Immune-check blocking combination multiple cytokines shown curative potential in mice tumor model
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Updated: Jul 30, 2025
Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors
Published on: December 16, 2022
Hongxia Su1, Hui Geng2, Linkang Cai3
1School of Pharmacy, Hubei University of Science and Technologyc, Xianning, China.
Objective:
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.
Methods:
A novel eukaryotic expression plasmid vector named pT7AMPCE containing T7RNA polymerase, T7 promoter, internal ribosome entry site (IRES), and poly A tailing signal was constructed by T4 DNA ligase, on which homologous recombination was used to clone and construct the vector carrying PD-1v, IL-2/15, IL-12, GM-CSF, and GFP. In vitro transfection of CT26 cells was performed, and the protein expression of PD-1v, IL-12 and GM-CSF was detected by Western blot and ELISA after 48 h. Mice were subcutaneously inoculated with CT26-IRFP tumor cells in the rib abdomen, and the tumor tissues were injected with PD-1v, IL-2/15, IL-12, and GM-CSF recombinant plasmids for treatment during the experimental period. The efficacy of the treatment was evaluated by assay tumor size and survival time of tumor-bearing mice during the experiment. Expression levels of IFN-γ, TNF, IL-4, IL-2, and IL-5 in mouse blood were measured using the CBA method. Tumor tissues were extracted and immune cell infiltration in tumor tissues was detected by HE staining and the IHC method.
Results:
The recombinant plasmids carrying PD-1v, IL-2/15, IL-12, and GM-CSF were successfully constructed, and the Western blot and ELISA results showed that PD-1v, IL-12, and GM-CSF were expressed in the supernatant of CT26 cells 48 h after in vitro cell transfection. The combined application of PD-1v, IL-2/15, 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.
Conclusion:
The combination of immune check blockade and multiple cytokine therapy can significantly activate the body's immune response and inhibit tumor growth.
Insights
This study developed a novel gene therapy vector combining immune checkpoint blockade (PD-1v) with multiple cytokines to activate immune responses and inhibit tumor growth. The combined therapy significantly reduced tumor size and improved survival in mice.
Area of Science:
- Immunology
- Molecular Biology
- Cancer Therapy
Background:
- Developing effective cancer therapies requires strategies to enhance the host immune response against tumors.
- Immune checkpoint inhibitors and cytokine therapies are promising but often used separately.
Purpose of the Study:
- To construct a eukaryotic expression vector encoding PD-1v and multiple cytokines (IL-2/15, IL-12, GM-CSF) for combined immunotherapy.
- To evaluate the efficacy of this combined therapy in activating immune responses and inhibiting tumor growth in a mouse model.
Main Methods:
- Construction of a novel eukaryotic expression plasmid (pT7AMPCE) carrying genes for PD-1v, IL-2/15, IL-12, and GM-CSF.
- In vitro transfection of CT26 cells and assessment of protein expression (PD-1v, IL-12, GM-CSF) via Western blot and ELISA.
- In vivo studies involving subcutaneous inoculation of CT26 tumor cells in mice, followed by intratumoral injection of recombinant plasmids and evaluation of tumor size, survival, immune cell infiltration, and cytokine levels.
Main Results:
- Successful construction and expression of recombinant plasmids encoding PD-1v, IL-12, and GM-CSF.
- Significant inhibition of tumor growth and improved survival rates in mice treated with the combined PD-1v and cytokine therapy compared to control groups.
- Demonstrated activation of immune cells and increased immune cell infiltration (IFN-γ, TNF, IL-4, IL-2, IL-5) within tumor tissues, alongside tumor cell necrosis.
Conclusions:
- The combination of immune checkpoint blockade (PD-1v) and multiple cytokine therapy effectively activates the immune system.
- This novel therapeutic strategy demonstrates significant potential for inhibiting tumor growth and warrants further investigation for cancer treatment.
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