BIOFUNCTIONALIZED NANOPARTICLES AND USES THEREOF IN ADOPTIVE CELL THERAPY

Abstract

The present invention relates to biofunctionalized nanoparticles and uses thereof in adoptive cell therapy. In particular, the present invention relates to a nanoparticle comprising an amount of at least one antigen and an amount of at least one antibody having specificity for a B cell receptor wherein the antigen and antibody are attached to the surface of the nanoparticle.

Claims

1. A nanoparticle comprising an amount of at least one antigen and an amount of at least one antibody having specificity for a B cell receptor wherein the antigen and antibody are attached to the surface of the nanoparticle.

2. The nanoparticle of claim 1 which is in the form of a sphere, needle, flake, platelet, tube, fiber, cube, prism, whiskers or which has an irregular shape and which is a mineral nanoparticle, is made of an organic polymer or is made of polysaccharides.

3. The nanoparticle of claim 1 wherein the at least one antigen is a viral antigen, a bacterial antigen, a fungal antigen, a protozoal antigen, a tumor-associated antigen, an auto-antigen, an allergen, a xenoantigen, an alloantigen, or a molecule that is exogenously administered for therapeutic or other purposes and may trigger an unwanted immune response.

4. The nanoparticle of claim 1 wherein the at least one antigen is a HLA molecule.

5. The nanoparticle of claim 1 wherein the antibody has specificity for the framework region of a kappa or lambda BCR light chain or for the framework region of a BCR heavy chain.

6. The nanoparticle of claim 1 wherein at least 2 or 3 anti-BCR antibodies are attached to the nanoparticles.

7. A method for preparing a population of antigen-presenting B cells comprising incubating a population of B cells with an amount of the nanoparticles of claim 1 for a time sufficient for allowing internalization of the nanoparticles into the B cells and isolating B cells that present the at least one antigen at their surface by MHCII molecules.

8. The method of claim 7 further comprising a step of conferring regulatory properties to said population of antigen-presenting B cells.

9. A method for expanding a population of antigen-specific T helper cells comprising i) providing a population of the antigen-presenting B cells prepared according to the method of claim 7 and ii) culturing a population of T cells in the presence of the population of the antigen presenting B cells of step i).

10. The method of claim 9 which further comprising a step of isolating the antigen-specific T helper cells.

11. The method of claim 9 further comprising a step of polarizing said population of antigen-specific T helper cells into a population of antigen-specific Th1, Th2 or Th17 cells.

12. The method of claim 9 further comprising a step of polarizing said population of antigen-specific T helper cells into a population of antigen-specific regulatory cells.

13. A method of treating a cancer, an infectious disease, an autoimmune disease, an allergy, an immune reaction against a molecule that is exogenously administered for a therapeutic purpose or an immune reaction against a grafted tissue or grafted hematopoietic cells or grafted blood cells in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the nanoparticles according to claim 1, a population of antigen-presenting B cells prepared by incubating B cells with an amount of the nanoparticles for a time sufficient to allow internalization of the nanoparticles into the B cells and isolating B cells that present the at least one antigen at their surface, or a population of antigen-specific T cells prepared by culturing T cells in the presence of the antigen presenting B cells.

Description

FIGURES

[0065] FIG. 1A-F. Biofunctionnalization of nanospheres to generate synthetic particulate antigens (SPAg).

[0066] (a) Schematic representation of the system used to vectorize chosen antigen into non-cognate B cell: 400 nm fluorescent streptavidin-coated nanospheres are decorated with biotinylated-antigens and biotinylated-monoclonal antibody (mAb) directed against a framework region of B cell receptor's kappa light chains (anti-K mAb).

[0067] (b) (Top) Verification of nanospheres coating with anti-K mAb by electron microscopy (EM) using gold particle-coupled anti-IgG secondary antibody. (Bottom) Coating efficiency using biotinylated (bonds; black histogram) versus purified (adsorption; grey histogram) anti-K mAb was compared in flow cytometry using a PE-conjugated anti-IgG secondary antibody.

[0068] (c) Verification of double coating with biotinylated-anti-K mAb and antigen (ovalbumin) by flow cytometry.

[0069] (d, left) Impact of polybiotinylation (blue histogram) vs monobiotinylation (orange histogram) of the proteins used for coating on nanospheres flocculation assessed by flow cytometry analysis of the particle size (The Forward-Scattered Light parameter (FSC) is proportional to the size of the analyzed event).

[0070] (d, right) After coating of nanospheres with mono-biotinylated proteins, aggregates were excluded by filtration through a low-binding membrane with a pore size of 650 nm. Histogramms and representative images of the SPAg fluorescence analyzed by imaging flow cytometry in the upper (orange) and lower (red) chambers of centrifugal devices.

[0071] (e) Estimation of the number of antigen molecules coated on each nanosphere by the saturation curve method. Nanospheres were incubated with increasing amounts of biotinylated ovalbumin. Ovalbumin binding was measured by flow cytometry with a FITC-conjugated anti-ovalbumin mAb.

[0072] (f) Numeration of SPAg by spectrophotometry using the standard curve method. Uncoated nanospheres samples of known concentrations are used as standards.

[0073] FIG. 2A-D. SPAg behave like genuine particulate antigen.

[0074] (a) Scheme representing the first steps of B cell activation by a particulate antigen. The binding of cognate epitope to B cell receptors (BCR) provides the first signal of activation (signal 1) to B cells and leads to the internalization of the antigen in the endosomal compartment where the antigen is then processed and loaded onto MHC class II molecules.

[0075] (b) B cells from wild type mice were incubated 10 minutes at 4 C. with SPAg and washed. (Left) Flow cytometry dot plots showing SPAg fluorescence according to B220 and lambda-chain expression. (Upper right) Flow cytometry dot plots showing the percentages of follicular (FO: CD21.sup.intCD23.sup.high) and marginal zone (MZ: CD21.sup.highCD23.sup.low) subsets among kappa positive B cells without (left, blue) and with (right, red) SPAg. (Lower right) EM images of B cells without (left, blue) or with (right, red) SPAg bound to their surface.

[0076] (c) SPAg triggers BCR signalling cascade. B cells from wild type mice were incubated 1, 2 or 3 minutes at 37 C. with either anti-IgM soluble Fab2 (positive control) or SPAg. Imaging flow cytometer was used to detect the phosphorylated form of the B cell linker protein (p-BLNK). (Left) Representative images showing p-BLNK staining in lambda positive and lambda negative B220+ B cells after anti-IgM (upper) or SPAg (lower) stimulation. (Right) The percentage of SPAg that have triggered BCR signalling at 1, 2 and 3 minutes is shown.

[0077] B cells from wild type mice were incubated 10 minutes at 37 C. with SPAg.

[0078] (d) Kinetic of SPAg internalization was defined using Image flow cytometer. (Upper) B cells were incubated at various time points with a fluorescent anti-ovalbumin mab, only non-internalized SPAg were accessible to the staining. (Lower) The percentages of SPAg internalized (red) and bound to B cell surface (green) is shown at various time points.

[0079] FIG. 3A-D. SPAg-loaded B cells induce antigen-specific T cells activation and proliferation.

[0080] (a) Scheme representing B cell presenting the antigen to CD4+ T cell. Internalized antigen is processed in late endosomal compartment, loaded onto MHC class II molecules, and presented on B cell surface. Together with the expression of costimulation molecules (CD80/86), antigen presentation leads to activation and proliferation of cognate CD4+ T cells.

[0081] (b) SPAg-induced MHC class II and CD86 upregulation. B cells from wild type mice were incubated (red histogram) or not (black histogram) with SPAg 10 minutes at 37 C. and cultured overnight. MHC class II (upper) and CD86 (lower) expression was assessed by flow cytometry.

[0082] SPAg-loaded B cells are potent antigen-presenting cells to CD4+ T lymphocytes.

[0083] (c) Bone marrow-derived dendritic cells (BMDC) or B cells from wild type mice were incubated 4 hours with soluble ovalbumin or with SPAg before being cocultured 72 hours at a 1:1 ratio with CTV-labelled ova-specific CD4+ T cells from OTII transgenic mice. Expression of the activation marker CD25 by OTII CD4+ T cells was assessed at 72 hours by flow cytometry.

[0084] (d) OTII CD4+ T cell proliferation was measured at 72 hours by flow cytometry in the various culture conditions.

[0085] FIG. 4A-C. SPAg-based approach is applicable in humans.

[0086] Purified B cells from a healthy volunteer were incubated 30 minutes at 37 C. with SPAg coated with anti-human kappa-light chain mAbs (hSPAg) and washed.

[0087] (a) Flow cytometry dot plots showing hSPAg fluorescence according to lambda-chain expression by B cells.

[0088] (b) Image flow cytometer was used to evaluate hSPAg internalization after 12 hours culture. (Left) Representative images showing hSPAg internalized in CD19+ B cells. (Right) Quantification of cells that have internalized hSPAg among lambda+ and kappa+ B cells.

[0089] (c) (Top) Schematic representation of the experimental procedure. Purified B cells from an HLA-DR01/01 healthy volunteer were incubated 30 minutes at 37 C. with hSPAg coated with either ovalbumin (negative control, hSPAg-ova) or a peptide made of 3 repetitions of a sequence of HIV-GAG protein (hSPAg-GAG). After washing, B cells were rested 12 hours and then coculture 6 hours with HLA-DR1-restricted CD4+ T cell clones specific for the HIV-GAG protein. Clones activation was assessed by flow-cytometry measurement of the intracellular cytokine MIP-1. (Bottom) Flow cytometry dot plots showing hSPAg fluorescence according to lambda-chain expression by B cells (left) and MIP-1 staining in CD4+ T cells clones (right) under three different experimental conditions (clones cocultured with B cells without hSPAg, with B cells-hSPAg-ova and with B cells-hSPAg-GAG).

[0090] FIG. 5A-C. SPAg-loaded B cells to expand rare cognate CD4+ T cells.

[0091] SPAg-loaded B cells from WT mice were cocultured 72 hours at a 1:2 ratio with a mix of cognate OTII CD4+ T cells (CD45.2) and polyclonal T cells (CD45.1). CD45.2/CD45.1 ratios were (a) 1/100, (b) 1/1000 or (c) 1/10000. (Far left) Flow cytometry analysis at 72 hours showing CD45.1 and CD45.2 staining of CD4+ T cells and (middle left) their respective proliferation profiles assessed by CTV staining. (Middle right) Bar charts representing the proportion of CD45.1 and CD45.2 CD4+ T cells that have undergone division. (Far right) CD45.2/CD45.1 ratios at the beginning (HO) and at the end (72 hours) of the coculture.

[0092] FIG. 6A-D. SPAg-loaded B cells to polarize cognate CD4+ T cells.

[0093] (a) B cells from wild type mice were incubated with SPAg 10 minutes at 37 C., washed and cocultured 5 days at a 1:1 ratio with OTII CD4+ T cells in media without exogenous cytokines (Th0, black plots), with recombinant IL12 (Th1, red plot, 20 ng/ml) or with TGF (Treg, blue plot, 1 ng/ml). Flow cytometry dot plots showing IFN staining under Th0 and Th1 polarizing conditions (left) and Foxp3 staining under Th0 and Treg polarizing conditions (right) in CD4+ T cells at 5 days of coculture.

[0094] (b) A scheme describing how regulatory properties can be conferred to SPAg-loaded B cells. B cells are loaded with SPAg and cultured with CpG. SPAg-loaded B reg produce IL-10 (c), do not induce significant proliferation of antigen-specific Foxp3 effector T cells (Teff) but rather promote the proliferation of antigen-specific Foxp3+ regulatory T cells (Treg) (d, e).

[0095] (c) B cells from IL10-IRES-eGFP mice were incubated with SPAg 10 minutes at 37 C. and cultured 48 hours in medium supplemented with either anti-CD40 agonist (10 g/ml; black curve) or with CpG (12.5 g/ml; green curve). The proportion of eGFP-positive SPAg-positive B cells was measured by flow cytometry at various time points.

[0096] (d) B cells from wild type mice were incubated or not with SPAg 10 minutes at 37 C. and cultured 36 hours in medium supplemented with either anti-CD40 agonist or with CpG. B cells without SPAg and sorted SPAg-positive B cells were cocultured 5 days at a 1:1 ratio with CTV-labelled OTII CD4+ T cells. (left) The proportions of effector (Foxp3.sup.neg) and regulatory (Foxp3.sup.pos) CD4+ T cells were determined by flow cytometry at 3 and 5 days of coculture in the various culture conditions. (right) The proliferation profiles of effector (Foxp3.sup.neg) and regulatory (Foxp3.sup.pos) CD4+ T cells were determined by flow cytometry at 5 days of coculture in the various culture conditions. Data are representative of two independent experiments.

EXAMPLE 1: MATERIAL AND METHODS

[0097] Surrogate Particulate Antigens (SPAg)

[0098] Proteins Biotinylation

[0099] Ovalbumin (Ova, Sigma), purified rat anti-mouse K Light Chain (anti-K mAb, clone 187.1, Becton Dickinson (BD)) and purified mouse anti-human Light Chain (clone G20-361, BD) were monobiotinylated using the EZ-Link Sulfo-NHS-LC-LC-Biotin kit (Thermo Scientific). Briefly, proteins suspended in phosphate-buffered saline (PBS) were incubated for 30 minutes at room temperature with Sulfo-NHS-LC-LC-Biotin at a 1:1 molar ratio. Excess non-reacted biotin was eliminated with Zeba Spin Desalting Columns, 7K MWCO (Thermo Scientific). Biotin Quantitation Kit (Pierce, Thermo Scientific) was used to determine the level of biotin incorporation.

[0100] HIV-GAG Trimer Peptide

[0101] Custom monobiotinylated trimer peptide was synthetized by Lifetein. Purity was 95.82%. Sequence was: Biotin-IILGLNKIVRMYSPTSILDIRQGPK-IILGLNKIVRMYSPTSILDIRQGPK-IILGLNKIVRMYSPTSILDIRQGPK, 75aa.

[0102] Coupling Procedure

[0103] 400 nm flash red (660/690) streptavidin nanospheres (Bangs Laboratories) were washed two times (PBS, 1% bovine serum albumin (BSA; Sigma), 0.0005% Tween 20 (Sigma)) with 100 nm pore size Ultrafree-MC DV Centrifugal Filters (2000 G/2 minutes, Durapore, Merck Millipore). Nanospheres were incubated for 30 minutes at room temperature with excess monobiotinylated-ova or monobiotinylated-trimer HIV-GAG peptide and with anti-mouse or anti-human mAb with constant mixing.

[0104] Coated nanospheres (SPAg) were washed two times in Tween 20-free PBS-1% BSA buffer prior to filtration through 650 nm pore size Ultrafree-MC DV Centrifugal Filters (2000 G/2 minutes, Durapore, Merck Millipore).

[0105] SPAg Quantification

[0106] The standard curve method was used to numerate SPAg. Briefly, SPAg samples and serial dilutions of uncoated nanospheres of known concentrations were pipetted into 96 wells black plates with transparent bottom (Greiner Bio One) to establish a standard curve. Fluorescence was measured with a microplate reader (Infinite Reader M200, Tecan) at 660/690 nm. Data were analyzed with the i.control (v1.6) and Excel softwares.

[0107] Mice

[0108] Wild type C57BL/6 (CD45.2), Ly5.1 C57BL/6 (CD45.1) and OTII TCR transgenic C57BL/6 mice aged 8-15 weeks were purchased from Charles River Laboratories (Saint Germain sur l'Arbresle, France). IL10-reporter mice (IL10-IRESeGFP) were purchased from Jackson Laboratories (Bar Harbor, Me., USA). All mice were maintained under EOPS condition in our animal facility (PBES, Lyon, France). All experimental protocols were approved by the local ethical committee (CECCAPP).

[0109] Healthy Volunteer Blood Donation

[0110] Two HIV-seronegative healthy volunteer donors provided peripheral blood after informed consent. Blood collection was organized at the Etablissement Francais du Sang, Lyon, France.

[0111] Cell Preparations and Cultures

[0112] Mouse B Cells

[0113] After spleen cells were harvested and erythrocytes lysed (ACK Lysing Buffer, Invitrogen), B cells were enriched to >95% purity by negative selection using magnetic enrichment kits (R&D system or Milteny Biotec).

[0114] Human B Cells

[0115] Peripheral Blood Mononuclear Cells (PBMC) were isolated by centrifugation on Ficoll density gradient (Histopaque, 10777, Sigma). B cells were enriched to >95% purity by negative selection using magnetic enrichment kits (R&D system).

[0116] B Cells Loading with SPAg and Culture

[0117] Pre-warmed B cells were incubated with SPAg for 10, 30 minutes or 6 hours (according to the experiments) at 37 C. in 5% CO2 at a 100 SPAg to B cell ratio. After two washes, cells were cultured at 37 C. in 5% CO2, in complete media (mouse: RPMI 1640 media Glutamax (Invitrogen) supplemented with 10% FCS, 50 M -mercaptoethanol (Sigma), 25 mM Hepes (Invitrogen), and 10 units/mL penicillin/streptomycin (Invitrogen); humans: Yssel medium as previously described.sup.1). When indicated, anti-CD40 agonist (clone FGK45, 10 g/ml; Enzo Life Sciences Inc;) or CPG (1668, 12.5 g/ml; mWG Biotech) were added.

[0118] Bone Marrow-Derived Dendritic Cells (BMDC) Preparation and Loading

[0119] Dendritic cells were generated from bone marrow of C57Bl/6 mice as described previously.sup.2. Briefly, bone marrow was prepared from femurs and tibiae of mice. Cells were cultured in 6 wells plates at a concentration of 10.sup.6 cells/ml in 4 ml complete medium supplemented with 1% culture supernatant containing GM-CSF (4 ng/ml final concentration). After 3 days, medium was removed and replaced with 4 ml of fresh medium. On days, 6 and 9, 4 ml of fresh medium was added without disrupting cells. Cells were collected on day 12, washed and resuspended in complete medium without GM-CSF. Purity was assessed by flow cytometry and was >90% (CD11c+MHC-II+ cells). BMDC were incubated with soluble ovalbumin (200 g/ml) or with SPAg for 6 h at 37 C. in 5% CO2 before being used as stimulators in the presentation assay.

[0120] Mouse Presentation Assay

[0121] CD4+ T cells were enriched from spleens of Ly5.1 C57BL/6 (CD45.1) or OTII transgenic mice (CD45.2) to >95% purity by negative selection using magnetic enrichment kits (R&D system or Milteny Biotec). CD4+ T cells were labelled with cell trace violet according to the manufacturer's protocol (Molecular probes, Life technology) before being cocultured with B cells or BMDC at a 1:1 ratio (96 wells plates, 2.10.sup.5 cells per well). T cells proliferation was measured by flow cytometry at 3, 4 or 5 days.

[0122] Human Presentation Assay

[0123] Purified B cells from an HLA-DR01/01 healthy volunteer were incubated 30 minutes at 37 C. with hSPAg coated with trimeric HIV-GAG peptide. After washing, B cells were rested 12 hours in complete medium at 370 and then coculture 6 hours at a 1:1 ratio with HLA-DR01-restricted CD4+ T cell clones specific for the HIV-GAG protein. Clones were generated as previously described.sup.3. After one hour of coculture, monensin (Biolegend) was added to culture media. Clone activation was assessed by flow cytometry measurement of the intracellular cytokine MIP-1 at H6.

[0124] Flow Cytometry

[0125] Antibodies

[0126] In mice experiments, antibodies directed against the following targets were used: CD3 (clone 452C11, BD), CD4 (clone RM-44, BD), CD8 (clone 53-6.7, BD), CD11c (clone HL3, BD), CD19 (clone ID3, BD), CD21 (clone 7G6, BD), CD23 (clone B3B4, BD), CD25 (clone PC61, BD), CD45.1 (clone A20, BioLegend), CD45.2 (clone 104, eBiosciences), CD80 (clone 16-10A1, BD), CD86 (clone GL1, BD), B220 (clone RA3-6B2, BD), F4/80 (clone BM8, eBiosciences), lambda light-chain (clone JC5-1, Abcam), pBLNK (clone J117-1278, BD), MHC-II (clone 2G9, BD), foxp3 (clone FJK-16s, ebiosciences), rat IgG1 (clone G1 7E7, Abcam), ovalbumin (Abcam).

[0127] In human experiments, antibodies directed against the following targets were used: CD4 (clone RPA-T4, BD), DC-SIGN (clone DCN46, BD), CD19 (clone HIB19, BD), lambda light-chain (CLONE JDC-12, BD), MIP1b (Clone 24006, R&D system).

[0128] Procedures

[0129] Single cell suspensions were incubated with a blocking anti-mouse Fc receptor antibody (clone 2.4G2) to prevent non-specific antibody binding and then with relevant fluorescent monoclonal antibodies for 15 minutes at 4 C. in flow buffer (PBS-Azide 0.01%-SVF 2%-EDTA 0.5 mM). Dead cells were excluded by staining with Fixable Viability Dye (eBiosciences) or 4,6-diamidino-2-phenylindole (DAPI). For cytokine intracellular staining, cells were then fixed and permeabilized before being incubated for 30 minutes at 4 C. with the relevant antibodies. For foxp3 intranuclear staining, the mouse regulatory T Cell staining kit was used according to the manufacturer's protocol (eBiosciences).

[0130] For phosphoflow analysis, the phospho-epitopes exposure kit (Beckman Coulter) was used. Briefly, 1.10.sup.6 prewarmed B cells were stimulated with either anti-IgM soluble F(ab)2 (15 g/ml) or SPAg at 37 C. for 1, 2 or 3 minutes before being incubated 10 minutes at room temperature with fixative reagent and then 5 minutes at 37 C. with permeabilizing reagent. Before incubation with anti-lambda light chain, anti-B220 and mouse anti-pBLNK mAbs (30 minutes at room temperature), permeabilized cells were incubated for 15 minutes with kappa light-chain positive mouse IgG isotype control (clone X40, BD) in order to avoid reactivity between anti-K mAb present on SPAg and mouse PE anti-pBLNK mAb.

[0131] Data Collection

[0132] Data were collected on LSRII or LSR Fortessa flow cytometers (BD Biosciences, San Jose, Calif., USA) and analyzed with FlowJo software (v10.0). For image analysis, samples were acquired on a 4 laser ImageStream X Mark II (Amnis-EMD Millipore) with 60 magnification and analyzed with IDEAS software (v6.0).

[0133] Electron Microscopy

[0134] Ultrastructural Analysis of B Cells

[0135] B cells were incubated 10 minutes at 37 C. with SPAg, cultured overnight and fixed in glutaraldhyde 4% and cacodylate 0.2M. Samples were washed three times in saccharose 0.4M/0.2 M Na CHCl-Cacodylate-HCl Ph7.4 0.2M for 1 hour at 4 C., and postfixed with 2% OsO4/0.3M Na CHCl Cacodylate-HCl pH 7.4 for 1 hour at 4 C. Then, cells were dehydrated with an increasing ethanol gradient (5 minutes in 30%, 50%, 70%, 95%, and 3 times for 10 minutes in absolute ethanol). Impregnation was performed with Epon A (50%) plus Epon B (50%) plus DMP30 (1.7%). Inclusion was obtained by polymerisation at 60 C. for 72 hours. Ultrathin sections (approximately 70 nm thick) were cut on a Reichert ultracut E (Leica) ultramicrotome, mounted on 200 mesh copper grids coated with 1:1,000 polylisine, and stabilized for 1 day at room temperature (RT) and, contrasted with uranyl acetate and lead citrate. Sections were examinated with a Jeol 1400JEM (Tokyo, Japan) transmission electron microscope equipped with a Orius 1000 camera and Digital Micrograph (CIQLE-Centre d'Imagerie Quantitative Lyon Est-Universit Claude Bernard Lyon).

[0136] Verification of Nanospheres Coating with Biotinylated Rat Anti-Mouse Kappa-Light Chain mAb

[0137] Immunogold labelling was performed by floating the grids on drops of reactive media. Nonspecific sites were coated with 1% BSA and 1% normal goat serum in 50 mM Tris-HCl, pH8.2 for 20 min at room temperature. Thereafter, incubation was carried out 45 min at room temperature in wet chamber with 10 nm gold-conjugated goat anti-rat Ab (British Bio Cell international, 1/80) in 1% BSA+50 Mm Tris-HCl pH 8.2. The immunocomplexe was successively washed three times in 50 Mm Tris-HCl pH8.2 and pH 7.4 and three times with infiltrated distilled water and fixed by a wash in glutaraldehyde 4% for 3 min. Sections were stained with 0.5% uranyl acetate in ethanol 50% for 5 min in darkness and observed with a Jeol 1400JEM (Tokyo, Japan) transmission electron microscope equipped with a Orius 1000 camera and Digital Micrograph (CIQLE-Centre d'Imagerie Quantitative Lyon Est-Universite Claude Bernard Lyon).

[0138] Confocal Microscopy

[0139] B cells loaded with SPAg were cultured overnight. 1.10.sup.5 B cells were plated on 17 mm glass coverslips (Zeiss) preincubated 4 hours with 0.01% poly-L-lysine (Sigma). Cells were permeabilized with 0.05% saponin, then incubated for 30 minutes at room temperature with blocking solution (PBS-5% BSA) and stained with Alexafluor488-conjugated anti-B220 (clone RA3-6B2, BD) and PE-conjugated anti-LAMP1 (clone 1D4B) mAbs for 45 minutes at room temperature. After 3 washes with PBS-5% BSA, cells were stained with DAPI (1/5000) for 10 minutes. After 3 additional washes, coverslips were mounted on glass slides with fluoromount aqueous mounting medium (Sigma). Confocal 3D image stacks were acquired with confocal spectral SP5 microscope (Leica). Images were analyzed with FIJI software.

[0140] Data Analysis Statistical analyses and graphs were performed using Prism software (GraphPad, V6.0). Unless noted otherwise, the data are represented as mean valuesSD. p values <0.05 were considered significant (Mann-Whitney test).

REFERENCES

[0141] (1) Yssel, H.; de Vries, J. E.; Koken, M.; Van Blitterswijk, W.; Spits, H. J. Immunol. Methods 1984, 72, 219-227. [0142] (2) Lutz, M. B.; Kukutsch, N.; Ogilvie, A. L.; Rossner, S.; Koch, F.; Romani, N.; Schuler, G. J. Immunol. Methods 1999, 223, 77-92. [0143] (3) Moris, A.; Pajot, A.; Blanchet, F.; Guivel-Benhassine, F.; Salcedo, M.; Schwartz, O. Blood 2006, 108, 1643-1651.

EXAMPLE 2: B CELLS LOADED WITH SYNTHETIC PARTICULATE ANTIGENS: A VERSATILE PLATFORM TO GENERATE ANTIGEN-SPECIFIC HELPER T CELLS FOR CELL THERAPY

[0144] We have developed an innovative strategy for biofunctionalization of nanoparticles. Bioengineered nanospheres were specifically designed to simultaneously: i) provide activation signal, and ii) deliver non-cognate antigens to endosomes of B cells. Using these Synthetic Particulate Antigens (SPAg), we were able to turn resting polyclonal B cells into potent stimulators of antigen-specific CD4+ T cells.

[0145] To generate SPAg, proteins of interest were biotinylated and immobilized on fluorescent streptavidin-coated nanospheres of 400 nm in diameter, a size comparable to the one of a typical pathogen which offers a good compromise between the possibility to be internalized by B cells and binding capacity.sup.39. BCR is composed of 2 pairs of polypeptides chains: 2 heavy chains and 2 light chains. Light chains can be of two types: lambda or kappa. More than 90% of murine B cells express kappa light chains. Each chain comprises both a constant domain, which is a shared framework independent of the antigenic specificity, and a highly variable domain, which is specific to each B cell clone and involved in the recognition of antigenic epitopes. We assumed that coating the nanospheres with a biotinylated monoclonal antibody directed against a framework region of kappa light chain (anti-K mAb) would confer them the capacity to target any non-cognate kappa positive BCR while behaving like genuine particulate antigens (FIG. 1a). The coating of nanospheres with anti-K mAb was verified by electron microscopy (EM) using gold particle-coupled anti-IgG secondary antibody (FIG. 1b, top). To ensure that anti-K mAb were attached to nanospheres by streptavidin-biotin solid bonds rather than unspecific adsorption, coating efficiency using biotinylated versus purified anti-K mAb was compared in flow cytometry with a PE-conjugated anti-IgG antibody (FIG. 1b, bottom). The successful double coating of nanospheres with biotinylated anti-K mAb and ovalbumin, which was used herein as model antigen, was verified by flow cytometry (FIG. 1c). To limit flocculation of nanospheres caused by the coating procedure, anti-K mAb and ovalbumin had to be monobiotinylated rather than polybiotinylated (FIG. 1d, left). A final filtration of coated particles appeared necessary to exclude residual aggregates (FIG. 1d, right). An estimation of the mean number of antigen molecules that can be coated on each nanosphere was obtained by incubating nanospheres with increasing amounts of biotinylated ovalbumin and quantifying ovalbumin binding by flow cytometry with a FITC-conjugated anti-ovalbumin mAb (FIG. 1e). Based on the saturation curve obtained by this method, we evaluated that the number of ovalbumin molecules than can be coated on each SPAg was approaching 10000 (FIG. 1e).. The final concentration of SPAg after filtration was measured by spectrophotometry using the standard curve method (FIG. 1f).

[0146] To test the hypothesis that SPAg would bind to any non-cognate kappa positive BCR and behave like genuine antigens, SPAg were incubated in vitro with B cells purified from the spleen of a wild type mice at a 100:1 SPAg to B cell ratio. Each of the steps necessary for antigen-presentation by B cells were analyzed: (i) attachment to surface BCR, (ii) triggering of activation signal and (iii) internalization in late endosomal compartment.sup.37 (FIG. 2a). Flow cytometry analysis showed that SPAg bound exclusively to B220+ lambda chain-B cells, confirming that SPAg interaction with B cells was dependent upon anti- mAb (FIG. 2b, left). Noteworthy, SPAg were able to bind with equal efficiency to all B cells with kappa positive BCR, regardless the subset (follicular or marginal zone; FIG. 2b, upper right). Flow cytometry results were confirmed by EM analyses (FIG. 2b, lower right). To assess whether SPAg were able to trigger BCR signaling cascade, as would cognate antigen, imaging flow cytometer was used to detect the phosphorylated form of the B cell linker protein (p-BLNK), an adaptor protein that is phosphorylated upon BCR crosslinking.sup.40 (FIG. 2c). A circular p-BLNK signal was detected in both lambda and kappa positive B lymphocytes, when the cells were activated with anti-IgM soluble Fab2 (positive control, FIG. 2c upper left). In contrast, incubation of B lymphocytes with SPAg resulted in a punctiform p-BLNK signal, which colocalized with SPAg fluorescence and was only detected in kappa-positive B cells (FIG. 2c lower left). Sixty percent of SPAg bound to B cell surface had triggered BCR signaling at 3 minutes post-incubation (FIG. 2c right). The internalization of SPAg, which is indispensable for antigen presentation, was analyzed by EM (data not shown) and quantified kinetically with imaging flow cytometry (FIG. 2d). After fifteen hours in culture, 98% of SPAg had been internalized in B cells. A confocal microscopy analysis demonstrated the co-localization of SPAg with a marker of late endosomes (Lamp 1) and demonstrated that internalized SPAgs were localized in late endosomal compartment, where antigens are processed and loaded onto MHCII (data not shown). In ordinary conditions, the subsequent migration of antigen-MHCII complexes at the surface of B cells and the expression of costimulation molecules (CD80/86) lead to the activation and proliferation of cognate CD4+ clones (FIG. 3a). Consistently, flow cytometry analyses after overnight culture showed a higher expression of MHCII and CD86 in B cells that had internalized SPAg, as compared with B cells that had not been incubated with SPAg (FIG. 3b). The ability of SPAg-loaded B cells (B cells-SPAg-ova) to activate antigen-specific T cells was assessed by measuring the expression of the activation marker CD25 on ova-specific CD4+ T cells from OTII transgenic mice after 72 hours of coculture at a 1:1 B cell to T cell ratio (FIG. 3c). B cells loaded with SPAg without ova (B cells-SPAg-no ova) and B cells preincubated with soluble ova (B cells-soluble ova) were used as negative controls whereas bone marrow-derived dendritic cells pulsed with SPAg (BMDC-SPAg) or with soluble ovalbumin (BMDC-soluble ova) were used as positive controls. In contrast with negative controls, B cells-SPAg-ova did activate OTII CD4+ T cells. Importantly, CD25 expression by OTII CD4+ T cells was higher when B cells-SPAg-ova were used as APCs than when BMDC-SPAg or BMDC-soluble ova were used (CD25 MFI=5406311061 vs 58641038 and 74011000 respectively, p<0.05, FIG. 3c). SPAg-loaded B cells also induced a higher OTII CD4+ T cells proliferation than BMDC-SPAg-ova or BMDC-soluble ova did (FIG. 3d, % of divided T cells at 72 hours: 51.37.4 versus 19.33.0 and 36.93.3 respectively, p<0.05). As expected, B cells-SPAg-no ova or B cells-soluble ova did not induce CD4+ T cell proliferation (FIG. 3d).

[0147] To test the applicability of our approach in humans, B cells purified from the peripheral blood of an healthy volunteer were incubated 30 minutes at 37 C. with SPAg coated with anti-human kappa-light chain mAbs (hSPAg). Flow cytometry analysis revealed that hSPAg bound exclusively to CD19+ lambda chain B cells, showing that the interaction of hSPAg with B cells is dependent upon anti- mAb (FIG. 4a). The internalization of hSPAg by humans B cells was verified by EM (data not shown) and quantified by imaging flow cytometry (FIG. 4b). After 12 hours culture, the proportion of B cells with internalized hSPAg was 38.5%. This relatively low percentage as compared with murine experiments is explained by the fact that the proportion of kappa-positive B cells is lower in humans than in mice (50% versus 90% respectively). Optimizing the efficiency of human B cells loading with hSPAg would simply require to replace anti-human kappa-light chain mAbs with mAbs targeting a framework region present on heavy chains (for example, clone G20-127) or to add anti-lambda mAb (for example, clone 1-155-2) in addition to anti- mAb.

[0148] HIV alters CD4+ T cell compartment of patients, hampering the production of broadly neutralizing antibodies.sup.41. Because educating HIV-specific CD4+ T cells seems particularly relevant in the clinic, we decided to use HIV-GAG protein antigen to test the ability of polyclonal human B cells loaded with hSPAg to present internalized antigen (FIG. 4c). B cells purified from the peripheral blood of a HLA-DR01/01 healthy volunteer were incubated 30 minutes at 37 C. with hSPAg coated with either ovalbumin (use here as negative control, hSPAg-ova) or a peptide made of 3 repetitions of a sequence of HIV-GAG protein (hSPAg-GAG). B cells were then rested 12 hours at 37 C. and coculture 6 hours with HLA-DR01-restricted CD4+ T cell clones specific for the HIV-GAG protein.sup.42. CD4+ T cell clones activation was assessed by flow-cytometry measurement of the intracellular cytokine MIP-1. The percentage of B cells loaded with hSPAg-ova and hSPAg-GAG were comparable (44.8% and 39.1% respectively). As expected, unloaded B cells and B cells loaded with hSPAg-ova did not induce production of MIP-1 by CD4+ T cell clones (0.39% and 0.59% of positive clones respectively). In contrast, 24.1% of clones did produce MIP-1 when cocultured with B cells loaded with hSPAg-GAG. This result demonstrates that SPAg can be used to induce efficient antigen presentation by human polyclonal B cells to cognate CD4+ T cells.

[0149] Because CD4+ T cells specific for a given epitope are rare in the general CD4+ T cell population (as low as 1/100 in the memory compartment and below 1/10000 in the nave repertoire.sup.20), the capacity of SPAg-loaded B cells to expand rare antigen-specific T cells populations was tested in our murine experimental setting (FIG. 5). B cells-SPAg-no ova and B cells-SPAg-ova were cultured 72 hours with a mix of cognate OTII CD4+ T cells (CD45.2) and polyclonal CD4+ T cells (CD45.1). Various cognate to polyclonal T cells ratios were tested: 1/100 (FIG. 5a), 1/1000 (FIG. 5b) or 1/10000 (FIG. 5c). At any ratios, B cells-SPAg-ova, but not B cells-SPAg-no ova, induced specific proliferation of cognate T cells. CD45.2/CD45.1 ratios increased more than two times during the 72 hours of cocultures. Thus, SPAg-loaded B cells can induce proliferation and expansion of rare cognate CD4+ T cells.

[0150] As discussed above, CD4+ T cells exert different roles according to the pathophysiological context. Upon TCR-mediated activation, the nature of the microenvironment guide them towards particular functions, a processed called polarization (for recent review see: .sup.16). There is compelling evidences in the literature that Th1 polarized effector CD4+ T cells, which produce high levels of the proinflammatory cytokine IFN, are more efficient in promoting immune responses against virus and cancers.sup.10,43,44. In contrast, regulatory CD4+ T cells (CD4+ Treg), which are characterized by the expression of the transcription factor Forkhead box P3 (Foxp3).sup.45, are specialized in promoting the tolerance towards the auto- and allo-antigens for which they are specific.sup.46,47. Be able to polarize antigen-specific CD4+ T cell toward the adequate profile is therefore essential for the success of ACT. To test whether it is possible to polarize antigen-specific CD4+ T cells upon activation with SPAg-loaded B cells, a presentation assay was performed under polarizing conditions (FIG. 6a). SPAg-loaded B cells were cocultured 5 days with OTII CD4+ T cells in media without exogenous cytokines (Th0), with recombinant IL12 (Th1 polarizing media) or with TGF (Treg polarizing media). Flow cytometry analyses showed that CD4+ T cells produced IFN only under Th1 polarizing conditions and expressed Foxp3 only under Treg polarizing conditions. Even if adequate polarization could be obtained by adding exogenous cytokine to the culture media, it is likely that this method does not entirely reproduce the complexity of the polarization process that takes place in vivo that depends not only on the nature of cytokines present in the microenvironment but also on the type of costimulation signals delivered by APCs.sup.16. B cells have been shown, both in animal models.sup.48 and in humans.sup.49, to be endowed with unique regulatory functions.sup.50,51. Regulatory B cells (Breg) inhibit effector CD4+ T cell (Teff) responses and promotes Treg differentiation through the production of suppressive cytokines (in particular IL10) and cell-to-cell crosstalk.sup.48,50,52. Importantly, it has been shown that B cells stimulated in vitro with high doses of toll like receptors (TLR) ligands acquire regulatory properties.sup.53,54. Based on these observations, we assumed that regulatory properties could be conferred to SPAg-loaded B cells by adding CpG (the ligand of TLR9) to the culture (FIG. 6b). We hypothesized that Bregs loaded with SPAg (Breg-SPAg) would generate antigen-specific Tregs ex vivo without the need for addition of exogenous cytokines (FIG. 6b). Beyond its simplicity and reduced cost, this approach would also harness all the physiological mechanisms used by Breg to inhibit Teff and induce Treg. To define whether SPAg-loaded B cells secrete IL10 upon CpG stimulation, B cells-SPAg from IL10-reporter mice (IL10-IRES-eGFP transgenic mice.sup.55) were cultured 48 hours in medium supplemented with either anti-CD40 agonist (negative control, B cells-SPAg-40) or CpG (B reg-SPAg) (FIG. 6c). The proportion of SPAg-positive B cells expressing eGFP was measured by flow cytometry kinetically. In contrast with anti-CD40 stimulation, CpG stimulation resulted in IL10 production by B cells-SPAg (FIG. 6c, black and green curves respectively). The number of eGFP+ SPAg+ B cells peaked at 36 hours of culture (24.6%). We next went on analyzing whether Breg-SPAg could be used to promote the expansion of antigen-specific Treg. OTII CD4+ T cells were cultured with sorted B cells-SPAg that had been prestimulated 36 hours with either anti-CD40 agonist (Beff-SPAg) or CpG (Breg-SPAg) (FIG. 6d). B cells without SPAg prestimulated with anti-CD40 or CpG (Beff-noSPAg and B reg-noSPAg respectively) were used as negative controls. The proportion and proliferation profiles of Treg (CD4+ Foxp3+) and Teff (CD4+ Foxp3) were kinetically assessed by flow cytometry over 5 days culture (FIG. 6d). In line with our hypothesis, a progressive increase in the proportion of Treg was observed only when OTII CD4+ T cells were cocultured with Breg-SPAg (FIG. 6d, left). This was due to the fact that Breg-SPAg did not induce a significant proliferation of Teff but promoted instead the proliferation of antigen-specific Treg (FIG. 6d right). Contrariwise, Beff-SPAg induced the proliferation of Teff but not of Treg.

[0151] In this study, we present a novel versatile approach to expand and polarize antigen-specific CD4+ T cells that could be used in ACT. Our work indeed demonstrates that nanospheres can be easily biofunctionalized to behave like synthetic particulate antigen (SPAg) able to simultaneously i) activate polyclonal B lymphocytes, and ii) deliver any antigen of interest into the endosomal compartment of these B lymphocytes, thus turning non-cognate B cells into highly efficient stimulators of antigen-specific CD4+ T cells. Furthermore, this technique offers the possibility to harness the unique ability of B cells to polarize CD4+ T cells into either effectors or regulators.

[0152] In contrast with DCs, which currently represent the gold standard for ex vivo stimulation of antigen-specific CD4+ T cells, B cells are readily accessible in peripheral blood and can be conveniently and cheaply expanded by logs in vitro, offering an almost inexhaustible fresh source of highly pure autologous APCs.sup.31,32. These decisive advantages had led several groups to explore the possibility of exploiting the antigen presentation function of B cells for purpose of cell therapy.sup.31-33, 56-67. In organ transplantation, donor's B cells have been shown to be more efficient than donor's DC to expand ex vivo graft-specific Tregs.sup.68. Since donor's HLA (which is the molecular target in rejection) is expressed on the surface of donor's B lymphocytes, these cells can be directly used to expand graft-specific CD4+ Treg.sup.56,57. However, in all other pathophysiological situations, B cells must first be loaded with the chosen exogenous antigen before being able to interact with CD4+ T cells. This point is challenging because cognate interactions of antigen with the BCR are required for effective internalization, processing and presentation of the antigen by B cells.sup.36-38. Several strategies have been tested to overcome this limitation. The group of Dr. Scott pioneered this field and showed that cloning the target protein in frame with an immunoglobulin heavy chain and delivering it via retrovirus to an activated B cell could be a strategy to induce tolerance to multiple epitopes.sup.58-60. This strategy has however some drawbacks that limit its translation in the clinic. The generation of vectors can be technically challenging, the size of the inserts coding for the antigenic sequences is limited, and long-term gene transfer into primary human B cells is known to be notoriously difficult.sup.69-71. Finally, this approach lack versatility since the whole process needs to be set up again each time the antigen is changed. More recently, Lee Szeto et al have used a microfluidic device to deliver antigens in solution to polyclonal B cells via mechano-poration.sup.63. With this method, whole proteins cross the plasma membrane through transient pores without any selective uptake by BCR. As a result, the antigen, which is not vectorized in the endosomal compartment of B cells, can only be loaded into the MHCI, limiting the technique to the generation of antigen-specific cytotoxic CD8+ T cells. SPAg offer several advantages: i) the antigen is delivered to polyclonal B cells through the BCR, i.e. the physiological route of antigen uptake, ii) antigens can be delivered to B cells in their native form without any important engineering, and iii) SPAg-loaded B cells process the whole antigen, ensuring a natural and exhaustive generation of distinct antigenic peptides. As a result, SPAg-based technology is cheap, fast (<1 hour) and accessible to all biologists (no need for specialized skills or equipments) while remaining highly efficient to promote the presentation of any antigen in the MHCII of polyclonal B cells.

[0153] Finally, although the present work only details the use of SPAg to generate antigen-specific CD4+ T cell ex vivo, the possibility to directly use SPAg-loaded B cells in vivo to treat patients shouldn't be ignored. B cells have indeed the ability to home to patient's secondary lymphoid organs.sup.72. There, SPAg-loaded B cells could act as a cellular vaccine to promote the development of an endogenous response against cancers or persistent virus infections. Alternatively, the transfer of SPAg-loaded regulatory B cells could promote tolerance in transplant recipients or patients with autoimmune diseases. Further studies in animal models are warranted to assess the therapeutic potentials and risks of such strategies.

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REFERENCES

[0226] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.