Immune‐check blocking combination multiple cytokines shown curative potential in mice tumor model

In order to ensure the stable transcription of target genes, we constructed a eukaryotic high expression vector carrying an immune‐check inhibitor PD‐1v and a variety of cytokines, and studied their effects on activating immune response to inhibit tumor growth.

IL-12, and GM-CSF recombinant plasmids significantly inhibited tumor growth in mice, and the tumor growth rate was significantly lower than that in the blank control group and GFP plasmid control group (p < 0.05). Cytometric bead array data suggested that the combination of PD-1v and various cytokines can effectively activate immune cells. HE and IHC analysis revealed plenty of immune cell infiltrates in the tumor tissue, and a large proportion of tumor cells showed the necrotic phenotype in the combination treatment group.

| INTRODUCTION
Tumor microenvironment (TME) is an important site that regulates tumor development, growth, invasion and distant metastasis, and plays a key role in inducing tumor immune escape. [1][2][3] Many types of tumor cells, such as melanoma, colon cancer, lung cancer, ovarian cancer, head and neck squamous cell carcinoma and glioma, can inhibit the killing function of T cells, increase the apoptosis of tumor-specific cytotoxic T lymphocyte (CTL), and facilitate tumors to escape from immune surveillance and killing by overexpressing PD-Ls (including PD-L1 and PD-L2), which interact with the negative regulatory receptor programmed death 1 (PD-1) on the surface of T lymphocytes. 4-7 PD-1 is an important negative regulatory receptor on the surface of T cells after the discovery of CTLA-4. The binding of PD-1 and PD-Ls is intended to prevent excessive activation of T cells and avoid inflammatory damage caused by excessive activation of T cells. However, tumor cells can induce the dysfunction and depletion of infiltrating lymphocytes through high expression of PD-Ls, so that tumors can escape immune surveillance and killing. [8][9][10] In recent years, studies have shown that antibody blocking targeting PD-1/PD-Ls, namely T-cell immune check point blocking, cutting off the PD-1/PD-Ls signaling pathway, can reverse the dysfunction and depletion of effector T cells and restore the antitumor activity of T cells. [11][12][13][14] In addition to T lymphocytes, other innate immune cells, including natural killer (NK) cells, NKT cells, macrophages, dendritic cells, mast cells, and neutrophils, also play an important role in controlling tumor growth. 15 As is known, tumor cells downregulated or lacked MHC class I molecules, and lacked the ability to present tumor-related antigens, making tumor cells present a low immunogenicity phenotype and thus escape the immune surveillance of the body. [16][17][18][19] MHC class I molecules expressed on the surface of normal cells are the main molecules to avoid killing attack of self-NK cells. 20 Therefore, NK cells recognize tumor cells with low MHC expression, and have a strong anti-tumor effect in the tumor microenvironment. 21,22 It was found that, on the one hand, NK cells can migrate to the tumor microenvironment under the effect of IL-15 and IL-2, improving the cytotoxicity of NK cells and the recognition of tumor cells. [23][24][25] On the other hand, NK cells have immunomodulatory potential and can secrete cytokines and chemokines IFN-γ, TNF-α, CCL5, XCL1, and granulocyte macrophage colony stimulating factor (GM-CSF) to facilitate innate and adaptive cellular antitumor responses. [26][27][28] Attracting immune cells into the tumor microenvironment, as well as the interaction between immune cells, cell relocation, and functional activation greatly affect the anti-tumor immune response. 29,30 In early research, people tried to use single cytokines such as IL-12 and IFN-γ treatment of tumor, but the effect is not significant. Recent studies have shown that delivering multiple cytokines such as IL-12, IL-15, and GM-CSF with herpes simplex virus type 1 or type 2 as the carrier can effectively mobilize the anti-tumor immune response of the body, suggesting that the combined application of multiple cytokines is a promising strategy to mobilize the anti-tumor immune response of the body. [31][32][33][34] Some studies have shown that although the viral vector has high transfection efficiency, its application is greatly limited due to the potential security risks, such as the occurrence of leukemia induced by some retroviruses, and the limited capacity of the viral vector. 35 In contrast, nonviral vectors have the advantages of low cost, simple preparation, easy mass production, high safety, and unlimited length of exogenous genes. 36 Plasmid vector is an attractive in vivo transfection vector in animals that can be injected directly into specific tissues and can achieve effective higher levels of gene expression in vivo. A large number of studies have reported that IRES structures from some viruses help eukaryotic mRNA lacking the 5 'cap structure to translate and synthesize proteins. 37 Also, studies have shown that adding poly(A) to T7 promoterinitiated transcription units can prolong mRNA life and improve translation efficiency. 38 In order to improve the efficiency of gene expression, especially the translation of target gene, we used molecular cloning technology to construct a new dual promoter plasmid expression vector pT7AMPCE carrying CMV promoter, T7 RNA polymerase (T7RNAP), BGHpA, T7 promoter, IRES structure and poly(A), aiming to express T7RNAP with CMV promoter, T7RNAP transcribes IRES, target genes and poly(A), and used to carry the immune check point inhibitor PD-1v and a variety of cytokines. To explore its role in attracting and driving immune cells into tumor tissue and restoring antitumor immune response.

Conclusion:
The combination of immune check blockade and multiple cytokine therapy can significantly activate the body's immune response and inhibit tumor growth.

| Ethics statement
All animal experiments were conducted under the protocol approved by the Hubei Province, P.R. China, Biological Studies Animal Care and Use Committee.

| Cell lines and mice
Mouse colon cancer CT26 cells were purchased from the National Biomedical Experimental Cell Repository. CT26 cells stably expressing near-infrared fluorescent protein (IRFP) (referred to as CT26-IRFP) were constructed in our laboratory by using the PiggyBac transposon system to transfer the near-infrared fluorescent protein IRFP 720 into the murine colon cancer cell line CT26 (System Bioscience), and the CT26-IRFP 720 monoclonal stable cell line was obtained by puromycin screening. CT26 and CT26-IRFP were cultured in Dulbecco's modified Eagle's medium/Nutrient Mixture F-12 Ham (DME/F-12) supplemented with 10% fetal bovine serum at 37°C in a 5% CO 2 incubator.
Six-to eight-week-old female BALB/C mice were purchased from the Hubei Provincial Center for Disease Control and Prevention (No. 42000600043578). Each experimental group had five BALB/c mice. Animals were housed at a controlled temperature of 20-25°C, relative humidity of 40-70%, with a 12-h light/dark cycle interval, and adequate supply of water and food. The study was approved by the Ethics Committee of Hubei University of Science and Technology (No. [2021] HBSTC-05 (981)). All animal experiments were performed in strict accordance with the requirements of the Hubei Provincial Laboratory Animal Management and Use Committee.

| Plasmid construction
pT7AMPCE plasmid construction: Primer pairs were designed to amplify fragment 1 (containing immediate early cytomegalovirus promoter, CMVp in short), fragment 2 (containing T7 RNA polymerase, T7RNApol in short), and fragment 3 (containing bovine growth hormone polyadenosine, BGHpA in short). The templates for fragments 1 and 3 were from pcDNA3 (Invitrogen), and the template for fragment 2 was from T7 phage (Invitrogen). Fragment 4 containing CMVp, T7RNA pol , and BGHpA was overlap-PCR amplified using the mixture of fragments 1-3 as the template and with primer pair. Fragment 5 containing pUC plasmid replication origin and ampicillin resistance gene (ampR) was amplified from pSP73 (Promega) using primer pair. Fragment 4 treated with T4 kinase (NEB) and fragment 5 were ligated with T4 DNA ligase (NEB). The ligation reaction was transformed into Top 10 competent cells to generate plasmid pCMVp-T7RNA pol -BGHpA followed by sequencing verification. Fragment 6 containing T7 promoter (T7p) and encephalomyocarditis virus internal ribosome entry site (ECMV-IRES) sequence amplified from pcDNA3-IRES (in house) using primer pair was then cloned into the pCMVp-T7RNA pol -BGHpA SspI site to generate pT7AMPCE.
Recombinant plasmid construction: PD-1v, IL-12, GM-CSF, and GFP gene templates were obtained from the recombinant HSV2 virus constructed earlier in our laboratory, and the IL-2/15 gene template was obtained from the pHG52D34.5-CMV-IL-2/15 recombinant plasmid preserved earlier in our laboratory. 18,21 The primer sequences used for cloning are shown in Table 1. The pT7AMPCE plasmid was digested by BglII and ligated with the above three gene fragments via ClonExpress II recombinase. All cDNAs inserted into the constructed plasmids were confirmed by DNA sequencing.

| Plasmid purification
The plasmids were extracted by conventional alkaline lysis and selectively purified by the spermine method. The endotoxin content was detected by the LAL method below 5 EU/Hg. The purity and conformation of the plasmids were detected by agarose electrophoresis, and the plasmid concentration was quantified and the purity was calculated by a UV spectrophotometer (OD260/ OD280). The purity of the plasmids used was greater than 98%.

| Cell transfection
The CT26 cells were inoculated in 6-well plates at 2 × 10 5 cells/well. When the cell confluence reached 60-70% confluence, DMEM-F/12 fresh medium without BSA was added into cells. Using lipofectamine 3000 transfection reagent, 3 μg of PD-1v, IL-12, and GM-CSF recombinant plasmids were mixed with lipofectamine 3000 transfection reagent in equal volumes, the mixture was added slowly along the well wall, shaken gently, and incubated at 37°C with 5% CO 2 , and GFP recombinant plasmid was used as positive control in this experiment. After 48 h, the cell supernatant was collected and the expression of the target protein was detected by Western blot and ELISA.

| CT26-IRFP tumor model
Five BALB/c mice were inoculated subcutaneously with 2 × 10 5 CT26-IRFP cells (in 100 μL physiological saline) in each group on the right flank. When the tumors grew to an average diameter of 60-80 mm 3 , 80 μg of four different plasmids, including PD-1v, IL-2/15, IL-12, and GM-CSF, was injected intra-tumorally into each mouse in the single treatment group, 80 μg of the four plasmid mixtures was injected into the combination treatment group, 100 μg of GFP recombinant plasmid was injected into the empty vector control group, and 100 μL of 0.9% NaCl was injected into the blank control group. Gene delivery was conducted every 3 days for four times in all. Tumor diameter was measured by a Vernier caliper every other day from Day 6. Tumor volume was calculated according to the formula: [(larger axis) × (smaller axis) 2 ×0.5]. When the tumor volume reached 1500 mm 3 , tumor volume measurement was stopped and the mice were euthanized to avoid unnecessary suffering.

| In vivo animal imaging detection
The mice were anesthetized with isoflurane and then grouped into small animal live imaging system chambers to collect CT26-IRFP signal intensity. The CT26 fluorescence signal intensity was measured every 7 days and continued until the end of the experiment.

| Detection cytokine levels in blood
On Day 14, blood was collected from the orbital vein and serum was separated. Cytometric bead array (CBA) was used to detect the levels of various cytokines in the serum of mice. After the CBA reagent was placed at room temperature for 30 min, the trapping microspheres of IL-2, IL-4, IL-5, IFN-γ, and TNF were mixed. In brief, 50 μL of capture microspheres, 50 μL of serum to be tested, and 50 μL of PE-labeled fluorescent antibody were added to each FACS tube. Each tube was washed with 1 mL of washing solution, centrifuged at 200g for 5 min, and the supernatant was carefully aspirated. Three hundred microliters of washing solution was added to each tube to resuspend the cells, then analyzed on a NovoCyte Flow Cytometer by using NovoExpress software (Agilent). The obtained data were used to calculate the content of each cytokine in the samples by CBA software FCAPArrayv3.
T A B L E 1 Primers in plasmid construction.

| Construction of expression vector
A new plasmid expression system vector carrying CMV promoter, T7 RNA polymerase (T7RNAP), bovine growth hormone polyadenosine signal (BGHpA), internal ribosome entry site (IRES), T7 promoter, and Poly A was constructed using the T4 DNA ligase cloning method. It was named pT7AMPCE, and the construction process is shown in Figure 1. With pT7AMPCE as the backbone, the cDNA of PD-1v, IL-2/15, IL-12, GM-CSF, and GFP was inserted between IRES and poly A sequences. There is a BglII restriction site at both ends of the target gene insertion for ease of digestion identification. The five recombinant plasmids obtained were confirmed by DNA sequencing to be consistent with the sequence published by GenBank, indicating that the plasmid construction was correct.

| Transgene expression in vitro
The recombinant plasmids described above were transfected into CT26 cells, and the supernatant of CT26 cells was collected 48 h later to detect the expression of the target protein. In the in vitro transfection experiment, GFP recombinant plasmid was used as the positive control of transfection. Fluorescence microscopy showed that many cells expressed green fluorescent protein after 48 h transfection with GFP recombinant plasmid (Figure 2A). The expression of PD-1v was detected by Western blot, and the expressions of GM-CSF and IL-12 were detected by ELISA.
Western blot results showed a band around 30 kDa in the CT26 cell supernatant after 48 h transfection with PD-1v recombinant plasmid, which was consistent with the expected molecular weight of PD-1v ( Figure 2B). ELISA detection showed that the concentration of IL-12 reached 600 pg/mL in the supernatant of CT26 cells transfected with the IL-12 recombinant plasmid ( Figure 2C). The concentration of GM-CSF reached 700 pg/mL in the supernatant of CT26 cells transfected with the GM-CSF recombinant plasmid ( Figure 2D). The above results suggest that the new pT7AMPCE vector can effectively express the target protein.

| Local application of PD-1v and various cytokines in tumor tissue inhibited tumor growth
In order to observe the effect of PD-1v and the combined application of various cytokines, CT26-IRFP cells were subcutaneously inoculated into the right flank of mice to establish a transplanted tumor model. When the average tumor volume reached 60-80 mm 3 , plasmids PD1-v, IL-2/15, IL-12, and GM-CSF were inoculated into the tumor, four times every 3 days ( Figure 3A). The continuous observation for 21 days was performed. Compared with the single-plasmid treatment group, the vehicle control group (pT7AMPCE GFP), and the solvent control group, the growth trend of tumor in the combined treatment group was much more flat ( Figure 3B). Statistical analysis of the tumor volume on Day 21 showed that the tumor volume of the combined treatment group was significantly smaller than that of the PD-1v and GM-CSF single plasmid treatment groups ( Figure 3C), while the IL-12 plasmid groups did not show significant therapeutic effect compared with the vehicle control group ( Figure 3C). The survival rate of tumor-bearing mice for 69 consecutive days showed that all the mice in the solvent control group and the vehicle control group died in 29 days. PD-1v, IL-12, and IL-2/15 single plasmid treatment groups showed the mortality rate teached 100% at 39 days, 42 days, and 46 days, respectively. The mortality rate of 60 days after GM-CSF treatment was 100%, while the survival rate of 69 days in the combined treatment group was 20%, which was significantly different from the control group and the single particle control group (p = 0.0019, Figure 3D).

| Comparison of fluorescence signal intensity of tumor IRFP gene expression in vivo
Small animal biopsy allows for long and staged, noninvasive quantitative detection of tumors in mice, and most critically, small animal biopsy allows for more accurate and intuitive results. Because CT26-IRFP cells carry fluorescence signal, we used small animal imaging system to observe the changes in fluorescence signal intensity in different treatment groups to indicate the growth of CT26 cells in vivo. Compared with the single plasmid treatment group, vehicle control group, and buffer control group (Buffer Ctrl), the IRFP fluorescence signal intensity of CT26 tumor cells in the combined treatment group increased slowly ( Figure 4A, B). The analysis of IRFP fluorescence signal intensity of CT26 tumor cells in each group on Day 21 showed that the fluorescence signal intensity of the combined treatment group was lower than that of plasmid treatment ( Figure 4C). However, there was no statistically significant difference between IL-12 and the vector control group in relation to tumor IRFP gene fluorescence signal intensity ( Figure 4C). The above data indicate that the combined therapy has a better antitumor effect than the single-plasmid therapy.

| Combined application of multiple plasmids can effectively stimulate the secretion of Th1 and Th2 cytokines
On Day 14 of treatment, CBA flow cytometry was used to analyze the secretion levels of Th1 and Th2 cytokines in the peripheral blood of mice in each experimental group. The results showed that there was no significant difference in IL-2 secretion in the IL-12 treatment group compared with the empty vector control group and the buffer control group (Buffer Ctrl) ( Figure 5D). The ability to secrete cytokines IFN-γ, TNF, and IL-5 in the single treatment group was increased to a certain extent ( Figure 5A, B, E). Compared with the IL-12 treatment group, the IL-2 secretion level in the combination treatment group was lower than that in the IL-12 treatment group ( Figure 5D). Cytokines IFN-γ, TNF, IL-4, and IL-5 were upregulated in the combination treatment group compared with the control group and the plasmid treatment group ( Figure 5). Our data suggested that the combination of PD-1v and various cytokines can effectively activate immune cells.

| Combined application of multiple plasmids carrying therapeutic genes can effectively stimulate immune cell infiltration
We also used HE staining to observe the infiltration of immune cells in tumor tissues. The microscopic sections stained with HE showed that immune cell infiltration into the tumor tissue in the combined treatment group was significantly enhanced compared with the control group and the single-particle treatment groups. In the combined treatment group, there were dense immune cells in the tumor tissue, and some tumor cells showed nucleolus and tumor cell necrosis ( Figure 6). It is suggested that the combination of multiple plasmids can effectively attract immune cells into the tumor tissue to play an anti-tumor role. Quantitative data are exhibited as mean ± SD. Statistical data were analyzed by Student's t-test. Differences were considered significant at p < 0.05 (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
In order to further observe the types of infiltrating cells, we carried out immunohistochemical detection of CD3 (indicating T lymphocytes), CD11b (indicating NK cells), and CD11c (indicating monocytes and macrophages). Figure 7 shows that there are a large number of T lymphocytes, NK cells, monocytes, and macrophages in the tumor tissue of the combined treatment group. It is further suggested that the combination of PD-1v and various cytokines can effectively enhance the chemoattractant effect on a variety of F I G U R E 5 Detection of Th1 and Th2 cytokines in peripheral blood of mice by CBA flow cytometry. Data were analyzed with FlowJo VX software and FCAP Array v3.0 (n = 3 biological replicates per experiment; mean ± SD is depicted; unpaired, two-tailed t-test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001). Secretion level of (B) TNF, (C) IL-4, (D) IL-2, and (E) IL-5.

| DISCUSSION
With more and more in-depth research on tumor immune escape, tumor immunotherapy has received more attention and has become a treatment method that cannot be ignored in tumor treatment. At present, the most difficult problem of tumor treatment is incompleteness and recurrence, mainly due to tumor immune tolerance and escape, especially the low function of the immune system in the tumor microenvironment. PD-1 is a negative regulatory receptor on the surface of T cells, which mediates activated T-lymphocyte depletion by delivering inhibitory signals to activated T cells. Therefore, the therapeutic strategy of blocking the inhibitory signal pathway is particularly important. In previous studies, antibody was used to block the inhibitory signal transmitted by PD-1, which can enhance the positive immune response against tumors. The anti-tumor effects achieved in animal experiments and clinical trials have proved this. 1,30 Unfortunately, the effect of intravenous injection is often systemic, facing serious side effects, and easy to induce autoimmune reaction when treating tumors. 27,38 Therefore, this paper studies the role of soluble PD-1 as a local gene therapy agent. PD-1v recombinant plasmid is used for local injection of tumor tissue, and the expression area is relatively limited, mainly by playing a role in the local role in tumor tissue.
In the process of tumor treatment, it is difficult to achieve good therapeutic effect with single gene therapy. The main reason is that the immune escape mechanism of tumor is multifaceted, especially in the process of treatment, the phenotype of tumor cells in the tumor microenvironment changes, and some escape mechanisms have evolved. Local gene therapy for tumor immune escape mechanism provides a new way to solve this problem. In particular, combined gene therapy can inhibit tumor growth by targeting different tumor escape mechanisms from several aspects. Studies have found that in addition to T lymphocytes, other innate immune cells, including natural killer (NK) cells, NKT cells, macrophages, dendritic cells, mast cells, and neutrophils, also play a key role in controlling tumor growth. 5 The results of some recent clinical studies combining cytokine gene therapy with different cancer treatments suggest that strategies expressing immunomodulatory genes in solid tumors have some clinical potential. In this study, we used pT7AMPCE as a vector to construct PD-1v, GM-CSF, IL-12, and IL-2/IL-15 expression plasmids. In vitro transfection experiments showed that the recombinant plasmid could efficiently express the target gene. After 48 h of transfection, WB detection showed that the PD-1v expression product formed a soluble molecule and was secreted outside the cell. The concentration of IL-12 in the cell supernatant reached 600 pg/mL, and the concentration of GM-CSF reached 700 pg/mL. Animal experiments showed that the local application of PD-1v, IL-2/15, IL-12, and GM-CSF can synergistically produce a stronger anti-tumor immunotherapeutic effect. Our studies further confirmed that the combined application can effectively stimulate the expression of IFN-γ, TNF, IL-4, IL-2, IL-5, and other cytokines, suggesting that multiple gene combination therapy can effectively enhance immune response, reverse immune tolerance, and inhibit tumor growth. Although the data of receiving combination therapy are encouraging, the limitations of recombinant plasmids in patients' primary tumor cells are still not negligible. Of note, this study is limited to single cell lines, and additional cell lines and comparative studies with viral vectors are needed.

DATA AVAILABILITY STATEMENT
The data that support the findings of this study are available the article.