Compositions Comprising Nanosilica Particles and Their Use in Methods of Activating T-Lymphocytes for Therapy - Patent application

Abstract

Compositions are provided for use in activating lymphocytes in culture or in whole blood. [Solution] A composition comprising silica particles having an average diameter of 0.5 to 20 nm for use in a method for activating T lymphocytes for therapy of a subject / individual, wherein the activation of the T lymphocytes is characterized by an increase in the expression of CD69 and / or CD25 by the T lymphocytes.

Description

[Technical field]

[0001] The present invention relates to compositions comprising nanosilica particles and their use in methods of activating T lymphocytes for therapy, in particular to biomarkers that characterize the efficacy of silica particle compositions for use in therapy. Assays and screening methods based on the use of these markers are also disclosed, as are devices and kits for generating and / or delivering nanosilica compositions or T lymphocytes that have been activated ex vivo prior to administration to a subject. [Background technology]

[0002] Silicon is an environmentally ubiquitous element, with adults in the Western world currently ingesting about 15-50 mg per day. In nature, it exists as silicates, where silicon is bonded to oxygen atoms. Silicic acid and silica are also terms used for such structures. These range from the simplest monosilicic acid (also called ortho) to silica particles. In this specification, the terms silicate and silica can be used interchangeably to mean materials containing silicon, oxygen and hydrogen, which may contain other ions but contain primarily silicon and oxygen, with the hydrogen content determined by factors such as size, degree of aggregation, pH, etc. The exact biological role of soluble silicates is not yet understood, but a large body of evidence points to an important role in connective tissue health (Jugdaohsingh et al., 2008). A typical connective tissue is There is also considerable evidence of the dietary, nutraceutical or therapeutic benefits of soluble or polymeric silicates in a variety of medical conditions, including bones, joints, blood vessels, skin, hair and nails, but also osteoporosis, osteopenia and other musculoskeletal and joint disorders, all types of cancer, various skin conditions, vascular, cardiovascular and coronary heart disease, inflammatory diseases, autoimmune diseases, Alzheimer's disease and various forms of cognitive impairment, various types of infections, wounds and ulcers, gastrointestinal, hepatic, renal and immune-related disorders and hormone-related changes and disorders. The beneficial nutritional and therapeutic effects of silicates appear to extend to other animals, particularly other mammals.

[0003] Silicates have been used as oral nutritional supplements, but achieving a formulation that allows for effective uptake (absorption) after administration is not straightforward. Silicon in its naturally occurring inorganic form is soluble as orthosilicic acid. However, its concentration in, for example, drinking water needs to be relatively low (≦1.7 mM) to prevent the initiation of polymerization of particles that gradually aggregate and / or grow in size and thus are not easily redissolved, since under natural conditions this is the maximum equilibrium solubility of aqueous silicates at pH<9. This behavior has severely plagued the development of nutritional silicon, since concentrated forms do not dissolve in the digestive tract to allow absorption, while dilute forms result in the need for large amounts of supplement to be ingested (e.g., 20-100 ml / day).

[0004] Typically, certain chemical moieties, e.g., ligands, can be used to bind and solubilize cations / anions that would otherwise precipitate at physiological pH, but silicates are tricky because the monomer typically has a greater affinity for itself (i.e. undergoes self-assembly) than for any other molecule, and the higher the concentration of silicon, the more difficult it becomes to stop its self-assembly in aqueous solution. This has led to alternative strategies to generate bioavailable silicate compositions.

[0005] But in addition, both intentionally and accidentally, researchers have found that the precipitated silicate composition For example, the effects of polymeric nano- and micron-sized silicates on cells have been investigated.

[0006] US Patent Application Publication No. 2011 / 0229577 (Kerek) discloses silicic acid particles with a shell-like structure, where the particles aggregate under conditions where the pH of the reaction mixture is first decreased and then increased, resulting in a composition that is said to have a pH of 2.1 or a pH greater than 9.2. The aggregated silicate compositions described in US Patent Application Publication No. 2011 / 0229577 are described as having low toxicity to in vitro cell cultures, as inhibitors of calcium ATPase pumps, and as inducing apoptosis in all cell types. The use of such aggregated silicates to reduce tumor size has been reported.

[0007] Silica compositions that are stabilized against agglomeration by high levels of choline are sold as food supplements and are called "choline stabilized orthosilicic acid" (Biosil™; orthosilicic acid stabilized with choline chloride, see WO 95 / 21124 and EP 1 371 289 A).

[0008] US Patent Application Publication No. 2009 / 0130230 (Stanley) describes their use for the treatment of inflammatory diseases, cancer, bacterial and viral infections, and for the treatment of infected and non-infected wounds. The reference hypothesizes that treatment occurs through activation of the innate immune system.

[0009] WO 2015 / 121666 (Medical Research Council) discloses nanosilica compositions containing stabilizers such as polyols, sugars and / or quaternary ammonium salts, such as choline and carnitine.In particular, WO 2015 / 121666 provides a process for producing stabilized polymeric silicate compositions, including polymeric silicic acid and nanosilica particles, where the polymerization and particle size growth of silicate are controlled, and the resulting particles are size stabilized by a combination of silicate concentration, pH and / or stabilizer.The use of these compositions for the treatment of a range of medical conditions and for silicon supplementation is described.

[0010] T lymphocytes are cells of the adaptive immune system that are directly responsible for both organizing (CD4+ helper T cells) and executing (CD8+ cytotoxic T cells) the elimination of infected and cancerous cells from the body. They are therefore a vital part of the immune system. Polyclonal human T cell activation by silicates in vitro was first reported in 1994 (Ueki et al., 1994), and despite the ongoing association of silicates with T cell activation in diseases such as silicosis, the magnitude, structure, extent of T cell interactions and the mechanisms of action that govern silicate and T cell interactions remain elusive (Hayashi et al., 2010, Lee et al., 2012, Kusaka et al., 2014). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] US Patent Application Publication No. 2011 / 0229577 [Patent Document 2] International Publication No. 95 / 21124 [Patent Document 3] European Patent No. 1 371 289 A [Patent Document 4] US Patent Application Publication No. 2009 / 0130230 [Patent Document 5] International Publication No. 2015 / 121666 [Non-patent literature]

[0012] [Non-Patent Document 1] Jugdaohsingh et al., 2008 [Non-Patent Document 2] Ueki et al., 1994 [Non-Patent Document 3] Hayashi et al., 2010 [Non-Patent Document 4] Lee et al., 2012 [Non-Patent Document 4] Kusaka et al., 2014 Summary of the Invention

[0013] In general, the present invention is based on the discovery that compositions comprising silica particles, also called polymeric silica or silica polymers, in particular silica structures with an average size according to standard particle size measurements of 0.5 nm to 20 nm, can be used to activate lymphocytes in culture or in whole blood. This newly observed technical effect opens the possibility that T cell activation can be used as a marker and biomarker for the effectiveness of silica treatments for cancer, infections and other chronic diseases. Without wishing to be bound by any particular theory, the inventors believe that the silica particles bind to T cell receptors and partially activate them, detectable for example by increased CD69 and / or CD25 expression. This means that T cells are activated when they encounter their cognate antigen (for example on cancer cells or infected cells). This means that T cells are less likely to be inactive and thus activation may be useful to overcome T cell exhaustion induced in cancer or certain chronic diseases in both veterinary and human medicine.

[0014] Conventionally, primary particles less than 100 nm in diameter in one direction are loosely referred to as “nano”, even though they may range from 1 to 100 nm and span a million times by volume. Furthermore, most researchers have focused on aggregates and agglomerates that are merely “nanostructured”. Truly dispersed nanoparticles have been less thoroughly studied, and nanoparticles less than 10 nm in diameter (i.e. in the protein size range) have received relatively little attention in biomedical applications. For example, extensive studies have found that silica particles (-Si-O-Si-, called SiO2 in their most aggregated form) exhibit differential cellular activity depending on size and crystallinity, ranging from mild activity for amorphous (poorly aggregated) larger particles to strong pro-inflammatory activity for crystalline quartz (an effect partially mimicked by nanosilica regardless of crystallinity). However, only a few studies based on ultrafine silica particles (less than 10 nm in diameter) exist. Despite the paucity of data, ultrafine silica particles are ubiquitous because their formation occurs when the concentration of aqueous monomeric Si(OH)4 exceeds about 1.7 mM total [Si]. Using such 'supersaturated' silicate formulations, Stanley et al. teach that cancer can be treated with the resulting mixture. Kerek reiterates that ultrafine silicate particles may be useful in the treatment of cancer and lists several possible methods for their synthesis.

[0015] Thus, in a first aspect, the present invention provides a composition comprising silica particles having an average diameter of 0.5-20 nm for use in a method of activating T lymphocytes for therapy in a subject / individual, wherein the activation of T lymphocytes is characterized by an increase in expression of CD69 and / or CD25 by the T lymphocytes. By way of example, preferably the increase in expression of CD69 and / or CD25 is at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70% or at least 80% compared to, for example, an unstimulated baseline subject. A cellular assay for determining CD69 and / or CD25 expression using peripheral blood mononuclear cells (PBMCs) is described in the examples provided below, although alternative assays for these biomarkers will be apparent to the skilled artisan.

[0016] In general, the inventors have found that activation of T lymphocytes can be achieved using the silica particle compositions described herein, but not using compositions described in the prior art, such as compositions made according to the teachings of Kerek (supra) or choline-stabilized orthosilicic acid (Biosil™).

[0017] Generally, the T lymphocytes activated by contact with the nanosilica composition used in the present invention are T cells such as helper T cells, cytotoxic T cells, cytotoxic natural killer T cells or gamma delta T cells. This is supported in experiments using helper (CD4+) T cells and cytotoxic (CD8+) T cells. Alternatively, or in addition, the activation of T lymphocytes increases the expression of one or more additional markers of T cell activation selected from CD40L, LAP / GARP and / or FoxP3.

[0018] CD25 (cluster of differentiation 25) is the alpha chain of the IL-2 receptor. It is a type I transmembrane protein present on activated T cells. CD69 (cluster of differentiation 69) is a human transmembrane C-type lectin protein. Activation of T lymphocytes and natural killer T (NKT) cells induces the expression of CD69 both in vivo and in vitro.

[0019] CD40 ligand or CD40L, also called CD154, is a protein that is expressed primarily on activated T lymphocytes and is a member of the TNF superfamily of molecules. GARP (glycoprotein A repeats dominant), also known as LRRC32 (leucine-rich repeats containing 32), mediates platelet and activated FOXP3 + It is a protein essential for the surface expression of latent TGF-β on regulatory T cells. Increased expression of GARP and latency-associated peptide (LAP) is an indicator of activation and expansion of regulatory T lymphocytes (Tregs).

[0020] FoxP3 (Forkhead box P3), also known as scurfin, is a protein involved in immune system responses. A member of the FOX protein family, FOXP3 appears to function as a master regulator of regulatory pathways in the development and function of regulatory T cells (Tregs).

[0021] Alternatively, or in addition, in some cases, lymphocyte activation can be identified by an increase in expression of IFN-γ. The experiments described herein further demonstrate that, although the compositions cause lymphocyte activation, they do not substantially induce lymphocyte cell proliferation.

[0022] As is well known in the art, an equilibrium exists between soluble silicic acid and the increasingly aggregated silicate compositions. Thus, in the present invention, a "stabilized polymeric silicate composition" includes soluble forms of silicic acid and polysilicic acid that are in equilibrium with the polymeric silicic acid and nanosilicate particles having the properties described herein and in the compositions or formulations in which they are included.

[0023] Evidence has emerged in the art that suggests that silicic acid is beneficial for health and disease prevention or healing in humans and other animals.Generally, the composition of the present invention comprises a polymeric silicate composition, in which the natural tendency of nanosilicates to grow and form higher polysilicates and silicate particles is inhibited by the inclusion of a substance such as an organic compound that can act as a growth inhibitor, i.e. inhibit the natural tendency of polysilicic acid to grow and form gels and more aggregated silicate particles or polymers and particles larger than the desired size of polymers and particles.Furthermore, in one aspect, the inventors have found that this approach means that the composition is stable at a physiologically acceptable pH, particularly at a neutral or slightly acidic pH or slightly alkaline pH.

[0024] A further advantage of the method described herein is that by selectively controlling pH, silicon concentration and stabilizer concentration during synthesis, particle size can be adjusted from small polymers with diameters of less than 0.5 nm up to 10 or 10 nm depending on the desired particle size, which can then be stabilized according to the invention outlined to allow administration to subjects or animals at a selected particle size.It will be clear to those skilled in the art that particle size refers to a range of sizes, and the numbers cited herein refer to the average diameter, most commonly the average diameter of the particle in that range.

[0025] In a further aspect, the present invention provides the use of a composition comprising silica particles having an average diameter of 0.5-20 nm in the preparation of a medicament for activating T lymphocytes for therapy relating to a subject / individual, wherein the activation of T lymphocytes is characterised by an increase in expression of CD69 and / or CD25 by the T lymphocytes.

[0026] In a further aspect, the present invention provides a method for determining the efficacy of a silica particle composition for therapeutic administration to a subject or individual, the method comprising the steps of: (a) contacting a sample of T lymphocytes with a silica particle composition; (b) determining the expression of CD69 and / or CD25 by T lymphocytes present in the sample; and (c) Silica particle compositions that cause an increase in expression of CD69 and / or CD25 by T lymphocytes are identified as suitable for use in therapy.

[0027] In a further aspect, the present invention provides an assay for determining the efficacy of a silica particle composition for therapeutic administration to a subject or individual, the assay comprising the steps of: (a) contacting a sample of T lymphocytes with a silica particle composition; (b) determining expression of CD69 and / or CD25 by T lymphocytes present in the sample; (c) The amount of CD69 and / or CD25 expression is measured or quantified relative to a control value, and if the amount of CD69 and / or CD25 expression is increased compared to the control value, the silica particle composition is selected as suitable for therapeutic administration.

[0028] In a further aspect, the present invention provides a method for determining the efficacy of a therapy comprising administration of a silica particle composition or a protocol for its administration to a subject or individual, the method comprising the steps of: (a) contacting a sample of T lymphocytes from a subject or individual treated with a silica particle composition or a protocol for its administration; (b) determining the expression of CD69 and / or CD25 by T lymphocytes present in the sample; and (c) identifying a subject or individual in which an increase in expression of CD69 and / or CD25 by T lymphocytes is observed in step (b) as having been successfully treated with the silica particle composition or a protocol of administration thereof; or (d) identifying a subject or individual in which an increase in CD69 and / or CD25 expression by T lymphocytes is not observed in step (b) and modifying the silica particle composition provided to that subject or individual or the protocol for its administration; (e) Optionally, steps (a) through (d) are repeated until the subject or individual is successfully treated with the silica particle composition or its administration protocol.

[0029] In the assays and methods described herein, an increase in expression of CD69 and / or CD25 by T lymphocytes is determined relative to a reference value, eg, an unstimulated baseline control.

[0030] In a further aspect, the present invention provides the use of expression of CD69 and / or CD25 by T lymphocytes as a marker or biomarker for the therapeutic effectiveness of a silica particle composition.

[0031] In a further aspect, the present invention provides a device for producing a silica composition for therapeutic use, the device comprising: a first vessel containing a silicate solution having a pH>10.5, a second vessel containing an acidic buffer solution; flow control means for controlling the flow of the silicate solution and the acidic buffer solution; and An outlet in fluid communication with the first and second containers for delivering the colloidal silica composition such that the two solutions are mixed prior to administration to a subject.

[0032] In one embodiment, the device is used for batch synthesis of silica compositions and further comprises a mixing chamber in fluid communication with the first and second containers for mixing solutions to produce a colloidal silica composition. Alternatively, if the device is for flow synthesis of the silica compositions of the present invention, the mixing of the solutions can occur in situ as they are delivered, for example in an intravenous line prior to administration to a subject.

[0033] In a further aspect of the invention, the device may be adapted for ex vivo T lymphocyte therapy. In this aspect, the device may further comprise a chamber for contacting a biological sample containing T lymphocytes obtained from a subject with the colloidal silica composition to cause activation of the T lymphocytes for therapy and / or a means for delivering a sample containing T lymphocytes extracted from a subject to the contact chamber and for returning the activated T lymphocytes to the subject.

[0034] In a further aspect, the present invention provides a kit comprising (i) a first container comprising a silicate solution having a pH>10.5 and (ii) a second container comprising an acidic buffer solution and (iii) instructions for mixing the silicate solution and the acidic buffer solution to produce a therapeutically effective colloidal silica composition having a pH of 4.0-8.5, more preferably 4.0-6.5. Preferably, the acidic solution comprises hydrochloric acid, a carboxylic acid, such as citric acid, or an acidic amino acid, such as glycine.

[0035] Aspects of the present invention will now be described by way of example, and not limitation, with reference to the accompanying drawings in which: Figure 1 is a schematic diagram of a method for manufacturing a semiconductor device according to the present invention;

[0036] "And / or," as used herein, should be taken as a specific disclosure of each of the two specified features or components, with or without the other. For example, "A and / or B" should be taken as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0037] Unless the context dictates otherwise, the descriptions and definitions of features set forth above are in no way limited to any particular aspect or embodiment of the invention, but apply equally to all aspects and embodiments described. [Brief description of the drawings]

[0038] [Figure 1] T cell activation with nanosilica particles (Si(OMe)4) synthesized according to the silica particle composition of the present invention and Example 4 of Kerek (US Patent Application Publication No. 2011 / 0229577). Cells were treated with nanoparticles and activation was assessed after 24 hours. [Diagram 2] T cell activation with silica particle compositions of the invention and ch-OSA® (choline stabilized orthosilicic acid, BioSil™). [Diagram 3] CD4+ and CD8+ expression with silica particle compositions of the present invention compared to Kerek. [Figure 4] CD4+ and CD8+ expression with silica particle compositions of the invention compared to Kerek (+ positive control, SEB). [Diagram 5] CD4+ and CD8+ expression with silica particle compositions of the invention compared to BioSil™. [Figure 6] CD4+ and CD8+ expression with silica particle compositions of the invention compared to BioSil™ (+ positive control, SEB). [Figure 7]Activation of CD4 and CD8 T cells with ultrafine nanosilica of different sizes (n=>8) prepared by pH neutralization of alkaline sodium silicate solution to a final concentration of 23-40 mM Si±NaCl (pH 7) and incubation for about 20 hours (for dispersions larger than 3 nm) or by adjustment of alkaline silicate solution to pH 0.8-1 and incubation for about 20 hours (dispersions smaller than 3 nm) and then added to cell cultures. The mean diameter of the particles (DV0.5) is reported. [Figure 8] Activation of different Th lineage cell types with ultrafine nano-silica, termed 'HS-7' and prepared by pH neutralization (pH 7) of an alkaline sodium silicate solution to a final concentration of 23 mM Si and incubation for approximately 20 hours before adding to cell cultures (n=6-7). The particles had a mean diameter (DV0.5)=3.4-3.8 nm from replicate analyses. [Figure 9] Effect of suc.uSANS (a), in situ uSANS (b) and their vehicle control (c) on the confluence of HUVEC cells. Note that data cannot be shown for the third nanosilica studied, called HS7, since it induced cytotoxicity (i.e. changes in cell shape and / or cell death) after short periods of incubation (less than 1 h) at all three concentrations (1, 2 and 4 mM Si), and at the 4 mM concentration most of the cells were dead after 4-5 h of incubation. In follow-up experiments with lower concentrations of HS7 (0.1, 0.25 and 0.5 mM Si), toxicity was present even at 0.5 mM Si. [Figure 10] pZap70 measured over a time course demonstrates rapid activation of T cells derived from nanosilica interacting with the T cell receptor leading to phosphorylation of ZAP-70. [Figure 11]Chemical instability of uSANS during acidic storage as determined by molybdate assay. All materials were stored at room temperature or 4°C. Sucrose stabilized uSANS (Suc uSANS) was stored at pH 2.15 ± 0.15 or 1.5 ± 0.1, while non-stabilized material (NS uSANS) was stored at pH 1.5 ± 0.1. Higher OD indicates greater chemical instability (i.e. less aggregated silicates). [Figure 12] Chemical instability of uSANS over time upon neutralization (pH 7). A molybdate assay was used to compare the instability of soluble silicate (Si(OH)4) with uSANS before (pH 2.3) and after (t 0 h to t 6 h) neutralization. [Figure 13] Chemical instability of in situ formed uSANS (40 mM Si) after various incubation periods (1 min to 1 h). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0039] The biological role of silicon and the chemistry of silicates Evidence suggests that silicic acid (either monomeric or polymeric) is beneficial for health and disease prevention or cure in humans and other animals. However, as mentioned above, a fundamental problem in the art is that silicic acid (whose monomer is represented as Si(OH)4) self-assembles and forms insoluble species at pH below 9.0 and at concentrations above the maximum solubility of aqueous silicates (1.7 mM at 25°C, see Figure 1 in Jugdaosingh et al., supra). As is well known in the art, an equilibrium exists between soluble silicic acid and increasingly aggregated silicate species, namely mono-, di- and tri-silicic acid, polysilicic acid and nanosilica particles. The process of growth of silicic acid from solution involves the development of single units that grow in size to an increasingly evolved (i.e. less unstable, soluble and / or dissolvable) state, and thus less able to revert to Si(OH)4 in the absence of added alkali. Growth can involve an increase in size by polymerization, agglomeration, aggregation or surface deposition of soluble species. Growth of polysilicic acid ultimately leads to the formation of a gel under appropriate conditions. These factors make it extremely difficult to stabilize silicate compositions above these concentrations of aqueous silicate and at physiologically relevant pH.

[0040] Therefore, the administration of silicate is a challenge, because the dosage must be delivered as required for the desired effect, both in terms of concentration and chemical form, at a pH that is compatible with physiological health, and in a way that will avoid persistent nanoparticles of silicate that may have adverse effects on health.It is particularly noteworthy that during the application of the dosage, three significant changes generally occur due to the physiological environment.First, there will be dilution by physiological fluids, second, there will be pH changes, and third, there will be changes in ionic strength.The net effect of these effects will determine the behavior of the administered silicate.

[0041] Nanosilica Compositions and T-Lymphocyte Activation We have found that the nanosilica compositions of the present invention, more preferably ultra-fine nanoparticles of silica (diameter less than 10 nm), can stimulate the activation of T lymphocytes, for example as measured by an increase in the markers CD25 and CD69 on the surface of T lymphocytes. Without wishing to be bound by any particular theory, the inventors believe that this occurs through direct binding of nanosilica to the CD3-T lymphocyte receptor complex (herein referred to as the CD3-TCR complex). Importantly, while size appears to be one predictor of whether a nanosilica composition can activate T lymphocytes, other factors may contribute to this property. For example, ultrafine nanoparticles of silica stabilized against agglomeration by high levels of choline are sold as food supplements and are called choline-stabilized orthosilicic acid (Biosil™; orthosilicic acid stabilized with choline chloride, see WO 95 / 21124 and EP 1 371 289 A), but they failed to stimulate T-lymphocyte activation in our assays (see Figures 2 and 5). In another example, the nanosilica reported by Kerek (US Patent Application Publication No. 2011 / 0229577), prepared as a comparison in Example 4 herein, was a very weak inducer of CD4 T-lymphocyte activation and a moderate inducer of CD8 T-lymphocyte activation, compared to some freshly precipitated nanosilicas prepared by pH adjustment of an alkaline solution of silicate salts (see Figures 1, 3 and 4). A further example is that the sucrose-stabilized nanosilica particles reported in WO 2015 / 121666 can activate CD4 and CD8 T lymphocytes in the same way as non-stabilized particles.Thus, in one aspect, the present invention provides an assay for determining the effectiveness of silica particle compositions to stimulate T lymphocytes, since this is not clear from simple physicochemical determinations.In turn, this can allow the activity of silica nanoparticles to be selected, including any desired differential effects on CD8 and CD4 T lymphocytes, which can allow for better targeted T lymphocyte activation therapy.

[0042] Nevertheless, the inventors have also found that certain subtypes or 'lineages' of T lymphocytes can be simultaneously activated by their exposure to ultrafine nano-silica. For example, CD4+ helper T (Th) cells can be conveniently divided into several different Th lineages depending on the type of immune response they are involved in regulating (Geginat et al., 2015). The so-called Thl CD4+ helper cells mediate cytotoxic T lymphocyte responses and can activate monocytes, and they are characterized by the production of IFN-γ (Geginat et al., 2015). On the other hand, the so-called regulatory CD4+ helper T cells (Tregs) are responsible for suppressing immune responses, and they can be identified by the surface expression of forkhead box P3 (FoxP3) protein as well as LAP and GARP (Sakaguchi et al., 2010, Gauthy et al., 2013; Stockis et al., 2009). On the other hand, so-called Th2 CD4+ helper T cells may be responsible for mediating B cell responses, for example: CD40 ligand, also called CD154, is expressed on Th2 cells and associates with the CD40 receptor on B cells, which may promote B cell proliferation and survival ( Crotty, 2015 ; Elgueta et al., 2009 ).

[0043] We have found that multiple lineage types of T lymphocytes can be susceptible to T lymphocyte activation by these ultrafine nanosilica particles (Figure 8). This allows T lymphocytes to be used in several therapeutic applications. First, the assay can be used to identify specific types / forms of nanosilica that select or polarize this action toward one lineage of T lymphocytes, so that regulatory, Th1 or Th2 can be preferentially selected by treatment. For example, selective activation of Th1 cells may be desirable for cancer or viral diseases. Selective activation of Tregs may be desirable for autoimmune or inflammatory diseases.

[0044] Secondly, by chronic administration, activation of T lymphocytes can be used therapeutically to achieve 'exhaustion', in other words reversal of activation and therefore dampening (deactivation) of all, or even selected, types of T lymphocytes. For example, this may be therapeutically useful in 'inflammatory diseases', whether classical autoimmunity, e.g. multiple sclerosis or rheumatoid arthritis, classical Th1, e.g. Crohn's disease or rheumatoid arthritis, or Th2, e.g. asthma or ulcerative colitis. Autoimmune diseases may have a Th1 or Th2 polarisation, hence rheumatoid arthritis is mentioned twice.

[0045] Third, the use of silica particles to activate or deplete all or selected T lymphocytes can be used in conjunction with the co-administration of therapies that suppress or enhance specific T cell lineages or phenotypes, thereby selecting the type of response. For example, the sequestration of Treg T lymphocyte lineages for cancer therapy with cyclophosphamide (Becker et al., 2013) thus allows for the selection of effector T lymphocyte lineages by nanosilica therapy. 'growth', which could be implemented in a regimen that avoids chronic T-lymphocyte exhaustion. Co-administration for such purposes could be before and / or during and / or after administration of nanosilica and could include one or more agents.

[0046] It would be desirable to deliver therapeutic nanosilica parenterally, since it is poorly absorbed from the gastrointestinal tract (Jugdaohsingh et al., 2000). It has been recognized that intravenous administration may be particularly desirable (WO2015121666). However, this would require that the nanosilica be minimally toxic to the endothelial cells lining the vascular system, which could be injected in high concentrations to achieve an effective therapeutic dose in the bloodstream. Indeed, one form of nanosilica disclosed in WO2015 / 121666, the so-called uSANS particles, is minimally persistent and therefore less toxic to endothelial cells than the nanosilica previously disclosed by Stanley in US2009 / 0130230 (herein referred to as HS7 particles). HS7 particles are relatively more toxic above 0.5 mM, thus reducing their clinical application at therapeutic doses. However, despite being better tolerated than HS7 particles, uSANS particles still show moderate toxicity to endothelial cells (Figure 9). However, we have found that there is a very rapid interaction of nanosilica particles with the TCR, as demonstrated by the rapid phosphorylation of zeta chain-associated protein kinase 70 (pZap70) of T lymphocytes by nanosilica (Figure 10), which means that ex vivo treatment of blood or isolated cells is a useful method for delivering nanosilica. In particular, cells that have been somewhat isolated from their tissues or biofluids or that remain present in whole blood do not need to leave the body for long periods of time and can then be treated with nanosilica and reinjected for therapeutic use. This would spare endothelial cell damage, as immune cell and protein binding by nanosilica and some dissolution of nanosilica particles would likely minimize endothelial cell interaction and damage by nanosilica upon its re-administration.

[0047] Thus, in one aspect, the invention provides a composition comprising silica particles having an average diameter of 0.5-20 nm for use in a method of activating T lymphocytes for therapy in a subject / individual, the method comprising generating the composition of silica particles by in situ synthesis prior to administration to the subject. As in other aspects of the invention, activation of T lymphocytes is generally characterised by an increase in expression of CD69 and / or CD25 by the T lymphocytes.

[0048] Alternatively, or in addition, the invention provides a composition for use in a method of T cell therapy comprising obtaining a sample comprising T cells from a subject, contacting the T cell sample with a silica composition of the invention to activate the T cells in the sample, and administering (i.e. returning) the activated T cells to the subject.

[0049] As a further aspect to the present invention, the inventors have discovered that minimally toxic uSANS can be produced by in situ synthesis (i.e., uSANS produced almost immediately prior to clinical application by either batch or flow synthesis approaches). These in situ uSANS can be produced in a quasi-neutral buffer system by batch synthesis. In situ synthesis involves two solution systems: (i) a silicate solution at a pH greater than 10.5 (i.e., the silicate is soluble but not colloidal), which is neutralized by (ii) an acidic buffer solution (containing a carboxylate, e.g., citrate, or an amino acid, e.g., glycine). Mixing of the two solutions results in a weakly acidic pH of 4.0 and 6.5 (more preferably pH 5.0-6.0), and then clusters / colloidal silicates (i.e., nanosilica) are rapidly formed. These colloids gradually become more aggregated and can then be administered at the selected point of instability. Preferably, the incubation will be carried out at pH 4.0-6.5 for slow incubation (batch in situ synthesis), or pH 6.5-8.5 for fast incubation (flow in situ synthesis). The concentration of silicate during the incubation is preferably in the range of 10-100 mM, preferably 20-60 mM. The incubation time is preferably 15-60 minutes for in situ batch synthesis or 10 seconds to 5 minutes (i.e. the length of time between the points of mixing and intravenous introduction) for in situ flow synthesis. For example, a 30 minute incubation upon mixing the acid and silicate components would be suitable for a batch synthesis at (i) 40 mM Si, (ii) room temperature, (iii) pH 5.3 (measured as pH 4.0-5.0 using pH paper). Additionally, for illustrative purposes, flow synthesis may require only a 1 minute incubation at room temperature (in the reaction tube) and may include 50 mM Si (upon mixing of the two components) at pH 7.0-8.0.Usefully, the inventors have discovered that synthesis conditions can be fine-tuned for optimal particle instability by use of the molybdate assay (lability assay) described herein.

[0050] As a further embodiment to the present invention, in situ uSANS can be produced by a flow system. This is advantageous because the flow system can be coupled to an intravenous delivery system or a cell isolation / treatment system, thus minimizing handling (and possible contamination) during synthesis and delivery. In this flow synthesis, two solutions are mixed in a line: (i) a silicate solution with a pH greater than 10.5 (i.e., soluble silicates rather than colloidal) and (ii) an acidic solution. Upon combining the two streams, a near-neutral pH (pH 6.5-8.0) is achieved, which results in the rapid formation of unstable silicates that can be administered ex vivo directly to a subject or to a subject's cells. Usefully, this flow synthesis system is highly tunable. For example, more aggregated particles (if required) can be produced by using a longer reaction tube, which results in a longer incubation time (in the reaction tube) before injection into a subject or device.

[0051] Usefully, in situ uSANS is as effective as most standard uSANS in activating T cells, but is less toxic. Notably, over the short exposure period typical of intravenous delivery, where there is rapid dilution of the active agent, in situ uSANS shows a complete lack of toxicity even at the highest concentration tested (4 mM; FIG. 9). These particles would need to be permanently present with the endothelial cells to cause damage, which would not occur in treatment protocols. A further advantage of the in situ uSANS system is that the shelf life of its component solutions is virtually 'infinite', as both the buffer (e.g., citrate) and silicate solutions are extremely stable. This is in contrast to standard uSANS, which, despite being stabilized by a low storage pH and possibly the presence of a stabilizing agent, becomes progressively less unstable as can be demonstrated by molybdate assays (FIG. 11). A further limitation to standard uSANS is that, due to their low pH, these solutions must be neutralized prior to administration (e.g., intravenous delivery). Upon neutralization, however, instability rapidly decreases, a clinically undesirable characteristic. In fact, this effect is measurable immediately after neutralization; after 12 minutes the colloid is no longer clinically usable (FIG. 12).

[0052] Nanosilica Composition The present invention relates to a composition comprising nanosilica in the form of silica particles having an average diameter of 0.5 nm to 20 nm, optionally stabilized by one or more stabilizers. The discoveries described in this application indicate that the nanosilica composition of the present invention can be used in a method of activating T cells, thus providing a therapy for a subject or individual, particularly in the treatment of cancer and infectious diseases, as discussed in more detail below.

[0053] Alternatively, compositions including stabilized nanosilica compositions with stabilizers and processes for their production are described in WO 2015 / 121666 (Medical Research Council), which is incorporated by reference in its entirety. Examples of stabilizers suitable for use with the nanosilica compositions of the present invention include polyols, sugars and / or quaternary ammonium salts, such as choline and carnitine. In particular, WO 2015 / 121666 provides a process for producing stabilized polymeric silicate compositions including polymeric silicic acid and nanosilica particles, where the polymerization and particle size growth of the silicates are controlled, and the resulting particles are size stabilized by a combination of silicate concentration, pH and / or stabilizer. In some embodiments, the composition can be further doped with metal cations, as these can induce particle size growth and provide the composition with additional useful properties.

[0054] The nanosilica composition of the present invention comprises a soluble polysilicic acid and nanosilica particles having an average diameter of 20 nm or less, in some cases more preferably less than 10 nm, more preferably less than 5 nm, 4 nm, 3 nm, 2 nm, 1 nm or 0.5 nm. In some embodiments, the particles can range from about 0.5 nm to about 2 nm, or from about 0.5 nm to about 3 nm, or from about 0.5 nm to about 4 nm, or from about 0.5 nm to about 5 nm, or from about 0.5 nm to about 10 nm, or from about 0.5 nm to about 15 nm, or from about 0.5 nm to about 20 nm, or from about 5 nm to about 20 nm, or from about 5 nm to about 15 nm, or from about 5 nm to about 10 nm, or from about 10 nm to about 15 nm, or from about 10 nm to about 20 nm, or from about 15 nm to about 20 nm. A preferred composition comprises silica particles having an average diameter of 0.5 to 10 nm and silica particles having an average diameter of 2 to 5 nm.

[0055] The insoluble nature of the polymeric silicic acid and / or nanosilica particles can be indirectly confirmed by the molybdic acid assay described above, which determines the soluble silicic acid fraction. In general, the material is considered to be in equilibrium with soluble silicic acid, with typical soluble silicic acid concentrations being less than about 2 mM at pH less than 9.0. The nanosilica compositions of the present invention can be contrasted with more aggregated forms of silicates, including larger nanoparticles (e.g., preferably having an average size greater than 50 nm, more preferably greater than 20 nm), polysilicic acid gels, and silicon dioxide (SiO2), which is a fully aggregated form of silicic acid with virtually no -OH groups. The particle size of polysilicic acid can be determined using dynamic light scattering, and the measurements are preferably made on freshly prepared samples if not stabilized. As will be understood by those skilled in the art, polysilicic acid will be in equilibrium with other silicate species. For example, and depending on the exact conditions present, it may contain less soluble silicic acid.

[0056] The compositions of nanosilica particles used in the present invention are generally aquated, i.e., water is present at least to some extent throughout their synthesis (e.g., at least 5% by weight, more preferably at least 10% by weight, at least 20% by weight), and preferably in the final formulation, i.e., the material is not dried or significantly heated before formulation and subsequent administration.However, it will be clear that stabilizers or other formulation agents can be used in such high concentrations that they displace water molecules from the silicate particles.Thus, water can be displaced, but the formulation does not dry.

[0057] The stabilization of the compositions of nanosilica particles used according to the present invention preferably extends from their synthesis through to their storage, formulation and / or administration (eg lack of unwanted agglomeration).

[0058] The compositions of nanosilica particles used according to the present invention are metastable, i.e., the compositions have a suitable stability for the shelf life purpose of their intended use. By way of illustration, the polymeric silicate compositions of the present invention are storage stable, e.g., stable for 3 months or more, more preferably 6 months or more, more preferably 12 months or more, more preferably 24 months or more. Thus, the polymeric silicate compositions of the present invention can be produced by partial aggregation of silicic acid (or silicate) molecules. These materials are not separate aggregates, but rather, the polymeric silicate compositions of the present invention are stable for storage for 3 months or more, more preferably 6 months or more, more preferably 12 months or more, more preferably 24 months or more. Not as clusters or colloids but metastable.

[0059] In some embodiments of the present invention, the composition of nanosilica particles comprises a stabilizing agent, preferably a sugar and / or a polyalkylene glycol. The saccharide comprises oligosaccharides consisting of 8 or less monosaccharides, such as monomeric, dimeric or trimeric saccharides. A preferred sugar is sucrose. The maximum number of monomeric units in the sugar is selected so that its administration does not induce an immune response in a subject upon administration. Polyalkylene glycols are a family of polyether compounds including polyethylene glycol (PEG) and polypropylene glycol. Examples of stabilizing agents that are sugars (saccharides) include monomeric, dimeric, trimeric and polymeric sugars (saccharides) or the corresponding sugar alcohols, such as glucose, fructose, mannose, sucrose, threitol, erythritol, sorbitol, mannitol, galactitol or adonitol. In some embodiments where the stabilizing agent is a sugar, it is an oligosaccharide other than lactose. In some embodiments where a sugar alcohol is used, it is other than mannitol. The use of sugars as stabilizers for internally administered compositions is preferred in the present invention as they are safe for administration to human and animal subjects.

[0060] In some embodiments, it is possible to use more than one different sugar(s) or polyalkylene glycol(s), for example a combination of two, three, four or five or more sugars or polyalkylene glycols, for example by adding them in steps (a) and / or (b). The sugar and / or polyalkylene glycol stabilizers are generally added at a concentration of 0.01M to 3.0M, more preferably 0.03M to 2.0M, most preferably 0.1M to 1.5M. One skilled in the art can perform routine testing to determine which combination of sugars and / or polyalkylene glycols works best in any given situation.

[0061] The stabilized nanosilica compositions of the present invention are distinct from those disclosed in U.S. Patent Publication No. 2003 / 0206967 (Choi et al.), which describes a composition comprising sodium metasilicate, borax, sodium thiosulfate, potassium carbonate, and refined sugar in water. This results in a very alkaline composition having a pH of about pH 13, as opposed to the pH of the stabilized polymeric silicate compositions of the present invention, which is preferably pH 3.0-9.0, more preferably 3.0-8.0, more preferably 5.5-7.5. The process used to make the compositions of Choi et al. is different from the present invention, since the present invention produces compositions by lowering the pH to produce stable silicate polymers. In view of the above, it is preferred that the nanosilica compositions used in the present invention do not contain one or more of sodium metasilicate, borax, sodium thiosulfate, potassium carbonate, and preferably do not contain borax. The preferred nanosilica compositions of the present invention can be choline-free and / or ethanol-free, and thus differ from the Biosil™ and silicate compositions described in Kerek, respectively.

[0062] In another aspect, the present invention can use a carboxylic acid as a stabilizer, the carboxylic acid being C 2-10It can be a carboxylic acid, such as a dicarboxylic acid, such as oxalic acid, malonic acid, glutaric acid, tartaric acid, succinic acid, adipic acid or pimelic acid, or an ionized form thereof (i.e., the corresponding carboxylate), such as adipate. Or, for example, a monocarboxylic acid, such as gluconic acid. Further examples of stabilizers are dicarboxylic acids, which can be represented by the formula HOOC-R1-COOH (or its ionized form), where R1 is an optionally substituted C 1-10 Alkyl, C 1-10 Alkenyl or C 1-10 It is an alkynyl group. Generally, R1 is C 1-10 Alkyl More preferably, it is a C 2-6 The use of carboxylic acids which are alkyl groups is preferred.

[0063] The inventors have also surprisingly found that the nanosilica composition of the present invention can be further stabilized by adding a non-aqueous solvent, such as an alcohol. A preferred example of an alcohol is ethanol. Illustratively, the non-aqueous solvent can be added at 10-50% v / v or 20-50% v / v or 10-20% v / v. Furthermore, in some cases, the inventors have found that the combination of sucrose with an alcohol is particularly effective for stabilizing the nanosilica composition.

[0064] In the following discussion of the process steps of the present invention, it will be apparent to one of skill in the art that it may be possible to rearrange some of the process steps and / or perform some of the steps simultaneously, others of the steps being optional, as indicated above and further described below.

[0065] In the research leading to the present invention, the inventors have found that several factors contribute to the stability of nanosilica compositions, including the rate at which the pH of the starting alkaline silicate solution drops, the inclusion of stabilizers, particularly sugars or polyalkylene glycols, the addition of metal cations, and / or the addition of non-aqueous solvents. The process for producing nanosilica compositions can use these approaches alone or in any combination to produce nanosilica compositions with sufficient stability for use, for example, as a nutritional supplement or therapeutic agent.

[0066] In some cases, particularly with respect to the production of nanosilica ultrasmall particles ("uSANS"), the rate at which the pH of the alkaline silicate solution is lowered can have a significant effect on the stability of the resulting nanosilica composition. Preferably, the pH is lowered (e.g., to pH 4.0 or a pH below 3.0) over a period of less than 60 seconds, more preferably less than 30 seconds, more preferably less than 10 seconds, or most preferably less than 5 seconds. The production of an alternative silica particle composition, HS-7, is provided in the examples below.

[0067] The concentration of the alkaline silicate solution is preferably 0.05M to 1.5M, more preferably 0.03 to 2.0M, and most preferably 0.1M to 1.0M. The use of a pH higher than 9.5 is also preferred to maintain the solubility of the silicates, and preferably the pH of the alkaline silicate solution in step (a) is about pH 10.5 or higher, even more preferably about pH 11.5 or higher. In the final polymeric silicate composition, the concentration of silicon can be 2.5mM or higher, 5.0mM or higher, 25mM or higher, 30mM or higher, 40mM or higher, 50mM or higher, 100mM or higher, 250mM or higher, 500mM or higher. In the final stabilized polymeric silicate composition, the concentration of silicon can be 1.5M or lower, 1.0M or lower, 500mM or lower, and ranges between these lower and upper limits.

[0068] In some embodiments, the lowered pH can have an effect on the type of stabilized silicate nanoparticles that can be produced. As shown in the examples, uSANS or very small particles with an average diameter of 5 nm or less can be formed by rapidly lowering the pH from a pH of more than 10 to a pH of 3.0 or less, allowing silicon concentrations up to 1M to be used. Alternatively, SANS or small nanoparticles with an average diameter of 10 nm or less can be formed by lowering the pH to about 7.4. In this case, lower concentrations of about 50 mM or less can be used. Thus, the lowered pH can be 7.4 or less, or 3.0 or less. This allows the preparation of uSANS at low pH, and as described, if this is required, the pH is raised to grow uSANS to SANS of a determined particle size, and the size is stabilized by lowering the pH again. In some cases, a stabilizer is required at some stage during this process. These processes are an important part of the technology.

[0069] In some cases, for long-term storage of the stabilized aqueous suspension, the pH can be lowered and / or the suspension can be diluted. Alternatively, or in addition, upon long-term storage at a non-physiological pH and prior to administration to a subject, the nanosilicate suspension can be adjusted to a physiological pH and / or diluted and / or a stabilizer can be added.

[0070] The composition can be stabilized so that the particle size of the nanoparticles will remain sufficiently stable (less than 20 nm) for the intended application. For example, in the case of a formulation for intravenous administration, the particle size of a first stock solution (e.g., pH<3 and 100 mM Si) will be stable for the duration of the storage period, and then once diluted with a buffered intravenous solution, it will first remain stable for several hours before application, and then once administered, it will not undergo agglomeration.

[0071] Synthetic Methodology for Batch Synthesis of In Situ uSANS(Citrate) Preparation of stock solutions Component A (HCl + Citric Acid): (i) 0.307 g citric acid (MW 192.124) is dissolved in 180 ml UHP water; (ii) 0.783 ml 37% HCl is added under stirring; (iii) the volume is adjusted to 200 ml with UHP water.

[0072] Component B (80 mM sodium silicate): Dilute 2.56 ml of sodium silicate (approximately 6.25 M Si) with water to a final volume of 200 mL. Component C (Saline; optional): Dissolve 4.5 g NaCl in 45 ml UHP water and adjust final volume to 50 ml with UHP water.

[0073] Sterilization of components A, B and C (optional): Under sterile conditions (flow cabinet), syringe filter the component solutions through 0.22 μm filters into sterile containers.

[0074] Preparation of colloids Step 1: Component A (2.5 mL) is mixed with Component B (2.5 mL). The resulting pH is approximately 5.4 as measured by a pH electrode (4.0-5.0 with pH paper).

[0075] Step 2: Incubate for 30 minutes. Step 3 (optional): After incubation and immediately prior to administration, Component C (0.425 mL) is added and mixed.

[0076] Synthesis methodology for in-line synthesis of in situ uSANS (unbuffered) Preparation of stock solutions Component A (HCl): (i) 1.2 ml of 37% HCl is diluted with water to a final volume of 100 ml.

[0077] Component B (50 mM Sodium Silicate): Dilute 0.8 ml of Sodium Silicate (Sigma-338443, approximately 6.25 M Si) with water to a final volume of 100 mL.

[0078] Preparation of colloids Colloids are prepared in a flow system as depicted in Figure 6 using a reaction tube with a length of 30 cm. Syringe pumps A and B were operated at 1.05 and 48.5 mL / h, respectively, which resulted in a pH of 7.55.

[0079] Formulation and Use of the Composition The nanosilica composition of the present invention can be formulated for use as a silicate-containing therapeutic agent.The composition can contain, in addition to nanosilica particles, pharma- ceutically acceptable excipients, carriers, buffers, stabilizers or other materials known to those skilled in the art.Such materials should be non-toxic and should not interfere with the efficacy of the nanosilica particle composition.

[0080] The experiments described herein demonstrate that the nanosilica particle compositions of the present invention can directly activate T lymphocytes as determined, for example, by an increase in expression of the biomarkers CD69 and / or CD25 compared to untreated control samples of T lymphocytes. The assays and methods of the present invention can be used in screening and development of effective therapeutic silica particle compositions and to determine the effectiveness of therapies administered to a subject or individual.

[0081] The activation of T lymphocytes using the present invention can be used in the treatment of a range of diseases or disorders that will have therapeutic benefit to the subject, whether as a treatment or as a prevention, and has the advantage that when administered at appropriate concentrations, the silica particle composition used in the present invention is not substantially directly cytotoxic to cancer cells.Cancer can include, but is not limited to, melanoma, skin cancer, lung cancer, pancreatic cancer, colorectal and other visceral cancer, gastric cancer, breast cancer, lymphoma, leukemia, uterine cancer, prostate cancer, esophageal cancer, bone cancer, bladder cancer, cervical cancer, endometrial cancer, brain cancer, eye cancer, ovarian cancer, testicular cancer, liver cancer, kidney cancer, head and neck cancer, including metastatic cancer and primary cancer.

[0082] However, although one preferred use of the present invention is in the treatment of cancer, the T cell therapy described herein can be used to treat other diseases, particularly infections, such as bacterial or viral infections.Infections include, but are not limited to, viruses, retroviruses, and bacteria, such as mycobacteria, gram-positive and gram-negative bacteria, as well as helminths, parasites, and other infectious agents.Transiently stable silicate nanoparticles can also serve as a reservoir for the release of silicic acid, which is effective in promoting connective tissue health and may be useful in osteoporosis, fracture healing, joint diseases, skin diseases, and vascular disorders.

[0083] Thus, administration may be by parenteral administration, the latter in particular by intravenous administration. Other medical uses of the compositions of the invention include the treatment of hypertension, diabetes, bone diseases, cardiovascular diseases, neurodegenerative pathologies, all types of cancer not mentioned above, hyperacidity, osteoporosis, dental tartar, Alzheimer's disease, Creutzfeldt-Jakob disease, as well as wound healing. Other medical uses of the compositions of the invention include the treatment of burns, wounds or skin affected by the action of pathogens or caustic chemicals, including the treatment of sunburn or any skin disease, including psoriasis, eczema and other types of dermatitis.

[0084] In one aspect, the medical use of the invention using activated T lymphocytes is for use in the treatment of human subjects. However, as well as having application for the treatment or prevention of diseases in human subjects, the invention also finds use in the veterinary field, for example in the treatment of non-human animals, more particularly non-human mammals, such as dogs, cats and horses. This article has application in relation to the above.

[0085] Liquid pharmaceutical compositions generally contain a liquid carrier, such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other sugar solution or glycols, such as ethylene glycol, propylene glycol or polyethylene glycol, can be included. For example, when the silicate-containing nutritional supplement needs to be maintained in solid form to control the delivery of the material components, it may be necessary to select the formulation components appropriately (for example, when a liquid formulation of the material is made). Preservatives, stabilizers, buffers, antioxidants and / or other additives can be included as necessary in the embodiment of the present invention, for example, when the polymeric silicate composition is suitable for administration to a subject by infusion.

[0086] In therapeutic applications, the nanosilica compositions used according to the present invention are preferably given to an individual in a "prophylactically effective amount" or "therapeutically effective amount" (although in some cases prevention may be considered therapy), which is sufficient to show benefit (e.g., bioavailability) to the individual. The actual amount administered and the rate and time course of administration will depend on the nature and severity of what is being treated. Decisions regarding the prescription of treatment, e.g., dosage, etc., are within the responsibility of general practitioners and other physicians, and typically take into account the disorder to be treated, the individual patient's illness, the site of delivery, the method of administration, and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington's Pharmaceutical Sciences, 20th Edition, 2000, Lippincott, Williams & Wilkins. The compositions can be administered alone or in combination with other treatments, either simultaneously or sequentially, depending on the illness to be treated. EXAMPLES

[0087] material and method Preparation of Silica Particle Composition HS-7 HS-7 was prepared by diluting 0.1 g of sodium silicate (catalog no. 338443; Sigma-Aldrich Chemical Co., Gillingham, UK) in 20 g of distilled deionized water (DDW; 18 μΩ / cm) and adjusting the pH to 6.7–7.0 with hydrochloric acid (HCl, 4–5 M) (pH paper, Sigma-Aldrich Co., catalog no. P3536). The dispersion was left at room temperature for 16–24 to equilibrate.

[0088] Preparation of silica particle composition "uSANS" Ultrasmall amorphous nanosilica (uSANS) nanoparticle dispersions (1.9 nm) were prepared according to WO 2015 / 121666. uSANS was prepared by diluting 4 mL of sodium silicate (catalog no. 338443; Sigma-Aldrich Chemical Co., Gillingham, UK) in 30 mL of distilled deionized water (DDW; 18 μΩ / cm). 2 mL of HCl (37%) was added rapidly with mixing. The solution was diluted to 50 mL with HO before use and incubated at room temperature for 16–24 h.

[0089] Comparative silica composition according to Example 4 of US Patent Application Publication No. 2011 / 0229577 (Kerek) 7.4 ml of TMOS was mixed with 25 ml of UHP water. The pH was adjusted to 3.6 with acetic acid (0.5 M) and the resulting mixture was incubated for 5 min at 45 C under stirring in an oil bath. Then the suspension was cooled at room temperature for 40 min and the pH was adjusted to 2.0 with 1 M HCl. Finally, the methanol was removed using a rotavapor (T=40-45 C).

[0090] Cellular assays Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation from single white blood cell cones purchased from the National Blood Service (Cambridge, UK). Cells were frozen in freezing media (10% DMSO, 50% FBS, 40% RPMI 1640). Frozen cells were thawed, washed in RPMI and rested for 2 hours in RPMI containing 10% FBS, 0.3 g / L L-glutamine, 1% penicillin-streptomycin and 0.01 μg / mL DNase. Cells were resuspended to 2×106 cells / mL in RPMI+20% FCS and 0.5 mL was placed in a FACS tube followed by 0.5 mL RPMI+ / -1600 uM nanosilica treatment (giving 1.0×106 cells / mL, 800 uM nanosilica). Cells were incubated for 24 hours, washed with PBS+1% BSA, and stained with FACS antibodies and viability markers (CD3, CD4, CD8, CD25, CD69, 7-AAD viability marker). Cells were analyzed on a real-time Cyan-ADP flow cytometer using Summit software for acquisition and analysis (Beckman Coulter) acquiring a minimum of 400,000 events per sample.

[0091] Activation of CD4 and CD8 T cells by ultrafine nanosilica Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation from single white blood cell cones purchased from the National Blood Service (Cambridge, UK). Cells were frozen in freezing medium (10% DMSO, 50% FBS, 40% RPMI 1640). Frozen cells were thawed, washed in RPMI, and incubated at 4°C for 1 h at 37°C for 2 h at 37°C. Cells were left for 2 hours in RPMI containing FBS, 0.3 g / L L-glutamine, 1% penicillin streptomycin and 0.01 μg / mL DNase. Cells were resuspended at 2×106 cells / mL in RPMI+20% FCS and 0.5 mL was placed in a FACS tube followed by 0.5 mL RPMI+ / -1600 uM nanosilica treatment (1.0×106 cells / mL, giving 800 uM nanosilica). Cells were incubated for 24 hours, washed with PBS+1% BSA and stained with FACS antibodies and viability markers (CD3, CD4, CD8, CD25, CD69, 7-AAD viability marker). Cells were analyzed on a real-time Cyan-ADP flow cytometer using Summit software for acquisition and analysis (Beckman Coulter) acquiring a minimum of 400,000 events per sample.

[0092] Activation of different Th lineage cell types by ultrafine nanosilica Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation from single white blood cell cones purchased from the National Blood Service (Cambridge, UK). Cells were frozen in freezing medium (10% DMSO, 50% FBS, 40% RPMI 1640). Frozen cells were thawed, washed in RPMI, and incubated at 4°C for 1 h at 37°C for 2 h at 37°C. Cells were left for 2 hours in RPMI containing FBS, 0.3 g / L L-glutamine, 1% penicillin streptomycin and 0.01 μg / mL DNase. Cells were resuspended at 2×106 cells / mL in RPMI+20% FCS and 0.5 mL was placed in a FACS tube followed by 0.5 mL RPMI+ / -17 uL or 35 uL nanosilica treatment (giving 1.0×106 cells / mL, 400 and 800 uM nanosilica, respectively). Cells were incubated for 24 hours, washed with PBS+1% BSA and stained with FACS antibodies and viability markers (CD3, CD4, CD8, CD25, CD69, CD40L, LAP, GARP, 7-AAD viability markers). For intracellular staining of FoxP3, cell membranes were treated with cell membrane permeabilization buffer prior to intracellular staining. Cells were analyzed on a real-time Cyan-ADP flow cytometer using Summit software for acquisition and analysis (Beckman Coulter) acquiring a minimum of 400,000 events per sample. For IFN gamma cytokine analysis, cell supernatants collected at the end of incubation were assayed for the presence of IFN gamma using a standard ELISA kit according to the manufacturer's instructions.

[0093] Toxicity of three different forms of nanosilica to endothelial venous cells Human umbilical vein endothelial cells (HUVECs; ATCC, Manassas, USA) were resuscitated from cryopreservation in liquid nitrogen and maintained in complete growth medium: F-12K medium (ATCC) supplemented with 10% fetal bovine serum (FBS 'Gold'; PAA Laboratories, UK), 50 μg / mL penicillin and streptomycin (Invitrogen Ltd, Life Technologies UK), 0.1 mg / mL heparin (Sigma Aldrich Chemical Co, UK) and 0.05 mg / mL endothelial cell growth supplement (Alfa Aesar, UK). Cells were grown at 37°C in a humidified atmosphere containing 5% CO2 in Nunc T75 culture flasks (75 cm2; VWR International, UK). For experiments, cells were detached with 0.1% trypsin / 0.02% EDTA solution at 70% confluence and plated in 48-well plates with 1 mL of complete growth medium per well. The cells grew to a confluent monolayer.

[0094] Nanosilica Treatment. Three different nanosilica preparations were tested. These were called HS7, In Situ uSANS and Sucrose uSANS, which refer to nanosilica prepared in the following manner, respectively: Sue.uSANS: One-day-old uSANS was prepared by rapidly acidifying 500 mM Si(OH)4 + 1.5 M sucrose to pH 0.8-1 and diluting to 40 mM with instant H20+ glucose (final glucose concentration 136 mM). The dispersion was incubated overnight and neutralized to pH 7 immediately before use.

[0095] In situ uSANS (pH 5.0; batch synthesis; buffered with citrate) was prepared by mixing 1 mL of 80 mM Si(OH)4 and 1 mL of citrate (8 mM, +47 mM HCl). The solution was incubated for 30 min and 0.17 mL of 10x saline was added before use.

[0096] These were compared to complete growth medium at final silicon concentrations of 1, 2 and 4 mM total Si in complete growth medium - three vehicles (one for each nanosilica preparation) were tested at equal doses relative to the 4 mM nanosilica treatment. Each treatment and control was tested in sextuplicate (i.e. n=6). Briefly, medium was aspirated from the sextuplicate wells and nanosilica was diluted in complete growth medium and immediately added to the wells (0.5 ml per well).

[0097] After addition of nanosilica or vehicle-treated cells, the 48-well plates were placed in an IncuCyte Zoom live cell imaging system (Essen Bioscience Inc., USA) and phase contrast images were acquired every 2 hours over a period of 68 hours.

[0098] Nanosilica-mediated pZap70 delivery in T cells Peripheral blood mononuclear cells (PBMCs) were isolated using density gradient centrifugation from single white blood cell cones purchased from the National Blood Service (Cambridge, UK). Cells were frozen in freezing medium (10% DMSO, 50% FBS, 40% RPMI 1640). Frozen cells were thawed, washed in RPMI, and incubated at 4°C for 1 h at 37°C for 2 h at 37°C. Cells were left for 2 hours in RPMI containing FBS, 0.3 g / L L-glutamine, 1% penicillin streptomycin and 0.01 μg / mL DNase. T cells were enriched using an untouched pan T cell isolation kit. Cells were resuspended at 2×106 cells / mL in RPMI+20% FCS and 0.5 mL was placed in a FACS tube followed by 0.5 mL RPMI+ / -1600 uM nanosilica treatment (1.0×106 cells / mL, giving 800 uM nanosilica). Cells were incubated for 0-6 hours, spun down and lysed using NuPage LDS sample buffer. Western blot analysis was performed on the lysates staining for vinculin (internal control, (An et al., 2012; Solan et al., 2003)) and pZap70 (Tyr319). Band intensities were quantified by integration and expressed as percentages of the internal control.

[0099] After engagement of the TCR / CD3 complex by pMHC complexes, superantigens or mitogenic antibodies, T cell activation is initiated by phosphorylation of zeta chain-associated protein kinase 70 (Zap70) located on phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) (Love and Hayes, 2010). Phosphorylated Zap70 (pZap70) then phosphorylates downstream proteins, which result in signaling by nuclear factor of activated T cells (NFAT) and activated T cell responses (Chakraborty and Weiss, 2014; Fathman and Lineberry, 2007; Rossy et al., 2012; Wang et al., 2010). .

[0100] Molybdate dissolution assay The molybdate dissolution assay involves taking a sample of the polymeric silicate composition and diluting it in a buffer. The molybdate assay can be used to determine the concentration of soluble silicate present in an aliquot of buffer over the time course of the assay. As shown in the examples, the composition can be diluted in 50 mM HEPES buffer at pH 7.0-7.4. A typical molybdate assay uses 100 μL of test solution or standard (prepared from Sigma Aldrich Si ICP 30 standard, 1000 mg / L) and 200 μL of molybdate color solution (34.5 mM Mo (added as NH4Mo74H20) and 0.15 M H2S04). The assay solution is transferred to a well plate and mixed for 10 minutes. After incubation, the absorbance (400 nm) can be measured and the concentration of soluble silicate can be determined using a standard curve.

[0101] Molybdate lability assay 7.5 μl of 40 mM Si suspension was transferred to a 96-well plate and very quickly 100 μl of UHP water was added followed by 200 μl of molybdic acid solution (34.5 mM NH4Mo7O244H20 in 0.15 M H2S04). The absorbance at 400 nm was measured every 60 seconds for 17 hours. A solution of 40 mM silicic acid was used as a control in the assay.

[0102] Lysis assay Whole blood was collected in heparin tubes and mixed briefly. Additional heparin was added (to a concentration of 0.5mg / mL) to prevent clotting. Nanosilica was then diluted according to the following schedule: 16mM for 25 seconds, 4mM for 1 hour, 2mM for 3 hours, and 1mM for 20 hours. After treatment and incubation, red blood cells were lysed, stained, and FACS was performed immediately. For a positive control, SEB was added to whole blood at 2ug / mL. This assay showed that in situ uSANS (citrate buffered, batch synthesis) had the same stimulation profile as standard uSANS, as determined by readouts of CD4(+)-CD25, CD4(+)-CD69, CD8(+)-CD25, and CD8(+)-CD69.

[0103] result The above experiments demonstrated that only ultrafine nano-silica induces chronic activation of T lymphocytes (soluble It has been demonstrated that neither silicic acid (Si(OH)4) nor larger silica particles (e.g. >10 nm) induce this. Figures 1 and 2 show the expression of the activation marker CD69 on helper T cells after 24 hours of silicic acid and ultrafine nanosilica treatment compared to unstimulated baseline controls and silicates produced according to US Patent Application Publication No. 2011 / 0229577 (Kerek, Figure 1) and Biosil™, a commercially available choline-stabilized orthosilicic acid composition (Figure 2). Figures 3-6 show a similar comparison with the addition that the T lymphocyte superantigen SEB (Staphylococcus enterotoxin B) was used as a positive control. The silica particle composition of the present invention was shown to induce T cell activation by increasing CD25 and CD69 activation marker expression levels. Also, silicic acid had no effect on the expression of activation markers on T cells (data not shown). The ultrafine nanosilica of the present invention also induced chronic activation of T cells comparable to that of the superantigen SEB. The figure shows that nanosilica activates both helper (CD4+) and cytotoxic (CD8+) T cells (i.e., they become CD25 and CD69 positive upon nanosilicate exposure).

[0104] The experiments described herein demonstrate that multiple lineage types of T cells may be susceptible to T cell activation by these ultrafine nanosilica particles (Figure 8). However, despite being better tolerated than HS7 particles, uSANS particles still exhibit moderate toxicity to endothelial cells (Figure 9). However, minimally toxic uSANS can be produced by in situ synthesis. This can be batch synthesis in a quasi-neutral buffer system or by flow synthesis. These results can also be reproduced using uSANS produced using the in situ synthesis method described herein.

[0105] Without being limited thereto, the present invention includes the following aspects. [Aspect 1] A composition comprising silica particles having an average diameter of 0.5 to 20 nm for use in a method of activating T lymphocytes for therapy in a subject / individual, wherein the activation of the T lymphocytes is characterized by an increase in expression of CD69 and / or CD25 by the T lymphocytes. [Aspect 2] 2. A composition for use in a method of treatment according to embodiment 1, wherein the increase in CD69 and / or CD25 expression is at least 20% compared to unstimulated baseline subjects. [Aspect 3] 3. A composition for use in the method of treatment according to embodiment 1 or embodiment 2, wherein said T lymphocytes are T cells. [Aspect 4] 4. A composition for use in the method of treatment according to any one of aspects 1 to 3, wherein the T cells are helper T cells, regulatory T cells, cytotoxic T cells, natural killer T cells (NKT) or gamma delta T cells. [Aspect 5] 20. A composition for use in the method of treatment of any one of the preceding aspects, wherein the lymphocyte activation comprises inducing an inflammatory Th1-type response. [Aspect 6] 20. A composition for use in the method of treatment of any one of the preceding embodiments, wherein the activation of said T lymphocytes comprises increasing expression of one or more additional markers selected from CD40L, LAP / GARP and / or FoxP3. [Aspect 7] 2. A composition for use in the method of treatment of any one of the preceding aspects, wherein the activation of lymphocytes comprises increasing expression of IFN-γ. [Aspect 8] 20. A composition for use in the method of treatment of any one of the preceding aspects, wherein the composition does not substantially induce lymphocyte proliferation. [Aspect 9] 13. A composition for use in the method of treatment according to any one of the preceding aspects, wherein the silica particles have an average diameter of from 0.5 to 10 nm. [Aspect 10] 10. A composition for use in the method of treatment according to embodiment 9, wherein the silica particles have an average diameter of 2 to 5 nm. [Aspect 11] 20. A composition for use in the method of treatment according to any one of the preceding embodiments, wherein the composition optionally comprises a stabilizing agent selected from polyols, sugars and / or quaternary ammonium salts, such as choline and carnitine. [Aspect 12] 11. A composition for use in the method of treatment according to any one of aspects 1 to 10, wherein the composition does not comprise a stabilizing agent. [Aspect 13] 2. A composition for use in the method of treatment according to any one of the preceding aspects, wherein the silica composition is obtainable by precipitation. [Aspect 14] 20. The composition for use in the method of treatment of any one of the preceding aspects, wherein the activated T lymphocytes are for use in the treatment of cancer. [Aspect 15] A composition for use in a method of treatment according to embodiment 14, wherein the silica particle composition is not substantially directly cytotoxic to cancer cells. [Aspect 16] 14. A composition for use in the method of treatment according to any one of aspects 1 to 13, wherein the composition is for use in a T cell therapy other than the treatment of cancer. [Aspect 17] A composition for use in the method of treatment according to embodiment 16, wherein the activated T lymphocytes are for use in the treatment of an infection. [Aspect 18] A composition for use in the method of treatment according to any one of aspects 1 to 13, wherein the activated T lymphocytes are for use in the treatment of an inflammatory disease. [Aspect 19] 20. A composition for use in a method of treatment according to embodiment 18, wherein the inflammatory disease is multiple sclerosis, rheumatoid arthritis, Crohn's disease, asthma or ulcerative colitis. [Aspect 20] 20. A composition for use in the method of treatment of any one of the preceding aspects, the method comprising administering to the subject an agent capable of suppressing or enhancing one or more particular T cell lineages or phenotypes. [Aspect 21] 21. A composition for use in the method of treatment according to embodiment 20, wherein the agent is cyclophosphamide. [Aspect 22] A composition for use in the method of treatment of any one of the preceding embodiments, the method comprising generating the silica particle composition by in situ synthesis prior to administration to the subject. [Aspect 23] 2. A composition for use in the method of treatment of any one of the preceding aspects, wherein the in situ synthesis comprises mixing (i) a silicate solution having a pH greater than 10.5 and (ii) an acidic buffer solution to produce a colloidal silica composition having a pH between 4.0 and 8.5. [Aspect 24] 24. A composition for use in the method of treatment according to embodiment 23, wherein the in situ synthesis comprises mixing (i) a silicate solution having a pH greater than 10.5 and (ii) an acidic buffer solution in a batch in situ synthesis process to produce a colloidal silica composition having a pH between 4.0 and 6.5. [Aspect 25] 24. A composition for use in the method of treatment according to embodiment 23, wherein the in situ synthesis comprises mixing (i) a silicate solution having a pH greater than 10.5 and (ii) an acidic buffer solution in a flow in situ synthesis process to produce a colloidal silica composition having a pH between 6.0 and 8.5. [Aspect 26] 20. A composition for use in a method of treatment according to any one of the preceding aspects, wherein the colloidal silica composition is for administration to a subject. [Aspect 27] 27. A composition for use in the method of treatment according to any one of embodiments 26, wherein the method comprises administering the colloidal silica composition to the subject less than 12 minutes from mixing the solution. [Aspect 28] 20. A composition for use in the method of treatment according to any one of the preceding embodiments, wherein the composition is for intravenous administration. [Aspect 29] 29. A composition for use in the method of treatment according to any one of aspects 23 to 28, wherein the acidic solution comprises hydrochloric acid, a carboxylic acid, such as citric acid, or an acidic amino acid, such as glycine. [Aspect 30] 20. A composition for use in the method of treatment according to any one of aspects 1 to 19, the method being a method of T cell therapy comprising obtaining a sample from a subject comprising T cells, contacting the T cell sample with the silica composition to activate the T cells in the sample, and administering the activated T cells to the subject. [Aspect 31] 20. The composition for use in the method of treatment of any one of the preceding aspects, wherein the activated T lymphocytes are for use in the treatment of a human subject. [Aspect 32] A composition for use in the method of treatment according to any one of aspects 1 to 30, wherein the activated T lymphocytes are for use in the treatment of a non-human animal. [Aspect 33] 33. A composition for use in the method of treatment according to embodiment 32, wherein the activated T lymphocytes are for veterinary use. [Aspect 34] 34. A composition for use in a method of treatment according to embodiment 33, wherein the activated T lymphocytes are for use in the treatment of dogs, cats or horses. [Aspect 35] 1. A method for determining the efficacy of a silica particle composition for therapeutic administration to a subject or individual, the method comprising the steps of: (a) contacting a sample of T lymphocytes with said silica particle composition; (b) determining the expression of CD69 and / or CD25 by the T lymphocytes present in the sample; and (c) identifying silica particle compositions that cause an increase in expression of CD69 and / or CD25 by said T lymphocytes as suitable for use in therapy; A method comprising: [Aspect 36] 1. An assay for determining the efficacy of a silica particle composition for therapeutic administration to a subject or individual, the assay comprising the steps of: (a) contacting a sample of T lymphocytes with said silica particle composition; (b) determining expression of CD69 and / or CD25 by the T lymphocytes present in the sample; (c) measuring or quantitating the amount of CD69 and / or CD25 expression relative to a control value, and selecting the silica particle composition as suitable for therapeutic administration if the amount of CD69 and / or CD25 expression is increased compared to the control value; The assay comprises: [Aspect 37] 1. A method for determining the efficacy of a therapy comprising administration of a silica particle composition or a protocol for its administration to said subject or individual, said method comprising the steps of: (a) contacting a sample of T lymphocytes from said subject or individual treated with said silica particle composition or a protocol for its administration; (b) determining the expression of CD69 and / or CD25 by the T lymphocytes present in the sample; and (c) identifying a subject or individual in which an increase in expression of CD69 and / or CD25 by said T lymphocytes is observed in step (b) as having been successfully treated with said silica particle composition or its administration protocol; or (d) identifying a subject or individual in which an increase in CD69 and / or CD25 expression by the T lymphocytes is not observed in step (b) and modifying the silica particle composition provided to that subject or individual or the protocol for its administration; (e) optionally repeating steps (a)-(d) until the subject or individual is successfully treated with the silica particle composition or its administration protocol; A method or protocol comprising: [Aspect 38] A method or assay according to any one of embodiments 35 to 37, wherein the increase in expression of CD69 and / or CD25 by said T lymphocytes is determined relative to a reference value. [Aspect 39] 39. A method or assay according to embodiment 38, wherein the reference value is an unstimulated baseline control. [Aspect 40] The use of CD69 and / or CD25 expression by T lymphocytes as a marker or biomarker for the therapeutic effectiveness of a silica particle composition. [Aspect 41] 1. A device for producing a silica composition for therapeutic use, the device comprising: a first vessel containing a silicate solution having a pH>10.5, a second vessel containing an acidic buffer solution; flow control means for controlling the flow of the silicate solution and the acidic buffer solution; and an outlet in fluid communication with the first and second containers for delivering the colloidal silica composition such that the two solutions are mixed prior to administration to a subject; Including, the device. [Aspect 42] 42. The device of embodiment 41, further comprising a mixing chamber in fluid communication with the first and second containers for mixing the solutions to produce a colloidal silica composition. [Aspect 43] 42. The device of embodiment 41, wherein the solutions are mixed in situ in an intravenous injection line prior to administration to a subject. [Aspect 44] 43. The device of embodiment 41 or embodiment 42, further comprising a chamber for contacting a biological sample comprising T lymphocytes obtained from a subject with the colloidal silica composition to cause activation of the T lymphocytes for therapy. [Aspect 45] 45. The device of embodiment 44, wherein the device comprises means for delivering a sample comprising T lymphocytes extracted from the subject to the contact chamber, and for returning the activated T lymphocytes to the subject. [Aspect 46] 46. ​​The device according to any one of aspects 41 to 45, wherein the outlet is connected to an intravenous injection line for delivery of the colloidal silica composition or the activated T lymphocytes to the subject. [Aspect 47] A kit comprising: (i) a first container comprising a silicate solution having a pH>10.5; and (ii) a second container comprising an acidic buffer solution; and (iii) instructions for mixing the silicate solution and the acidic buffer solution to produce a therapeutically effective colloidal silica composition having a pH of 4.0 to 8.5. [Aspect 48] 48. The kit according to embodiment 47, wherein the acidic solution comprises hydrochloric acid, a carboxylic acid, such as citric acid, or an acidic amino acid, such as glycine. References: All documents mentioned herein are hereby incorporated by reference in their entirety.

[0106] [ka]

[0107] International Publication No. 2009 / 052090. U.S. Patent Publication No. 2009 / 0130230. U.S. Patent Publication No. 2013 / 0149396.

[0108] U.S. Patent No. 5,807,951 (Nippon Zoki Pharmaceutical Co., Ltd.). WO 95 / 21124. European Patent No. 1 371 289 A.

[0109] U.S. Patent Publication No. 2011 / 0229577 (Kerek).

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Claims

1. A composition comprising amorphous silica particles having an average diameter of 0.5-10 nm for use in a method of activating T lymphocytes for therapy for a subject / individual, wherein the activated T lymphocytes are for use in the treatment of an infectious disease, and wherein the activation of the T lymphocytes is characterized by an increase in expression of CD69 and / or CD25 by the T lymphocytes.

2. 2. A composition for use in the method of treatment of claim 1, wherein the increase in CD69 and / or CD25 expression is at least 20% compared to unstimulated baseline subjects.

3. 3. A composition for use in the method of treatment according to claim 1 or claim 2, wherein the T lymphocytes are T cells.

4. 4. A composition for use in the method of treatment according to any one of claims 1 to 3, wherein the T lymphocytes are helper T cells, cytotoxic T cells, natural killer T cells (NKT) or gamma delta T cells.

5. 5. A composition for use in a method of treatment according to any one of claims 1 to 4, wherein activation of the T lymphocytes comprises inducing an inflammatory Th1-type response.

6. 6. A composition for use in a method of treatment according to any one of claims 1 to 5, wherein activation of said T lymphocytes comprises increasing expression of one or more further markers selected from CD40L, LAP / GARP and / or FoxP3.

7. 7. A composition for use in the method of treatment according to any one of claims 1 to 6, wherein activation of lymphocytes comprises increasing expression of IFN-γ.

8. 8. A composition for use in the method of treatment according to any one of claims 1 to 7, wherein the composition does not substantially induce lymphocyte proliferation.

9. 9. A composition for use in a method of treatment according to any one of claims 1 to 8, wherein the silica particles have an average diameter of 2 to 5 nm.

10. 10. A composition for use in the method of treatment according to any one of claims 1 to 9, the composition optionally comprising a stabilising agent selected from polyols, sugars and / or quaternary ammonium salts, such as choline and carnitine.

11. 10. A composition for use in the method of treatment according to any one of claims 1 to 9, wherein the composition does not comprise a stabilizing agent.

12. 12. A composition for use in a method of treatment according to any one of claims 1 to 11, wherein the silica particle composition is obtainable by precipitation.

13. A composition for use in a method of treatment according to any one of claims 1 to 11, wherein the infection is a bacterial infection.

14. A composition for use in a method of treatment according to any one of claims 1 to 11, wherein the infectious disease is a viral infection.

15. 15. A composition for use in a method of treatment according to any one of claims 1 to 14, said method comprising administering to said subject an agent capable of suppressing or enhancing one or more particular T cell lineages or phenotypes.

16. 16. A composition for use in the method of treatment of claim 15, wherein the agent is cyclophosphamide.

17. A method for preparing a composition for use in the method of treatment according to any one of claims 1 to 16, comprising the steps of: (i) an in situ synthesis of a colloidal silica composition by mixing a silicate solution having a pH greater than 10.5 and (ii) an acidic buffer solution, the colloidal silica composition having a pH of 4.0 to 8.5; Methods of preparing compositions for use in methods of treatment.

18. 18. A method for preparing a composition for use in the method of treatment of claim 17, comprising: (i) an in situ synthesis of a colloidal silica composition by mixing a silicate solution having a pH greater than 10.5 and (ii) an acidic buffer solution, the colloidal silica composition having a pH of 4.0 to 6.5; Methods of preparing compositions for use in methods of treatment.

19. 20. A method for preparing a composition for use in the method of treatment of claim 17, the method comprising the steps of: (i) an in situ synthesis of a colloidal silica composition by mixing a silicate solution having a pH greater than 10.5 and (ii) an acidic buffer solution, the colloidal silica composition having a pH of 6.5 to 8.5; Methods of preparing compositions for use in methods of treatment.

20. 17. The composition for use in the method of treatment according to any one of claims 1 to 16, wherein the pH is from 4.0 to 8.

5.

21. 21. A composition for use in a method of treatment according to claim 20, wherein the composition is for administration to a subject.

22. 22. A composition for use in a method of treatment according to claim 21, the method comprising administering the composition to the subject less than 12 minutes from mixing the solution.

23. 23. A composition for use in a method of treatment according to any one of claims 1 to 16, 20 to 22, wherein the composition is for intravenous administration.

24. 20. A method of preparing a composition for use in a method of treatment according to any one of claims 17 to 19, wherein the acidic buffer comprises hydrochloric acid, a carboxylic acid, such as citric acid, or an acidic amino acid, such as glycine.

25. 15. A composition for use in a method of treatment according to any one of claims 3 to 14, the method being a method of T cell therapy comprising obtaining a sample from a subject comprising T cells, contacting the sample with the silica particle composition to activate T cells in the sample, and administering the activated T cells to the subject.

26. 23. A composition for use in a method of treatment according to any one of claims 1 to 16, 20 to 22, wherein the activated T lymphocytes are for use in the treatment of a human subject.

27. 23. A composition for use in a method of treatment according to any one of claims 1 to 16, 20 to 22, wherein the activated T lymphocytes are for use in the treatment of a non-human animal.

28. 28. A composition for use in the method of treatment of claim 27, wherein the activated T lymphocytes are for veterinary use.

29. 30. A composition for use in the method of treatment of claim 28, wherein the activated T lymphocytes are for use in the treatment of dogs, cats or horses.

30. In vitro studies were performed to determine the efficacy of compositions comprising amorphous silica particles having an average diameter of 0.5-10 nm for therapeutic administration to a subject or individual for use in the treatment of infectious diseases. The Toro method, comprising the steps of: (a) contacting a sample of T lymphocytes with said silica particle composition; (b) determining the expression of CD69 and / or CD25 by the T lymphocytes present in the sample; and (c) identifying said silica particle compositions that cause an increase in expression of CD69 and / or CD25 by said T lymphocytes as suitable for use in therapy; A method comprising:

31. 1. An in vitro assay for determining the effectiveness of a composition comprising amorphous silica particles having an average diameter of 0.5-10 nm for therapeutic administration to a subject or individual for use in the treatment of an infectious disease, the assay comprising the steps of: (a) contacting a sample of T lymphocytes with said silica particle composition; (b) determining the expression of CD69 and / or CD25 by the T lymphocytes present in the sample; (c) measuring or quantitating the amount of CD69 and / or CD25 expression relative to a control value, and selecting the silica particle composition as suitable for therapeutic administration if the amount of CD69 and / or CD25 expression is increased compared to the control value; The assay comprises:

32. 32. The method or assay of claim 30 or 31, wherein an increase in expression of CD69 and / or CD25 by said T lymphocytes is determined compared to a reference value.

33. 33. The method or assay of claim 32, wherein the reference value is an unstimulated baseline control.

34. Use of a composition comprising amorphous silica particles having a mean diameter of 0.5-10 nm as a marker or biomarker of expression of CD69 and / or CD25 by T lymphocytes for the therapeutic effectiveness in the treatment of infectious diseases.

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