WO2023147504A1 - Systèmes particulaires à base de polysaccharide a pour atténuation d'auto-immunité, d'allergie et de rejet de greffe - Google Patents

Systèmes particulaires à base de polysaccharide a pour atténuation d'auto-immunité, d'allergie et de rejet de greffe Download PDF

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WO2023147504A1
WO2023147504A1 PCT/US2023/061496 US2023061496W WO2023147504A1 WO 2023147504 A1 WO2023147504 A1 WO 2023147504A1 US 2023061496 W US2023061496 W US 2023061496W WO 2023147504 A1 WO2023147504 A1 WO 2023147504A1
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nanoparticle
antigen
psa
cell
subject
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PCT/US2023/061496
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Jamal S. LEWIS
Hamilton KAKWERE
Rian HARRIMAN
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The Regents Of The University Of California
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/48—Preparations in capsules, e.g. of gelatin, of chocolate
    • A61K9/50—Microcapsules having a gas, liquid or semi-solid filling; Solid microparticles or pellets surrounded by a distinct coating layer, e.g. coated microspheres, coated drug crystals
    • A61K9/51—Nanocapsules; Nanoparticles
    • A61K9/5107—Excipients; Inactive ingredients
    • A61K9/513—Organic macromolecular compounds; Dendrimers
    • A61K9/5161—Polysaccharides, e.g. alginate, chitosan, cellulose derivatives; Cyclodextrin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/35—Allergens
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/385—Haptens or antigens, bound to carriers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55511—Organic adjuvants
    • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/57—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2
    • A61K2039/577—Medicinal preparations containing antigens or antibodies characterised by the type of response, e.g. Th1, Th2 tolerising response
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6087—Polysaccharides; Lipopolysaccharides [LPS]

Definitions

  • the nanoparticles may include a crosslinked polysaccharide A (PSA) encapsulating an antigen.
  • PSA polysaccharide A
  • the PSA is a purified PSA that has a specific mTLR2 activity at least twice the activity of lipopolysaccharide (LPS), and in some embodiments, the PSA is devoid of or substantially devoid of LPS.
  • the PSA is crosslinked with a crosslinker that reacts with an amine and/or carboxylic acid group in the PSA (e.g., a di- or multi-functional aldehyde such as glutaraldehyde.
  • a nanoparticle of the invention may induce increased tolerogenicity in a cell or subject.
  • the nanoparticle(s) may induce dendritic cells to form or co-express greater than 5% CD86+, MHCII+ and ICOSL+ cells.
  • the nanoparticle(s) may further increase the formation of interleukin 10 (IL- 10) in a cell (e.g., a T cell).
  • IL- 10 interleukin 10
  • composition that includes a nanoparticle of the invention and a pharmaceutically acceptable carrier.
  • kits for treating, reducing, or inhibiting an immune reaction to an antigen in a subject wherein the kit includes at least one nanoparticle or composition of the invention.
  • methods of generating an antigen specific anti-inflammatory regulatory T cell that include contacting a T cell with at least one nanoparticle or composition of the invention for a time and under conditions sufficient to generate an antigen specific regulatory T cell that is capable of inhibiting a pro-inflammatory response against the antigen.
  • Also provided according to embodiments of the present invention are methods of generating an antigen specific anti-inflammatory regulatory T cell that include contacting a T cell with at least one nanoparticle or composition of the invention for a time and under conditions sufficient to generate an antigen specific regulatory T cell that is capable of inhibiting a pro-inflammatory response against the antigen.
  • Also provided according to embodiments of the present invention are methods of generating an antigen specific anti-inflammatory regulatory T cell that include contacting an antigen presenting cell with at least one nanoparticle or composition of the invention for a time and under conditions sufficient to generate an antigen specific regulatory T cell that is capable of inhibiting a pro- inflammatory response against the antigen.
  • Also provided according to embodiments of the present invention are methods of inhibiting antigen specific inflammation in a subject that include administering to the subject at least one nanoparticle or composition of the invention for a time and under conditions sufficient to induce an antigen specific anti-inflammatory regulatory T cell in the individual, thereby inhibiting the antigen specific inflammation in the subject.
  • Also provided according to embodiments of the present invention are methods of of inhibiting or reducing an immune response to an antigen in a subject that include administering to the subject at least one nanoparticle or composition of the invention for a time and under conditions sufficient to inhibit or reduce the immune response to the antigen in the subject.
  • Also provided according to embodiments of the present invention are methods of inducing the formation of CD86+, ICOSL+ and/or MHCII+ cells from dendritic cells that include contacting the dendritic cells with at least one nanoparticle or composition of the invention for a time and under conditions sufficient to induce the formation of the CD86+, ICOSL+ and/or MHCII+ cells.
  • Also provided according to embodiments of the present invention are methods of inducing the formation of interleukin 10 in a T cell that include contacting the T cell and/or a dendritic cell therewith with at least one nanoparticle or composition of the invention for a time and under conditions sufficient to induce or increase the formation and/or secretion of the interleukin- 10 in the T cell.
  • FIG. 1 is a schematic describing an exemplary method of purifying polysaccharide A (PSA).
  • FIG. 2 is a schematic describing a method of forming a nanoparticle according to an embodiment of the invention.
  • FIG. 3 provides SEM images of ovalbumin nanoparticles (OVA NPs), nanoparticles including PSA and OVA (PSA-OVA NPs) and PSA only nanoparticles (PSA NPs)
  • OVA NPs ovalbumin nanoparticles
  • PSA-OVA NPs PSA only nanoparticles
  • PSA NPs PSA only nanoparticles
  • FIG. 4A is a graph of the particle size distribution of the PSA NPs, PSA-OVA NPs, and OVA NPs from FIG. 3, as determined by dynamic light scattering.
  • FIG. 4B is a graph showing the zeta potential of the PSA NPs and PSA-OVA NPs from FIG. 3.
  • FIGS. 5A and 5B are graphs illustrating that the particle size and distribution of the PSA NPs can be varied by varying the emulsion conditions.
  • the average particle size at the lower homogenizer setting 3 (FIGS. 5A) is larger than the average particle size obtained at the higher homogenizer setting 6 (FIGS. 5B).
  • FIGS. 6A and 6B provide SEM images of PSA-OVA NPs with different ratios of PSA:OVA.
  • the PSA-OVA NPs in FIGS. 6A have a PSA:OVA mass ratio of 30: 1 and the PSA- OVA NPs in FIGS. 6B have a PSA:OVA mass ratio of 3 : 1.
  • FIG. 7 is a schematic describing general procedures for a simulated gastric fluid (SGF) treatment and nitric oxide (NONOate) treatment of nanoparticles.
  • SGF gastric fluid
  • NONOate nitric oxide
  • FIG. 8 provides SEM images of SGF treated PSA NPs at different time points (0 to 5 hours).
  • FIG. 9 provides graphs showing the particle size distribution of the PSA NPs in FIG. 8 at different time points in the SGF treatment, as measured by dynamic light scattering.
  • FIG. 10A shows SEM images of PSA NPs and OVA NPs after 1-3 hours of SGF treatment.
  • FIG. 10B provides the particle size distribution of the PSA NPs and OVA NPs after 1-3 hours of simulated gastric fluid treatment.
  • FIGS. 11A and 11B provide SEM images of PSA NPs before (FIG. 11 A) and after (FIG. 11B) treatment with nitric oxide (NONOate).
  • NONOate nitric oxide
  • FIGS. 12A and 12A provide particle size distributions for PSA NPs before (FIG. 12A) and after (FIG. 12B) treatment with nitric oxide (NONOate).
  • NONOate nitric oxide
  • FIGS. 13A and 13B provide SEM images of PSA-0 VA NPs (30: 1 PSA:OVA mass ratio) before (FIG. 13A) and after (FIG. 13B) treatment with nitric oxide (NONOate).
  • FIGS. 14A and 14A provide particle size distributions for PSA-OVANPs (30: 1 PSA:OVA mass ratio) before (FIG. 14A) and after (FIG. 14B) treatment with nitric oxide (NONOate).
  • PSA-OVANPs (30: 1 PSA:OVA mass ratio) before (FIG. 14A) and after (FIG. 14B) treatment with nitric oxide (NONOate).
  • NONOate nitric oxide
  • FIGS. 15A and 15B provide SEM images of PSA-OVA NPs (3: 1 PSA:OVA mass ratio) before (FIG. 15A) and after (FIG. 15B) treatment with nitric oxide (NONOate).
  • FIGS. 16A and 16B provide particle size distributions for PSA-OVA NPs (3: 1 PSA:OVA mass ratio) before (FIG. 16A) and after (FIG. 16B) treatment with nitric oxide (NONOate).
  • PSA-OVA NPs 3: 1 PSA:OVA mass ratio
  • NONOate nitric oxide
  • FIG. 17 is a schematic of a mTLR2 specificity assay.
  • FIG. 18A is a graph showing the specific mTLR2 activity (normalized to HzO) of PSA, PSA NPs, PSA-OVANPs, PSA+OVA, and OVA compared to the controls of LPS and untreated sample.
  • FIG. 18A is a graph showing the specific mTLR2 activity (normalized to HzO) of PSA, PSA NPs, PSA-OVANPs, PSA+OVA, and OVA compared to the controls of LPS and untreated sample.
  • 18B is a graph showing the specific mTLR2 activity (normalized to untreated) of PSA, PSA NPs, PSA-OVA NPs, PSA NPs after NO treatment (PSA NPs_NO), and the PSA-OVA NPs after NO treatment (PSA-OVA NPs NO) compared to the controls of LPS and untreated sample (* P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, **** P ⁇ 0.0001).
  • FIG. 19 is a schematic of a dendritic cell immunomodulation assay.
  • FIG. 20 is a graph showing the percent of MHCII+/CD86+/ ICOSL+ cells after treatment with the PSA, PSA NPs, PSA-OVA NPs at 30: 1 mass ratio (PSA NPs 30: 1), PSA-OVA NPs at 3 : 1 mass ratio (PSA NPs 3: 1), PSA+OVA, LPS, OVA, and water (* P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0 001, **** P ⁇ 0.0001).
  • FIG. 21A is a graph showing the percent of singlet/alive CD86+ cells after treatment with PSA, PSA NPs, PSA-OVA NPs, untreated and LPS + OVA.
  • FIG. 21B is a graph showing the percent of singlet/alive ICOSL+ cells after treatment with PSA, PSA NPs, PSA-OVA NPs, untreated and LPS + OVA.
  • FIG. 21C is a graph showing the percent of singlet/alive MHCII+ cells after treatment with PSA, PSA NPs, PSA-OVA NPs, untreated and LPS + OVA.
  • FIG. 21A is a graph showing the percent of singlet/alive CD86+ cells after treatment with PSA, PSA NPs, PSA-OVA NPs, untreated and LPS + OVA.
  • 21D is a graph showing the percent of singlet/alive MHCII+/CD86+/ ICOSL+ cells after treatment with PSA, PSANPs, PSA-OVANPs, untreated and LPS + OVA (* P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, **** P ⁇ 0.0001).
  • FIG. 22 is array of representative dot plots showing the expression of CD86, MHC II and ICOSL on bone marrow-derived dendritic cells treated for 48 h with PSA NPs, OVA-loaded PSA NPs and other relevant controls. The plots show that the inherent immunosuppressive activity of PSA is retained after physical and chemical manipulation of PSA to form nanoparticles.
  • FIG. 23 is a schematic of a T cell immunomodulation assay.
  • FIG. 24 A is a graph showing the percent of CD39+ cells (% of CD4) after treatment with PSA, PSA NPs, PSA-OVA NPs, untreated and LPS + OVA.
  • FIG. 24B is a graph showing the percent ICOS+ cells (% of CD4) after treatment with PSA, PSA NPs, PSA-OVA NPs, untreated and LPS + OVA.
  • FIG. 24C is a graph showing the percent of Lag3+ cells (% of CD4) after treatment with PSA, PSA NPs, PSA-OVA NPs, untreated and LPS + OVA.
  • FIG. 24 A is a graph showing the percent of CD39+ cells (% of CD4) after treatment with PSA, PSA NPs, PSA-OVA NPs, untreated and LPS + OVA.
  • FIG. 24B is a graph showing the percent ICOS+ cells (% of CD4) after treatment with PSA, PSA NPs, PSA-OVA NPs
  • FIG. 24D is a graph showing the percent of CD39/ ICOS+ cells (% of CD4) after treatment with PSA, PSA NPs, PSA- OVA NPs, untreated and LPS + OVA.
  • FIG. 24E is a graph showing the IL-10 (pg/ml) after treatment with PSA, PSANPs, PSA-OVANPs, untreated and LPS + OVA (* P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, **** P ⁇ 0.0001).
  • FIG. 25 is array of representative dot plots showing the expression of CD4, CD39, ICOS and Lag3 on T cells incubated for 72 h with bone marrow-derived dendritic cells previously cultured with PSA NPs, OVA-loaded PSA NPs and other relevant controls. The plots show that the dendritic cells after exposure to OVA-loaded PSANPs drive an OVA-specific Tri regulatory cell phenotype.
  • FIG. 26A is a graph showing the IL-10 (pg/ml) detected after treatment with PSA, PSA NPs, PSA-OVA NPs (30: 1), PSA-OVA NPs (3: 1), PSA+OVA, LPS, OVA, and water.
  • FIG. 1 is a graph showing the IL-10 (pg/ml) detected after treatment with PSA, PSA NPs, PSA-OVA NPs (30: 1), PSA-OVA NPs (3: 1), PSA+OVA, LPS, OVA, and
  • 26B is a graph showing the %CD73+/CD39+/ICOS+ after treatment with PSA, PSA NPs, PSA-OVA NPs (30: 1), PSA-OVA NPs (3: 1), PSA+OVA, LPS, OVA, and water.
  • FIG. 26C is a graph showing the percent of Lag3+ cells after treatment with PSA, PSA NPs, PSA-OVA NPs (30: 1), PSA-OVA NPs (3: 1), PSA+OVA, LPS, OVA, and water (* P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001, **** P ⁇ 0.0001).
  • transitional phrase “consisting essentially of’ (and grammatical variants) is to be interpreted as encompassing the recited materials or steps “and those that do not materially affect the basic and novel characteristic(s)” of the claimed invention. See, In re Herz, 537 F.2d 549, 551-52, 190 U.S.P.Q. 461, 463 (CCPA 1976) (emphasis in the original); see also MPEP ⁇ 2111.03.
  • a measurable value such as an amount or concentration and the like, is meant to encompass variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of the specified value as well as the specified value.
  • “about X” where X is the measurable value is meant to include X as well as variations of ⁇ 10%, ⁇ 5%, ⁇ 1%, ⁇ 0.5%, or even ⁇ 0.1% of X.
  • a range provided herein for a measurable value may include any other range and/or individual value therein.
  • the terms “increase,” “increases,” “increased,” “increasing,” “improve,” “enhance,” and similar terms indicate an elevation in the specified parameter of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 100%, 150%, 200%, 300%, 400%, 500% or more.
  • the terms “reduce,” “reduces,” “reduced,” “reduction,” “inhibit,” and similar terms refer to a decrease in the specified parameter of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, or 100%.
  • treating by the term “treat,” “treating,” or “treatment of’ (or grammatically equivalent terms), it is meant that the severity of the subject’s condition is reduced or at least partially improved or ameliorated and/or that some alleviation, mitigation or decrease in at least one clinical symptom is achieved.
  • Treating in reference to a disorder or condition may refer to: (i) inhibiting a disorder or condition, e.g., arresting its development, (ii) relieving a disorder or condition, e.g., causing regression of the clinical symptoms; and/or (iii) stabilizing or controlling the progression of a disorder or condition.
  • an allergenic or immune response to an allergen or antigen may be considered a disorder or condition.
  • the terms “inflammatory response”, “pro-inflammatory response” and “inflammation” as used herein indicate the complex biological response of vascular tissues of an individual to harmful stimuli, such as pathogens, damaged cells, or irritants, and includes secretion of cytokines and more particularly of pro-inflammatory cytokines, i.e. cytokines which are produced predominantly by activated immune cells such as microglia and are involved in the amplification of inflammatory reactions.
  • Reducing an inflammatory response, a pro-inflammatory response, and/or inflammation may include reducing or inhibiting the expression of any pro- inflammatory molecule (e.g., a pro-inflammatory cytokine) and/or may include increasing the expression of any anti-inflammatory molecule (e.g., an anti-inflammatory cytokine, e.g., interleukin- 10).
  • a pro-inflammatory molecule e.g., a pro-inflammatory cytokine
  • an anti-inflammatory cytokine e.g., interleukin- 10
  • the gut microbiota comprises trillions of bacteria, which are mostly commensal.
  • the presence of these bacteria is central for maintenance of physiological homeostasis within the gut, particularly with respect to inflammatory responses, gut immune system function, and metabolic homeostasis.
  • commensal bacterial are crucial to maintaining the desired tolerogenic microenvironment prevalent in the gut.
  • the special ability of these bacteria to quiesce the immune system in the gut is, in part, due to the functionalization of their cell walls with carbohydrate molecules such as polysaccharide A (PSA), which is a capsular zwitterionic polysaccharide (CZP).
  • PSA polysaccharide A
  • CZPs capsular zwitterionic polysaccharides
  • CZPs bear alternating zwitterionic charges within a repeating unit which is an important structural motif for their immunomodulatory activity.
  • CZPs which include types 5 and 8 polysaccharides of Staphylococcus aureus, type 1 Streptococcus pneumoniae polysaccharide and polysaccharide A (PSA) from Bacteroides fragilis (B. fragiHs). produce a T cell dependent immune response upon processing by antigen presenting cells (APCs).
  • APCs antigen presenting cells
  • the CZPs are processed by APCs through interacting with the Toll-like receptor 2 (TLR2) and undergoing presentation via the major histocompatibility complex II (MHC II) to activate CD4+ T cells.
  • TLR2 Toll-like receptor 2
  • MHC II major histocompatibility complex II
  • CZPs represent an interesting class of biologically active carbohydrates with potential for generating material constructs with immunomodulatory activity that are applicable for the treatment of diseases or usable as vaccines.
  • particulates e.g., nanoparticles.
  • chemical modification of immunomodulatory materials may reduce or eliminate such properties, prior to the present invention, it was unclear whether it was possible to form PSA particulates that retain their immunomodulatory properties.
  • a nanoparticle or a plurality of nanoparticles, for inducing antigen-specific immune tolerance
  • the nanoparticle includes a crosslinked polysaccharide A (PSA) encapsulating an antigen.
  • PSA polysaccharide A
  • Polysaccharide A or “PSA” is a molecule produced by the PSA locus of Bacteroides fragilis and derivatives thereof which include but are not limited to polymers of the repeating unit ⁇ —>3) a-d-AAT Galp( l ⁇ 4)-[P-d-Galf( l ⁇ 3)] a-d-GalpNAc(l—>3)-[4,6-pyruvate]- P-d-Galp(l— ⁇ , where AATGal is acetamido-amino-2,4,6-trideoxygalactose, and the galactopyranosyl residue is modified by a pyruvate substituent spanning 0-4 and 0-6.
  • a derivative polysaccharide of PSA retains one or more functional activities in connection with PSA in association with the anti-inflammatory activity of PSA.
  • the PSA may be purified, for example, by a method described herein and/or a method described in Kakwere et al., Engineering immunomodulatory nanoplatforms from commensal bacteria-derived polysaccharide A, J. Mater. Chem. B, 2022, 10, 1210-1225.
  • the PSA is devoid or substantially devoid (less than 1% or 0.1% by weight) of lipopolysaccharide (LPS).
  • the purified PSA has an average molecular weight in a range of about 70 kd to about 150 kd (e.g., about 100 kd to about 140 kd, or about 130 kd).
  • the nanoparticle may also include at least one additional polymer encapsulating the antigen.
  • additional polymer encapsulating the antigen.
  • biopolymers such as polypeptides/proteins (e.g., TGF-betal) and other polysaccharides, particularly those having immunoregulatory or tolerogenic properties (e.g., P- glucan/galactan polysaccharides derived from the cell wall of B. bifidum, see, e.g, Verma et al., Cell surface polysaccharides of Bifidobacterium bifidum induce the generation of Foxp3 + regulatory T cells, Science Immunology, Vol. 3, 28, 2018).
  • the at least one additional polymer has a specific mTLR2 activity that is at least 2 times greater than the activity of LPS and/or induces the production or secretion of IL-10.
  • the PSA is the only polymer in the nanoparticle that encapsulates the antigen.
  • the PSA in the nanoparticle(s) of the invention is crosslinked (e.g., via covalent and/or ionic bonds).
  • a number of different crosslinkers and crosslinking methods may be used.
  • the crosslinkers may bind with the amine and/or carboxylic acid groups of the PSA.
  • the nanoparticle is crosslinked with a di- or poly-functional aldehyde (e.g., glutaraldehyde), wherein the aldehyde moieties may react with amino groups in the PSA to form imine linkages.
  • crosslinkers include but are not limited to dihydrazide crosslinkers (see, e.g., Mohammadi et al., Anal. Chem. 2021, 93, 4, 1944-1950), N- hydroxysuccinimide-based crosslinkers, and carboiimide-based crosslinkers.
  • the PSA may also be modified so that other crosslinkers may be used.
  • the crosslinker should crosslink the PSA without substantially altering its tolerogenicity/immunoregulatory properties.
  • the specific mTLR2 activity of the PSA after crosslinking is greater than, substantially equivalent to, or less than 5%, 10%, or 20% of the specific mTLR2 activity of the PSA before the crosslinking (under the same conditions).
  • the amount of crosslinking may vary but is sufficient to encapsulate at least one antigen within the nanoparticle.
  • a nanoparticle of the invention may have a diameter in a range of about 1 nm to about 500 nm.
  • the diameter may be the average diameter of multiple nanoparticles of the invention, for example, as determined by dynamic light scattering (DLS).
  • the “diameter” of the particle(s) may also be expressed in terms of the longest diameter or cross-section for non- spherical particles.
  • the nanoparticles are substantially spheroidal. However, the nanoparticles are not limited to a spherical shape, and may be elliptical, oval, oblong, or irregularly shaped.
  • a nanoparticle may have an average diameter in the range of 1 about nm to about 20 nm, about 1 nm to about 40 nm, about 1 nm to about 60 nm, about 1 nm to about 80 nm, about 1 nm to about 100 nm, about 50 nm to about 100 nm, about 50 nm to about 200 nm, about 50 nm to about 300 nm, about 50 nm to about 400 nm, about 50 nm to about 500 nm, about 50 nm to about 600 nm about 50 nm to about 700 nm, about 50 nm to about 800 nm, about 50 nm to about 900 nm, about 50 nm to about 1000 nm, about 100 nm to about 200 nm, about 100 nm to about 300 nm, about 100 nm to about 400 nm, or about 100 nm to about 500 nm.
  • the nanoparticle may be about 1 nm, about 5 nm, about 10 nm, about 20 nm, about 30 nm, about 40 nm, about 50 nm, about 60 nm, about 70 nm, about 80 nm, about 90 nm, about 100 nm, about 110 nm, about 120 nm, about 130 nm, about 140 nm, about 150 nm, about 160 nm, about 170 nm, about 180 nm, about 190 nm, about 200 nm, about 210 nm, about 220 nm, about 230 nm, about 240 nm, about 250 nm, about 260 nm, about 270 nm, about 280 nm, about 290 nm, about 300 nm, or any range defined therebetween.
  • the nanoparticle includes at least one antigen encapsulated or bound therein.
  • the term “encapsulated” refers to antigens that are fully or partially bound and included within the PSA. In some embodiments, no portion of the antigen is present on the surface of the nanoparticle, but in some embodiments, at least a portion of the antigen is present on the outer surface of the nanoparticle.
  • the phrase “at least a portion” means at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, but less than 100%.
  • a least a portion of at least one antigen is positioned internal to the outer surface of the nanoparticle.
  • a nanoparticle of the invention may include multiple antigens encapsulated therein.
  • the nanoparticle includes at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more of the same or different antigens, including all integers in between.
  • a nanoparticle of the invention may include a single type of antigen, or combinations of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 or more (including all integers in between) different antigens.
  • the term “antigen” is a molecule recognized by a subject's immune system (e.g., elicits or stimulates an immune response in the subject).
  • the type of antigen is not limited.
  • Exemplary antigens include molecules that bind specifically to an antibody, and any molecule or molecular fragment that can be bound by a major histocompatibility complex (MHC) and presented to a T-cell receptor.
  • MHC major histocompatibility complex
  • Biomarkers that can be used to identify and/or detect antigen specific Treg include but are not limited to those described herein and CD25, GITR, CTLA-4, or nuclear expression of Foxp3.
  • the antigen may be a naturally occurring antigen or a modified antigen.
  • antigen types that may be included in a nanoparticle of the invention, include, but are not limited to, microbial antigens, cancer antigens, autoimmune antigens (autoantigens), environmental antigens, viral antigens, parasitic antigens, fungal antigens, allergens (e.g., food allergens), and the like.
  • An antigen included in a nanoparticle of the invention may comprise a natural or synthetic peptide, lipid, glycolipid, lipopeptide, or carbohydrate molecule.
  • the antigen includes a food allergen, such as an allergen from peanuts, tree nuts, or shellfish, including but not limited to tropomyosin, arginase kinase, troponin C, cupins, prolamin, profilin, and Bet-v-1 and related proteins, and other proteins listed in A. Lopata et al., Aller go J Ini., 2016; 25(7):210-218 and G. Mueller et al., Curr Allergy Asthma Rep . 2014 May; 14(5):429.
  • a food allergen such as an allergen from peanuts, tree nuts, or shellfish, including but not limited to tropomyosin, arginase kinase, troponin C, cupins, prolamin, profilin, and Bet-v-1 and related proteins, and other proteins listed in A. Lopata et al., Aller go J Ini., 2016; 25(7):210-218 and G. Mueller et al., Curr Allergy
  • the antigen is an antigen that plays a role in an autoimmune disorder (an auto-antigen) such as rheumatoid arthritis, including but not limited to Type II collagen and citrullinated fibrinogen.
  • an auto-antigen such as rheumatoid arthritis
  • the antigen is an antigen that plays a role in diabetes (e.g., type 1 diabetes) including but not limited to insulin, GAD, carboxypeptidase H, and the like (see, e.g., Roep et al., Cold Spring Harb Perspect Med., 2012 Apr; 2(4): a007781).
  • a nanoparticle of the invention may have toleranceinducing properties.
  • the nanoparticle may induce dendritic cells having a tolerogenic phenotype such as increased expression of CD86+, ICOSL+ and/or MHCII+ biomarkers (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more of the total dendritic cells, or in a range of 5% to 90%, including 5%, 10%, or 30% to 60%, 70%, 80%, or 90%).
  • a tolerogenic phenotype such as increased expression of CD86+, ICOSL+ and/or MHCII+ biomarkers (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more of the total dendritic cells, or in a range of 5% to 90%, including 5%, 10%, or 30% to 60%, 70%, 80%, or 90%).
  • a nanoparticle of the invention may induce the formation of tolerogenic T cells having increased expression of CD39+, ICOS+ and/or Lag+ biomarkers (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more, or in a range of 5% to 90%, including 5%, 10%, or 30% to 60%, 70%, 80%, or 90%).
  • a nanoparticle of the invention may induce the formation of tolerogenic T cells that produce and/or secrete interleukin 10 at a concentration of at least 500 pg/ml or at least 1000 pg/ml.
  • compositions that include a nanoparticle, or a plurality of nanoparticles, of the invention.
  • Such compositions may include nanoparticle(s) of the invention and a pharmaceutically acceptable carrier.
  • a “pharmaceutically acceptable carrier” can mean any substance, not itself a therapeutic agent, used as a carrier, diluent, adjuvant, binder, and/or vehicle for delivery of a therapeutic agent to a subject, or added to a pharmaceutical composition to improve its handling or storage properties or to permit or facilitate formation of a compound or composition into a unit dosage form for administration.
  • Non-limiting examples of pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials that are well-known in the art. Such compositions may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention.
  • Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like.
  • pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium, or calcium salts.
  • the composition is an aqueous solution or dispersion.
  • Aqueous suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of nanoparticles of the invention may be prepared as oil-based suspensions.
  • Suitable solvents or vehicles may include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides.
  • a pharmaceutical composition of the invention may be in the form of a tablet, pill, capsule, liquid, gel, syrup, slurry, film, suspension, lozenge, spray, powder (e.g, lyophilized powder) and the like.
  • Suitable excipients that may also be included in formulations for administration of nanoparticles of the invention may include, but are not limited to fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol
  • cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP).
  • PVP polyvinylpyrrolidone
  • compositions of the invention can be prepared using known materials and techniques.
  • a composition of the invention may include an antigen at a concentration in a range of about 1 microgram to 1 milligram, 1 microgram to 5 milligrams, 50 micrograms to 1 milligram, 50 micrograms to 5 milligrams, 500 micrograms to 5 milligrams, 1 femtogram to 5 nanograms, 1 femtogram to 500 nanograms, 1 femtogram to 100 nanograms, 1 femtogram to 10 nanograms, 1 femtogram to 1 nanograms, 50 femtograms to 100 nanograms, 50 femtograms to 50 nanograms, 50 femtograms to 5 nanograms, 50 femtograms to 1 nanogram, 500 femtograms to 500 nanograms, 500 femtograms to 500 nanograms, 500 femtograms to 500 nanograms, 500 femtograms to 100 nanograms, 50 femt
  • compositions of the invention may further include at least one additional therapeutic agent, including immunomodulating agents, vaccines, and/or drug therapies,
  • a composition includes at least one other tolerogenic agent (e.g., vaccine), anti-inflammatory cytokine, such as IL-10, INF-gamma, and INF-lambda; and transforming growth factor beta 1 (TGF-pi), oral anti-histamine, alpha/beta adrenergic agonists, and the like.
  • a composition of the invention may include two or more different nanoparticles of the invention, including nanoparticles having different antigens encapsulated therein.
  • a composition of the invention may include a first nanoparticle of the invention including a first antigen, a second nanoparticle of the invention including a second antigen, and so on.
  • a composition of the invention may include nanoparticles of the invention having two or more different sizes and/or polymer compositions.
  • a variety of administration routes are available with which to administer a nanoparticle or composition of the invention to a subject.
  • the particular mode selected will depend, at least in part, on the particular nanoparticle selected, the particular condition being treated, and the dosage required for therapeutic efficacy.
  • the methods of administering a nanoparticle or composition of the invention may include any mode of administration that is medically acceptable, meaning any mode that produces effective levels of a desired response without causing clinically unacceptable adverse effects.
  • Modes of administration that may be used with nanoparticles and methods of the invention include, but are not limited to parenteral administration, such as, subcutaneous injections, intravenous, intramuscular, intraperitoneal, intraventricular, intracranial, intrathecal, intrasternal injection or infusion techniques.
  • nanoparticles of the invention include, but are not limited to, oral, mucosal, rectal, vaginal, sublingual, intranasal, intratracheal, inhalation, ocular, transdermal, etc.
  • the composition may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion.
  • the composition of the invention is an oral composition.
  • a nanoparticle or composition of the invention can be administered at least daily, every other day, weekly, every other week, monthly, yearly, every at least 2, 3, 4, 5, 10, 15, 20, 30, 40 years, etc. to a subject. Doses may be administered once per day or more than once per day, for example, 2, 3, or more times in one 24-hour period.
  • Nanoparticle(s) or compositions of the invention may be administered alone, in combination with other nanoparticle(s) or compositions of the invention, and/or in combination with other immunomodulating agents, vaccines, drug therapies, or other treatment regimens.
  • the doses of the nanoparticle or composition of the invention can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors may include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery method or route) may be employed to the extent that subject tolerance permits.
  • the dose administered is a dose included in a composition of the invention as described herein (e.g., 1 femtogram to 1 milligram).
  • a subj ect shall mean a human or vertebrate mammal including but not limited to a dog, cat, horse, cow, goat, and primate, e.g., monkey.
  • the invention can be used to treat diseases or conditions in human and non-human (animal) subjects.
  • methods and compositions of the invention can be used in veterinary or livestock applications as well as in human prevention and treatment regimens.
  • the subject is a human.
  • Non-limiting examples of subjects to which the present methods and nanoparticles of the invention can be applied are subjects who are diagnosed with, suspected of having, or at risk of having, a disease or condition that may be treated by administering an antigen to stimulate an immune response.
  • Methods of the invention may be applied to a subject who, at the time of treatment, has been diagnosed as having a disease or condition, or a subject who is considered to be at risk for having or developing the disease or condition.
  • a nanoparticle or composition of the invention may also be administered to a subject at the same time as, or prior to, or subsequent to administration of at least one other therapeutic agent (e.g., another immunomodulatory agent).
  • the nanoparticle or composition of the invention is administered with an oral anti-histamine, alpha/beta adrenergic agonists (e.g., phenylephrine), and the like.
  • the invention in part, pertains to eliciting an immune response to treat a subject in need of immune stimulation.
  • a nanoparticle or composition of the invention may be used to stimulate an immune response to prevent or treat a disease or condition in a subject having, or at risk of having, the disease or condition.
  • diseases or conditions that may be treated or prevented by immune stimulation by nanoparticles of the invention include, but are not limited to an infection, an auto-immune response, a cancer, tissue or organ rejection, allergen, etc.
  • the presence of or risk of having a disease or condition in a subject may be determined using standard diagnostic means, known and routinely practiced in the medical and veterinary arts.
  • Immune cells that can be targeted according to the present invention include, but are not limited to, dendritic cells, macrophages, lymphocytes, monocytes, neutrophils, mast cells, B cells, T cells, T helper cells, and antigen-presenting cells.
  • dendritic cells, macrophages, T cells, and B cells are targeted.
  • dendritic cell and/or Treg cells are targeted by the nanoparticles of the invention.
  • a nanoparticle or composition of the invention is contacted with a cell in vitro or in vivo or administered to a subject in vivo.
  • the methods include administering to a subject to which the induction of antigen-specific immune tolerance is needed, an effective amount of the nanoparticle(s) or composition of the present invention.
  • the therapeutic composition of the present invention specifically targets dendritic cells, induces dendritic cells with a tolerogenic phenotype, promotes induction of Treg cells, and/or suppresses T cell proliferation in vivo or in vitro.
  • the nanoparticle or composition of the invention treats the subject and/or prevents, reduces, or inhibits an immune response or inflammation in the subject, for example, as compared to an untreated control subject.
  • the term “tolerance,” as used herein, refers to a failure to respond, or a reduced response, to an antigen, including auto-antigens and allergens.
  • the term “tolerogenic” or “toleranceinducing, as used herein, refers to a phenotype that induces tolerance to an antigen directly or indirectly, or is capable of silencing or down-regulating an adaptive immunological response to an antigen. Tolerogenic dendritic cells have a low ability to activate effector T cells but have a high ability to induce and activate regulatory T cells.
  • tolerogenic dendritic cells have increased expression of CD86+, ICOSL+ and/or MHCII+ biomarkers (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more, or in a range of 5% to 90%, including 5%, 10%, or 30% to 60%, 70%, 80%, or 90%).
  • CD86+, ICOSL+ and/or MHCII+ biomarkers e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more, or in a range of 5% to 90%, including 5%, 10%, or 30% to 60%, 70%, 80%, or 90%).
  • tolerogenic T cells have increased expression of CD39+, ICOS+ and/or Lag+ biomarkers (e.g., at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, or more, or in a range of 5% to 90%, including 5%, 10%, or 30% to 60%, 70%, 80%, or 90%).
  • tolerogenic T cells produce and/or secrete interleukin 10 at a concentration of at least 500 pg/ml or at least 1000 pg/ml.
  • the present invention provides methods for the prevention and/or treatment of autoimmune diseases, allergenic reactions, transplant rejection, chronic inflammation, and other diseases or disorders in which induction of specific tolerance would be beneficial.
  • the method comprises administering, to a subject, who has been diagnosed to be in need of such treatment, an effective amount of a nanoparticle or composition of the invention.
  • subjects treated in accordance with the present invention have been diagnosed with an autoimmune disease, allergy, chronic inflammation, and other disease or disorder in which induction of specific tolerance would be beneficial.
  • the present invention is useful to treat a disease or condition associated with immune disorders and autoimmune diseases.
  • the diseases or conditions include, but are not limited to, asthma, allergies, rhinitis, chronic urticaria, and atopic dermatitis.
  • the present invention can also be used to inhibit macrophage or T cell associated aspects of an immune response.
  • the present invention can be used to inhibit macrophage or T cell activities including, but not limited to, macrophage antigen-presenting activity, macrophage-associated cytokine production, T cell cytokine production, T cell adhesion, and T cell proliferative activities.
  • the present invention may also be useful to suppress or inhibit humoral and/or cellular immune responses, including those present after transplantation.
  • Also provided according to embodiments of the present invention are methods of generating an antigen specific anti-inflammatory regulatory T cell that include contacting a T cell with at least one nanoparticle or composition of the invention for a time and under conditions sufficient to generate an antigen specific regulatory T cell that is capable of inhibiting a pro-inflammatory response against the antigen.
  • the time and conditions for such assays are generally known in the art. Such methods may be performed in vivo or in vitro.
  • the antigen specific anti-inflammatory regulatory T cell is injected into a subject, thereby reducing inflammation and/or an immune response in the subject.
  • Also provided according to embodiments of the present invention are methods of generating an antigen specific regulatory T cell that include contacting an antigen presenting cell with at least one nanoparticle or composition of the invention for a time and under conditions sufficient to generate an antigen specific regulatory T cell that is capable of inhibiting a pro-inflammatory response against the antigen. Such methods may be performed in vivo or in vitro. In some embodiments, the generated antigen specific regulatory T cell is injected back into the subject thereby reducing inflammation and/or an immune response in the subject.
  • Also provided according to embodiments of the present invention are methods of inhibiting antigen specific inflammation in a subject that include administering to the subject at least one nanoparticle or composition of the invention for a time and under conditions sufficient to induce an antigen specific anti-inflammatory regulatory T cell in the individual, thereby inhibiting the antigen specific inflammation in the subject.
  • Also provided according to embodiments of the present invention are methods of of inhibiting or reducing an immune response to an antigen in a subject that include administering to the subject at least one nanoparticle or composition of the invention for a time and under conditions sufficient to inhibit or reduce the immune response to the antigen in the subject.
  • Also provided according to embodiments of the present invention are methods of inducing the formation (or co-expression) of CD86+, ICOSL+ and/or MHCII+ cells from dendritic cells that include contacting the dendritic cells with at least one nanoparticle or composition of the invention for a time and under conditions sufficient to induce the formation or co-expression of the CD86+, ICOSL+ and/or MHCII+ cells.
  • the time and conditions for such assays are generally known in the art. Such methods may be performed in vivo or in vitro.
  • Also provided according to embodiments of the present invention are methods of inducing the formation or secretion of interleukin 10 in a T cell that include contacting the T cell and/or a dendritic cell therewith (a dendritic cell in contact with the T cell) with at least one nanoparticle or composition of the invention for a time and under conditions sufficient to induce or increase the formation or secretion of the interleukin- 10 in the T-cell.
  • the time and conditions for such assays are generally known in the art.
  • methods of inducing the formation (and/or co-expression) of CD39+, ICOS+, and Lag3+ T cells are provided.
  • Such methods include contacting the T cell or a dendritic cell therewith with at least one nanoparticle or composition of the invention for a time and under conditions sufficient to form CD39+, ICOS+, and Lag3+ T cells. Such methods may be performed in vivo or in vitro.
  • kits that include a composition of the invention.
  • the kits may include a composition or compositions in unit dosage form, e.g., in ampoules or in multi-dose containers.
  • the methods of the invention are performed using a kit comprising at least one unit dosage form comprising a composition of the invention.
  • a kit may include the solid oral dosage forms for one day’s dose of the composition.
  • a kit may also include unit dosage forms for more than one day of the cycle (e.g., a week) or for the full cycle.
  • a kit may include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or more solid oral dosage forms comprising a nanoparticle or composition of the invention.
  • the kit may further comprise a container and/or a package suitable for commercial sale.
  • the container can be in any conventional shape or form known in the art which is made of a pharmaceutically acceptable material, such as a paper or cardboard box, a glass or plastic bottle or jar, a re-sealable bag, or a blister pack with individual dosages for pressing out of the pack according to a therapeutic schedule. More than one container can be used together in a single package. For example, tablets may be contained in a blister pack which is in turn contained within a box.
  • the container is a bottle, e.g., a 30-cc white high-density polyethylene bottles containing unit dosage forms.
  • the bottle may further contain desiccant, e.g., silica desiccant canisters.
  • desiccant e.g., silica desiccant canisters.
  • the container is a blister pack, e.g., formed by aluminum foil on foil lidding containing one tablet per cavity.
  • the blister packs may be present in a carton.
  • the kit may further comprise information.
  • the information may be provided on a readable medium.
  • the readable medium may comprise a label.
  • the information may be directed towards a physician, pharmacist, or patient.
  • the information may indicate that the unit dosage form may cause one or more adverse effects.
  • the information may comprise instructions for administering the unit dosage form, such as in a manner described herein. These instructions may be provided in a variety of ways.
  • the information can be associated with the container, for example, by being: written on a label (e.g., the prescription label or a separate label) adhesively affixed to a container; included inside a container as a written package insert; applied directly to the container such as being printed on the wall of a box or blister pack; or attached as by being tied or taped, for example as an instructional card affixed to the neck of a bottle via a string, cord or other line, lanyard or tether type device.
  • a label e.g., the prescription label or a separate label
  • the nanoparticles may be formed by a number of different methods. However, in some embodiments of the invention, a nanoparticle is formed using a water in oil emulsion.
  • an aqueous solution including PSA and an aqueous solution including the antigen maybe added to an oil (e.g., a paraffin oil).
  • the ratio of the PSA to the antigen is in a range of 3: 1 to 30: 1 by weight. In some embodiments, the ratio of PSA to the antigen is in a range of 5: 1 to 15: 1 (e.g., about 10: 1).
  • the water is deionized water. Any suitable oil may be used but, in some embodiments, the oil is a paraffin oil. In some embodiments, a surfactant is also to the oil and/or water to facilitate the formation of the emulsion.
  • the water and oil may be homogenized and/or sonicated to form aqueous droplets in the oil.
  • the crosslinking agent such as a di- or multi-functional aldehyde (e.g., glutaraldehyde) may then be added to the emulsion, thereby crosslinking the PSA and encapsulating the antigen within the crosslinked PSA.
  • the crosslinker is added in excess (e.g., at 5 to 15, e.g., 8- 10) times more than the PSA by weight.
  • the nanoparticles may precipitate from the emulsion.
  • the aqueous phase may then be evaporated under vacuum or upon exposure to the atmosphere for a sufficient time.
  • a quencher is added to the nanoparticles to ensure that all crosslinker has reacted.
  • the resulting nanoparticles may then be washed, for example, a hexane, diethyl ether, and water wash, to purify the particles.
  • the nanoparticles are dialyzed in water (e.g., for 1-5 days) for further purification.
  • Also provided according to embodiments of the invention are methods of purifying PSA that include using high pressure liquid chromatography (e.g., via method described herein). Such methods may produce a purified PSA that is devoid or substantially devoid of LPS.
  • PSA was purified from B. fragilis strain DmpiM44, an engineered strain of Bacteroides fragilis that only expresses PSA in its capsule, and quality controlled before using in these studies.
  • B. fragilis was grown in twelve 1 L bottles at 37 °C under anaerobic condition in BHI broth containing 5 pg mL' 1 hemin for 4-5 days. Soluble materials were isolated from bacterial pellets with phenol/chloroform extraction, and organic solvents removed via ether wash and rotary evaporation. Nucleic acids and proteins were degraded by DNase/RNase and pronase, respectively, and removed via dialysis.78 PSA was then purified from other contaminants, via ion exchange high performance liquid chromatography (IEX-HPLC).
  • IEX-HPLC ion exchange high performance liquid chromatography
  • the mobile phase consisted of eluents A (Tris buffer 25 mM, pH 7.3), eluent B (0.1% CHAPS (w/v) in Tris buffer 25 mM, pH 7.3), eluent C (170 mM NaCl in Tris buffer 25 mM, pH 7.3) and D (500 mM NaCl in Tris buffer 25 mM, pH 7.3).
  • Solvent D was substituted with 2000 mM NaCl solution for washing.
  • the fractions collected were dialyzed against MilliQ water for 2 days under stirring with the water being changed every few hours.
  • the in vitro TLR2 (Toll-like receptor-2) assay provides an indication of the level of specificity of the analyte towards activation of the TLR2 pathway, and thus, the greater the fold difference (constant concentration), the more specific the analyte is towards TLR2 activation (the greater the purity).
  • the fold difference of the PSA isolated via the isocratic TBS-NaCl system was only about 3 which is relatively low and indeed only a hint of blue color was observed for cells treated with the PSA in the presence of an anti-TLR2 antibody.
  • the signal should be negligible since the TLR2 receptors are blocked and a signal is only observed if activation is occurring via another pathway independent of the TLR2 pathway (e.g.
  • PSA nanoparticles were synthesized using a water in oil emulsion.
  • the PSA was dissolved in water (1-3 mg/ml) and added via syringe to an oil bath with sonication and/or homogenization to form the water in oil emulsion with the PSA substantially present in the water droplets.
  • a crosslinker that is miscible or soluble in the aqueous droplets (such as glutaraldehyde) was then added to the emulsion via syringe, thereby crosslinking the PSA in the water droplets. The addition of the crosslinker resulted in the nanoparticle formation.
  • the aqueous phase was then allowed to evaporate (e.g., for 24 hours), followed by the addition of a quencher of the crosslinker (e.g., propylamine). Once the quenching reaction was deemed complete (e.g., after 24 hours), the nanoparticles were washed (e.g., with hexane and ether) to remove any residual water or reactants. The nanoparticles were then dialyzed in water to further purify the nanoparticles. For antigen- encapsulated PSA nanoparticles, a similar approach was followed. See FIG. 2. The PSA was dissolved in water and the antigen (e.g., ovalbumin, OVA) was dissolved in water.
  • the antigen e.g., ovalbumin, OVA
  • the two aqueous solutions (or one solution including both the PSA and OVA) were added via syringe to an oil bath with sonication and/or homogenization to form the water in oil emulsion with the PSA and OVA substantially present in the water droplets.
  • the method described above was then performed.
  • the crosslinker is added to the water in oil emulsion, the OVA protein becomes entangled, bound, and/or encapsulated by the PSA in the nanoparticle.
  • 1-3 mg/ml PSA(aq) with or without antigen (3-30x less antigen than PSA) is resuspended in 1ml diH20 and added dropwise in to 30ml paraffin oil with 1% span80 (surfactant) and homogenized. After homogenization, the emulsion is transferred to a beaker and glutaraldehyde (8-1 Ox more glutaraldehyde than PSA) is slowly added dropwise with continuous stirring. The homogenization is stirred for 48 hours to allow the aqueous phase to evaporated and for crosslinking to occur.
  • Residual glutaraldehyde is quenched with the addition of propylamine (8- lOx more propylamine than glutaraldehyde) while stirring for 24 hours.
  • the resulting particles are centrifuged and washed with hexane, diethyl ether, and water (3 times each) and then dialyzed in water 1-5 days.
  • FIG. 3 shows the SEM images of the ovalbumin only nanoparticles (OVA NPs), the OVA-encapsulated PSA nanoparticles (PSA-OVA NPs) and the PSA only nanoparticles (PSA NPs).
  • FIG. 4A shows the particle diameter distribution for the PSA NPs, the PSA-OVA NPs, and the OVA NPs.
  • FIG. 4B shows the zeta potential measurements for the PSA and PSA-OVA nanoparticles. The particles were suspended in water and zeta potential measured.
  • the zeta potential denotes surface charge.
  • a negative surface charge will help to minimize or prevent adhesion to negatively charged mucus and allow the particles to reach intestinal wall.
  • the size of the nanoparticles may be varied by varying the emulsion conditions. For example, as shown in FIGS. 5A and 5B, when the homogenization speed was increased, smaller nanoparticles may be obtained.
  • the ratio of the PSA to antigen may be varied.
  • PSA-OVA NPs having a PSA:OVA weight ratio of 30: 1 were formed, and as shown in the SEM image in FIG. 6B, PSA-OVANPs having a weight ratio of 3: 1 were also formed.
  • the nanoparticles were evaluated before and after simulated gastric fluid treatment and before and after nitric oxide deamination. See FIG. 7 for a general schematic for these protocols. For the simulated gastric fluid treatment experiment, the particles were incubated with pepsin supplemented simulated gastric fluid and incubated at 37 °C for 5 hrs.
  • FIG. 8 shows the SEM images of simulated gastric fluid treated particles (PSA NPs) at different time points during the treatment (0 to 5 hr) and FIG. 9 provides particle size/distribution measurements via DLS for the nanoparticles at the different time periods during the treatment.
  • FIG. 10A shows SEM images comparing PSA NPs and OVA NPs after 1, 2 and 3 hrs of the simulated gastric fluid treatment.
  • FIG. 10B shows the size distribution for the PSA NPs and OVA NPs under the same time periods. It can be seen from these figures that the PSA NPs and the PSA OVA NPs were relatively stable during the SGF treatment while the OVA only NPs were degraded under the same conditions.
  • the PSA While the nanoparticles are desirably stable in gastric fluids, it is desirable for the PSA to break down/ depolymerize in nitric oxide to ensure that the nanoparticle is broken down in the phagosome of dendritic cells so that the antigen can be released and processed for antigen presentation.
  • FIG. HA and 11B provide SEM images of PSA NPs before and after NONOate treatment, and FIG. 12A and 12B provide their size distribution measurements obtained by DLS.
  • FIG. 13A and 13B provide SEM images of PSA-OVA NPs (30:1 mass ratio) particles before and after NONOate treatment and FIG. 14A and 14B provide their size distribution measurements obtained by DLS.
  • FIG. 15A and 15B provide SEM images of PSA-OVA NPs (30: 1 mass ratio) particles before and after NONOate treatment and FIG. 16A and 16B provide their size distribution measurements obtained by DLS. It can be seen from this data that the PSA NPs (with or without OVA) were able to be depolymerized by the NO during the treatment.
  • HEK-BlueTM TLR2 cells Important to the application of the PSA-based nanomaterials is their ability to exhibit the T cell dependent immunomodulatory behavior via activation of TLR2.
  • the immunomodulatory activity of nanoparticles was first evaluated by monitoring their ability to activate the TLR2 in HEK-BlueTM TLR2 cells in vitro. See FIG. 17 for a general procedure.
  • HEK-Blue mTLR 2 reporter cells Invivogen were treated with 25 pg/ml PSA, PSA NPs, or PSA-OVA NPs for 16 hours at 37 °C in the absence or presence of blocking anti mTLR 2 antibody. Absorbance was measured at 625 nm.
  • FIG. 18A and 18B provide graphs showing that all of the NPs formed show specificity towards TLR2 activation.
  • PSA-NPs and OVA-loaded PSA-NPS showed slightly lower specific mTLR2 specific activity than unaltered PSA, but significantly higher activity than both LPS and water controls.
  • the specific mTLR2 activity shows that the nanoparticles retain their immunogenic properties even after the nanoparticle synthetic procedure and the encapsulation of the OVA antigen.
  • FIG. 19 is a schematic of dendritic cell immunomodulation assay.
  • Flt3-L bone marrow derived dentritic cells BMDCs
  • PSA NPs Flt3-L bone marrow derived dentritic cells
  • PSA- OVA NPs Flt3-L bone marrow derived dentritic cells
  • FIG. 20 and FIGS 21A-21D Enumeration of CD 86+/ICOSL+/MHCII+ cells after treatment via flow cytometry is shown in FIG. 20 and FIGS 21A-21D.
  • This data shows that PSA-OVA NPs successfully delivers OVA to dendritic cells and induces downstream tolerogenic phenotypes in dendritic cells.
  • FIG. 22 further describes the results obtained from these experiments.
  • One of the beneficial effects of PSA in biological systems is the induction of host regulatory T cells to produce IL- 10 after CD4 + T cell activation by PSA-exposed dendritic cells.
  • the secretion of IL-10 from CD4 + T cells co-cultured with splenic DCs in vitro was evaluated and quantified via ELISA.
  • FIG. 23 is a schematic of T cell immune modulation assay.
  • CD4+ T cells and Flt3L BMDCs (5: 1) from OT-II mice were incubated with 50 ug/ml PSA, PSA NPs, or PSA-OVA NPs for 4 days before performing flow cytometry analysis and the IL- 10 ELISA assay.
  • FIGS 24A-24D and FIG 25 show that the PSA-OVA NPs induce downstream tolerogenic phenotypes in DCs and OVA- specific T-cells and induce the production of IL-10.
  • TCRs T cell receptors
  • DCs take up OVA-loaded PSA particles, break down the particles, and present OVA- peptides on their MHCII complexes.
  • the OT-II T cells then contact the OVA:MHCII complexes on DCs using their TCRs and become activated.
  • the T cells receive immunomodulatory cues from the PSA-exposed DCs to induce a unique differentiation characterized by the upregulation of CD39 and ICOS, and IL-10 production.
  • Lag3 is also upregulated, its upregulation seems to be most dependent on presence of OVA. It may also be noted the lack of activation in treatment groups where OVA is omitted (i.e., PSA, PSA nps, untreated).

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Abstract

L'invention concerne des nanoparticules permettant d'induire une tolérance immunitaire spécifique d'un antigène qui comprennent un polysaccharide réticulé A (PSA) encapsulant un antigène. L'invention concerne également des méthodes de préparation et d'utilisation desdites nanoparticules.
PCT/US2023/061496 2022-01-29 2023-01-27 Systèmes particulaires à base de polysaccharide a pour atténuation d'auto-immunité, d'allergie et de rejet de greffe WO2023147504A1 (fr)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080095810A1 (en) * 2005-03-14 2008-04-24 Advanced In Vitro Cell Technologies, S.L. Nanoparticles Of Chitosan And Polyethyleneglycol As A System For The Administration Of Biologically-Active Molecules
US20140314865A1 (en) * 2007-10-12 2014-10-23 Massachusetts Institute Of Technology Vaccine Nanotechnology
US20180117171A1 (en) * 2015-04-01 2018-05-03 President And Fellows Of Harvard College Immunoconjugates for programming or reprogramming of cells
US20200085756A1 (en) * 2018-09-14 2020-03-19 Massachusetts Institute Of Technology Nanoparticle vaccine adjuvant and methods of use thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20080095810A1 (en) * 2005-03-14 2008-04-24 Advanced In Vitro Cell Technologies, S.L. Nanoparticles Of Chitosan And Polyethyleneglycol As A System For The Administration Of Biologically-Active Molecules
US20140314865A1 (en) * 2007-10-12 2014-10-23 Massachusetts Institute Of Technology Vaccine Nanotechnology
US20180117171A1 (en) * 2015-04-01 2018-05-03 President And Fellows Of Harvard College Immunoconjugates for programming or reprogramming of cells
US20200085756A1 (en) * 2018-09-14 2020-03-19 Massachusetts Institute Of Technology Nanoparticle vaccine adjuvant and methods of use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WANG QUN, MCLOUGHLIN RACHEL M., COBB BRIAN A., CHARREL-DENNIS MARIE, ZALESKI KATHLEEN J., GOLENBOCK DOUGLAS, TZIANABOS ARTHUR O., : "A bacterial carbohydrate links innate and adaptive responses through Toll-like receptor 2", JOURNAL OF EXPERIMENTAL MEDICINE, ROCKEFELLER UNIVERSITY PRESS, US, vol. 203, no. 13, 25 December 2006 (2006-12-25), US , pages 2853 - 2863, XP093083498, ISSN: 0022-1007, DOI: 10.1084/jem.20062008 *

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