Therapeutic cell engineering with surface-conjugated synthetic nanoparticles

Abstract

A major limitation of cell therapies is the rapid decline in viability and function of the transplanted cells. Here we describe a strategy to enhance cell therapy via the conjugation of adjuvant drug-loaded nanoparticles to the surfaces of therapeutic cells. With this method of providing sustained pseudoautocrine stimulation to donor cells, we elicited marked enhancements in tumor elimination in a model of adoptive T cell therapy for cancer. We also increased the in vivo repopulation rate of hematopoietic stem cell grafts with very low doses of adjuvant drugs that were ineffective when given systemically. This approach is a simple and generalizable strategy to augment cytoreagents while minimizing the systemic side effects of adjuvant drugs. In addition, these results suggest therapeutic cells are promising vectors for actively targeted drug delivery.

Figure 1

Stable conjugation of nanoparticles (NPs) to the surfaces of T-cells and HSCs via cell-surface thiols. (a) Flow cytometry analysis of cell surface thiols on mouse splenocytes detected by fluorophore-conjugated malemide co-staining with lineage surface markers for erythrocytes (Ter-119), T-cells (CD3), B-cells (B220) and hematopoietic stem cells (c-kit). (b) Schematic of maleimide-based conjugation to cell surface thiols. (c) Confocal microscopy images of CD8⁺ effector T-cells and lineage^-Sca-1⁺c-kit⁺ HSCs immediately following conjugation with fluorescent DiD-labeled multilamellar lipid NPs (left panel) and after four day in vitro expansion (right panel). Scale bars, 2 μm. (d) Flow cytometry analysis of CD8⁺ T-cells after incubation with DiD-labeled multilamellar lipid NPs synthesized with or without maleimide-headgroup lipids. (e) Quantification of nanoparticle internalization. Immature dendritic cells (DCs), effector CD8⁺ T-cells, or HSCs were conjugated with carboxyfluorescein-tagged maleimide-bearing liposomes. Extracellular trypan blue quenching was used to differentiate surface-bound and internalized liposomes immediately following conjugation or after four days in culture.

Figure 2

Nanoparticle conjugation does not impact key T-cell functions. OT-1 ova-specific CD8⁺ effector T-cells were conjugated with 100 DiD-labeled multilamellar lipid NPs per cell or left unmanipulated as controls. (a) CFSE dilution of unmodified or NP-conjugated T-cells stimulated in vitro with mature ova peptide-pulsed dendritic cells. DiD Mean Fluorescence Intensities (MFI) for distinct CFSE lymphocytes populations are indicated on the right. (b) Standard 4 h ⁵¹Cr release assay comparing cytotoxicity of unmanipulated (open symbols) and particle-conjugated (filled symbols) OT-1 cells targeting ova peptide-pulsed (circles) or control (triangles) EL4 tumor cells. (c,d) Transmigration of OT-1 T-cells (with or without surface-bound particles) seeded onto MS1 endothelial cell monolayers in the upper well of a transwell chamber, following addition of the chemoattractant MCP-1 to the lower chamber. The fraction of transmigrating T-cells (c) and the profile of cell-bound NP fluorescence before (UW) and after (LW) transmigration (d) were quantitated by flow cytometry. (DiD MFI±s.e.m. from triplicate samples shown in blue).

Figure 3

Nanoparticle-decorated T cells efficiently carry surface-tethered NPs into antigen-expressing tumors. (a,b) Comparative whole-animal in vivo bioluminescence (tumors, T-cells) and fluorescence imaging (NPs) of mice bearing established s.c. Gaussia luc-expressing EG7-OVA and EL4 tumors on opposite flanks, two days after i.v. infusion of firefly luc-transgenic Thy1.1⁺ effector OT-1 T-cells (with or without attached DiD-labeled NPs), or an equivalent number of free NPs. Thy1.1⁺ OT-1 T-cells recovered from the EG7-OVA tumors were analyzed for surface-bound DiD NPs by flow cytometry (a), and the mean bioluminescent T-cell and fluorescent NP signals from groups of 6 mice are shown in (b). NS, no significance. (c) In an independent experiment, CellTracker green-labeled OT-1 T-cells conjugated with rhodamine-labeled NPs were transferred into mice bearing established s.c. EG7-OVA tumors, and tumors were excised and sectioned for confocal histological analysis two days later. Scale bar, 10 μm. A higher magnification image of NP-carrying tumor infiltrating T-cells is shown in the right panel. Scale bar, 1.5 μm. Yellow arrowheads highlight evidence for surface localization of NPs. Shown is 1 of 2 independent experiments. (d) Groups of 3 C57Bl/6 mice bearing s.c. EG7-OVA tumors were i.v. injected with 15 × 10⁶ OT-1 effector T-cells bearing surface-conjugated with DiD-labeled NPs (100 NPs/cell, filled bars), an equivalent number of DiD-labeled particles alone (open bars). After 48 h indicated tissues were removed, weighed, and macerated with scissors. We quantified specific DiD tissue fluorescence for each organ using the IVIS Spectrum imaging system and calculated the mean percentage of injected dose per gram of tissue (%ID g^-1) as final readout (d). Data shown are pooled from three independent experiments.

Figure 4

Pmel-1 T-cells conjugated with IL-15Sa/IL-21-releasing NPs robustly proliferate in vivo and eradicate established B16 melanomas. Lung and bone marrow tumors were established by tail vein injection of 1×10⁶ Gaussia luciferase-expressing B16F10 cells in C57Bl/6 mice. Tumor-bearing animals were treated after 1 week by sublethal irradiation followed by i.v. infusion of 10×10⁶ Click beetle red luciferase-expressing Vβ13⁺CD8⁺ Pmel-1 T-cells. One group of mice received Pmel-1 T-cells conjugated with 100 NPs/cell carrying a total dose of 5 μg IL-15Sa/IL-21 (4.03 μg IL-15Sa + 0.93 μg IL-21), control groups received unmodified Pmel-1 T-cells and a single systemic injection of the same doses of IL-15Sa/IL-21 or Pmel-1 T-cells alone. (a) Dual longitudinal in vivo bioluminescence imaging of Gaussia luc-expressing B16F10 tumors and CBR-luc-expressing Pmel-1 T-cells. (b) Frequencies of Vβ13⁺CD8⁺ Pmel-1 T-cells recovered from pooled lymph nodes of representative animals 16 days after T-cell transfer. (c) CBR-luc T-cell signal intensities from sequential bioluminescence imaging every two days after T-cell transfer. Every line represents one animal with each dot showing the whole animal photon count. (d) Survival of animals following T-cell therapy illustrated by Kaplan-Meier curves. Shown are six mice/treatment group pooled from three independent experiments.

Figure 5

HSCs carrying GSK-3β inhibitor-loaded nanoparticles reconstitute recipient animals with rapid kinetics following bone marrow transplants without affecting multilineage differentiation potential. (a,b) Engraftment kinetics of luciferase-transgenic HSC grafts in lethally-irradiated nontransgenic syngeneic recipients. Mice were treated with a single bolus injection of the GSK-3β inhibitor TWS119 (1.6 ng) on the day of transplantation, an equivalent TWS119 dose encapsulated in HSC-attached NPs, or no exogenous adjuvant compounds. Transplanted mice were imaged for whole-body bioluminescence every seven days for three weeks. Shown are representative IVIS images (a) and whole animal photon counts (b) for nine mice total/treatment condition. (c) Percentage of donor-derived cells two weeks after transplantation of GFP⁺ HSCs into lethally-irradiated recipients with or without TWS119 adjuvant drug. *P < 0.001. (d) Average frequency of donor-derived GFP⁺ B-cells, T-cells, and myeloid cells in recipient mice three months after transplantation. five mice/group were analyzed.