Receptor-mediated gene delivery to human peripheral blood mononuclear cells using anti-CD3 antibody coupled to polyethylenimine

Purpose: Primary lymphoid cells, especially human peripheral blood mononuclear cells (PBMC), are very difficult to transfect with viral and nonviral vectors. There is a need for efficient nonviral, receptor-mediated gene delivery to PBMC for cell-based therapies and for studying gene function in primary lymphocytes.

Hypothesis: If polyethylenimine (PEI) is coupled to an anti-CD3 antibody, then gene delivery to human PBMC via the CD3 receptor will be efficient, particularly in activated PBMC, and signalling through CD3 may contribute to transfection efficacy beyond mere receptor binding and endocytosis.

Aims: Evaluate anti-CD3-PEI conjugates for receptor-mediated gene delivery to human PBMC. - Compare anti-CD3-PEI with anti-ICAM1-PEI, anti-MHC class I-PEI, nontargeted PEI, and electroporation. - Determine the effects of PBMC prestimulation with PHA and IL-2 on transfection efficiency. - Assess the kinetics of transgene expression and the role of CD3 signalling using immobilised anti-CD3 antibody.

Delivery system: Platform: Receptor-targeted nonviral polyplex. - Polymer: Polyethylenimine (PEI) conjugated to anti-CD3 antibody (anti-CD3-PEI). Also tested: anti-ICAM1-PEI, anti-HLA class I (W6/32)-PEI, and nontargeted PEI. - Payload: pGL3-control plasmid DNA (luciferase reporter) and pEGFP plasmid DNA (GFP reporter). - Targeting ligand: Anti-CD3 antibody for CD3 receptor on T cells. - Formulation: DNA/anti-CD3-PEI complexes prepared in HBS; typical transfection used 10 µg DNA and 6 µg anti-CD3-PEI (PEI concentration) per sample.

Approach: In vitro human system: PBMC isolated from healthy volunteers by Ficoll-Hypaque density gradient centrifugation. Cells were either naive/unstimulated or activated with PHA (1 µg/mL) for 48–72 h; some cultures received human rIL-2 (10 U/mL) 24 h before transfection. - Transfection: anti-CD3-PEI/DNA complexes incubated with cells for 4 h, then replaced with complete medium with IL-2. Electroporation used as a comparison. PMA (20 ng/mL) was used in some experiments as an additional activation stimulus. - No in vivo experiments.

Key methods: Luciferase assay for reporter gene expression (RLU/1 × 10⁵ cells/sec). - Flow cytometry (FACS) for GFP-positive cells and mean channel fluorescence (MCF). - Comparison of transfection with different ligand-PEI conjugates. - Costimulation with immobilised anti-CD3 antibody in plate wells. - TNF-α measurement in transfected cultures.

Key results: Naive vs activated PBMC: Unstimulated PBMC did not express transgene; PHA-activated PBMC did. Optimal transfection occurred 48–72 h after PHA stimulation. Adding IL-2 24 h before transfection increased expression; adding IL-2 at day 0 with PHA reduced expression. - Kinetics: Transgene expression peaked at 24–48 h and was detectable until 96 h, gradually decreasing after 48 h. GFP-positive cells by FACS: 56.05% at 24 h, 73.27% at 48 h, 62.27% at 72 h, and 57.35% at 96 h. - Comparison with electroporation: Anti-CD3-PEI gave significantly higher luciferase activity than electroporation. PMA further augmented anti-CD3-PEI-mediated expression by ~60% (from 45,406 ± 851 to 72,361 ± 378 RLU). - Ligand specificity: Anti-ICAM1-PEI and anti-HLA class I-PEI were much less efficient than anti-CD3-PEI. However, anti-ICAM1-PEI transfection was significantly augmented when cells were cultured with immobilised anti-CD3 antibody, reaching or exceeding anti-CD3-PEI levels. - TNF-α/apoptosis: Anti-CD3-PEI-transfected cells produced 50.83 pg/mL TNF-α, whereas nontransfected cells had undetectable levels. Some apoptosis was observed within 24 h, possibly due to activation-induced cell death.

Interpretation: The authors conclude that anti-CD3-PEI is an effective nonviral vector for gene delivery to activated human PBMC and that CD3 receptor engagement provides both targeting and signalling benefits. They suggest this approach is useful for genetically modifying primary lymphocytes, studying gene expression/signaling in primary lymphoid cells, and potentially developing cell-based therapies.

Limitations: Purely in vitro; no in vivo animal or human therapeutic data. - Only activated PBMC were efficiently transfected; naive PBMC did not express transgene. - Transfection efficiency varied between PBMC samples. - Anti-CD3-PEI induced TNF-α and some apoptosis within 24 h, indicating a balance between beneficial expression and activation-induced cell death. - Mechanism of enhanced delivery (internalisation vs transcriptional activation) was not fully resolved. - No dose-response or long-term expression/stability data beyond 96 h. - No direct comparison with viral vectors in the same study.