Cell expansion

Abstract

The present invention relates to a microsphere or population of microspheres, and associated methods.

Description

[0001] CELL EXPANSION

[0002] Field of invention

[0003] The invention relates to a microsphere or population of microspheres, and associated methods.

[0004] Background of the invention

[0005] The in vitro expansion and differentiation of cells such as T-cells is a fundamental process in both basic immunological research and the clinical application of cellular therapies, such as CAR T-cells. Traditional methods to achieve T-cell expansion involve activation using monoclonal antibodies targeting the T-cell receptor complex subunit CD3 and the co-receptor CD28, typically presented on the surface of rigid cell-sized particles such as Dynabeads. This technique facilitates the activation and proliferation of T-cells, generating large cell quantities for subsequent assays.

[0006] While large numbers of cells can be generated using Dynabeads, their phenotypic composition is often skewed towards a terminally differentiated cell type of limited proliferative and functional capacity with elevated expression of exhaustion markers. While this may be desirable in some cases, when considering the downstream application of the generated cells, this highly differentiated and exhausted phenotype may be detrimental in other contexts. One notable case is cellular therapies, where it is of critical importance that the engineered cells are highly functional and ideally retain a naive or memory phenotype.

[0007] The function of T-cells is dependent on mechanics. During the formation of the immunological synapse, T-cells actively generate mechanical forces, exposing the TCR-pMHC interaction, and other co-receptor ligand interactions (for example CD8, CD28) to mechanical forces. The mechanics of this interaction depend on both the mechanical properties of the T-cell and its target, the dynamics of the T-cell actin-myosin cytoskeleton and the concentration and affinity of receptor-ligand interactions. The TCR-pMHC binding kinetics is influenced by force, thus implicating mechanics as a key regulator of T-cell fate, influencing their ability to discriminate antigen, differentiate into subtypes andcarry out their effector function. Existing T-cell expansion platforms such as Dynabeads poorly mimic the mechanics of the immunological synapse.

[0008] It is therefore an object of the invention to provide further and / or improved products and methods for the expansion and differentiation of cells, such as T-cells.

[0009] Summary of the invention

[0010] The inventors identified and developed a novel cell expansion and activation platform based on soft (e.g. 100-5000Pa) polymeric microspheres, such as hydrogel microspheres. The effectiveness of such soft microspheres is surprising. The microspheres may be tuned to mimic the mechanochemical environment of a physiological cell-target interaction, such as a physiological T-cell-target interaction. This is in stark contrast to prior art microspheres such as commercially available Dynabeads which have a stiffness in the MPa-GPa range.

[0011] By optimising the microsphere stiffness, ligand density, and / or the specific composition of the presented ligands, the inventors surprisingly achieved a dramatic improvement of cell expansion compared to well-established prior art systems, such as those utilising Dynabeads. Specifically, expansion via the microsphere platform of the invention leads to increased proliferation and thus an increased number of cells. The cells exhibit a less differentiated and / or less exhausted phenotype. Further, on re-stimulation, the cells show an enhanced functional capacity as quantified by, for example, cytokine secretion.

[0012] The development of soft polymeric microspheres that mimic the mechanochemical environment of the physiological cell-target interactions, such as T-cell-target interactions, represents a surprising, significant and advantageous advancement in the field of cell expansion and differentiation, offering a robust alternative to the prior art systems, such as widely used Dynabeads. While these prior art systems have been instrumental in advancing immunological research and clinical applications, they come with notable limitations, particularly the propensity to generate terminally differentiated T-cells with limited proliferative and functional capacities and increased expression of exhaustion markers

[0013] During T-cell activation, mechanical forces are generated that influence TCR-pMHC and co-receptor ligand interactions, which in turn affect T-cell fate, includingdifferentiation and effector functions. However, existing T-cell expansion platforms such as Dynabeads do not mimic the mechanochemical properties of natural antigen-presenting cells, thus failing to replicate the physiological conditions necessary for optimal T-cell activation and expansion.

[0014] The novel microsphere platform of the present invention addresses these shortcomings in the prior art by incorporating relatively soft hydrogel microspheres that may have tuneable stiffness to mimic the mechanical properties of physiological cell-target interactions, such as T-cell-APC interactions. This innovation significantly enhances cell proliferation, such a T-cell proliferation, while maintaining a less differentiated and less exhausted phenotype. This system allows for a more physiologically relevant activation of cells, such as T-cells. In the context of CAR T-cell therapy, the potential of this platform is particularly promising. CAR T-cell therapy has revolutionised the treatment of certain cancers, particularly B-cell malignancies. However, the generation of CAR T-cells using traditional expansion methods often results in cells with suboptimal phenotypic and functional profiles, limiting their efficacy and persistence in vivo. The ability of the microspheres of the invention to produce cells, such as T-cells, that are less differentiated and less exhausted, yet highly functional, is a significant advantage. This could, in particular, translate to CAR T-cells with enhanced proliferation, persistence, and antitumour activity, addressing some of the key challenges currently faced in the field.

[0015] Furthermore, the improved cytokine secretion observed with microsphere-expanded T-cells indicates a higher functional capacity, which is critical for effective tumour eradication. This enhanced functionality could lead to better clinical outcomes, with more durable responses and lower relapse rates. Additionally, the maintenance of a more naive and central memory phenotype in microsphere-expanded T-cells suggests that these cells have better longevity and self-renewal capabilities, further enhancing the therapeutic potential of CAR T-cells.

[0016] In summary, the development of the microsphere platform marks a significant improvement over traditional cell expansion methods and results in superior microspheres. By better mimicking the physiological conditions of, in particular, T-cell activation, microspheres generate T-cells that are more suited for effective immunotherapies. This innovation holds great promise for CAR T-cell therapy, improving the efficacy, persistence, and overall outcomes of these cutting-edge treatments. More generally, the microspheres of the invention are useful as models (e.g. in vitro, in vivo or ex vivo) withmany uses, such as for studying tissue biology (e.g. development, homeostasis or regeneration), immunology, regenerative medicine (the production of cell therapies), and disease modelling (e.g. disease mechanism, drug screening, or personalised medicine).

[0017] Accordingly, the invention provides a microsphere or population of microspheres, wherein: the microsphere has a stiffness of 10-10,000 Pa; or the population of microspheres has a mean stiffness of 10-10,000 Pa.

[0018] The invention also provides a method of producing the microsphere or population of microspheres, comprising:

[0019] (a) forming an emulsion, such as an inverse emulsion, between an aqueous phase comprising a pre-polymer solution and optionally a free radical generator, and an oil phase comprising a surfactant;

[0020] (b) polymerising the pre-polymer solution to provide a microsphere or population of microspheres, such as by chemical and / or photopolymerization; and

[0021] (c) optionally functionalising the microsphere or population of microspheres by attaching one or more moiety to the surface of the microsphere or population of microspheres, wherein the one or more moiety is each selected from a protein, a polysaccharide chain, and / or an extracellular matrix protein.

[0022] The invention also provides a method of culturing a cell, comprising culturing the cell in a suspension comprising the microsphere or population of microspheres, to provide a cultured cell.

[0023] The invention also provides a composition comprising the microsphere and a cell. The invention also provides a cell or cell population obtained by or obtainable by the method of the invention.

[0024] The invention also provides a use of the cultured cell, cell or cell population of the invention, to identify agents capable of preventing or treating a disease, comprising treating a disease model before, during or after induction of the disease, optionally wherein the disease is cancer.

[0025] The invention also provides the cultured cell, cell or cell population of the invention, for use in a method of therapy, optionally wherein the therapy is an immunotherapy and / or is for use in treating cancer.

[0026] The invention also provides a method of treating a disease in a subject comprising administering to the subject the cultured cell, cell or cell population of the invention, optionally wherein the method is an immunotherapy and / or wherein the disease is cancer.Brief description of the figures

[0027] Figure 1: Microsphere production and functionalization, a) An inverse emulsion is formed between an aqueous phase containing the pre-polymer solution (acrylamide, bisacrylamide, acrylic acid) and a free radical generator (APS-TEMED), and an oil phase composed of mineral oil and the surfactant Span80 (sorbitan monooleate), b) Following polymerisation, microspheres are functionalized using EDC / NHS chemistry. First an amine-PEG-biotin linker is covalently attached to the microspheres, followed by streptavidin and lastly biotinylated antibodies, c) Representative fluorescent images showing both, “soft” (245 Pa) and “stiff’ (5470 Pa) microspheres labelled with a fluorescent biotin-PE. Scale bar is 30 pm and applies to both the “soft” and “stiff’ microspheres (i.e. the magnification of the “soft” and “stiff’ microspheres is the same), d) Quantification of biotin-PE binding for soft and stiff microspheres, e) Quantification of soft and stiff microsphere stiffness by AFM indentation.

[0028] Figure 2: T-cell proliferation, a) T-cells isolated from human blood were cocultured with both soft and stiff microspheres and commercially available Dynabeads (polystyrene beads), and the cultures maintained for 14 days prior to quantification, b) Brightfield imaging of T-cell expansion induced by microsphere stimulation at both Day 3 and Day 14 demonstrates by way of example significant expansion of T-cells between Day 3 and Day 14 of culture. Scale bar is 1 mm. c) Flow cytometry-based quantification of the number of CD4+ and CD8+ T-cells at Day 3, 7 and 14 of co-culture (“Soft” has the lowest count at day 7; “Dyna” has an intermediate count at day 7; “Stiff’ has the highest count at day 7). d) Stacked bar plot showing both absolute counts of CD4+ cells (lighter / higher bars) and CD8+ cells (darker / lower bars), as well CD4+ cells (lighter / higher bar) and CD8+ cells (darker / lower bar) as a fraction of all CD3+ T-cells. Data shown is for 3 human donors.

[0029] Figure 3: T-cell activation, a) Flow cytometry-based quantification of the fraction of CD8+ T-cells that are CD25+ at Day 3, 7 and 14 following stimulation by “soft” and “stiff’ microspheres and Dynabeads (polystyrene beads), b) 2D scatter plot of cell expression of CD25 and the proliferation stain CTV. Decreasing CTV signal indicates cell division and proliferation. The scatter plots are shown for Day 3, 7 and 14 for all stimulation conditions, c) Flow cytometry -based quantification of the fraction of CD8+ T-cells that are CD69+ at Day 3, 7 and 14 following stimulation by “soft” and “stiff’ microspheres and Dynabeads (polystyrene beads). Data shown is for 3 human donors. For both a) and c), “Dyna” has the highest fractions at days 7 and 14; “Soft” has intermediate fractions at days 7 and 14; and “Stiff’ has the lowest fractions at days 7 and 14.

[0030] Figure 4: T-cell phenotypes, a) Flow cytometry -based quantification of the fraction and absolute counts of CD8+ T-cells that exhibit a Naive (N) (CCR7+ / CD45RA+), Central Memory (CM) (CCR7+ / CD45RA), Effector Memory (EM) (CCR7- / CD45RA-), and Effector Memory cells re-expressing CD45RA (TEMRA) (CCR7- / CD45RA+) phenotypes at Day 14 following estimation by “soft” and “stiff’ microspheres and Dynabeads (polystyrene beads), b) The equivalent analysis as shown in a) but for CD4+ T-cells. For both a) and b), bars from top to bottom (and darkest to lightest) are: N; CM; EM; and TEMRA. c) 2D scatter plot (left) and corresponding quantification of CD8+ T-cell fraction expression both CD45RA and CD62L (right), d) 2D scatter plot (left) and corresponding quantification of CD8+ T-cell fraction expression both CD45RA and CD27. e) 2D scatter plot (left) and corresponding quantification of CD8+ T-cell fraction expression both CD45RA and TCF1 (right). Data shown is for 3 human donors and pooled across 2 technical repeats. P value thresholds - *** (0.001), ** (0.01), * (0.05).

[0031] Figure 5: T-cell exhaustion, a) Flow cytometry -based quantification of the expression of exhaustion markers TIGIT, PD1 and CTLA-4 at Day 14 in CD8+ T-cells. The distribution of expression is shown for all stimulation conditions (histograms: top is “soft”; middle is “stiff’; and bottom is “Dyna”), as well as a quantification of the MFI of each marker, b) The corresponding quantification for CD4+ T-cells. Data shown is for 3 human donors. P value thresholds - *** (0.001), ** (0.01), * (0.05).

[0032] Figure 6: T-cell cytokine profile, a) Flow cytometry-based quantification of the fraction of CD8+ T-cells expressing the cytokines, TNF, IL2 and INFy following 14 days initial stimulation by “soft” and “stiff’ microspheres and Dynabeads (polystyrene beads) and a subsequent re-stimulation at Day 14 by PMA-ionomycin. All stimulated conditions are compared to an unstimulated control “U” which is PMA-ionomycin treated T-cells directly following isolation from the blood. Data shown is for 3 human donors. P value thresholds - *** (0.001), ** (0.01), * (0.05).

[0033] Detailed description of the inventionGeneral Definitions

[0034] Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this invention belongs.

[0035] In general, the term “comprising" is intended to mean including but not limited to. For example, the phrase “the pre-polymer solution comprises PEG, alginate, acrylamide, bis-acrylamide and / or acrylic acid" should be interpreted to mean that the pre-polymer solution includes at least one of PEG, alginate, acrylamide, bis-acrylamide and acrylic acid, but the pre-polymer solution may comprise further components. The pre-polymer solution may therefore for example comprise at least two, at least three, at least four or at least five of PEG, alginate, acrylamide, bis-acrylamide and acrylic acid, and optionally one or more further components.

[0036] The terms “protein" and “polypeptide" are used interchangeably herein, and are intended to refer to a polymeric chain of amino acids of any length.

[0037] The term “around", “about" and “~" and their grammatical equivalents are used interchangeably here, and are intended to refer to a reference numerical value and its grammatical equivalents as used herein can include the numerical value itself, such as a point value or the end point(s) of a range, and a range of values plus or minus 10% from that numerical value. The term “around" in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. For example, a final composition of around -60-70% CD8+ refers to percentage values of 54%-66%, 63%-77%, and 54% to 77%.

[0038] The term “between ” in relation to a pair of reference numerical values and its grammatical equivalents as used herein can include the numerical values themselves and the range of values between the reference numerical values. For example, the term between “Day 3 and Day 14" may refer to Day, 3, Day 14, or any value falling within the range of Day 3 to Day 14, such as Day 3.5 or Day 9.

[0039] The term “mean" and its mathematical and grammatical equivalents as used herein can be calculated by counting the total of all data points within a population and then dividing by the total number of data points. For example, the term “population of microspheres having a mean stiffness of 100 Pa" may refer to a population of 100microspheres wherein each microsphere has a stiffness of 100 Pa. Alternatively, the term may refer to a population of 1000 microspheres wherein 250 microspheres in the population each have a stiffness of 50 Pa, 500 microspheres in the population each have a stiffness of 100 Pa, and 250 microspheres in the population of microspheres each have a stiffness of 150 Pa.

[0040] The singular forms “a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “a microsphere” includes two or more microspheres.

[0041] All publications, patents and patent applications cited herein, whether supra or infra, are hereby incorporated by reference in their entirety.

[0042] General description of a microsphere and population of microspheres

[0043] Microspheres are small, substantially spherical particles that can range in size from 1 to 1000 micrometres (pm). The term particle may be used interchangeably herein. A microsphere of the invention may be used to deliver one or more bioactive molecule. The bioactive molecule may be partially or fully encapsulated by the microsphere.

[0044] Alternatively or additionally, the bioactive molecule may be partially or fully on the surface of the microsphere. A bioactive molecule may be attached to a microsphere by dissolving or dispersing within the microsphere, covalent linkage to the microsphere, or non-covalent linkage to the microsphere.

[0045] Microspheres of the invention may be from a variety of materials, including natural and synthetic polymers, glass, metal, and ceramics. Microspheres of the invention may be solid or substantially solid, comprising or consisting of a solid matrix. Alternatively, microspheres of the invention may be hollow, having a lumen that may contain a cargo such as a bioactive molecule. Microspheres of the invention may comprise a shell surrounding a core, wherein the shell and core comprise different materials. Microspheres or the invention may have one or more pore on the surface and / or core.

[0046] The invention relates to a single microsphere or a population comprising a plurality of microspheres. The population may comprise or consist of a homogenous population of microspheres, a substantially homogenous population of microspheres, or a heterogenous population of microspheres. A “substantially homogenous population of microspheres” is a population of microspheres within which a reference numerical value for at least 85%, atleast 90%, at least 95%, at least 98% or at least 99% of each individual microsphere in the population has a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. For example, a substantially homogenous population of microspheres may comprise microspheres in which 95% of the microspheres have a stiffness within 5% of 500 Pa (i.e. 475 Pa to 525 Pa) and / or 98% of the microspheres have a size of within 10% of 100 pm (i.e. 90 pm to 110 pm).

[0047] Thus, the present invention relates both to individual microspheres and populations comprising individual microspheres, as will be clear to the skilled person. A population of microspheres according to the invention may comprise at least 2, at least 10, at least 100, at least 1000, at least 10000, at least 100000, at least 1000000, at least 10000000, at least 100000000, at least 1000000000 or at least 10000000000 individual microspheres. A population of microspheres according to the invention may comprise at most 2, at most 10, at most 100, at most 1000, at most 10000, at most 100000, at most 1000000, at most 10000000, at most 100000000, at most 1000000000 or at most 10000000000 individual microspheres. Thus, the population of microspheres according to the invention may have 2-10, 2-100, 2-1000, 2-10000, 2-100000, 2-1000000, 2-10000000, 2-100000000, 2-1000000000, 2-10000000000, 10-100, 10-1000, 10-10000, 10-100000, 10-1000000, 10-10000000, 10-100000000, 10-1000000000, 10-10000000000, 100-1000, 100-10000, 100-100000, 100-1000000, 100-10000000, 100-100000000, 100-1000000000, 100-10000000000, 1000-10000, 1000-100000, 1000-1000000, 1000-10000000, 1000-100000000, 1000-1000000000, 1000-10000000000, 10000-100000, 10000-1000000, 10000-10000000, 10000-100000000, 10000-1000000000, 10000-10000000000, 100000-1000000, 100000-10000000, 100000-100000000, 100000-1000000000, 100000-10000000000, 1000000-10000000, 1000000-100000000, 1000000-1000000000, 1000000-10000000000, 10000000-100000000, 10000000-1000000000, 10000000-10000000000, 100000000-1000000000, 100000000-10000000000 or 1000000000-10000000000 individual microspheres.

[0048] Where the invention relates to a heterogenous population of microspheres, there may be at least 2, at least 10, at least 100, at least 1000 or at least 10000 different homogenous microspheres in said heterogenous population. Thus, the population of microspheres according to the invention may have 2-10, 2-100, 2-1000, 2-10000, 10-100, 10-1000, 10-10000, 100-1000, 100-10000 or 1000-10000 different homogenous microspheres.The microsphere and populations thereof of the invention may be characterised by one or more particular features, such as stiffness, size, composition, and surface moiety and copies thereof. Where the feature relates to a population of microspheres, said feature may be defined as a mean value or range of values.

[0049] The microsphere of the invention may have a diameter of 1 to 1000 pm, such as 5-500 pm. For example, the microsphere of the invention may have a diameter of at least 1 pm, at least 5 pm, at least 10 pm, at least 50 pm, at least 100 pm, at least 250 pm, or at least 500 pm. Alternatively or in addition, the microsphere of the invention may have a diameter of at most 5 pm, at most 10 pm, at most 50 pm, at most 100 pm, at most 250 pm, at most 500 pm or at most 1000 pm. Thus, the microsphere of the invention may have a diameter of 5-10 pm, 5-25 pm, 5-50 pm, 5-100 pm, 5-250 pm, 5-500 pm, 10-25 pm, 10-50 pm, 10-100 pm, 10-250 pm, 10-500 pm, 25-50 pm, 25-100 pm, 25-250 pm, 25-500 pm, 50-100 pm, 50-250 pm, 50-500 pm, 100-250 pm, 100-500 pm or 250-500 pm. In one preferable embodiment, the microsphere may have a diameter of 5-50 pm. The diameter of the microsphere may be measured by any suitable means known to the skilled person. For example, the diameter of a microsphere may be measured by any suitable microscopy technique, such as by analysing fluorescent microscopy images such as those set out in Figure 1c. The diameter of the microsphere may be measured manually and / or using a suitable automated algorithm such as a suitable ImageJ algorithm.

[0050] The population of microspheres of the invention may have a mean diameter of 1 to 1000 pm, such as 5-500 pm. For example, the population of microsphere of the invention may have a mean diameter of at least 1 pm, at least 5 pm, at least 10 pm, at least 50 pm, at least 100 pm, at least 250 pm, or at least 500 pm. Alternatively or in addition, the population of microspheres of the invention may have a mean diameter of at most 5 pm, at most 10 pm, at most 50 pm, at most 100 pm, at most 250 pm, at most 500 pm or at most 1000 pm. Thus, the population of microspheres of the invention may have a mean diameter of 5-10 pm, 5-25 pm, 5-50 pm, 5-100 pm, 5-250 pm, 5-500 pm, 10-25 pm, 10-50 pm, 10-100 pm, 10-250 pm, 10-500 pm, 25-50 pm, 25-100 pm, 25-250 pm, 25-500 pm, 50-100 pm, 50-250 pm, 50-500 pm, 100-250 pm, 100-500 pm or 250-500 pm. In one preferable embodiment, the population of microspheres may have a mean diameter of 5-50 pm.

[0051] The microsphere according to the invention may comprise one or more of any suitable polymer, such as a hydrophilic polymer. A hydrophilic polymer is a polymer thatdissolves in water or swells in it. Hydrophilic polymers contain polar or charged functional groups that allow them to interact with water, which is polar. They can be natural, semisynthetic or synthetic. The hydrophilic polymer may be one or more of poly(vinyl alcohol) (PVA), poly(acrylamide) (PAAm), poly(2 -hydroxypropyl methacrylamide) (PHPMA), polyvinylpyrrolidone) (PVP), poly(N,N-dimethylacrylamide) (PDMA), poly(ethylene glycol) (PEG), poly(ethylene imine) (PEI), or poly(2-methyl-2-oxazoline) (PMeOx). Alternatively or additionally, the microsphere may comprise a hydrogel microsphere such as a synthetic hydrogel. A hydrogel microsphere is a colloidal particle made up of a cross-linked network of hydrophilic or amphiphilic polymer chains. In one preferred embodiment, the microsphere may be a PEG microsphere, an alginate microsphere, or a polyacrylamide microsphere. In one preferred embodiment, the microsphere is a polyacrylamide microsphere. Preferably, the microsphere is not a polystyrene bead such as a Dynabead. A Dynabead is a polystyrene bead that has been made magnetic. The polystyrene beads typically have a stiffness of 100,000 Pa or more. Dynabeads are superparamagnetic, meaning that they are only magnetic when in a magnetic field and can be resuspended when the magnetic field is removed.

[0052] Similarly, the population of microspheres according to the invention may comprise one or more of any suitable polymer, such as a hydrophilic polymer. Hydrophilic polymers can be natural, semisynthetic or synthetic. The hydrophilic polymer may be one or more of poly(vinyl alcohol) (PVA), poly(acrylamide) (PAAm), poly(2-hydroxypropyl methacrylamide) (PHPMA), poly(vinylpyrrolidone) (PVP), poly(N,N-dimethylacrylamide) (PDMA), poly(ethylene glycol) (PEG), poly(ethylene imine) (PEI), or poly(2-methyl-2-oxazoline) (PMeOx). Alternatively or additionally, the population of microspheres according to the invention may comprise a hydrogel microsphere such as a synthetic hydrogel. In one preferred embodiment, the population of microspheres of the invention may comprise one or more a PEG microsphere, an alginate microsphere, or a polyacrylamide microsphere. In one preferred embodiment, the population of microspheres comprises polyacrylamide microspheres. Preferably, the population of microspheres does not contain any polystyrene beads such as a Dynabead.

[0053] StiffnessStiffness is the extent to which an object resists deformation in response to an applied force. The term “stiffness” is used interchangeably with the term “Young’s modulus” . The SI unit of a pascal (Pa) may be used to measure stiffness, and a lower value corresponds to a less stiff material (i.e. a material that is more deformable). Therefore, quoted values in Pa correspond to the Young’s modulus in Pa. Thus, a microsphere of the invention having a stiffness of 100 Pa (i.e. a Young’s modulus of 100 Pa) is less stiff and less resistant to deformation than a microsphere having a stiffness of 5000 Pa (i.e. a Young’s modulus of 5000 Pa).

[0054] Atomic force microscopy (AFM) is a standard method that can be used to measure the stiffness of materials, such as microspheres. The material, such as a microsphere of the invention, is indented with a tip, and the resulting force measured.

[0055] A particular advantage of the present invention is the relatively low stiffness of the microsphere, in particular compared to the stiffness of polystyrene beads such as Dynabeads known to the skilled person. Specifically, the invention relates to microspheres having a stiffness in the general range of 10-10,000 Pa. In contrast, known microspheres typically have a stiffness several orders of magnitude higher, such as in the MPa or GPa range.

[0056] The microsphere of the invention may have a stiffness of at most 10 Pa, at most 100 Pa, at most 250 Pa, at most 500 Pa, at most 1000 Pa, at most 2500 Pa, at most 5000 Pa or at most 10000 Pa. The microsphere of the invention may, for example, therefore have a stiffness of 10-100 Pa, 10-250 Pa, 10-500 Pa, 10-1000 Pa, 10-2500 Pa, 10-5000 Pa, 10-10000 Pa, 100-250 Pa, 100-500 Pa, 100-1000 Pa, 100-2500 Pa, 100-5000 Pa, 100-10000 Pa, 250-500 Pa, 250-1000 Pa, 250-2500 Pa, 250-5000 Pa, 250-10000 Pa, 500-1000 Pa, 500-2500 Pa, 500-5000 Pa, 500-10000 Pa, 1000-2500 Pa, 1000-5000 Pa, 1000-10000 Pa, 2500-5000 Pa, 2500-10000 Pa or 5000-10000 Pa.

[0057] A particular advantage of the present invention is the relatively low stiffness of the microsphere. In in particular, the invention relates to microspheres having a stiffness in the general range of 10-10,000 Pa, as measured for example by AFM. In contrast, known microspheres typically have a stiffness several orders of magnitude higher, such as in the MPa or GPa range, as measured for example by AFM.

[0058] The microsphere of the invention may have a stiffness of at most 10 Pa, at most 100 Pa, at most 250 Pa, at most 500 Pa, at most 1000 Pa, at most 2500 Pa, at most 5000 Pa or at most 10000 Pa., as measured for example by AFM. The microsphere of the inventionmay, for example, therefore have a stiffness of 10-100 Pa, 10-250 Pa, 10-500 Pa, 10-1000 Pa, 10-2500 Pa, 10-5000 Pa, 10-10000 Pa, 100-250 Pa, 100-500 Pa, 100-1000 Pa, 100-2500 Pa, 100-5000 Pa, 100-10000 Pa, 250-500 Pa, 250-1000 Pa, 250-2500 Pa, 250-5000 Pa, 250-10000 Pa, 500-1000 Pa, 500-2500 Pa, 500-5000 Pa, 500-10000 Pa, 1000-2500 Pa, 1000-5000 Pa, 1000-10000 Pa, 2500-5000 Pa, 2500-10000 Pa or 5000-10000 Pa, as measured for example by AFM.

[0059] The microsphere of the invention may have a stiffness of less than 5000 Pa, less than 2500 Pa, less than 1000 Pa, less than 500 Pa, less than 250 Pa, less than 200 Pa, or less than 150 Pa, as measured for example by AFM. The microsphere of the invention may therefore have a stiffness 100-250 Pa, 100-500 Pa, 100-1000 Pa, 100-2500 Pa, 100-5000 Pa, 250-500 Pa, 250-1000 Pa, 250-2500 Pa, 250-5000Pa, 500-1000 Pa, 500-2500 Pa, 500-5000 Pa, 1000-2500 Pa, 1000-5000 Pa or 2500-5000 Pa, as measured for example by AFM.

[0060] The population of microspheres of the invention may have a mean stiffness of at most 10 Pa, at most 100 Pa, at most 250 Pa, at most 500 Pa, at most 1000 Pa, at most 2500 Pa, at most 5000 Pa or at most 10000 Pa., as measured for example by AFM. The population of microspheres of the invention may therefore have a mean stiffness of, for example, 10-100 Pa, 10-250 Pa, 10-500 Pa, 10-1000 Pa, 10-2500 Pa, 10-5000 Pa, 10-10000 Pa, 100-250 Pa, 100-500 Pa, 100-1000 Pa, 100-2500 Pa, 100-5000 Pa, 100-10000 Pa, 250-500 Pa, 250-1000 Pa, 250-2500 Pa, 250-5000 Pa, 250-10000 Pa, 500-1000 Pa, 500-2500 Pa, 500-5000 Pa, 500-10000 Pa, 1000-2500 Pa, 1000-5000 Pa, 1000-10000 Pa, 2500-5000 Pa, 2500-10000 Pa or 5000-10000 Pa, as measured for example by AFM.

[0061] The population of microspheres of the invention may have a mean stiffness of less than 5000 Pa, less than 2500 Pa, less than 1000 Pa, less than 500 Pa, less than 250 Pa, less than 200 Pa, or less than 150 Pa, as measured for example by AFM. The population of microsphere of the invention may therefore have a mean stiffness of, for example, 100-250 Pa, 100-500 Pa, 100-1000 Pa, 100-2500 Pa, 100-5000 Pa, 250-500 Pa, 250-1000 Pa, 250-2500 Pa, 250-5000Pa, 500-1000 Pa, 500-2500 Pa, 500-5000 Pa, 1000-2500 Pa, 1000-5000 Pa or 2500-5000 Pa, as measured for example by AFM.

[0062] The skilled person would be able to make use of AFM to measure stiffness of the microspheres. For example, microspheres whose stiffness is to be measured can be adhered to a solid support. Under an atomic force microscope, indentation experiments may be conducted using a suitable indenter by applying a suitable force, such as a peakforce of 1 nN, and generating and fitting data to force displacement curves. Any suitable AFM fitting software may be used, such as PyJibe. A Hertzian model with a geometric correction factor accounting for the finite size of the indenter and microspheres may be applied.

[0063] Moieties

[0064] A moiety may be associated with the surface of the microsphere, and therefore exposed to the environment that is external to the microsphere. The term “associated” may be used in its broadest sense to describe a moiety associated with a microsphere such that the moiety and microsphere move substantially together as one unit when in bulk solution. The moiety may, for example, be associated with the outer surface of the microsphere (i.e. on the surface of the microsphere). The moiety may be associated through a non-covalent interaction. Examples of non-covalent interactions include electrostatic interactions such as ionic interactions, hydrogen bonding and halogen bonding. Examples of non-covalent interactions also include Van der Waals forces and hydrophobic effects. In one preferred embodiment, the moiety is biotinylated and associated with streptavidin (e.g. streptavidin conjugated to a moiety) on the surface of the microsphere. The moiety may alternatively be fused to the surface of the microsphere by a covalent interaction. In one preferred embodiment, the moiety is associated with the surface of the microsphere, such as a polyacrylamide microsphere, by a cross-linking reaction such as l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry.

[0065] One or more linkers can optionally be included, such as to the surface of the microsphere. The linker may result in a fusion of two or more molecules. The linker physically separates and preferably preserves the functionality of at least one of the two more molecules that are fused. For example, the presence of a linker, may allow flexibility and enables the fusion to be positioned optimally for display on the surface of the microsphere or luminally according to what is required. Linkers according to the invention are useful in providing increased flexibility, improving pharmacokinetics (PK), increasing expression and improving biological activity of the fusion polypeptide constructs, and may also be used to ensure avoidance of steric hindrance and maintained functionality of the at least one of the two or more molecules. The linker may preferably be 10 amino acids or fewer in length. In some aspects, the linker is 9, 8, 6, 5, 4, 3, 2 amino acids in length orshorter. The linker can include any amino acids, such as Ala, Pro, Cys, and Gly. In one preferred embodiment, the linker is a PEG linker, such as a PEG-biotin linker. In another preferred embodiment, the linker results in a fusion between an amino group and streptavidin. One or more moiety may be directly or indirectly attached to the linker. For example, the one or more moiety may be attached indirectly to the linker via streptavidin. Thus, the microsphere may comprise a linker, such as an amine-PEG-biotin linker, wherein one end of the linker is attached to an amino group on the surface of the microsphere; optionally wherein streptavidin or a moiety is attached directly to the other end of the linker; and optionally wherein one or more moiety is attached to the streptavidin, such that the amino group, linker, streptavidin and / or moiety are on the external surface of the microsphere.

[0066] The moiety on the surface of the microsphere may be selected from any suitable molecule. For example, the moiety may be selected from one or more of a peptide and / or protein, a targeting peptide and / or targeting protein which binds to a molecule present on a cell to be targeted, a therapeutic moiety such as a receptor decoy, an endosomal escape moiety, an enzyme, a nuclease, a CRISPR-associated protein (Cas) such as SaCas9, SpCas9, Cas9, Casl2, Casl3 and variants and fusions thereof, a Transcription activatorlike effector nucleases (TALEN) and variants and fusions thereof, a meganuclease and variants and fusions thereof, a zinc finger nuclease and variants and fusions thereof, an antibody and / or antigen-binding variant or fragment thereof which may be biotinylated, a single chain variable fragment (scFv), VHH, a nanobody, a nucleic acid-binding protein and / or peptide, a RNA- and / or DNA-binding protein, viral-binding protein, a small molecule drug, a nucleic acid, a nucleic acid analogue, an unnatural nucleic acid, gRNA, miRNA, shRNA, siRNA, piRNA, PMO, DNA and a DNA plasmid encoding a therapeutic peptide and / or protein. In one preferable embodiment, the moiety is a targeting moiety. A targeting moiety is any moiety which confers tropism or increases the tropism of the microsphere to a desired target, such as a desired cell type or tissue. The moiety may have therapeutic utility.

[0067] In one particular, embodiment, the microsphere has on its surface one or more moiety each selected from a protein, a polysaccharide chain, and / or an extracellular matrix protein. Similarly, the population of microspheres may comprise microspheres each having on their surface one or more moiety each selected from a protein, a polysaccharide chain, and / or an extracellular matrix protein. The moiety may be an agonist. An agonist isany substance that binds to a receptor on a cell and causes a physiological response, such as making the cell more active. Alternatively or in addition, the moiety may be an antagonist. An antagonist is any substance that binds to a receptor and prevents the effect of an agonist and / or blocks or dampens a biological response by binding to the receptor.

[0068] Agonists and / or antagonists may be endogenous, meaning they are natural compounds, or exogenous, meaning they are synthetic medications or compounds.

[0069] Agonists and / or antagonists may be competitive, non-competitive, and uncompetitive.

[0070] In one embodiment, the microsphere or population of microspheres may:

[0071] (a) comprises an antibody or antigen-binding fragment thereof;

[0072] (b) comprises one or more of an antibody or antigen-binding fragment thereof which targets CD3, such as antibody 0KT3; an antibody or antigen-binding fragment thereof which targets CD28, such as antibody CD28.2; and comprises an antibody or antigen-binding fragment thereof which targets CD2, such as antibody TS1 / 8 orRPA-2.10; and / or

[0073] (c) comprises an antibody or antigen-binding fragment thereof which targets CD3, an antibody or antigen-binding fragment thereof which targets CD28, and an antibody or antigen-binding fragment thereof which targets CD2; and / or

[0074] (d) comprises a moiety that binds to a target on the surface of a T-cell.

[0075] In a preferred embodiment the moiety may be an antibody or antigen-binding fragment thereof and / or bind to a target on the surface of a T-cell. The moiety may be biotinylated. In advantageous embodiments, the moiety may target primary stimulation of a T-cell, co-stimulation of a T-cell, and / or adhesion of a T-cell. For example, the moiety may:

[0076] (a) target primary stimulation of a T-cell by targeting a TCR complex such as targeting CD3, a T-cell receptor, and / or subdomain thereof;

[0077] (b) comprise an antibody or antigen-binding fragment thereof which targets CD3, such as antibody 0KT3;

[0078] (c) target co-stimulation of a T-cell by targeting CD28, CD27, CD4, CD8 and / or 4- IBB;

[0079] (d) comprise an antibody or antigen-binding fragment thereof which targets CD28, such as antibody CD28.2;(e) target adhesion of a T-cell by targeting CD2, LFA-1, CD44, CXCR3, CCR7, CD31, VLA-4 and / or CD62L;

[0080] (f) comprise an antibody or antigen-binding fragment thereof which targets CD2, such as antibody TS1 / 8 orRPA-2.10; and / or

[0081] (g) comprise an antibody or antigen-binding fragment thereof which targets CD3, an antibody or antigen-binding fragment thereof which targets CD28, and an antibody or antigen-binding fragment thereof which targets CD2. Examples of primary stimulators of a T-cell include CD3, a T-cell receptor and subdomain(s) thereof. Examples of co-stimulators of a T-cell include CD28, CD27, CD4, CD8 and 4-1BB. Examples of moieties targeting adhesion of a T-cell include CD2, LFA-1, CD44, CXCR3, CCR7, CD31, VLA-4 and / or CD62L.

[0082] The microsphere according to the invention may have at least 100, at least 1000, at least 10,000, at least 100,000 or at least at least 1,000,000 copies of said moiety. Thus, the microsphere may have 100-1000 copies, 100-10,000 copies, 100-100,000 copies, 100-1,000,000 copies, 1000-10,000 copies, 1000-100,000 copies, 1000-1,000,000 copies, 10,000-100,000 copies, 10,000-1,000,000 copies or 100,000-1,000,000 copies of each moiety. Similarly, the population of microsphere according to the invention may comprise microspheres each having at least 100, at least 1000, at least 10,000, at least 100,000 or at least at least 1,000,000 copies of said moiety. Thus, the population of microspheres according to the invention may comprise microspheres each having 100-1000 copies, 100-10,000 copies, 100-100,000 copies, 100-1,000,000 copies, 1000-10,000 copies, 1000-100,000 copies, 1000-1,000,000 copies, 10,000-100,000 copies, 10,000-1,000,000 copies or 100,000-1,000,000 copies of each moiety.

[0083] Targeting

[0084] The microsphere of the present invention may be targeted, for example to a desired cell type or tissue. This targeting is achieved by expressing on the surface of the microsphere a targeting moiety which binds to the target, such as a target expressed on the surface of a cell or tissue to be targeted. The targeting moiety may be any moiety identified above. The targeting moiety may be agonists or antagonists of receptors on the cell to be targeted, such as an immune cells, and more preferably a T-cell.In more detail, the microsphere of the invention can be targeted, for example to particular cell types or tissues, by expressing on their surface a targeting moiety such as a peptide. Suitable peptides are those which bind to cell surface moieties such as receptors or their ligands found on the cell surface of the cell to be targeted. Examples of suitable targeting moieties are short peptides, scFv and complete proteins, so long as the targeting moiety can be expressed on the surface of the microsphere and preferably does not interfere with cargo carrying capacity of the microsphere. Peptide targeting moieties may typically be less than 100 amino acids in length, for example less than 50 amino acids in length, less than 30 amino acids in length, to a minimum length of 10, 5 or 3 amino acids. For example, peptide targeting moieties may be 3 to 5 amino acids, 3 to 10 amino acids, 3 to 30 amino acids, 3 to 50 amino acids, 3 to 100 amino acids, 5 to 10 amino acids, 5 to 30 amino acids, 5 to 50 amino acids, 5 to 100 amino acids, 10 to 30 amino acids, 10 to 50 amino acids, 10 to 100 amino acids, 30 to 50 amino acids, 30 to 100 amino acids or 50 to 100 amino acids in length.

[0085] Targeting moieties can be selected to target particular cells, subcellular locations, tissues, organs or other bodily compartments. Organs and cell types that may be targeted include: the brain, neuronal cells, the blood brain barrier, muscle tissue, the eye, lungs, liver, kidneys, heart, stomach, intestines, pancreas, red blood cells, white blood cells including B-cells and T-cells, lymph nodes, bone marrow, spleen and cancer cells.

[0086] Alternatively, or in addition, targeting moieties may target a diseased tissue such as a tumour. In a preferred embodiment, the microsphere are targeted to brain tissue. The targeting moiety targets the liver, the heart, the brain or neuronal tissue, hepatocytes, cardiomyocytes, cardiac smooth muscle cells, sensory neurons, motor neurons, interneurons or glia cells.

[0087] In a particularly preferred embodiment, the targeting moiety targets an immune cell for example by binding a receptor on the cell to be targeted. The targeting moiety may be an agonist or an antagonist. The receptor may be present on an immune cell, such as a neutrophil, an eosinophil, a basophil, a mast cell, a macrophage, a histocyte, a Kupffer cell, an alveolar macrophage, a dendritic cell, a B cell, a plasma cell, a memory B cell, a Killer T cell, a memory T cell, a T helper cell such as a population of TH1, TH2, TH3, TH17, TH9, or TFH cells, a Natural killer T cell, a CAR T-cell, an innate lymphoid cell and / or a natural killer cell. More preferably, the cell is a T-cell, optionally a CAR T-cell. By better mimicking the physiological conditions of, in particular, T-cell activation, microspheres ofthe invention generate T-cells that are more suited for effective immunotherapies. In particular, the microspheres of the invention can be used for activating T cell in vitro for subsequent administration as a cell therapy.

[0088] Cargo Molecule

[0089] The microsphere may be loaded with a cargo molecule (i.e. cargo). The terms “load”, “loaded”, “loading”, “onto a microsphere” and “into a microsphere” may be used in their broadest sense to describe a cargo molecule associated with a microsphere such that the microsphere and its cargo molecule move substantially together as one unit when in bulk solution. Thus, the cargo molecule may be encapsulated in the interior (i.e. within the lumen) of the microsphere. Alternatively, or in addition, the cargo molecule may be present on the surface (i.e. outside) of the microsphere.

[0090] The cargo molecule may be inside and / or outside the microsphere. When the cargo molecule is inside the microsphere, it may also be possible to reduce or eliminate recognition by the innate immune system of the cargo molecule and thus reduce or eliminate acute inflammatory responses associated with the naked delivery of the cargo molecule.

[0091] The cargo molecule may be added to an isolated microsphere. The cargo molecule may be selected from any suitable molecule. For example, the cargo molecule may be selected from one or more of a therapeutic cargo, a peptide and / or protein, an enzyme, a nuclease, a CRISPR-associated protein (Cas) such as SaCas9, SpCas9, Cas9, Cast 2, Cast 3 and variants and fusions thereof, a Transcription activator-like effector nucleases (TALEN) and variants and fusions thereof, a meganuclease and variants and fusions thereof, a zinc finger nuclease and variants and fusions thereof, an antibody and / or antigen-binding variant or fragment thereof, a single chain variable fragment (scFv), VHH, a nanobody, a nucleic acid-binding protein and / or peptide, a RNA- and / or DNA-binding protein, a small molecule drug, a nucleic acid, a nucleic acid analogue, an unnatural nucleic acid, gRNA, miRNA, shRNA, siRNA, piRNA, PMO, DNA and a DNA plasmid encoding a therapeutic peptide and / or protein. In a preferred embodiment, the cargo molecule has therapeutic utility.

[0092] The cargo molecule may alternatively or in addition be selected from enzymes, receptors such as decoy receptors, membrane proteins, transporters, cytokines, antigens,neoantigens, immune effector molecules, ribonuclear proteins, nucleic acid binding proteins, antibodies, nanobodies, antibody fragments, antibody-drug conjugates, gene editing proteins such as CRISPR effector proteins including Cas proteins, transcription activator-like effector nucleases (TALENs), meganucleases.

[0093] The cargo molecule may alternatively or in addition be selected from antibodies, intrabodies, nanobodies, single chain variable fragments (scFv), VHHs, affibodies, bi- and multispecific antibodies or binders including bispecific T-cell engagers (BiTEs), receptors, ligands, transporters, enzymes for e.g. ERT or gene editing, tumour suppressors, viral or bacterial inhibitors, cell component proteins, DNA repair inhibitors, nucleases, proteinases, integrases, transcription factors, growth factors, apoptosis inhibitors and inducers, toxins (for instance pseudomonas exotoxins), structural proteins, neurotrophic factors such as NT3 / 4, brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) and its individual subunits such as the 2.5S beta subunit, ion channels, membrane transporters, proteostasis factors, proteins involved in cellular signaling, translation- and transcription related proteins, nucleotide binding proteins, protein binding proteins, lipid binding proteins, glycosaminoglycans (GAGs) and GAG-binding proteins, metabolic proteins, cellular stress regulating proteins, inflammation and immune system regulating proteins such as cytokines and inhibitors of such cytokines (cytokines may include: CXCL8, GMCSF, interleukins including: IL-1 family, IL-2, IL-4, IL-6, IL-6-like, IL-9, IL-10, IL12, IL-13, IL-17, Interferons including INF-alpha / beta / gamma, TNF family members, CD40 and CD40L, TRAIL, and TGF-beta family) mitochondrial proteins, and heat shock proteins, etc. The cargo protein may be a reporter protein such as green fluorescent protein (GFP) or nanoLuc. In one embodiment, the encoded protein is a CRISPR-associated (Cas) polypeptide (such as Cas9) with intact nuclease activity which is associated with (i.e. carries with it) an RNA strand that enables the Cas polypeptide to carry out its nuclease activity in a target cell once delivered by the peptide. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) components according to the present invention include CRISPR components that are derived from any bacterial source. The CRISPR components may come from class 1 or class 2, specifically the Cas type may be Cas type I, II, III, IV, V or VI. The specific Cas protein may be Cas9, Cas 12 (Cas 12a or Cas 12b), C2c2, Cpfl, CaslO, Cas 13 (cas 13 a, Cas 13b or Cas 13c), Cas3, a Cas 14 protein, a CasX protein, or a CasY protein, CasMINI, or SuperFi-Cas9. The CRISPR protein may be a CRISPR nuclease, a CRISPR nickase, or a nuclease deficient CRISPR variant.Alternatively, in another embodiment, the Cas polypeptide may be catalytically inactive, to enable targeted genetic engineering. Yet another alternative may be any other type of CRISPR effector such as the single RNA guided endonuclease Cpf 1.

[0094] The cargo molecule may alternatively or in addition be selected from the group comprising enzymes or transporters for lysosomal storage disorders, for instance glucocerebrosidases such as imiglucerase, alpha-galactosidase, alpha-L-iduronidase, iduronate-2-sulfatase and idursulfase, aryl sulfatase, galsulfase, acid-alpha glucosidase (GAA), sphingomyelinase, galactocerebrosidase, galactosylceramidase, ceramidase, alpha-N-acetylgalactosaminidase, beta-galactosidase, lysosomal acid lipase, acid sphingomyelinase, NPC1, NPC2, heparan sulfamidase, N-acetylglucosaminidase, heparan-a-glucosaminide-N-acetyltransferase, N-acetylglucosamine 6-sulfatase, galactose-6-sulfate sulfatase, galactose-6-sulfate sulfatase, hyaluronidase, alphaN -acetyl neuraminidase, GlcNAc phosphotransferase, mucolipinl, palmitoylprotein thioesterase, tripeptidyl peptidase I, palmitoyl-protein thioesterase 1, tripeptidyl peptidase 1, battenin, linclin, alpha-D-mannosidase, beta-mannosidase, aspartylglucosaminidase, alpha-L-fucosidase, cystinosin, cathepsin K, sialin, LAMP2, and hexoaminidase.

[0095] The cargo molecule may alternatively or in addition be selected from the group comprising enzymes associated with Urea cycle disorders including N-Acetylglutamate synthase, carbamoyl phosphate synthetase, ornithine transcarbamoylase, argininosuccinic acid synthase, argininosuccinic acid lyase, arginase, mitochondrial ornithine transporter, citrin, y+L amino acid transporter 1, uridine monophosphate synthase UMPS.

[0096] The cargo molecule may be e.g. an intracellular protein that modifies inflammatory responses, for instance epigenetic proteins such as methylases and bromodomains, or an intracellular protein that modifies muscle function, e.g. transcription factors such as MyoD or Myf5, proteins regulating muscle contractility e.g. myosin, actin, calcium / binding proteins such as troponin, or structural proteins such as Dystrophin, mini-dystrophin, micro-dystrophin, utrophin, titin, nebulin, dystrophin-associated proteins such as dystrobrevin, syntrophin, syncoilin, desmin, sarcoglycan, dystroglycan, sarcospan, agrin, and / or fukutin. The cargo is typically a protein or peptide of human origin unless indicated otherwise by their name, any other nomenclature, or as known to a person skilled in the art, and they can be found in various publicly available databases such as Uniprot, RCSB, etc.

[0097] The cargo molecule may alternatively or in addition be an antigen / neoantigen, optionally wherein the antigen / neoantigen is suitable for use in cancer immunotherapy.Any antigen / neoantigen may be incorporated into the microspheres of the present invention. The antigens may be suitable for raising immune responses against pathogens such as bacteria, viruses, funguses or the antigen may be a tumour antigen useful in eliciting an immune response against a tumour for cancer immunotherapy. There may be one or more antigens / neoantigens present in any microsphere according to the invention.

[0098] Where the cargo molecule is a genetic cargo molecule such as a nucleic acid, the cargo molecule may have utility in gene therapy and / or gene editing. Nucleic acids are routinely used in gene therapy for the replacement of non-functional genes and for neutralization of disease-causing mutations via RNA interference (RNAi) effector molecules such as miRNAs, shRNAs and siRNAs. Exemplary genetic material cargos include: messenger RNA (mRNA), circular mRNA, Doggybone® DNA (dbDNA®), linear DNA, circular DNA, plasmid DNA, linear RNA, circular RNA, self-amplifying RNA or DNA, a viral genome either “naked” or within a capsid or a modified version of any of the above. As naked DNA and RNA are difficult to deliver in vivo due to rapid clearance, nucleases, lack of organ-specific distribution and low efficacy of cellular uptake, specialized gene delivery vehicles, such as viral vectors and cationic liposomes, are usually used for delivery. Loading microspheres with genetic material cargo has a number of advantages, such as overcoming mutagenic integration associated with viruses such as lentiviruses; and inflammatory toxicity and rapid clearance associated with liposomes.

[0099] The genetic material to be loaded into the microspheres is chosen on the basis of the desired effect of that genetic material on the cell into which it is intended to be delivered and the mechanism by which that effect is to be carried out. For example, the genetic material may be useful in gene therapy, for example in order to express a desired gene in a cell or group of cells. Such genetic material is typically in the form of plasmid DNA or viral vector encoding the desired gene and operatively linked to appropriate regulatory sequences such as promoters, enhancers and the like such that the plasmid DNA is expressed once it has been delivered to the cells to be treated. Exemplary viral cargos include: a viral vector which is an AAV vector or a lentiviral vector. In some embodiments, the viral vector is an AAV vector. In some embodiments, the AAV vector comprises a capsid from human AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV 10, AAV11 or AAV12. In some embodiments, the AAV vector comprises an AAV viral genome comprising inverted terminal repeat (ITR) sequences from human AAV serotype AAV1, AAV2, AAV3, AAV4, AAV5, AAV6,AAV7, AAV8, AAV9, r AAV10. In some embodiments, the AAV capsid and the AAV ITR are from the same serotype or from different serotypes. In some embodiments the viral vector comprises a viral capsid and a viral genome, the viral genome comprising one or more heterologous transgenes. In preferred embodiments, the heterologous transgene encodes a polypeptide or protein. The protein encoded with in the viral genome may be any one of the protein cargos according to the invention allowing the viral cargo to act as a gene replacement therapy.

[0100] Examples of diseases susceptible to gene therapy include genetic diseases such as haemophilia B (Factor IX), cystic fibrosis (CTFR), Phenylketonuria (PKU), ALS and spinal muscular atrophy (SMN-1).

[0101] Genetic material can also be used in gene silencing. Such gene silencing may be useful in therapy to switch off aberrant gene expression or in animal model studies to create single or more genetic knock outs. Typically, such genetic material is provided in the form of siRNAs.

[0102] Genetic material can also be used for example in immunisation to express one or more antigens against which it is desired to produce an immune response. Thus, the nucleic acid cargo to be loaded into the microsphere can encode one or more antigens against which is desired to produce an immune response, including but not limited to tumour antigens, antigens from pathogens such as viral, bacterial or fungal pathogens.

[0103] Genetic material, for example nucleic acid cargo molecules and / or nucleic acid that may be bound to a RNA / DNA binding protein or exogenously loaded into the microsphere may in addition be selected from the group comprising shRNA, siRNA, saRNA, miRNA, an anti-miRNA, mRNA, modified mRNA, gRNA, pri-miRNA, pre-miRNA, circular RNA, piRNA, tRNA, rRNA, snRNA, IncRNA, ribozymes, mini-circle DNA, plasmid DNA, RNA / DNA vectors, trans-splicing oligonucleotides, splice-switching oligonucleotides, CRISPR guide strands, morpholinos (PMO) antisense oligonucleotides (ASO), peptidenucleic acids (PNA), a viral genome and viral genetic material (for instance a naked AAV genome), but essentially any type of nucleic acid molecule can be delivered by the microspheres of the present invention. Both single-stranded and double-stranded nucleic acid molecules are within the scope of the present invention, and the nucleic acid molecule may be naturally occurring (such as RNA or DNA) or may be a chemically synthesised RNA and / or DNA molecule which may comprise chemically modified nucleotides such as 2’-0-Me, 2’-O-Allyl, 2’-0-M0E, 2’-F, 2’-CE, 2’-EA 2’-FANA, LNA, CLNA, ENA,PNA, phosphorothioates, tricyclo-DNA, thionucleotides, phosphoramidate, PNA, PMO, etc.

[0104] The cargo molecule, such as the genetic material, may be modified. For example, the genetic cargo molecule may comprise: (i) single stranded 2’-O-methyl ribose modifications, single stranded 2’ methoxy-ethyl backbone chemistry, single stranded phosphoroamidate backbone chemistry, single stranded methylphosphonate backbone chemistry and / or single stranded phospohorothioate backbone chemistry; (ii) antisense modified oligonucleotides, antisense modified oligonucleotides comprising 2’-0-Me ribose modifications, antisense modified oligonucleotides comprising peptide nucleic acids, antisense modified oligonucleotides designed to induce exon skipping, antisense modified oligonucleotides which inhibit hairpin loops, and / or trans-splicing antisense modified oligonucleotides; and / or (iii) unnatural oligonucleotides selected from 2’-0-Me ribose modified oligonucleotides, morpholino, thio-nucleotides, chemically modified siRNAs and / or peptide nucleic acids.

[0105] The cargo molecule may be a small molecule. Non-limiting examples of small molecules include anticancer agents such as doxorubicin, methotrexate, 5 -fluorouracil or other nucleoside analogues such as cytosine arabinoside, proteasome inhibitors such as bortezomib, or kinase inhibitors such as imatinib or seliciclib, or NSAIDs such as naproxen, aspirin, or celecoxib, antibiotics such as heracillin, or antihypertensives such as ACE inhibitors such as enalapril, ARBs such as candesartan, cyclic dinucleotides, etc. The present invention is naturally applicable also to other small molecules without departing from the gist of the invention, as would be clear to a person skilled in the art.

[0106] Production Method

[0107] Provided is a method of producing a microsphere or population of microspheres as defined above. The method may be a step-wise method comprising first forming an emulsion and then a polymerisation step to form the microsphere or population of microspheres. Optionally, the microsphere or population of microspheres may be functionalised. Thus the invention provides a method of producing a microsphere or population of microspheres according to the invention, comprising:(a) forming an emulsion, such as an inverse emulsion, between an aqueous phase comprising a pre-polymer solution and optionally a free radical generator, and an oil phase comprising a surfactant;

[0108] (b) polymerising the pre-polymer solution to provide a microsphere or population of microspheres, such as by chemical and / or photopolymerization; and

[0109] (c) optionally functionalising the microsphere or population of microspheres by attaching the one or more moiety as defined in any one of claims 5-8 to the surface of the microsphere or population of microspheres.

[0110] Any one or more of the steps, such as the step of forming an emulsion, polymerising and / or functionalising, may be carried out in a dry atmosphere, such as a dry nitrogen (N2) atmosphere. Additionally or alternatively, any one or more of the steps, such as the step of forming an emulsion, polymerising and / or functionalising, may comprise stirring of the liquid phase, for example at least 10 rpm, at least 100 rpm, at least 1000 rpm or at least 1300 rpm.

[0111] An emulsion is a mixture of two or more liquids that are normally un-mixable, where one liquid is dispersed into the other as tiny droplets. The emulsion may be an inverse emulsion or an oil-in-water emulsion. Preferably, the emulsion is an inverse emulsion. An inverse emulsion is a type of emulsion where a continuous phase is oil and an internal phase is water. This is the opposite of a conventional emulsion, also known as an oil-in-water emulsion, where water is the continuous phase. Inverse emulsions are also known as water-in-oil emulsions.

[0112] The inverse emulsion may be formed between an aqueous phase comprising any suitable pre-polymer solution and optionally any suitable free radical generator, and any suitable oil phase comprising any suitable surfactant such as an anionic surfactant, an amphoteric surfactant or a non-ionic surfactant. The pre-polymer solution may comprise one or more of PEG, alginate, acrylamide, bis-acrylamide and acrylic acid. The free radical generator may comprises one or more of APS-TEMED; ammonium persulfate, riboflavin, dibenzoyl peroxide, azobis(isobutyronitrile), ceric ammonium nitrate, potassium persulphate, and potassium permanganate. The oil phase may comprises mineral oil and / or the surfactant sorbitan monooleate (Span®80).

[0113] The pre-polymer solution may be polymerised to provide a microsphere or population of microspheres using any suitable means, such as by chemical and / orphotopolymerization. Polymerization is a reaction that creates three-dimensional networks or polymer chains. The resulting polymers are generally resilient and have adjustable mechanical and thermal properties. Chemical polymerisation may comprise addition polymerisation, condensation polymerisation or radical reactions. In one preferable embodiment, polymerisation is a radical reaction in which a polymer forms by the successive addition of free-radical building blocks. Photopolymerization is a process in which small monomers are linked to become a chainlike polymer through a photochemical reaction, typically with the help of a catalyst. The skilled person would be able to select appropriate photopolymerization conditions. For example, the photo-initiator LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) may be used under 365 nm UV irradiation to induce polymerisation, such as polymerisation of PAA (poly(acrylic acid)).

[0114] The microsphere or population of microspheres may be functionalised by any suitable reaction, such as a cross-linking reaction. For example, l-ethyl-3 -(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry may be used to functionalise the microsphere or population of microspheres. Here, the carboxyl group present on the polymer backbone is activated using EDC / NHS chemistry, forming an amine reactive NHS ester. This ester may be used to covalently attach a small PEG-biotin linker. Streptavidin may then be conjugated to the PEG-biotin linker.

[0115] Biotinylated moieties, such as antibodies descried herein that have been biotinylated may then be conjugated to the streptavidin on the surface of the microsphere or population of microspheres.

[0116] The stiffness of the microsphere may be tuned by the production method. For example, a desired stiffness of the microsphere may be achieved by controlling the overall concentration of the monomer (such as the overall concentration of acrylamide) and / or the overall concentration of the cross-linker (such as the overall concentration of bisacrylamide). By increasing the concentration of the monomer alone, the density of polymerised chains becomes higher, resulting in more chain interactions and network entanglements, increasing the Young’s modulus. Similarly, increasing the concentration of cross-linker increases chain interactions, and leads to increased Young’s modulus. The skilled person would readily be able to make use of the present disclosure and their common general knowledge to determine the resulting Young’s modulus for a given microsphere.The monomer concentration may be present at a total concentration of up to 0.1% (v / v), up to 1% (v / v), up to 2% (v / v), up to 3% (v / v), up to 5% (v / v), up to 10% (v / v), up to 15% (v / v), up to 20% (v / v), up to 30% (v / v) or up 50% (v / v). Any of these percentage values may be combined to obtain a percentage range for the concentration of the monomer. Thus, the monomer concentration may be selected from 0.1-1% (v / v), 0.1-2% (v / v), 0.1-3% (v / v), 0.1-5% (v / v), 0.1-10% (v / v), 0.1-15% (v / v), 0.1-20% (v / v), 0.1-30% (v / v), 0.1-50% (v / v), 1-2% (v / v), 1-3% (v / v), 1-5% (v / v), 1-10% (v / v), 1-15% (v / v), 1-20% (v / v), 1-30% (v / v), 1-50% (v / v), 2-3% (v / v), 2-5% (v / v), 2-10% (v / v), 2-15% (v / v), 2-20% (v / v), 2-30% (v / v), 2-50% (v / v), 3-5% (v / v), 3-10% (v / v), 3-15% (v / v), 3-20% (v / v), 3-30% (v / v), 3-50% (v / v), 5-10% (v / v), 5-15% (v / v), 5-20% (v / v), 5-30% (v / v), 5-50% (v / v), 10-15% (v / v), 10-20% (v / v), 10-30% (v / v), 10-50% (v / v), 15-20% (v / v), 15-30% (v / v), 15-50% (v / v), 20-30% (v / v), 20-50% (v / v) and 30-50% (v / v). In one preferred embodiment, the monomer is selected from 1-10% (v / v), and more preferably from 3-5% (v / v).

[0117] The cross-linker concentration may be present at a total concentration of up to 0.01,% (v / v), up to 0.03% (v / v), up to 0.05% (v / v), up to 0.1% (v / v), up to 1% (v / v), up to 2% (v / v), up to 5% (v / v), up to 10% (v / v) or up to 20% (v / v). Any of these percentage values may be combined to obtain a percentage range for the concentration of the crosslinker. Thus, the cross-linker concentration may be selected from 0.01-0.03% (v / v), 0.01-0.05% (v / v), 0.01-0.1% (v / v), 0.01-1% (v / v), 0.01-2% (v / v), 0.01-5% (v / v), 0.01-10% (v / v), 0.01-20% (v / v), 0.03-0.05% (v / v), 0.03-0.1% (v / v), 0.03-1% (v / v), 0.03-2% (v / v), 0.03-5% (v / v), 0.03-10% (v / v), 0.03-20% (v / v), 0.05-0.1% (v / v), 0.05-1% (v / v), 0.05-2% (v / v), 0.05-5% (v / v), 0.05-10% (v / v), 0.05-20% (v / v), 0.1-1% (v / v), 0.1-2% (v / v), 0.1-5% (v / v), 0.1-10% (v / v), 0.1-20% (v / v), 1-2% (v / v), 1-5% (v / v), 1-10% (v / v), 1-20% (v / v), 2-5% (v / v), 2-10% (v / v), 2-20% (v / v), 5-10% (v / v), 5-20% (v / v) and 10-20% (v / v). In one preferred embodiment, the cross-linker is selected from 0.01-2% (v / v), and more preferably from 0.03-0.1% (v / v).

[0118] Also provided is a microsphere or population of microspheres obtained or obtainable by the method of the invention.

[0119] Cells and Culturing

[0120] Provided is a method of culturing a cell, comprising culturing the cell in a suspension comprising the microsphere or population of microspheres according theinvention, to provide a cultured cell. The cultured cell may be maintained and / or expanded. The ratio of the microsphere to the cell may be:

[0121] (a) about 1:1000; about 1:500; about 1:250; about 1:100: about 1:50; about 1:25; about 1:10; about 1:5; about 1:4, about 1:2; about 1:1; about 2:1; about 4:1; about 5:1; about 10:1; about 25:1; about 50:1; about 100:1; about 250:1; about 500:1; or about 1000:1;

[0122] (b) at least 1 : 1000; at least 1 :500; at least 1 :250; at least 1 : 100: at least 1:50; at least 1:25; at least 1:10; at least 1:5; at least 1:4, at least 1:2; at least 1:1; at least 2:1; at least 4:1; at least 5:1; at least 10:1; at least 25:1; at least 50:1; at least 100:1; at least 250:1; at least 500:1; or least 1000:1;

[0123] (c) at most 1:1000; at most 1:500; at most 1:250; at most 1:100: at most 1:50; at most 1:25; at most 1:10; at most 1:5; at most 1:4, at most 1:2; at most 1:1; at most 2:1; at most 4:1; at most 5:1; at most 10:1; at most 25:1; at most 50:1; at most 100:1; at most 250:1; at most 500:1; or ,most 1000:1; or (d) 1:1 to 1:10; 1:1 to 1:5; 1:1 to 1:4; 1:1 to 1:2; 1:2 to 1:10; 1:2 to 1:5; 1:2 to 1:4; 1:4 to 1:10; 1:4 to 1:5; or 1:5 to 1:10.

[0124] The microsphere or population of microspheres may be obtained according to any one or more of the methods set out herein or by an alternative method. As used herein, “cultured” is to be construed broadly as any method by which any cell is sustained and / or grown. Typically, cells will be cultured under controlled conditions that would be apparent and / or can be determined by the skilled person. Alternatively, or additionally, once cells having a desired characteristic are obtained, the cells may then be “maintained”, meaning that defining characteristics of the cell are not altered. For example, a maintained stem cell would not differentiate. Alternatively, or additionally cells may be “expanded” meaning that the number of cells is increased. When being expanded, cells may maintain their defining characteristics or acquire new defining characteristics (for example, if during expansion, the cell also differentiates). When being cultured, maintained and / or expanded, cells may be passaged as necessary. Passaging comprises replenishing the culture medium with new culture medium, for example as components in the culture medium are used up and / or degrade. Passaging may involve adding new culture medium without removing the old culture medium. Passaging may also involve removal of some or all of the culture medium, and adding new culture medium. Cells may also be transferred from a previous culture vessel to a new culture vessel during passaging. During passaging, a mediumhaving the same composition may be used. Alternatively, a medium having a different composition may be used (i.e. the media switched in whole or in part). Therefore, during passaging, any of the components in the culture medium may be replenished. The cells described herein may be cultured, maintained and / or expanded under adherent conditions (i.e. being attached to a solid support) or suspension conditions (i.e. as a colloid in the culture medium).

[0125] The cell may be an immune cell such as a neutrophil, an eosinophil, a basophil, a mast cell, a macrophage, a histocyte, a Kupffer cell, an alveolar macrophage, a dendritic cell, a B cell, a plasma cell, a memory B cell, a Killer T cell, a memory T cell, a T helper cell such as a population of TH1, TH2, TH3, TH17, TH9, or TFH cells, a Natural killer T cell, a CAR T-cell, an innate lymphoid cell and / or a natural killer cell. In one preferable embodiment, the cell is a T-cell, optionally wherein the cell is a CAR T-cell. The cell may be a CD4+ T-cell or a CD8+ T-cell.

[0126] The cultured cell may be an immune cell such as a neutrophil, an eosinophil, a basophil, a mast cell, a macrophage, a histocyte, a Kupffer cell, an alveolar macrophage, a dendritic cell, a B cell, a plasma cell, a memory B cell, a Killer T cell, a memory T cell, a T helper cell such as a population of TH1, TH2, TH3, TH17, TH9, or TFH cells, a Natural killer T cell, a CAR T-cell, an innate lymphoid cell and / or a natural killer cell. In one preferable embodiment, the cultured is a T-cell, optionally wherein the cultured cell is a CAR T-cell. The cultured cell may be a CD4+ T-cell or a CD8+ T-cell.

[0127] The cell and cultured cell may be an immune cell such as a neutrophil, an eosinophil, a basophil, a mast cell, a macrophage, a histocyte, a Kupffer cell, an alveolar macrophage, a dendritic cell, a B cell, a plasma cell, a memory B cell, a Killer T cell, a memory T cell, a T helper cell such as a population of TH1, TH2, TH3, TH17, TH9, or TFH cells, a Natural killer T cell, a CAR T-cell, an innate lymphoid cell and / or a natural killer cell. In one preferable embodiment, the cell and cultured cell is a T-cell, optionally wherein the cell and cultured cell is a CAR T-cell. The cell and cultured cell may be a CD4+ T-cell or a CD8+ T-cell.

[0128] Sources of immune cells will be known to persons skilled in the art, illustrative examples of which include peripheral blood, peripheral blood mononuclear cells, bone marrow, lymph nodes tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumours.The immune call may be derived from an autologous cell. The immune cell may be derived from an allogeneic cell. The term "autologous" refers to any material derived from the same individual to whom the material is later to be re-introduced to the individual. The term "allogeneic" refers to any material derived from a different individual of the same species as the individual to whom the material is introduced. Two or more individuals are said to be allogeneic to one another when the genes at one or more loci are not identical. In some aspects, allogeneic materials from individuals of the same species may be sufficiently genetically distinct to interact antigenically.

[0129] The immune cell may be obtained from any suitable mammalian or nonmammalian animal. Non-mammalian animals include Zebrafish, Drosophila and Xenopus. Mammalian animals include human, rodent (e.g. mouse or rat), pig (e.g. Sus scrofa), monkey, marmoset (Callithrix jacchus). dog, rabbit, llama or camelid. In one embodiment, the immune cell is preferably a mammalian cell, and more preferably a human cell.

[0130] The cultured cell may be obtained by culturing a cell as defined above, which may be a naive / non-activated immune cell such as a T-cell, in a suspension comprising the microsphere or population of microspheres according the invention, to provide a cultured cell. The cultured cell may have:

[0131] (a) a lower expression level of CD25 and / or CD69 compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres: cell of 1 : 1;

[0132] (b) a lower expression level of PD1, CTLA4 and / or TIGIT compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres: cell of 1 : 1;

[0133] (c) a higher expression level of TNF, IL2 and / or IFNy compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres: cell of 1 : 1;

[0134] (d) a higher expression level of CD45RA and / or TCF1 compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres: cell of 1 : 1;(e) a higher expression level of CD45RA and / or CD27 compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres: cell of 1 : 1;

[0135] (f) a higher expression level of CD45RA and / or CCR7 compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres: cell of 1 : 1;

[0136] (g) a T-cell which has a higher expression level of CD62L and / or CD27 compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1:1; and / or

[0137] (h) a population of a T-cells which has a higher proportion of CD4+ T-cell and / or a CD8+ T-cell compared to an equivalent cell population, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres: cell of 1 : 1;

[0138] wherein in any one of (a)-(h) the population of equivalent polystyrene microspheres have a mean stiffness greater than 100,000 Pa.

[0139] Lower expression levels may be at least 1%, at least 2%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90% or at least 95% lower than the equivalent expression in an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres: cell of 1 : 1. Expression may be measured by any suitable technique such as qRT-PCR or immunostaining techniques known to the skilled person.

[0140] Alternatively or additionally, higher expression levels may be at least 1%, at least 2%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90% or at least 95% higher than the equivalent expression in an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1. Expression may be measured by any suitable technique such as qRT-PCR or immunostaining techniques known to the skilled person.

[0141] Thus provided is a method for producing a cell comprising a lower expression level of CD25 and / or CD69. Optionally the lower expression level of CD25 and / or CD69 islower when compared to an equivalent cell which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres: cell of 1 : 1.

[0142] Also provided is a method for producing a cell comprising a lower expression level of PD1, CTLA4 and / or TIGIT. Optionally, the lower expression of PD1, CTLA4 and / or TIGIT is lower when compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1.

[0143] Also provided is a method for producing a cell comprising a higher expression level of TNF, IL2 and / or TFNy. Optionally, the higher expression of TNF, IL2 and / or IFNy is higher when compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres: cell of 1 : 1.

[0144] Also provided is a method for producing a cell comprising a higher expression level of CD45RA and / or TCF1. Optionally, the higher expression of CD45RA and / or TCF1 is higher when compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1.

[0145] Also provided is a method for producing a cell comprising a higher expression level of CD45RA and / or CD27. Optionally, the higher expression of CD45RA and / or CD27 is higher when compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1.

[0146] Also provided is a method for producing a cell comprising a higher expression level of CD45RA and / or CCR7. Optionally, the higher expression of CD45RA and / or CCR7 is higher when compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1.

[0147] Also provided is a method for producing a cell comprising a higher expression level of CD62L and / or CD27. Optionally, the higher expression of CD62L and / or CD27 is higher when compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres: cell of 1 : 1.Also provided is a method for producing a population of T-cells which have a higher proportion of CD4+ T-cell and / or a CD8+ T-cell. Optionally, the higher proportion of CD4+ T-cell and / or a CD8+ T-cell is higher when compared to an equivalent cell population, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1.

[0148] The cultured cell may have the same or greater functional activity and / or be less differentiated compared to an equivalent cell which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microsphere:cell of 1 : 1, and wherein the population of equivalent polystyrene microspheres have a mean stiffness greater than 100,000 Pa.

[0149] The cell may be cultured for: 1-2 days, 1-5 days, 1-7 days, 1-10 days, 1-12 days, 1-14 days, 1-20 days, 1-30 days, 2-5 days, 2-7 days, 2-10 days, 2-12 days, 2-14 days, 2-20 days, 2-30 days, 5-7 days, 5-10 days, 5-12 days, 5-14 days, 5-20 days, 5-30 days, 7-10 days, 7-12 days, 7-14 days, 7-20 days, 7-30 days, 10-12 days, 10-14 days, 10-20 days, 10-30 days, 12-14 days, 12-20 days, 12-30 days, 14-20 days, 14-30 days, or 20-30 days. The cell may be cultured for at least 1 day, at least 2 days, at least 5 days, at least 7 days, at least 10 days, at least 12 days, at least 14 days, at least 20 days, or at least 30 days.

[0150] The cell may then be isolated and formulated for use in a T-cell therapy. By better mimicking the physiological conditions of, in particular, T-cell activation, microspheres of the invention allow for the generation of cultured cell, such as cultured T-cells, that are more suited for effective immunotherapies. In particular, the microspheres of the invention can be used for activating T cell in vitro for subsequent administration as a cell therapy.

[0151] Also provided is a composition comprising a microsphere or population thereof as defined herein, and the cell as defined herein. Also provided is a cell or population thereof obtained or obtainable by a method defined herein.

[0152] Also provided is the use of the cultured cell as defined herein to identify agents capable of preventing or treating a disease, comprising treating a disease model before, during or after induction of the disease, optionally wherein the disease is cancer. Also provided is the use of the cell or cell population as defined herein to identify agents capable of preventing or treating a disease, comprising treating a disease model before, during or after induction of the disease, optionally wherein the disease is cancer.Therapy and Delivery / Administration

[0153] Provided is a cell composition (i.e. a cultured cell as defined here and / or a cell or cell population as defined herein), for use in a method of therapy. Therapy encompasses the treatment and / or prevention of a disease. The therapy may be an immunotherapy. The therapy may be a method of treating cancer. The cancer may be selected from one or more of a blood cancer such as Acute lymphocytic leukaemia, Acute myelogenous leukaemia; Chronic lymphocytic leukaemia; Chronic myeloid leukaemia; Hodgkin lymphoma;

[0154] Lymphoma; Myelodysplasia; Myeloma; and / or Myeloproliferative neoplasm.

[0155] Alternatively or additionally, the cancer may be selected from one or more of a solid tumour such as Breast cancer; Carcinosarcoma; Lung cancer; Lymphoma; Melanoma; Colon cancer; a Germ cell tumour; Prostate cancer; Brain cancer; Carcinoma;

[0156] Chondrosarcoma; and / or Neuroblastoma.

[0157] Also provided is a method of treating or preventing a disease in a subject comprising administering to the subject a cultured cell as defined herein and / or a cell or cell population as defined herein. The method may be a method of immunotherapy. The method may be a method of treating cancer. The cancer may be selected from one or more of a blood cancer such as Acute lymphocytic leukaemia, Acute myelogenous leukaemia; Chronic lymphocytic leukaemia; Chronic myeloid leukaemia; Hodgkin lymphoma;

[0158] Lymphoma; Myelodysplasia; Myeloma; and / or Myeloproliferative neoplasm.

[0159] Alternatively or additionally, the cancer may be selected from one or more of a solid tumour such as Breast cancer; Carcinosarcoma; Lung cancer; Lymphoma; Melanoma; Colon cancer; a Germ cell tumour; Prostate cancer; Brain cancer; Carcinoma;

[0160] Chondrosarcoma; and / or Neuroblastoma.

[0161] Also provided is a the use of a cultured cell as defined herein and / or a cell or cell population as defined herein in the manufacture of a medicament for the treatment or prevention of a disease. The use may be immunotherapy. The disease may be cancer. The cancer may be selected from one or more of a blood cancer such as Acute lymphocytic leukaemia, Acute myelogenous leukaemia; Chronic lymphocytic leukaemia; Chronic myeloid leukaemia; Hodgkin lymphoma; Lymphoma; Myelodysplasia; Myeloma; and / or Myeloproliferative neoplasm. Alternatively or additionally, the cancer may be selected from one or more of a solid tumour such as Breast cancer; Carcinosarcoma; Lung cancer;Lymphoma; Melanoma; Colon cancer; a Germ cell tumour; Prostate cancer; Brain cancer; Carcinoma; Chondrosarcoma; and / or Neuroblastoma.

[0162] The cell composition may be administered by any suitable means. Administration to a human or animal subject may be selected from parenteral, intramuscular, intracerebral, intravascular (including intravenous), subcutaneous, intranasal, intracardiac, intracerebroventricular, intraperitoneal or transdermal administration. Typically, the method of delivery is by injection. Preferably the injection is intramuscular or intravascular (e.g. intravenous). A physician will be able to determine the required route of administration for each particular patient.

[0163] The cell composition may be formulated for any suitable means of administration, including parenteral, intramuscular, intracerebral, intravascular (including intravenous), intracardiac, intracerebroventricular, intraperitoneal, subcutaneous, intranasal or transdermal administration. Compositions for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. The cell composition of the invention may be formulated in a pharmaceutical composition, which may include pharmaceutically acceptable carriers, thickeners, diluents, buffers, preservatives, and other pharmaceutically acceptable carriers or excipients and the like.

[0164] A "pharmaceutically acceptable carrier" (excipient) is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to a subject. Typical pharmaceutically acceptable carriers include, but are not limited to, binding agents (e.g. pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc); fillers (e.g. lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc); lubricants (e.g. magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, corn starch, polyethylene glycols, sodium benzoate, sodium acetate, etc); disintegrates (e.g. starch, sodium starch glycolate, etc); or wetting agents (e.g. sodium lauryl sulphate, etc).

[0165] The term "excipient" or "carrier" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of a compound. The terms encompass any of the agents approved by a regulatory agency such as the FDA or EMEA or listed in the U.S. Pharmacopeia for use in animals, including humans, as well as any carrier or diluent that does not cause significant irritation to a subject and does not abrogatethe biological activity and properties of the therapeutic cargo. Included are excipients and carriers that are useful in preparing a pharmaceutical composition and are generally safe and non-toxic.

[0166] Further exemplary excipients include degradation or loss of activity stabiliser excipients such as proteins such as human serum albumin, polyols such as glycerol, sorbitol and erythritol, amino acids such as arginine, aspartic acid, glutamic acid, lysine, proline, glycine, histidine and methionine, polymers such as polyvinylpyrrolidone and hydroxypropyl cellulose, surfactants such as polysorbate 80, polysorbate 20 and pluronicF68, antioxidants such as ascorbic acid and alpha-tocopherol (vitamin E), buffers such as acetate, succinate, citrate, phosphate, histidine, tri s(hydroxymethyl)aminom ethane (TRIS), metal ion / chelators such as Ca2+, Zn2+ and EDTA, Cyclodextrin based such as hydroxypropyl B-cyclodextrin and others such as polyanions and salts, stabilisers or bulking agents such as lactose, trehalose, dextrose, sucrose, sorbitol, glycerol, albumin, gelatin, mannitol and dextran, or preservatives such as benzyl alcohol, m-cresol, phenol, 2-phenoxy ethanol.

[0167] The compositions provided herein may additionally contain other adjunct components conventionally found in pharmaceutical compositions. Thus, for example, the compositions may contain additional compatible pharmaceutically-active materials or may contain additional materials useful in physically formulating various dosage forms of the composition of present invention, such as dyes, flavouring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions provided herein.

[0168] A therapeutically effective amount of composition is administered. The dose may be determined according to various parameters, especially according to the severity of the condition, age, and weight of the patient to be treated; the route of administration; and the required regimen. A physician will be able to determine the required route of administration and dosage for any particular patient. Optimum dosages may vary depending on the relative potency of the cell composition, and can generally be estimated based on EC50s found to be effective in vitro and in in vivo animal models. Different dosages of the cell composition may be administered depending on whether administration is by intramuscular injection or systemic (intravenous or subcutaneous) injection.It will be clear to the skilled artisan that when describing medical and scientific uses and applications of the cell composition, the present invention normally relates to a plurality of the cell composition, i.e. a population which may comprise thousands, millions, billions or even trillions of individual cells. The cell composition may be present in concentrations such as about 105, 108, IO10, 1011, 1012or 1013individual cells per unit of volume (for instance per ml), or any other number larger, smaller or anywhere in between, such as 105-l 012individual cells per unit volume, or 108to IO10cells per unit volume. The desired cell (e.g. cultured cell as defined here and / or cell or cell population as defined herein) may be at least 50% of the total cells in the composition, such as at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.9% of the total cells in the composition.

[0169] The composition according to the present invention may be formulated by any known method of formulation including but not limited to

[0170] • Oral formulations - Tablet, Capsule, Sustained release, liquid

[0171] • Intravenous Formulations

[0172] • Parenteral Formulations

[0173] • Topical Formulations - cutaneous administration: cream, ointment, gel, paste, powder

[0174] • Modified release Formulations - sustained release formulation

[0175] • Liquid or lyophilized formulations

[0176] • Nebulized formulations

[0177] Additional routes of administration by which the cell compositions of the invention may be administered to a human or animal subject include auricular (otic), buccal, conjunctival, cutaneous, dental, electro-osmosis, endocervical, endosinusial, endotracheal, enteral, epidural, extra-amniotic, extracorporeal, hemodialysis, infiltration, interstitial, intra-abdominal, intra-amniotic, intra-arterial, intra-articular, intrabiliary, intrabronchial, intrabursal, intracardiac, intracartilaginous, intracaudal, intracavemous, intracavitary, intracerebral, intracerebroventricular, intracistemal, intracorneal, intracoronal (dental), intracoronary, intracorporus cavernosum, intradermal, intradiscal, intraductal, intraduodenal, intradural, intraepidermal, intraesophageal, intragastric, intragingival, intraileal, intralesional, intraluminal, intralymphatic, intramedullary, intrameningeal, intramuscular, intraocular, intraovarian, intraperi cardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrasinal, intraspinal, intrasynovial, intratendinous,intratesticular, intrathecal, intrathoracic, intratubular, intratumour, intratym panic, intrauterine, intravascular, intravenous, intravenous bolus, intravenous drip, intraventricular, intravesical, intravitreal, iontophoresis, irrigation, laryngeal, nasal, nasogastric, occlusive dressing technique, ophthalmic, oral, oropharyngeal, other, parenteral, percutaneous, periarticular, peridural, perineural, periodontal, rectal, respiratory (inhalation), retrobulbar, soft tissue, subarachnoid, subconjunctival, subcutaneous, sublingual, submucosal, topical, transdermal, transmucosal, transplacental, transtracheal, transtympanic, ureteral, urethral, and / or vaginal administration, and / or any combination of the above administration routes, which typically depends on the disease to be treated and / or the characteristics of the cell composition.

[0178] Due to clearance of the cell composition (and breakdown of any cargo molecule), the patient may have to be treated repeatedly, for example once or more daily, weekly, monthly or yearly. Persons of ordinary skill in the art can easily estimate repetition rates for dosing based on measured residence times and concentrations of the cell composition in bodily fluids or tissues. Following successful treatment, it may be desirable to have the patient undergo maintenance therapy, wherein the cell composition is administered in maintenance doses, once or more daily, to once every 20 years. Thus, it is envisaged that any dosage regime would be applicable to the engineered cell composition of the invention. The dosage regime chosen will depend on the cargo being delivered by the cell composition and the disease to be treated and any additional therapies being administered which will be determined by the skilled physician. It is envisaged that the cell composition of the present invention will be administered multiple times, i.e. more than 1 time but normally more than 2 times or potentially for chronic, long-term treatment (i.e. administered tens to hundreds to thousands of times). Preferably, if the cargo is an antigen that is being administered as a vaccine, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks. Similarly, if the cargo is e.g. an RNA agent such as an siRNA or mRNA or a protein such as an antibody or an enzyme or a transporter, the cell composition comprising the cargo in question may be administered more than once, normally multiple times as part of a chronic treatment regimen.

[0179] A composition of the invention may be co-administered with one or more other agent. The one or more other agent may be administered separately to the composition of the invention, at substantially the same time as the composition of the invention. Thus,combination therapy comprising the cell composition of the invention and one or more other agent is envisaged.

[0180] The following non-limiting examples illustrate the invention.

[0181] Example 1 - Microsphere production

[0182] To generate large numbers of spherical, cell sized polymer particles that form the basis of the technology, the inventors employed an inverse emulsion method. Specifically, a solution containing the monomer acrylamide, acrylic acid, a cross-linker such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), bis-acrylamide and a free radical initiator (APS / TEMED) were dispersed in a volume of mineral oil containing the surfactant Span80 (sorbitan monooleate) (Fig. la). Rapid agitation of the aqueous and oil phase led to the generation of an emulsion of droplets containing the polymer components. The presence of the free radical initiator led to the rapid polymerization of the droplets under a nitrogen flow to minimise the effects of absorbed oxygen which would quench the radical reaction. Once polymerisation was complete, the polymerised particles were removed from the oil phase via a solvent wash.

[0183] The polymerised particles were then functionalized using EDC / NHS chemistry (Fig. lb). Specifically, the co-polymerised acrylic acid presented carboxylic acid groups on the polymer backbone, which on reacting with EDC / NHS is converted into an NHS ester. The NHS ester was then conjugated to an amine-PEG-biotin linker, providing a flexible biotin group on the polymer backbone. Microspheres were then subsequently incubated with streptavidin solution followed by biotinylated antibodies (Fig.lc). For optimal T-cell stimulation, the inventors incorporated antibodies targeting CD3, CD28 and CD2. At all stages of the protocol, microspheres were quality controlled for size, ligand binding (Fig. Id) and stiffness (via AFM indentation, Fig.le). Notably, no difference was observed in the concentration of bound ligand between the “soft” (245+ / - 143Pa) and “stiff’ (5470+ / - 1390Pa) microspheres.

[0184] To examine the performance of the microspheres as a T-cell activation platform, primary human T-cells were isolated from blood and co-cultures set-up in parallel comparing Dynabeads and both “soft” and “stiff’ microspheres. In a 96 well plate, 100,000 Pan T-cells freshly isolated from blood were combined with either CD3 / CD28Dynabeads (Catalogue number: 11132D, bead:cell ratio of 1 : 1 as per the manufacturer's protocol) or “Soft” and “Stiff’ CD3 / CD28 / CD2 microspheres (microsphere:cell ratio of 1:4, optimised ratio) (Fig.2a, b). All co-cultures were supplemented with 100 Units of IL2. Cultures were maintained for a maximum of 14 days, with fresh media applied every 2-3 days.

[0185] Example 2 - Microsphere stimulation led to enhanced T-cell proliferation

[0186] To assess the differences between T-cells stimulated using Dynabeads and custom made microspheres, a number of flow cytometry based assays were undertaken. Firstly, absolute counts of both CD4+ and CD8+ T-cells were quantified at 3 timepoints during the experiments, Day 3, 7and 14 from 3 different donors (Fig.2c). From this, it was clear that all conditions led to a rapid proliferation and expansion of the both CD4+ and CD8+ T-cells. The “stiff’ microspheres showed the highest level of proliferation for CD8+ cells at all time points, and for CD4+ cells up until Day 7, with Dynabeads showing the highest number of CD4+ cells at Day 14. The increased number of CD8+ cells generated using the microspheres of both stiffnesses compared to Dynabeads was very striking, and furthermore, by looking at the fraction of CD4+ / CD8+ cells over time, it was possible to see that while the original fractions present at Day 3 persist in the Dynabead condition (~20-30%CD8+ / 60-70% CD4+), a clear enrichment of CD8+ cells was evident with microsphere stimulation, leading to a final composition at Day 14 of around -60-70% CD8+ / 20-30% CD4+ (Fig.2d). By quantifying the expression of CD25 and CD69, it was possible to track the activation state of the cells as a function of their proliferation across the three time points (Fig.3a,b,c). Interestingly, Dynabeads stimulated CD8+ T-cells showed a sustained high level of CD25 and CD69 throughout the 2-week time period, only showing a slight reduction of -20% at Day 14. In contrast, microsphere-stimulated CD8+ T-cells maintained a lower CD25 and CD69 level throughout the time period, indicating a lower overall level of activation.

[0187] Example 3 - Microsphere stimulation led to a less differentiated T-cell phenotype

[0188] In addition to quantifying the number of CD4+ and CD8+ T-cells over time, the inventors were also able to quantify the phenotypic composition of the cells within eachstimulation condition. Using the expression of both CCR7 and CD45RA, the inventors applied commonly identified phenotypes representing the differentiation state of the T-cell, Naive (CCR7+ / CD45RA+), Central Memory (CCR7+ / CD45RA-), Effector Memory (CCR7- / CD45RA-), and Terminally differentiated re-expressing CD45RA (CCR7- / CD45RA+). By plotting these populations as a fraction and absolute count of all CD8+ cells, the inventors saw a trend towards increased differentiation in all conditions at Day 14 (>60 % Effector Memory and TEMRA) (Fig.4a). However, for both microsphere conditions, the inventors saw a maintenance of both >10% Central Memory and Naive like cells, in contrast to Dynabeads where <1% remain. This trend was also observed in CD4+T-cells. Interestingly, when the qualification to other markers that characterise a less differentiated phenotype (CD62L, Naive cells, Fig.4c), CD27 (Memory Cells, Fig.4d) and TCF1 (Stemness / proliferative capacity, Fig.4e) was carried out, the inventors again saw an enhancement in cells expressing these markers when stimulating with microspheres (“stiff’ and “soft”) compared to Dynabeads. This striking result indicated that in combination with an increased number of cells generated by microsphere stimulation, the resulting cells were less differentiated, retaining features associated with naive and memory T-cells.

[0189] Example 4 - Microsphere stimulation led to T-cells exhibiting decreased exhaustion

[0190] The use of Dynabeads in the expansion of T-cells leads to the generation of exhausted cells, as characterised by markers such as PD1, CTLA-4, and TIGIT. To assess if this was also the case using microsphere mediated expansion, the inventors quantified the distribution of these markers across all three stimulation conditions at Day 14. In both CD4+ and CD8+ cells, there was an elevated expression of PD1 and CTLA-4 in Dynabead stimulated cells compared to “soft” and “stiff’ microspheres, as measured by their mean fluorescence intensity (MFI) (Fig.5a, b). TIGIT was elevated for CD4+Dynabeads stimulated cells but not for CD8+T-cells.

[0191] Example 5 - Microsphere stimulation led to T-cells with an increased functional capacity

[0192] The inventors explored how the cells resulting from the three stimulation conditions differed in their functional capacity, as measured by their production of threekey cytokines, IL2, TNF and ZFNy. After 14 days of expansion, cells were re-stimulated using PMA-ionomycin in the presence of Brefeldin A and Monensin to inhibit the trafficking of cytokines to the cell surface and their subsequent release into the surrounding media. Intracellular staining for the presence of IL2, TNF and IFNy revealed significant differences between the stimulation conditions (Fig.6a) with soft and stiff microspheres showing enhanced production of TNF, IL2 and INFy when compared to Dynabead stimulated cells. This increase in cytokine secretion was indicative of an enhanced functional capacity of the cells stimulated by microspheres.

[0193] Example 6 - In vivo testing

[0194] The inventors will also explore how microsphere stimulation leads to enhanced T-cell proliferation, a less differentiated T-cell phenotype, T-cells exhibiting decreased exhaustion, and / or T-cells with an increased functional capacity in an in vivo model. In particular, the inventors will make use of a xenografted murine model to test cells, and in particular T-cell obtained after microsphere stimulation. More specifically, the inventors will make use of a CD 19+ Nalm-6 murine model of acute lymphoblastic leukemia (ALL). The CD 19+ Nalm-6 murine model is a leukemia model that uses Nalm-6 cells to create an aggressive form of ALL in mice. The model is commonly used to test the efficacy of immunotherapies and CAR T-cells in vivo. The mice may be immunodeficient mice, such as an NSG® mouse (a NOD SCID Gamma mouse, also known as NOD-.scvt / IL2Rgammanu11, NOD-.scvt / IL2Rgnu11, NOD scid gamma).

[0195] Example 7 - Materials and Methods

[0196] Additional materials and methods are provided below.

[0197] Microsphere preparation

[0198] Polyacrylamide microspheres were prepared by inverse emulsion polymerisation, which is an example of chemical polymerisation. Pre-polymerisation solutions containing acrylamide, bis acrylamide and acrylic acid were first prepared in PBS and the pH adjusted to 7.4 pH using NaOH. The specific concentration of acrylamide and bis-acrylamide wasused to tune the resulting stiffness of the microsphere (3% acrylamide, 0.03% bisacrylamide - 400 Pa. 5% acrylamide, 0.1% bis-acrylamide - 5kPa). Acrylic acid was maintained at 0.1% for all compositions. The oil phase was composed of mineral oil containing 0.5% Span80. Prior to forming the emulsion, both the oil phase and the prepolymerisation mixture was purged of oxygen by bubbling with N2 for 15 mins. To initiate free radical polymerisation 0.5 pL TEMED (N,N,N,N’-tetramethyl-ethylenediamine) and 5 pL APS (Ammonium persulfate) were added to ImL of the aqueous phase and mixed thoroughly. Working quickly, this volume was added to 20mL of the oil phase and manually agitated to produce a cloudy emulsion. The emulsion was transferred to a glass flask containing a stir bar and capped with a rubber septum. N2 gas was delivered to the emulsion via needle, and the emulsion mixed at 1000 rpm. The polymerisation was allowed to continue for 2hrs at room temperature. Following completion, the emulsion was removed from the flask and transferred to a 50mL falcon tube. To remove the oil phase, the emulsion was centrifuged at 1000g for 10 mins, allowing the oil phase to be poured off leaving a dense pellet of polymerised material. To further remove the oil, a 1 : 1 mixture of ddEEO and diethyl ether were used to resuspend the palette. Once the ether and aqueous phases has separated, the ether was removed via Pasteur pipette, leaving a water phase containing the hydrated and polymerised microspheres.

[0199] Photopolymerization was also used to induce polymerisation. For example, the photo-initiator LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate) used under 365 nm UV irradiation to induce polymerisation of PAA (poly(acrylic acid)).

[0200] Microspheres were stored in PBS for no more than 6 months.

[0201] Microsphere functionalisation

[0202] The polymerised particles functionalized using EDC (l-ethyl-3-(-3-dimethylaminopropyl) carbodiimide) / NHS (N-hydroxysuccinimide) chemistry. First, 5xl06microspheres were washed in ImL of lOOmM MES (2-(N-morpholino)ethanesulfonic acid) buffer (pH6). The wash was performed by centrifuging the microspheres in a 1.5mL microcentrifuge tube at 9000g for 4 mins and discarding the storage buffer. ImL of MES was then used to resuspend the microspheres before a further centrifugation step. Next, a reaction buffer containing 100 mM EDC, and 20 mM NHS was prepared in MES, 200 uL of which was used to resuspend the palette of microspheres.The solution was then incubated at room temperature for 15 minutes. The solution was washed twice in PBS at pH 8. On the final wash, the microspheres were resuspended with a solution containing 10 uM amine-PEG7-biotin in PBS pH8. The reaction was allowed to proceed for 2 hrs at room temperature. On completion, the microspheres were washed a further 2 times in PBS pH 7.4. Next, the microspheres were resuspended in a lOug / mL solution of streptavidin and incubated at RT for 2hrs. Following a further 2 wash steps to remove free streptavidin, the microspheres were incubated in a solution containing the following biotin-conjugated monoclonal antibodies for a further 2 hours: 3.3 ug / mL antihuman CD3 (clone OKT3; BioLegend® Cat No. 317320), 3.3 ug / mL anti-human CD28 (clone CD28.2; BioLegend® Cat No. 302904), 3.3 ug / mL and anti-human CD2 (clone TS1 / 8; or RPA-2.10 BioLegend® Cat No. 300204). The isotype of clone OKT3 is mouse IgG2a, K (see, for example US patent application 5,929,212A). The isotype of clone CD28.2 is mouse IgGl, K. The isotype of clone RPA-2.10 is mouse IgGl, K.

[0203] Microspheres were then washed in PBS and stored at 4°C for use.

[0204] Microsphere characterisation - Stiffness

[0205] To quantify the stiffness of the microspheres, atomic force microscopy (AFM) indentation was used. A glass 35 mm dish was first coated with a solution of poly-L-lysine, followed by a solution containing the microspheres. After allowing the microspheres to adhere to the surface for 15 minutes, the sample was transferred to the Bruker Nanowizard 4. Indentation experiments were conducted using a 10 um diameter spherical tipped indenter (sQube CP-CONT-BSG-B-5), applying and peak force of InN.

[0206] 30-50 microspheres were sampled per stiffness condition, with 5 indentation measurements per microsphere. The resulting force-displacement curves were fit using the open source AFM fitting software PyJibe (https: / / github.com / AFM-analysis / PyJibe), applying a hertzian model with a geometric correction factor accounting for the finite size of the spherical indenter and microsphere.

[0207] T-cell isolation

[0208] T-cells were isolated from blood cones provided by the NHSBT at the John Radcliffe hospital, Oxford, UK. The cone material was first diluted 1:1 in PBS before theaddition of RosetteSep™ Human T Cell Enrichment Cocktail following the manufacturers protocol. Following purification, T-cells were cryopreserved in FBS containing 10% DMSO.

[0209] Microsphere-T-cell co-culture

[0210] T-cell-microspheres stimulation co-culture experiments were all carried out in 96 well u-shaped plastic tissue culture plates. Freshly isolated T-cells were combined with microspheres at a ratio of 4: 1, 2xl05T-cells per well, in a 200 uL volume of RIO medium containing 100 Units of IL2. Dynabead stimulation was carried out in parallel following the manufacturers protocol, using a T-cell-Dynabead ratio of 1 : 1. Co-cultures were maintained for a maximum of 14 days, with fresh media applied every 2-3 days.

[0211] Flow cytometry staining and analysis

[0212] Following stimulation, T-cells were stained for flow cytometry. T-cells were stained with markers to indicate phenotype, degree of activation following stimulation, exhaustion, and functional capacity (see Table below). For all surface markers, 2 x 105cells per condition were deposited in a u-shaped 96 well plate and washed two times in 200 uL of PBS + 2% FBS. Following the wash, cells were resuspended in 50 uL of staining buffer containing all the listed antibodies at pre-optimised concentrations. Following an incubation for 15 mins at 37°C, cells were washed once in PBS, followed by a resuspension in PBS containing 1 : 1000 dilution of Zombie NIR Live dead stain.

[0213] Following incubation at room temperature for 15 mins, cells were washed a further two times in PBS + 2% FBS. This was followed by cell fixation in Biolegend Fixation Buffer.

[0214] To assess T-cell function following the stimulation period, at day 14, T-cells were re-stimulated using PMA-Ionomycin for 4hrs in the presence of monensin and brefeldin to limit cytokine secretion. Intracellular staining followed to quantify the level of cytokine production.

[0215] Flow cytometry was performed using a Cytek Aurora spectral flow cytometer equipped with 5 lasers. This allowed for high dimensional flow cytometry using up to 30 markers. Single stained cell controls for each channel were acquired, including anunstained T-cell control, permitting unmixing between fluorescent markers using the SpectroFlo® Software. Analysis of the acquired data was performed in FlowJo.Table - markers to indicate phenotype, degree of activation following stimulation, exhaustion, and functional capacity

[0216]

Claims

1. Claims1. A microsphere or population of microspheres, wherein:(a) the microsphere has a stiffness of 10-10,000 Pa; or(b) the population of microspheres has a mean stiffness of 10-10,000 Pa.

2. The microsphere or population of microspheres of claim 1, wherein:(a) the microsphere has a stiffness of 10-100 Pa, 10-250 Pa, 10-500 Pa, 10- 1000 Pa, 10-2500 Pa, 10-5000 Pa, 10-10000 Pa, 100-250 Pa, 100-500 Pa, 100-1000 Pa, 100-2500 Pa, 100-5000 Pa, 100-10000 Pa, 250-500 Pa, 250- 1000 Pa, 250-2500 Pa, 250-5000 Pa, 250-10000 Pa, 500-1000 Pa, 500-2500 Pa, 500-5000 Pa, 500-10000 Pa, 1000-2500 Pa, 1000-5000 Pa, 1000-10000 Pa, 2500-5000 Pa, 2500-10000 Pa or 5000-10000 Pa;(b) the microsphere has a stiffness at most 10 Pa, at most 100 Pa, at most 250 Pa, at most 500 Pa, at most 1000 Pa, at most 2500 Pa, at most 5000 Pa or at most 10000 Pa;(c) the population of microspheres has a mean stiffness of 10-100 Pa, 10-250 Pa, 10-500 Pa, 10-1000 Pa, 10-2500 Pa, 10-5000 Pa, 10-10000 Pa, 100-250 Pa, 100-500 Pa, 100-1000 Pa, 100-2500 Pa, 100-5000 Pa, 100-10000 Pa, 250-500 Pa, 250-1000 Pa, 250-2500 Pa, 250-5000 Pa, 250-10000 Pa, 500- 1000 Pa, 500-2500 Pa, 500-5000 Pa, 500-10000 Pa, 1000-2500 Pa, 1000- 5000 Pa, 1000-10000 Pa, 2500-5000 Pa, 2500-10000 Pa or 5000-10000 Pa; or(d) the population of microspheres has a mean stiffness of at most 10 Pa, at most 100 Pa, at most 250 Pa, at most 500 Pa, at most 1000 Pa, at most 2500 Pa, at most 5000 Pa or at most 10000 Pa.

3. The microsphere or population of microspheres of claim 1 or 2, wherein:(a) the microsphere has a diameter of 5-500 pm; or(b) the population of microspheres has a mean diameter of 5-500 pm.

4. The microsphere or population of microspheres of any one of the preceding claims, wherein:48(a) the microsphere is a hydrophilic polymer microsphere, a hydrogel microsphere such as a synthetic hydrogel, a PEG microsphere, an alginate microsphere, or a polyacrylamide microsphere; or(b) the microsphere is a hydrophilic polymer microsphere, a hydrogel microsphere such as a synthetic hydrogel, a PEG microsphere, an alginate microsphere, or a polyacrylamide microsphere; and wherein the microsphere is not a polystyrene bead such as a Dynabead; or (c) the population of microspheres comprises hydrophilic polymer microspheres, hydrogel microspheres such as synthetic hydrogels, PEG microspheres, alginate microspheres, and / or polyacrylamide microspheres; (d) the population of microspheres comprises hydrophilic polymer microspheres, hydrogel microspheres such as synthetic hydrogels, PEG microspheres, alginate microspheres, and / or polyacrylamide microspheres; and wherein the population of microspheres does not comprise polystyrene beads, such as Dynabeads.

5. The microsphere or population of microspheres of any one of the preceding claims, wherein:(a) the microsphere has associated with its surface one or more moiety each selected from a protein, a polysaccharide chain, and / or an extracellular matrix protein; or(b) the population of microspheres comprises microspheres each having associated with their surface one or more moiety each selected from a protein, a polysaccharide chain, and / or an extracellular matrix protein;optionally wherein the moiety is associated with the surface of the microsphere by a cross-linking reaction such as l-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry.

6. The microsphere or population of microspheres according to claim 5, wherein:(a) the microsphere has 100-1000 copies, 100-10,000 copies, 100-100,000 copies, 100-1,000,000 copies, 1000-10,000 copies, 1000-100,000 copies, 1000-1,000,000 copies, 10,000-100,000 copies, 10,000-1,000,000 copies, or 100,000-1,000,000 copies of each of said one or more moiety; or(b) the population of microspheres comprises microspheres each having 100- 1000 copies, 100-10,000 copies, 100-100,000 copies, 100-1,000,000 copies, 1000-10,000 copies, 1000-100,000 copies, 1000-1,000,000 copies, 10,000- 100,000 copies, 10,000-1,000,000 copies, or 100,000-1,000,000 copies of each of said one or more moiety.

7. The microsphere or population of microspheres of claim 5 or 6, wherein at least one of the one or more moiety:(a) comprises an antibody or antigen-binding fragment thereof;(b) comprises one or more of an antibody or antigen-binding fragment thereof which targets CD3, such as antibody OKT3; an antibody or antigen-binding fragment thereof which targets CD28, such as antibody CD28.2; and comprises an antibody or antigen-binding fragment thereof which targets CD2, such as antibody TS1 / 8 orRPA-2.10; and / or(c) comprises an antibody or antigen-binding fragment thereof which targets CD3, an antibody or antigen-binding fragment thereof which targets CD28, and an antibody or antigen-binding fragment thereof which targets CD2; and / or(d) comprises a moiety that binds to a target on the surface of a T-cell.

8. The microsphere or population of microspheres of claim 7, wherein the one or more moiety targets primary stimulation of a T-cell, co-stimulation of a T-cell, and / or adhesion of a T-cell; optionally wherein the one or more moiety:(a) targets primary stimulation of a T-cell by targeting a TCR complex such as targeting CD3, a T-cell receptor, and / or subdomain thereof;(b) comprises an antibody or antigen-binding fragment thereof which targets CD3, such as antibody OKT3;(c) targets co-stimulation of a T-cell by targeting CD28, CD27, CD4, CD8 and / or 4- IBB;(d) comprises an antibody or antigen-binding fragment thereof which targets CD28, such as antibody CD28.2;(e) targets adhesion of a T-cell by targeting CD2, LFA-1, CD44, CXCR3, CCR7, CD31, VLA-4 and / or CD62L;(f) comprises an antibody or antigen-binding fragment thereof which targets CD2, such as antibody TS1 / 8 orRPA-2.10; and / or(g) comprises an antibody or antigen-binding fragment thereof which targets CD3, an antibody or antigen-binding fragment thereof which targets CD28, and an antibody or antigen-binding fragment thereof which targets CD2.

9. A method of producing a microsphere or population of microspheres according to any one of claims 1-8, comprising:(a) forming an emulsion, such as an inverse emulsion, between an aqueous phase comprising a pre-polymer solution and optionally a free radical generator, and an oil phase comprising a surfactant;(b) polymerising the pre-polymer solution to provide a microsphere or population of microspheres, such as by chemical and / or photopolymerization; and(c) optionally functionalising the microsphere or population of microspheres by attaching the one or more moiety as defined in any one of claims 5-8 to the surface of the microsphere or population of microspheres.

10. The method according to claim 9, wherein:(a) the pre-polymer solution comprises PEG, alginate, acrylamide, bisacrylamide and / or acrylic acid;(b) the free radical generator comprises APS-TEMED; ammonium persulfate, riboflavin, dibenzoyl peroxide, azobis(isobutyronitrile), ceric ammonium nitrate, potassium persulphate, and / or potassium permanganate;(c) the oil phase comprises mineral oil and the surfactant such as sorbitan monooleate;(d) functionalising the microsphere or population of microspheres comprises a cross-linking reaction such as 1 -ethyl -3 -(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry; and / or(e) functionalising the microsphere or population of microspheres comprises attaching a linker, such as an amine-PEG-biotin linker, to the surface of the microsphere; optionally attaching streptavidin to the linker; and optionally attaching the one or more moiety as defined in any one of claims 5-8 to the streptavidin.

11. A microsphere or population of microspheres obtained or obtainable by the method of claim 9 or 10.

12. A method of culturing a cell, comprising culturing the cell in a suspension comprising the microsphere or population of microspheres according to any one of claims 1-8 or 11, to provide a cultured cell.

13. The method according to claim 12, wherein the ratio of the microsphere to the cell is:(a) about 1:1000; about 1:500; about 1:250; about 1:100: about 1:50; about 1:25; about 1:10; about 1:5; about 1:4, about 1:2; about 1:1; about 2:1; about 4:1; about 5:1; about 10:1; about 25:1; about 50:1; about 100:1; about 250:1; about 500:1; or about 1000:1;(b) at least 1 : 1000; at least 1 :500; at least 1 :250; at least 1 : 100: at least 1:50; at least 1:25; at least 1:10; at least 1:5; at least 1:4, at least 1:2; at least 1:1; at least 2:1; at least 4:1; at least 5:1; at least 10:1; at least 25:1; at least 50:1; at least 100:1; at least 250:1; at least 500:1; or least 1000:1;(c) at most 1:1000; at most 1:500; at most 1:250; at most 1:100: at most 1:50; at most 1:25; at most 1:10; at most 1:5; at most 1:4, at most 1:2; at most 1:1; at most 2:1; at most 4:1; at most 5:1; at most 10:1; at most 25:1; at most 50:1; at most 100:1; at most 250:1; at most 500:1; or ,most 1000:1; or (d) 1:1 to 1:10; 1:1 to 1:5; 1:1 to 1:4; 1:1 to 1:2; 1:2 to 1:10; 1:2 to 1:5; 1:2 to 1:4; 1:4 to 1:10; 1:4 to 1:5; or 1:5 to 1:10.

14. The method according to claim 12 or 13, wherein the cultured cell is maintained and / or expanded.

15. The method according to any one of claims 12-14, wherein the cell and / or cultured cell is an immune cell such as a neutrophil, an eosinophil, a basophil, a mast cell, a macrophage, a histocyte, a Kupffer cell, an alveolar macrophage, a dendritic cell, a B cell, a plasma cell, a memory B cell, a Killer T cell, a memory T cell, a T helper cell, a Natural killer T cell, a CAR T-cell, an innate lymphoid cell and / or a natural killer cell.

16. The method according to any one of claims 12-15, wherein the cell and / or cultured cell is a T-cell, optionally wherein the cell and / or cultured cell is a CAR T-cell.

17. The method according to any one of claims 12-16, wherein the cell and / or cultured cell is a CD4+ T-cell or a CD8+ T-cell.

18. The method according to claim 16 or 17, wherein the cultured cell is:(a) a T-cell which has a lower expression level of CD25 and / or CD69 compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1;(b) a T-cell which has a lower expression level of PD1, CTLA4 and / or TIGIT compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1;(c) a T-cell which has a higher expression level of TNF, IL2 and / or fFNy compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1;(d) a T-cell which has a higher expression level of CD45RA and / or TCF1 compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1;(e) a T-cell which has a higher expression level of CD45RA and / or CD27 compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1;(f) a T-cell which has a higher expression level of CD45RA and / or CCR7 compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1;(g) a T-cell which has a higher expression level of CD62L and / or CD27 compared to an equivalent cell, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1:1; and / or (h) a population of a T-cells which has a higher proportion of CD4+ T-cell and / or a CD8+ T-cell compared to an equivalent cell population, which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microspheres:cell of 1 : 1;wherein in any one of (a)-(h) the population of equivalent polystyrene microspheres have a mean stiffness greater than 100,000 Pa;optionally wherein the lower expression level is at least 1%, at least 2%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90% or at least 95% lower; and / or the higher expression level is at least at least 1%, at least 2%, at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 90% or at least 95% higher.

19. The method according to any one of claims 12-18, wherein:(a) the cultured cell has functional activity and / or is less differentiated compared to an equivalent cell which has been cultured with a population of equivalent polystyrene microspheres according to an equivalent method using a ratio of polystyrene microsphere:cell of 1 : 1, and wherein the population of equivalent polystyrene microspheres have a mean stiffness greater than 100,000 Pa; and / or(b) the cell is cultured for: 1-2 days, 1-5 days, 1-7 days, 1-10 days, 1-12 days, 1-14 days, 1-20 days, 1-30 days, 2-5 days, 2-7 days, 2-10 days, 2-12 days, 2-14 days, 2-20 days, 2-30 days, 5-7 days, 5-10 days, 5-12 days, 5-14 days, 5-20 days, 5-30 days, 7-10 days, 7-12 days, 7-14 days, 7-20 days, 7-30 days, 10-12 days, 10-14 days, 10-20 days, 10-30 days, 12-14 days, 12-20 days, 12-30 days, 14-20 days, 14-30 days, or 20-30 days; and / or(c) the cell is cultured for: at least 1 day, at least 2 days, at least 5 days, at least 7 days, at least 10 days, at least 12 days, at least 14 days, at least 20 days, or at least 30 days.

20. A composition comprising a microsphere according to any one of claims 1-8 or 11 and the cell and / or cultured cell as defined in any one of claims 12-19.

21. A cell or cell population obtained by or obtainable by the method of any one of claims 12-19.

22. Use of the cultured cell as defined in any one of claims 12-17, or the cell or cell population of claim 21, to identify agents capable of preventing or treating a disease, comprising treating a disease model before, during or after induction of the disease, optionally wherein the disease is cancer.

23. The cultured cell as defined in any one of claims 12-17, or the cell or cell population of claim 21, for use in a method of:(a) therapy,(b) immunotherapy; or(b) treating cancer.

24. A method of treating a disease in a subject comprising administering to the subject the cultured cell as defined in any one of claims 12-17, or the cell or cell population of claim 21, optionally wherein the method is an immunotherapy, and optionally wherein the disease is cancer.

25. The use according to claim 22, the cultured cell, cell or cell population for use according to claim 23, or method according to claim 24, wherein the cancer is selected from one or more of:(a) a blood cancer such as Acute lymphocytic leukaemia, Acute myelogenous leukaemia; Chronic lymphocytic leukaemia; Chronic myeloid leukaemia; Hodgkin lymphoma; Lymphoma; Myelodysplasia; Myeloma; and / or Myeloproliferative neoplasm; and / or(b) a solid tumour such as Breast cancer; Carcinosarcoma; Lung cancer;Lymphoma; Melanoma; Colon cancer; a Germ cell tumour; Prostate cancer; Brain cancer; Carcinoma; Chondrosarcoma; and / or Neuroblastoma.56

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