title: Encapsulation of two different TLR ligands into liposomes confer protective immunity and prevent tumor development.
abstract: Nucleic acid-based Toll-like receptor (TLR) ligands are promising adjuvants and immunotherapeutic agents. Combination of TLR ligands potentiates immune response by providing synergistic immune activity via triggering different signaling pathways and may impact antigen dependent T-cell immune memory. However, their short circulation time due to nuclease attack hampers their clinical performance. Liposomes offer inclusion of protein and nucleic acid-based drugs with high encapsulation efficiency and drug loading. Furthermore, they protect cargo from enzymatic cleavage while providing stability, and enhancing biological activity. Herein, we aimed to develop a liposomal carrier system co-encapsulating TLR3 (polyinosinic-polycytidylic acid; poly(I:C)) and TLR9 (oligodeoxynucleotides (ODN) expressing unmethylated CpG motifs; CpG ODN) ligands as immunoadjuvants together with protein antigen. To demonstrate that this depot system not only induce synergistic innate immune activation but also boost antigen-dependent immune response, we analyzed the potency of dual ligand encapsulated liposomes in long-term cancer protection assay. Data revealed that CpG ODN and poly(I:C) co-encapsulation significantly enhanced cytokine production from spleen cells. Activation and maturation of dendritic cells as well as bactericidal potency of macrophages along with internalization capacity of ligands were elevated upon incubation with liposomes co-encapsulating CpG ODN and poly(I:C). Immunization with co-encapsulated liposomes induced OVA-specific Th1-biased immunity which persisted for eight months post-booster injection. Subsequent challenge with OVA-expressing tumor cell line, E.G7, demonstrated that mice immunized with liposomes co-encapsulating dual ligands had significantly slower tumor progression. Tumor clearance was dependent on OVA-specific cytotoxic memory T-cells. These results suggest that liposomes co-encapsulating TLR3 and TLR9 ligands and a specific cancer antigen could be developed as a preventive cancer vaccine.
authors.affiliation: Thorlab. Therapeutic ODN Research Lab, Department of Molecular Biology and Genetics, Bilkent University, Bilkent, 06800, Ankara, Turkey.
authors.forename: Banu
authors.initials: B
authors.lastname: Bayyurt
authors.affiliation: Thorlab. Therapeutic ODN Research Lab, Department of Molecular Biology and Genetics, Bilkent University, Bilkent, 06800, Ankara, Turkey.
authors.forename: Gizem
authors.initials: G
authors.lastname: Tincer
authors.affiliation: Thorlab. Therapeutic ODN Research Lab, Department of Molecular Biology and Genetics, Bilkent University, Bilkent, 06800, Ankara, Turkey.
authors.forename: Kubra
authors.initials: K
authors.lastname: Almacioglu
authors.affiliation: Department of Biological Sciences, Middle East Technical University, 06800, Ankara, Turkey.
authors.forename: Esin
authors.initials: E
authors.lastname: Alpdundar
authors.affiliation: Department of Biological Sciences, Middle East Technical University, 06800, Ankara, Turkey.
authors.forename: Mayda
authors.initials: M
authors.lastname: Gursel
authors.affiliation: Thorlab. Therapeutic ODN Research Lab, Department of Molecular Biology and Genetics, Bilkent University, Bilkent, 06800, Ankara, Turkey. Electronic address: ihsangursel@bilkent.edu.tr.
authors.forename: Ihsan
authors.initials: I
authors.lastname: Gursel
mesh_terms: D000276:Adjuvants, Immunologic / Q000008:administration & dosage* / Q000494:pharmacology / Q000627:therapeutic use; D000818:Animals; D019496:Cancer Vaccines / Q000008:administration & dosage* / Q000494:pharmacology / Q000627:therapeutic use; D005260:Female; D007109:Immunity / Q000187:drug effects; D007114:Immunization; D007372:Interferons / Q000276:immunology; D015850:Interleukin-6 / Q000276:immunology; D008081:Liposomes / Q000737:chemistry*; D051379:Mice; D008810:Mice, Inbred C57BL; D009369:Neoplasms / Q000276:immunology / Q000517:prevention & control*; D009838:Oligodeoxyribonucleotides / Q000008:administration & dosage* / Q000494:pharmacology / Q000627:therapeutic use; D011070:Poly I-C / Q000008:administration & dosage* / Q000494:pharmacology / Q000627:therapeutic use; D000067996:RAW 264.7 Cells; D051196:Toll-Like Receptor 3 / Q000276:immunology; D051217:Toll-Like Receptor 9 / Q000276:immunology
chemical_list: D000276:Adjuvants, Immunologic; C408982:CPG-oligonucleotide; D019496:Cancer Vaccines; D015850:Interleukin-6; D008081:Liposomes; D009838:Oligodeoxyribonucleotides; D051196:Toll-Like Receptor 3; D051217:Toll-Like Receptor 9; D007372:Interferons; D011070:Poly I-C
keywords: Immune response; Immunoadjuvant; Ligands; Liposomes; Preventive cancer vaccine; TLR3; TLR9