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Vaccine2008ResearchNon-viral Gene Delivery

Co-delivery of cancer-associated antigen and Toll-like receptor 4 ligand in PLGA nanoparticles induces potent CD8⁺ T cell-mediated anti-tumor immunity

S. Hamdy, O. Molavi, Z. Ma, A. Haddadi, A. Alshamsan, Z. Gobti, S. Elhasi, J. Samuel, A. LavasanifarDOI 10.1016/j.vaccine.2008.07.035

Summary

Most PLGA-based cancer vaccine studies used foreign model antigens such as ovalbumin (OVA), which are not subject to central/peripheral tolerance and can overestimate therapeutic efficacy. There is a need to evaluate PLGA vaccines against a realistic, clinically relevant self/tumor antigen (TRP2) and to overcome self-tolerance and the immunosuppressive tumor microenvironment. Co-delivery of a TLR4 ligand (7-acyl lipid A) with a tumor antigen in. Nanoparticle Characterization: - Size 350–410 nm, PDI < 0.2. - TRP2 EE 5.2 ± 0.6%; loading 0.94 ± 0.11 µg/mg. - 7-acyl lipid A EE 67.3 ± 6.9%; loading 1.79 ± 0.18 µg/mg. Normal Mice Vaccination (ELISPOT): - Empty-NP: no.

Keywords

T cellsPLGANanoparticlesTumor microenvironmentPolymericDendritic cellsNanocarriers
Purpose: Most PLGA-based cancer vaccine studies used foreign model antigens such as ovalbumin (OVA), which are not subject to central/peripheral tolerance and can overestimate therapeutic efficacy. There is a need to evaluate PLGA vaccines against a realistic, clinically relevant self/tumor antigen (TRP2) and to overcome self-tolerance and the immunosuppressive tumor microenvironment. Co-delivery of a TLR4 ligand (7-acyl lipid A) with a tumor antigen in the same PLGA nanoparticle may provide the necessary danger signals to induce potent CD8⁺ T cell-mediated anti-tumor immunity.
Hypothesis: If TRP2₁₈₀₋₁₈₈ (a self/tumor-associated melanoma antigen) and 7-acyl lipid A (a TLR4 ligand) are co-encapsulated in the same PLGA nanoparticle and delivered to dendritic cells, then the formulation will break self-tolerance, induce potent TRP2-specific CD8⁺ T cell responses, reverse the immunosuppressive tumor microenvironment, and mediate therapeutic anti-tumor efficacy in the B16 melanoma model—superior to antigen alone or empty nanoparticles.
Aims: Primary aim: Develop PLGA nanoparticles co-encapsulating TRP2₁₈₀₋₁₈₈ and 7-acyl lipid A.
  • Secondary aim 1: Quantify encapsulation efficiency and loading of both TRP2 peptide and 7-acyl lipid A using LC-MS.
  • Secondary aim 2: Assess induction of TRP2-specific IFN-γ-secreting CD8⁺ T cells in lymph nodes and spleens of vaccinated normal and tumor-bearing C57BL/6 mice.
  • Secondary aim 3: Evaluate therapeutic anti-tumor efficacy in B16-F10 melanoma-bearing mice (tumor size, tumor weight, survival/morbidity).
  • Secondary aim 4: Measure vaccine-induced changes in pro-inflammatory/Th1 cytokines and immunosuppressive factors (VEGF) in the tumor microenvironment.
Delivery system:

Component: Polymer; Description: PLGA (50:50 lactide:glycolide), MW ≈ 7,000 Da

Component: Nanoparticle Type; Description: Double-emulsion (W/O/W) solvent evaporation

Component: Targeting Ligand; Description: None (non-targeted passive delivery to APCs)

Component: Payload 1; Description: TRP2₁₈₀₋₁₈₈ peptide (self/tumor-associated melanoma antigen; H-2Kᵇ-restricted CD8⁺ T cell epitope)

Component: Payload 2; Description: 7-acyl lipid A (synthetic TLR4 ligand; also called BC1-005; MW 1955.5 Da)

Component: Size; Description: 350–410 nm; polydispersity < 0.2

Component: TRP2 Encapsulation; Description: EE = 5.2 ± 0.6%; loading = 0.94 ± 0.11 µg/mg NP

Component: 7-acyl lipid A Encapsulation; Description: EE = 67.3 ± 6.9%; loading = 1.79 ± 0.18 µg/mg NP

Component: Dose per Injection; Description: ~10 µg TRP2 ± ~17 µg 7-acyl lipid A

Component: Formulations; Description: Empty-NP, TRP2-NP, TRP2/7-acyl lipid A-NP

Approach: In Vivo Mouse Models (C57BL/6 mice):
  • Normal vaccination study: 5 mice/group; subcutaneous (s.c.) vaccination in right flank with Empty-NP, TRP2-NP, or TRP2/7-acyl lipid A-NP; booster 11 days later; lymph nodes and spleens isolated 7 days after boost.
  • Tumor therapy study: B16-F10 melanoma cells (0.1 × 10⁶) injected s.c. on day 0. On day 3, mice randomized into 3 groups (8–10 mice/group): Empty-NP, TRP2-NP, TRP2/7-acyl lipid A-NP. Vaccinated s.c. on days 3, 7, and 13. Tumor size measured from day 7 every 2–3 days until day 21. Mice euthanized when tumor area > 300 mm² or ulceration. Tumors, lymph nodes, and spleens harvested at day 21.
  • Controls: Empty-NP; antigen-only NP; TLR ligand-only NP (in some experiments).
  • Doses: ~10 µg TRP2 and/or ~17 µg 7-acyl lipid A per immunization.

No in vitro cell line–based T cell activation assay was used; immune readouts were ex vivo ELISPOT and ELISA from vaccinated mice.

Key methods:

Technique: LC-MS (SIR mode); Purpose: Quantify TRP2₁₈₀₋₁₈₈ encapsulation; lansoprazole as internal standard

Technique: Dynamic light scattering (DLS); Purpose: Particle size and polydispersity

Technique: IFN-γ ELISPOT; Purpose: Quantify antigen-specific IFN-γ-secreting CD8⁺ T cells in lymph nodes, splenocytes, and isolated CD8⁺ T cells

Technique: ELISA; Purpose: Measure TNF-α, IL-12, IFN-γ, IL-2, IL-6, and VEGF in tumor supernatants

Technique: Tumor size/weight measurements; Purpose: Assess therapeutic efficacy; tumor weight < 0.3 g defined as controlled growth

Technique: CD8⁺ T cell negative selection; Purpose: Eliminate non-specific IFN-γ background in ELISPOT

Key results: Nanoparticle Characterization: - Size 350–410 nm, PDI < 0.2. - TRP2 EE 5.2 ± 0.6%; loading 0.94 ± 0.11 µg/mg. - 7-acyl lipid A EE 67.3 ± 6.9%; loading 1.79 ± 0.18 µg/mg.

Normal Mice Vaccination (ELISPOT): - Empty-NP: no measurable IFN-γ. - TRP2-NP and TRP2/7-acyl lipid A-NP induced comparable IFN-γ-secreting T cells in lymph nodes (113.5 ± 5 vs 145.6 ± 10 spots/million lymphocytes). - In spleens, co-delivery of 7-acyl lipid A led to 3-fold (ACK-lysed splenocytes) and 12-fold (isolated CD8⁺ T cells) increases in TRP2-specific IFN-γ-secreting CD8⁺ T cells compared to TRP2-NP.

Tumor Therapy in B16 Melanoma: - Average tumor size in TRP2/7-acyl lipid A-NP group was approximately half that of TRP2-NP group at all time points. - Controlled tumor growth (< 0.3 g) at day 21: 85% for TRP2/7-acyl lipid A-NP, 40% for TRP2-NP, 16% for Empty-NP. - No mice in TRP2/7-acyl lipid A-NP group required euthanasia; 1 mouse each in Empty-NP and TRP2-NP groups were euthanized due to large tumors.

Ex Vivo ELISPOT in Tumor-Bearing Mice: - TRP2-NP induced higher responses in draining lymph nodes (141.5 ± 10.8 vs 107.25 ± 8.5 spots; p < 0.05). - TRP2/7-acyl lipid A-NP induced superior responses in spleens, suggesting better T cell migration.

Tumor Microenvironment (ELISA): - TRP2/7-acyl lipid A-NP significantly increased pro-inflammatory/Th1 cytokines: IFN-γ, TNF-α, IL-2, IL-6, IL-12. - Decreased VEGF (immunosuppressive/pro-angiogenic factor).

Interpretation: The authors claim this is the first report showing that co-delivery of a real cancer antigen (TRP2) along with a TLR4 ligand (7-acyl lipid A) in PLGA nanoparticles can break self-tolerance, induce potent and specific CD8⁺ T cell-mediated anti-tumor immunity without concomitant autoimmunity, and reverse the immunosuppressive tumor microenvironment (increased Th1 cytokines, decreased VEGF). They highlight 7-acyl lipid A as a powerful adjuvant for future cancer vaccine trials and support PLGA-NPs as competent antigen delivery systems.
Limitations: Large intra-group variability in tumor size/weight prevented statistically significant differences between TRP2-NP and TRP2/7-acyl lipid A-NP groups; higher n needed. - Only one tumor model (B16-F10 melanoma) and one antigen (TRP2) tested. - Short follow-up (21 days); no long-term survival or memory response assessment. - No direct in vitro human DC/T cell validation; immune readouts are murine ex vivo. - Non-specific IFN-γ background observed in lymph node/splenocyte ELISPOT wells, requiring pure CD8⁺ T cell isolation to eliminate. - NK cell activation and relative contribution to anti-tumor effects were not directly assessed (stated as future goal). - No autoimmunity evaluation beyond absence of overt toxicity.

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Co-delivery of cancer-associated antigen and Toll-like receptor 4 ligand in PLGA nanoparticles induces potent CD8⁺ T cell-mediated anti-tumor immunity | Brilliant Blue Biosciences