In situ T-cell transfection by anti-CD3-conjugated lipid nanoparticles leads to T-cell activation, migration, and phenotypic shift

Purpose: Ex vivo T-cell engineering is effective but complex, costly, and difficult to scale. A method to transfect T cells directly in situ could simplify T-cell immunotherapy, but T-cell targeting may also trigger activation, depletion, cytokine release, and phenotypic changes that must be understood.

Hypothesis: Anti-CD3-conjugated lipid nanoparticles (aCD3-LNPs) can deliver mRNA specifically to T cells in vitro and in vivo, but this targeting will be accompanied by T-cell activation, temporary depletion, migration, cytokine secretion, and phenotypic shifts.

Aims: Develop and optimize aCD3-LNPs for T-cell-specific mRNA delivery. - Evaluate in vitro transfection, activation, and depletion in Jurkat T cells. - Assess in vivo biodistribution, transfection efficiency, activation, depletion, migration, cytokine release, and phenotype in spleen and blood. - Test whether transfected T cells localize to tumors and tumor-draining lymph nodes after immunotherapy.

Delivery system: Platform: Ionizable lipid nanoparticles (LNPs). - Key components: DLin-MC3-DMA, DSPC, cholesterol, DSPE-PEG2k, DSPE-PEG5k-maleimide. - Targeting ligand: Anti-CD3 antibody or anti-mouse CD3 F(ab′)₂ fragment; isotype IgG controls. - Payloads: mCherry mRNA, firefly luciferase (Fluc) mRNA. - Tracking label: DSPE-PEG2k-Cy7 for LNP tracking. - Physicochemical properties: ~70 nm; PDI ~0.13; zeta potential ~ −9 mV; ~90% mRNA encapsulation. - Route: Intravenous tail-vein injection.

Approach: In vitro: Human Jurkat T cells; aCD3-LNPs with mCherry mRNA; flow cytometry, fluorescence imaging, viability. - In vivo: C57BL/6 and FVB/n mice; IV injection at ~4 × 10¹³ particles/kg, ~0.6 mg mRNA/kg, ~25 mg lipid/kg. - Models: Healthy mice for immune profiling; NDL and E0771 tumor models for tumor/TDLN accumulation with CpG + anti-PD-1 immunotherapy. - Groups: 16% aCD3-LNPs, 1% or 2% aCD3-LNPs, non-targeted LNPs, isotype-LNPs, aCD3-liposomes, free aCD3, untreated controls; n = 3–28 depending on experiment.

Key methods: DLS, zeta potential, NanoSight, electron microscopy. - Flow cytometry for mCherry, CD3e, CD4, CD8a, CD69, CD25, OX40, CD44, CD62L, CD45, CD19, F4/80, CD11c. - IVIS bioluminescence and Cy7 fluorescence imaging. - RT-qPCR for mCherry mRNA in liver and spleen. - Luminex assay for plasma cytokines. - Immunohistochemistry/confocal microscopy for splenic architecture and tumor infiltration.

Key results: In vitro: >80% of Jurkat cells expressed mCherry with 16% aCD3-LNPs; ~97% became CD69⁺; aCD3 coating caused T-cell depletion and CD3e internalization. - In vivo 24 h: aCD3-LNPs transfected ~2–4% of splenic T cells and ~2–7% of circulating T cells; efficiency depended on aCD3 coating density. - Activation: >85% splenic CD4⁺ and >90% CD8a⁺ T cells expressed CD69 by 5 h; CD25 and OX40 were also induced but returned near baseline by 48 h. - Depletion: circulating CD4⁺ and CD8a⁺ T cells dropped sharply at 5 h but recovered by 48 h; spleen weight increased ~2-fold at 24 h and normalized by 1 week. - Migration/phenotype: CD8⁺ T cells moved from white pulp/PALS to marginal zone/red pulp; naive T cells decreased and CD44⁺/CD62L⁺ activated phenotypes increased. - Cytokines: Th1 and Th2 cytokines increased in plasma, plus myeloid-derived chemokines. - Tumors: mCherry⁺ T cells were detected in tumors and TDLNs mainly after CpG + anti-PD-1 immunotherapy.

Interpretation: CD3-targeted LNPs can transfect T cells in situ, but the approach causes reversible T-cell activation, depletion, migration, cytokine release, and phenotypic shifts. The authors conclude that monovalent or non-activating CD3 ligands will likely be needed to translate this strategy toward cancer immunotherapy.

Limitations: T-cell targeting caused activation, depletion, and systemic cytokine release, limiting immediate therapeutic safety. - Transfection efficiency in vivo was modest (2–7% of T cells). - Tumor/TDLN accumulation was preliminary and depended on prior immunotherapy. - No long-term functional studies of antigen response, tolerance, or memory. - No large-animal validation. - Bivalent aCD3 F(ab′)₂ still crosslinked TCR and caused activation; monovalent formats were not tested. - No therapeutic efficacy endpoint such as tumor growth inhibition or survival.