Programmable multispecific DNA-origami-based T-cell engagers

All of the experiments and experimental circumstances described all through this examine adjust to the moral rules set forth by the institutional evaluate board of the medical college of the Ludwig-Maximilians-Universität and the Regierung von Oberbayern (approval of animal experiments).

Chemical substances, antibodies and cell strains

Except in any other case talked about, chemical substances used inside this work have been bought from Sigma-Aldrich and all of the IgG antibodies, RPMI, PBS, FBS and penicillin–streptomycin have been bought from Thermo Fisher.

The human cell strains Jurkat (T-cell leukaemia, DSMZ, no. ACC-282), NALM-6 (B-cell precursor leukaemia, DSMZ, no. ACC-128), MCF-7 (breast adenocarcinoma, DSMZ, no. ACC-115) and Molm-13 (acute myeloid leukaemia, DSMZ, no. ACC 554) have been obtained from DMSZ. Human cell line LNCaP (metastatic lesion of prostate adenocarcinoma, CLS, 300265) was obtained from CLS. All of the cell strains have been saved in liquid nitrogen. Profitable cell-line authentication was completed by way of polymerase chain response for Jurkat, NALM-6, LNCap and MCF-7.

Jurkat, NALM-6 and Molm-13 cells have been grown in RPMI 1640 medium, supplemented with 10% FBS, penicillin (200 U ml^–1), streptomycin (200 µg ml^–1) and with or with out extra 20 mM l-glutamine for Jurkat and NALM-6, respectively. The MCF-7 cells have been grown in high-glucose (25 mM d-glucose) Dulbecco’s modified Eagle’s medium medium, supplemented with 5% FBS, penicillin (200 U ml^–1), streptomycin (200 µg ml^–1) and 4 mM l-glutamine and passaged utilizing Trypsin-EDTA (0.05%). Each SK-BR-3 and LNCaP cells have been grown in RPMI 1640 medium, supplemented with 20% FBS, penicillin (200 U ml^–1), streptomycin (200 µg ml^–1) and a pair of mM l-glutamine and passaged utilizing TrypLE Choose Enzyme.

The murine A20 cell line was saved in liquid nitrogen and grown in RPMI 1640, supplemented with 10% FBS, penicillin (200 U ml^–1), streptomycin (200 µg ml^–1), 200 µM l-glutamine, 25 mM d-glucose, 10 mM HEPES and 1 mM sodium pyruvate.

The cells have been cultivated in T-75 cell tradition flasks at 37 °C and 5% CO₂. The cells have been maintained in line with the directions from DSMZ and used for circulate cytometric and cell-based assays as much as a most of 15 passages. This cell line was used as a murine B-cell lymphoma mannequin.

For in vivo fashions, NALM-6 tumour cells have been lentivirally transduced with a pCDH-EF1a-eFly-eGFP plasmid as beforehand described^49,50. Quick tandem repeat profiling was used to confirm the origin of this cell line.

For circulate cytometric experiments, the next antibodies have been used as bought.

Folding of DNA origami objects (chassis)

The response mixtures contained scaffold DNA at a focus of fifty nM and oligonucleotide strands at 200 nM every. The response buffer included 5 mM Tris, 1 mM EDTA, 5 mM NaCl (pH 8) and 20 mM MgCl₂. The response mixtures have been subjected to a thermal annealing ramp utilizing Tetrad (MJ Analysis, now Bio-Rad) thermal biking gadgets. Oligonucleotides have been obtained from IDT. DNA scaffolds have been produced in-house in line with one other work⁵¹. The desk under exhibits the folding ramps used to assemble the objects described within the manuscript.

Gel electrophoresis of PTEs

Folded DNA nanostructures have been electrophoresed on 1.5% to three.5% agarose gels containing 0.5× TBE and MgCl₂ at completely different concentrations for round 2 h at 70 V bias voltage in a gel field immersed in a water tub, until specified in any other case. The electrophoresed agarose gels have been scanned utilizing a Storm FLA 9500 laser scanner (GE Healthcare) at 50 µm per pixel decision. The ensuing 16-bit TIFF photos have been analysed utilizing ImageJ v. 1.440. For every lane that contained the pattern, a cross-sectional depth profile was calculated by averaging over grey-scale values inside a 50-pixel-wide field. The height intensities of the monomers and higher-ordered bands have been decided within the goal band. These depth values have been used for additional evaluation.

Purification, enrichment and in vitro stabilization of PTEs

After the folding response, all of the response merchandise have been purified utilizing PEG precipitation⁵². For concentrating DNA origami objects (chassis or PTEs), PEG precipitation or ultrafiltration was used. All of the procedures have been carried out as beforehand described⁵³. Concentrations of DNA origami objects have been analysed with a Nanodrop 8000 instrument (Thermo Fisher). Earlier than utilizing the objects in cell tradition assays, the objects have been stabilized to be used in low-ionic-strength buffers and the presence of nucleases. To that finish, we used the protocol from one other work²² and coated all our constructions with K10-PEG oligolysine, bought from Alamanda Polymers.

Antibody–DNA conjugation, antibody digestion and attachment to DNA origami objects

Conjugation of full-sized IgG antibodies: oligonucleotides modified with 5’- or a 3’-thiol modification have been bought, HPLC purified and dried (Biomers). The oligos have been dissolved in PBS (100 mM NaPi, 150 mM NaCl, pH 7.2) with 5 mM TCEP and incubated for 1 h at RT. After purification, 10 nmol of the lowered thiol oligo was blended with 10 equivalents of sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Sulfo-SMCC; dissolved in ddH₂O) (Thermo Fisher) for 15 min. After purification, together with buffer change to PBS (pH 8), 100 µg of antibody in PBS (pH 8) was added. The conjugate was subsequently purified by ion change chromatography (proFIRE, Dynamic Biosensors) utilizing a NaCl gradient of 150–1,000 mM in PBS (pH 7.2). Purified oligo–antibody conjugates have been analysed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and agarose gel electrophoresis.

Preparation of Fab fragments: full-sized IgGs have been digested and purified utilizing the Pierce Fab Preparation Package (Thermo Fisher, 44985) in line with the provider’s guide. Briefly, IgGs have been incubated with papain beads and purified from Fc fragments utilizing Protein-A affinity beads. Fab era was checked utilizing sodium dodecyl sulfate–polyacrylamide gel electrophoresis.

Conjugation of Fab fragments: to keep away from an orientation the place the F(ab)’s paratope factors in the direction of the DNA origami chassis, we relied on a site-specific conjugation methodology just like one other work⁵⁴. Briefly, maleimide-modified DNA was bought from Biomers or ready by mixing amine-modified DNA with an SMCC crosslinker and subsequent ultracentrifugation (10k filters, Amicon). Fab fragments, which have been produced utilizing the papaine digestion of IgG (Pierce Fab Preparation Package, Thermo Fisher, 44985) have been lowered with 5 mM TCEP for 30 min. Extra TCEP was eliminated utilizing ultracentrifugation (10k filters, Amicon), and blended with maleimide-modified DNA strands in 50 mM HEPES with 200 mM NaCl at pH 6.7. Reactions have been carried out in a single day at room temperature. This conjugation method ends in single- and double-labelled Fab fragments. Single-labelled F(ab)–DNA conjugates have been purified utilizing ion change chromatography.

Conjugation of scFv: maleimide-modified DNA was bought from Biomers or ready by mixing amine-modified DNA with SMCC crosslinker. scFv contained a free N-terminal cysteine and have been expressed and bought from Genscript or Icosagen. The response was carried out as described for the Fab fragments.

Attachment of antibody–DNA conjugates to DNA origami objects: antibody–DNA conjugates and DNA origami objects with the corresponding binding websites have been incubated in equimolar ratios for 1 h at 37 °C.

Adverse-stain TEM

Preparation, acquisition and information processing

Purified response merchandise have been adsorbed on glow-discharged Cu400 TEM grids (Science Companies) and stained utilizing a 2% aqueous uranyl formate answer containing 25 mM sodium hydroxide. The samples have been incubated for 30 s at 20–25 mM Mg²⁺. Magnifications between ×10,000 to ×30,000 have been used for buying the info.

Imaging was carried out on completely different microscopes.

TEM micrographs used within the figures have been high-pass filtered to take away long-range staining gradients and the distinction was auto-levelled (Adobe Photoshop CS6).

Fluorescence microscopy

Fluorescence microscopy experiments have been obtained on a Thermo Fisher CX7 confocal microscope with an on-stage incubator. Incubation circumstances for time-resolved measurements have been similar to the cell tradition circumstances used for the respective cell strains. The samples have been incubated on 96-well ibidi plates (89626).

Cell-based assays

Cell floor binding experiments

For circulate cytometric experiments, the cells have been grown to a cell density of 1.5–2.0 × 10⁶ cells ml⁻¹ in T-75 cell tradition flasks. The cells have been centrifuged for five min at 160×g and washed with ice-cold PBS, twice. All of the circulate cytometric experiments have been executed at a cell density of two × 10⁷ cells ml⁻¹ in PBS or medium. The pattern (chassis or PTE) was added at a remaining focus of 1 nM and incubated for the completely different time factors. Earlier than circulate cytometric evaluation, the cells have been centrifuged for five min at 500×g and resuspended to a remaining cell focus of two × 10⁶ cells ml⁻¹ in PBS. Move cytometric evaluation was carried out on a Cytoflex (Beckman Coulter) or Attune Nxt (Thermo Fisher), measuring the fluorescent depth by excitation at 640 nm and band-pass filter detection at 660/20 nm. The one cells have been gated primarily based on the ahead scatter versus aspect scatter. For every measurement, fluorescent intensities of fifty,000 particular person cells have been analysed with an in-house MATLAB (R2021b) script.

T-cell activation assay

The expression of interleukin 2, as an indicator for T-cell activation, was analysed utilizing T-Cell Activation Bioassays (Promega, J1655)³⁵. The experiment was carried out in line with the provider’s directions. Briefly, CD19-expressing goal cells (NALM-6) have been added to 96-well microtiter plates at a remaining focus of 5 × 10⁵ cells ml⁻¹. Then, a serial dilution of various samples (in RPMI 1640 medium) was added. Ultimately, the genetically modified TCR/CD3 effector cells have been added at a remaining focus of 1.3 × 10⁶ cells ml⁻¹. The response combination was incubated for six h at 37 °C and 5% CO₂. The genetically modified effector cells (Jurkat-NFAT) intercellularly specific a luciferase if the interleukin 2 promoter is activated. By the addition of the Bio-Glo reagent, which features a substrate for the luciferase, the luminescence sign is a direct proportional sign for the activation of the TCR/CD3 effector cells, which was analysed in a microtiter plate reader (Clariostar Plus, BMG). Knowledge have been normalized and the background sign was corrected.

Internalization assay

The internalization assay was carried out in line with one other work⁵⁵. Every DNA origami brick (chassis) carried a fluorescence internalization probe (FIP) comprising a protruding sequence with a terminal-attached Cy5 dye (Supplementary Fig. 4, proper inset). This FIP will be quenched utilizing a quenching strand with a complementary sequence to the FIP and an connected Black Gap Quencher-2. On hybridization, Black Gap Quencher-2 quenches the fluorescence of the FIP. Because the quencher strand can solely attain the chassis on the cell floor, and never the internalized chassis, the quantity of quenched sign is proportional to the quantity of surface-exposed chassis.

NALM-6 cells (1 × 10⁷ cells ml^–1) have been incubated with 1 nM chassis for 1 h at 37 °C within the cell tradition medium after which washed to take away the surplus chassis. After washing, the cells have been resuspended within the cell tradition medium and incubated at 37 °C. At every time level, a measurement consists of taking a pattern and incubating it for 10 min at 4 °C on ice. Half of the pattern is incubated with no quencher strand and the opposite half is incubated with a 100 nM quencher strand. Each samples have been incubated for 10 min on ice to permit for quencher hybridization (if added) and to cease internalization. The fraction of the internalized chassis with two anti-CD19 antibodies was calculated from the median fluorescence F as follows.

Cytotoxic T-cell-killing assay of liquid tumour cells

Preparation of goal cells

Goal cells (for instance, NALM-6) have been fluorescently stained utilizing CellTrace CSFE Cell Proliferation Package (Thermo Fisher, C34554) in line with the producer’s protocol.

Preparation of PBMC (effector)

For the cytotoxic T-cell-killing assay, we used frozen human PBMC (STEMCELL, 70025.1 or CTL, CTL-UP1). We dealt with the PBMC cells in line with the provider’s instruction.

Assay

Right here 2 × 10⁵ CSFE-stained goal cells per millilitre have been incubated with 1 × 10⁶ PBMC ml^–1 within the cell tradition medium at 37 °C (5% CO₂) and the PTEs in numerous concentrations or with out an extra recruiter. Cell fluorescence and scattering depth have been decided utilizing an Attune Nxt circulate cytometry with a Cytkick Max autosampler (Thermo Fisher). Residual contaminations, equivalent to salts or endotoxins might trigger the non-specific lysis of goal cells. These results are notably pronounced at excessive PTE concentrations and depend upon the cell sort.

Cytotoxic T-cell-killing assay of stable tumour cells

Preparation of goal cells: goal cells (for instance, MCF-7) have been seeded 24 h prematurely to from a confluent monolayer.

Preparation of PBMC (effector): for the cytotoxic T-cell-killing assay, we used frozen human PBMC (STEMCELL, 70025.1 or CTL, CTL-UP1). We dealt with the PBMC cells in line with the provider’s instruction.

Assay: confluent goal cells have been incubated with PBMC (E:T 2:1 or 4:1) in cell tradition medium at 37 °C (5% CO₂) and the PTEs in numerous concentrations or with out an extra recruiter. The fraction of alive goal cells was decided by quantifying the quantity of ATP after 48 h by way of Bio-Glo Cell Titer System (Promega) of samples with and with out PTEs. Residual contaminations, equivalent to salts or endotoxins, might trigger the non-specific lysis of goal cells. These results are notably pronounced at excessive PTE concentrations and depend upon the cell sort.

In vivo animal fashions

Endotoxin willpower of in vivo prepared constructs

The endotoxin focus was measured with a Charles River nexgen-PTS150 V10.2.3 instrument. We used cartridges with a variety between 5.00 and 0.05 EU ml^–1. The samples have been diluted 25-fold to suit into the delicate vary of the cartridges. The endotoxin threshold degree for mouse research was set to 36 EU ml^–1. This worth is in accordance with the specs given by the FDA⁴².

In vivo mouse experiments

Approval for all of the animal experiments was granted by the native regulatory authorities (Regierung von Oberbayern).

NOD.Cg-Prkdc^scid Il2rg^tm1WjI/SzJ (NSG) have been bought from Janvier or bred in-house. NSG mice carry a mutation within the Pkrdc DNA restore gene, related to extreme mixed immunodeficiency, resulting in a T- and B-cell deficiency. The entire null allele of the IL-2 receptor gamma chain (ll2rg) abrogates the cytokine signalling of crucial homoeostatic cytokines equivalent to IL-2, IL-4, IL-7, IL-9 and IL-15, stopping the event of practical NK cells. Lastly, the NOD background additional compromises the innate department of the immune system (lowered performance of dendritic cells and macrophages). Generally, these extremely immunodeficient mice assist secure and reproducible engraftment of human tumour and T cells in mice and are at present thought to be the state-of-the-art mannequin for human xenograft fashions in mice^56,57,58.

All of the animals have been housed in particular pathogen-free amenities.

BLI and fluorescence imaging have been carried out utilizing the in vivo imaging platform Lumina X5 (IVIS, PerkinElmer), as beforehand described⁵⁹. Briefly, mice have been anaesthetized with a 1.5–2.5% isoflurane–oxygen combination for all of the live-imaging procedures. For BLI, the substrate (Xenolight d-luciferin potassium salt, PerkinElmer) was intraperitoneally injected in line with the producer’s directions. For organ evaluation, the background fluorescence of every organ was subtracted.

NALM-6-luc⁺-GFP⁺ xenograft fashions have been established by i.v. tail-vein injection.

No information factors (mice) have been excluded within the animal research.

Move cytometry for in vivo experiments

Move cytometric information have been generated utilizing a BD LSRFortessa II, a Beckman Coulter CytoFLEX LX or a Thermo Fisher Attune Nxt with an autoloader. Move cytometric evaluation of organs was carried out as beforehand reported⁶⁰. Single-cell suspensions of harvested organs have been stained with human anti-CD3 BV711 (clone, OKT3), anti-CD4 PerCP-Cy5.5 (clone, OKT4), anti-CD8 PE (clone, HIT8a), anti-CD19 BV786 (clone, HIB19) and anti-CD69 PE-Cy7 (clone, FN50) or mouse anti-CD45 pacific blue (clone, 30-F11) antibodies (Biolegend). Fixable viability dye (eFluor 780, eBioscience) was used to exclude the useless cells. The utmost tumour burden permitted by the native regulatory authorities was not exceeded.

Software program and statistical analyses

Move cytometric information have been analysed utilizing FlowJo v. 10.3 to v. 10.8.1 software program. Quantifying bioluminescence and fluorescence intensities was completed utilizing Dwelling Picture 4.4 (PerkinElmer). Statistical evaluation was carried out with GraphPad Prism v. 9.4.0. Energy calculations (for in vivo experiments) have been carried utilizing G*Energy 3.1 with given alpha, energy and impact dimension.

Statistics and reproducibility

Statistics and reproducibility are acknowledged within the legend of the figures, for instance, organic or technical replicates. Agarose gel photos proven within the figures are consultant examples of experiments that yielded the identical or related outcomes. For TEM evaluation: the overall variety of equally carried out TEM evaluation of samples ready following the identical protocols assorted between experiments—in case of replicates, reproducibility was noticed.

Reporting abstract

Additional data on analysis design is offered within the Nature Portfolio Reporting Abstract linked to this text.