VAX and Click O₂-Cryogel_VAX. These vaccine groups also exhibited the highest number of APCs and mature B cells in the draining LNs. Improving DC activity at the vaccine site should promote DC-T cell interactions as well as DC-B cell interactions, which are required for germinal center formation, B cell affinity maturation, and the generation of high-titer, class-switched antibody responses.⁷⁹ Surprisingly, we found that Cryogel_VAX and Click Cryogel_VAX did not increase anti-PAP antibody production compared with our control, Bolus_VAX. However, O₂ delivery was consistent between O₂-Cryogel_VAX and Click O₂-Cryogel_VAX, in increasing IgG1 titers as previously shown with O₂-Cryogel_VAX for COVID-19.⁵⁹ Additionally, we showed that cryogel delivery skewed stronger IgG2c titers, but our findings contrast with previous studies showing that HA-based cryogel delivery improved IgG1 and IgG2b titers; although these studies used strongly immunogenic proteins and CpG-ODN as an adjuvant.^23,59 It would be necessary to further investigate the adjuvant effect in skewing the immune response.

Interestingly, despite Click O₂-Cryogel_VAX delivering around 4 times more PAP than O₂-Cryogel_VAX, O₂-Cryogel_VAX also increased levels of PAP-positive DCs after 1 week and higher PAP-specific antibody titers compared to Click Cryogel_VAX, suggesting that O₂ generation plays a role in the humoral immune response. While we have previously shown that O₂-Cryogel restores the functions of hypoxic DCs, which could mediate the co-adjuvant effects of O₂-Cryogel_VAX, future studies should examine the proinflammatory effects of residual H₂O₂ and calcium hydroxide formed during CaO₂ decomposition to determine whether preventing hypoxia is the key factor in enhancing vaccine efficacy.^62,80,81 Additionally, the O₂-Cryogel_VAX formulations exhibited the best release of PAP, and subsequent DC phagocytosis, future work should identify if O₂ generation or improved release led to the improved DC response. However, Click O₂-Cryogel_VAX showed a potential synergistic effect, resulting in the highest levels of PAP-specific binding antibodies and overall IgM, IgG1, IgG2b, and IgG3 antibodies, suggesting that both improved antigen delivery and O₂ were key in skewing a Th1 antibody response.

Immunization with Cryogel_VAX and Click Cryogel_VAX skewed the generation of central memory T cells over effector memory T cells in both CD4⁺ and CD8⁺ T cells in the spleen. This finding suggests that scaffold-based delivery plays an important role in generating central memory relative to Bolus_VAX. Unexpectedly, while Click Cryogel_VAX slightly increased the mean percentages of central memory T cells over Cryogel_VAX it was not a significant increase, suggesting that protein dose may not have influenced the general memory profile. However, our results support previous studies that reported that sustained antigen release with rapid adjuvant release can drive Th1 responses.³⁷ Additionally, when collected splenocytes were exposed to TAA-derived peptide epitopes and TRAMP-C2 cells, Click O₂-Cryogel_VAX promoted the strongest expression of the Th1-biased proinflammatory cytokines TNF-α and IL-2, consistent with our findings that Click O₂-Cryogel_VAX improved TAA delivery to DCs. Interestingly, Click Cryogel_VAX elicited the strongest IFN-γ response but was hindered by O₂ production. This finding warrants further investigation into whether the decreased response is attributable to O₂ generation or to the kinetics of antigen release from Click O₂-Cryogel_VAX compared with Click Cryogel_VAX. Control over cryogel degradation in vivo presents an opportunity to explore how the timing of antigen release relative to local oxygenation skews IFN-γ responses, and future studies should be conducted to uncover these mechanistic insights.

Optimizing Cryogel_VAX with click-mediated covalent conjugation to deliver the entire protein dose ensured enough exposure to the TAA, generated a TAA-specific response, and supported the development of central memory T-cell populations. Additionally, O₂ release performed as a co-adjuvant to create a stronger Th1-biased response. The presence of central memory T cells is critical to the efficacy of anti-cancer immunotherapies, and a robust Th1 response is essential for mounting an effective, durable cellular attack against cancer.^82–84 An increased presence of memory T cells has also been shown to improve therapeutic chimeric antigen receptor (CAR)-T cell therapy outcomes. CAR-T cell products enriched for naïve and early memory subsets correlate with superior CAR-T cell expansion, persistence, and efficacy.^85,86 Using multiplex gene editing approaches, we recently demonstrated that CAR-T cells can be engineered to be resistant to hypoxia-driven biochemical and immunological negative regulators, thereby enabling an allogeneic CAR-T therapy that eliminates immunosuppressive solid tumors in a humanized xenograft mouse model.⁸⁷ Findings of this study suggest that treatment with cryogel-based vaccines could be an effective strategy to prime naïve and central memory anti-cancer T cells to synergize with CAR-T therapies, or be used to generate a source of memory or Th1 T cells to be used for CAR T-cell generation.

Finally, treatment with Click O₂-Cryogel_VAX elicited a potent anti-cancer response in the immunologically cold TRAMP-C2 tumor model and extended the median lifespan of mice by 62%. Further research is needed to assess the duration of the vaccine-induced immune responses and the optimal dosing required to maintain tumor-free survival. Additionally, although our earlier biocompatibility studies suggest that O₂-Cryogels are safe to use, further research, including toxicological analyses, is necessary to evaluate their long-term safety. Moreover, these findings support the potential of Click O₂-Cryogel_VAX as a more effective delivery vehicle for therapeutic cancer vaccines and could increase the immunogenicity of delivered neoantigens for personalized vaccines.⁸⁸ The next steps should investigate the performance of Click O₂-Cryogel_VAX in multiple tumor models, as click-mediated conjugation enables platform modularity for various vaccine applications, as any antigenic protein or peptide can be readily substituted. Furthermore, Click O₂-Cryogel_VAX should be explored in combination with other immunotherapies, such as ICIs and ACT.

Materials and Methods

Materials

HA sodium salt (~1.5 MDa), Dulbecco’s phosphate buffered saline (PBS), sodium bicarbonate (NaHCO₃), MES hydrate (C₆H₁₃NO₄S · xH₂O), glycidyl methacrylate (GM), N, N-dimethylformamide (DMF), triethylamine (TEA), tetramethylethylenediamine (TEMED), ammonium persulfate (APS), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC), N-hydroxysuccinimide (NHS), fetal bovine serum, Catalase from Aspergillus niger, and Insulin solution from bovine pancreas were acquired from MilliporeSigma (St. Louis, MO, USA). Deuterium oxide (D₂O), penicillin/streptomycin, (+)-Dehydroisoandrosterone, and eBioscience^™ Fixable Viability Dye eFluor^™ 506 were purchased from Thermo Fisher Scientific (Waltham, MA, USA). The TRAMP-C2 cells were purchased from American Type Culture Collection (Manassas, VA, USA). Recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF), Dulbecco’s Modified Eagle Medium (DMEM), and Roswell Park Memorial Institute 1640 medium (RPMI) were procured from Gibco (Waltham, MA, USA). Poly(I:C) (HMW) VacciGrade^™ was purchased from InvivoGen (San Diego, CA, USA). Acrylate-polyethylene glycol (PEG)-succinimidyl (AP-NHS, 3.4 kDa) was purchased from Laysan Bio (Arab, AL, USA). Recombinant murine PAP was purchased from Abcam (Cambridge, UK). Azido-PEG3-amine and DBCO-PEG4-NHS Ester were from Vector Labs (Newark, CA, USA).

Methods

Synthesis of HAGM and HAGM-PEG3-Azido

First, HAGM was synthesized as previously reported.^89,90 Briefly, HA (Sigma) was reacted with GM in a co-solvent mixture of PBS and DMF for 10 d, and then subsequently precipitated in acetone and vacuum dried. To functionalize with an azido group, HAGM (1 % w/v) was dissolved in MES buffer (100 mM, pH 6.5). EDC and NHS were sequentially added to the polymer solution at 5 and 6 equivalents, respectively, and allowed to react for 15 minutes. Azido-PEG3-amine (Vector Labs) was dissolved in solution at a 1:10 molar ratio relative to carboxyl groups, and the reaction was conducted for 24 h protected from light. The polymer solution was transferred to dialysis tubing (10 kDa MWCO) and dialyzed against deionized H₂O for 24 h, with the H₂O changed 3 times. The polymer solution was then stored at −80 °C and lyophilized. HAGM and HAGM-PEG3-Azido were verified through ¹H NMR spectroscopy and ATR-FTIR.

DBCO and dye conjugation to PAP

Murine recombinant PAP (Abcam) (1 mg/mL) was dissolved in PBS and functionalized with DBCO-PEG4-NHS ester (Vector Labs) in a 1:30 molar excess as per the manufacturer’s recommendation. The protein solution was incubated for 2 h at room temperature, protected from light, and then purified for DBCO-PEG4-PAP using Pierce^™ Dye and Biotin Removal Spin columns (Thermo Scientific).

For fluorescent microscopy imaging or phagocytosis analysis, PAP was incubated with Alexa Fluor^™ 488 NHS ester or pHrodo^™ Green STP ester (Invitrogen), respectively, at a 1:15 molar excess, as recommended by the manufacturer. The labeled PAP was purified using Pierce^™ dye removal columns. DBCO-PEG4-PAP was labeled with the above fluorescent compounds simultaneously with the reaction with DBCO-PEG4-NHS ester.

Fabrication of Cryogel_VAX and O₂-Cryogel_VAX

Cryogel vaccine scaffolds against prostate cancer were fabricated using HAGM (Cryogel_VAX) or HAGM-PEG3-Azido (Click Cryogel_VAX) (4 % w/v) by cryo-polymerization (TEMED, 0.07 % w/v; APS, 0.28 % w/v) at −20 °C. HAGM and HAGM-PEG3-Azido were dissolved in deionized H₂O with 10 μg PAP and DBCO-PEG4-PAP, respectively, overnight at 4 °C before cryo-polymerization. Immunomodulatory factors (25 μg/cryogel Poly(I:C) and 1.5 μg/cryogel GM-CSF) were incorporated into the polymer solution immediately prior to cryo-polymerization. To produce O₂-generating cryogel vaccine scaffolds (O₂-Cryogel_VAX, Click O₂-Cryogel_VAX), cryogels were hybridized with ball-milled calcium peroxide (CaO_2, 1 % w/v), and grafted with chemically modified catalase (CAT, 1 % w/v) as previously reported.^57,59 For Bolus_VAX, the equivalent amounts of PAP, GM-CSF, and Poly(I:C) were formulated in 100 μL of PBS. Further details on the methodology for cryogel physical characterization are provided in the Supplemental Methods.

Loading and release of immunomodulatory factors and proteins from cryogels

To determine the loading efficiency and in vitro release kinetics of PAP, GM-CSF, and Poly(I:C) from Cryogel_VAX and O₂-Cryogel_VAX, gel samples were briefly washed in 70 % ethanol followed by 2 PBS washes. Each washed gel was dissociated to measure loading or incubated in sterile PBS in a microcentrifuge tube under orbital shaking at 4 °C. Partial supernatant was removed periodically and replaced with an equal volume of fresh buffer. GM-CSF and PAP were detected by either ELISA (Uncoated mGM-CSF, Invitrogen) or bicinchoninic acid assay (micro-BCA, Thermo Scientific). The concentration of Poly(I:C) was quantified using a NanoDrop^™ One UV-Vis spectrophotometer (Thermo Scientific).

O₂ release kinetics (H₂O₂ depletion)

O₂ release from O₂-Cryogel_VAX was monitored using O₂ optical sensor spots (OXSP5-ADH, PyroScience, GmbH, Aachen, Germany). O₂-Cryogel_VAX scaffolds were individually placed into a 96-well plate containing 200 μL of PBS at room temperature. Dissolved O₂ (Torr) within the well was recorded for 48 h using FireString software. To ensure adequate H₂O₂ depletion through the inclusion of CAT, O₂-Cryogels were incubated in PBS at 37 °C for 48 h and H₂O₂ concentration in the supernatant was measured using the Pierce^™ Quantitative Peroxide Assay Kit (Thermo Scientific).

Mouse model and study design

Vaccination studies were performed on 6–8-week-old male C57BL/6 mice (Charles River). Bolus_VAX, O₂-Cryogels, and Cryogel_VAX scaffolds were injected subcutaneously (s.c.) in both flanks close to the inguinal LNs (total of 2 injections/mouse). All cryogel scaffolds were sterilized in 70 % ethanol for 10 min, and washed 3 times with sterile PBS for 5 min before use in animals.

Phagocytosis and hypoxia characterization of cryogel-infiltrating cells

Mice received injections of Cryogel_VAX, Click Cryogel_VAX, O₂-Cryogel_VAX, or Click O₂-Cryogel_VAX and were euthanized after 3 or 7 d, and cryogels were explanted. The samples were homogenized over a 70 μm cell strainer to create single-cell suspensions, which were then washed with protein blocking buffer (PBA) (PBS + 1 % BSA) and stained for flow cytometry analysis. For characterization of phagocytosis, cryogels were loaded with pHrodo^™ Green-conjugated PAP or DBCO-PEG4-PAP prior to cryopolymerization. The following fluorescent antibodies were used for extracellular staining: eFluor^™ 450-conjugated anti-mouse CD11c (clone N418), APC/Cyanine 7-conjugated anti-mouse I-A/I-E (clone M5/114.15.2), PerCP/Cy 5.5-conjugated anti-mouse CD45 (clone S18009F), and eBioscience^™ Fixable Viability Dye eFluor 506. Further details on the methodology for phagocytosis studies are provided in the Supplemental Methods. For hypoxia characterization, mice were injected intraperitoneally with Pimonidazole hydrochloride (Hypoxyprobe^™) in PBS (dosage: 60 mg kg⁻¹), 1 h prior to euthanasia. Collected infiltrating cells were first labeled with extracellular markers (PerCP/Cy5.5-conjugated anti-mouse CD45 (clone S18009F), and eBioscience^™ Fixable Viability Dye eFluor 506) and then fixed and permeabilized using a FoxP3/Transcription Factor Fixation/Permeabilization Kit (Cell Signaling Technology) according to the manufacturer’s protocol. Cells were then incubated with Rabbit IgG1 polyclonal anti-pimonidazole antibody (Pab27, Hypoxyprobe^™) for 45 min, washed, and stained with Alexa Fluor 488 goat anti-rabbit IgG (H+L) antibody (Invitrogen) for 45 min. Cells were washed 3 times with PBA and analyzed using the NovoCyte Advanteon flow cytometer (Agilent Technologies).

Antigen-presenting cell characterization in draining LNs

Mice received immunization (day 0) and boost (day 14) doses of Bolus_VAX, Cryogel_VAX, Click Cryogel_VAX, O₂-Cryogel_VAX, Click O₂-Cryogel_VAX, or O₂-Cryogel alone. Blood samples were collected every 7 d. The inguinal LNs were explanted 28 d after initial immunization and dissociated into single-cell suspensions of equivalent volume. Cells were stained with fixable viability dye eFluor 506 (eBioscience, 1:400 dilution in PBS) for 30 min at 4 °C. The cells were subsequently washed with PBA before being stained overnight with fluorochrome-conjugated antibodies (CD11c-FITC (Clone N418), I-A/I-E-APC-Cy7 (Clone: M5/114.15.2), IgD-Alexa Fluor 700 (Clone 11–26c.2a), CD45-PerCP-Cy5.5 (Clone S18009F), CD11b-PE-Cy7 (Clone: M1/70), F4/80-APC (Clone: BM8), CD19-BV605 (Clone 6D5), BioLegend) in PBA at 4 °C. Cells were washed 3 times with PBA prior to analysis. Flow cytometry measurements of total cell counts were done using the Attune NxT flow cytometer (Thermo Fisher Scientific).

Antibody titration by immunoassays

Mouse sera were collected 14 and 28 d after initial immunization. Immunoglobulin isotyping was evaluated using the Mouse Immunoglobulin Isotyping LegendPlex kit (BioLegend) and Uncoated IgG2c Mouse ELISA kit (Thermo Fisher Scientific). Specific IgG to murine PAP was evaluated using a custom ELISA protocol. Specifically, PAP-IgG was detected using recombinant PAP (Abcam) as the capture and anti-IgG (H+L)-biotin (Invitrogen) and streptavidin Poly-HRP (Thermo Scientific) as the detection. Mouse sera were serially diluted to calculate the half-maximal effective concentration (EC50) as the titer for each treatment group. LegendPlex bead PE-Geometric Mean Fluorescence Intensity (MFI) or ELISA Absorbance was analyzed using a nonlinear fit model (log(concentration) vs. response, four-parameter variable slope) with top baseline constraints to determine LogEC50 values.

T cell-characterization in spleens

Mice received immunization (D0) and boost (D14) doses of Bolus_VAX, Cryogel_VAX, Click Cryogel_VAX, O₂-Cryogel_VAX, Click O₂-Cryogel_VAX, or O₂-Cryogel alone. The spleens were explanted 28 d after initial immunization and dissociated into single-cell suspensions. Cells were stained with fixable viability dye eFluor 506 (eBioscience, 1:400 dilution in PBS) for 30 min at 4 °C. The cells were subsequently washed with PBA before being stained overnight with fluorochrome-conjugated antibodies (CD3ε-FITC (Clone: 500A2), CD44-PE (Clone: IM7), CD4-PerCP-Cy5.5 (Clone: RM4–5), CD8a-APC (Clone: 53–6.7), CD62L-BV605 (Clone MEL-14), BioLegend) in PBA at 4 °C. Cells were washed 3 times with PBA prior to analysis. Flow cytometry measurements were done using the Attune NxT flow cytometer (Thermo Fisher Scientific).

Splenocyte activation and intracellular cytokine staining

Splenocytes (40 × 10⁶ cells mL⁻¹) were incubated with either Cell Stimulation Cocktail (eBioscience), 30 μg mL⁻¹ PAP protein-derived peptides mPAP_114–122 and mPAP_128–136 (Biosynth), 0.5 × 10⁶ cells mL⁻¹ TRAMP-C2 or control (no stimulation) in Protein Transport Inhibitor Cocktail (eBioscience) solutions for 6 h at 37 °C. Next, the cells were washed with PBS and incubated for 30 min with Fixable Viability Dye eFluor 506 (eBioscience) in PBS (1:400 dilution) at 4 °C. Cells were then washed once with PBS and twice with PBA before being stained for 1 h with fluorochrome-conjugated antibodies (CD3ε-FITC (Clone: 500A2), CD4-APC-Cy7 (Clone: RM4–5), CD8-PerCp-Cy5.5 (Clone: 53–6.7), CD44-BV421 (Clone: IM7), BioLegend) in PBA at 4 °C. Cells were subsequently washed 3 times with PBA, fixed and permeabilized using a FoxP3/Transcription Factor Fixation/Permeabilization Kit (Cell Signaling Technology) according to the manufacturer’s protocol. Intracellular staining was performed by incubating the cells with fluorochrome-conjugated antibodies (IL-2-PE (Clone: JES6–5H4), TNFα-AF700 (Clone: MP6-XT22), BioLegend) in permeabilization buffer for 30 min at 4 °C. Lastly, cells were washed 3 times with PBA and analyzed using the NovoCyte Advanteon flow cytometer (Agilent Technologies).

Splenocyte activation and IFN-γ ELISpot

Splenocytes (3 × 10⁶ cells mL⁻¹) were incubated with either Cell Stimulation Cocktail (eBioscience), 30 ug mL⁻¹ PAP protein-derived peptides mPAP_114–122 and mPAP_128–136 (Biosynth), 0.5 × 10⁶ cells mL⁻¹ TRAMP-C2, or control (no stimulation) in a mouse IFN-γ ELISpot plate (R&D Systems) overnight at 37 °C. Following incubation, the manufacturer’s protocol was followed. Spots were counted manually using a dissection microscope and normalized to the number of cells initially seeded per well.

TRAMP-C2 therapeutic model

Male C57BL/6 mice (8–10-week-old) were subcutaneously injected with 2.5 × 10⁶ TRAMP-C2 cells mixed 1:1 with Matrigel Matrix Basement Membrane HC (Corning, 18.97 MG/ML). Mice were immunized with Bolus_VAX, O₂-Cryogels, O₂-Cryogel_VAX, or Click O₂-Cryogel_VAX when tumors reached a mean volume of 50 mm³. Mice were boosted 14 days later, and tumor volume was measured with calipers and recorded two or three times per week. Animals were euthanized when tumors reached a volume greater than 1500 mm³ or became ulcerated. To assess the antitumor immune response, tumors were explanted and dissociated, and infiltrating T cells were analyzed for immunosuppressive markers through flow cytometry using the NovoCyte Advanteon (Agilent Technologies). Cells were stained for 1 h with fluorochrome-conjugated antibodies (CD4-PerCP/Cy5.5 (Clone: RM4–5), CD8-APC (Clone: 53–6.7), CD25-BV785 (Clone: PC61), and CD279 (PD-1)-BV510 (Clone: 29F.1A12), BioLegend) in PBA at 4 °C. Cells were subsequently washed 3 times with PBA, fixed and permeabilized using a FoxP3/Transcription Factor Fixation/Permeabilization Kit (Cell Signaling Technology) according to the manufacturer’s protocol. Intracellular staining was performed by incubating the cells with fluorochrome-conjugated antibody (FOXP3-PE (Clone: MF-14), BioLegend) in permeabilization buffer for 30 min at 4 °C. Lastly, cells were washed 3 times with PBA and analyzed.

Statistical analysis

Flow cytometry data were processed using FlowJo software and manual gating was performed. Statistical analysis was conducted using GraphPad Prism 10 software (La Jolla, CA, USA). Significant differences between groups were assessed using one-way analysis of variance (ANOVA), mixed-effects analysis, or log-rank (Mantel-Cox) with Bonferroni post hoc tests. Tumor growth data were analyzed using a Mixed-effects model with a compound symmetry covariance matrix, fit by Restricted Maximum Likelihood (REML), and the Geisser-Greenhouse correction was applied. Pairwise comparisons between treatment groups were performed using Bonferroni’s multiple comparisons test. Differences were considered statistically significant at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Regulatory compliance and approval for animal studies

All experiments requiring mice were conducted in compliance with the National Institutes of Health (NIH) guidelines and approved by the Division of Laboratory Animal Medicine (DLAM) and Northeastern University Institutional Animal Care and Use Committee (protocol number 23–0512R).

Resource availability

Lead contact

Further information and requests for resources and materials should be directed to the lead contact, Stephen M. Hatfield (s.hatfield@northeastern.edu).

Materials availability

Newly generated materials described in this study are available upon reasonable request.

Data and code availability

The main data supporting the findings of this study are available within the article and its supplemental information. This study did not generate any new code. Other raw data generated in this study are available from the lead contacts upon reasonable request.

The Bigger Picture.

Therapeutic cancer vaccines have achieved limited clinical success, largely because they fail to generate strong antitumor immune responses to overcome immunological tolerance and immunosuppression. Although innovative biomaterial-based delivery systems have been developed to enhance adaptive immunity, they often provide insufficient sustained exposure to tumor-associated antigens and remain constrained by local hypoxia-mediated immunosuppression and HIF-1α-regulated anti-inflammatory pathways. To address these major physiological and immunological barriers, we engineered an injectable cryogel-based vaccine that combines covalent antigen bioconjugation via click chemistry, controlled release of immunomodulatory factors, and local oxygen (O₂) generation. We show that Click O₂-Cryogel_VAX, targeting the tumor-associated antigen prostatic acid phosphatase, enhances adaptive immune responses and provides strong therapeutic benefits in a prostate cancer model. This work demonstrates how sustained antigen delivery through cryogel bioconjugation, together with the O₂-mediated reversal of hypoxia-driven immunosuppression, can reinforce antitumor immunity and improve therapeutic outcomes.

Highlights.

• Click chemistry improves tumor-antigen loading and release in cryogel vaccines.

• Cryogel vaccination promotes cell-mediated antitumor immunity.

• Oxygen acts as a powerful co-adjuvant to boost antibody-mediated immunity.

• Click O₂-Cryogel_VAX induces an effective therapeutic antitumor response.