Therapeutic cell engineering with surface-conjugated synthetic nanoparticles

Journal name:
Nature Medicine
Volume:
16,
Pages:
1035–1041
Year published:
DOI:
doi:10.1038/nm.2198
Received
Accepted
Published online

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.

At a glance

Figures

  1. Stable conjugation of nanoparticles (NPs) to the surfaces of T cells and HSCs via cell-surface thiols.
    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-specific 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. MPB-PE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-[4-(p-maleimidophenyl)butyramide]. (c) Confocal microscopy images of CD8+ effector T cells and lineageSca-1+c-Kit+ HSCs immediately after conjugation with fluorescent 1,1-dioctadecyl-3,3,3,3-tetramethylindodicarbocyanine (DiD)-labeled multilamellar lipid nanoparticles (left) and after 4-d in vitro expansion (right). Scale bars, 2 μm. (d) Flow cytometry analysis of CD8+ T cells after incubation with DiD-labeled multilamellar lipid nanoparticles 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 (CFSE)-tagged maleimide-bearing liposomes. Extracellular trypan blue quenching was used to differentiate surface-bound and internalized liposomes immediately after conjugation or after 4 d in culture. Data represent the mean ± s.e.m. of two independent experiments conducted in triplicate.

  2. Nanoparticle conjugation does not affect key T cell functions.
    Figure 2: Nanoparticle conjugation does not affect key T cell functions.

    OT-1 ovalbumin-specific CD8+ effector T cells were conjugated with 100 DiD-labeled multilamellar lipid nanoparticles per cell or left unmanipulated as controls. (a) CFSE dilution of unmodified or nanoparticle-conjugated T cells stimulated in vitro with mature ovalbumin peptide–pulsed dendritic cells. DiD mean fluorescence intensities (MFI)s for distinct CFSE lymphocytes populations are indicated in the bottom right quadrant. (b) 51Cr release assays of unmanipulated and particle-conjugated OT-1 cells targeting ovalbumin peptide–pulsed or control 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 after addition of the chemoattractant monocyte chemoattractant protein-1 to the lower chamber. The fraction of transmigrating T cells (c) and the profile of cell-bound nanoparticle fluorescence before (UW) and after (LW) transmigration (d) were quantified by flow cytometry (DiD MFI ± s.e.m. from triplicate samples shown in blue).

  3. Nanoparticle-decorated T cells efficiently carry surface-tethered nanoparticles into antigen-expressing tumors.
    Figure 3: Nanoparticle-decorated T cells efficiently carry surface-tethered nanoparticles into antigen-expressing tumors.

    (a,b) Comparative whole-mouse in vivo bioluminescence (tumors and T cells) and fluorescence imaging (nanoparticles) of mice bearing established subcutaneous extG-luc–expressing EG7-OVA and EL4 tumors on opposite flanks, 2 d after i.v. infusion of firefly luciferase–transgenic Thy1.1+ effector OT-1 T cells (with or without attached DiD-labeled nanoparticles) or an equivalent number of free nanoparticles. Thy1.1+ OT-1 T cells recovered from the EG7-OVA tumors were analyzed for surface-bound DiD nanoparticles by flow cytometry (a), and the mean bioluminescent T cell and fluorescent nanoparticle signals from groups of 6 mice are shown in b. Respective differences in the bioluminescent or fluorescent photon counts of EL4 compared to EG7-OVA tumors were analyzed by the Student's t test. NS, not significant. ↑ refers to higher fluorescent signal strength of the data points on the right in comparison to respective values on the left. (c) Confocal images of nanoparticle-decorated OT-1 T cells infiltrating EG7-OVA tumor 2 d after adoptive transfer. Scale bar, 10 μm. Higher magnification images of nanoparticle-carrying tumor-infiltrating T cells are shown at right. Scale bars, 1.5 μm. Yellow arrowheads highlight evidence for surface localization of nanoparticles. Shown is one of two independent experiments. (d) Biodistribution of an equivalent number of DiD-labeled nanoparticles injected systemically (open bars) or conjugated to adoptively transferred OT-1 T cells (filled bars) after 48 h. The mean fluorescent signal intensities (± s.d.) of nine mice from three independent experiments are graphed. ID, injected dose. Data shown are pooled from three independent experiments.

  4. Pmel-1 T cells conjugated with IL-15Sa- and IL-21-releasing nanoparticles robustly proliferate in vivo and eradicate established B16 melanomas.
    Figure 4: Pmel-1 T cells conjugated with IL-15Sa– and IL-21–releasing nanoparticles robustly proliferate in vivo and eradicate established B16 melanomas.

    Lung and bone marrow tumors were established by tail vein injection of 1 × 106 extG-luc–expressing B16F10 cells into C57BL/6 mice. Tumor-bearing mice were treated after 1 week by sublethal irradiation followed by i.v. infusion of 1 × 107 CBR-luc–expressing Vβ13+CD8+ Pmel-1 T cells. One group of mice received Pmel-1 T cells conjugated with 100 nanoparticles per cell carrying a total dose of 5 μg IL-15Sa and 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 and IL-21 or Pmel-1 T cells alone. (a) Dual longitudinal in vivo bioluminescence imaging of extG-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 mice 16 d after T cell transfer. (c) CBR-luc T cell signal intensities from sequential bioluminescence imaging every 2 d after T cell transfer. Every line represents one mouse, with each dot showing the whole-mouse photon count. (d) Survival of mice after T cell therapy illustrated by Kaplan-Meier curves. Shown are six mice per treatment group pooled from three independent experiments.

  5. HSCs carrying GSK-3[beta] inhibitor-loaded nanoparticles reconstitute recipient animals with rapid kinetics after bone marrow transplants without affecting multilineage differentiation potential.
    Figure 5: HSCs carrying GSK-3β inhibitor–loaded nanoparticles reconstitute recipient animals with rapid kinetics after 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 nanoparticles or no exogenous adjuvant compounds. Transplanted mice were imaged for whole-body bioluminescence every 7 d for 3 weeks. Shown are representative in vivo imaging system images (a) and whole-mouse photon counts (b) for nine mice total per 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 five recipient mice per group (± s.d.) three months after transplantation are shown.

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Author information

Affiliations

  1. Department of Material Science and Engineering, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts, USA.

    • Matthias T Stephan,
    • James J Moon,
    • Soong Ho Um,
    • Anna Bershteyn &
    • Darrell J Irvine
  2. Koch Institute for Integrative Cancer Research, MIT, Cambridge, Massachusetts, USA.

    • Matthias T Stephan,
    • James J Moon,
    • Soong Ho Um,
    • Anna Bershteyn &
    • Darrell J Irvine
  3. Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

    • Soong Ho Um &
    • Darrell J Irvine
  4. Ragon Institute of Massachusetts General Hospital, MIT and Harvard University, Boston, Massachusetts, USA.

    • Darrell J Irvine
  5. Howard Hughes Medical Institute, Chevy Chase, Maryland, USA.

    • Darrell J Irvine

Contributions

M.T.S. designed and conducted all experiments and wrote the manuscript. J.J.M. assisted in T cell transmigration assays, optimization of multilamellar lipid nanoparticle synthesis and in vivo nanoparticle biodistribution assays. S.H.U. assisted optimization of multilamellar lipid nanoparticle synthesis. A.B. assisted in initial in vitro T cell assays, collected electron microscopy images and contributed experimental suggestions. D.J.I. supervised all experiments and wrote the manuscript.

Competing financial interests

The authors declare no competing financial interests.

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Supplementary information

PDF files

  1. Supplementary Text and Figures (4M)

    Supplementary Figures 1–17 and Supplementary Methods

Movies

  1. Supplementary Movie 1 (644K)

    Maleimide-bearing NPs are not internalized after cell surface conjugation. CD8+ T cells were surface-conjugated with fluorescent DiD-tagged NPs (Fig. 1c). Sequences of single confocal z-sections acquired at 0.4 1m intervals throughout the T cells are shown here and in Supplementary Movie 2.

  2. Supplementary Movie 2 (716K)

    Maleimide-bearing NPs are not internalized after cell surface conjugation. CD8+ T cells were surface-conjugated with fluorescent DiD-tagged NPs (Fig. 1c). Sequences of single confocal z-sections acquired at 0.4 1m intervals throughout the T cells are here and in Supplementary Movie 1.

  3. Supplementary Movie 3 (672K)

    Membrane-tethered NPs are retained on the cell surface of tumor homing T lymphocytes. Explanted cross-sectioned EG7-OVA tumors 2 d after T cell injection (Fig 3c). CellTracker green–labeled OT-1 T cells and rhodamine lipid–coated NPs (magenta) were visualized by confocal microscopy. Sequences of single confocal z-sections acquired at 0.4-1m intervals throughout the T cells are shown here and in Supplementary Movie 4. Scale bar, 1.5 1m.

  4. Supplementary Movie 4 (1M)

    Membrane-tethered NPs are retained on the cell surface of tumor homing T lymphocytes. Explanted cross-sectioned EG7-OVA tumors 2 d after T cell injection (Fig 3c). CellTracker green–labeled OT-1 T cells and rhodamine lipid–coated NPs (magenta) were visualized by confocal microscopy. Sequences of single confocal z-sections acquired at 0.4-1m intervals throughout the T cells are shown here and in Supplementary Movie 3. Scale bar, 1.5 1m.

Additional data