Immune checkpoint inhibitors can help some cancer patients, but many tumors barely respond. This study asks whether a muscle-injected DNA vaccine and a plasmid that produces a PD-1 antibody can work better together. Immune checkpoint inhibitors have moderate response rates of about twenty to thirty percent in some malignancies, according to clinical evidence described here.

DNA tumor vaccines could optimize the efficacy of cancer treatment when combined with other immunotherapeutic strategies, but the study found that mice receiving either pDNA-OVA or pDNA-a-PD-1 alone showed weak tumor inhibition in the MC38-OVA-bearing model. The central problem is that immune checkpoint inhibitor therapy is still ineffective in most cancers, with the majority of cancer patients receiving little benefit from it.

The proposed response is combination treatment, which is considered a good treatment protocol and could have a tangible curative effect. DNA vaccines have good prospects for cancer prevention and treatment because of their ability to promote extensive immune activation, but they have not received enough attention in clinical applications because of insufficient immune response so far.

The transfection of plasmid DNA in vivo enables specific tissues to produce desired proteins, and plasmid DNA can encode antigens or therapeutic proteins. The method uses DNA vectors as vaccine and immune checkpoint blockade protein-encoding gene carriers, with the pluronic L64, electroporation, and epigallocatechin gallate system used to promote gene delivery efficiency.

The combination of plasmid DNA encoding ovalbumin vaccine with pVAX-a-PD-1 caused enormous tumor regression, and the combination prevented lung metastasis in B16F10-ovalbumin-bearing mice models. The mouse PD-1 antibody sequence was cloned onto the pVAX1 vector, while the OVA gene was cloned onto pcDNA3.1 and the efficient muscle-specific plasmid pEMS to construct the DNA vaccine.

The pEMS is a reconstructed plasmid backbone that replaces the CMV promoter of pcDNA3.1 with EMS, and plasmid purity and concentration were determined using agarose gel electrophoresis and spectrophotometry. Intramuscular gene delivery used the combined pluronic L64, electroporation, and EGCG system, called L/E/G.

Plasmid DNA was dissolved in EGCG working solution and incubated for thirty minutes at room temperature, then the EGCG and plasmid DNA complex was mixed with L64 until L64 reached a concentration of zero point one percent. The EGCG, plasmid DNA, and L64 complex was injected into the right anterior tibial muscle, and one hour later an electrical pulse was used to promote gene transfer.

The DNA molecule for in vivo antibody production contained the single-chain variable fragment of anti-mouse PD-1 and the mouse Fc region with a D265A point mutation that leads to total loss of cytolytic function. In human 293T cells transfected with pVAX-a-PD-1-Fc, western blot analysis detected secretion of a one-hundred-twenty kilodalton protein.

An ELISA assay showed that anti-PD-1 from cell culture media bound to PD-1 protein in a dose-dependent manner. In panel D, the blue binding curve rises to roughly one-point-five absorbance units before declining toward baseline as antibody concentration increases; the black control remains close to baseline throughout.

In the MC38-OVA-bearing mice, the treatment groups included the pCMV-empty vector, pCMV-OVA, and pEMS-OVA, and each group was divided after tumor formation to receive either pVAX-Ctrl or pVAX-a-PD-1. The pCMV-OVA vaccination showed little tumor inhibition, while the pEMS-OVA vaccination caused the greatest delay in tumor growth.

All mice receiving pVAX-a-PD-1 displayed slower tumor growth than mice receiving DNA-only vaccine treatment, and the pEMS-OVA combined with pVAX-a-PD-1 group showed significant tumor regression. All mice displayed stable weight gain irrespective of group, a preliminary finding that indicated these treatments were safe enough.

All mice in the empty-vector and pVAX-Ctrl group died within twenty-three days, whereas about sixty percent of the pEMS-OVA and pVAX-a-PD-1 group survived for more than forty-five days. Approximately forty percent of the pCMV-OVA and pVAX-a-PD-1 group and approximately twenty percent of the pVAX-a-PD-1 single-treatment group survived for more than forty-five days.

The separation is clearest in panel F, where the pEMS-OVA-plus-alpha-PD-1 curve remains near the bottom while the EV-plus-alpha-PD-1 curve rises sharply, with pCMV-OVA-plus-alpha-PD-1 between them. Panel H shows the corresponding spacing among survival curves, while panel G shows the six weight trajectories clustered within a relatively narrow upward band.

The concentrations of IL-2, IL-4, and IL-10 were very low in the vaccine-only treatment groups, while the DNA vaccine combined with pVAX-a-PD-1 significantly increased all three cytokines in serum. Without the vaccine, very few IFN-gamma spots were detected, but splenocytes from DNA vaccine-treated mice showed a significant increase, and combined treatment resulted in a significantly increased number of spots.

The levels of CD8-positive and CD4-positive T cells were higher in tumor tissues from the therapeutic groups than in tissues from the empty-vector group. In panel A, the red pEMS-OVA plus alpha-PD-1 bars are visibly the tallest across all three cytokine plots, with the IL-4 and IL-10 bars extending far above the other groups.

In panel C, the blue combination-treatment bar rises to roughly the mid-twenties, while the corresponding black bar is only around the low teens; panels D and E likewise show denser brown-stained T-cell areas in the therapeutic columns. The B16-F10 lung metastasis model tested whether pOVA vaccines could enhance the inhibitory effect of intramuscular plasmid-DNA-based PD-1 antibody therapy.

The weakest inhibition occurred in the empty-vector group, and DNA vaccination without L/E/G also produced weak suppression of lung metastasis. pCMV-OVA combined with pVAX-a-PD-1 strongly suppressed lung metastasis, while pEMS-OVA combined with pVAX-a-PD-1 further prevented the formation of pulmonary nodules.

In panel C, the black and purple bars remain near twenty lung nodules, whereas the orange and red bars are only a few nodules high. Panel D shows the same visual separation in lung weight, while panel E shows all four body-weight curves staying close together and generally rising by day fourteen.

Hematoxylin and eosin staining identified intensive metastases in control mice and in mice receiving DNA vaccination without L/E/G, while notably small numbers of nodules appeared after pCMV-OVA or pEMS-OVA combined with pVAX-a-PD-1. The combination of pVAX-a-PD-1 and pEMS-OVA produced almost normal lung histopathology, and the pEMS-OVA combined with pVAX-a-PD-1 group had CD8-positive T-cell proportions increased by up to forty-five percent in blood and up to twenty-seven percent in spleen.

There was no demonstrable difference in the proportion of CD4-positive T cells across treatment groups in either blood or spleen, while CD8-positive T cells in spleen and peripheral blood were significantly elevated. Looking across the bottom row, spleen weight and ALT show overlapping bars with non-significant comparisons.

In the AST panel, the purple treatment bar is visibly higher than the black control and is marked with a single asterisk, while the orange and red bars are lower and fall within a non-significant overall comparison. The pEMS-OVA expressed vaccine had the highest tumor-suppressing efficiency among the immunization methods examined, and pEMS-OVA combined with pVAX-a-PD-1 ameliorated survival rate and reduced tumor growth.

The combination of pOVA and pVAX-a-PD-1 increased IL-2, IL-4, IL-10, and IFN-gamma production, and also increased CD8-positive and CD4-positive T cells. The production of cytokines is only one of many factors affecting tumor volume, so changes in tumor volumes cannot be completely attributed to the observed cytokine trends.

In mice, the pEMS-OVA DNA vaccine combined with plasmid-encoded PD-1 antibody produced the strongest tumor control, improved survival, limited lung metastasis, and increased CD8+ T cells. The result supports combination treatment, while remaining preclinical.