Vaccines targeting p53 mutants elicit anti-tumor immunity

Abstract

The p53 tumor suppressor is commonly mutated in cancer; however, there are no effective treatments targeting p53 mutants. A DNA vaccine gWIZ-S237G targeting the p53 S237G mutant, which is highly expressed in A20 murine tumor cells, was developed and administered intramuscularly via electroporation, either alone or in combination with PD1 blockade. The anti-p53-S237G immunization elicited a robust protective response against subcutaneous A20 tumors and facilitated the infiltration of immune cells including CD8⁺ T cells, NK cells, and DCs. The vaccine enhanced the induction and maturation of CD11c⁺, CD103⁺CD11c⁺, and CD8⁺CD11c⁺ cells, which in turn promoted tumor-specific antibody production, as well as Th1 and CD8⁺ T cell-mediated immune responses. Several antigenic epitopes of p53-S237G effectively stimulated multifunctional CD8⁺ T cells to secrete IFN-γ and TNF-α. The vaccine showed long-term anti-tumor effects that were dependent on memory CD8⁺ T cells. Furthermore, the anti-p53-S237G vaccine exhibited significant protective efficacy in the A20 liver metastasis models. When combined with PD-1 inhibition, the vaccine showed superior inhibition of tumor growth and liver metastasis. Targeting p53 mutants by vaccination represents a potential precision medicine strategy against cancers harboring p53 mutations.

1. Introduction

The p53 tumor suppressor regulates critical cellular processes such as cell cycle, DNA repair, and apoptosis [1]. Mutations in the p53 gene are prevalent in many cancers and often lead to the loss of tumor-suppressive function [2, 3]. Most p53 mutations are missense mutations that alter the amino acid sequence of the protein and disrupt its conformation [4]. In addition to loss of function, some p53 mutants display “gain-of-function” [1, 5]. Due to the prevalence and impact of p53 mutations in cancer, it has become a critical target for the development of cancer therapeutics using small molecule inhibitors and biologics.

The p53 is dysregulated in most human cancers, making it a highly compelling target for cancer vaccines [6, 7]. In a phase II trial, a p53 synthetic long peptide (p53-SLP) vaccine containing 10 peptides of 25–30 amino acid residues covering WT p53 from position 70–248, was administered to 20 ovarian cancer patients [8]. This vaccine induced p53-specific CD4⁺ T cell immune responses, but not CD8⁺ T cell responses. It was tested in patients with multiple cancers in phase I/II clinical trials [9–12]. The adenoviral vaccine rAd/hup53, encoding full-length human WT p53, successfully primed A2.1-restricted and human p53 epitope-specific CTLs in vivo, but did not induce p53-specific antibodies [13]. Long-peptide vaccines targeting p53 and KRAS induce p53 mutation-specific T cell immunity, although this response is associated with increased Treg cell levels [14]. DC vaccines expressing full-length p53-WT or pulsed with p53 peptides elicited p53-specific T cell responses and demonstrated a high rate of objective clinical responses to chemotherapy in more than half of the patients with extensive-stage small cell lung cancer or breast cancer [15, 16]. While a DNA vaccine encoding full-length human p53 provided protection against the MC38 tumor challenge, the mouse p53 DNA vaccine did not [17]. Despite these developments, no anti-p53 vaccines passed Phase 2 trials, underscoring the challenges in vaccine design and therapeutic potential. In recent years, DNA vaccines have emerged as a promising cancer therapeutic option in preclinical studies [18, 19]. These vaccines function by expressing tumor-associated or tumor-specific antigens in vivo to elicit targeted immune responses [20, 21]. The design flexibility of DNA plasmids enables the incorporation of multiple tumor antigens or immunostimulatory factors for an optimized immune response. Compared to protein-based or viral vector vaccines, DNA vaccines have the advantage of greater stability during storage, transport, and simplified production processes [22, 23]. DNA vaccines can be personalized to include patient-specific tumor mutations, enabling precision medicine to target a wide range of cancers [24].

In this study, we developed a DNA vaccine gWIZ-S237G targeting the p53 mutation S237G. This mutation was found in a mouse B-cell lymphoma cell line (A20), in which serine (S) was substituted with glycine (G) at position 237 of the p53 protein. The human p53 ortholog S240G is a rare mutation found in approximately 0.03% of human cancers [25]. The anti-p53-S237G vaccine was delivered intramuscularly via electroporation to ensure efficient gene transfer. The vaccine significantly inhibited the growth of A20 lymphoma tumors and prevented liver metastasis in syngeneic mice. The vaccine’s long-term anti-tumor effects were primarily mediated by dendritic cell (DC)-driven multifunctional CD8⁺ T cell responses. Additionally, when combined with anti-PD-1 immune checkpoint blockade (ICB) therapy, the p53-S237G vaccine exhibited enhanced efficacy in controlling tumor growth and metastasis compared to monotherapy alone. These findings suggest that a DNA vaccine targeting the p53-S237G mutation is a potent precision therapeutic agent against cancers carrying a specific mutation.

2. Material and methods

2.1. Animals, cell lines, and antibodies

Six- to eight-week-old female BALB/c mice were purchased from the Jackson Laboratory and housed at the Center for Comparative Medicine (CCM) of Baylor College of Medicine under pathogen-free conditions. All animal procedures and protocols were conducted according to the guidelines and approved by the Institutional Animal Care and Use Committee (IACUC) of the Baylor College of Medicine.

The mouse B-cell lymphoma cell line A20, melanoma cell lines B16-F0 and B16-F10, embryonal testicular teratoma cell line F9, hepatoma cell line Hepal1-6, lung carcinoma cell line LLC1, colon carcinoma cell line CT26, and human non-small cell lung cancer cell line H1299 were obtained from the American Type Culture Collection (ATCC). The mouse colon adenocarcinoma cell line MC38 was purchased from Sigma-Aldrich. A20 cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco), 0.05 mM 2-mercaptoethanol (Cat# 21985023, Gibco), and 1× antibiotics/antimycotics (anti/anti) solution. CT26, CT26-p53-R172H (p53-R172H knock-in), and H1299 cells were cultured in complete RPMI-1640 medium. B16-F0, B16-F10, F9, Hepal1-6, and LLC1 cells were cultured in Dulbecco’ DMEM medium (Gibco) supplemented with 10% FBS and 1× anti/anti solution.

The antibodies used in this study are as follows: anti-p53 (Cat# sc-126, Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-GAPDH (Cat# 60004–1-Ig, Proteintech), anti-mouse IgG-HRP (Cat# 7076, Cell Signaling Technology, Danvers, MA, USA), anti-mouse CD49b (Cat# ab181548, abcam, Waltham, MA, USA), Anti-rabbit IgG-HRP (Cat# 7074, Cell Signaling Technology), anti-mouse CD8 (Cat# 14-0081-82, eBioscience), anti-Rat IgG2a-HRP (Cat NBP2–69583, Novus Biologicals, Centennial, CO, USA), αPD-1 (Cat# BE0146 RMP1–14, BioXcell, West Lebanon, NH, USA), αCD4 (Cat# BE0119, BioXcell), αCD8 (Cat# BP0117, BioXcell), anti-Asialo GM1 (Cat# 146002, BioLegend), anti-mouse CD16/32 (Cat# 101302, BioLegend, San Diego, CA, USA), APC-BrdU (Cat# 364114, BioLegend,), APC/Cy7-CD45 (Cat# 103116, BioLegend), Brilliant Violet 750-CD45 (Cat# 103157, BioLegend), PE/Cy7-CD45 (Cat# 103114, BioLegend), PE-CD3 (Cat# 100206, BioLegend), FITC-CD4 (Cat# 100405, BioLegend), Pacific Blue-CD4 (Cat# 100531, BioLegend), PerCP/Cy5.5-CD8α (Cat# 100734, BioLegend), APC-CD49b (Cat# 108910, BioLegend), PE/Cy7-IFN-γ (Cat# 25-7311-82, Invitrogen), APC-IFN-γ (Cat# 505810, BioLegend), APC-TNF-α (Cat# 17-7321-82, Invitrogen), Alexa Fluor 488-TNF-α (Cat# 506313, BioLegend), PE-IL-2 (Cat# 503808, Biolegend), APC-CD11c (Cat# 117310, BioLegend), FITC-CD11b (Cat# 101206, BioLegend), PE-CD103 (Cat# 121406, BioLegend), PerCP-F4/80 (Cat# 123126, BioLegend), Alexa Fluor 647-FOXP3 (Cat# 126408, BioLegend), APC/Cy7-Ly-6G/Ly-6C (Cat# 108424, BioLegend), PE-CD44 (Cat# 103024, BioLegend), FITC-CD62L (Cat# 104406, BioLegend), PE-CD80 (Cat# 104708, BioLegend), FITC-CD86 (Cat# 105006, BioLegend), Pacific Blue-I-A/I-E (Cat# 107620, BioLegend), PerCP/Cy5.5-H-2K^d/H-2D^d (Cat# 114716, BioLegend).

2.2. Plasmid and vaccine preparation

An N-terminal signal peptide derived from bovine prolactin (MDSKGSSQKGSRLLLLLVVSNLLLPQGVLA) linked to full-length mouse p53-WT and p53-S237G, with or without C-terminal Fc or Ferritin, was subcloned into gWIZ using SalI and BglII. The plasmids gWIZ-p53-Wild type (gWIZ-WT), gWIZ-WT-Fc, gWIZ-WT-Ferritin, gWIZ-p53-S237G (gWIZ-S237G), gWIZ-S237G-Fc, gWIZ-S237G-Ferritin, gWIZ-p53-R172H (gWIZ-R172H), gWIZ-R172H-Fc, and gWIZ-R172H-Ferritin were constructed through Epoch Life Science (Houston, TX, USA). These plasmids were then transformed into Escherichia coli (DH5a) competent cells, propagated in LB broth supplemented with 50 μg/ml kanamycin and purified using the ZymoPURE^™ II Plasmid Maxiprep Kit (Cat# D4203, Zymo Research, Irvine, CA, USA;), which is endotoxin-free, following the protocol provided by the manufacturer. For the vaccine formulation, 25 μg of DNA in 10 μl of water was combined with 10 μl of AddaS03^™ adjuvant (Cat# vac-as03–10, InvivoGen, San Diego, CA, USA) to prepare the immunization dose for each mouse.

2.3. Cell transfection

H1299 cells were seeded in a 6-well plate at a density of 1 × 10⁶ cells/well. After overnight incubation when the cells reached 70%–80% confluency, they were transfected with 5 μg of plasmid DNA per well using 15 μl of TransIT-LT1 transfection reagent (Cat# MIR 2305, Mirus Bio, Madison, WI, USA) in Opti-MEM medium (Gibco). After transfection, the cells were incubated for 48 h before harvesting for subsequent experiments.

2.4. Western blot

Transfected cells were collected and treated with RIPA lysis buffer (Cat# 89901, Invitrogen) containing protease and phosphatase inhibitors (Cat# 78443, Invitrogen). Equal quantities of protein from each sample were then separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes (Cat# IPVH00010, Millipore). The membranes were blocked using 5% bovine serum albumin (BSA) and incubated overnight at 4 °C with primary antibodies targeting p53 or GAPDH. After several washes with phosphate-buffered saline containing 0.1% Tween 20 (PBST), the membranes were treated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 2 h at room temperature. Finally, immunoreactive bands were developed using Immobilon ECL Ultra Western HRP Substrate (Cat# WBULS0500, Millipore) and captured using an automatic chemiluminescence imaging system (Bio-Rad).

2.5. Animal experiments

For prophylactic vaccine experiments, mice were intramuscularly immunized with gWIZ, gWIZ-WT, or gWIZ-S237G vaccine (25 μg/mouse) by electroporation on days −35, −21, and −7. Following immunization, mice were challenged with subcutaneous or liver metastatic tumors. For subcutaneous tumors, A20 cells (1 × 10⁶ cells/mouse) were injected subcutaneously on day 0. For liver metastasis, A20 cells (2 × 10⁶ cells/mouse) were injected intravenously on day 0. Additionally, cell deletion therapy with 500 μg/mouse of αCD4, αCD8, or anti-Asialo GM1 was administered twice weekly following tumor inoculation.

For the therapeutic vaccine experiments, subcutaneous tumor models were established by injecting A20 cells (1 × 10⁶ cells/mouse) on day 0. Mice were then intramuscularly immunized with gWIZ, gWIZ-WT, or gWIZ-S237G vaccine (25 μg/mouse) on days 1, 11, and 21 post tumor inoculation. Additionally, combination therapy with αPD-1 (200 μg/mouse) was administered after electroporation on days 7 and 14 post-tumor inoculation. Liver metastasis models were established by intravenous injection of A20 cells (2 × 10⁶ cells/mouse) on day 0. Mice were intramuscularly immunized with 25 μg of gWIZ, gWIZ-WT, or gWIZ-S237G vaccine on days −10, 1, and 11 following tumor inoculation. Moreover, αPD-1 (200 μg/mouse) was administered as a combination therapy following electroporation on days 7 and 14 after tumor inoculation. Tumor growth and survival were evaluated at the end of the treatment period. Tumor volume was calculated using the following formula: V = (length × width²)/2.

2.6. Flow cytometry (FACS)

Single-cell suspensions of spleen cells, tumor-draining lymph nodes (DLN), or tumor-infiltrating leukocytes (TILs) were prepared by gently homogenizing the spleen or enzymatically digesting tumor tissues using a Mouse Tumor Dissociation Kit (Cat# 130-096-730, Miltenyi Biotec, Auburn, CA, USA). After blocking Fc receptors and removing dead cells with the Zombie Aqua Fixable Viability Kit (Cat# 423102, BioLegend, San Diego, CA, USA), the cells were surface stained by incubating them with specific antibodies for 30 min at 4°C. For further intracellular staining, cells were fixed with 4% paraformaldehyde (Cat# sc-281692, Santa Cruz Biotechnology), permeabilized using permeabilization buffer (Cat# 421002, BioLegend) and washed thoroughly. Finally, the cells were incubated overnight at 4 °C with antibodies specific to the intracellular targets to complete the staining process. Isotype-matched immunoglobulins were used as controls. Data collection was performed on a Cytek^® NL-3000 flow cytometric system (Cytek Biosciences, Fremont, CA, USA) and it was further analyzed using the FlowJo V10 software (Tree Star Inc., Ashland, OR, USA).

2.7. Antibody titers

Enzyme-linked immunosorbent assay (ELISA) was performed to measure the titers of serum totally IgG. The plates were coated with 5 μg/ml of the antigen peptides and incubated overnight at 4°C. After incubation, the plates were washed with PBS containing 0.05% Tween 20, and then blocked with 1% BSA in 0.05% Tween 20/PBS for 2 h at room temperature. Serum samples were serially diluted ten-fold, added in duplicate to the plates, and incubated at 37 °C for 2 h. Following this, HRP-conjugated anti-mouse IgG antibody was applied for 1 h at room temperature. The assay was developed using TMB substrate, and the absorbance was measured at 450 nm using a CLARIOstar Plus microplate reader (BMG LABTECH, Ortenberg, Germany).

2.8. T lymphocyte proliferation

Splenocytes were plated in 48-well plates at a density of 1×10⁶ cells per well, cultured in a medium supplemented with IL-2 (100 U/ml) and a synthesized p53-S237G peptide pool (10 μg/ml; PEPTIDE 2.0 Inc., Chantilly, VA, USA). The plates were maintained at 37°C in a humidified incubator with 5% CO₂ for 5 days, and the medium was refreshed on day 3. On day 5, cells were treated with 10 μM BrdU (Cat# 19–160, Sigma-Aldrich) for 2 h at 37°C. After incubation, the cells were harvested and subjected to surface staining with anti-CD8α or anti-CD4 antibodies for 30 min. After fixation, permeabilization, and washing, intracellular staining was performed using APC-conjugated BrdU antibody (Cat# 364114, BioLegend) for an additional 30 min. After staining, the cells were washed three times with the permeabilization buffer. The percentage of BrdU⁺ cells in the CD4⁺ and CD8⁺ T cell populations was analyzed using FACS, wherein the BrdU⁺CD8⁺ T cell percentage representing the proliferation rate.

2.9. ELISPOT

Mouse IFN-γ/TNF-α Double-Color Enzyme-linked immunospot (ELISPOT) Kit (Cellular Technology Limited (CTL), Cleveland, OH, USA) was used to detect the T cells secreting IFN-γ/TNF-α according to the manufacturer’s protocol. Splenocytes were plated in ELISPOT plates at a concentration of 1 × 10⁶ cells per well with a medium supplemented with IL-2 (100 U/ml) and a recombinant p53-S237G peptide pool (10 μg/ml) for 60 h at 37°C in a 5% CO₂ incubator. The cells were then washed and treated with detection antibodies, including biotinylated-TNF-α and FITC-IFN-γ. After an additional washing step, streptavidin-AP conjugate or FITC-HRP (diluted 1:1000) was added. The spots were visualized on the plates, and spot-forming colonies (SFCs) per well were scanned and quantified using a CTL reader. IFN-γ-producing cells appeared as red spots, while TNF-α-producing cells were visualized as blue spots.

2.10. Cytotoxic T lymphocyte (CTL) activity

Splenocytes were cultured in RPMI 1640 medium supplemented with 100 U/ml IL-2 and 10 μg/ml p53-S237G peptide pool, and incubated at 37°C in a humidified incubator with 5% CO₂ for 7 days. Subsequently, the splenocytes were washed, resuspended in fresh medium, and prepared as effector cells. A20 tumor cells were used as target cells. Effector and target cells were then plated in U-bottom 96-well plates at various effector-to-target ratios (50:1, 25:1, and 12.5:1), with 1 × 10⁴ target cells added to each well. The plates were incubated at 37 °C for 72 h. Cytotoxicity was assessed using the Cytotoxicity Detection Kit PLUS (Cat# 4744926001, Roche, Palo Alto, CA, USA), following the manufacturer’s instructions.

2.11. Cytokine detection by ELISA

Splenocytes isolated from immunized mice were plated in a 24-well culture plate at a concentration of 4 × 10⁶ cells/ml and stimulated with 10 μg/ml p53-S237G peptide pool at 37°C in a 5% CO₂ incubator for 72 h. Cytokine levels of IFN-γ, TNF-α, IL-10, and IL-4, were measured in the culture supernatants using ELISA kits from R&D Systems (Minneapolis, MN, USA). Additionally, serum was collected from the immunized mice, and IL-6 and TNF-α levels were quantified in the serum through ELISA following the manufacturer’s instructions.

2.12. Pathological analyses and immunohistochemistry (IHC) staining

Liver tumor tissues were fixed in 10% formalin, embedded in paraffin, sectioned into 5 μm slices, and stained with hematoxylin and eosin (H&E) following the manufacturer’s protocol at the Pathology & Histology Core, Baylor College of Medicine. For IHC staining, the paraffin-embedded sections were first deparaffinized and antigen retrieval was performed by heating the rehydrated sections to 95°C in 10 mmol/L sodium citrate buffer (pH 6.0) for 30 min. Endogenous peroxidase activity was blocked by treating slides with 3% hydrogen peroxide for 1 h. The sections were then blocked with 10% BSA and incubated with an anti-mouse CD8 antibody, and then incubated with a secondary antibody. The staining was developed using the Pierce^™ DAB substrate kit (Cat# 34002, Thermo Scientific, Waltham, MA, USA) and it was counterstained with hematoxylin (Cat# ab220365, Abcam, Waltham, MA, USA) to visualize the nuclei. Images of stained tissues were captured using an Olympus IX51 microscope (Olympus America Inc., Center Valley, PA, USA).

2.13. p53 structure prediction

After performing amino acid mutations using the Mutagenesis tool in PyMOL, the structure was imported into Swiss-PdbViewer 4.1.0 for Energy Minimization. The optimized structure was saved as a PDB file and re-imported into PyMOL for visualization. The motif Seq 30 was highlighted in red, motif Seq 31 in blue, and motif Seq 63 in green.

2.14. Statistical analyses

Statistical analyses were performed using the GraphPad Prism 8.0 (GraphPad Software). Data are expressed as mean ± standard deviation (SD). An unpaired Student’s t-test was used to compare two groups, whereas one-way analysis of variance (ANOVA) was employed for comparisons among multiple groups. Differences in survival were evaluated using the log-rank test (Mantel-Cox). A p-value of less than 0.05 was considered statistically significant, with significance levels indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3. Results

3.1. The gWIZ-S237G immunization induces a potent anti-tumor effect by promoting the infiltration of immune cells

An effective vaccine relies on the expression of its target antigen [26]. As shown in Fig. S1A–S1C, p53-R172H and p53-S237G are mouse orthologs to human p53 mutations R175H (the most frequent mutation in human cancers) and S240G (a rare p53 mutation in human). We first evaluated p53 expression in several commonly used murine syngeneic tumor cell lines: A20, B16-F0, B16-F10, F9, Hepa1-6, LLC1, CT26, and MC38. We found that p53 had the highest expression in A20 cells, which carries the p53-S237G mutant (Fig. S1D). In vaccine design based on the gWIZ vector, the secretion peptide from bovine prolactin was fused to the N-terminus of the p53-WT, p53-S237G, or p53-R172H protein, allowing the mutant protein to be secreted outside the cells and released in the circulation to stimulate immune responses. Additionally, we fused either human IgG1 Fc or ferritin (FR)[27] to the C-terminal of p53. Fc and ferritin are commonly used to enhance antigen stability and valency. p53 expression was validated in H1299 cells (p53-null) transfected with the individual constructs (Fig. S1E). We delivered p53-S237G or -WT into mice intramuscularly and found that the animals produced p53 proteins in their sera (Fig. S1F). Mice were immunized intramuscularly with gWIZ, gWIZ-WT, or gWIZ-S237G on days 35, 21, and 7 before inoculation with A20, CT26, or CT26-R172H cells (Fig. S1G). CT26-R172H is an isogenic recombinant cell line carrying the p53 R172H mutation [28]. The p53-WT vaccine did not inhibit the growth of CT26, CT26-R172H, or A20 tumors (Fig. 1A, S3A, S3B, 1E, S3G, S3H, 1F, S3K, and S3L). Although gWIZ-R172H or gWIZ-R172H-FR partially inhibited tumor progression in the CT26-R172H and A20 models, the difference was not significant (Fig. 1B, S3C, S3D, 1F, S3K, and S3L), and gWIZ-S237G did not inhibit tumor progression in the CT26 and CT26-R172H models (Fig. 1D, S3G, S3H, 1E, S3I, and S3J). Particularly for A20 tumor, significant inhibition was induced by all forms of the S237G-specific vaccines (gWIZ-S237G, gWIZ-S237G-Fc, and gWIZ-S237G-FR), with two, one, and two of the five mice completely rejecting the tumors, that is, complete response (CR). (Fig. 1C and 1F). The gWIZ-S237G immunization did not result in increased inflammation in the kidneys, lungs, spleen, liver, or heart of immunized mice compared to the control group (Figure S2A). All the mice displayed normal weight gain over time (Fig. S2B). IL-6 and TNF-α levels increased in the serum of vaccinated mice on day 2 post-vaccination but returned to baseline by day 7 (Fig. S2C and S2D). These findings suggest that gWIZ-S237G immunization effectively prevents subcutaneous A20 tumor growth with minimal toxicity.

Fig. 1. The prophylactic activity of the gWIZ-S237G vaccine in subcutaneous syngeneic tumor models.

Mice were intramuscularly immunized with the vaccine or the control three times (25 μg of DNA per animal in 10 μl of water) in combination with the AddaS03 adjuvant (10 μl), and animals were subcutaneously inoculated with 1 × 10⁶ tumor cells (CT26, CT26-R172H, or A20) 7 days after the last immunization (A-F) Average volumes of the tumors. (A) CT26 tumors in mice immunized with the p53-WT vaccines (gWIZ-WT, gWIZ-WTFc, gWIZ-WT-FR, or the vector control gWIZ). (B) CT26-R172H tumors in mice immunized with the p53-R172H vaccines. (C) A20 tumors in mice immunized with the p53-S237G vaccines. (D-F) CT26 (D), CT26-R172H (E), or A20 (F) tumors in mice immunized with the WT p53 vaccine (gWIZ-WT or mutants (gWIZ-R172H or -S237G). (G-I) Percentages of immune cells in spleens (G), DLNs (H), and tumors (I) (n=5 per group). Data were presented as mean ±SD. Multiple group comparisons were conducted using one-way ANOVA. Statistical significance was set at *p < 0.05, **p < 0.01, and ****p < 0.0001; ns indicated

To elucidate the possible mechanisms underlying the anti-tumor effects of the vaccine, we first analyzed the percentages of CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, natural killer (NK) cells, NKT cells, DCs, macrophages, regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSCs) in the spleen, DLN, and tumor. Compared with the gWIZ control group, the gWIZ-S237G group showed increased percentages of CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, NK cells, NKT cells, and DCs, along with reduced percentages of Tregs and MDSCs, whereas macrophages showed no significant difference in the spleen (Fig. 1G). Correspondingly, the percentages of CD3⁺ T cells, CD8⁺ T cells, NK cells, NKT cells, and DCs increased, whereas those of Tregs and MDSCs decreased in DLNs and tumors (Fig. 1H and 1I). Similar levels of macrophages were observed in the DLNs, with fewer macrophages in the tumors (Fig. 1I). These results indicate that the gWIZ-S237G vaccine induces immune cell infiltration and shifts them towards anti-tumor subpopulations.

3.2. The gWIZ-S237G vaccine induces DC maturation and promotes robust antigen-specific protective immunity

DCs are primary pivotal antigen-presenting cells (APCs) initiating and regulating immune responses by presenting antigens to T cells, thereby triggering potent antigen-specific antibody and T cell-mediated immunity [29, 30]. The induction and maturation of DCs are vital processes influenced by vaccines and determine the strength and specificity of the immune responses [23, 31]. We examined the proportions of various DC subpopulations in the spleens and tumors from immunized mice. There was a significant increase in the number of CD11b⁺CD11c⁺ cells in the spleens of mice immunized with gWIZ-S237G (Fig. 2A and 2B), indicating enhanced DC recruitment or proliferation in the spleen. Moreover, the proportion of these cells was reduced in the tumor microenvironment (Fig. 2A and 2C), suggesting a potential redistribution or functional specialization of DCs within different tissues. The gWIZ-S237G immunization group exhibited a marked increase in CD103⁺CD11c⁺ cells in the spleens and tumors (Fig. 2A–2C), highlighting the ability of the vaccine to promote the expansion and activation of conventional DCs, which transport antigens to the lymph nodes and prime antigen-specific CD8⁺ T cells. CD8⁺CD11c⁺ cells, also considered conventional DCs, significantly increased in the spleens and tumors (Fig. 2D–2F). The gWIZ-S237G vaccination led to a substantial upregulation of activation markers on CD11c⁺ cells, including CD80, CD86, MHC-II, and MHC-I, in the spleens and tumors (Fig. 2G, S4A–S4D, 2H, and S4E–S4H). These cell surface markers are indicative of DC maturation and enhanced antigen presentation to CD8⁺ T cells. Collectively, these data support that the gWIZ-S237G vaccine promotes the induction and maturation of DCs to facilitate the activation of CD8⁺ T cell-mediated immune responses.

Fig. 2. The characterization of DCs induced by the gWIZ-S237G vaccine in vivo.

Mice were sacrificed 20 days post immunization and tumor inoculation, and DCs subsets in spleens or tumors were analyzed by FACS. (A) Percentages of CD11c⁺CD11b⁻CD103⁺ DC and CD11c⁺CD11b⁺CD103⁻ DC subsets in spleens or tumors. One representative FACS data from each group was shown. (B and C) Statistical analysis of the percentages of DCs subpopulations shown in (A) (n=5 per group). (D) Percentages of CD8⁺CD11c⁺ DCs in spleens or tumors. One representative FACS data from each group is shown. (E and F) Statistical analysis of the percentages of DC subsets shown in (D). (G and H) Expression levels of CD80, CD86, MHC-II, and MHC-I on CD11c⁺ cells in spleens or tumors. One representative FACS data from each group was shown. Data were presented as means ± SD. Multiple group comparisons were conducted using one-way ANOVA. Statistical significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns indicated not significant.

Next, we assessed the vaccine-induced antigen-specific humoral response and observed significantly higher serum titers of IgG antibodies against the p53 peptide pool (Supplemental Table 1) containing the S237G mutation in mice immunized with gWIZ-S237G 35 days after the first immunization (Figure 3A). A p53-S237G peptide pool consisting of 68 peptides was used to evaluate T cell activation. gWIZ-S237G group demonstrated significantly enhanced CD8⁺ T cell proliferation compared to those immunized with either gWIZ or gWIZ-WT (Fig. 3B and 3C). A similar trend was observed in the proliferation of CD4⁺ T cells within the activated splenocytes of gWIZ-S237G-immunized mice, further indicating the broad immunostimulatory effects of the vaccine (Fig. S5A and S5B). The ELISPOT assay revealed elevated levels of IFN-γ-secreting CD8⁺ T cells in the gWIZ-S237G vaccinated group, indicating a strong antigen-specific cellular immune response (Fig. 3D and 3E). Splenocytes from gWIZ-S237G immunized mice exhibited potent tumor cell-killing activity in a CTL assay (Fig. 3F). Higher levels of Th1 cytokines, such as IFN-γ and TNF-α, were observed in the gWIZ-S237G group than those in the controls (Fig. 3G). No significant changes in the levels of the Th2 cytokines (IL-4 and IL-10) were observed. The gWIZ-S237G vaccine induced a higher percentage of functional CD8⁺ or CD4⁺ T cells expressing IFN-γ, IL-2, and TNF-α in the spleens and tumors of gWIZ-S237G-immunized mice compared to the controls (Fig. 3H–3K; Fig. S5C–S5F). Thus, these results suggest that gWIZ-S237G immunization effectively promotes DC-mediated antibody production and antigen-specific T cell responses skewed towards Th1.

Fig. 3. Antigen-specific immunity induced by the gWIZ-S237G vaccine.

(A) Anti-p53-S237G total IgG antibody levels in sera. Mice were intramuscularly immunized with the vaccines three times at 2-week intervals. Serum IgG antibody levels were measured at 35 days after the first immunization using ELISA. (B and C) Percentages of BrdU⁺ cells of total CD8⁺ T cells from splenocytes stimulated with a p53-S237G peptide pool. (B) One representative FACS data from each group is shown. (C) Statistical analysis of the data from (B) (n=5 per group). (D and E) ELISPOT analysis of IFN-γ-secreting splenic T cells. (F) the CTL activity of splenic T cells co-cultured with A20 tumor cells. (G) Secretory cytokine levels from activated splenocytes. Splenocytes from immunized mice were cultured with IL-2 (100 U/ml) and the p53-S237G peptide pool (10 μg/ml) for 72 h, followed by ELISA analysis of IFN-γ, TNF-α, IL-10, and IL-4 from supernatants. (H-K) FACS analysis of IFN-γ⁺, IL-2⁺, TNF-α⁺ CD8⁺ T cells from spleens (H and J) or tumors (I and K). Splenocytes were first stimulated with the p53-S237G peptide pool (10 μg/ml) protein for 67 h, before adding 500 ng/ml ionomycin, 50 ng/ml PMA, and 5 μg/ml BFA for another 5 h (H and J). One representative FACS from each group is shown (H and I) and statistical analyses were performed (J and K). Experiments were performed with 5 mice per group. Data were represented as means ± SD. Multiple group comparisons were performed using one-way ANOVA. Statistical significance was set at ***p < 0.001 and ****p < 0.0001.

3.3. Multiple antigen epitopes of gWIZ-S237G trigger antigen-specific T cell responses

Antigen epitopes are recognized by the immune system to elicit targeted immune reactions and form immunological memory [32–34]. We used the VaxiJen v3.0 server to predict the toxicity and immunogenicity of the 68 peptides of the p53-S237G pool [35, 36] (Fig. 4A; Supplemental Table 1). The ELISPOT assay was performed to examine the ability of each peptide to induce IFN-γ and TNF-α secretion for splenocytes from mice immunized with gWIZ-S237G (Fig. 4B). Notably, 3 peptides (30, 31, and 63) significantly induced IFN-γ secretion, while 5 peptides (13, 30, 31, 61, 63, 64, and 67) strongly induced TNF-α secretion (Fig. 4C). Peptides Seq 67 and Seq 68 contained the S237G mutation, but only Seq 67 was active during cytokine stimulation. Seven peptides (13, 30, 31, 61, 63, 64, and 67) did not significantly stimulate cytokine production in splenocytes of gWIZ-WT and gWIZ-R172H immunized mice (Fig. S6A and S6B).

Fig. 4. Identification of T cell antigen epitopes.

(A) Prediction of the immunogenicity (IMM) and cytotoxicity the immunogenicity (IMM) and cytotoxicity (TOX) of each antigen epitope from the p53-S237G peptide pool using the VaxiJen v3.0 server. (B) ELISPOT analysis of splenic IFN-γ- or TNF-α-secreting T cells. Splenocytes from immunized mice were cultured with IL-2 (100 U/ml) and each individual peptide from the p53-S237G peptide pool (10 μg/ml) for 60 h before the ELISPOT analysis. (C) Sequences of each peptide that stimulated IFN-γ- and/or TNF-α secretion in splenic T cells, along with the predicted 3D structures of Seq 30 (red), Seq 31 (blue), and Seq 63 (green) in p53-S237G. (D) FACS analysis of IFN-γ- or TNF-α-secreting CD4⁺ or CD8⁺ T cells in splenocytes stimulated with each peptide. One representative FACS data from each group is shown. (E) Statistical analysis of the percentages of IFN-γ⁺CD4⁺ T cells, TNF-α⁺CD4⁺ T cells, and IFN-γ⁺TNF-α⁺CD4⁺ T cells shown in (D). (F) Statistical analysis of the percentages of IFN-γ⁺CD8⁺ T cells, TNF-α⁺CD8⁺ T cells, and IFN-γ⁺TNF-α⁺CD8⁺ T cells shown in (D). Data were presented as mean ± SD. Statistical significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001; ns indicated not significant.

We performed FACS analysis of splenocytes from gWIZ-S237G-immunized mice incubated with individual peptides. Compared to the controls, 3 peptides (30, 31, and 63) significantly stimulated CD4⁺ splenocytes to secrete IFN-γ and TNF-α, while 3 other peptides (13, 64, and 67) induced TNF-α expression only (Fig. S6C; Fig. 4E and 4F). Six peptides (Seqs 30, 31, 61, 63, 64, and 67) induced both IFN-γ and TNF-α expression in CD8⁺ splenocytes, while Seq 13 induced TNF-α expression only (Figures S6D; Figure 4E and 4F). These results suggest that multiple peptide epitopes induce specific CD4⁺ or CD8⁺ T cell immune responses for tumor eradication.

3.4. The long-term anti-tumor effects of gWIZ-S237G are mainly dependent on multifunctional CD8⁺ T cells

Multifunctional CD8⁺ T cells simultaneously secrete several cytokines (IFN-γ, TNF-α, and IL-2) that synergistically enhance their anti-tumor activity[37, 38]. The percentages of antigen-specific CD8⁺ T cells secreting two or three cytokines in spleens and tumors of immunized mice were measured using FACS. The proportions of CD8⁺ T cells secreting two or three cytokines were the highest in the antigen-stimulated splenocytes from the gWIZ-S237G immunization group (Fig. 5A). A similar trend was observed for tumor-infiltrating CD8⁺ T cells (Fig. 5B). We also used anti-CD8 mAb to deplete CD8⁺ T cells in mice. CD8⁺ T cell depletion completely abolished the anti-tumor effects of the gWIZ-S237G vaccine (Fig. 5C). The depletion of NK or CD4⁺ T cells caused a slight reduction in the anti-tumor activity of the S237G vaccine; however, the differences were not statistically significant (Fig. 5D and 5E). As expected, the percentages of CD8⁺ T cells and CD8⁺CD11c⁺ DCs in the tumors were significantly reduced after CD8⁺ T cell depletion (Fig. 5F). These results indicate that multifunctional CD8⁺ T cells induced by the S237G vaccine are crucial for the anti-tumor immune responses.

Fig. 5. Multifunctional CD8⁺ T cells were essential for the anti-tumor effect of the gWIZ-S237G vaccine.

(A and B) Proportions of IFN-γ⁺TNF-α⁺CD8⁺, IFN-γ⁺IL-2⁺CD8⁺, IL-2⁺TNF-α⁺ CD8⁺, and IFN-γ⁺IL-2⁺TNF-α⁺CD8⁺ T cell subsets in splenocytes and tumors in mice vaccinated with gWIZ-WT or -S237G (n=5 per group). (C-F) Survival of tumor-bearing mice (n=10 per group) treated with gWIZ-S237G after deletion of CD8⁺ T cells, NK cells, or CD4⁺ T cells. Mice were immunized and inoculated with tumor cells, and treatment with mAbs (0.5 mg per animal directed against mouse CD8α, GM1, or CD4) started 7 days after the first dose of gWIZ-S237G, followed by two more on days 14 and 21. (C-E) Animal survival; (F) Percentages of CD8⁺ T cells and CD8⁺ DCs within tumors. (G and H) Tumor cell rechallenge. Five mice with complete response were rechallenged on the opposite flank with the same amounts of A20 tumor cells. A separate group of naive mice was challenged with A20 tumor cells as the control. (G) Individual tumor growth curves for each animal, recorded until 5 weeks post-rechallenge. (H) Survival curves of the rechallenged mice compared to the control. (I) Representative dot plots showing CD8⁺ memory T cell populations in splenocytes from immunized mice 20 days post-tumor inoculation (n=5 per group). (J) Statistical analysis of the percentages of naive (CD44^lowCd62L⁺), central memory (CD44^highCD62L⁺), and effector memory (CD44^highCD62L⁻) CD8⁺ T cells in (J). Data were presented as mean ± SD. Survival analysis was conducted using the log-rank (Mantel-Cox) test. Comparisons between two groups were performed using a two-tailed independent Student’s t-test. Multiple group comparisons were performed using one-way ANOVA. Statistical significance was set at **p < 0.01, ***p < 0.001 and ****p < 0.0001; ns indicated not significant.

To evaluate long-term anti-tumor immune responses, mice that were previously immunized with gWIZ-S237G and rejected A20 tumors were rechallenged contralaterally with A20 cells. The rechallenged animals had substantially smaller tumors and survived longer compared to the naive mice, and three of the five rechallenged mice rejected the tumors (Fig. 5G and 5H). We analyzed the changes observed in the memory CD8⁺ T cells in the spleens of mice immunized with gWIZ-S237G and inoculated with A20 tumor cells using FACS. Effector memory (EM) CD8⁺ T cells (CD44^highCD62L⁻CD8⁺ T, T_EM) and central memory (CM) CD8⁺ T cells (CD44^highCD62L⁺CD8⁺ T, T_CM) significantly increased in the gWIZ-S237G group compared to the gWIZ or gWIZ-WT group (Fig. 5I and 5J). The percentage of naive CD8⁺ T cells (CD44^lowCD62L^highCD8⁺ T, T_N) was lower in the gWIZ-S237G group than in the control group (Fig. 5I and 5J). These findings suggest that the gWIZ-S237G vaccine elicits a memory CD8⁺ T cell response against A20 tumors.

3.5. The gWIZ-S237G vaccine induces protective immunity against tumor liver metastasis by enhancing CD8⁺ T cell immune responses

To evaluate the protective effect of the gWIZ-S237G vaccine against A20 liver metastasis, mice were immunized before intravenously administration of A20 cells. The vaccine caused a significant reduction in the metastatic tumor nodules on the liver surface (Fig. 6A and 6B). A histological examination confirmed the protective effects of gWIZ-S237G (Fig. 6C). The mice in the gWIZ-S237G group survived longer than those in the control group (Fig. 6D). IHC data indicated that the numbers of tumor-infiltrating NK cells and CD8⁺ T cells in the gWIZ-S237G group were higher than those in the control group (Fig. 6E). CD8⁺ T cells from the spleens of gWIZ-S237G-immunized mice displayed increased proliferation when stimulated with the p53-S237G peptide pool (Fig. 6F). The stimulated splenocytes from the gWIZ-S237G group had stronger CTL activity against A20 target cells (Fig. 6G), and the number of cells secreting IFN-γ was higher than the controls, as measured using the ELISPOT assay (Fig. 6H). The proportion of CD8⁺ T cells secreting two or three cytokines was higher in the gWIZ-S237G group than in the controls (Fig. 6I). Thus, gWIZ-S237G immunization effectively inhibits tumor liver metastasis by inducing a strong CD8⁺ T cell-mediated anti-tumor immune response.

Fig. 6. The protective effects of the gWIZ-S237G vaccine against A20 liver metastasis.

A20 tumor cells (2 × 10⁶ tumor cells) were intravenously injected 7 days after the last immunization. (A) Two representative images of liver tumor metastasis from each group. (B) The number of metastatic nodules counted on the liver surface in each group (n=5 mice per group). (C) Two representative images of H&E staining of liver tissues. (D) Animal survival (n = 10 mice per group). (E) Immunohistochemical staining of tumor-infiltrating CD8⁺ T cells and CD49b⁺ cells. (F) The percentages of BrdU⁺ cells from total splenic CD8⁺ T cells. (G) The CTL activity of splenic T cells incubated with A20 tumor cells. (H) The number of IFN-γ-secreting splenocytes measured using the ELISPOT assay. (I) Percentages of multifunctional splenic CD8⁺ T cells producing IFN-γ, IL-2, and/or TNF-α. Splenocytes were first stimulated with the p53-S237G peptide pool (10 μg/ml) protein for 67 h, before addition of 500 ng/ml ionomycin, 50 ng/ml PMA, and 5 μg/ml BFA for another 5 h. Survival analysis was conducted using the log-rank (Mantel-Cox) test. Multiple group comparisons were performed using one-way ANOVA. Data were presented as means ± SD. Statistical significance was set at **p < 0.01, ***p < 0.001, and ****p < 0.0001.

3.6. PD-1 blockade enhances the therapeutic activity of the gWIZ-S237G vaccine

To investigate the therapeutic benefit of combining the gWIZ-S237G vaccine with an anti-PD-1 mAb against A20 tumors, mice were immunized intramuscularly with the gWIZ-S237G vaccine on days 1, 11, and 21 and were administered PD-1-mAb intraperitoneally on days 7 and 14 after subcutaneous injection of tumor cells (Fig. 7A). Combination of gWIZ-S237G vaccine and anti-PD-1 mAb exhibited stronger tumor inhibition than either monotherapy, achieving CR in three out of five mice (Fig. 7B). FACS analysis of tumor-infiltrating CD8⁺ cells showed that the combination did not lead to more CD8⁺ T cells or CD8⁺CD11c⁺ DCs than the gWIZ-S237G vaccine (Fig. 7C and 7D), yet it did markedly enhance the percentages of IFN-γ-positive CD8⁺ T cells within the tumor microenvironment (Fig. 7E and 7F). For splenocytes obtained from treated mice, the combination resulted in more IFN-γ-secreting cells observed using the ELISPOT assay, more BrdU-positive cells observed through FACS analysis, and higher CTL activity against A20 tumor cells than either monotherapy (Fig. 7G, 7H, and 7I).

Fig. 7. The therapeutic effects of the gWIZ-S237G vaccine in combination with PD-1 blockade in subcutaneous and metastatic A20 tumor models.

(A) Schematic representation of the subcutaneous tumor model treated with gWIZ-S237G and/or an anti-PD-1 mAb (n = 5 mice). (B) Tumor volumes in treated mice. (C and D) Percentages of CD8⁺CD11c⁺ DC subsets within tumors from mice treated with gWIZ-S237G and/or the anti-PD-1 mAb. (E and F) Percentages of IFN-γ⁺CD8⁺ T cells within tumors. (G) ELISPOT analysis of splenic IFN-γ-secreting T cells. (H) CD8⁺ T cell proliferation. (I) The CTL activity. (J) Schematic representation of the A20 liver metastatic model treated with gWIZ-S237G and/or the anti-PD-1 mAb. (K) Animal survival (n = 10 mice per group). (L) Histological analysis of liver tissues. (M) The proliferation of splenic CD8⁺ T cells. (N) The CTL activity. Survival analysis was conducted using the log-rank (Mantel-Cox) test. Multiple group comparisons were performed using one-way ANOVA. Data were presented as means ± SD. Statistical significance was set at *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001.

Next, mice were immunized intramuscularly with the gWIZ-S237G vaccine on days −10, 1, and 11, and received intraperitoneal injections of the anti-PD-1 mAb on days 7 and 14 after A20 tumor cell inoculation intravenously on day 0 (Fig. 7J). The combination significantly improved animal survival, with 90% remaining alive 7 weeks after tumor cell inoculation, compared to 0% survival in the vector control group, 40% in the PD-1-mAb group, and 50% in the gWIZ-S237G vaccine alone group (Fig. 7K). Combination therapy reduced the number of tumors in the liver (Fig. 7L) and increased the proliferation and CTL activity of CD8⁺ splenocytes in treated mice (Fig. 7M and 7N). These findings suggest that the gWIZ-S237G vaccine combined with PD-1 inhibition effectively attenuates tumor development and metastasis.

4. Discussion

The A20 cell line carries an endogenous p53-S237G mutation and exhibits the highest expression levels among various mouse syngeneic cell lines. CT26 tumors with p53-WT are known to be immunogenic, meaning they can provoke a robust immune response, which is valuable for evaluating the efficacy of immune-based treatments. The cell line’s predictable growth patterns and well-documented behavior in mice make CT26 a feasible model for preclinical studies focused on tumor immunity and therapeutic interventions. CT26-R172H is an engineered CT26 line carrying p53-R172H [28], so the recombinant line is likely not addicted to R172H. In this study, we developed a DNA vaccine gWIZ-S237G targeting the p53 S237G mutation in the A20 B-cell lymphoma cell line and demonstrated that the p53 antigen was expressed in vaccinated mice. The gWIZ-S237G vaccine had a specific and effective protective immune response against A20 subcutaneous tumors and liver metastases, but not against CT26 tumors with WT p53 or CT26-R172H tumors. In contrast, gWIZ-R172H exhibited no significant anti-tumor activities against CT26-R172H, indicating that the vaccines may only work for cells with higher expression of p53 endogenous mutants. We did not test other aggregation mutants against A20 cells, only p53-R172H and p53-S237G aggregation mutants were tested against A20 cells. We used the vaccine in combination with PD-1 checkpoint blockade to treat mice with established A20 subcutaneous and liver metastatic tumors. The combination significantly inhibited tumor growth and metastasis, with some animals achieving CR. The promising anti-tumor activity observed in prophylactic and therapeutic models indicates that the p53-S237G vaccine effectively prevents tumor development, and when used in combination with PD-1 checkpoint blockade, it robustly triggered an immune response to counteract tumor progression.

The gWIZ-S237G vaccine immunization promoted T cells and DCs infiltration while reducing the infiltration of immunosuppressive Tregs and MDSCs in subcutaneous A20 tumors. DCs are the most potent antigen-presenting cells that play a critical role in phagocytosis and processing of the antigen proteins expressed by the vaccine, ultimately presenting them to T cells and promoting T cell-mediated tumor killing [39]. CD103⁺CD11c⁺ and CD8⁺CD11c⁺ DC subpopulations are essential for antigen presentation and activation of CD8⁺ T cells for anti-tumor immunity [40, 41]. Immunization with the gWIZ-S237G vaccine enhanced the induction of DCs, particularly the CD103⁺CD11c⁺ and CD8⁺CD11c⁺ subpopulations cells. Expression of maturation and activation markers such as CD80, CD86, MHC-I, and MHC-II on the surface of DCs was significantly upregulated in the spleens and tumors of gWIZ-S237G-immunized mice. CD4⁺ T cells and antibody-secreting B cells assist antigen-specific CD8⁺ T cells in triggering anti-tumor immune responses [42]. We found that the gWIZ-S237G-immunized mice produced antibodies targeting the p53-S237G mutant peptide. Vaccination did not increase the percentage of tumor-infiltrating CD4⁺ T cells but increased cytokine production in these cells. Moreover, vaccine-induced T cell immune responses were skewed towards a Th1-type anti-tumor response. These results demonstrate that the vaccine effectively induces DC maturation, leading to efficient antigen presentation and activation of both humoral and cellular immune responses.

As well-known executors of anti-tumor immune responses, multifunctional CD8⁺ T cells directly target tumor cells and interact with other immune cells by secreting various cytokines like IFN-γ, IL-2, and TNF-α [43]. The gWIZ-S37G vaccine significantly increased the numbers of multifunctional CD8⁺ T cells expressing two or more cytokines in the spleen and tumors of immunized mice. Depletion of CD8⁺ T cells, but not CD4⁺ or NK cells, significantly abolished the prophylactic anti-tumor activity of the vaccine, underlying the pivotal role of CD8⁺ T cells in tumor control. In addition, memory CD8⁺ T cells are indispensable for the long-term anti-tumor effects of the vaccines.

Fersht and colleagues studied the limited proteolysis of soluble WT p53 and G245S mutant aggregates using trypsin in vitro [44]. We assumed that in vivo WT p53 is soluble, most mutant p53 aggregates irreversibly [45, 46], and mutant p53 degradation products differ from WT p53 beyond the mutant epitopes. There would be three types of peptides from p53-G245S degradation that MHCs could present for TCR recognition: (I) WT peptides at a supraphysiological level, given mutant p53 is upregulated in the tumor cells; (II) mutant peptides containing the mutated residue G245S; (III) peptides with a WT amino acid sequence that are produced only from mutant p53 degradation. Experimental proteolysis of p53-G245S aggregates has identified several Type III peptides [44]. Vaccines with WT p53 as the antigen may only generate Type I peptides that elicit CTLs with low avidity, owing to self-tolerance [47, 48]. However, the latter two peptides (neoantigens) from mutant p53 degradation are not subjected to self-tolerant, when mutant p53 is used as an immunogen. We recognize that in vivo proteolysis of tumor antigens is a complex process mediated by proteasomes, lysosomes, and endosomes. The human T cell repertoire does not necessarily lack low- and even residual high-avidity p53-specific CTLs; however, self-tolerance certainly limits the number of high-avidity CD8⁺ CTLs binding to MHC-restricted WT p53 peptides (i.e., Type I peptides) [47, 48]. Type II p53 peptides (neoepitopes with mutant amino acid sequences) in mice were detected as early as 1994 [49], yet the TCRs recognizing such MHC-restricted neoantigens in cancer patients were not cloned until 2019 [50–53]. However, Type III peptides (neoantigens with WT amino acid sequences) have not been reported or tested in vivo. Vaccination with p53-S237G clearly produced a Type II peptide (Seq 67) and several Type III peptides that stimulated cytokine production in splenocytes. We did not clone the TCRs specific to these peptides, yet our data demonstrates the potential of using p53 mutants as immunogens for vaccine design.

We reviewed the clinical and immunological effects of p53-targeting vaccines for cancer patients in over 20 clinical trials [7]. These vaccines predominantly used WT p53 full-length protein or peptides as antigens, which may reduce the cytotoxic T cell avidity due to self-tolerance. These anti-p53 vaccines, developed before the ICB era, elicited p53-specific immune responses and demonstrated safety, yet patients undergoing adjunctive chemotherapy sometimes experienced high-grade adverse events. However, the immune responses induced by these vaccines are insufficient to effectively inhibit tumor growth in patients. Historically, antiviral vaccines are designed for disease prophylaxis rather than for the treatment of active infections. Similarly, in preclinical models, many anti-tumor vaccines have demonstrated greater efficacy in preventing tumor development in prophylactic settings than in treating established tumors. The failure of earlier anti-p53 vaccines as monotherapies is comparable to attempting to treat an active viral infection with a vaccine. Currently, multiple promising cancer vaccines targeting neoantigens and tumor antigens have been developed using synthetic long peptide (SLP) [54], exosome [55, 56], RNA [57–60], or DNA [61–63]. In most cases, continuous infusion of anti-PD-1 or anti-PD-L1 ICB antibodies is employed to enhance therapeutic efficacy. We believe the success of cancer vaccines is dependent on ICB to amplify T cell responses. In this study, we incorporated two improvements in vaccine design: (1) designing a specific vaccine for each p53 mutant and (2) combining the vaccine with anti-PD-1 ICB for the treatment of established tumors. Our work presents DNA-encoded vaccines tailored for cancer patients with tumors expressing high levels of p53 mutants, which, in future clinical trials, should be administered alongside adjunctive ICB therapy for optimal efficacy.

5. Conclusions

In summary, the p53-S237G vaccine effectively prevented and treated A20 tumorigenesis and metastasis. PD-1 blockade may be required to enhance the activity of the vaccine to treat cancer. Our findings suggest that DNA vaccines targeting individual p53 mutations represent a promising personalized therapeutic approach for cancers highly expressing p53 mutant proteins.

Availability of data and materials

All data supporting the findings of this study have been included in this article. Supplementary information is available upon request from the corresponding authors.