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WO2025027013A1 - Composition et procédés de traitement du lupus érythémateux du système - Google Patents

Composition et procédés de traitement du lupus érythémateux du système Download PDF

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Publication number
WO2025027013A1
WO2025027013A1 PCT/EP2024/071530 EP2024071530W WO2025027013A1 WO 2025027013 A1 WO2025027013 A1 WO 2025027013A1 EP 2024071530 W EP2024071530 W EP 2024071530W WO 2025027013 A1 WO2025027013 A1 WO 2025027013A1
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Prior art keywords
plant
composition
based nanoparticle
virus
plant virus
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Valentina GARONZI
Linda Avesani
Roberta ZAMPIERI
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Diamante Srl
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Diamante Srl
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • 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
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/517—Plant cells
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5256—Virus expressing foreign proteins
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/525—Virus
    • A61K2039/5258—Virus-like particles
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/18011—Comoviridae
    • C12N2770/18041—Use of virus, viral particle or viral elements as a vector
    • C12N2770/18043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
    • C12N2770/00011—Details
    • C12N2770/38011—Tombusviridae
    • C12N2770/38041—Use of virus, viral particle or viral elements as a vector
    • C12N2770/38043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention is directed to a composition and methods of an engineered virus-based 10 nanoparticle for the treatment of system lupus erythematosus (SLE).
  • SLE system lupus erythematosus
  • This specification includes a sequence listing submitted herewith, which includes the file entitled 1266-003PCT1.xml having the following size: 1,716 bytes which was created July 19, 2024, the contents of which are incorporated by reference herein.
  • 15 BACKGROUND System lupus erythematosus (SLE) is an autoimmune disease wherein the body’s immune system erroneously attacks its own healthy tissues, leading to systemic inflammation and damage. Current therapeutic strategies often fail to fully control SLE or may induce significant side effects.
  • compositions for treating system lupus erythematosus are described.
  • Composition includes a plant virus-based nanoparticle engineered to express at least a tolerogenic peptide associated with SLE and a buffer.
  • a method of manufacturing composition for treating SLE is described.
  • Method includes infecting a plant with a virus to produce a plant virus-based nanoparticle, sampling symptomatic leaves from the plant, homogenizing the plant virus-based nanoparticle, incubating the plant virus-based nanoparticle, centrifuging the plant virus-based nanoparticle, and filtrating the plant virus-based nanoparticle.
  • another method of manufacturing composition for treating SLE includes infecting a plant with a virus to produce a plant virus-based 1 Attorney Docket No.1266-003PCT1 nanoparticle, sampling symptomatic leaves from the plant, homogenizing the plant virus-based nanoparticle, incubating the plant virus-based nanoparticle, and filtrating the plant virus-based nanoparticle.
  • Method includes agroinfiltrating a plant with a virus to produce a plant virus-based nanoparticle, sampling symptomatic leaves of the plant, homogenizing the plant virus-based nanoparticle, centrifuging the plant virus-based nanoparticle, and filtrating the plant virus-based nanoparticle.
  • FIG.1 is a schematic illustration of an exemplary embodiment of a composition for treating system lupus erythematosus (SLE).
  • FIGS.2A-B are schematic illustrations of exemplary embodiments of a plant virus-based 20 nanoparticle;
  • FIG.3A is a schematic illustration of a Tomato bushy stunt virus (TBSV);
  • FIG.3B is a schematic illustration of a Cowpea mosaic virus (CPMV);
  • FIG.4 is a flow diagram illustrating an exemplary embodiment of a method of manufacturing the composition for treating SLE;
  • FIG.5 is a flow diagram illustrating an exemplary embodiment of another method of manufacturing the composition for treating SLE;
  • FIG.6 is an exemplary embodiment of a method of using plant virus-based nanoparticle for treating SLE;
  • FIG.7 is an exemplary embodiment of an extraction process of a plant virus-based nanoparticle.
  • compositions and methods for treating system lupus erythematosus may include a plant virus-based nanoparticle engineered to express at least a tolerogenic peptide associated with SLE and a buffer.
  • method may include identifying tolerogenic peptide associated with SLE, modifying plant-virus based 10 nanoparticle to express the identified tolerogenic peptide, and synthesizing the plant virus-based nanoparticle containing the identified tolerogenic peptide.
  • identifying tolerogenic peptide associated with SLE modifying plant-virus based 10 nanoparticle to express the identified tolerogenic peptide, and synthesizing the plant virus-based nanoparticle containing the identified tolerogenic peptide.
  • aspects of the present disclosure can be used to prevent and treat autoimmune diseases such as SLE using plant virus-based nanoparticles. Referring now to FIG.1, an exemplary embodiment of a composition 100 for treating 15 SLE is illustrated.
  • system lupus erythematosus is a complex, multisystemic autoimmune disease that is often characterized by a production of autoantibodies that target a body’s own tissues, dysregulation of cytokines, apoptosis, and B- and T-cell functions, and/or systemic clinical manifestations that include inflammation and tissue damage.
  • This complex disorder involves intricate interactions between 20 genetic, hormonal, immunological, and environmental factors.
  • Clinical manifestations of SLE may range from milder forms such as skin rash and arthritis, to severe life-threatening systemic diseases involving the kidneys, heart, lungs, and nervous system.
  • composition 100 lies in harnessing the immunomodulatory effect of a virus-based nanoparticle, which carries a tolerogenic peptide designed to recalibrate aberrant immune responses observed in SLE, as described below.
  • an “immunomodulatory effect” is an effect of modulating the immune system.
  • a plant virus-based nanoparticle 30 may modulate an immune system to induce tolerance and therefore prevent or treat a case of autoimmune disease, such as without limitation SLE.
  • composition 100 comprises a plant virus-based nanoparticle 104.
  • a “nanoparticle” is a particle with a size that ranges between 1 and 500 nanometers.
  • a “plant virus- based nanoparticle” is a virus nanoparticle that is capable of infecting and replicating in plants. 5
  • plant virus-based nanoparticle 104 may include at least a unit or subunit of a plant viral protein.
  • a “plant virus protein” is a protein that constitutes or is produced by a plant virus.
  • Nanoparticles created from subunits of plant virus proteins may be genetically engineered to 10 express, on their external surface, an antigen-specific peptide related to an autoimmune disease and then be grown in a plant host, as described in detail below in this disclosure.
  • a “virus nanoparticle” is a proteinaceous and often infectious nanoscale structure that is capable of delivering its nucleic acid efficiently into a host cell, enabling production of new viruses therein.
  • Virus” and “virus nanoparticle” may be used 15 interchangeably throughout this disclosure.
  • plant virus and “plant virus-based nanoparticle” may be used interchangeably as well.
  • Virus includes a capsid.
  • a “capsid” is a protein shell exposed at the exterior of a virus that possesses a specific geometric pattern.
  • capsid may possess an icosahedral shape.
  • an “icosahedron” is a geometric shape with 20 sides, each 20 composed of an equilateral triangle.
  • capsid may include a filamentous structure.
  • a “filamentous structure” is an elongated, thread-like formation that makes up a capsid of certain viruses.
  • capsid may include a rod-shaped structure.
  • capsid may include a helical structure.
  • a “helical 25 structure” is a type of structure characterized by a cylindrical, elongated shape with a helical symmetry. This structure may be formed by the regular, repeating arrangement of protein subunits around a viral nucleic acid, providing protection, structural integrity, and aiding in the infectivity of a virus.
  • capsid may include a spherical structure. Additionally, and/or alternatively, capsid may adopt any geometry not disclosed herein yet 30 deemed possible by a person of ordinary skill in the art upon reviewing the entirety of this disclosure.
  • plant virus protein may be produced through a process of molecular farming.
  • molecular farming is a process of producing pharmaceutically important and commercially valuable proteins in plants. Once extracted, plant virus proteins may be used to create plant 5 virus-based nanoparticle 104. Plant virus-based nanoparticle 104 and/or plant virus protein may be sourced from a variety of plant hosts.
  • Suitable host plants for such purpose may include a Nicotiana benthamiana plant, a Nicotiana tabacum plant, a Solanum lycopersicum or Lycopersicon esculentum plant, a Cycorium intybus plant, a Brassica oleracea var. capitata plant, a Beta vulgaris var cicla plant, a Ocimum basilicum plant, a red beet plant, a spinach plant, 10 or the like.
  • a “Nicotiana benthamiana plant” is a close relative of tobacco and a species of Nicotiana indigenous to Australia. It may be used for farming monoclonal antibodies and other recombinant proteins.
  • a “Nicotiana tabacum plant”, commonly known as cultivated tobacco, is a plant species that belongs to the Nicotiana genus in the Solanaceae family. It is a widely grown herbaceous plant 15 primarily used to produce tobacco products. This species is characterized by large, broad leaves and is cultivated in various climates worldwide.
  • a Nicotiana tabacum plant may serve as a model organism and a host for genetic engineering, enabling the expression and study of recombinant proteins, vaccines, and other biologically significant compounds.
  • a “Solanum lycopersicum plant” or “Lycopersicon esculentum plant”, 20 commonly known as the tomato plant is a plant species that belongs to the Solanaceae family and characterized by its production of edible, fleshy fruits. Solanum lycopersicum is noted for its agricultural significance and its utility in genetic engineering and plant breeding. This species may serve as a model organism for studying plant genetics, disease resistance, and metabolic pathways. It may also be utilized in biotechnological applications for the production of 25 recombinant proteins, novel traits, and improved cultivars through genetic modification techniques.
  • a “Cycorium intybus plant” is a hardy plant widely used in folklore medicine to treat various ailments ranging from wounds to diabetes. It is believed to have antimicrobial, anthelmintic, antimalarial, hepatoprotective, antidiabetic, gastroprotective, anti-inflammatory, analgesic, antioxidant, tumor-inhibitory, and antiallergic 30 activities across several different cultures.
  • capitata plant is a is a biennial plant grown as an annual for its dense-leaved 5 Attorney Docket No.1266-003PCT1 heads and characterized by a short stem and a rosette of green, purple, or white leaves that form a tight, globular, and compact head. It is known as wild cabbage in its uncultivated form.
  • Some cultivated forms of Brassica oleracea var. capitata plant include cabbage, broccoli, cauliflower, kale, brussels sprouts, collard greens, savoy cabbage, kohlrabi and gai lan. It is native to costal 5 southern and western Europe.
  • a “Beta vulgaris var cicla plant” is a plant that is more commonly known as chard or spinach beet. It originates from the Mediterranean and has some medicinal properties, mainly in boosting the immune system and lowering blood pressure.
  • an “Ocimum basilicum plant” is a member of the Lamiaceae (mint) family and is more commonly referred to as basil.
  • a “red beet plant” is a biennial plant grown as an annual for its edible root and leafy greens and characterized by its swollen root, which is typically deep red or purple in color. It is a promising candidate for some medicinal uses.
  • the phytochemicals present in red beet may provide protection against diseases including cancer and cardiovascular diseases.
  • a “spinach plant” is a leafy green 15 that belongs to the amaranth family and is closely related to beets and quinoa.
  • plant virus-based nanoparticle 104 may contain one or more viruses, such as without limitation Tomato bush stunt virus (TBSV), Cowpea mosaic virus (CPMV), and/or the like.
  • TBSV Tumor bush stunt virus
  • TBSV is a plant virus from the Tombusvirus group that 20 primarily infects vegetable crops and causes stunting of growth, leaf mottling, and deformed or absent fruit in an infected plant.
  • TBSV is a prototypic member of the Tombusviridae family.
  • TBSV may provide a scaffold for plant virus-based nanoparticle 104 to enable a stable C- terminal display of peptides.
  • CP may include an RNA binding domain (R), a shell domain (S) forming a capsid backbone, and a C-terminal protruding (P) domain that can accommodate exogenous peptides for display.
  • the structure of TBSV may include a single-stranded RNA with a linear genome of about 4,800 nucleotides.
  • TBSV may also possess three symmetrically distinct coat protein monomers.
  • TBSV may replicate, without limitation, using cytoplasmic replication. This 30 virus may penetrate a host cell by uncoating and releasing viral RNA into cytoplasm.
  • TBSV may also spread indirectly, such as without limitation through water, soil, and/or infected seeds.
  • a “Cowpea 5 mosaic virus (CPMV)” or “Sunn-hemp mosaic virus” is a non-enveloped plant virus of the Comovirus group. Infection of a susceptible cowpea leaf may cause a "mosaic" pattern in the leaf and result in high virus yields.
  • CPMV genome may include 2 strands of RNA which are separately encapsulated.
  • CPMV may be approximately 30 nanometers in diameter and contain 60 copies each of a Large (L) and Small (S) coat proteins, as explained below in this disclosure. 10
  • the structure of CPMV is well-characterized to an atomic resolution.
  • CPMV nanoparticles may be thermostable and readily isolated from plants. There are many stable mutants of CPMV already prepared that allow specific modification of their capsid surfaces. In some cases, CPMV may include an icosahedral capsid, as described above.
  • plant virus-based nanoparticle 104 is configured to 15 express at least a tolerogenic peptide 108.
  • a “tolerogenic peptide” is a short chain of amino acids designed to induce immune tolerance, particularly in the context of autoimmune diseases, allergies, and transplant rejection. By promoting tolerance, a tolerogenic peptide may prevent an immune system from attacking a body's own tissues or introduced antigens without broadly suppressing immune function.
  • 20 tolerogenic peptide 108 may include hCDR1. For the purposes of this disclosure.
  • hCDR1 is a tolerogenic peptide with a sequence of HGYYWSWIRQPPGKGEEWI (SEQ ID NO: 1) that may significantly down-regulate the mRNA expression of pathogenic cytokines and pro- apoptotic molecules, while up-regulating the expression of TGF-beta and FoxP3. This may result in a significant decrease in SLEDAI-2K and BILAG scores, suggesting that hCDR1 may be a 25 disease-specific treatment for SLE patients. hCDR1 has been used on mice and has shown potential beneficial effect in human lupus.
  • a treatment with hCDR1 may result in significant reduction in the gene expression of inflammatory cytokines IL-1b, TNF- a, IFN-g, and IL-10, and B Lymphocyte Stimulator (BLyS), a protein believed to play a role in the development of SLE. This reduction was observed in all patients treated with hCDR1.
  • 30 hCDR1 is also known as edratide and may be designed based on complementarity determining regions 1 (CDR1) of a human ant-DNA autoantibody. 7 Attorney Docket No.1266-003PCT1 With continued reference to FIG.1, in one or more embodiments, tolerogenic peptide 108 may include an immunodominant peptide.
  • an “immunodominant peptide” is a peptide that is a representative epitope of a given protein antigen to an immune system in the context of a specific autoimmune disease.
  • immunodominant peptide may include a cytoplasmically located protein.
  • a “cytoplasmical location” is a location inside the cytoplasm of a cell.
  • a coat protein of plant virus-based nanoparticle 104 may be engineered to display immunodominant peptides.
  • a “coat protein” is a protein that is a constituent of the capsid of a virus, as described above.
  • At least an immunodominant peptide may be fused to a protein of CPMV and/or TBSV.
  • the presence of immunodominant peptide may reduce the yield of plant virus-based nanoparticles 104 in plants.
  • the presence of hCDR1 may reduce the yield of CPMV and/or TBSV nanoparticles.
  • the presence of immunodominant peptide may improve the yield of plant virus- 15 based nanoparticles 104 in plants.
  • the presence of hCDR1 may improve the yield of CPMV and/or TBSV.
  • composition 100 further comprises a buffer 112.
  • Buffer may stabilize plant virus-based nanoparticle 104 and maintain a pH for increased stability and functionality.
  • a “buffer” is a solution or mixture that 20 contains at least a pair of weak acid, HA, and its conjugate base, A-, (i.e., the weak acid minus one proton) in a molar ratio between 10:1 and 1:10, wherein the solution maintains a stable pH close to the pK a (i.e., the negative log of the acid dissociation constant, K a ) of the weak acid, against addition of acidic or basic chemical species.
  • A- the conjugate base
  • K a the negative log of the acid dissociation constant
  • buffer may include any type of buffer deemed suitable by a person of ordinary skill in the art upon reviewing the entirety of this disclosure.
  • buffer may include an acetate buffer (i.e., 30 CH3COONa/CH3COOH).
  • buffer may include a borate buffer 8 Attorney Docket No.1266-003PCT1 (i.e., Na2B4O7 ⁇ 10H2O/H3BO3).
  • buffer may include a bicarbonate buffer (i.e., NaHCO 3 /H 2 CO 3 or Na 2 CO 3 /NaHCO 3 , depending on the desired pH).
  • composition 100 may include a cacodylate buffer (i.e., NaC2H6AsO2/HC2H6AsO2).
  • buffer may include a Good’s buffer.
  • “Good’s buffers” are a group of more than 20 conjugate acid/base pairs selected and described by Norman Good and colleagues for biochemical and biological research during 1966–1980. For simplicity, only the conjugate acid may be shown for each conjugate acid/base pair.
  • Good’s buffers include MES (C6H13NO4S), ACES (C4H9NO4S), PIPES (C8H18N2O6S2), MOPS (C7H15NO4S), TES (C6H15NO6S), HEPES (C8H18N2O4S), Tricine (C6H13NO5), TRIS (C4H11NO3), Bicine (C6H13NO4), TAPS (C7H17NO6S), CHES (C8H17NO3S), CAPS (C9H19NO3S), AMPSO (C9H19NO4S), Gly-Gly (C4H8N2O3), ADA (C4H7NO4), BES (C6H15NO5S), MOPSO (C7H15NO5S), EPPS (C9H20N2O4S), HEPPS (C11H24N2O4S), CAPSO (C9H19NO4S), HEPPSO (C9H20N2O
  • buffer may include a phosphate buffer (i.e., NaH2PO4/H3PO4, Na2HPO4/NaH2PO4, or Na3HPO4/ Na2HPO4, depending on the desired pH).
  • buffer may include a phosphate- buffered saline (PBS) solution, a commonly used buffer in biological research and pharmaceutical formulations that typically contains 137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, and 1.8 mM KH2PO4.
  • PBS phosphate- buffered saline
  • composition 100 may include sodium acetate.
  • sodium acetate is organic sodium salt with a chemical formula of CH 3 COONa. It may also be known as acetic acid, sodium salt, sodium acetate anhydrous, or acetic acid sodium salt. Sodium acetate is the anhydrous, sodium salt form of acetic acid. Sodium acetate may be a hygroscopic powder that absorbs moisture from air.
  • Sodium acetate is a strong electrolyte that fully dissolves and disassociates in water to 9 Attorney Docket No.1266-003PCT1 form sodium ions (Na + ) and acetate ions (CH3COO-). Na + is the principal cation of extracellular fluid and plays a large part in fluid and electrolyte replacement therapies.
  • Sodium acetate may be used as an electrolyte replenisher in isosmotic solution for parenteral replacement of acute losses of extracellular fluid without disturbing normal electrolyte balance.
  • a solution of sodium acetate 5 and acetic acid may act as a buffer to maintain a relatively constant pH. This may be useful especially in biochemical applications where reactions are pH-dependent in a mildly acidic range.
  • composition 100 may be taken in any suitable form of dosage and/or delivery, including without limitation oral dosage and intravenous dosage.
  • oral dosage is an ingestion of a composition through the mouth.
  • Oral dosage of composition 100 may include use of pills, syrup, tablets, thin film, liquid solution, powder, solid crystals, natural or herbal plants, seeds, or food, pastes, or the like.
  • intravenous dosage is an administration of a composition using injection. Intravenous dosage may include without limitation intravenous injection.
  • composition 100 may be given intravenously in any suitable manner, including as a bolus and/or as an infusion. Alternatively, other injection methods such as intramuscular injection, intraperitoneal injection, subcutaneous injection, and/or transcutaneous injection may be used. With continued reference to FIG.1, in one or more embodiments, composition 100 may include one or more anti-lupus ingredients.
  • an “anti-lupus20 ingredient” is a medically active chemical that may be used to treat and/or prevent SLE.
  • Anti- lupus ingredient may include a non-steroidal anti-inflammatory drug (NSAID), a corticosteroid, methotrexate, hydroxychloroquine, sulfasalazine, leflunomide, a tumor necrosis factor inhibitor, a T-cell costimulatory blocking agent, a B-cell depleting agent, an Interleukin-1 receptor antagonist therapy, and/or any other immunomodulatory and/or cytotoxic agent.
  • NSAID non-steroidal anti-inflammatory drug
  • corticosteroid methotrexate
  • hydroxychloroquine hydroxychloroquine
  • sulfasalazine hydroxychloroquine
  • sulfasalazine hydroxychloroquine
  • leflunomide hydroxychloroquine
  • sulfasalazine hydroxychloroquine
  • sulfasalazine hydroxychloroquine
  • plant virus-based nanoparticle 104 may include 60 copies of large coat protein subunit 204.
  • plant virus- 30 based nanoparticle 104 may include a small coat protein subunit 208.
  • plant virus-based nanoparticle 104 may include 60 copies of small coat protein subunit 208.
  • plant virus-based nanoparticle 104 may include at least a tolerogenic peptide 108.
  • at least a tolerogenic peptide 108 may include immunodominant peptide 212.
  • immunodominant peptide 212 may be embedded within coat protein.
  • immunodominant peptide 212 5 may be located outside of coat protein. In one or more embodiments, immunodominant peptide 212 may be exposed on the surface of large coat protein subunit 204. In one or more embodiments, immunodominant peptide 212 may be located inside of a nanoparticle. In one or more embodiments, immunodominant peptide 212 may be placed inside of coat protein.
  • plant virus-based nanoparticle 104 may include an icosahedral structure. 10 In one or more embodiments, plant virus-based nanoparticle 104 may include a filamentous structure. In one or more embodiments, plant virus-based nanoparticle 104 may include a rod- shaped structure.
  • plant virus-based nanoparticle 104 may include a helical structure. In one or more embodiments, plant virus-based nanoparticle 104 may include a spherical structure. In one or more embodiments, plant virus-based nanoparticle 104 may be 15 homogeneous or uniform in size. In some embodiments, plant virus-based nanoparticle 104 may be homogeneous or uniform in shape. Additionally, and/or alternatively, plant virus-based nanoparticle 104 may adopt any geometry not disclosed herein yet deemed possible by a person of ordinary skill in the art upon reviewing the entirety of this disclosure. Referring now to FIG.2B, an exemplary embodiment 200b of plant virus-based 20 nanoparticle 104 is illustrated.
  • Plant virus-based nanoparticle 104 disclosed herein may be consistent with details described with respect to FIG.2A.
  • Embodiment 200b may be a structural representation of TBSV.
  • plant virus-based nanoparticle 104 may include TBSV, as described above.
  • plant virus-based nanoparticle 104 may include a single protein subunit 216.
  • plant virus- 25 based nanoparticle 104 may include 180 copies of a single protein subunit 216.
  • plant virus-based nanoparticle 104 may include at least a tolerogenic peptide 108.
  • At least a tolerogenic peptide 108 may include immunodominant peptide 212.
  • immunodominant peptide 212 may be exposed on the surface of a single protein subunit 216.
  • FIGS.3A-B exemplary structures of TBSV (300a) and CPMV (300b) are illustrated in FIG.3A and 3B, respectively.
  • 11 Attorney Docket No.1266-003PCT1 Referring now to FIG.4, a flow diagram illustrating an exemplary embodiment of method 400 for manufacturing plant virus-based nanoparticle 104 is illustrated.
  • Plant virus-based nanoparticle 104 may include any type of plant virus-based nanoparticle described in this disclosure without limitation.
  • method 400 includes infecting a plant with a virus to 5 produce plant virus-based nanoparticle 104.
  • infection is a process of delivering viral genes into a host, wherein the viral genes are capable of replication to produce new copies of the corresponding virus. Infection allows a plant to produce plant virus- based nanoparticle 104. In one or more embodiments, infection may be done by spontaneous infiltration.
  • spontaneous infiltration is a type of infiltration 10 that is associated with a negative Gibbs free energy change and occurs naturally without energy input or intervention.
  • infiltration is a process through which one or more substances penetrate or permeate from the surface of a plant into its tissues.
  • spontaneous infiltration may include spraying infectious viral genes onto a plant, spraying viral solution obtained from previously infected leaves, immersion of 15 plants into a solution obtained from previously infected leaves and the like.
  • infection may be done by infiltration of viral genes into a plant.
  • infection may be done by infiltration of viral solution obtained from previously infected leaves.
  • infection may be done by forced infiltration.
  • forced infiltration is a type of infiltration that 20 requires a force, a pressure, an energy input, or a similar form of intervention to be applied.
  • forced infiltration may include syringe infiltration, vacuum infiltration, and the like.
  • vacuum infiltration may include vacuum infiltration using a vacuum pump, vacuum infiltration using a syringe, and the like.
  • “sampling” is an action of taking samples from the leaves of plant to inspect whether they have produced plant virus- 30 based nanoparticles 104. This may happen after a certain time period subsequent to a successful infection step, as described above. Samples may be taken from leaves that are symptomatic.
  • “symptomatic” is a descriptor that describes one or more characteristics of an object that appear different due to viral infection. As a nonlimiting example, it may take three to six days for a CPMV-based nanoparticle and/or for a TBSV-based nanoparticle to form and accumulate in a plant.
  • method 400 includes homogenizing the 10 plant virus-based nanoparticle 104.
  • homogenizing is a process of blending elements into a uniform mixture with a consistent or substantially consistent composition across its entirety.
  • a homogenization process may include combining a tissue of plant containing plant virus-based nanoparticles 104 with an extraction buffer solution.
  • an “extraction buffer solution” is 15 an aqueous solution capable of breaking open cells and releasing elements therein.
  • extraction buffer solution may include salts to regulate its acidity.
  • a sodium acetate solution may be used as extraction buffer solution.
  • method 400 includes incubating plant virus-based nanoparticle 104. A homogenous mixture of plant leaves and extraction buffer is 20 incubated in ice for a period of time.
  • “incubating” is a process of subjecting an item to a hot or cold temperature for a certain period of time, until a certain goal is accomplished.
  • method 400 may include centrifuging plant virus-based nanoparticle 104.
  • 25 “centrifuging” is a process of separating multiple components in a mixture based on their difference in density by applying a centrifugal force to the mixture using a centrifuge device.
  • a “centrifuge device” is a device comprising a rotating element attached to a stationary axis and configured to a spin sample under a high rotational speed in order to achieve separation between various elements therein.
  • the amount of time to apply for 30 this centrifuging step may vary from one type of plant virus-based nanoparticle 104 to another. 13 Attorney Docket No.1266-003PCT1 In some cases, this step may be repeated multiple times to achieve an improved yield. As a nonlimiting example, for TBSV, centrifuging step may be repeated two or three times.
  • method 400 includes filtrating plant virus-based nanoparticle 104.
  • filtrating plant virus-based nanoparticle 104 may 5 include filtrating the plant virus-based nanoparticle 104 using tangential flow filtration (TFF), nanofiltration (NF), and/or gel-filtration chromatography, among other similar separation/purification techniques.
  • tangential flow filtration also known as cross-flow filtration
  • a feed solution may flow tangentially across the 10 surface of a filter membrane, while an applied pressure forces some of the fluid through the membrane as filtrate (permeate).
  • a tangential motion may help reduce membrane fouling and allow continuous filtration.
  • TFF is commonly employed in bioprocessing for concentrating proteins, clarifying cell lysates, and purifying biopharmaceuticals, providing an efficient method for separating components based on size and molecular weight.
  • NF nanofiltration
  • NF may utilize a semi- permeable membrane to selectively allow certain molecules, such as monovalent ions and small organic molecules, to pass through while rejecting larger molecules, multivalent ions, and contaminants.
  • NF may operate under moderate pressure and is commonly employed in water 20 treatment, pharmaceutical purification, and food processing, providing a means for removing impurities, softening water, and concentrating valuable substances with high efficiency.
  • gel-filtration chromatography is a type of separation and purification technique based on a differing ability of chemical species to retain in pores of a gel- filtration medium.
  • Gel-filtration chromatography is also known as size-exclusion 25 chromatography.
  • a column used for gel-filtration chromatography may be packed with fine, porous beads composed of dextran polymers, agarose, polyacrylamide, and/or the like. The pore sizes of these beads are used to estimate the dimensions of macromolecules and separate them accordingly.
  • chemical species of smaller sizes tend to retain in gel-filtration medium for a longer period of time (i.e., separates from a column later)
  • chemical species of 30 larger sizes tend to retain in gel-filtration medium for a shorter period of time (i.e., separates from a column earlier).
  • FIG.5 a flow diagram illustrating another exemplary embodiment of 5 method 400 for manufacturing plant virus-based nanoparticle 104 is illustrated.
  • Plant virus-based nanoparticle 104 may include any type of plant virus-based nanoparticle described in this disclosure without limitation.
  • method 500 includes agroinfiltrating a plant with a virus to produce plant virus-based nanoparticle 104.
  • agroinfiltration is a method used in plant biology to induce transient expression of genes in a 10 plant in order to produce a desired protein.
  • agroinfiltration may be used for CPMV and/or for TBSV.
  • Agrobacterium tumefaciens may be directly injected into a plant leaf or brought into association with plant cells immobilized on a porous support. Subsequently, bacteria may transfer a desired gene into plant cells via transfer of T-DNA.
  • Agroinfiltration may be beneficial when compared 15 to more traditional plant transformations due to its speed and convenience, although yields of the recombinant protein may generally also be higher and more consistent.
  • method 500 includes sampling leaves of the plant. This step may be performed utilizing any process of sampling consistent with details described above.
  • method 500 includes homogenizing the plant virus-based nanoparticle. This step may be performed utilizing any process of 25 homogenization as explained above. As a nonlimiting example, for CPMV and TBSV, a phosphate buffer or a PBS buffer may be used, consistent with details described above.
  • method 500 includes centrifuging the plant virus-based nanoparticle 104. This step may be performed utilizing any process of centrifugation as described above. It is worth noting that production of CPMV-based and TBSV- 30 based nanoparticles may not necessarily involve an incubation step described above for method 400. 15 Attorney Docket No.1266-003PCT1 With continued reference to FIG.5, at step 525, method 500 includes filtrating plant virus-based nanoparticle 104. This step may be performed utilizing any process of filtration as described above. Referring now to FIG.6, an exemplary embodiment of a method 600 of using plant virus- 5 based nanoparticle 104 for treating SLE is illustrated.
  • hCDR1 HGYYWSWIRQPPGKGEEWI, SEQ ID NO: 1
  • an “expression vector” is typically a plasmid or virus designed for gene expression in cells. Expression vector is also known as expression construct.
  • hCDR1 10 genes may be isolated from TBSV, an expression vector that is infective, and incorporated into CPMV, an expression vector that is non-infective. The obtained expression vector may then undergo either in vitro retro-transcription or an Agrobacterium tumefaciens transformation.
  • plant may undergo infection using an infective RNA.
  • plant may undergo agroinfiltration, as previously 15 described.
  • FIG.7 an exemplary embodiment of a production process 700 of a plant virus-based nanoparticle 104 is presented.
  • pVNP plant virus-based nanoparticle 104
  • the plant may start to produce genetically modified plant viruses.
  • plant may function as a bioreactor.
  • 20 samples containing such plant virus-based nanoparticles 104 may be homogenized with extraction buffer solution, as described above.
  • samples may be centrifuged and filtrated following procedures described above to yield plant virus-based nanoparticles 104, which may be used for downstream therapeutic applications, such as treatment of SLE. 25
  • the phrases “at least one of A, B, and C;” “one or more of A, B, and C;” and “A, B, and/or C” are each intended to mean “A alone, B alone, C alone, A and B 15 together, A and C together, B and C together, or A and B and C together.”
  • use of the term “based on,” above and in the claims is intended to mean, “based at least in part on,” such that an unrecited feature or element is also permissible.
  • the subject matter described herein can be embodied in systems, apparatus, methods, and/or articles depending on the desired configuration. The implementations set forth in the 20 foregoing description do not represent all implementations consistent with the subject matter described herein.

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Abstract

L'invention concerne une composition pour le traitement du lupus érythémateux du système (SLE) qui comprend une nanoparticule à base de virus de plante modifiée pour exprimer au moins un peptide tolérogène associé au SLE et un tampon, dont un procédé de fabrication comprend l'infection d'une plante avec un virus pour produire la nanoparticule à base de virus de plante, l'échantillonnage de feuilles symptomatiques à partir de la plante, l'homogénéisation de la nanoparticule à base de virus de plante, l'incubation de la nanoparticule à base de virus de plante, la centrifugation de la nanoparticule à base de virus de plante, et la filtration de la nanoparticule à base de virus de plante.
PCT/EP2024/071530 2023-08-01 2024-07-30 Composition et procédés de traitement du lupus érythémateux du système Pending WO2025027013A1 (fr)

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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20100322928A1 (en) * 2007-01-19 2010-12-23 Yeda Research And Development Co., Ltd. Methods of diagnosing, monitoring treatment and treating systemic lupus erythematosus (sle)
US7858738B2 (en) * 2001-02-26 2010-12-28 Yeda Research And Development Co., Ltd. Synthetic human peptides and pharmaceutical compositions comprising them for the treatment of systemic lupus erythematosus

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7858738B2 (en) * 2001-02-26 2010-12-28 Yeda Research And Development Co., Ltd. Synthetic human peptides and pharmaceutical compositions comprising them for the treatment of systemic lupus erythematosus
US20100322928A1 (en) * 2007-01-19 2010-12-23 Yeda Research And Development Co., Ltd. Methods of diagnosing, monitoring treatment and treating systemic lupus erythematosus (sle)

Non-Patent Citations (1)

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Title
STHOEGER ZEV ET AL: "The Tolerogenic Peptide, hCDR1, Down-Regulates the Expression of Interferon-[alpha] in Murine and Human Systemic Lupus Erythematosus", PLOS ONE, vol. 8, no. 3, 28 March 2013 (2013-03-28), US, pages e60394, XP093222078, ISSN: 1932-6203, DOI: 10.1371/journal.pone.0060394 *

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