Nanoscale artificial antigen presenting cells for T cell immunotherapy

Abstract

Artificial antigen presenting cells (aAPC), which deliver stimulatory signals to cytotoxic lymphocytes, are a powerful tool for both adoptive and active immunotherapy. Thus far, aAPC have been synthesized by coupling T cell activating proteins such as CD3 or MHC-peptide to micron-sized beads. Nanoscale platforms have different trafficking and biophysical interaction properties and may allow development of new immunotherapeutic strategies. We therefore manufactured aAPC based on two types of nanoscale particle platforms: biocompatible iron-dextran paramagnetic particles (50-100 nm in diameter) and avidin-coated quantum dot nanocrystals (~30 nm). Nanoscale aAPC induced antigen-specific T cell proliferation from mouse splenocytes and human peripheral blood T cells. When injected in vivo, both iron-dextran particles and quantum dot nanocrystals enhanced tumor rejection in a subcutaneous mouse melanoma model. This is the first description of nanoscale aAPC that induce antigen-specific T cell proliferation in vitro and lead to effective T cell stimulation and inhibition of tumor growth in vivo.

From the clinical editor: Artifical antigen presenting cells could revolutionize the field of cancer-directed immunotherapy. This team of investigators have manufactured two types of nanoscale particle platform-based aAPCs and demonstrates that both iron-dextran particles and quantum dot nanocrystals enhance tumor rejection in a melanoma model, providing the first description of nanoscale aAPCs that lead to effective T cell stimulation and inhibition of tumor growth.

Keywords: Artificial antigen presenting cell; Immunotherapy; Nanoparticle; T cell.

Figure 1

Synthesis and Characterization of Iron-Dextran Nano-aAPC . Nano-aAPC were synthesized in one of two ways: (A) Direct chemical coupling of soluble MHC-Ig dimer (signal 1) and B7.1-Ig (signal 2) in a 1:1 molar ratio to the surface of a paramagnetic iron-oxide, dextran-coated particle. (B) Binding of biotinylated MHC-Ig dimer (signal 1) and biotinylated anti-CD28 (signal 2) in a 1:1 molar ratio to anti-biotin coated particles. (C) Nanoparticle tracking analysis confirms that nano-aAPC are a monodisperse mixture of particles with a mean diameter of 50–100 nm suspended at a concentration of 8.3 nM.

Figure 2

Nano aAPC Induced Proliferation is Antigen-Specific and Dose-Dependent (A) Antigen specific nano-aAPC induce proliferation. T cells were counted seven days after stimulation with anti-biotin coated nano-aAPC to calculate fold expansion from day 0. TCR transgenic 2C (grey) and pMEL (white) T cells proliferated only when incubated with nanoparticles bearing cognate MHC/peptide (22-fold and 16-fold, respectively), and not in the presence of naoparticles bearing either non-cognate peptide or non-cognate MHC (<3-fold). (B) Addition of both signal 1 and signal 2 leads to optimal T cell expansion. At a dose of 10 µL particles per 1*10⁶ T cells, only anti-biotin particles bearing both MHC-Ig and anti-CD28 induced robust T cell expansion. Proliferation of CD8+ CTL induced by Low Density, LD (16 µg protein/mL particles), and High Density, HD (65 µg protein/mL), particles. Results are representative of three experiments. (C) Equivalent doses of HD and LD particles were used to stimulate pMEL T cells. Proliferation was measured by dilution of CFSE three days after stimulation. Decreased fluorescence indicates increased proliferation. Equivalent volumes of HD particles induce greater proliferation than LD particles, with 0.5 uL LD particles inducing almost no expansion. (D) Fold expansion on day 7 of dose equivalent samples shows a similar pattern. Proliferation is dose-dependent and 2–4 fold greater for HD particles compared to an equivalent dose of LD particles (21-fold compared to 7-fold at 5 µL). (E) Day 3 CFSE dilution of CD8+ CTL induced by LD and HD particles at equivalent protein concentrations, with approximately 5.5-fold more LD than HD at a given dose. When particle doses are normalized to equivalent protein concentrations, particles induce similar amounts of CFSE dilution. (F) Fold expansion on day 7 demonstrates equivalent expansion for HD and LD particles at an equivalent protein dose (17-fold at 3.5 µL of HD and 20 µL of LD). A threshold of about 0.5 uL LD particles or 0.08 uL HD particles is required to induce detectable expansion.

Figure 3

Synthesis and Characterization of Quantum Dot Nano-aAPC (A) Quantum Dot (Qdot) Nano-aAPC were constructed by avidin-biotin mediated coupling of soluble MHC-Ig dimer (signal 1) and anti-CD28 antibody (signal 2) in a 1:1 ratio to the surface of a polymer-coated quantum dot particle. (B) Qdot Nano-aAPC expansion in whole CD8+ T cells. Fold expansion on Day 7 is dose dependent and antigen-specific. Non cognate particles did not induce any expansion, whereas the highest dose of cognate QD aAPC (D^b-GP100) induced approximately 15 fold expansion of CTL. Results are representative of 3 experiments.

Figure 4

Antigen-specific Human T Cell Expansion From Endogenous Precursors (A) CD8+ T cells were isolated from PBMC by magnetic enrichment and incubated with increasing doses of iron-dextran nano-aAPC bearing A2-Ig complexes loaded with antigen derived from the immunodominant epitope of the influenza M1 protein, and assessed for antigen-specificity by tetramer staining before stimulation (PBMC, top row) and after one (middle row) or two (bottom row) weeks of stimulation. Numbers in top left represent percentage of CD8+ cells that were tetramer positive (gated).The size of the M1 specific population increases with repeated rounds of stimulation (top to bottom) and increasing dose of nano-aAPC (left to right), from 0.36% of CD8+ PBMC to 77.7% at the highest dose. Plots are representative of results from three separate experiments, summarized in panel B. (B) Percentage of CD8+ PBMC binding HLA-A2 M1 tetramers increases with repeated stimulation and increasing dose of nano-aAPC (left panel). The total number of tetramer positive cells (right panel) similarly increases with rounds of stimulation and particle dose, expanding up to 800-fold over the initial precursor population.

Figure 5

Enhanced Drainage of Nano- Compared to Micro-aAPC. (A) Visualization of drainage of near infrared labeled micro-aAPC (left) compared to nano-aAPC (right) after subcutaneous injection on right flank. Right flank views are shown for representative mice (3 mice/group) at the indicated timepoints after injection. Legend at right relates color in image to arbitrary fluorescence units. Micro-aAPC are confined largely to injection site, whereas local drainage of Nano-aAPC is more pronounced. (B) Biodistribution is quantified as area of visible drainage at indicated timepoints. Nano-aAPC have five-fold greater area of drainage than micro-aAPC at equivalent timepoints. (C) Simultaneous NIR images of biodistribution for pMEL T cells (green) and aAPC (red). Forty-eight hours after intravenous injection, T cells are visible in axillary lymph nodes, spleen, inguinal lymph nodes (white arrows, left to right) and cervical lymph nodes (not pictured). Right flank view (top row) shows nano-aAPC which were injected in right hindlimb 48 hrs. earlier reach inguinal lymph node, whereas micro-aAPC do not. This is even more pronounced after dissection (bottom row); aAPC signal is stronger and can be seen in the area of the inguinal lymph node (white arrow) for nano- but not micro-aAPC. Images are representative of three mice.

Figure 6

Nano-aAPC Inhibit Tumor Growth In Vivo (A) QD aAPC. B16 Tumors were injected subcutaneously on day 0, with injection of naive pMEL T cells on the same day. One day later, QD aAPC were injected intravenously (iv). IL-2 was administered on days 3, 4, and 5. Tumor size was measured as surface area (mm²) on indicated days, with area under the curve (AUC) shown at right. Mice treated with pMEL T cells and cognate QD aAPC (black bars) had less tumor growth compared to no treatment (white), T cells alone (light grey), and T cells + noncognate QD aAPC (checkered) (4 mice per group). Significance was characterized over entire experiment by AUC (p<0.001 by ANOVA with Tukey’s Post-Test, * indicates significant difference from no treatment group). (B) Iron-Dextran aAPC. Naive pMEL T cells were injected intravenously on day -7. One day later, iron-dextran aAPC were injected either iv or subcutaneously (sc) on the right flank. B16 tumors were injected sc on right flank on day 0. Mice in treatment arms were given an additional injection on day 4 post tumor injection either iv or sc, to form four treatment groups: noncognate aAPC iv (day -6) then sc (day 4) (checkered), cognate aAPC iv then iv (light grey), cognate aAPC iv then sc (dark grey), and cognate aAPC sc then sc (black). Mice treated with pMEL T cells and cognate Iron-Dextran aAPC iv/sc or sc/sc (filled squares) had less tumor growth compared to noncognate aAPC (7 mice per group, p<0.02 by ANOVA with Tukey’s Post-Test, * indicates significant difference from no treatment group).