In situ programming of leukaemia-specific T cells using synthetic DNA nanocarriers

Abstract

An emerging approach for treating cancer involves programming patient-derived T cells with genes encoding disease-specific chimeric antigen receptors (CARs), so that they can combat tumour cells once they are reinfused. Although trials of this therapy have produced impressive results, the in vitro methods they require to generate large numbers of tumour-specific T cells are too elaborate for widespread application to treat cancer patients. Here, we describe a method to quickly program circulating T cells with tumour-recognizing capabilities, thus avoiding these complications. Specifically, we demonstrate that DNA-carrying nanoparticles can efficiently introduce leukaemia-targeting CAR genes into T-cell nuclei, thereby bringing about long-term disease remission. These polymer nanoparticles are easy to manufacture in a stable form, which simplifies storage and reduces cost. Our technology may therefore provide a practical, broadly applicable treatment that can generate anti-tumour immunity 'on demand' for oncologists in a variety of settings.

Figure 1. Design and manufacture of lymphocyte-programming nanoparticles

a, Schematic of the T-cell-targeted DNA nanocarrier used in our experiments. The inset shows a transmission electron micrograph of a representative nanoparticle. Scale bar, 100 nm. Also depicted are the two plasmids that were encapsulated into the nanoparticles; these encode an all-murine 194-1BBz CAR and the hyperactive iPB7 transposase. EF1A, eukaryotic translation elongation factor 1 alpha 1; BGH PA, bovine growth hormone polyadenylation signal; ampicillin resistance gene; ORI, origin of replication. b, Diagram describing the fabrication of the poly(β-amino ester) nanoparticles. Also shown are the chemical structures of the PBAE 447 polymer and polyglutamic acid, as well as the amino acid sequence of the microtubule-associated-nuclear localization (MTAS-NLS) peptide.

Figure 2. DNA nanocarriers choreograph robust and persistent CAR production by lymphocytes in vitro

a, Flow cytometry of nanoparticles binding to T cells. Splenocytes from naive C57BL/6 mice were mixed with CD3-targeted nanoparticles carrying Cy5-labelled plasmid DNA. After a 20-min incubation, cells were washed to remove unbound particles, and analysed by flow cytometry. The profiles shown here are representative of eight independent experiments. b, Confocal microscopy establishes that nanoparticles loaded with Cy5-labelled DNA (magenta) are rapidly (within 120 min) internalized from the cell surface. To provide contrast, T cells were labelled with CellTracker Green prior to nanoparticle exposure. The images are representative of 20 randomly chosen fields. Scale bars, 2 μm. c, Flow cytometry of T cells 30 h after incubation with nanoparticles bearing 194-1BBz_2A_GFP genes. The graph displays CD3⁺-gated lymphocyte populations. d, Comparison of T-cell transfection efficiencies achieved with DNA nanocarriers that contain microtubule-associated and nuclear localization signalling peptide sequences with those that do not, based on 10 independent experiments. e, Flow cytometry of T cells 30 h after transduction with a lentiviral vector encoding the same 194-1BBz_2A_GFP construct. The numbers within the graphs in c and e show the percentage of CAR + T cells (using GFP as surrogate marker). f, In vitro assay comparing cytotoxicity of nanoparticle-transfected versus lentivirus-transfected T cells against Eμ-ALL01 leukaemia cells, or the B16F10 cell line as a control. To ensure equal CAR expression levels, transfected T cells were sorted using FACS to GFP mean fluorescence intensities (as a surrogate reporter for CAR expression) between 10³ and 10⁴ before using them in the functional assays. Each point represents the mean ± s.e.m. pooled from independent experiments conducted in triplicate. g, ELISA measurements of IL-2 (at 24 h), and IFN-γ and TNF-α (at 48 h) secretion by transfected cells following co-culture with Eμ-ALL01 leukaemia cells. Unstimulated, lentivirus-transduced 194-1BBz CAR T-cells were analysed for comparison. h, Co-delivery of plasmids encoding the hyperactive piggyBac transposase iPB7 promotes persistent CAR gene expression. After transfection and sorting, CAR-positive T cells were cultured and the persistence of their 194-1BBz_2A_GFP expression was measured with flow cytometry. Data are representative of two independent experiments.

Figure 3. CD3-targeted nanoparticles bind to circulating T cells in mice

a, Flow cytometry demonstrating fluorescent nanoparticle binding to peripheral T cells 4 h after a 3 × 10¹¹ dose was injected. The right panel is confocal microscopy of CD3-sorted T cells, establishing that, like in vitro, the particles are rapidly internalized from the surfaces of circulating cells. Shown below are the phenotypes of other circulating cell subtypes that non-specifically bound the injected nanoparticles, as measured by flow cytometry: neutrophils (Ly6G⁺, CD11b⁺, CD11c⁻), monocytes (Ly6C⁺, CD11b⁺, CD11c⁻), eosinophils (CD11b⁺, CD193⁺, F4/80⁺), natural killer (NK) cells (CD49b⁺, NKp46⁺), B cells (B220⁺). Data are representative of two independent experiments with two animals per treatment group. Scale bar, 3 μm. b, Phenotypes of circulating T cell subtypes internalizing injected nanoparticles, as measured by flow cytometry: naive T cells (CD62L⁺, CD44⁻), effector T cells (CD62L⁻, CD44⁺), central memory (CM) T cells (CD62L^high, CD44⁺), effector memory (EM) T cells (CD62L^low, CD44⁺), and regulatory T (Treg) cells (CD4⁺, Foxp3⁺, CD25⁺). The CD4:CD8 ratio of nanoparticle-transfected T cells is shown as a pie chart in the centre; the bar graphs on the sides reflect percentages of each T-cell subtype. c, Biodistribution of fluorescent T-cell-targeted or non-targeted nanoparticles 4 h after tail-vein injection. Data are expressed as injected dose (ID) per gram of tissue. Bl, blood; Li, liver; Sp, spleen; Ln, lymph node; Bm, bone marrow; Lu, lung; Si, small intestine; Ki, kidney; Mu, muscle. Data are from ten mice per treatment condition pooled from two independent experiments. Each bar represents the mean percentage of ID per gram tissue ± s.e.m. d, Bioimaging of nanoparticle distributions. One representative mouse from each cohort (n = 10) is shown. A bar graph on the right reflects percentages of splenocytes positive for fluorescent nanoparticles in animals treated with CD3-targeted nanoparticles, as measured by flow cytometry: T cells (CD3⁺), macrophages (F4/80⁺, CD11b⁺, CD11c⁻), monocytes (CD11b⁺, Gr1⁺, F4/80^low), and B cells (B220⁺).

Figure 4. Reprogramming host T cells with leukaemia-specific CAR genes

a, Top: flow cytometry of peripheral T cells following injection of nanoparticles delivering DNA that encodes 194-1BBz_2A_GFP or tumour-irrelevant P4-1BBz_2A_GFP genes. The profiles shown here are representative of two independent experiments consisting of five mice per group. Bottom: to determine whether persistent CAR expression in actively dividing T cells requires co-delivery of plasmid encoding the hyperactive transposase, we also compared 194-1BBz transgene-loaded nanocarriers containing or lacking iPBS7 transgenes. b, Sequential bioimaging of nanoparticle-programmed CAR⁺ T cells. In this experiment, nanoparticles were loaded with plasmids that co-express the click beetle red luciferase (CBR-luc) reporter along with the CAR transgene. As in the previous experiments (panel a), 194-1BBz CAR-encoding nanoparticles were prepared with or without iPB7 transgenes. Five representative mice from each cohort (n = 10) are shown. c, Plots of CBR-luc signal intensities after nanoparticle injections. Each line represents one animal and each dot reflects its whole animal photon count. Pairwise differences in photon counts between treatment groups were analysed using the Wilcoxon rank-sum test. Shown are data for ten mice per treatment condition pooled from three independent experiments.

Figure 5. Nanoparticle-programmed CAR lymphocytes can cause tumour regression with efficacies similar to adoptive T-cell therapy

a, Sequential bioimaging of firefly luciferase-expressing Eμ-ALL01 leukaemia cells systemically injected into albino C57BL/6 mice. One week after this injection (Day 0), the animals were treated with five sequential injections (Day 0-Day 5) of 3 ×10¹¹ lymphocyte-targeting nanoparticles carrying 194-1BBz or P4-1BBz CAR-encoding transgenes. To test whether integration of nanoparticle-delivered CAR transgenes into the chromosomes of in situ reprogrammed T cells is a requirement to achieve anti-leukaemia effects, we injected 194-1BBz transgene-loaded nanocarriers with or without iPBS7 transgenes into two different treatment groups. Controls were not treated. An additional group of mice was first given cyclophosphamide, then a day later treated with a single dose of 5 million CAR⁺ T cells that had been transduced ex vivo with 194-1BBz-encoding lentiviral vectors. Five representative mice from each cohort (n = 10) are shown. b, Quantification of the results shown in a. Every line represents one animal and each dot reflects the whole animal photon count. c, Survival of animals following therapy, depicted as Kaplan-Meier curves. Shown are ten mice per treatment group pooled from three independent experiments. ms, median survival. Statistical analysis between the treated experimental and the untreated control group was performed using the Log-rank test; P <0.05 was considered significant. d, Flow cytometry plots showing killing of malignant and normal B cells 12 days after treatment with 194-1BBz-encoding nanoparticles. The numbers labelling the peaks represent percentage of GFP-negative (left) and GFP-positive (right) cells. The leukemia cells were GFP-positive, whereas the endogenous B cells are GFP-negative. To distinguish leukaemia from healthy B lymphocytes, Eμ-ALL01 cells were genetically tagged with GFP. The respective subsets are illustrated in separate histogram plots that are gated on CD19⁺ cell populations. Data are representative of ten mice per treatment group pooled from three independent experiments.