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Nature Communications2021ResearchNon-viral Gene Delivery

PLGA-particle vaccine carrying TLR3/RIG-I ligand Riboxxim synergizes with immune checkpoint blockade for effective anti-cancer immunotherapy

Julia Koerner, Dennis Horvath, Valerie L. Herrmann, Anna Mackeracher, Bruno Gander, Hideo Yagita, Jacques Rohayem, Marcus GroettrupDOI 10.1038/s41467-021-23244-3

Summary

Cancer immunotherapy needs potent, pharmaceutically defined, GMP-compatible adjuvants for clinical translation. Poly(I:C) is a widely used TLR3 agonist but has ill-defined structure, heterogeneity, pyrogen contamination, and toxicity concerns. Riboxxim is a well-defined 100-bp double-stranded RNA with a 5′-triphosphate moiety that activates both endosomal TLR3 and cytosolic RIG-I. PLGA particles can co-deliver antigen and adjuvant to dendritic. Particle properties: MPs ~1–1.5 µm; NPs ~250 nm; negative zeta potential; OVA release burst within 24 h followed by sustained release. MPs showed better release profile than NPs. - Route comparison: Subcutaneous.

Keywords

PLGACancer immunotherapyT cellsMacrophagesNanoparticlesAntigen presentationPolymeric
Purpose: Cancer immunotherapy needs potent, pharmaceutically defined, GMP-compatible adjuvants for clinical translation. Poly(I:C) is a widely used TLR3 agonist but has ill-defined structure, heterogeneity, pyrogen contamination, and toxicity concerns. Riboxxim is a well-defined 100-bp double-stranded RNA with a 5′-triphosphate moiety that activates both endosomal TLR3 and cytosolic RIG-I. PLGA particles can co-deliver antigen and adjuvant to dendritic cells (DCs), potentially improving cross-presentation and CD8⁺ T cell priming.
Hypothesis: If Riboxxim is co-encapsulated with antigen in PLGA particles, then it will potently activate murine and human DCs via TLR3 and RIG-I/MAVS, induce type I interferons and pro-inflammatory cytokines, generate superior tumor-specific CD8⁺ T cell responses compared with classical dsRNA analogues such as poly(I:C), and synergize with immune checkpoint blockade to enhance antitumor efficacy, including tumor ablation and long-term memory.
Aims: Primary aim: Evaluate Riboxxim-containing PLGA particles as an anticancer vaccine platform.
  • Secondary aim 1: Compare PLGA nanoparticles (NP) and microparticles (MP), and different administration routes, for induction of tumor-specific CD8⁺ T cells.
  • Secondary aim 2: Characterize particle size, release, uptake by DCs/macrophages, DC maturation, cytokine secretion, and signaling pathways (TLR3/TICAM-1 and RIG-I/MAVS).
  • Secondary aim 3: Assess CD8⁺ T cell priming, proliferation, cytotoxicity, memory, and responses to human tumor peptide antigens.
  • Secondary aim 4: Evaluate antitumor efficacy alone and in combination with anti-CTLA-4 or anti-PD-1 in E.G7-OVA and MO5 melanoma models.
Delivery system:

Component: Polymer; Description: PLGA (Resomer RG502H)

Component: Particle Type; Description: Microparticles (MP) by spray-drying; nanoparticles (NP) by double-emulsion solvent evaporation

Component: Targeting Ligand; Description: None

Component: Payloads; Description: Ovalbumin (OVA) protein or peptides; Riboxxim (TLR3/RIG-I ligand); poly(I:C) or pyrogen-free poly(I:C) as comparators; QuantumDot705 for imaging

Component: Riboxxim; Description: Defined 100-bp dsRNA with uncapped 5′-triphosphate; TLR3 and RIG-I agonist

Component: Size; Description: MP: ~1–1.5 µm; NP: ~250 nm

Component: Zeta Potential; Description: Negative (−30 to −56 mV depending on formulation)

Component: Encapsulation/Release; Description: OVA release showed burst within 24 h followed by sustained release over days; MPs superior to NPs in release profile

Component: Key Design Feature; Description: Co-encapsulation of antigen and Riboxxim in the same PLGA particle for simultaneous delivery to DCs and dual TLR3/RIG-I activation

Approach: In Vitro: - Murine bone marrow-derived DCs (BMDCs), peritoneal macrophages. - Human CD14⁺ monocyte-derived DCs/macrophages, THP-1-derived DC-like cells, primary CD1c⁺ and CD141⁺ myeloid DCs from healthy donors. - Readouts: DC maturation markers, cytokine secretion, antigen presentation (B3Z, DOBW hybridomas), T cell proliferation (CFSE), cytotoxicity.

In Vivo: - Mice: C57BL/6J, BALB/c, OT-1 transgenic, AAD (HLA-A*0201) transgenic, Mavs⁻/⁻, Tlr3⁻/⁻. - Vaccination routes: subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular (i.m.), intranodal (i.nd.), intranasal (i.n.). - Typical dose: 5 mg PLGA particles containing 250 µg OVA and 2.5 µg dsRNA adjuvant per mouse (s.c.). - Tumor models: E.G7-OVA-luc thymoma (protective and therapeutic), MO5 melanoma lung metastasis, B16BL6-OVA. - Combination therapy: anti-CTLA-4 (cumulative 250 µg/mouse) or anti-PD-1 (total 1500 µg/mouse). - CD8⁺ T cell depletion with anti-CD8β mAb.

Key methods:

Technique: SEM, DLS, zeta potential; Purpose: Particle size, morphology, surface charge

Technique: SDS-PAGE/silver stain, MicroBCA; Purpose: Antigen release/encapsulation

Technique: Flow cytometry; Purpose: DC uptake, maturation markers, T cell phenotyping, IFNγ intracellular staining

Technique: ELISA; Purpose: Cytokine quantification (IFNα/β, IL-6, TNF, IL-12, IL-10, etc.)

Technique: ELISPOT; Purpose: Antigen-specific IFNγ-producing T cells

Technique: In vivo cytotoxicity assay; Purpose: Peptide-specific killing of target cells

Technique: CFSE dilution; Purpose: T cell proliferation

Technique: B3Z/DOBW hybridoma assays; Purpose: MHC class I/II antigen presentation

Technique: IVIS bioluminescence/NIR imaging; Purpose: Particle distribution, tumor burden

Technique: Histology/IHC; Purpose: CD8⁺ T cell infiltration, organ safety

Technique: Tumor volume/survival; Purpose: Therapeutic efficacy

Key results: Particle properties: MPs ~1–1.5 µm; NPs ~250 nm; negative zeta potential; OVA release burst within 24 h followed by sustained release. MPs showed better release profile than NPs. - Route comparison: Subcutaneous immunization gave the strongest CD8⁺ T cell responses, though i.p., i.m., and i.nd. were also immunogenic. - DC activation: Riboxxim in PLGA particles upregulated CD80, CD86, and MHC-I on murine and human DCs, superior to poly(I:C). It induced type I IFN (IFNα/β) and IL-6/TNF, dependent on both TLR3/TICAM-1 and MAVS/RIG-I signaling. - T cell priming: MP-OVA/Riboxxim elicited significantly more IFNγ⁺ antigen-specific CD8⁺ T cells than MP-OVA/poly(I:C), irrespective of adjuvant dose. Cytotoxic killing was more pronounced and lasted at least 6 weeks after a single vaccination. - Antitumor efficacy: In protective E.G7 model, MP-OVA/Riboxxim-vaccinated mice remained tumor-free for 60 days. In therapeutic E.G7 model, delayed tumor growth and prolonged survival. In MO5 lung metastasis model, reduced lung metastases and increased CD8⁺ T cell infiltration. - Combination with checkpoint blockade: Anti-CTLA-4 plus MP-OVA/Riboxxim led to complete remission in 6/10 mice and long-term memory; anti-PD-1 alone was less effective but supported tumor eradication in combination. - Safety: No serum cytokine response post-vaccination, no histological damage in major organs.
Interpretation: The authors conclude that Riboxxim is a potent, pharmaceutically defined, GMP-grade dsRNA adjuvant for PLGA particle-based cancer vaccines. Co-encapsulation of antigen and Riboxxim activates both TLR3 and RIG-I/MAVS, induces type I interferons, and generates superior tumor-specific CD8⁺ T cell responses compared with poly(I:C). Combining this vaccine with immune checkpoint blockade reinvigorates cytotoxic T lymphocytes and can ablate tumors, supporting clinical translation and personalized cancer vaccine strategies using tumor lysates or neoantigens.
Limitations: Preclinical only: All efficacy and safety data are from mouse models; no human clinical trial data. - Human DC activation was demonstrated in vitro, not in vivo. - Model antigen bias: Much of the mechanistic work uses OVA, a foreign antigen; however, human tumor peptide antigens (STEAP1, PSMA, PAP, NY-ESO-1, HER2, TRP-2) were also tested in AAD mice, which partially addresses self-tolerance. - Tumor models: E.G7 and MO5 are transplantable models; no spontaneous or genetically engineered autochthonous tumor models. - No large-animal validation. - Manufacturing/scalability of GMP-grade Riboxxim-loaded PLGA particles was not fully addressed. - Long-term autoimmunity and toxicity beyond the study period were not extensively evaluated, though no acute systemic cytokine response or organ damage was observed. - Dependence on intranasal/s.c. prime-boost for lung metastasis model; clinical feasibility of repeated intranasal dosing remains to be established.

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PLGA-particle vaccine carrying TLR3/RIG-I ligand Riboxxim synergizes with immune checkpoint blockade for effective anti-cancer immunotherapy | Brilliant Blue Biosciences