WO2025229172A1

WO2025229172A1 - Live cell encapsulation

Info

Publication number: WO2025229172A1
Application number: PCT/EP2025/062057
Authority: WO
Inventors: Diego CATTONI; Quentin BOUSSAU; Jérôme BONNET; Horacio Cabral; Pengwen CHEN; Keita Masuda
Original assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Montpellier; University of Tokyo NUC
Current assignee: Centre National de la Recherche Scientifique CNRS; Institut National de la Sante et de la Recherche Medicale INSERM; Universite de Montpellier; University of Tokyo NUC
Priority date: 2024-05-03
Filing date: 2025-05-02
Publication date: 2025-11-06
Anticipated expiration: 2026-11-03

Abstract

Translating bacterial-based therapies to clinical success has yet been proven challenging, facing targeting inconsistencies, formulation issues and immune system noncompliance that can lead up to sepsis. To counter these limitations, the invention provides the Smart Polymer Shield (SPS) for the encapsulation of individual live cells.

Description

Live cell encapsulation

Technical Field

[0001] This disclosure pertains to the field of cell encapsulation, in particular for use in therapy.

Background Art

[0002] Cancers are one of the leading causes of mortality worldwide with almost 10 million deaths occurring in 2020 and an estimation of 19.3 million new cases the same year (1). This global cancer burden is expected to reach 28.4 million cases in 2040, representing a 47% increase from 2020 (1).

[0003] Despite the overall increase of survival rate of cancer-diagnosed patients during the latest decades, actual treatments remain improvable (2,3). Recent advances in synthetic biology and nano deliveries unveil a new cancer therapeutic option based on live bacteria which hold promise both in terms of efficacy and patient compliance (4). To date, the attenuated live strain of Mycobacterium bovis, Bacillus Calmette-Guerin (BCG) is the only live bacterial-based treatment that have been clinically approved by the US Food and Drug Administration (FDA) and is now the choice of care to treat high-grade non-muscle invasive bladder cancer (NMIBC) (5).

[0004] However, an increasing number of recent studies have developed innovative ways of using bacteria to fight tumors, either by improving their natural abilities to cope with cancer cells or by creating synthetic biological machines that can be used to deliver therapeutic payloads to the tumor microenvironment (TME), with some of them reaching the clinical step (4,6,7).

[0005] Yet, despite encouraging proof of concepts and early clinical trials, translating bacterial-based therapies to clinical success has yet been proven challenging, facing targeting inconsistencies, formulation issues and immune system noncompliance that can lead up to sepsis (8).

Detailed description

[0006] The inventors disclose an encapsulated cell wherein the cell is encapsulated with a block copolymer as described in W02020262550A1 which is incorporated by reference, referred to as the Smart Polymer Shield (SPS) herein.

[0007] The inventors have demonstrated that cells grafted with SPS forms stable capsules that release encapsulated cells in a pH-sensitive manner, mimicking the tumor microenvironment.

[0008] The cells are each individually encapsulated, and encapsulated cells exhibit enhanced resistance to host immune recognition, significantly reducing the immune response compared to nonencapsulated cell. It has also the advantage of allowing for precise tumor targeting, reduced toxicity, and enhanced therapeutic payload delivery. In a preferred embodiment, the encapsulated cell is an individual live cell. The encapsulated cell is protected from the immune environment so as it remains alive.

[0009] In a first aspect, the invention relates to an encapsulated cell wherein the cell is encapsulated with a block copolymer represented by the following formula (1): wherein R1 and R2 each independently represent a hydrogen atom, or an optionally substituted linear or branched alkyl group containing 1 to 12 carbon atoms, or an azide, an amine, maleimide, a ligand or a labeling agent,

R3 represents a compound represented by the following formula (I): wherein Ra represents a hydrogen atom, or an optionally substituted alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a heterocyclic group, a heterocyclic alkyl group, a hydroxy group, an alkoxy group or an aryloxy group.

L1 represents NH, CO, or a group represented by the following formula (11):

-(CH2)p1-NH- (11) wherein p1 represents an integer of 1 to 6, or a group represented by the following formula (12):

-L2a-(CH2)q1-L3a- (12) wherein L2a represents OCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO, L3a represents NH or CO, and q1 represents an integer of 1 to 6, ml and m2 each independently represent an integer of 0 to 500 (provided that the sum of ml and m2 represents an integer of 10 to 500), m3, m4 and m5 each independently represent an integer of 1 to 5, and n represents an integer of 0 to 500, and the symbol "/" means that [m1 +m2] units of the respective monomer units shown on the left and right sides of this symbol may be in any sequence.

[0010] The block copolymer of formula (1) comprises a poly-ethylene glycol (PEG), a poly-lysine moiety, and a pH-dependent reactive moiety R3. [0011] PEG is the goal standard in the field of therapeutic protein delivery as it is known to be nontoxic, highly soluble in water and FDA-approved (9-11). Moreover, the PEG polymerization capacity allows to form a mesh that advantageously protect the cell and hide its epitopes.

[0012] Poly-lysine moiety is composed of L-lysine. This moiety advantageously allows to create bonds to stabilize the polymerized structure by linking the monomers to each other and to the carboxyl groups of cell membrane proteins (Figure 1).

[0013] The pH-dependent reactive moiety R3 is a maleic anhydride derivative represented by the following formula (I):

This moiety creates covalent bonds between the block copolymers and amide groups for the cell membrane poteins. This moiety forms reversible covalent bonds. As a matter of fact, it forms a stable and covalent interaction of the block copolymer SPS to the cell at physiological pH of 7.4 but cleaves at more acidic pH of 6.5 and lower. In particular, it may form a reversible covalent bond with an amino group of a membrane protein.

[0014] This three-part composition makes the block copolymer SPS able to covalently bind and protect cells at physiological pH of 7.4 while releasing it when encountering a lower pH as found in solid tumors.

[0015] In the block copolymer of formula (1), R1 and R2 each independently represent a hydrogen atom, or an optionally substituted linear or branched alkyl group containing 1 to 12 carbon atoms, or an azide, an amine, maleimide, a ligand or a labeling agent. In a preferred embodiment, R2 represents a ligand or labeling agent.

[0016] A ligand refers to a compound used with the aim of targeting a certain biomolecule, and examples include an antibody, a pH-responsive antibody, a variable domain on a heavy chain (VHH) antibody, an aptamer, a protein, a peptide, an amino acid, a sugar, a low molecular compound, a single-stranded and double stranded DNA and a monomer of a biological macromolecule. Examples of a labeling agent include, but are not limited to, a fluorescent labeling agent such as succinimidyl ester (NHS ester) more particularly Alexa Fluor™ 488 NHS Ester, a rare earth fluorescent labeling agent, coumarin, dimethylaminosulfonyl benzoxadiazole (DBD), dansyl, nitrobenzoxadiazole (NBD), pyrene, fluorescein and a fluorescent protein, in particular a succinimidyl ester (NHS ester) more particularly Alexa Fluor™ 488 NHS Ester.

[0017] According to an embodiment, the compound represented by formula (I) is at least one of compounds represented by the following formulae (la) to (Ic):

[0018] According to an embodiment, the compound represented by formula (I) is a compound of formula (lb):

[0019] An encapsulated cell according to any one of the preceding claims, wherein the block copolymer represented by formula (1) is a block copolymer represented by the following formula (2): wherein m2/(m1 +m2) is from 0.05 to 0.5, in particular from 0.25 to 0.5, more particularly from 0.35 to 0.5, and n represents an integer of 1 to 500, in particular of 100 to 400, more particularly of 200 to 300.

[0020] As explained in the background art, cancer therapeutic option based on live bacteria holds promise both in terms of efficacy and patient compliance (4). As explained by Sedighi et al. (Cancer Med. 2019;8(6):3167-3181 bacteria can be used in cancer therapy by taking advantage of different strategies that include native bacterial toxicity, combination with other therapies, bacteria that can control expression of anticancer agents, expression of tumor-specific antigens, gene transfer, RNA interference, and pro-drug cleavage. In addition, studies have developed approach for cancer therapy using genetically engineered bacteria designed to express reporter genes, cytotoxic protein and/or anticancer agents, and tumor-specific antigens. The most common bacteria used in cancer therapy are the genera Salmonella, Clostridium, Bifidobacterium, Lactobacillus, Escherichia, Pseudomonas, Caulobacter, Listeria, Proteus and Streptococcus.

[0021] According to an embodiment, the encapsulated cell is a prokaryote. [0022] According to an embodiment, the prokaryote is a Gram-positive (+) bacterium, in particular of the genera Lactobacillus, Clostridium, Bifidobacterium, Corynebacterium, Listeria or Streptococcus more particularly of the genera Lactobacillus.

[0023] According to an embodiment, the prokaryote is Gram-negative (-) bacterium, in particular of the genera Escherichia, Salmonella, Pseudomonas, Caulobacter or Proteus, more particularly of the genera Escherichia.

[0024] According to an embodiment, the prokaryote is selected from Escherichia coli, Lactobacillus gasseri, Streptococcus pyogenes, Bacillus Calmette-Guerin, Salmonella typhimurium, Listeria monocytogenes, Clostridium acetobutylicum, Clostridium novyi, Clostridium sporogenes, Bifidobacterium longum, Salmonella enterica, Serratia marcescens, Corynebacterium diphtheriae, Clostridium perfringens, Clostridium botulinum and Pseudomonas aeruginosa, in particular from Escherichia coli and Lactobacillus gasseri

[0025] In a preferred embodiment, the prokaryote is selected from Escherichia coli Nissle 1917, Escherichia coli K12 MG1655 and Lactobacillus gasseri ATCC 33323.

[0026] As described in the literature, eukaryotic cells, in particular immune cells hold immense potential for targeted cancer treatment.

[0027] According to an embodiment, the encapsulated cell is a eukaryote, in particular an immune cell, more particularly an innate lymphoid cell (ILC) in particular a natural killer cell (NK cell), a T lymphocyte (T cell), B lymphocyte (B cell), a macrophage, a neutrophile, a dendritic cell, an eosinophil, a basophile, a mast cell, a monocyte, or a myeloid-derived suppressor cell (MDSC).

[0028] According to an embodiment, the encapsulated cell is a natural killer cell (NK cell).

[0029] In another aspect, the invention relates to the method of encapsulating a cell with the block copolymer of formula (I) as defined above, said method comprising the following steps of: a) placing the cell in an acidic buffer at a pH from 4.5 to 5.5, in particular from 4.7 to 5.3, more particularly a pH of about 5; b) adding the block copolymer to the cell in the acidic buffer, so as to obtain a mixture; and c) adjusting the pH of the mixture to a value from 6.8 to 9, in particular from 7.0 to 8, more particularly a value of about 7.4, so as to obtain an encapsulated cell.

[0030] In the method of the invention, the acidic buffer is required for the block polymer to be in free form. The mixture between the cell and the polymer is thus carried on in an acidic buffer. The overall pH of the mixture is then adjusted to a value from 6.8 to 9, in particular from 7.0 to 8, more particularly a value of about 7.4 in order to self-assemble the block copolymer around the cell into a full SPS coating.

[0031] In step b), the block copolymer may be added as a block copolymer solution having a concentration from 0.5 mg/ml to 15 mg/ml, in particular from 1 mg/ml to 10 mg/ml, in particular from 1 mg/ml to 6 mg/ml, more particularly a concentration of about 2 mg/ml, and having a pH from 4.5 to 5.5, in particular from 4.7 to 5.3, more particularly a pH of about 5. This solution allows the polymer to be in free form when added to the cell in the acidic buffer so as to encapsulate the cell.

[0032] The skilled person will select conditions that are required throughout the method of encapsulating the cell to keep it alive.

[0033] Optionally, after having adjusted the pH in step c), the method of the invention may comprise a step of purification by centrifugation so as to eliminate empty micelles and micelles encapsulating free proteins that may be present in the mixture.

[0034] In another aspect, the invention relates to an encapsulated cell as described above for use in therapy.

[0035] In another aspect, the invention relates to an encapsulated cell as described above for use in treating a cancer, in particular a solid tumor cancer.

[0036] As used herein, a “solid tumor cancer” refers to one or more cells which are growing or have grown in an uncontrolled manner to form cancertissue. As used herein, the term “solid tumor cancer” includes, but is not limited to “carcinomas”, “adenocarcinomas” and “sarcomas”. “Sarcomas” are cancers of the connective tissue, cartilage, bone, muscle, and so on. “Carcinomas” are cancers of epithelial (lining) cells. “Adenocarcinoma” refers to carcinoma derived from cells of glandular origin.

Brief Description of Drawings

[0037] Figure 1 : Scheme of the encapsulation and the associated protocol. (A) Scheme of the SPS concept and its interaction with the bacterial membrane proteins. (B) Scheme of the encapsulation protocol.

[0038] Figure 2: Various bacteria can be coated with a complete layer of SPS with a near hundred percent efficacy. (A) Bright field observation (60X) of non-encapsulated bacteria and SPS-E.coli. (B) 3D-SIM observation of SPS-E. coli expressing mKate2 fluorescent protein and stained with DAPI. (C) Flow cytometry analysis of the percentage of bacteria displaying SPS green fluorescence after encapsulation. (D) Flow cytometry analysis of the SPS green fluorescence means of different bacterial populations with or without SPS encapsulation. (E) Cryo-EM micrographs of nonencapsulated E.coli and SPS-E.coli. Red arrows are showing the bacterial double membrane. (F) Scheme of the experiment aiming to assess the impermeability of the SPS to antibodies. (G) Percentage of E.coli and SPS-E.co//displaying red fluorescence from the anti-E.co//antibodies (n=3). (H) Fluorescence microscopy images of E.coli and SPS-E.coli after being confronted to the anti- E.coli antibodies. Green fluorescence corresponds to the SPS, red fluorescence corresponds to anti- E.coli antibodies.

[0039] Figure 3: Characterization of the SPS-bacteria properties. (A) Comparison of the SPS stability around E.coli Nissle and fixed E.coli Nissle over two weeks at 4°C in HEPES (n=3). (B) Comparison of the evolution of the permeability of the SPS to anti-E.co// antibodies on fixed and non fixed E.coli over two weeks at 4°C in HEPES (n=3). (C) Colony forming unit (CFU) count of E.coli, SPS-E.coli and fixed SPS-E.coli after 2 week incubation in HEPES at 4°C compared to freshly cultured E.coli. (D) Effect of pH and salt on SPS-E.coli stability over 24h at 4°C in HEPES (n=3). (E) Propidium Iodide (PI) staining of SPS- control E.coli, SPS-E.coli, released SPS-E.coli and heat- shocked E.coli (n=3) 14 days post encapsulation. (F) Growth curves comparison of E.coli and SPS- E.coli (n=3) 14 days post encapsulation.

[0040] Figure 4: The smart polymer shield (SPS) improves bacterial survival and decreases bacterial immunogenicity in vitro. (A) Scheme of the experiment allowing cytokines levels quantification produced by PBMC after incubation with bacteria. (B) Cytokines levels comparison of human PBMC after being incubated for 12h with different EcN and SPS combinations (n=2).

[0041] Figure 5: The smart polymer shield is decreasing the immunogenicity of the bacteria while maintaining their tumor colonization capacities in an immunocompetent tumor-bearing mice model. (A) Scheme of the experimental plan of the in vivo experiment. (B) Tumor volume means of mice treated with EcN, SPS-EcN or PBS across time. (C) Relative weight loss of mice groups after receiving the bacterial or PBS injection. (D) Kinetic of bioluminescence levels expressed by bacteria found at the tumor site of mice injected with EcN, SPS-EcN or PBS. (E) Bacterial bioluminescence levels and localization in mice 1 and 7 days post EcN, SPS-EcN or PBS injection. (F) CFU per gram of tumor found on day 8 post EcN, SPS-EcN or PBS injection. (G) Quantification of TNF-a, IFN-g, IL-6 & IL-17 cytokines levels in the blood of mice 1 and 6 days after being injected with EcN, SPS- EcN or PBS.

[0042] Figure 6: (A) Scheme of the experimental plan of the in vivo experiment. (B) Bacterial bioluminescence levels and localization in mice 1 and 7 days post EcN, SPS-EcN or PBS injection.

Examples

[0043] Live Bacteria encapsulation

[0044] Materials and Methods

[0045] Bacterial strains and growth conditions

[0046] Escherichia coll Nissle 1917 mKate-Lux (EcNAfim_mKATE2 Amat ux) and Escherichia coll K-12 MG1655 WT used in this study were kindly gifted by Luis Angel Fernandez and are described respectively in Seco et al. 2022 and Pinero-Lambea et al. 2014.

Lactobacillus gasseri ATCC 33323 were obtained from LGC STANDARDS.

E.coli strains were grown in M9 media supplemented with 22 mM glucose at 37°C with 200 RPM agitation in aerobic conditions. M9 composition is as follow: 1X Bacto M9 minimal salts (Difco), 1 mM thiamine hydrochloride, 0,2% Casamino acids (Difco), 2 mM MgSO4 and 0,1 mM CaCI2 into sterile deionized water.

L.gasseri were grown MRS media as described in Fristot et al. 2023.

[0047] SPS polymer synthesis

[0048] The Smart Polymer Shield (SPS) corresponds to CDM-Modified Polyethylene glycol)-Poly(L- lysine) and was synthesized as described in Chen et al. 2023. After synthesis, PEG-pLL(CDM) were labeled with Alexa Fluor™ 488 NHS Ester (Succinimidyl ester) and shipped on dry ice to the CBS (Montpellier, France) where all the experiments were conducted.

[0049] Encapsulation protocol

[0050] Overnight cultures of bacteria were grown in M9 media at 37°C and shaking at 200 RPM. Bacteria were centrifuged at 3.000 Xg for 3 minutes and washed 3 times with cold sterile HEPES free acid 0.1 M buffered at pH 7.4 (EUROMEDEX). Bacterial concentration was counted by flow cytometry and adjusted to 1x10⁹ bacteria/ml. Polymers were resuspended into HEPES 0.1 M buffered at pH 5 at a concentration of 2 mg/ml and vortexed vigorously. Bacteria were washed a 4^th time and resuspended on HEPES 0.1 M pH 5. 250 pl of polymer solution was then slowly added for each ml of washed bacterial culture to have a ratio of 500 pg of polymer per 1x10⁹ bacteria. For negative controls, polymer solution was replaced by the addition of the same volume of HEPES 0.1 M pH 5. The mixed solution was vigorously homogenized by a couple series of vortexing before increasing the pH of the bacterial/polymer solution back to physiological by adding 2 times the volume of the solution of HEPES 0.1 M buffered at pH 8. Bacterial/polymer solution was incubated 10 minutes at room temperature with repeated vortexing along the incubation. Bacteria were washed 3 times with HEPES 0.1 M pH 7.4 to rinse the excess of polymer. Lastly, SPS-bact and negative control solutions were counted in 3 technical replicates by flow-cytometry and adjusted to the desired concentration.

[0051] Flow cytometry analysis

[0052] Flow cytometry was performed on an Attune NxT flow cytometer (Thermo Fisher) equipped with an autosampler and Attune NxT Version 2.7 Software. Experiments on Attune NxT were performed in 96-well plates or single tubes with the following settings: FSC: 440 V; SSC: 440 V; green intensity BL1 : 400 V (488 nm laser and 510/10 nm filter), red intensity YL2: 400 V (561 laser and 620/15 nm filter), flow rate = 25 pl/min. All events were collected with a cutoff of 20.000 singlecell. Every experiment included a negative control consisting of bacteria grown in the same condition and without fluorescence from bacteria or polymer to delineate the gates. See “supplementary figure n°X” for gating strategy. Data were analyzed using Flowjo (Treestar, Inc.) and Prism (GraphPad).

[0053] Colony forming units (CFU) count

[0054] 50 to 200 pl of bacterial solution or dissociated and filtered organs were aseptically serial diluted 1 :10 seven times in a 96 well plate filled with PBS in 3 technical replicates. Using a 8x6 replica plater (Sigma-Aldrich), a 1 pl drop of each homogenized dilution was transferred onto a LB agar plate and dried before being incubated at 37°C overnight (15 hours). Colony forming units (CFU) were counted in each drop of the first countable dilution (count of 10 to 60 colonies per drop). The bacterial concentration was deduced by taking the mean number of CFU of the three technical replicates of each condition. For tumors and organs, CFU were normalized by the weight of the sample to obtain CFU/g.

[0055] Microscopy [0056] Bright field images of EcN and SPS-EcN were acquired with a EVOS® FL imaging system (ThermoFisher Scientific) equipped with a 60x oil immersion objective (Olympus). The fluorescence microscopy images were acquired with a super-resolution DeltaVision OMX microscope (Image Solution Ltd) equipped with 405, 488, 561 and 642 nm lasers, 100x Plan SuperApochromat 1.4 oil immersion objective (Zeiss) and 4 EMCCD Evolve 512B cameras (Teledyne Photometries). Image acquisition was done using the 3D-SIM mode of the microscope.

[0057] Cryo-transmission electron microscopy

[0058] EcN and SPS-EcN were concentrated in PBS at an OD of 9 before placing 3 pl of the solution onto Lacey carbon grids (300 mesh; 50 pm; Delta Microscopy) previously glow-discharged for 10 s using PELCO easiGlow system (Ted Pella). Grids were then blotted for 2 s before being flash-frozen in liquid ethan using the Vitrobot Mark IV (FEI) at 20°C and 100% humidity. Data was collected using a LaB6 JEOL 1400 transmission electron microscope operating at 120kV and equipped with a One View camera (Gatan Inc).

[0059] Human PBMC cytokines production quantification

[0060] Peripheral Blood Mononuclear Cells (PBMCs) were isolated with Ficoll-Hypaque gradients from blood samples of anonymized healthy donors provided by the French Establishment for blood donations (EFS, Montpellier, France). EcN and SPS-EcN were incubated in PBMCs at a multiplicity of infection (MOI) of % at 37°C and 5% CO2 for 12h. Controls were made with the replacements of the EcN by PBS, SPS at 20 pg/ml and LPS at 200 ng/ml (Sigma). Proinflammatory cytokines secretion was quantified from the supernatant of the different conditions using LegendPLex™ beadbased immunoassays (BioLegend) and analyzed on the BD Bioscience-LSR Fortessa flow cytometer. The final quantification was established using the LEGENDplex™ data analysis software provided by BioLegend.

[0061] Animal models

[0062] Animal procedures were performed according to protocols approved by the French research ministry and the Languedoc-Roussillon ethical committee for animal experimentation, CEEA-LR-036. Animal experiments were conducted on 6-8 weeks-old female C57BL/6J mice (Charles River Laboratories) kept in clear plastic boxes and subjected to classical light cycles in the animal facility of the Research Institute in Cancerology of Montpellier (IRCM). Tumor models were established by subcutaneous injections of 5x105 murine colon adenocarcinoma cells (MC38) mixed 1 :1 in matrigel in the flank of the mice. MC38 cells were cultured in Dulbecco’s modified MEM supplemented with 10% fetal bovine serum, 2mM glutamine, 0,1 mM nonessential amino acids, 1 mM sodium pyruvate, 1 mM HEPES and 50pg/ml gentamicin sulfate. MC38 cell line is certified materials and was kindly gifted by the SIRIC (Integrated Research Center on Cancer). Tumors volume was calculated from its length and width (V = L * W * W/2) measured using an electronic caliper. Tumors were grown to an approximate volume of 200 mm3 and mice were randomized into groups with the same mean tumor volume before intravenous bacterial injection. Euthanasia of animals was required when the tumor volume reached 1500 mm3, when the weight loss was superior to 20% or after advice from the veterinary staff.

[0063] Bacterial administration for in vivo experiments

[0064] After proceeding with the encapsulation protocol, EcN and SPS-EcN concentration were measured using flow-cytometry in 3 technical replicates. Bacterial concentrations were then adjusted to the desired concentration to be injected in HEPES 0.1 M pH 7.4 and re-counted by flow-cytometry in 3 technical replicates for confirmation. Bacteria were kept on ice during the whole process. Intravenous injections were performed through the tail vein of the mice in a total volume of 100 pl with various bacterial concentrations. Following bacteria injection, leftover bacterial solutions were plated on LB agar plates to count CFU to further validate the bacterial concentration that had been injected.

[0065] In vivo imaging and biodistribution

[0066] E.coli Nissle used in the in vivo experiments are chromosomally integrated with luxCDABE bioluminescence cassette as described in Seco et al. 2022 allowing their visualization using the MS Lumina III spectrum imaging system and Living Image® software (PerkinElmer). The total flux (photons/second) subtracted from background flux was used to quantify the bioluminescence emitted by EcN in gates of identical size for each mouse.

[0067] At the study endpoint, mice were euthanized by cervical dislocation. The tumor and selected organs were collected, weighted and homogenized using the gentleMACS tissue dissociator (Miltenyi Biotec; C-tubes) in 5 ml PBS containing 50 pl collagenase B and 5 pl DNase I. Homogenates were filtered through 70 pm cell strainers (Corning), serial dilute in PBS and plated on LB agar plates as described in the CFU count section in order to quantify the bacterial colonization. CFU were counted the next day and bioluminescence production of the colonies were assessed with the Amersham imager 600 (GE Healthcare Life Science) to assure the retrieved bacteria correspond to the injected EcN-lux.

[0068] Mice blood cytokines production quantification

[0069] On the day following bacterial injection and one day before mice sacrifice, 120 pl of blood was aseptically sampled from the retro-orbital sinus of the mice in a 800pl silica and gel microtube (Vacutest Kima). 20 pl of the blood were directly transferred into an Eppendroff tube containing 180 pl PBS and were used to determine the presence of blood circulating bacteria by CFU count. The remaining 100 pl were kept at room temperature for an hour to clot before being centrifuged at 5.000 Xg for 10 minutes to isolate the serum. Proinflammatory cytokines secretion was quantified from the serum using LegendPLex™ bead-based immunoassays (BioLegend) and analyzed on the BD Bioscience-LSR Fortessa flow cytometer. The final quantification was established using the LEGENDplex™ data analysis software provided by BioLegend.

[0070] Results

[0071] Smart Polymeric Shield (SPS) composition and characteristics [0072] The polymers used in this study can be decomposed in several parts with specific properties in the SPS-encapsulation of live bacteria. In the first place, poly-ethylene glycol (PEG) is used for its polymerization capacity to form a mesh that will protect the bacteria and hide its epitopes. PEG is the goal standard in the field of therapeutic protein delivery as it is known to be non-toxic, highly soluble in water and FDA-approved (9-11).

[0073] Secondly, polymers are composed of poly L-lysine that is used to create ionic bonds to stabilize the polymerized structure by linking the monomers to each other and to the carboxyl groups of bacterial membrane proteins (Figure 1.A). Lastly, the polymer is composed of a pH-dependent reactive group that creates covalent bonds between the polymers and amide groups from the bacterial membrane proteins (Figure 1.A). This group, the carboxymethyl-maleic anhydride (CDM), allows the stable and covalent interaction of the SPS to the bacteria at physiological pH of 7.4 but cleaves at more acidic pH of 6.5 and lower. As demonstrated with cancer targeting protein delivery in Tao et al. 2020 (12), this three-part composition then makes the SPS theoretically able to covalently bind and protect bacteria at physiological pH of 7.4 while releasing it when encountering a lower pH as found in solid tumors.

[0074] Preparation and characterization of SPS-Bacteria

[0075] After an overnight growth in complete LB medium, 2x108 stationary growth phase bacteria are washed and placed in 200 pl of protein-free 100 mM HEPES buffer at pH 5. In parallel, polymer is resuspended at 2 mg/ml into 100 mM HEPES buffer at pH 3.5 to depolymerize it. Then, 50pl of the polymer solution is slowly added to the bacterial mix and homogenized. The overall pH of the solution is then adjusted to 7.4 in order to self-assemble the polymer around the bacterial into a full SPS coating. As the polymer is able to create empty micelles or to encapsulate free proteins that might be still present in the mix, the SPS-Bacteria are washed 3 times by centrifugation with HEPES 100 mM at pH 7.4 (Figure 1.B). The efficiency of the encapsulation is finally assessed by flowcytometry analysis and fluorescent microscopy observation.

[0076] SPS can be grafted onto various bacterial membranes to form a continuous layer

[0077] Initially, the inventors aimed to assess the capability of SPS to coat the surface of E. coll bacteria using an Alexa 488-labeled version of SPS. Under bright field observation, the bacteria exhibited a normal phenotype with varying sizes, indicative of different stages of the dividing cycle, and no discernible morphological alterations (Figure 2.A). To gain a comprehensive view of bacterial characteristics, the inventors utilized the expression of a cytoplasmic protein (mKate2) and simultaneously imaged it with DAPI, which labeled the bacterial nucleoid, along with SPS (Figure 2.B). Polymer integrity and continuity were observed in all axial planes collected. Notably, the polymer coating could follow the invagination of the division septum in dividing bacteria. To approximate the thickness of the SPS coating, the inventors conducted line profile measurements of the polymeric structure at various axial depths. The results indicated that the coating thickness is homogeneous across all imaging depths and is either equal to or lower than the resolution limit of 3D-SIM. Finally, the inventors validated the coating strategy in three different bacterial species, including Gram-positive and Gram-negative strains (Figure 2.C-D). The inventors found that SPS efficiently coated both Gram-positive B. subtilis and Gram-negative E.coli and Lactobacillus gasseri with equivalent efficiency (Figure 2.C-D). Additionally, for E.coli bacteria, the inventors tested the coating on two widely employed strains, E.co// Nissle 1917 and K12 MG1655 that are the main E.coli strains used for the development of bacterial therapies (13-14). In all tested strains, the inventors observed a uniform and continuous coating with equivalent stability.

[0078] SPS efficiently encapsulates over 99% of bacteria

[0079] Next, the inventors assessed the efficiency of SPS encapsulation. To achieve this, the inventors utilized Alexa 488-labeled SPS and cytoplasm-expressing mKate2 E.coli, and quantified the samples using flow cytometry. The data demonstrates that more than 99 % of red fluorescent bacteria exhibit green polymer encapsulation (Figure 2.C). Additionally, the inventors showed that using forward scattering as a gating mode allowed us to obtain identical results as when using red fluorescence detection. Furthermore, the inventors performed simultaneous imaging of identical samples via microscopy and quantified them using flow cytometry, obtaining consistent results. The reproducibility of the encapsulation method was confirmed through the percentage of encapsulated cells and their SPS-fluorescence mean on independent replicates (Figure 2.C-D). The inventors extended the evaluation by utilizing flow cytometry to assess the efficiency of encapsulation in other gram-negative and gram-positive species, obtaining comparable results (Figure 2.C-D). This comprehensive analysis supports the robustness and reliability of the encapsulation technique across independent replicates and different bacterial species.

[0080] SPS forms a fine continuous film around bacteria

[0081] To further characterize the interactions between SPS and the bacterial membrane, as well as its properties, the inventors employed cryo-electron microscopy. The inventors observed a noticeable contrast between non-encapsulated and SPS-E.coli. The double membrane, which was visible for non-encapsulated bacteria, was completely masked by the polymer shield in encapsulated bacteria (Figure 2.E). The electron microscopy images suggest that the thickness of the polymer layer is on the order of tens of nanometers.

[0082] SPS-encapsulated bacteria are invisible to anti-E coli antibodies in vitro.

[0083] The inventors aimed to assess the efficacy of SPS in isolating E.coli from antibody recognition. To achieve this, the inventors utilized a fluorescently labeled antibody designed to recognize E.coli epitopes. This antibody was then exposed to both non-encapsulated E.coli and SPS-E.coli (Figure 2.F). Simultaneously, the inventors quantified the presence of SPS and antibody recognition using flow cytometry and microscopy. The results revealed that over 99% of non-encapsulated bacteria were successfully recognized by the antibody. In contrast, when SPS was present, around 10% of bacteria exhibited detectable antibody binding (Figure 2.G). Microscopy images corroborate these findings, demonstrating that the 10% of bacteria displaying non-specific displayed as small patches still retained a complete SPS capsule (Figure 2.H). These findings underscore the potential of SPS in shielding bacteria from antibody recognition in vivo.

[0084] Bacterial encapsulation is stable for several weeks [0085] The inventors aimed to assess the stability of bacterial encapsulation in the study. To achieve this, the inventors encapsulated live E.coli and previously PFA-fixed E.coli with SPS. Both preparations were stored at 4°C for the duration of the experiment. The inventors quantified both the encapsulation degree and the recognition of anti-E.co// antibodies. The data indicates that both living and fixed E.coli remained stably encapsulated for over two weeks (Figure 3.A). Importantly, antibody recognition demonstrated that the shielding effect was maintained throughout the entire observation period (Figure 3.B). Viability of the E.coli, SPS-E.coli and fixed SPS-E.coli was assessed by CFU count at the end of the two week period and compared to fresh culture, showing no loss of viability (Figure 3.C).

Further experiments were conducted by placing SPS-EcN in different culture media. It was observed that the encapsulation of replication halted SPS-EcN remained stable for over 8 hours. In nutrient deprived media (Hepes buffer), bacteria remain encapsulated and fully viable for over 2 weeks.

In further experiments, SPS-EcN were placed in patient serum at 37° C. The results show that the bacteria remain encapsulated and that encapsulation protects the bacteria from immune response.

[0086] Encapsulated bacteria are released in a pH dependent manner

[0087] The inventors next tested the capacity of SPS to detach from the bacterial membrane and release bacteria from capsules based on pH of the environment. For this the inventors encapsulated E.coli with fluorescently labeled SPS and followed by flow cytometry the SPS release during incubation at different pH and salt concentration, mimicking the physiological and tumor microenvironment. When incubating SPS-E.coli in HEPES at a pH of 7.4 without salts, SPS remains fully coated for 24 hours (Figure 3.D) and can be kept in this state up to 2 weeks (Figure 3.A). As the bond between the SPS and bacterial membrane proteins can be sensitive to salt concentrations, the inventors incubated SPS-E.coli in HEPES pH 7.4 with 600 mM NaCI for 24 h. In this condition, the SPS is remaining stably anchored to the bacterial membrane as only 10% of the bacteria have been released after 24 h. Lastly, when SPS-E.coli is placed in the acidic condition of HEPES at pH 6.5 and in presence of salt, 40 % of the bacteria loses their SPS coating after the first hour of incubation and 75 % of them are freed after 24 h (Figure 3.D). This highlights the high specificity and fastresponding ability of the SPS to free the bacteria only when located in an acidic environment as found in tumors. The inventors then believe that this SPS capacity will be the key to a better localization of the bacterial delivery in vivo as well as an increased safety and compliance with patients.

[0088] Encapsulated bacteria remain viable, and their fitness is not altered

[0089] To assess bacterial viability during encapsulation and after release, the inventors conducted propidium iodide (PI) stain and measurements by flow cytometry. The findings reveal that E.coli, SPS-E.coli and SPS-released E.coli exhibit equivalent PI staining (Figure 3.E), suggesting that neither the encapsulation protocol northe pH-sensitive release affects bacterial viability. Interestingly, the results indicate that upon bacterial replication, the organisms can be released from the polymer capsule. To gain a deeper understanding of bacterial fitness, the inventors evaluated the growth kinetics of E.coli and SPS-E.coli in culture media. Importantly, the results demonstrate equivalent growth curves for both conditions, implying that the interaction with SPS does not impact bacterial fitness (Figure 3. F).

[0090] SPS encapsulation decreases bacterial immunogenicity ex-vivo

[0091] To assess the effectiveness of the SPS to decreases the immunogenicity of its cargo, the inventors incubated E.coli and SPS-E.coli into isolated human Peripheral Blood Monocellular Cells (PBMCs) and quantified the secretion of 6 proinflammatory cytokines (Figure 4.A). From here, the inventors decided to focus the bacterial strain on E.coli Nissle 1917 (EcN) as the inventors believe it is the most suitable chassis for bacterial therapy development, displaying both complete engineering toolbox and probiotic specificities. Incubation of PBS, Lipopolysaccharide (LPS), and SPS micelles were used as controls. First of all, cytokines levels secreted by the PBMCs incubated with either SPS micelles or PBS are identical, confirming the non-immunogenicity of the SPS polymer and the coherence of its use as an immune shield for bacteria (Figure 4.B). Most importantly, cytokines levels secreted by the PBMCs are systematically lower when they are incubated with SPS-EcN than with EcN alone (Figure 4.B). Similar results were observed regarding CXCL8 and CXCL1 levels with other cell types (data not shown). Together with the effective impermeability to antibodies, these results represent the first proof of concept of the immunological shielding effect of the SPS for live bacteria in a diverse, complex and competent immune environment.

[0092] SPS encapsulation do not interfere with tumor colonization in vivo

[0093] For the next step, the inventors aimed to find out if the SPS encapsulation does bring actual benefits in the tumor colonization ability of EcN in vivo. To do so, the inventors injected intravenously 1x10^A7 EcN or SPS-EcN into fully immunocompetent tumor-bearing mice and monitored the bacterial localization in the mice via bioluminescence expression, blood sampling and tumor dissociation followed by CFU count (Figure 5.A). First of all, EcN injection either SPS-encapsulated or not did not have any effect on the tumor growth in mice, which is expected at this point as no therapeutic effector has been engineered in this strain (Figure 5.B). Secondly, mice injected with EcN lost around 8% of their weight in the first day post injection before retrieving it 5 days later (Figure 5.C). This kinetics is identical for mice injected with SPS-EcN, indicating no significant effect of the SPS on the loss of weight post bacterial injection. In the same way, mice seem to behave identically to both conditions when looking at tumor colonization. Indeed, EcN and SPS-EcN both localize quickly at the tumor site with already detectable bioluminescence signatures 24h post injection and increasing to what it seems to be a concentration threshold a week later (Figure 5.D-E). Also, CFU count made from circulating blood samples from the mice at D+1 and D+7 post bacterial injection was all inconclusive indicating that bacteria are cleared from mice blood in less than 24 h by the immune system. Lastly, CFU count from homogenized tumors on day 8 post bacterial injection confirms the overall identical tumor colonization of EcN and SPS-EcN with 100% colonization in both groups and with similar concentration (Figure 5.F). Interestingly, CFU counts found in the tumors of both mice groups are higher than the injected concentration, proving that EcN is growing inside the tumor and that the SPS encapsulation does not block bacterial access to tumors nor growth inside it. Further experiments were conducted (Figure 6.A). As shown in Figure 6.B, EcN and SPS-EcN specifically colonize the tumor.

[0094] SPS encapsulation decreases bacterial immunogenicity in vivo

[0095] Lastly, the inventors measured the circulating levels of 4 proinflammatory cytokines from the blood samples retrieved from mice on day 1 and day 7 post EcN or SPS-EcN intravenous injection (Figure 5.G). Despite heterogeneous cytokine levels between mice, the means of the mice group injected with non-encapsulated EcN is always higher than the group that received SPS-EcN. This phenomenon seems to be due to the presence in the EcN injected group of high-responding mice that display significantly higher cytokines levels than the rest of the group, especially for TNF-a, IL-6 and IL-17. As these “high responding” mice with higher immune activation are not found in the group injected with the SPS encapsulated EcN, the inventors believe that the SPS does reduce the immunogenicity of the bacteria it carries. Furthermore, one of the mice that were injected with nonencapsulated EcN died of sepsis on day 2 post bacterial injection. After blood cytokines analysis, the inventors then figured that this septic mouse was one of the high-responding mice, highlighting the successful role of the Smart Polymer Shield (SPS) to decrease the immunogenicity of E.coli Nissle when injected in vivo, thus limiting both proinflammatory cytokines response and sepsis occurrence.

[0096] SPS encapsulation decreases sepsis occurrence.

[0097] Observations have been made of all the experiments conducted by the inventors on mice. Among all EcN-injected mice, 8.5% developed septicemia (n=178). Among all mice injected with SPS-EcN, no sepsis was observed (n=47). These results highlight the successful role of the Smart Polymer Shield (SPS) to decrease the immunogenicity of E.coli Nissle when injected in vivo, thus limiting sepsis occurrence.

[0098] Live eukaryote cell encapsulation

[0099] Materials and Methods

[0100] Natural killer (NK-92) cells were cultured in RPMI 1640/Glutamax supplemented with 10% FBS and 100 lU/mL of IL-2 (PeproTech) at 37°C in a 95% air and 5% CO₂ atmosphere. Cultures were maintained in Corning T-25 flasks, and the medium was renewed every three days prior to encapsulation.

[0101] For encapsulation, cells were counted and concentration was adjusted to 3 x 10^A5 cells/mL by diluting in culture media. Next, 2 mL of cells were centrifuged at 130 xg for 5 minutes in a benchtop centrifuge and resuspended in sterile HEPES free acid, 0.1 M, buffered at pH 7.4 (EUROMEDEX). This washing procedure was repeated three times, and the final resuspension was in 150 pL of HEPES 0.1 M buffered at pH 5. Then, 150 pL of polymer solution (2 mg/mL in HEPES 0.1 M pH 5) was slowly added in ten 15 pL aliquots, allowing 2 minutes of incubation with manual shaking at room temperature after each addition. Subsequently, the samples were incubated for 90 minutes at room temperature in a microtiter incubator (SI505, Cole-Parmer) at 600 RPM. Next, the inventors added 400 pL of HEPES 0.1 M pH 8 and incubated for 30 minutes in a microtiter incubator with shaking at 1200 RPM. The samples were then centrifuged at 130 x g for 3 minutes and resuspended in 0.5 mL of HEPES 0.1 M pH 7.4, 0.15 M NaCI; this step was repeated three times. Cells were either kept alive or fixed for subsequent imaging.

[0102] Results

[0103] The inventors have efficiently encapsulated NK-92 cells with SPS. Live cell images reveal that cells maintain the integrity of their membranes, and their morphology remains unaffected by SPS coating when compared with controls. The inventors do not observe internalization of polymers through endocytosis or intracellular aggregates of SPS.

[0104] Wide-field microscopy images of live cells do not provide sufficient resolution in the axial plane to distinguish the cell membrane. To address this, the inventors fixed cells using paraformaldehyde. The slight dehydration that naturally occurs during fixation allows to obtain thinner cells, thereby making their membrane contours coated with SPS more easily discernible when using wide-field microscopy.

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Claims

[Claim 1] An encapsulated cell wherein the cell is encapsulated with a block copolymer represented by the following formula (1): wherein R1 and R2 each independently represent a hydrogen atom, or an optionally substituted linear or branched alkyl group containing 1 to 12 carbon atoms, or an azide, an amine, maleimide, a ligand or a labeling agent,

R3 represents a compound represented by the following formula (I): wherein Ra represents a hydrogen atom, or an optionally substituted alkyl group, an alkenyl group, a cycloalkyl group, an aryl group, an aralkyl group, an acyl group, a heterocyclic group, a heterocyclic alkyl group, a hydroxy group, an alkoxy group or an aryloxy group. ,

L1 represents NH, CO, or a group represented by the following formula (11):

[Claim 2] An encapsulated cell according to claim 1 , wherein the compound represented by formula

(I) is a compound of formula (lb):

[Claim 3] An encapsulated cell according to claim 2, wherein the block copolymer represented by formula (1) is a block copolymer represented by the following formula (2): wherein m2/(m1 +m2) is from 0.05 to 0.5, in particular from 0.25 to 0.5, more particularly from 0.35 to 0.5, and n represents an integer of 1 to 500, in particular of 100 to 400, more particularly of 200 to 300.

[Claim 4] An encapsulated cell according to any one of claims 1 to 3, wherein the encapsulated cell is an individual live cell.

[Claim 5] An encapsulated cell according to any one of the preceding claims, wherein the encapsulated cell is a prokaryote.

[Claim 6] An encapsulated cell according to claim 5, wherein the prokaryote is a Gram-positive (+) bacterium, in particular of the genera Lactobacillus, Clostridium, Bifidobacterium, Corynebacterium Listeria or Streptococcus, more particularly of the genera Lactobacillus.

[Claim 7] An encapsulated cell according to claim 5, wherein the prokaryote is a Gram-negative (-) bacterium, in particular of the genera Escherichia, Salmonella, Pseudomonas, Caulobacter or Proteus, more particularly of the genera Escherichia.

[Claim 8] An encapsulated cell according to claim 5, wherein the prokaryote is selected from Escherichia coli Nissle 1917, Escherichia coli K12 MG1655 and Lactobacillus gasseri ATCC 33323.

[Claim 9] An encapsulated cell according to any one of claims 1 to 4, wherein the encapsulated cell is a eukaryote, in particular an immune cell.

[Claim 10] An encapsulated cell according to claim 9, wherein the immune cell is a natural killer cell.

[Claim 11] Method of encapsulating a cell with the block copolymer represented by formula (I) so as to obtain an encapsulated cell according to any one of claims 1 to 10 comprising the following steps of: a) placing the cell in an acidic buffer at a pH from 4.5 to 5.5, in particular from 4.7 to 5.3, more particularly a pH of about 5; b) adding the block copolymer to the cell in the acidic buffer, so as to obtain a mixture; and c) adjusting the pH of the mixture to a value from 6.8 to 9, in particular from 7.0 to 8, more particularly a value of about 7.4, so as to obtain the encapsulated cell.

[Claim 12] Method according to claim 11 further comprising a step of purification by centrifugation so as to eliminate empty micelles and micelles encapsulating free proteins that may be present in the mixture.

[Claim 13] An encapsulated cell according to any one of claims 1 to 10 for use in therapy.

[Claim 14] An encapsulated cell according to claim 13 for use in treating a cancer.

[Claim 15] An encapsulated cell according to claim 14 for use in treating a solid tumor cancer.