Immunostimulatory DNA Tetrahedron-Based Nanovaccine Combined With Immune Checkpoint PD-1 Blockade for Boosting Systemic Immune Responses Against Oral Squamous Cell Carcinoma

Abstract

Despite advancements in immune checkpoint blockade (ICB) therapies for treating various tumors, the immunosuppressive environment in oral squamous cell carcinoma (OSCC) significantly limits therapeutic efficacy. Tumor vaccines, which offer great potential for cancer immunotherapy, still face challenges like potential mutation risks, rapid elimination, and low in vivo delivery efficiency. In this study, we fabricate an immunostimulatory nanovaccine using tetrahedral framework nucleic acids (tFNAs) as a carrier for stable and efficient delivery of CpG oligonucleotide. Then an intensive tumor immunotherapeutic strategy by combining tFNA-CpG nanovaccine with PD-1 inhibitor is used in OSCC tumor-bearing mice. Intravenous administration of the tFNA-CpG nanovaccine effectively activates the antigen-presenting cells (APCs), resulting in an increased proportion of M1-like macrophages and mature dendritic cells, accompanied by heightened production of inflammatory cytokines IL-1β, IL-12, and IL-6. When combined with ICB therapy, the anti-PD-1 drug inhibits the PD-1/PD-L1 interaction within tumor microenvironment. Subsequently, the APCs activated by tFNA-CpG facilitate the phenotypic differentiation of T cells, resulting in a substantial boost in infiltration of cytotoxic T cells (expressing IFN-γ and Granzyme B) in both lymph nodes and tumor tissues, thereby executing a potent antitumor effect and inhibiting the progression of OSCC tumors in C3H mouse. Therefore, this study presents an attractive approach to overcoming current ICB limitations in OSCC immunotherapy and provides new avenues for future clinical practice.

Keywords

immune checkpoint blockade / nanovaccines / oral squamous cell carcinoma / tetrahedral framework nucleic acids / tumor immunotherapy

References

[1] R. L. Siegel, A. N. Giaquinto, and A. Jemal, “Cancer Statistics, 2024,” CA: A Cancer Journal for Clinicians 74 (2024): 12-49.

[2] S. Miyauchi, S. S. Kim, J. Pang, et al., “Immune Modulation of Head and Neck Squamous Cell Carcinoma and the Tumor Microenvironment by Conventional Therapeutics,” Clinical Cancer Research 25 (2019): 4211-4223.

[3] X. X. Hao, C. Y. Li, Y. J. Zhang, et al., “Programmable Chemotherapy and Immunotherapy Against Breast Cancer Guided by Multiplexed Fluorescence Imaging in the Second Near-Infrared Window,” Advanced Materials 30 (2018): 1804437.

[4] B. Sangro, P. Sarobe, S. Hervás-Stubbs, and I. Melero, “Advances in Immunotherapy for Hepatocellular Carcinoma,” Nature reviews Gastroenterology & Hepatology 18 (2021): 525-543.

[5] A. T. Ruffin, H. Li, L. Vujanovic, D. P. Zandberg, R. L. Ferris, and T. C. Bruno, “Improving Head and Neck Cancer Therapies by Immunomodulation of the Tumour Microenvironment,” Nature Reviews Cancer 23 (2023): 173-188.

[6] L. Feng, K. Yin, S. Zhang, Z. Chen, Y. Bao, and T. Li, “Anti-PD-1 Therapy Is Beneficial for the Survival of Patients With Oral Squamous Cell Carcinoma,” Cancer Management and Research 14 (2022): 2723-2731.

[7] J. D. Cramer, B. Burtness, and R. L. Ferris, “Immunotherapy for Head and Neck Cancer: Recent Advances and Future Directions,” Oral Oncology 99 (2019): 104460.

[8] A. von Witzleben, C. Wang, S. Laban, N. Savelyeva, and C. H. Ottensmeier, “HNSCC: Tumour Antigens and Their Targeting by Immunotherapy,” Cells 9 (2020): 2103.

[9] D. G. Wang, T. T. Wang, H. J. Yu, et al., “Engineering Nanoparticles to Locally Activate T Cells in the Tumor Microenvironment,” Science Immunology 4 (2019): eaau6584.

[10] A. Brandenburg, A. Heine, and P. Brossart, “Next-Generation Cancer Vaccines and Emerging Immunotherapy Combinations,” Trends in Cancer 10 (2024): 749-769.

[11] S. Pagliari, B. Dema, A. Sanchez-Martinez, G. Montalvo Zurbia-Flores, and C. S. Rollier, “DNA Vaccines: History, Molecular Mechanisms and Future Perspectives,” Journal of Molecular Biology 435 (2023): 168297.

[12] Y. Weng, C. Li, T. Yang, et al., “The Challenge and Prospect of mRNA Therapeutics Landscape,” Biotechnology Advances 40 (2020): 107534.

[13] X. B. Xi, T. Ye, S. Wang, et al., “Self-Healing Microcapsules Synergetically Modulate Immunization Microenvironments for Potent Cancer Vaccination,” Science Advances 6 (2020): eaay7735.

[14] T. Liu, W. Yao, W. Sun, et al., “Components, Formulations, Deliveries, and Combinations of Tumor Vaccines,” ACS Nano 18 (2024): 18801-18833.

[15] Y. Yang, J. Yang, J. Zhu, et al., “A DNA Tetrahedron-Based Nanosuit for Efficient Delivery of Amifostine and Multi-Organ Radioprotection,” Bioactive Materials 39 (2024): 191-205.

[16] T. Tian, T. Zhang, S. Shi, Y. Gao, X. Cai, and Y. Lin, “A Dynamic DNA Tetrahedron Framework for Active Targeting,” Nature Protocols 18 (2023): 1028-1055.

[17] S. Li, Y. Liu, T. Zhang, et al., “A Tetrahedral Framework DNA-Based Bioswitchable miRNA Inhibitor Delivery System: Application to Skin Anti-Aging,” Advanced Materials 34 (2022): e2204287.

[18] X. Qin, L. Xiao, N. Li, et al., “Tetrahedral Framework Nucleic Acids-Based Delivery of microRNA-155 Inhibits Choroidal Neovascularization by Regulating the Polarization of Macrophages,” Bioactive Materials 14 (2021): 134-144.

[19] C. Qi, Q. Sun, D. Xiao, et al., “Tetrahedral Framework Nucleic Acids/Hyaluronic Acid-Methacrylic Anhydride Hybrid Hydrogel With Antimicrobial and Anti-Inflammatory Properties for Infected Wound Healing,” International Journal of Oral Science 16 (2024): 30.

[20] T. Zhang, T. Tian, and Y. Lin, “Functionalizing Framework Nucleic-Acid-Based Nanostructures for Biomedical Application,” Advanced Materials 34 (2022): e2107820.

[21] A. Lacroix and H. F. Sleiman, “DNA Nanostructures: Current Challenges and Opportunities for Cellular Delivery,” ACS Nano 15 (2021): 3631-3645.

[22] J. Chen, H. Fang, Y. Hu, et al., “Combining Mannose Receptor Mediated Nanovaccines and Gene Regulated PD-L1 Blockade for Boosting Cancer Immunotherapy,” Bioactive Materials 7 (2021): 167-180.

[23] M. Li, H. Yao, K. Yi, Y. H. Lao, D. Shao, and Y. Tao, “Emerging Nanoparticle Platforms for CpG Oligonucleotide Delivery,” Biomaterials Science 12 (2024): 2203-2228.

[24] Q. Fan, Z. Li, J. Yin, et al., “Inhalable pH-Responsive DNA Tetrahedron Nanoplatform for Boosting Anti-Tumor Immune Responses Against Metastatic Lung Cancer,” Biomaterials 301 (2023): 122283.

[25] M. Liu, L. Hao, D. Zhao, J. Li, and Y. Lin, “Self-Assembled Immunostimulatory Tetrahedral Framework Nucleic Acid Vehicles for Tumor Chemo-Immunotherapy,” ACS Applied Materials & Interfaces 14 (2022): 38506-38514.

[26] L. Liang, J. Li, Q. Li, et al., “Single-Particle Tracking and Modulation of Cell Entry Pathways of a Tetrahedral DNA Nanostructure in Live Cells,” Angewandte Chemie International Edition 53 (2014): 7745-7750.

[27] D. Wang, Y. Wang, S. Zhang, et al., “Tetrahedral-DNA-Nanostructure-Modified Engineered Extracellular Vesicles Enhance Oral Squamous Cell Carcinomas Therapy by Targeting GPX4,” ACS Nano 19 (2025): 9351-9366.

[28] X. Qin, B. Zhang, X. Sun, et al., “Tetrahedral-Framework Nucleic Acid Loaded With MicroRNA-155 Enhances Immunocompetence in Cyclophosphamide-Induced Immunosuppressed Mice by Modulating Dendritic Cells and Macrophages,” ACS Applied Materials & Interfaces 15 (2023): 7793-7803.

[29] J. Xu, H. Wang, L. G. Xu, et al., “Nanovaccine Based on a Protein-Delivering Dendrimer for Effective Antigen Cross-Presentation and Cancer Immunotherapy,” Biomaterials 207 (2019): 1-9.

[30] C. E. Castro, F. Kilchherr, D. N. Kim, et al., “A Primer to Scaffolded DNA Origami,” Nature Methods 8 (2011): 221-229.

[31] D. G. DeNardo and B. Ruffell, “Macrophages as Regulators of Tumour Immunity and Immunotherapy,” Nature Reviews Immunology 19 (2019): 369-382.

[32] A. Mantovani, F. Marchesi, A. Malesci, L. Laghi, and P. Allavena, “Tumour-Associated Macrophages as Treatment Targets in Oncology,” Nature Reviews Clinical Oncology 14 (2017): 399-416.

[33] Y. Guo, S. Z. Wang, X. Zhang, et al., “In Situ Generation of Micrometer-Sized Tumor Cell-Derived Vesicles as Autologous Cancer Vaccines for Boosting Systemic Immune Responses,” Nature Communications 13 (2022): 6534.

[34] L. Kong, W. Smith, and D. Hao, “Overview of RAW264.7 for Osteoclastogenesis Study: Phenotype and Stimuli,” Journal of Cellular and Molecular Medicine 23 (2019): 3077-3087.

[35] I. Santos-Zas, J. Lemarié, I. Zlatanova, et al., “Cytotoxic CD8+ T Cells Promote Granzyme B-Dependent Adverse Post-Ischemic Cardiac Remodeling,” Nature Communications 12 (2021): 1483.

[36] D. E. Sanin and E. J. Pearce, “The Cell Identity of Cytotoxic T Lymphocytes,” Nature Immunology 17 (2016): 45-46.

[37] R. Yerushalmi, R. Woods, P. M. Ravdin, M. M. Hayes, and K. A. Gelmon, “Ki67 in Breast Cancer: Prognostic and Predictive Potential,” Lancet Oncology 11 (2010): 174-183.

[38] K. Augoff, A. Hryniewicz-Jankowska, R. Tabola, and K. Stach, “MMP9: A Tough Target for Targeted Therapy for Cancer,” Cancers 14 (2022): 1847.

[39] H. du Bois, T. A. Heim, and A. W. Lund, “Tumor-Draining Lymph Nodes: At the Crossroads of Metastasis and Immunity,” Science Immunology 6 (2021): eabg3551.

[40] P. A. ehnlich, R. M. Powell, M. J. W. Peeters, A. Rahbech, and P. T. Straten, “TAM Receptor Inhibition-Implications for Cancer and the Immune System,” Cancers 13 (2021): 1195.

[41] Y. You, Z. Tian, Z. Du, et al., “M1-Like Tumor-Associated Macrophages Cascade a Mesenchymal/Stem-Like Phenotype of Oral Squamous Cell Carcinoma via the IL6/Stat3/THBS1 Feedback Loop,” Journal of Experimental & Clinical Cancer Research 41 (2022): 10.

[42] X. Qu, X. Zhao, K. Lin, et al., “M2-Like Tumor-Associated Macrophage-Related Biomarkers to Construct a Novel Prognostic Signature, Reveal the Immune Landscape, and Screen Drugs in Hepatocellular Carcinoma,” Frontiers in Immunology 13 (2022): 994019.

[43] S. Singh, N. Singh, M. Baranwal, S. Sharma, S. S. K. Devi, and S. Kuma, “Understanding Immune Checkpoints and PD-1/PD-L1-Mediated Immune Resistance Towards Tumour Immunotherapy,” 3 Biotech 13 (2023): 411.

[44] T. S. K. Mok, Y. L. Wu, I. Kudaba, et al., “Pembrolizumab Versus Chemotherapy for Previously Untreated, PD-L1-Expressing, Locally Advanced or Metastatic Non-Small-Cell Lung Cancer (KEYNOTE-042): A Randomised, Open-Label, Controlled, Phase 3 Trial,” Lancet 393 (2019): 1819-1830.

[45] S. S. K. Devi, S. Singh, R. Joga, et al., “Enhancing Cancer Immunotherapy: Exploring Strategies to Target the PD-1/PD-L1 Axis and Analyzing the Associated Patent, Regulatory, and Clinical Trial Landscape,” European Journal of Pharmaceutics and Biopharmaceutics 200 (2024): 114323.

[46] R. S. Herbst, G. Giaccone, F. de Marinis, et al., “Atezolizumab for First-Line Treatment of PD-L1-Selected Patients With NSCLC,” New England Journal of Medicine 383 (2020): 1328-1339.

[47] L. A. Diaz, K. K. Shiu, T. W. Kim, et al., “Pembrolizumab Versus Chemotherapy for Microsatellite Instability-high or Mismatch Repair-Deficient Metastatic Colorectal Cancer (KEYNOTE-177): Final Analysis of a Randomised, Open-Label, Phase 3 Study,” Lancet Oncology 23 (2022): 659-670.

[48] A. Bhatia and B. Burtness, “Treating Head and Neck Cancer in the Age of Immunotherapy: A 2023 Update,” Drugs 83 (2023): 217-248.

[49] Y. Qian, H. L. Jin, S. Qiao, et al., “Targeting Dendritic Cells in Lymph Node With an Antigen Peptide-Based Nanovaccine for Cancer Immunotherapy,” Biomaterials 98 (2016): 171-183.

[50] X. Yu, Y. Dai, Y. Zhao, et al., “Melittin-Lipid Nanoparticles Target to Lymph Nodes and Elicit a Systemic Anti-Tumor Immune Response,” Nature Communications 11 (2020): 1110.

[51] E. A. Scott, A. Stano, M. Gillard, A. C. M. Liu, M. A. Swartz, and J. A. Hubbell, “Dendritic Cell Activation and T Cell Priming With Adjuvant- and Antigen-Loaded Oxidation-Sensitive Polymersomes,” Biomaterials 33 (2012): 6211-6219.

[52] Q. Liu, X. Chen, J. Jia, et al., “pH-Responsive Poly(d, l-Lactic-Co-Glycolic Acid) Nanoparticles With Rapid Antigen Release Behavior Promote Immune Response,” ACS Nano 9 (2015): 4925-4938.

[53] Y. Hu, L. Lin, J. Chen, A. Maruyama, H. Tian, and X. Chen, “Synergistic Tumor Immunological Strategy by Combining Tumor Nanovaccine With Gene-Mediated Extracellular Matrix Scavenger,” Biomaterials 252 (2020): 120114.

[54] X. Guan, J. Chen, Y. Hu, et al., “Highly Enhanced Cancer Immunotherapy by Combining Nanovaccine With Hyaluronidase,” Biomaterials 171 (2018): 198-206.

[55] C. B. Fox and J. Haensler, “An Update on Safety and Immunogenicity of Vaccines Containing Emulsion-Based Adjuvants,” Expert Review of Vaccines 12 (2013): 747-758.

[56] L. Zhang, S. Wu, Y. Qin, et al., “Targeted Codelivery of an Antigen and Dual Agonists by Hybrid Nanoparticles for Enhanced Cancer Immunotherapy,” Nano Letters 19 (2019): 4237-4249.

[57] N. Kayraklioglu, B. Horuluoglu, and D. M. Klinman, “CpG Oligonucleotides as Vaccine Adjuvants,” Methods in Molecular Biology 2197 (2021): 51-85.

[58] W. Chen, M. Jiang, W. Yu, et al., “CpG-Based Nanovaccines for Cancer Immunotherapy,” International Journal of Nanomedicine 16 (2021): 5281-5299.

[59] S. Jhunjhunwala, C. Hammer, and L. Delamarre, “Antigen Presentation in Cancer: Insights Into Tumour Immunogenicity and Immune Evasion,” Nature Reviews Cancer 21 (2021): 298-312.

[60] D. Ghosh, W. Jiang, D. Mukhopadhyay, and E. D. Mellins, “New Insights Into B Cells as Antigen Presenting Cells,” Current Opinion in Immunology 70 (2021): 129-137.

[61] L. B. Darragh and S. D. Karam, “Amateur Antigen-Presenting Cells in the Tumor Microenvironment,” Molecular Carcinogenesis 61 (2022): 153-164.

[62] A. V. Joglekar and G. Li, “T Cell Antigen Discovery,” Nature Methods 18 (2021): 873-880.

[63] D. A. Vignali and V. K. Kuchroo, “IL-12 Family Cytokines: Immunological Playmakers,” Nature Immunology 13 (2012): 722-728.

[64] P. P. Yang, Q. Luo, G. B. Qi, et al., “Host Materials Transformable in Tumor Microenvironment for Homing Theranostics,” Advanced Materials 29 (2017): 1605869.

[65] X. Zhang, C. Wang, J. Wang, et al., “PD-1 Blockade Cellular Vesicles for Cancer Immunotherapy,” Advanced Materials 30 (2018): e1707112.

[66] R. N. Germain, “T-Cell Development and the CD4-CD8 Lineage Decision,” Nature Reviews Immunology 2 (2002): 309-322.

[67] T. Parrot, R. Oger, M. Allard, et al., “Transcriptomic Features of Tumour-Infiltrating CD4lowCD8high Double Positive Αβ T Cells in Melanoma,” Scientific Reports 10 (2020): 5900.

[68] J. Desfrançois, A. Moreau-Aubry, V. Vignard, et al., “Double Positive CD4CD8 Αβ T Cells: A New Tumor-Reactive Population in Human Melanomas,” PLoS ONE 5 (2010): e8437.

[69] D. B. Sant'Angelo, B. Lucas, P. G. Waterbury, et al., “A Molecular Map of T Cell Development,” Immunity 9 (1998): 179-186.

[70] A. van Renterghem, J. van de Haar, and E. E. Voest, “Functional Precision Oncology Using Patient-Derived Assays: Bridging Genotype and Phenotype,” Nature Reviews Clinical Oncology 20 (2023): 305-317.

[71] F. Baharom, R. A. Ramirez-Valdez, K. K. S. Tobin, et al., “Intravenous Nanoparticle Vaccination Generates Stem-Like TCF1+ Neoantigen-Specific CD8+ T Cells,” Nature Immunology 22 (2021): 41-52.

[72] M. B. Lutz, N. Kukutsch, A. L. Ogilvie, et al., “An Advanced Culture Method for Generating Large Quantities of Highly Pure Dendritic Cells From Mouse Bone Marrow,” Journal of Immunological Methods 223 (1999): 77-92.

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