Cellular Immunology (Elsevier)2014ResearchNon-viral Gene Delivery

PLGA-nanoparticle mediated delivery of anti-OX40 monoclonal antibody enhances anti-tumor cytotoxic T cell responses

Mingshui Chen, Haichao Ouyang, Shangyong Zhou, Jieyu Li, Yunbin Ye29 September 2026DOI 10.1016/j.cellimm.2014.01.003

Summary

Agonistic anti-OX40 monoclonal antibody (mAb) can enhance anti-tumor immunity in preclinical models, but as monotherapy it showed only marginal activity and no objective clinical responses in a phase I trial. A delivery system that improves T cell access, multivalent OX40 engagement, and sustained release of anti-OX40 mAb could enhance cytotoxic T lymphocyte (CTL) responses for cancer immunotherapy. Nanoparticle properties: Size 86.0 ± 14.1 nm; zeta potential −12.8 ± 1.5 mV; encapsulation efficiency 65.8 ± 5.6%; loading ~25% (248 ± 16.3 µg mAb/mg polymer); sustained release ~55% over 20 days with no initial burst.

Keywords

T cells PLGA Nanoparticles Polymeric Cancer immunotherapy Dendritic cells Antigen presentation

Purpose: Agonistic anti-OX40 monoclonal antibody (mAb) can enhance anti-tumor immunity in preclinical models, but as monotherapy it showed only marginal activity and no objective clinical responses in a phase I trial. A delivery system that improves T cell access, multivalent OX40 engagement, and sustained release of anti-OX40 mAb could enhance cytotoxic T lymphocyte (CTL) responses for cancer immunotherapy.

Hypothesis: If anti-OX40 agonist mAb is conjugated to biodegradable PLGA nanoparticles, then the nanoparticle formulation will induce stronger CTL proliferation, activation, cytokine production, and tumor antigen-specific cytotoxicity than free anti-OX40 mAb.

Aims: Primary aim: Prepare and characterize anti-OX40 mAb-loaded PLGA nanoparticles (anti-OX40-PLGA-NP).

Secondary aim 1: Measure nanoparticle size, zeta potential, morphology, antibody loading, encapsulation efficiency, and release kinetics.

Secondary aim 2: Evaluate OX40 expression on activated CD8⁺ T cells and the effect of anti-OX40-PLGA-NP on T cell phenotype.

Secondary aim 3: Assess CTL proliferation, cytokine secretion, and antigen-specific cytotoxicity in vitro compared with free anti-OX40 mAb, empty PLGA-NP, and immobilized anti-OX40 mAb.

Delivery system:

Component: Polymer; Description: COOH-terminated PLGA (50:50 lactide:glycolide)

Component: Nanoparticle Type; Description: PLGA nanoparticles prepared by oil-in-water emulsion solvent extraction/evaporation

Component: Targeting Ligand; Description: None

Component: Payload; Description: Anti-human OX40 monoclonal antibody (agonist)

Component: Conjugation Chemistry; Description: Covalent attachment via EDC/NHS activation of PLGA carboxylic groups to antibody amine groups

Component: Size; Description: 86.0 ± 14.1 nm

Component: Zeta Potential; Description: −12.8 ± 1.5 mV

Component: Encapsulation Efficiency; Description: 65.8 ± 5.6%

Component: Loading Efficiency; Description: ~25% (248 ± 16.3 µg anti-OX40 mAb per mg polymer)

Component: Release; Description: Sustained; ~12% release in 1 day (no initial burst), ~55% cumulative over 20 days

Component: Stability; Description: Hydrodynamic diameter unchanged over 6 h at 25°C

Component: Key Design Feature; Description: Multivalent surface display of anti-OX40 mAb on PLGA-NP for enhanced OX40 engagement and sustained release

Approach: In vitro only; no in vivo animal studies. - Human cells: Peripheral blood mononuclear cells (PBMCs) from healthy donors; CD14⁺ cells differentiated into dendritic cells (DCs) with GM-CSF/IL-4 and matured with LPS; CD8⁺ T cells purified and stimulated with allogeneic DCs to generate CTLs. - Antigen-specific CTL generation: DCs pulsed with AFP₁₅₈₋₁₆₆ peptide (hepatocellular carcinoma antigen) or gp100₁₅₄₋₁₆₂ peptide (melanoma antigen, negative control). - Target cells: AFP-expressing human hepatocellular carcinoma HepG2 cells. - Treatment groups: Untreated CTL, PLGA-NP alone, free anti-OX40 mAb, immobilized anti-OX40 mAb, and anti-OX40-PLGA-NP. Dose: 10 µg/mL anti-OX40 equivalent. - Readouts: Flow cytometry for OX40, CD3/CD4/CD8, CD25, CD69; CFSE dilution for proliferation; ELISA for IFN-γ, IL-2, IL-4, IL-10, IL-17; LDH release for cytotoxicity at effector:target ratios 40:1, 20:1, 10:1. - n = 3 independent experiments for most assays; statistical significance by Student’s t-test (p < 0.05).

Key methods:

Technique: Scanning electron microscopy (SEM); Purpose: Nanoparticle morphology and size

Technique: Photon correlation spectroscopy / Zetasizer; Purpose: Hydrodynamic diameter and stability

Technique: Zeta potential measurement; Purpose: Surface charge

Technique: Bradford/BCA protein assays; Purpose: Antibody loading, encapsulation efficiency, release

Technique: Flow cytometry; Purpose: OX40 expression; T cell phenotype (CD3, CD4, CD8, CD25, CD69)

Technique: CFSE dilution assay; Purpose: CTL proliferation

Technique: ELISA; Purpose: Cytokine secretion (IFN-γ, IL-2, IL-4, IL-10, IL-17)

Technique: Lactate dehydrogenase (LDH) release assay; Purpose: Antigen-specific CTL cytotoxicity against HepG2

Key results: Nanoparticle properties: Size 86.0 ± 14.1 nm; zeta potential −12.8 ± 1.5 mV; encapsulation efficiency 65.8 ± 5.6%; loading ~25% (248 ± 16.3 µg mAb/mg polymer); sustained release ~55% over 20 days with no initial burst. - OX40 expression: Naive CD8⁺ T cells did not express OX40; allogeneic DC stimulation increased OX40 expression in a time-dependent manner, with ~half of cells positive by day 7. - T cell phenotype: Anti-OX40-PLGA-NP increased CD8⁺ T cells from 38.4% to 62.9%, CD25⁺ from 11.7% to 23.6%, and CD69⁺ from 1.6% to 5.9% (p < 0.05 for all). - Proliferation: Anti-OX40-PLGA-NP induced 48.9% proliferating CTLs vs 27.2% for free anti-OX40 mAb, 24.7% for PLGA-NP alone, and 43.2% for immobilized anti-OX40 mAb. - Cytokines: Anti-OX40-PLGA-NP significantly increased IFN-γ (~9-fold) and IL-2 (~13-fold) and IL-17 vs free anti-OX40 mAb; IL-4 and IL-10 were unchanged, favoring Th1/Th17-type responses. - Cytotoxicity: Anti-OX40-PLGA-NP produced significantly higher specific lysis of AFP-expressing HepG2 cells than free anti-OX40 mAb or PLGA-NP at all effector:target ratios (40:1, 20:1, 10:1). gp100-specific CTLs showed no killing of HepG2, confirming antigen specificity.

Interpretation: The authors conclude that PLGA nanoparticle delivery of anti-OX40 mAb provides an efficient formulation for cancer immunotherapy, enhancing CTL proliferation, activation, cytokine production, and tumor antigen-specific cytotoxicity more effectively than free anti-OX40 mAb. The multivalent surface display and sustained release from PLGA-NP may improve OX40 agonist therapy, especially where sustained T cell stimulation is needed.

Limitations: In vitro only: No in vivo tumor models, biodistribution, pharmacokinetics, or therapeutic efficacy data. - No toxicity assessment: Potential cytotoxicity of <100 nm nanoparticles and long-term safety were not evaluated. - Limited stability data: Nanoparticle integrity tested only up to 6 h; release study lasted 20 days, but no long-term storage stability. - Human PBMC-derived allogeneic DC stimulation may not fully reflect autologous tumor antigen presentation. - No comparison with PEGylated or targeted PLGA-NP or other antibody delivery systems. - Small sample size: Most assays n = 3; no power analysis. - No memory T cell or in vivo CTL recall response evaluation.

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PLGA-nanoparticle mediated delivery of anti-OX40 monoclonal antibody enhances anti-tumor cytotoxic T cell responses

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