METHODS FOR THE NANOCONFINED CULTIVATION OF T-, B- AND NK- CELLS

Abstract

The present invention is directed to a method for the cultivation, optionally activation and growth of lymphocytes (T-, B- and NK-cells) by culturing these cells in a suitable cell growth medium on a nanoporous substrate having a pore diameter in the range of about 100 to 500 nm, optionally about 150 to 250 nm.

Claims

1. A method for the cultivation of lymphocytes (T-, B- and NK-cells) comprising the step of culturing the cells in a suitable cell growth medium on a nanoporous substrate, characterized in that the nanoporous substrate has a pore diameter in the range of about 100 to 500 nm.

2. The method according to claim 1, wherein the nanoporous substrate has a pore diameter in the range of 150 to 400 or 150 to 250, optionally of about 200 nm.

3. The method according to claim 1 or 2, wherein the nanoporous substrate is based on, optionally consists of a material selected from the group consisting of polymers, optionally polystyrene, polycarbonate, polydimethyl sulfate (PDMS), aluminum oxide, anodic aluminum oxide (AAO), titanium oxide, anodic titanium oxide (ATO), nanotubes, silicon, silicon oxide, silicon nitride, silicon carbide, diamond, diamond-like carbon, glassy carbon, optionally selected from the group consisting of polydimethyl sulfate (PDMS), silicon oxide, and AAO, optionally AAO.

4. The method according to any of claims 1 to 3, wherein the nanoporous substrate is coated, (i) optionally coated for T-cells with (poly)peptides, optionally antibodies, antibody fragments, antibody derivatives or antibody analogues, optionally against CD3 and/or CD28 and/or CD19, and/or with a polymer selected from the group consisting of poly-D-Lysine (PDL), poly-L-Lysine (PLL), and polyethylene glycol (PEG); (ii) optionally coated for B-cells with (poly)peptides, optionally antibodies, antibody fragments, antibody derivatives or antibody analogues, optionally against CD40 and/or IgM while optionally supplemented with IL4 and/or CpG, and/or with a polymer selected from the group consisting of poly-D-Lysine (PDL), poly-L-Lysine (PLL), and polyethylene glycol (PEG); and (iii) optionally coated for NK-cells with (poly)peptides, optionally antibodies, antibody fragments, antibody derivatives or antibody analogues, optionally against CD16, NKG2D, SLAM family members and/or at least one of the natural cytotoxicity receptors NKp30, NKp44 and NKp46, and/or with a polymer selected from the group consisting of poly-D-Lysine (PDL), poly-L-Lysine (PLL), and polyethylene glycol (PEG).

5. The method according to any of claims 1 to 4 for activating T-cells, wherein the nanoporous substrate is coated with antibodies, antibody fragments, antibody derivatives or antibody analogues against CD3 and/or CD28.

6. The method according to any of claims 1 to 4 for activating B-cells, wherein the nanoporous substrate is coated with antibodies, antibody fragments, antibody derivatives or antibody analogues against aCD40 and/or IgM.

7. The method according to any of claims 1 to 4 for activating NK-cells, wherein the nanoporous substrate is coated with antibodies, antibody fragments, antibody derivatives or antibody analogues against CD16, NKG2D, SLAM family members and/or at least one of the natural cytotoxicity receptors NKp30, NKp44 and NKp46.

8. The method according to any of claims 1 to 4 and 5 for producing activated T-cells, comprising the steps of (i) providing and optionally surface cleaning the nanoporous substrate, optionally chemically and/or by plasma; (ii) optionally surface functionalization, optionally with antibodies, (poly)peptides and/or polymers; (iii) culturing T-cells, optionally genetically engineered T-cells, optionally CAR T-cells, on the surface of the porous substrate, optionally prior, during and/or after the activation; and (iv) optionally co-culturing the T-cells with further different cell types.

9. The method according to any of claims 1 to 4 and 6 for producing activated B-cells and/or for producing antibodies, comprising the steps of (i) providing and optionally surface cleaning the nanoporous substrate, optionally chemically and/or by plasma; (ii) optionally surface functionalization, optionally with antibodies, (poly)peptides and/or polymers; (v) culturing B-cells, optionally genetically engineered B-cells, on the surface of the porous substrate, optionally prior, during and/or after the activation; and (vi) optionally co-culturing the B-cells with further different cell types, optionally forming hybridoma cells.

10. The method according to any of claims 1 to 4 and 7 for producing activated NK-cells, comprising the steps of (i) providing and optionally surface cleaning the nanoporous substrate, optionally chemically and/or by plasma; (ii) optionally surface functionalization, optionally with antibodies, (poly)peptides and/or polymers; (vii) culturing NK-cells, optionally genetically engineered NK-cells, optionally CAR NK Cells on the surface of the porous substrate, optionally prior, during and/or after the activation; and (viii) optionally co-culturing the NK-cells with further different cell types.

Description

FIGURES

[0046] Figures for T-Cells

[0047] FIG. 1 a is a confocal fluorescent microscopy image of Jurkat T-cells on a porous AAO with 200 nm pore diameter (3D view), scale bar 10 μm. Bottom—shows a schematic presentation of the actin-rich protrusions in T-cells (not to scale).

[0048] FIG. 1b shows the confocal fluorescent microscopy images of TCR from T-cells on a porous surface at the basal membrane (upper panel) or inside the pores (lower panel).

[0049] FIG. 1 c are bar diagrams showing activation of human primary T-cells on porous and non-porous surfaces. +/− signs indicate the presence of activation antibodies (αCD3/CD28) on the surface. CD69 expression and IL-2 secretion were measured after 24 hours, and CD25 expression was measured after 4 days. Three independent experiments were performed in duplicates or triplicates. The p-values were determined by two-sided Mann-Whitney tests in R.

[0050] FIG. 2 a) are two photographs showing the proliferation of primary human T-cells activated on different surfaces with aCD3/CD28 immobilized on the surface. The representative microscope images show proliferation of T-cells 5 days after activation (left: activated on non-porous surface, and right: activated on porous surface). The dark areas are clusters of proliferated T-cells. T-cells were activated on the non-porous or porous surfaces and were transferred to a plastic culture dish on day 3.

[0051] FIG. 2 b) is a bar plot of the statistical analysis of fold expansion of T-cells that were activated on porous or non-porous surfaces (2 independent experiments with 3 or 5 replicates). The cells were cultured for expansion according to the standard protocol. A 100-fold expansion was obtained when cells were activated on the porous surfaces.

[0052] Figures for B Cells

[0053] FIG. 3 is a column graph demonstrating early activation as checked via CD69 surface expression and flow cytometry. The further three column graphs relate to activated B-cells after 5 days that were stained intracellularly for IgG and analyzed by flow cytometry according to Example II.1 below. The graphs show percentages and representative histograms of proliferated (CTV-) cells, IgG+ cells among proliferated CTV cells as well as mean fluorescent intensity (MFI) of IgG staining of CTV-B cells on porous and non-porous surfaces, which represents the enhanced antibody production.

[0054] FIG. 4 a) is a column graph demonstrating early activation as checked via CD69 surface expression and flow cytometry. Primary B cell are solely activating via physical constraints of the nanopores for different nanopore dimensions. CD69 levels are shown for flat AAO and nanopores ranging from 20 nm, 100 nm, 200 nm till 400 nm diameter.

[0055] FIG. 4 b) is a column graph demonstrating early activation as verified via CD69 surface expression and flow cytometry. Primary B cell are activated using anti CD40 and anti IgM antibodies, immobilized on the AAO surface via Streptavidin, furthermore IL2 and IL4 are supplemented.

[0056] FIG. 4 c) (left) is a confocal fluorescent microscopy image of a primary B cell on a porous AAO with 200 nm pore diameter. Actin cytoskeleton is stained post-fixation using phalloidin-Alexa647. The red dots represent protrusions reaching into the AAO substrate. The image plane is 1 micrometer below AAO surface. (right) are normalized fluorescence intensity versus nanopore depth plots of confocal microscopy data for at least 50 protrusions of 5 B cells. Primary B cells 1 hour post seeding on 200 nm AAO surfaces with antibodies against IgM and CD40 are stained for pSyk, BCR and actin. Data shows enrichment of pSyk, BCR and actin within the protrusions.

EXAMPLES

[0057] Materials & Methods

[0058] Porous anodic aluminum oxide (AAO) samples were round 13 mm diameter Whatman Anodisc Circles (Sigma-Aldrich), with 20 nm (WHA68097003), 100 nm (WHA68097013) and 200 nm (WHA68097023) pore diameter, or polystyrene/polycarbonate membranes (Isopore Membrane Filters, GTTP01300, HTTP01300) with 200 and 400 nm pore diameter.

[0059] High throughput RNA-sequencing, RNA extraction, differential expression analysis, plots (2D multidimensional scaling plot of filtered data with edgeR, volcano plots generated with EnhancedVolcano package (Blighe et al. EnhancedVolcano, R package version 1.4.0. (2019), gene-set enrichment analysis, flow cytometry, ELISA, immunostaining, optical microscopy, electron microscopy and statistical methods (using R, ±s.d., two-sided Mann-Whitney tests) were employed and routinely adapted to the T and B cells and the task.

[0060] ERK (Extracellular signal-regulated kinase) inhibition was achieved by incubating cells with U0126 (Abcam, ab120241) at different doses (0, 0.1, 1 and 10 μM diluted in Anhydrous DMSO (Sigma-Aldrich). The cells were then incubated with the porous/non-porous surfaces for 0-60 min and 24 hours. The cell concentration was 5×10.sup.5 cells/mL.

[0061] For Polymer coating the AAO membrane was first spin coated with photoresist (PR) AZ1518 (MicroChemicals GmbH) at 1750 rpm for 1 min and baked at 100° C. for 3 min. Reactive-ion etching (RIE) (Oxford, Plasmalab 80) was used to create and control different depths of nanohole on composite AAO-PR substrate. AAO-PR substrates were etched by RIE for 10, 15, 20 and 30 min with O.sub.2: 20 sccm and RF power of 100 watt, achieving the respective depths of 0.5, 1, 2 and 4 μm. The AAO-PR nanohole was sputter coated with a 5 nm layer of Pt for SEM (Zeiss ULTRA 55) analysis.

Example I T Cell Activation and Expansion

Example I.1—Preparation of Non-Porous Aluminum Oxide Samples

[0062] Non-porous aluminum oxide samples were prepared by deposition of 40 nm of Al.sub.2O.sub.3 on a 13 mm round coverslip (1.5 H, 0117530, Marienfeld) using atomic layer deposition (ALD, Picosun Sunale R-150B). Briefly, ultrahigh-purity nitrogen carrier gas was purged at a flow rate of 200 sccm and a pressure of about 1 Torr was maintained. Al.sub.2O.sub.3 ALD was conducted with alternating exposures to trimethylaluminum (TMA) and water. TMA exposure and purge times were 0.1 and 4 s, respectively. The deposition was conducted at 150° C. with 400 cycles (0.1 nm/cycle).

Example I.2 Cleaning the Surfaces

[0063] The samples were cleaned in an air-plasma (3 min at 18 W, using a PDC-32G; Harrick Plasma, USA). For glass coverslips, the samples were sonicated in acetone and isopropanol solutions (3 min each), rinsed with MilliQ water and dried with a nitrogen flow prior to the plasma cleaning. All samples were autoclaved at 120° C. for 2 h for experiments with primary human T-cells.

Example I.3 Antibody Coating of the Surfaces

[0064] To coat the surface of the samples with activating antibodies (aCD3 and aCD28), a streptavidin intermediate was used. Streptavidin (Thermo Fisher Scientific, 434301) was diluted in PBS (ROTI Cell PBS, Carl Roth) to the concentration of 10 μg/mL. 100 μL of diluted streptavidin was added directly on top of the samples and was incubated for 30 min at room temperature. The samples were washed with 100 μL PBS (three times) before adding the activating antibodies.

[0065] Monoclonal CD3 and CD28 antibodies were used as activating antibodies. 100 μl of 5 μg/mL biotinylated CD3 (Thermo Fisher Scientific, 13-0037-82) and CD28 (Thermo Fisher Scientific, 13-0289-82) antibodies were added on top of each sample, and were incubated for 20 min at room temperature. The samples were then washed with PBS three times and were transferred to 24-well plates for cell seeding.

Example 1.4 Cell Seeding

[0066] T-cells were freshly plated at a density of 2.5×10.sup.5 cells per well in a 24-well plate containing the 13 mm samples at the bottom. The medium consisted of RPMI (RPMI 1640, Invitrogen) with 10% fetal bovine serum (ATCC-LGC Standards). Cells were incubated for the planned duration at 37° C. and 5% CO 2, without any further supplements.

Example 1.5 Human Primary T-Cells

[0067] Collection of plasma and PBMCs was approved by the Kantonale Ethikkommission Zurich (KEK-ZH-Nr. 2012-0111), and written consent was obtained from all subjects. A total of 6 separate healthy donors participated in this study. The average age of healthy donors was 35.7±3.1 (mean±s.d.). All experiments were performed in accordance with relevant guidelines and regulations.

Example I.6 Isolation of the Primary Human T-Cells

[0068] Pan T-cells were purified from total peripheral blood from healthy adult volunteers. Freshly donated whole blood was first diluted to half with PBS (ROTI Cell PBS, Carl Roth) at room temperature. Then 35 mL of the diluted blood was added to 50 mL centrifuge tubes (SepMate, Stemcell Technologies) which were prefilled with 15 mL of density gradient medium (Lymphoprep, Stemcell Technologies). The samples were centrifuged at 1200×g for 10 min at room temperature. PBMCs in the top layer were washed with PBS and T-cells were isolated by negative selection using EasySep Human Naïve Pan T Cell Isolation Kit (Stemcell Technologies #17961), according to manufacturer's protocol. Briefly, 50 μL of isolation cocktail antibodies and 50 μL of TCR gamma/delta depletion cocktail were added to 1 mL of Lymphoprep-purified PBMCs for 5 min at room temperature. Next, 60 μL of RapidSpheres™ were added per 1 mL of sample for another 3 min at room temperature, the sample was topped up to 2.5 mL and placed on a magnet (EasySep magnet, StemCell Technologies) for 3 min. The unlabeled cells were poured into a new tube, washed, counted and used for further applications.

Example I.7 T Cell Culture

[0069] Primary T cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) (ATCC-LGC Standards), 1× penicillin/streptomycin, 2-ME (50 mM), nonessential amino acids (ThermoFischer Scientific), sodium pyruvate (ThermoFischer Scientific), HEPES (ThermoFischer Scientific) and glutamate (ThermoFischer Scientific). Jurkat cells (an immortalized line of human T-cell), were cultured in RPMI 1640 (Invitrogen) supplemented with 10% FBS and 1× penicillin-streptomycin (Invitrogen), and were kept in an incubator at 37° C. and 5% CO 2 for at least 3 days before measurements. To maintain the concentration of less than 10.sup.6 cells/mL, cells were split 1:4 with fresh media every 3-4 days. Prior to the experiments, cells were counted, centrifuged at 300×g for 3 minutes at room temperature and then resuspended in the fresh medium to a concentration of 5×10.sup.5 cells/mL. The amount of 0.5 mL of the cell suspension was then seeded into 24-well plates which contained the 13 mm samples in the bottom. The cells were incubated at 37° C. and 5% CO.sub.2 before further analysis.

Example I.8 Experimental Results and Conclusions

[0070] In summary, culturing T-cells on membranes with pores of about 100 to 500, in particular about 150 to 250 nm, for example about 200 nm, significantly boosts their activation and proliferation (see FIG. 2). Engineered nanoporous AAO membranes provide a guide for actin-rich nanoscale protrusions (see FIG. 1A), boosting T-cell activation and proliferation (see FIG. 1 C). The boosted activation of T-cells is facilitated via pore diameter-dependent segregation of membrane proteins, amplifying and sustaining the necessary signaling events. Kinetic segregation can be locally induced when T-cells protrude into nanoporous openings (see FIG. 1 B). Notably, cell signaling cascades and gene regulation are significantly altered in cells cultured on nanoporous membranes. ERK phosphorylation—a major component of MAP Kinase pathway and T-cell activation—is augmented and sustained in T-cells on nanoporous substrates, e.g. AAO, contributing to the activation of the cells. The synergistic combination of protrusion formation and antigen stimulation presents a simple and inexpensive strategy to optimize in vitro T-cell activation and can be used for adoptive T-cell therapy.

Example II—B Cell Activation and Expansion on Porous Surface

Example II.1 Surface Modification without IgM (Standard for B-Cell Activation)

[0071] Porous AAO with 200 nm pore diameter and non-porous glass surfaces were treated with oxygen plasma for 1 min, then coated with 10 ug/ml streptavidin containing PBS and subsequent incubation with 50 ug/ml ICAM for 30 min followed by biotinylated anti-CD40 for 15 min.

Example II.2 Surface Modification with IgM

[0072] Additionally to the above described surface modification the role of surface presentation of anti-IgM aside anti-CD40 was investigated. Therefore, the surface with anti-IgM and a-CD40 for 15 min was co-incubated prior to cell seeding.

Example II.3 B-Cell Seeding

[0073] Primary B-cells were purified from Peripheral Blood Mononuclear Cells (PBMCs) with an EasySep StemCell isolation kit, before seeding onto the modified AAO surface. B-cells were cultured for different times in the presence of 30 U/ml IL2 and 20 ng/ml IL4 (to compare porous with non-porous substrates.

Example II.4 B-Cell Activation Via aCD40

[0074] After 30 min B-Cell seeding, samples were fixed with PFA, stained with phalloidin-Alexa647 and analysed by confocal microscopy. Early activation was checked via CD69 surface expression and flow cytometry (FIG. 3). After 5 days, activated B cells were stained intracellularly for IgG and analysed by flow cytometry. Shown are percentages and representative histograms of proliferated (CTV-) cells, IgG+ cells among proliferated CTV-cells as well as mean fluorescent intensity (MFI) of IgG staining of CTV-B cells on porous and non-porous surfaces, which represents the enhanced antibody production (FIG. 3).

Example II.5 B-Cell Activation Via Anti-CD40 and Anti-IgM

[0075] Porous AAO with 200 nm pore diameter and non-porous glass surfaces were treated with oxygen plasma for 1 min, then coated with 10 ug/ml streptavidin for 30 min followed by biotinylated anti-CD40 and anti-IgM for 15 min. Primary B cells were purified from PBMCs with an EasySep StemCell isolation kit, labeled with CTV for 10 min at 37° C. and cultured on the coated surfaces in the presence of 30 U/ml IL2 and 20 ng/ml IL4. After 24 h, activated B cells were stained for CD69 surface expression and measured by flow cytometry.

Example II.6 Experimental Results and Conclusions

[0076] Similar to the above-described results for T cells it was demonstrated that B cells can form actin-rich protrusions in pores of about 100 to 500, in particular about 150 to 250 nm, for example about 200 nm. It was found that the surface topography on which B cells are cultured strongly enhances their activation (as measured and shown by CD69 expression), proliferation (as measured by CTV dilution) and most importantly antibody production (as measured by intracellular staining of IgG) (see FIG. 3). The activation for B-cells is similar to that of T-cells and is correlated with porous substrates directly regulating the gene expression program responsible for activation, proliferation and differentiation into a sub-species of B-cells with enhanced antibody production qualities.

[0077] In summary, culturing B-cells on membranes with pores of about 100 to 500, in particular about 150 to 250 nm, significantly boosts their activation, proliferation and antibody production (see FIG. 3). Engineered nanoporous AAO membranes provides a guide for actin-rich nanoscale protrusions (see FIG. 3), boosting B-cell activation and proliferation (see FIG. 3). The boosted activation of B-cells is facilitated via pore diameter-dependent segregation of membrane proteins, amplifying and sustaining the necessary signaling events (see FIG. 4). Kinetic segregation is locally induced when B-cells protrude into nanoporous openings. The synergistic combination of protrusion formation and antigen stimulation presents a simple and inexpensive strategy to optimize in vitro B-cell activation and is of value for protocols for adoptive cell therapy and antibody production.

[0078] Most current protocols for activating naive or memory B cells involve costimulation of CD40 and IL4 with or without the presence of soluble TLR ligands such as CpG. Similar to the findings on T cells, it was demonstrated that B cells can form actin-rich protrusions in specifically diameter-limited pores. Additionally, the surface topography on which B cells are cultured can strongly enhance their activation (as measured by CD69 expression), proliferation (measured by CTV dilution) and most importantly antibody production as measured by intracellular staining of IgG. The activation for B-cells is similar to that of T-cells and can be correlated with porous substrates directly regulating the gene expression program responsible for activation, proliferation and differentiation into a sub-species of B-cells with enhanced antibody production quality.

Example III NK-Cell Activation and Expansion

Example III.1 Surface Modification with aCD16 (Standard for NK-Cell Activation)

[0079] Porous AAO with about 200 nm pore diameter and non-porous glass surfaces can be treated with oxygen plasma for 1 min, then coated with 10 ug/ml streptavidin containing PBS and subsequently incubated with biotinylated anti-CD16 to activate NK cells.

Example III.2 Surface Modification with Antibodies Against NKG2D, SLAM Family Members and the Natural Cytotoxicity Receptors NKp30, NKp44 and NKp46

[0080] Additionally to the above described surface modification the role of surface presentation of antibodies against NKG2D, SLAM family members and the natural cytotoxicity receptors NKp30, NKp44 and NKp46 can be optionally used for NK cell activation. Therefore, the nanoporous surface can optionally be co-incubated with one or more of the mentioned antibodies, optionally against CD16 prior to cell seeding.

Example III.3 NK-Cell Seeding

[0081] Primary NK-cells can be purified from Peripheral Blood Mononuclear Cells (PBMCs) with an EasySep StemCell isolation kit, before seeding onto the modified AAO surface. NK-cells can be cultured for different times in the presence of TGF-β and/or IL15 and/or IL18 (to compare porous with non-porous substrates).