WO2017025889A1 - Polymeric nanoparticles with dec-205 ligand and co-encapsulating an antigen subject to an autoimmune response and a glucocorticoid receptor agonist - Google Patents

Polymeric nanoparticles with dec-205 ligand and co-encapsulating an antigen subject to an autoimmune response and a glucocorticoid receptor agonist Download PDF

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Publication number
WO2017025889A1
WO2017025889A1 PCT/IB2016/054769 IB2016054769W WO2017025889A1 WO 2017025889 A1 WO2017025889 A1 WO 2017025889A1 IB 2016054769 W IB2016054769 W IB 2016054769W WO 2017025889 A1 WO2017025889 A1 WO 2017025889A1
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nanoparticle
targeting ligand
nanoparticles
dec
poly
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PCT/IB2016/054769
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French (fr)
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Micah James BENSON
Ramin Darvari
Jason Nash EDMONDS
Michael Wai-Haung Look
Roger Hochoon PAK
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Pfizer Inc.
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Publication of WO2017025889A1 publication Critical patent/WO2017025889A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/36—Blood coagulation or fibrinolysis factors
    • A61K38/37—Factors VIII
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
    • A61K31/57—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone
    • A61K31/573—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids substituted in position 17 beta by a chain of two carbon atoms, e.g. pregnane or progesterone substituted in position 21, e.g. cortisone, dexamethasone, prednisone or aldosterone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6921—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere
    • A61K47/6927—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores
    • A61K47/6929—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle
    • A61K47/6931—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer
    • A61K47/6935—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol
    • A61K47/6937—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a particulate, a powder, an adsorbate, a bead or a sphere the form being a solid microparticle having no hollow or gas-filled cores the form being a nanoparticle, e.g. an immuno-nanoparticle the material constituting the nanoparticle being a polymer the polymer being obtained otherwise than by reactions involving carbon to carbon unsaturated bonds, e.g. polyesters, polyamides or polyglycerol the polymer being PLGA, PLA or polyglycolic acid
    • 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

Definitions

  • the present invention relates to polymeric nanoparticles and their use in suppressing or preventing antigen-specific immune responses, in particular those associated with protein or enzyme replacement therapies and autoimmune diseases.
  • a particularly important 5 application of the nanoparticles is in the treatment of certain types of haemophilia.
  • Haemophilia is the name given to a group of genetic disorders in which the mammalian body's ability to manufacture a blood clot at the site of a damaged blood vessel and prevent bleeding is impaired. The most common of these disorders is haemophilia A in which blood is unable to clot due to a deficiency in the blood-clotting protein factor VIII, most
  • Haemophilia A may also be acquired by the initiation of a neutralising antibody response to endogenously produced factor VIII, often as the result of an underlying autoimmune disease (see Bailliere's Clinical Haematology, 1998, 1 1 , 287-303; and Thrombosis and Haemostasis, 2013, 1 10, 1 1 14-
  • haemophilia A The inability of haemophilia A patients to produce factor VIII at sufficient levels leads to a deficiency in haemostasis with resulting haemorrhagic events that can lead to significant morbidity and mortality in untreated patients.
  • Current standard-of-care treatments for haemophilia A include the administration of exogenous factor VIII two to three times a week in order to maintain haemostasis (Haemophilia: the Official Journal of the World Federation
  • the mammalian immune system has evolved to distinguish between endogenously produced proteins and potentially pathogenic foreign proteins.
  • a major clinical problem encountered in the treatment of haemophilia A patients with factor VIII replacement therapy is the recognition of the exogenous factor VIII by the patient's immune system as foreign and
  • immunosuppressants such as cyclophosphamide together with intravenous immunoglobulin (New England Journal of Medicine, 1988, 318, 947-950) or rituximab (Journal of Thrombosis and Haemostasis, 2009, 7, 787-794), either alone or in combination with corticosteroids, are sometimes used as an adjunctive therapy in refractory patients.
  • immunosuppressants such as cyclophosphamide together with intravenous immunoglobulin (New England Journal of Medicine, 1988, 318, 947-950) or rituximab (Journal of Thrombosis and Haemostasis, 2009, 7, 787-794), either alone or in combination with corticosteroids, are sometimes used as an adjunctive therapy in refractory patients.
  • the use of such broad and non-selective immunosuppression is associated with an increased risk of adverse events including serious infection.
  • the present invention therefore provides a nanoparticle comprising a polymeric matrix and a covalently-bound surface-orientated targeting ligand for DEC-205, said polymeric matrix encapsulating an antigen which is the subject of an undesirable immune response and a glucocorticoid receptor agonist.
  • a protein antigen to DEC-205 expressing cells via polymeric nanoparticles induces an antigen-specific tolerance with a range of antigens.
  • the nanoparticle platform enables simultaneous delivery of the antigen and glucocorticoid receptor agonist to the particular antigen-presenting cells that are capable of triggering the desired tolerogenic response to the antigen, considerably enhancing the potency of both agents and both allowing a selective tolerogenic effect and avoiding any increase in immunogenic response to the antigen.
  • the invention provides as embodiment E1 a nanoparticle comprising a polymeric matrix and a covalently-bound surface-orientated targeting ligand for DEC-205, said polymeric matrix encapsulating an antigen which is the subject of an undesirable immune response and a glucocorticoid receptor agonist.
  • the invention provides as embodiment E2 a nanoparticle as defined in embodiment E1 wherein the nanoparticle is from about 20 to about 500 nm in diameter.
  • the invention provides as embodiment E3 a nanoparticle as defined in embodiment E2 wherein the nanoparticle is from about 60 to about 300 nm in diameter.
  • the invention provides as embodiment E4 a nanoparticle as defined in any one of embodiments E1 to E3 wherein the polymers from which the polymeric matrix are constructed are biocompatible.
  • the invention provides as embodiment E5 a nanoparticle as defined in any one of embodiments E1 to E4 wherein the polymers from which the polymeric matrix are constructed are biodegradable.
  • the invention provides as embodiment E6 a nanoparticle as defined in any one of embodiments E1 to E5 wherein the polymeric matrix comprises PLGA.
  • the invention provides as embodiment E7 a nanoparticle as defined in any one of embodiments E1 to E6 wherein the polymeric matrix comprises PEG.
  • the invention provides as embodiment E8 a nanoparticle as defined in any one of embodiments E1 to E7 wherein the polymeric matrix comprises a PLGA-PEG block copolymer.
  • the invention provides as embodiment E9 a nanoparticle as defined in any one of embodiments E1 to E6 wherein the polymeric matrix consists of PLGA.
  • the invention provides as embodiment E10 a nanoparticle as defined in any one of embodiments E1 to E8 wherein the polymeric matrix consists of a PLGA-PEG block copolymer.
  • the invention provides as embodiment E1 1 a nanoparticle as defined in any one of embodiments E1 to E8 wherein the polymeric matrix consists of a mixture of PLGA and a PLGA-PEG block co-polymer.
  • the invention provides as embodiment E12 a nanoparticle as defined in any one of embodiments E6, E8, E9, E10 or E1 1 wherein the ratio of lactic acid to glycolic acid is about 1 :1 .
  • the invention provides as embodiment E13 a nanoparticle as defined in any one of embodiments E6, E8, E9, E10, E1 1 or E12 wherein the average molecular weight of the PLGA in each chain is from about 15,000 to about 90,000, more specifically from about 15,000 to about 50,000 daltons, more specifically from about 15,000 to about 40,000 daltons.
  • the invention provides as embodiment E14 a nanoparticle as defined in embodiment E13 wherein the average molecular weight of the PLGA in each chain is about 20,000 daltons.
  • the invention provides as embodiment E15 a nanoparticle as defined in any one of embodiments E7, E8, E10 or E1 1 wherein the average molecular weight of the PEG in each chain is from about 2,500 to about 20,000 daltons.
  • the invention provides as embodiment E16 a nanoparticle as defined in embodiment E15 wherein the average molecular weight of the PEG in each chain is about 5,000 daltons.
  • the invention provides as embodiment E17 a nanoparticle as defined in any one of embodiments E6, E8, E9, E10, E1 1 , E12, E13 or E14 wherein the targeting ligand is attached to a PLGA polymer chain, preferably at the terminal position.
  • the invention provides as embodiment E18 a nanoparticle as defined in any one of embodiments E7, E8, E10, E1 1 , E15 or E16 wherein the targeting ligand is attached to a PEG chain.
  • the invention provides as embodiment E19 a nanoparticle as defined in embodiment E18 wherein the targeting ligand is attached at the terminal position of a PEG chain.
  • the invention provides as embodiment E20 a nanoparticle as defined in any one of embodiments E1 to E19 wherein the polymer chains are neutral at pH 7.
  • the invention provides as embodiment E21 a nanoparticle as defined in any one of embodiments E1 to E20 wherein the targeting ligand is located both inside and on the surface of the nanoparticle.
  • the invention provides as embodiment E22 a nanoparticle as defined in embodiment E21 wherein the targeting ligand is located exclusively on the surface of the nanoparticle.
  • the invention provides as embodiment E23 a nanoparticle as defined in any one of embodiments E1 to E22 wherein the targeting ligand binds to DEC-205 with a binding affinity (K d ) of less than about 1000 nM.
  • the invention provides as embodiment E24 a nanoparticle as defined in embodiment E23 wherein the targeting ligand binds to DEC-205 with a binding affinity (K d ) of less than about 100 nM.
  • the invention provides as embodiment E25 a nanoparticle as defined in embodiment E23 wherein the targeting ligand binds to DEC-205 with a binding affinity (K d ) of less than about 10 nM.
  • the invention provides as embodiment E26 a nanoparticle as defined in embodiment E23 wherein the targeting ligand binds to DEC-205 with a binding affinity (K d ) of less than about 1 nM.
  • the invention provides as embodiment E27 a nanoparticle as defined in any one of embodiments E1 to E26 wherein the targeting ligand binds to DEC-205 with high avidity.
  • the invention provides as embodiment E28 a nanoparticle as defined in any one of embodiments E1 to E27 wherein the targeting ligand is an antibody or an antigen-binding portion thereof.
  • the invention provides as embodiment E29 a nanoparticle as defined in embodiment E28 wherein the targeting ligand is a monoclonal antibody or antigen-binding portion thereof.
  • the invention provides as embodiment E30 a nanoparticle as defined in either of embodiments E28 and E29 wherein the targeting ligand is a humanized antibody or antigen- binding portion thereof.
  • the invention provides as embodiment E31 a nanoparticle as defined in either of embodiments E28 and E29 wherein the targeting ligand is a fully human antibody or antigen- binding portion thereof.
  • the invention provides as embodiment E32 a nanoparticle as defined in any one of embodiments E28 to E31 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 4.
  • the invention provides as embodiment E33 a nanoparticle as defined in any one of embodiments E28 to E32 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 5.
  • the invention provides as embodiment E34 a nanoparticle as defined in any one of embodiments E28 to E33 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 6.
  • the invention provides as embodiment E35 a nanoparticle as defined in any one of embodiments E28 to E34 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 10.
  • the invention provides as embodiment E36 a nanoparticle as defined in any one of embodiments E28 to E35 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 1 1 .
  • the invention provides as embodiment E37 a nanoparticle as defined in any one of embodiments E28 to E36 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 12.
  • the invention provides as embodiment E38 a nanoparticle as defined in any one of embodiments E28 to E37 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 26.
  • the invention provides as embodiment E39 a nanoparticle as defined in any one of embodiments E28 to E38 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 27.
  • the invention provides as embodiment E40 a nanoparticle as defined in any one of embodiments E28 to E39 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 28.
  • the invention provides as embodiment E41 a nanoparticle as defined in any one of embodiments E1 to E40 wherein the targeting ligand binds to human DEC-205.
  • the invention provides as embodiment E42 a nanoparticle as defined in any one of embodiments E1 to E41 wherein the targeting ligand density on the nanoparticle is from about 0.05 nmol to about 67 nmol targeting ligand per mg of nanoparticle polymer matrix.
  • the invention provides as embodiment E43 a nanoparticle as defined in embodiment E42 wherein the targeting ligand density on the nanoparticle is from about 0.1 nmol to about 26 nmol targeting ligand per mg of nanoparticle polymer matrix.
  • the invention provides as embodiment E44 a nanoparticle as defined in embodiment E43 wherein the targeting ligand density on the nanoparticle is from about 1 nmol to about 20 nmol targeting ligand per mg of nanoparticle polymer matrix.
  • the invention provides as embodiment E45 a nanoparticle as defined in any one of embodiments E1 to E44 wherein the targeting ligand is covalently bound to the polymeric matrix through a bond formed by the nucleophilic addition of a cysteine thiol group on the targeting ligand to a maleimide group on the polymeric matrix.
  • the invention provides as embodiment E46 a nanoparticle as defined in any one of embodiments E1 to E44 wherein the targeting ligand is covalently bound to the polymeric matrix through bonds formed by the cycloaddition of an alkyne group on the targeting ligand to an azide group on the polymeric matrix.
  • the invention provides as embodiment E47 a nanoparticle as defined in embodiment
  • alkyne is a strained alkyne such as a dibenzocyclooctyne.
  • the invention provides as embodiment E48 a nanoparticle as defined in any one of embodiments E1 to E47 wherein the undesirable immune response is to factor VIII.
  • the invention provides as embodiment E48a a nanoparticle as defined in any one of embodiments E1 to E47 wherein the undesirable immune response is to myelin oligodendrocyte glycoprotein (MOG).
  • MOG myelin oligodendrocyte glycoprotein
  • the invention provides as embodiment E49 a nanoparticle as defined in embodiment E48 wherein the antigen is human factor VIII having the amino acid sequence of SEQ ID NO: 24.
  • the invention provides as embodiment E50 a nanoparticle as defined in embodiment
  • antigen is B-domain deleted-human factor VIII having the amino acid sequence of SEQ ID NO: 25.
  • the invention provides as embodiment E51 a nanoparticle as defined in any one of embodiments E1 to E50 wherein the antigen comprises from about 0.25 to about 5% by weight of the total dry mass of the nanoparticle.
  • the invention provides as embodiment E52 a nanoparticle as defined in any one of embodiments E1 to E51 wherein the glucocorticoid receptor agonist comprises from about 1 to about 20% by weight of the total dry mass of the nanoparticle.
  • the invention provides as embodiment E53 a nanoparticle as defined in any one of embodiments E1 to E52 wherein the glucocorticoid receptor agonist has a logP of about 3 to about 7.
  • the invention provides as embodiment E54 a nanoparticle as defined in embodiment E53 wherein the glucocorticoid receptor agonist has a logP of about 3.5 to about 4.5.
  • the invention provides as embodiment E55 a nanoparticle as defined in embodiment E53 wherein the glucocorticoid receptor agonist is betamethasone-17-valerate.
  • the invention provides as embodiment E56 a nanoparticle as defined in embodiment E53 wherein the glucocorticoid receptor agonist is betamethasone-17,21 -dipropionate.
  • the invention provides as embodiment E57 a process for preparing a nanoparticle according to any one of embodiments E1 to E56, wherein: (i) the antigen is solubilized in an aqueous solution (w ⁇ at a concentration of at least 0.5 mg/mL (preferably at least 5mg/ml_); (ii) the antigen solution from step (i) is homogenised with an organic phase (o) that contains the glucocorticoid and polymer to form an w ⁇ o emulsion; (iii) the w ⁇ o emulsion is added to a second aqueous solution (w 2 ) containing an emulsifier with an HLB value greater than about 10 and homogenised to form a double emulsion; (iv) solvent is removed to form the nanoparticle; and (v) a targeting ligand is conjugated to the surface of the nanoparticle.
  • the invention provides as embodiment E58 a process for preparing a nanoparticle as defined in embodiment E57 wherein step (v) is performed at a pH between about 5 and about 7.
  • the invention provides as embodiment E59 a process for preparing a nanoparticle as defined in embodiment E58 wherein step (v) is performed at a pH between about 5.5 and about 6.5.
  • treatment include references to curative, palliative and prophylactic treatment.
  • treatment encompasses any objective or subjective improvement in a subject with respect to a relevant condition or disease.
  • prophylactic treatment means that the nanoparticle or pharmaceutical composition thereof is administered to a subject to inhibit or stop the relevant condition from occurring in a subject, particularly in a subject or member of a population that is significantly predisposed to the relevant condition.
  • nanoparticle or pharmaceutical composition thereof is administered to a subject to remedy signs and/or symptoms of a condition, without necessarily modifying the progression of, or underlying etiology of, the relevant condition.
  • curative treatment means that the nanoparticle or pharmaceutical composition thereof is administered to a subject for the purpose of bringing the disease or disorder into complete remission, or that the disease or disorder is undetectable after such treatment.
  • nanoparticle Whilst the present invention has been defined in terms of a "nanoparticle", it is understood that the invention will be practiced by making and using a population of such nanoparticles. Any reference to “nanoparticle” is therefore understood to apply equally to a “population of nanoparticles” wherein each member of the population has the characteristics of the single nanoparticle.
  • the nanoparticles within such a population may be heterogeneous in nature, e.g. having a range of sizes or targeting ligand density.
  • the nanoparticle is defined by a numerical parameter given as a range, it is understood that this parameter applies, in the case of a population of nanoparticles, to the distribution within the population as a whole, unless the context requires a different interpretation
  • the nanoparticle comprises a polymeric matrix that is capable of encapsulating the antigen and the glucocorticoid receptor agonist and onto which a targeting ligand for DEC- 205 is covalently attached.
  • the size of the nanoparticle is preferably from about 20 to about 500 nm in diameter, most preferably from about 60 to about 300 nm in diameter. Within a population of the nanoparticles, the mean size of the population is preferably from about 100 to about 150 nm.
  • the polymers used are preferably biocompatible (e.g. poly(ethylene glycol, PEG) and may optionally be biodegradable (e.g. poly(lactic-co-glycolic acid), PLGA).
  • Block co-polymers such as PEG-PLGA may also be used.
  • a sub-population of the polymer chains must be functionalised with a reactive group in order to facilitate attachment of the targeting ligand for DEC-205 to the exterior of the particles.
  • Functionalisation is preferably effected on a more hydrophilic part of the polymer chains in order to encourage the reactive groups to locate on the exterior of the nanoparticles when fabricated.
  • polymeric matrix means the three-dimensional fabric of the nanoparticle which consists of an entanglement of polymer chains from which the nanoparticle is constructed. The chains are not covalently attached to each other but held together by non- covalent interactions such as van der Waals forces, hydrogen bonds and hydrophobic effects.
  • the antigen and glucocorticoid receptor agonist are held within the polymer matrix by non-covalent means.
  • the antigen will initially be held within small droplets of aqueous phase that form in holes or channels within the polymer matrix during fabrication of the nanoparticles and will remain in these holes or channels when solvent is removed.
  • the glucocorticoid receptor agonist being more lipophilic, will be distributed throughout the nanoparticle embedded in spaces between the polymer chains.
  • the antigen and glucocorticoid agonist are free to diffuse out of the polymer matrix when it is suspended in solution and/or as the polymer matrix is degraded in vivo.
  • biocompatible means something that is biologically inert or non-reactive with intracellular and extra cellular biological molecules, and non-toxic.
  • Biocompatible polymers for use in the present invention have preferably been approved by regulatory authorities for use in human beings.
  • the polymer from which the nanoparticle is constructed is a block copolymer of poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) or a mixture of a PLGA-PEG co-polymer and PLGA.
  • PLGA poly(lactic-co-glycolic acid)
  • PEG poly(ethylene glycol)
  • PLGA-PEG co-polymer a block copolymer of poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) or a mixture of a PLGA-PEG co-polymer and PLGA.
  • PLGA poly(lactic-co-glycolic acid)
  • PEG poly(ethylene glycol)
  • the present invention therefore also provides a nanoparticle comprising a polymeric matrix and a covalently-bound surface-orientated targeting ligand for DEC-205, said polymeric matrix encapsulating an antigen which is the subject of an undesirable immune response and a glucocorticoid receptor agonist and said polymeric matrix comprising (and more preferably consisting of) a block co-polymer of PLGA-PEG or a mixture of a PLGA and a block co-polymer of PLGA-PEG.
  • the nanoparticles are fabricated from a PLGA-PEG block copolymer.
  • the ratio of lactic acid to glycolic acid is about 1 :1 and the average molecular weight of the PLGA and PEG in each chain is from about 15,000 to about 50,000 daltons (e.g. about 20,000 daltons) for the PLGA and from about 2,500 to about 20,000 daltons (e.g. about 5000 daltons) for the PEG.
  • the nanoparticles are fabricated from a mixture of 80% PLGA- PEG block co-polymer and 20% PLGA.
  • the ratio of lactic acid to glycolic acid is 1 :1 and the average molecular weight of the PLGA and PEG in each chain is from about 15,000 to about 50,000 daltons (e.g. about 20,000 daltons) for the PLGA and from about 2,500 to about 20,000 daltons (e.g. about 5000 daltons) for the PEG.
  • a proportion of the PEG chains have a moiety, preferably a terminal moiety, that facilitates attachment of the DEC-205 targeting ligand.
  • at least 50% of the PEG chains have a moiety (preferably a terminal moiety) that facilitates attachment of the DEC-205 targeting ligand.
  • about 50% of the PEG chains have a moiety (preferably a terminal moiety) that facilitates attachment of the DEC-205 targeting ligand.
  • the amount of PEG chains with a moiety (preferably a terminal moiety) that facilitates attachment of the DEC-205 targeting ligand is at least about 55% or 60% or 65% or 70% of 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99%.
  • the PLGA and PLGA-PEG polymer chains are neutral, i.e. do not contain any group that is charged at pH 7.
  • such neutral polymer chains terminate in a methoxy group, i.e. a methyl ester in the case of PLGA, a methyl ether in the case of PEG.
  • the DEC-205 targeting ligand is surface-oriented, i.e. attached to the surface of the nanoparticle and is covalently attached to the polymeric matrix from which the nanoparticle is constructed.
  • Surface-oriented in this context means that the binding region of the target ligand is solvent accessible and is not sterically or otherwise hindered by the rest of the nanoparticle from binding to its DEC-205 target
  • the DEC-205 targeting ligand is exclusively attached to the surface of the nanoparticle and does not form part of the interior of the nanoparticle or is only minimally internalised.
  • a proportion of the targeting ligand may be located inside the polymeric matrix of the nanoparticle as long as sufficient targeting ligand is located on the surface to facilitate targeting of the nanoparticles to DEC-205-expressing cells.
  • the percentage of DEC-205 targeting ligand that is surface-orientated is at least about 50% or 55% or 60% or 65% or 70% of 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99%.
  • DEC-205 is a surface protein expressed primarily by cells of the immune system, particularly by dendritic cells, which facilitates internalisation of nanoparticles that bind to it.
  • the full length sequence of human DEC-205 may be found under GenBank accession number NP_002340.
  • the targeting ligand must be specific for the DEC-205 protein expressed by the mammal undergoing treatment. For the treatment of humans, the DEC-205 targeting ligand must target human DEC-205.
  • the targeting ligand binds to DEC-205 with high affinity, for instance with a binding affinity (K d ) of less than about 1000 nM or with a K d of less than about 1 00 nM or with a K d of less than about 10 nM or with a K d of less that about 1 nM.
  • the targeting ligand binds to the DEC- 205 with high avidity as a result of the co-operative binding of targeting ligands on the nanoparticle surface.
  • DEC-205 may be determined using in vitro assays (biochemical or immunological based assays) known in the art for antigen-binding domain, including but not limited to enzyme- linked immu nosorbent assay (ELISA) assay, surface plasmon resonance (SPR) assay, Bio- Layer Interferometry, or immunoprecipitation assays.
  • ELISA enzyme- linked immu nosorbent assay
  • SPR surface plasmon resonance
  • Kd binding affinity
  • Kd binding affinity
  • Kd binding affinity
  • Kd values for antibodies can be determined using methods well established in the art.
  • One method for determining the Kd of an antibody is by using surface plasmon resonance (SPR), typically using a biosensor system such as a BIACORE® system.
  • BIAcore kinetic analysis comprises analyzing the binding and dissociation of an antigen from chips with immobilized molecules (e.g. molecules comprising epitope binding domains), on their surface.
  • Another method for determining the Kd of an antibody is by using Bio-Layer Interferometry, typically using OCTET® technology (Octet QKe system, ForteBio).
  • the targeting ligand may be an antibody that binds to DEC-205 or an antigen binding portion thereof, such as a full-length antibody for DEC-205 or a fragment of a full- length antibody (Fab) in which some of the non-binding domains have been deleted e.g. a fragment of a full-length antibody containing a portion of the hinge region (Fab').
  • Fab full-length antibody
  • Fab' fragment of a full-length antibody containing a portion of the hinge region
  • Other antibody-related entities that may be utilised as the targeting ligand include single-chain antibodies (scFv), diabodies (db), small immunoproteins (SIP), Vhh domains (Vhh) and other similar immunoproteins known to those skilled in the art.
  • An antibody is an immunoglobulin molecule capable of specific binding to a target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen-binding site, located in the variable region of the immunoglobulin molecule.
  • a target or antigen such as a carbohydrate, polynucleotide, lipid, polypeptide, etc.
  • the term is intended to encompass not only intact polyclonal or monoclonal antibodies comprising two identical full- length heavy chain polypeptides and two identical light chain polypeptides, but also fragments thereof (such as Fab, Fab', F(ab') 2 , Fv), single chain (ScFv) and domain antibodies (dAbs), including shark and camelid antibodies, and fusion proteins comprising an antibody portion, multivalent antibodies, multispecific antibodies (e.g.
  • bispecific antibodies so long as they exhibit the desired biological activity
  • antibody fragments as described herein, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site, for example without limitation, minibodies, maxibodies, monobodies, peptibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis- scFv.
  • Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies.
  • Antigen-binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an Ig that is sufficient to confer specific antigen binding to the polypeptide.
  • immunoglobulin is a heteromultimeric molecule.
  • each multimer is composed primarily of identical pairs of polypeptide chains, each pair having one "light” (about 25 kDa) and one "heavy” chain (about 50-70 kDa).
  • each chain includes a variable region, of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition.
  • the carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
  • Variable domains exhibit the same general structure of relatively conserved framework regions (FR) joined by 3 hypervariable regions, also called complementarity determining regions or CDRs.
  • the CDRs from the 2 chains of each pair are aligned by the framework regions, enabling binding to a specific epitope.
  • both light and heavy chains comprise the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4.
  • the targeting ligand is an antibody, it is preferably a human antibody or a humanized antibody.
  • human antibody includes all antibodies that have one or more variable and constant regions derived from human Ig sequences. In some embodiments of the present invention, all of the variable and constant domains of the antibody are derived from human Ig sequences (a fully human antibody). Fully human antibodies are expected to minimize the immunogenic and allergic responses intrinsic to mouse or mouse-derivatized monoclonal antibodies (Mabs) and thus to increase the efficacy and safety of the administered nanoparticles.
  • a humanized antibody is an antibody that is derived from a non-human species, in which certain amino acids have been mutated so as to avoid or abrogate an immune response in humans. Alternatively, a humanized antibody may be produced by fusing the constant domains from a human antibody to the variable domains of a non-human species.
  • the targeting ligand is an antibody that is specific for a protein comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments of the invention the targeting ligand is an antibody that is specific for a protein comprising the amino acid sequence of SEQ ID NO: 30.
  • Non-antibody based targeting ligands can also be used if they show specific binding to DEC-205.
  • Such molecules include aptamers and other similar oligonucleotides, peptides, small organic molecule ligands and other various non-covalent and covalent binding ligands with specificity towards DEC-205.
  • the preferred targeting ligand for DEC-205 is a fragment of a full-length monoclonal antibody (Fab) which is easier to prepare and conjugate than a full length antibody.
  • Fab monoclonal antibody
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Further, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • the targeting ligand is an antibody fragment comprising SEQ ID NO: 4. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 5. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 6. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 10. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 1 1 . In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 12.
  • the targeting ligand is an antibody fragment comprising SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO:6. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 10, SEQ ID NO: 1 1 and SEQ ID NO: 12. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 1 1 and SEQ ID NO: 12. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 26. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 27. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 26 and SEQ ID NO: 27. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 28.
  • a cysteine may be introduced in the antibody or antibody fragment providing the targeting ligand with a free nucleophilic thiol (-SH) group capable of being conjugated to the nanoparticle in various ways.
  • the thiol group can be directly added by nucleophilic addition to a maleimide group borne on the polymer chains from which the nanoparticle is fabricated or can be further functionalised with a small linker which itself bears a reactive functional group (e.g.
  • a strained alkyne tailored to participate in a conjugation reaction with a moiety attached to the polymer (e.g. in the case of a strained alkyne on the targeting ligand, an azide) .
  • a cysteine may be incorporated at any position on the antibody peptide chain or chains that is accessible for conjugation and does not interfere with the binding of the targeting ligand to DEC-205 such as, for example, at the C-terminus of one of the peptide chains, particularly the C-terminus of the heavy chain.
  • a unique "handle" suitable for bioconjugation may be engineered into the targeting ligand, such as an enzyme substrate sequence (e.g.
  • the density of the targeting ligand on the surface of the nanoparticle has been found to be an important factor in optimising the targeting of the nanoparticles to relevant cells of the immune system.
  • the density as measured by reverse phase high pressure liquid chromatography (RP-HPLC), typically ranges from about 0.05 nmol to about 67 nmol targeting ligand per mg of nanoparticle polymer matrix, preferably from about 0.1 nmol to about 26 nmol targeting ligand per mg of nanoparticle polymer matrix.
  • the optimal density was found to be from about 1 nmol to about 20 nmol targeting ligand per mg of nanoparticle polymer matrix.
  • the actual mass will differ according to the molecular weight of the targeting ligand.
  • a certain amount of targeting ligand may be adsorbed onto the surface of the nanoparticle, only covalently-bound targeting ligand has been found to play a significant role in achieving specific and targeted immune cell delivery of the nanoparticles.
  • Figure 14 shows the results of experiments carried out using the procedure of Example 9 to measure in vitro the amount of nanoparticle uptake by DEC-205 expressing cells using various kinds of nanoparticle at various target ligand densities.
  • a model nanoparticle was constructed using PLGA-PEG and a DEC-205 targeting ligand was conjugated to the surface using maleimide chemistry (see Example 3).
  • Nanoparticles with different targeting ligand density were prepared by varying the ratio of targeting ligand:nanoparticle polymer and the resulting target ligand density was measured using a reverse phase HPLC method.
  • Nanoparticles bearing a non-specific targeting ligand did not achieve an acceptable level of cellular uptake. Nanoparticles that did not have any covalently bound targeting ligand but had adsorbed targeting ligand on the surface ( Figure 14, DEC-205-specific Fab/NF-NP) also exhibited a very low level of cellular uptake.
  • the results show that the use of a covalently bound targeting ligand that is specific to DEC-205 at a target ligand density of from about 0.5 nmol to about 26 nmol or more preferably from about 1 nmol to about 20 nmol/mg nanoparticle polymer, leads to the optimal level of uptake by the target cells of the immune system and an optimal biological response.
  • a subpopulation of the polymer chains from which the polymeric matrix is constructed must be functionalised with a surface-orientated reactive moiety onto which the targeting ligand for DEC-205 may be attached.
  • the reactive moiety may be present at any position on the polymer chain which forms part of the surface of the fabricated particle but is typically at the terminus of the chain. It may be bound directly to the polymeric chain or by means of a short linker.
  • the targeting ligand is typically attached after fabrication of the nanoparticle (in which case it will be exclusively located on the exterior of the nanoparticle) but, in certain instances, where the targeting ligand is sufficiently robust to withstand the conditions used during fabrication of the nanoparticle, it may be attached to the polymer chains which make up the polymeric matrix prior to nanoparticle fabrication (in which case a certain proportion of the targeting ligand may be found in the interior of the nanoparticle). In the latter case, the targeting ligand is preferably attached to a more hydrophilic region of the polymer (e.g. the PEG part of a PEG-PLGA block co-polymer) in order to favour its location on the exterior of the nanoparticles when fabricated.
  • a more hydrophilic region of the polymer e.g. the PEG part of a PEG-PLGA block co-polymer
  • the nature of the reactive moiety will depend on the kind of conjugation chemistry that is employed in order to generate the covalent link between the polymeric chain and the targeting ligand.
  • the targeting ligand can be coupled directly to the reactive moiety on the nanoparticle or through a bifunctional linker.
  • the reactive moiety may comprise a number of different functional groups such as an amino group, a carboxylic acid group, a sulfhydryl group, a hydroxyl group, a hydroxylamine group, a hydrazine group, a tetrazine group or an azide group.
  • the link between the nanoparticle and the targeting ligand is to be constructed by forming an amide bond then the polymeric chain should bear a carboxylic acid or amine moiety, or a protected variant of either which can be deprotected prior to conjugation.
  • a maleimide, haloacetamide or methanesulfonylheterocycle moiety may be attached to the polymer chain and the conjugation of the targeting ligand achieved by means of the nucleophilic addition of a thiol group borne by the targeting ligand.
  • the polymer chain bears an azide moiety and the conjugation is achieved by means of the cycloaddition of an alkyne group borne on the targeting ligand or via reaction with a phosphine moiety through a Staudinger Ligation.
  • a cycloaddition will typically be a strain-promoted alkyne-azide cycloaddition (SpAAC) using a reagent such as dibenzocyclooctyne (DBCO) or bicycle[6.1 .Ojnonyne (BCN).
  • a copper- catalyzed alkyne-azide cycloaddition may also be used.
  • an inverse electron demand Diels-Alder reaction iEDDA
  • TCO tetrazine/trans-cyclooct-5-enol
  • a bifunctional linker may contain a cleavage element such as a disulphide bond, pH cleavable moiety or protease labile group which would enable facile release of the targeting group within the target cell.
  • a cleavage element such as a disulphide bond, pH cleavable moiety or protease labile group which would enable facile release of the targeting group within the target cell.
  • An additional route to targeting ligand conjugation is the pre-fabrication route whereby the targeting ligand is conjugated to the polymer chain prior to nanoparticle fabrication. This route is acceptable for targeting ligands, such as, but not limited to, organic small molecules, peptides, oligonucleotides or aptamers, that are robust and resistant to the organic solvent and high stress conditions of the nanoparticle emulsion process.
  • Such targeting ligands should have a covalent bond to the polymer chain, preferably connected at the terminal position of a hydrophilic region of the polymer (e.g. a PEG chain), so that the targeting ligand will be primarily surface-oriented after nanoparticle fabrication.
  • a hydrophilic region of the polymer e.g. a PEG chain
  • the antigen is the subject of an undesirable immune response.
  • Such an undesirable immune response may be associated with an autoimmune disease or may be the result of introducing an exogenous protein into the body, e.g. as part of an enzyme replacement therapy.
  • Antigens according to the invention include, but are not limited to, those listed in Table 1 below, and fragments thereof.
  • the antigen to be used in accordance with the present invention is preferably the whole protein. In other embodiments, however, the antigen may be a fragment of the protein which contains the epitopes giving rise to the unwanted immune response.
  • the encapsulated antigen preferably comprises from about 0.25 to about 5% by weight of the total dry mass of the nanoparticle.
  • the preferred antigen is the full-length human sequence of factor VIII commercially available as Advate® (GenBank accession number AAA52484, SEQ ID NO: 24) or the B-domain deleted human sequence commercially available as Refacto® (SEQ ID NO: 25).
  • the encapsulated glucocorticoid receptor agonist preferably comprises from about 1 to about 20% by weight of the total dry mass of the nanoparticle.
  • the glucocorticoid receptor agonist is a compound which, when delivered to cells, acts as an agonist at the glucocorticoid receptor.
  • the glucocorticoid receptor agonist may be a compound which acts directly as an agonist at the glucocorticoid receptor itself or a prodrug which has little or no agonist activity itself but which is hydrolysed to an active compound in vivo, for example, by hydrolytic cleavage (e.g. by esterase enzymes in the case of an ester prodrug).
  • prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the glucocorticoid receptor agonist with certain moieties known to those skilled in the art as 'pro-moieties' as described, for example, in "Design of Prodrugs" by H Bundgaard (Elsevier, 1985).
  • the glucocorticoid receptor agonist contains a hydroxyl group
  • a pharmaceutically acceptable acid for example a methanoate, ethanoate, propanoate (propionate), butanoate or pentanoate (valerate) ester.
  • the glucocorticoid receptor agonist contains 2 hydroxyl groups it may be possible to esterify either one or both in order to make prodrugs.
  • ester prodrugs may be particularly advantageous in adjusting the physicochemical properties of the glucocorticoid receptor agonist such as its lipophilicity.
  • the glucocorticoid receptor agonist must have a degree of lipophilicity that is conducive to its ready incorporation and retention between the chains of the polymer from which the nanoparticle is constructed but also allows for a reasonable rate of release when the nanoparticle is administered to a patient.
  • Lipophilicity is usually measured in terms of logP which is the logarithm of the ratio of an unionized compound's solubility in 1 -octanol to its solubility in water. A higher logP therefore correlates with a higher lipophilicity and an increased affinity between the glucocorticoid receptor agonist and the polymeric matrix.
  • the glucocorticoid receptor agonist should preferably have a logP from about 3 to about 7, most preferably from about 3.5 to about 4.5.
  • the present invention therefore also provides a nanoparticle comprising a polymeric matrix and a covalently-bound surface-orientated targeting ligand for DEC-205, said polymeric matrix encapsulating an antigen which is the subject of an undesirable immune response and a glucocorticoid receptor agonist, said polymeric matrix comprising (and more preferably consisting of) a block co-polymer of PLGA-PEG or a mixture of PLGA and a block co-polymer of PLGA-PEG, and said glucocorticoid receptor agonist having a logP in the range 3 to 7 (and more preferably being betamethasone-17-valerate or betamethasone- 17,21 -dipropionate).
  • glucocorticoid receptor agonists include dexamethasone 17-propionate (3.12), dexamethasone 21 -propionate (3.12), budesonide (3.186), dexamethasone isonicotinate (3.195), 21 -0-(1-ethoxy)ethyl desoxymetasone (3.648), clobetasol propionate (3.678), dexamethasone 17-, 21 -dipropionate (4.185), flumethasone pivalate (4.267), mometasone furoate (4.268), beclomethasone dipropionate (4.492), and fluticasone propionate (4.605).
  • glucocorticoid receptor agonists that can be used with the nanoparticles of the present invention include: dexamethasone, dexamethasone acetate, dexamethasone palmitate, fluticasone furoate, hydrocortisone, prednisone, mifepristone, desoximetasone-21 - cinnamate, desoxymetasone, fludrocortisone and Z-guggulsterone.
  • the antigen and glucocorticoid receptor agonist are slowly release from the matrix by diffusion and, if the polymeric matrix is biodegradable, by degradation of the polymer.
  • Drug release from nanoparticles can be measured in vitro using a dialysis method in which the amount of drug retained within a nanoparticle is measured to determine the cumulative amount of drug released over time. At select time points, a sample is removed from the dialysis unit and then lyophilized. The lyophilized samples are dissolved in DMSO, and the amount of glucocorticoid receptor agonist encapsulated is measured by reverse phase HPLC. The results demonstrate that drug release from particles varies from one glucocorticoid receptor agonist to another, a compound with a lower logP typically having faster rate of drug release.
  • the polymeric chains from which the nanoparticle is fabricated can also bear other moieties in addition to the attachment means for the DEC 205 targeting ligand. For instance, in some cases it may be desired to attach charged groups such as carboxyl groups or amino groups in order to fine-tune the surface properties of the nanoparticles and modify their pharmacokinetic and pharmacodynamics properties.
  • a label such as a fluorophore (e.g. FKR648) in order to be able to trace the movement of the particles in vitro and in vivo.
  • a label such as a fluorophore (e.g. FKR648)
  • haemophilia A is a preferred embodiment.
  • the treatment of multiple sclerosis is another preferred embodiment.
  • the invention therefore provides a nanoparticle, as defined in any of the preceding embodiments, for use as a medicament.
  • the invention also provides a nanoparticle, as defined in any of the preceding embodiments, for use in the treatment of an autoimmune disease (such one or more of the diseases listed in Table 1 above, notably haemophilia A).
  • the invention also provides a method of treating an autoimmune disease (such one or more of the diseases listed in Table 1 above, notably haemophilia A), in a mammal, including administering to said mammal an effective amount of a nanoparticle as defined in any of the preceding embodiments.
  • the invention also provides a pharmaceutical composition comprising a nanoparticle as defined in any of the preceding embodiments, for use in treating an autoimmune disease.
  • the nanoparticles may be stored by suspending them in an aqueous medium containing a cryoprotectant and freezing the suspension (typically at - 80°C).
  • a suitable cryoprotectant is a sugar or polyol.
  • the nanoparticles are suspended in an aqueous solution of at least 10 % (weight/volume) sucrose or trehalose, at a concentration of 1 -20 mg/mL (preferably at a concentration of about 5 mg/mL), and the suspension is frozen.
  • the frozen suspension may be stored without further treatment or lyophilized.
  • the nanoparticles are conveniently administered to a patient by as a suspension by sub-cutaneous injection or by intravenous injection/infusion. Administration by intravenous injection or infusion is preferred.
  • Suitable devices for intravenous administration include needle-based injectors and infusion techniques.
  • Suitable devices for subcutaneous injection include needle-based injectors.
  • the invention therefore also provides a pharmaceutical composition suitable for subcutaneous or intravenous administration comprising a nanoparticle, as defined in any of the preceding embodiments, and a pharmaceutically acceptable excipient.
  • compositions suitable for subcutaneous or intravenous administration are typically aqueous solutions which may contain excipients such as salts, amino acids, carbohydrates and buffering agents.
  • the nanoparticles of the invention may be administered in an isotonic aqueous buffer including, but not limited to, a phosphate buffer, an acetate buffer, a citrate buffer, a tartrate buffer or a bicarbonate buffer.
  • Suitable carbohydrates include anhydrous and hydrated forms of lactose, mannose, sucrose, glucose and fructose.
  • a freeze-dried formulation of the nanoparticles is reconstituted with sterile water for injection and then diluted with 0.9% aqueous sodium chloride or 5% dextrose solution prior to intravenous infusion.
  • a volume of 10-200 ml is typically used.
  • the preferred methods of preparation are vacuum drying and freeze drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof.
  • the proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. .
  • the composition may be administered once, but may also be administered multiple times.
  • the composition may be administered from once daily to once every six months or longer.
  • the administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months.
  • the composition may be administered once, at least twice or for at least the period of time until the disease is treated, palliated or cured.
  • the composition generally may be administered for as long as the disease is present.
  • compositions of the invention may include a therapeutically effective amount or a prophylactically effective amount of the nanoparticles of the invention.
  • the therapeutically effective amount of the nanoparticles present in the composition can be determined, for example, by taking into account the desired dose volumes and mode(s) of administration, the nature and severity of the condition to be treated, and the age and size of the subject.
  • Exemplary, non-limiting dose ranges for administration of the pharmaceutical compositions of the present invention to a subject are from about 0.01 mg/kg to about 200 mg/kg (expressed in terms of milligrams (mg) of nanoparticle administered per kilogram (kg) of subject weight), from about 0.1 mg/kg to about 100 mg/kg, from about 1 .0 mg/kg to about 50 mg/kg, from about 5.0 mg/kg to about 20 mg/kg, or about 15 mg/kg.
  • an average human subject weighs about 70 kg.
  • Ranges intermediate to any of the dosages cited herein, e.g., about 0.02 mg/kg - 199 mg/kg are also intended to be part of this invention. For example, ranges of values using a combination of any of the recited values as upper and/or lower limits are intended to be included.
  • Dosage regimens can also be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response) by administering several divided doses to a subject over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • Unit dosage forms of the nanoparticles of the inventions include freeze-dried compositions for reconstitution and dilution and liquid compositions.
  • a unit dosage per vial may contain from 1 to 1000 milliliters (mis) of different concentrations of the nanoparticle. In other embodiments, a unit dosage per vial may contain about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml or 100 ml of different concentrations of the nanoparticles. If necessary, these preparations can be adjusted to a desired concentration by adding a sterile diluent to each vial.
  • the liquid compositions of the present invention can also be prepared as unit dosage forms in sterile bags or containers, which are suitable for connection to an intravenous administration line or catheter.
  • the pharmaceutical composition may be packaged in a variety of ways depending upon the method used for administering the drug.
  • an article for distribution includes a container having deposited therein the pharmaceutical formulation in an appropriate form.
  • Suitable containers are well-known to those skilled in the art and include materials such as vials, bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and the like.
  • the container may also include a tamper-proof assemblage to prevent indiscreet access to the contents of the package.
  • the container has deposited thereon a label that describes the contents of the container. The label may also include appropriate warnings.
  • CTATAAGGCA TCTAGCCTGC AATCAGGGGT CCCATCAAGG TTCAGTGGCA
  • CTATAAGGCA TCTAGCCTGC AATCAGGGGT CCCATCAAGG TTCAGTGGCA
  • VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS
  • VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR WSVLTVLHQ
  • VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS
  • Figures Figure 1 shows that dendritic cells expressing DEC-205 selectively recognise and internalise nanoparticles with a covalently bound DEC-205 targeting ligand in vitro.
  • the graph plots geometric mean fluorescent intensity (MFI; a measure of nanoparticle uptake) against time of incubation (minutes) for non-functionalised nanoparticles (NF-NPS), DEC- 205-targeted nanoparticles (NLDC - targeting ligand densities of 1 :5 and 1 :1 used) and nanoparticles functionalised with a ligand that does not target DEC-205 (8.8).
  • MFI geometric mean fluorescent intensity
  • Figure 2 shows that dendritic cells expressing DEC-205 selectively recognise and internalise nanoparticles with a covalently bound DEC-205 targeting ligand in vivo. There is significant DEC-205-targeted nanoparticle uptake by CD8a + dendritic cells compared to control Fab-conjugated and non-functionalized (untargeted) nanoparticles.
  • Figure 3 shows that CD8 + dendritic cells isolated from mouse spleen have the highest level of DEC-205 expression in comparison to B cells (B220+), neutrophils, macrophages, myeloid dendritic cells (CD1 1 b+ DC) and monocytes.
  • Figure 4 shows that free glucocorticoid receptor agonists suppress the activation of JAWSM dendritic cells (measured in terms of TNFa release) when stimulated with LPS.
  • Figure 5 shows that nanoparticle encapsulated glucocorticoid receptor agonists suppress the activation of JAWSM dendritic cells (measured in terms of TNFa release) when stimulated with LPS.
  • Figure 6 shows that free glucocorticoid receptor agonists suppress the activation of human whole blood cells (measured in terms of TNFa release) when stimulated with LPS
  • Figure 7 shows that nanoparticle encapsulated glucocorticoid receptor agonists suppress the activation of human whole blood cells (measured in terms of TNFa release) when stimulated with LPS.
  • Figure 8 shows that nanoparticles co-encapsulating ovalbumin and betamethasone- 17-valerate (PF575) cause the greatest amount of Foxp3+ T regulatory cell induction in vitro as compared with nanoparticles encapsulating ovalbumin alone (PF556) and nanoparticles encapsulating ovalbumin alone + free beta-methasone-17-valerate in solution (PF556 + FD).
  • Figure 9 shows that nanoparticles co-encapsulating a protein antigen and a glucocorticoid receptor agonist reduce cognate T cell expansion in vivo.
  • Figure 10 shows the size distribution curve for a batch of DEC-205 targeted nanoparticles encapsulating B-domain deleted Factor VIM and betamethasone dipropionate
  • Figure 1 1 shows the selective delivery of DEC-205 targeted nanoparticles encapsulating factor VI II and betamethasone dipropionate to JAWSI I dendritic cells in vitro
  • Figures 12-13 show that nanoparticle agglomeration during the targeting ligand conjugation process can be prevented by controlling the pH during conjugation
  • Figure 14 shows that nanoparticle uptake by DEC-205-expressing cells varies with targeting ligand density
  • Figure 1 5 shows that targeting ligand density can be controlled by adjusting the ratio of targeting ligand to nanoparticle in the conjugation reaction
  • Figure 16 shows that the treatment of mice with nanoparticles containing the peptide MOG (relevant to multiple sclerosis) prior to immune challenge with MOG limited the production of MOG-specific antibodies.
  • the nanoparticles of the invention are preferably prepared by first fabricating the polymeric matrix in the presence of the antigen and glucocorticoid receptor agonist in order to form particles encapsulating both these agents.
  • the targeting ligand for DEC-205 is then attached to the exterior of the particles so formed.
  • the nanoparticles may be prepared using a double-emulsion process in which a first aqueous phase containing the antigen (referred to as “w ') is emulsified with an immiscible organic phase containing the polymer and the glucocorticoid receptor agonist (referred to as "o") to form a first water-in-oil emulsion. Formation of this w ⁇ o emulsion is achieved by coarse mixing with a rotor-stator, followed by homogenization on a homogenizer.
  • This first emulsion w-i/o is then further emulsified into a second aqueous phase (referred to as "w 2 ") to form a water-in-oil-in-water double emulsion
  • w 2 a second aqueous phase
  • Formation of a double emulsion is also achieved by coarse mixing with a rotor- stator, followed by homogenization on a homogenizer.
  • the solvent is then removed to leave discrete polymeric particles containing antigen and glucocorticoid receptor agonist.
  • the protein is retained within aqueous droplets that fill pores in the polymeric matrix and the glucocorticoid receptor agonist becomes lodged directly between the polymeric chains, being lipophilic.
  • the first aqueous phase (w ⁇ containing the antigen is generally an aqueous buffer in which is suitable for solubilising and stabilising the antigen.
  • factor VII I or B- domain deleted factor VIII may be formulated in an aqueous buffer containing 19 millimolar histidine, 3.4 millimolar calcium chloride, 250 millimolar arginine (pH 7.1).
  • the antigen is present at a concentration of at least 0.5 mg/mL, preferably at a concentration of 5 mg/mL or greater.
  • sodium chloride should be excluded from the aqueous phase in order to prevent the nanoparticles from becoming too porous.
  • the organic phase (o) preferably has a polymer concentration of 10-100 mg/mL.
  • An organic solvent such as dichloromethane, chloroform, or ethyl acetate is typically used and the glucocorticoid content is 5-30 % (weight/weight) with respect to the initial polymer content.
  • the organic phase may optionally contain an emulsifying agent with a low hydrophobic-lipophilic (HLB) value (typically less than 10).
  • HLB hydrophobic-lipophilic
  • the volume of the first aqueous phase is typically 1 to 20 times less than the volume of the organic phase (preferably 5 to 10 times less).
  • the double emulsion needs to be stabilised at the interface between the organic phase and the second aqueous phase (w 2 ) and the second aqueous phase therefore contains an stabilising emulsifier with a high HLB value, typically greater than about 10.
  • polyvinyl alcohol is used as the stabilising agent, more preferably polyvinyl alcohol) with a molecular weight of 30-70 kDa and 87-90% hydrolysed.
  • the amount of stabliliser required is typically 1 -10% (weight/volume).
  • the second aqueous phase generally has a volume 2-10 times greater than the w ⁇ o solution.
  • Emulsion formation is achieved in each case by high-energy mixing, e.g. using a rotor-stator or sonicator followed by high-pressure homogenisation (e.g. using a Microfluidics (Westwood, MA) processor).
  • high-energy mixing e.g. using a rotor-stator or sonicator followed by high-pressure homogenisation (e.g. using a Microfluidics (Westwood, MA) processor).
  • Solvent may be removed in order to generate the particles by means of evaporation or by particle wash through a tangential flow filtration device.
  • Example 1 Fabrication and characterization of nanoparticles containing the antigen ovalbumin and various glucocorticoid receptor agonists
  • the resulting emulsion was then added to 100 mL of 1 % aqueous polyvinyl alcohol), mixed under a rotor stator, and then homogenized for 5 cycles on the Microfluidics instrument with F20Y processor at 5000 psi for 4 passes.
  • This emulsion was transferred to an open beaker with 100 mL water, and then continuously stirred for approximately 18 hours to evaporate off the organic solvent.
  • the particles were pelleted by centrifugation at 35000 rcf for at least 10 minutes at 4 °C. The supernatant was removed and the particle pellet was resuspended with water and washed. The wash and centrifugation step was repeated 1 to 3 more times. After the final wash, the nanoparticles were resuspended at 5 mg/mL or less in 10% (w/v) sucrose, frozen, and then lyophilized.
  • the total yield of nanoparticle was determined by gravimetric measurement of an aliquot in water.
  • the encapsulation of glucocorticoid receptor agonist was measured by using reverse phase high performance liquid chromatography (RP-HPLC) with an absorbance detector. Specifically, nanoparticles were dissolved in 100% dimethyl sulfoxide (DMSO) or dimethylformamide (DMF), and then analysed by RP-HPLC using a gradient protocol consisting of 0.1 % trifluoroacetic acid (TFA) in acetonitrile and 0.1 % TFA in water.
  • Glucocorticoid receptor agonist absorbance was detected at 239 nm.
  • an SDS-PAGE was performed with Coomassie or silver stain, followed by a densitometric image analysis of the gel to quantify protein content.
  • the amount of fluourescence from a dissolved sample can be measured with a fluorimeter, such as a Safire2 (Tecan, Switzerland), and compared to a standard curve.
  • the size and charge of the nanoparticle is measured by dynamic light scattering, using a Malvern nanosizer.
  • FKR648 is a fluorophore allowing visualisation of the nanoparticles
  • Factor VIII and betamethasone dipropionate A 1 g polymer mixture of 50% (weight/weight) PLGA(20K)-PEG-maleimide, 30%
  • the resulting mixture was added to 100 mL of deionized water containing 1 % PVA, mixed with a rotor-stator at 5500 rpm, and then transferred to a Microfluidics homogenizer and processed for four passes at 5000 psi through an F20Y chamber.
  • the particulate suspension was stirred overnight in an open beaker to remove organic solvent, and then centrifuged at 30000 rcf for 25 min. The supernatant was decanted, and the pellet was washed with water. This centrifugation and wash step was repeated twice. The particles were finally suspended in a 10% (w/v) sucrose solution, frozen, and then lyophilized. Particle size was measured by dynamic light scattering, using a Malvern Zetasizer (Nano ZS model). The encapsulation efficiency of FVIII was measured with a BCA assay, and the betamethasone dipropionate loading was measured by reverse phase HPLC.
  • FKR648 is a fluorophore allowing visualisation of the nanoparticles
  • a DEC-205 targeting Iigand comprised of Fab protein with a single C-terminal cysteine ( ⁇ 50 kDa MW) was buffer exchanged into phosphate buffer, pH 6.5.
  • a reducing agent, tris(2-carboxyethyl)phosphine (TCEP) was used to reduce the C-terminal cysteine to give a free thiol (from any disulfide bound residual free cysteine or glutathione from protein expression).
  • TCEP tris(2-carboxyethyl)phosphine
  • NP 15 mg was added to a 100 kD MWCO centrifuge filter unit, CFU, and centrifuged (2100 rpm, 4°C) for at least 90 minutes and until the final volume was less than 750 ⁇ _.
  • the concentrated Mal-NP solution was removed from the CFU.
  • the filter membrane was washed with reaction buffer (20 mM phosphate, 150 mM NaCI, 1 mM EDTA, pH 7.0) to recover residual Mal-NP and pooled with the solution.
  • the final volume was brought to 750 ⁇ _ with reaction buffer, for a theoretical concentration of 20 mg/mL of Mal-NP.
  • the size and charge of the nanoparticles were measured using a Malvern Zetasizer instrument.
  • a concentrated 2x solution of targeting Iigand with a single free thiol was added in equal volume to the 20 mg/mL (2x) solution of Mal-NP.
  • the amount of accessible maleimide on the surface of the nanoparticle was assayed using a thiol-containing fluorophore reagent.
  • the concentration of the targeting ligand solution will be dependent on the molar ratio targeted. As an example, a 1 :20 (targeting ligand:Mal-NP) molar ratio was tested for conjugation.
  • the amounts needed in the example reaction are shown in Table 5 below:
  • the conjugation reaction was incubated on an orbital shaker (250 rpm) at ambient temperature (approx. 22°C) for 2 hours. The reaction was quenched with 0.4 mM Cysteine to quench any free maleimides.
  • the targeting ligand-conjugated nanoparticles, TL-NP were centrifuged to filter out all unreacted/un-associated targeting ligand using 100 kD MWCO CFUs. Three centrifugations were completed, each for 1 .5 hours (2100 rpm, 4°C) with subsequent addition of fresh storage buffer (20 mM histidine, pH 5.8, 8.5% sucrose) to the retentate solution. The TL-NPs in the retentate were analyzed for size and charge upon completion of each centrifugation. Each filtrate wash was collected for analysis (Reverse- phase HPLC method to determine targeting ligand conjugated-PEG-PLGA polymer).
  • TL-NPs include target ligand density by reverse-phase high performance liquid chromatography (HPLC) with a C4 column using in-line detectors such as UV-VIS, Fluorescence and Evaporative Light-Scattering Detection (ELSD) or Charged Aerosol Detection (CAD).
  • HPLC reverse-phase high performance liquid chromatography
  • ELSD Fluorescence and Evaporative Light-Scattering Detection
  • CAD Charged Aerosol Detection
  • Nanoparticle concentration was measured using either a freeze-dried weight, an HPLC assay of the polymer components or a plate based assay using a modified Dragendorff reagent for PEG quantitation. Nanoparticle morphology was analyzed using cryo-transmission electron microscopy (cryo-TEM).
  • ECD extracellular domain
  • HEK Freestyle 293 human embryonic kidney cells
  • Soluble protein of interest was purified using standard protein A chromatographic techniques (Protein A FF, GE Healthcare, Piscataway NJ) followed by gel filtration size exclusion chromatography (Superdex200, GE Healthcare, Piscataway NJ). Purified protein was characterized for purity and activity by binding ELISA using commercially-available anti-human DEC-205 monoclonal antibodies (eBiosciences, San Diego, CA).
  • Rat-anti-murine DEC-205 hybridoma NLDC-145 was obtained from American Type
  • cDNA complementary deoxyribonucleic acid sequence encoding the immunoglobulin gamma (IgG) variable regions (VH and VL) for NLDC-145 was determined using conventional molecular biological techniques and the manufacturer's protocols unless otherwise noted. Briefly, one million NLDC-145 hybridoma cells were homogenized for total RNA isolation with QIAGEN RNAeasy Mini kit (Valencia, CA). First strand cDNA was then produced using Superscript III RT kit (LifeTechnologies, Grand Island, NY).
  • Double stranded cDNAs for variable regions (VH and VL) of NLDC-145 were generated by PCR using primers from the rat IgG heavy chain (lgG1 , 2a, 2b) and light chain (kappa or lambda) constant regions, as described below. PCR cycling conditions: 1 cycle at 95°C for 1 min; 25 cycles at 95°C for 1 min, 63°C for 1 min and 72 °C for 1 min. The resulting RT-PCR products were cloned into TOPO-Blunt cloning vector (LifeTechnologies, Grand Island, NY) and sequenced by conventional methods.
  • a human chimeric Fab was generated as follows: rat constant domains CL1 and CH1 were replaced with human constant domains CL1 and CH1 .
  • the rat hinge region was also replaced with a modified human hinge region. Mutations for site-specific maleimide conjugation were made to the lower hinge region positions C226G, C229P, P230C (EU numbering). Additionally, a single point mutation was introduced in the VH position 62 (N62T; Kabat). Expression vectors for the chimeric NLDC-145 Fab VH and VL were then synthesized (Blue Heron, Bothell, WA) and sequence-confirmed.
  • CM conditioned media
  • CaptureSelect KappaXL column GE Healthcare, Piscataway, NJ
  • the CM was loaded at 25-30 mL/min overnight at 4°C.
  • the column was then washed with 10 column volumes (CV) of PBS pH 7.2.
  • the protein of interest was step-eluted with 20 mM citric acid, pH 3.5 for 5 CVs, then immediately neutralized with 5% of 1 .0 M Tris-HCI, pH 8.0.
  • the captured pools were loaded onto a 320 mL Superdex75 gel filtration column (GE Healthcare, Piscataway, NJ) to remove high molecular weight (HMW) species.
  • the peaks were pooled based on the analytical size exclusion (SEC) using a TSK G3000 SEC column (Tosoh Bioscience, King of Prussia, PA) and filtered through a 0.22 ⁇ PES membrane.
  • the final pool was analyzed by OD280 (NanoDrop, Thermo Fisher Scientific, Madison, CT), SDS-PAGE (LifeTechnologies, Grand Island, NY), analytical SEC, mass spectrometry and endotoxin level by Limulus Amebocyte Lysate (LAL) assay (Charles River, Wilmington, MA). Less than 1 % high HMW species was observed by analytical SEC.
  • Example 8 Conjugation of targeting ligand to nanoparticles and prevention of agglomeration
  • Targeting ligand (DEC-205 specific Fab bearing a C-terminal cysteine) was reduced using 2x molar excess of reducing agent (tris (2-carboxyethyl)phosphine (TCEP). The reduction step was allowed to proceed at ambient temperature for 30 minutes. After reduction, the excess TCEP was removed while the reduced targeting ligand was buffer exchanged to reaction buffer and concentrated using 10 kD MWCO centrifugal filter units (CFU) to 4 mg/ml.
  • the control reaction buffer was 20 mM sodium phosphate, 150 mM sodium chloride, 1 mM EDTA, pH 7.0. The final concentration of the targeting ligand was > 40 mg/ml.
  • Nanoparticles conjugated to DEC-205 targeting ligands were compared to nanoparticles conjugated to control targeting ligands (denoted by Fab) in a JAWSII uptake assay. To perform the assay, 200,000 cells of a JAWSII mouse dendritic cell line are added to a 96 well plate.
  • Nanoparticles are added at a final concentration of 0.2 mg/mL (based on Dragondorff assay concentrations), with the assay performed simultaneously at 37°C and on ice.
  • the 37°C plate is chilled in an ice bath, and washed 3x with ice-cold buffer (PBS/2mM EDTA/0.5% FBS/0.02%NaN3), with cells fixed in 2% formaldehyde for 20min before washing and reading on an LSR Fortessa cytometer.
  • line plots were constructed from cytometer geometric mean fluorescent intensity values using GraphPad software.
  • NLDC:NP DEC-205-targeted nanoparticles
  • Fab:NP ligand that is not specific for DEC-205
  • NF-NPS nanoparticle without any targeting ligand
  • nanoparticles with a higher density of attached targeting ligand (1 :1 NLDC:NP) were recognised and internalised faster than nanoparticles with a lower density of attached targeting ligand (1 :5 NLDC:NP).
  • Example 10 In vivo assay determining DEC-205 targeting
  • DEC-205 targeted nanoparticles target CD8a + DEC205 + CD1 1 c + dendritic cells in comparison to untargeted nanoparticles.
  • C57BL/6 mice were injected with 0.5mg of either DEC-205 targeted (NLDC145 1 :1 PF554,) or untargeted (PF129, PF130, 8.8 1 :1 PF554 and PF541 ) nanoparticles containing 20% FKR648-PLGA as a fluorescent label.
  • mice Upon sacrifice of mice, spleens were removed and stored at room temperature in PBS/2mM EDTA / 0.5% FBS in Miltenyi GentleMACS C-tubes until further processing. Spleens were reduced to single-cell suspensions using an OctoMACS, with red blood cell lysis subsequently performed on both blood and spleen single-cell suspension samples. Lymphocytes were washed extensively in fluorescent activated cell sorting (FACS) buffer (PBS/2mM EDTA/0.5% FBS), with conventional dendritic cells identified by FACS using CD8a-AF488 + and CD1 1 c-BV421 + antibodies.
  • FACS fluorescent activated cell sorting
  • FIG. 2 shows the engagement of DEC-205 targeted nanoparticles (denoted NLDC) by spleen CD8 + dendritic cells in comparison to untargeted nanoparticles, with each number representing the percent of cells having engaged a nanoparticle.
  • CD8a + dendritic cells are known to have the highest level of DEC-205 expression in comparison to B cells (B220+), neutrophils, macrophages, myeloid dendritic cells (CD1 1 b+ DC) and monocytes (see Figure 3). Of note, no untargeted nanoparticles were found to be associated with CD8a + dendritic cells.
  • Example 1 1 - In vitro assay determining functional tolerogenic activity of nanoparticles
  • Tolerogenic dendritic cells are characterized by their resistance to producing inflammatory cytokines in response to a strong inflammatory stimulus.
  • the potency of a therapeutic in maintaining a tolerogenic state can be ranked by its ability to inhibit the production of inflammatory cytokines by dendritic cells stimulated by lipopolysaccharide (LPS).
  • LPS lipopolysaccharide
  • JAWSII cells were pre-incubated with either nanoparticles or soluble glucocorticoids, with TNFa release assessed upon LPS stimulation. JAWSII cells were seeded the morning of the assay into 384-well plates.
  • nanoparticle preparations were normalized to 10 ⁇ of drug, with soluble glucocorticoids normalized to the same concentration.
  • Cells were pre-incubated for 2 hours with either DMSO vehicle, nanoparticles, or soluble glucocorticoids, whereupon LPS was added at a final concentration of 100 ng/ml for an additional 18 hours at 37°C, 5% C0 2 .
  • LPS was added at a final concentration of 100 ng/ml for an additional 18 hours at 37°C, 5% C0 2 .
  • supernatants were removed and stored, and TNFa concentrations were measured by a mouse TNFa R&D Systems ELISA. The results are shown in Figure 4 (soluble glucocorticoid receptor agonist) and Figure 5 (nanoparticle encapsulated glucocorticoid receptor agonist).
  • IC 50 in units of M
  • soluble dexamethasone was observed to be 2.41 -2.61 E-09, betamethasone diproprionate to be between 7.8-8.01 E-10, and betamethasone valerate to be 5.17-7.02E-10; while nanoparticle-encapsulated dexamethasone exhibited an IC 50 of 2.61 -3.74E-09, betamethasone diproprionate of 9.42-9.88E-10, and betamethasone valerate of 1 .72-1 .89E- 10.
  • Another method of assessing the potency of glucocorticoid-encapsulated nanoparticles is to co-culture either nanoparticles or soluble compound with human whole blood. Heparinized human whole blood from two different donors was diluted 1 :10 final with complete RPMI / 10%FBS. Diluted blood was aliquoted to a 96-well plate, with soluble glucocorticoids or nanoparticle-encapsulated glucocorticoids subsequently added and pre- incubated for 5 minutes. LPS was added at a concentration of 0.2ng/ml, with samples incubated for 18 hours, with TNFa levels assessed by ELISA (R&D Systems Quantkine) after 18hr incubation.
  • Example 12 In vitro assay demonstrating increased potency of nanoparticle- delivered glucocorticoid receptor agonist versus soluble compound and DEC-205 targeted nanoparticle-delivered glucocorticoid receptor agonist versus untargeted nanoparticles
  • nanoparticle encapsulating ovalbumin in the presence of free solubilised beta methasone-17 valerate also resulted in an approximate 2 fold increase in the percentage of FoxP3+ T cells observed compared with nanoparticle encapsulating ovalbumin alone (PF556, Figure 8).
  • bone marrow derived dendritic cells (0.5x10 6 /well) were incubated with titrating doses of nanoparticles encapsulating ovalbumin (PF-556), nanoparticles encapsulating ovalbumin and free solubilised betamethasone 17-valerate (PF-556+FD) and nanoparticles encapsulating both albumin and betamethasone 17-valerate for 4 hours followed by overnight maturation with LPS (50ng/ml).
  • dendritic cells were washed, counted and re-plated at 2x10 4 cells/well in a 96 well plate.
  • Violet labelled splenic CD4+ OT- II T-cells (1x10 5 cells/well) were co-cultured with the matured BM-dendritic cells in the presence of interleukin-2 (5ng/ml), and as a control, titrating doses of whole ovalbumin were normalized to encapsulated ovalbumin (nM) was included separately.
  • cells were harvested, and stained for Foxp3 expression and analysed in conjunction with Violet dilution. The data is presented as the replicate average for each NP/whole OVA dilution point.
  • Example 13 In vivo assay measuring the functional tolerogenic activity of nanoparticles Nanoparticles co-encapsulating protein antigen and a glucocorticoid receptor agonist suppress cognate T cell expansion in vivo (Figure 9).
  • CD45.1 + congenically marked C57BL/6 mice (strain B6.S J L-Pfprc a Pepc VBoyJ; Jackson Laboratory) were adoptively transferred with labelled 2x10E6 CD4 + CD45.2 + OT II Ova-specific T cells on Day 0.
  • spleens of B6.129S6-f?ag2 imiFwa Tg(TcraTcrb)425Cbn were harvested and reduced to single cell suspension using GentleMACS. After erythrocyte lysis, cells were counted and labelled with the proliferation tracking dye CellTracker Violet (Invitrogen) at a concentration of 5 ⁇ . Cells were then washed, the frequency of OT II cells determined by FACS, and mice injected intravenously in the contralateral tail vein using insulin syringes with cells in cold PBS.
  • CellTracker Violet Invitrogen
  • mice were euthanized, spleens harvested and reduced to single-cell suspensions by GentleMACS, and erythrocytes lysed.
  • Example 14 - DEC-205 targeted nanoparticles co-encapsulating Factor VIII and a glucocorticoid receptor agonist efficiently target DEC-205-expressing cells in vitro
  • DEC-205 targeted nanoparticles (PF584) loaded with B-domain deleted Factor VIII and betamethasone diproprionate were formulated using the double emulsion method and a polymer blend of 50% PLGA-PEG-Maleimide, 20% PLGA-FKR648, and 30% PLGA-PEG.
  • FKR648 is a fluorescent label.
  • B-domain deleted factor VIII was reformulated to ⁇ 20 fold higher concentration (based on the commercial formulation) to enable the desired loading of BDD-FVIII in the nanoparticles.
  • B-domain deleted Factor VIII was formulated at 10-12 mg/mL concentration in a sodium chloride-free medium consisting of 19mM histidine, 3.4mM calcium chloride and 250mM arginine (pH 7.1). This formulation was used as the inner aqueous phase (w ⁇ during the encapsulation process.
  • B-domain deleted Factor VIII was loaded at 1 .5% w/w loading, or 15.76 ⁇ g/mg loading as determined by BCA assay.
  • Betamethasone diproprionate was loaded at 9.4% w/w loading, or 94.29 +/- 4.2 ⁇ g/mg.
  • PF584 was conjugated either to NLDC-145 Fab (NT162), 8.8 Fab (NT161), or to free cysteine (Cys, NT163), with a targeting ligand density of 215.85 ⁇ g/mg for NLDC-145 and 176.32 g/mg for 8.8.
  • Example 15 - DEC-205 targeted particles containing myelin oligodendrocyte glycoprotein (MOG) and betamethasone dipropionate attenuated MOG-specific antibodies in mice challenged with Complete Freud's Adjuvant (CFAVMOG Anti-DEC-205 nanoparticles containing MOG35-55 peptide and betamethasone dipropionate (NT191 , NT192, NT193, NT196) were formulated as described in Table 8. These particles were administered i.v. two times, a week apart, to C57BL/6 female mice (day -14 and day - 7), at a dose of approximately 0.04 mpk MOG peptide and 1 .2 mpk betamethasone dipropionate.
  • MOG myelin oligodendrocyte glycoprotein
  • betamethasone dipropionate NT191 , NT192, NT193, NT196
  • mice were challenged with CFA/MOG35-55.
  • Plasma from days 10, 17, and 24 after challenge with CFA/MOG were analyzed by ELISA for the amount of MOG35-55 antibodies.
  • Treatment with DEC205 targeted particles that contained MOG35-55 peptide and betamethasone dipropionate led to reduced MOG-specific antibodies, relative to mice that did not receive any nanoparticles, and with greater effect than non-targeted particles observed on day 17 (Fig. 16). Treatment with non-targeted particles was eventually able to achieve similar reductions by day 24.
  • administration of nanoparticles containing betamethasone dipropionate and an irrelevant protein (ovalbumin, OVA) had less of an impact on reducing MOG-specific antibody titers.
  • the 10 treatment protocols illustrated in Fig. 16, from left to right are: (1) CFA challenge alone without MOG; (2) MOG immunization alone; (3) pre-treatment with DEC205-targeted (NLDC) nanoparticles containing MOG and betamethasone dipropionate before MOG immunization; (4) pre-treatment with Fab8 targeted nanoparticles containing MOG and betamethasone dipropionate before MOG immunization; (5) pre-treatment with untargeted nanoparticles containing MOG and betamethasone dipropionate before MOG immuniazation; (6) pre-treatment with DEC205-targeted nanoparticles containing MOG before MOG immunization; (7) pre-treatment with DEC205-targeted nanoparticles containing OVA and betamethasone dipropionate before MOG immunization.

Abstract

The present invention provides a nanoparticle comprising a polymeric matrix and a covalently-bound surface-orientated targeting ligand for DEC-205, said polymeric matrix encapsulating an antigen which is the subject of an undesirable immune response and a glucocorticoid receptor agonist. The nanoparticles of the invention can be used to treat autoimmune diseases such as haemophilia A and multiple sclerosis.

Description

POLYMERIC NANOPARTICLES WITH DEC-205 LIGAND AND CO-ENCAPSULATING AN ANTIGEN SUBJECT TO AN AUTOI M MUNE RESPONSE AND A GLUCOCORTICOID RECEPTOR AGONIST
The present invention relates to polymeric nanoparticles and their use in suppressing or preventing antigen-specific immune responses, in particular those associated with protein or enzyme replacement therapies and autoimmune diseases. A particularly important 5 application of the nanoparticles is in the treatment of certain types of haemophilia.
Haemophilia is the name given to a group of genetic disorders in which the mammalian body's ability to manufacture a blood clot at the site of a damaged blood vessel and prevent bleeding is impaired. The most common of these disorders is haemophilia A in which blood is unable to clot due to a deficiency in the blood-clotting protein factor VIII, most
10 usually as the result of the inactivation or reduced function of the gene encoding factor VIII (see Textbook of Haemophilia, 201 1 , John Wiley and Sons). Haemophilia A may also be acquired by the initiation of a neutralising antibody response to endogenously produced factor VIII, often as the result of an underlying autoimmune disease (see Bailliere's Clinical Haematology, 1998, 1 1 , 287-303; and Thrombosis and Haemostasis, 2013, 1 10, 1 1 14-
15 1 120). The inability of haemophilia A patients to produce factor VIII at sufficient levels leads to a deficiency in haemostasis with resulting haemorrhagic events that can lead to significant morbidity and mortality in untreated patients. Current standard-of-care treatments for haemophilia A include the administration of exogenous factor VIII two to three times a week in order to maintain haemostasis (Haemophilia: the Official Journal of the World Federation
20 of Haemophilia, 2007, 13 (supplement 2), 10-15).
The mammalian immune system has evolved to distinguish between endogenously produced proteins and potentially pathogenic foreign proteins. A major clinical problem encountered in the treatment of haemophilia A patients with factor VIII replacement therapy is the recognition of the exogenous factor VIII by the patient's immune system as foreign and
25 the subsequent generation of an anti-factor VIII immune response (Thrombosis and Haemostasis, 1998, 79, 762-766). It is estimated that between 25-30% of patients with haemophilia A develop neutralising antibodies to factor VIII replacement products, patients with severe haemophilia being most at risk (The Lancet, 1992, 339, 594-598). These neutralising antibodies cause partial or complete inhibition of the factor VIII replacement
30 therapy, leaving the patient once again at risk of haemorrhagic events and their associated morbidity.
The current standard of care for haemophilia A patients that exhibit an immune response to replacement therapy is immune tolerance induction in which factor VIII is administered daily at high doses until Factor VIII ceases to be neutralised and haemostasis 35 is maintained. This process normally lasts between one and two years and involves daily infusions of factor VIII, placing a large burden on the patient, and is not always effective (Blood, 2012, 1 19, 1335-1344). In order to increase the efficacy of the treatment, immunosuppressants such as cyclophosphamide together with intravenous immunoglobulin (New England Journal of Medicine, 1988, 318, 947-950) or rituximab (Journal of Thrombosis and Haemostasis, 2009, 7, 787-794), either alone or in combination with corticosteroids, are sometimes used as an adjunctive therapy in refractory patients. However, the use of such broad and non-selective immunosuppression is associated with an increased risk of adverse events including serious infection.
A significant unmet need therefore exists for an effective and convenient method of inducing tolerance to factor VIII which is selective and limits or avoids a general suppression of the immune system. Furthermore, the need for an effective and convenient method for inducing tolerance to therapeutic proteins and avoiding an undesired immune response is not limited to haemophilia but is also apparent in other diseases associated with protein and enzyme replacement therapies such as Pompe disease and autoimmune diseases such as type-1 diabetes and multiple sclerosis.
The present invention therefore provides a nanoparticle comprising a polymeric matrix and a covalently-bound surface-orientated targeting ligand for DEC-205, said polymeric matrix encapsulating an antigen which is the subject of an undesirable immune response and a glucocorticoid receptor agonist. We have found that delivering a protein antigen to DEC-205 expressing cells via polymeric nanoparticles induces an antigen-specific tolerance with a range of antigens. Crucially, it is possible to induce a tolerogenic response to the antigen, rather than an immunogenic response, by co-encapsulating the antigen in a targeted polymeric nanoparticle with a glucocorticoid receptor agonist. By such means, a general immunosuppressive effect is also avoided and tolerance is achieved in an antigen- specific manner. The nanoparticle platform enables simultaneous delivery of the antigen and glucocorticoid receptor agonist to the particular antigen-presenting cells that are capable of triggering the desired tolerogenic response to the antigen, considerably enhancing the potency of both agents and both allowing a selective tolerogenic effect and avoiding any increase in immunogenic response to the antigen. Summary of the invention
The invention provides as embodiment E1 a nanoparticle comprising a polymeric matrix and a covalently-bound surface-orientated targeting ligand for DEC-205, said polymeric matrix encapsulating an antigen which is the subject of an undesirable immune response and a glucocorticoid receptor agonist. The invention provides as embodiment E2 a nanoparticle as defined in embodiment E1 wherein the nanoparticle is from about 20 to about 500 nm in diameter.
The invention provides as embodiment E3 a nanoparticle as defined in embodiment E2 wherein the nanoparticle is from about 60 to about 300 nm in diameter. The invention provides as embodiment E4 a nanoparticle as defined in any one of embodiments E1 to E3 wherein the polymers from which the polymeric matrix are constructed are biocompatible.
The invention provides as embodiment E5 a nanoparticle as defined in any one of embodiments E1 to E4 wherein the polymers from which the polymeric matrix are constructed are biodegradable.
The invention provides as embodiment E6 a nanoparticle as defined in any one of embodiments E1 to E5 wherein the polymeric matrix comprises PLGA.
The invention provides as embodiment E7 a nanoparticle as defined in any one of embodiments E1 to E6 wherein the polymeric matrix comprises PEG. The invention provides as embodiment E8 a nanoparticle as defined in any one of embodiments E1 to E7 wherein the polymeric matrix comprises a PLGA-PEG block copolymer.
The invention provides as embodiment E9 a nanoparticle as defined in any one of embodiments E1 to E6 wherein the polymeric matrix consists of PLGA. The invention provides as embodiment E10 a nanoparticle as defined in any one of embodiments E1 to E8 wherein the polymeric matrix consists of a PLGA-PEG block copolymer.
The invention provides as embodiment E1 1 a nanoparticle as defined in any one of embodiments E1 to E8 wherein the polymeric matrix consists of a mixture of PLGA and a PLGA-PEG block co-polymer.
The invention provides as embodiment E12 a nanoparticle as defined in any one of embodiments E6, E8, E9, E10 or E1 1 wherein the ratio of lactic acid to glycolic acid is about 1 :1 .
The invention provides as embodiment E13 a nanoparticle as defined in any one of embodiments E6, E8, E9, E10, E1 1 or E12 wherein the average molecular weight of the PLGA in each chain is from about 15,000 to about 90,000, more specifically from about 15,000 to about 50,000 daltons, more specifically from about 15,000 to about 40,000 daltons.
The invention provides as embodiment E14 a nanoparticle as defined in embodiment E13 wherein the average molecular weight of the PLGA in each chain is about 20,000 daltons.
The invention provides as embodiment E15 a nanoparticle as defined in any one of embodiments E7, E8, E10 or E1 1 wherein the average molecular weight of the PEG in each chain is from about 2,500 to about 20,000 daltons.
The invention provides as embodiment E16 a nanoparticle as defined in embodiment E15 wherein the average molecular weight of the PEG in each chain is about 5,000 daltons.
The invention provides as embodiment E17 a nanoparticle as defined in any one of embodiments E6, E8, E9, E10, E1 1 , E12, E13 or E14 wherein the targeting ligand is attached to a PLGA polymer chain, preferably at the terminal position.
The invention provides as embodiment E18 a nanoparticle as defined in any one of embodiments E7, E8, E10, E1 1 , E15 or E16 wherein the targeting ligand is attached to a PEG chain.
The invention provides as embodiment E19 a nanoparticle as defined in embodiment E18 wherein the targeting ligand is attached at the terminal position of a PEG chain.
The invention provides as embodiment E20 a nanoparticle as defined in any one of embodiments E1 to E19 wherein the polymer chains are neutral at pH 7.
The invention provides as embodiment E21 a nanoparticle as defined in any one of embodiments E1 to E20 wherein the targeting ligand is located both inside and on the surface of the nanoparticle.
The invention provides as embodiment E22 a nanoparticle as defined in embodiment E21 wherein the targeting ligand is located exclusively on the surface of the nanoparticle.
The invention provides as embodiment E23 a nanoparticle as defined in any one of embodiments E1 to E22 wherein the targeting ligand binds to DEC-205 with a binding affinity (Kd) of less than about 1000 nM.
The invention provides as embodiment E24 a nanoparticle as defined in embodiment E23 wherein the targeting ligand binds to DEC-205 with a binding affinity (Kd) of less than about 100 nM. The invention provides as embodiment E25 a nanoparticle as defined in embodiment E23 wherein the targeting ligand binds to DEC-205 with a binding affinity (Kd) of less than about 10 nM.
The invention provides as embodiment E26 a nanoparticle as defined in embodiment E23 wherein the targeting ligand binds to DEC-205 with a binding affinity (Kd) of less than about 1 nM.
The invention provides as embodiment E27 a nanoparticle as defined in any one of embodiments E1 to E26 wherein the targeting ligand binds to DEC-205 with high avidity.
The invention provides as embodiment E28 a nanoparticle as defined in any one of embodiments E1 to E27 wherein the targeting ligand is an antibody or an antigen-binding portion thereof.
The invention provides as embodiment E29 a nanoparticle as defined in embodiment E28 wherein the targeting ligand is a monoclonal antibody or antigen-binding portion thereof.
The invention provides as embodiment E30 a nanoparticle as defined in either of embodiments E28 and E29 wherein the targeting ligand is a humanized antibody or antigen- binding portion thereof.
The invention provides as embodiment E31 a nanoparticle as defined in either of embodiments E28 and E29 wherein the targeting ligand is a fully human antibody or antigen- binding portion thereof. The invention provides as embodiment E32 a nanoparticle as defined in any one of embodiments E28 to E31 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 4.
The invention provides as embodiment E33 a nanoparticle as defined in any one of embodiments E28 to E32 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 5.
The invention provides as embodiment E34 a nanoparticle as defined in any one of embodiments E28 to E33 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 6.
The invention provides as embodiment E35 a nanoparticle as defined in any one of embodiments E28 to E34 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 10. The invention provides as embodiment E36 a nanoparticle as defined in any one of embodiments E28 to E35 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 1 1 .
The invention provides as embodiment E37 a nanoparticle as defined in any one of embodiments E28 to E36 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 12.
The invention provides as embodiment E38 a nanoparticle as defined in any one of embodiments E28 to E37 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 26. The invention provides as embodiment E39 a nanoparticle as defined in any one of embodiments E28 to E38 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 27.
The invention provides as embodiment E40 a nanoparticle as defined in any one of embodiments E28 to E39 wherein the targeting ligand is an antibody or antigen-binding portion thereof comprising SEQ ID NO: 28.
The invention provides as embodiment E41 a nanoparticle as defined in any one of embodiments E1 to E40 wherein the targeting ligand binds to human DEC-205.
The invention provides as embodiment E42 a nanoparticle as defined in any one of embodiments E1 to E41 wherein the targeting ligand density on the nanoparticle is from about 0.05 nmol to about 67 nmol targeting ligand per mg of nanoparticle polymer matrix.
The invention provides as embodiment E43 a nanoparticle as defined in embodiment E42 wherein the targeting ligand density on the nanoparticle is from about 0.1 nmol to about 26 nmol targeting ligand per mg of nanoparticle polymer matrix.
The invention provides as embodiment E44 a nanoparticle as defined in embodiment E43 wherein the targeting ligand density on the nanoparticle is from about 1 nmol to about 20 nmol targeting ligand per mg of nanoparticle polymer matrix.
The invention provides as embodiment E45 a nanoparticle as defined in any one of embodiments E1 to E44 wherein the targeting ligand is covalently bound to the polymeric matrix through a bond formed by the nucleophilic addition of a cysteine thiol group on the targeting ligand to a maleimide group on the polymeric matrix. The invention provides as embodiment E46 a nanoparticle as defined in any one of embodiments E1 to E44 wherein the targeting ligand is covalently bound to the polymeric matrix through bonds formed by the cycloaddition of an alkyne group on the targeting ligand to an azide group on the polymeric matrix. The invention provides as embodiment E47 a nanoparticle as defined in embodiment
E46 wherein the alkyne is a strained alkyne such as a dibenzocyclooctyne.
The invention provides as embodiment E48 a nanoparticle as defined in any one of embodiments E1 to E47 wherein the undesirable immune response is to factor VIII.
The invention provides as embodiment E48a a nanoparticle as defined in any one of embodiments E1 to E47 wherein the undesirable immune response is to myelin oligodendrocyte glycoprotein (MOG).
The invention provides as embodiment E49 a nanoparticle as defined in embodiment E48 wherein the antigen is human factor VIII having the amino acid sequence of SEQ ID NO: 24. The invention provides as embodiment E50 a nanoparticle as defined in embodiment
E48 wherein the antigen is B-domain deleted-human factor VIII having the amino acid sequence of SEQ ID NO: 25.
The invention provides as embodiment E51 a nanoparticle as defined in any one of embodiments E1 to E50 wherein the antigen comprises from about 0.25 to about 5% by weight of the total dry mass of the nanoparticle.
The invention provides as embodiment E52 a nanoparticle as defined in any one of embodiments E1 to E51 wherein the glucocorticoid receptor agonist comprises from about 1 to about 20% by weight of the total dry mass of the nanoparticle.
The invention provides as embodiment E53 a nanoparticle as defined in any one of embodiments E1 to E52 wherein the glucocorticoid receptor agonist has a logP of about 3 to about 7.
The invention provides as embodiment E54 a nanoparticle as defined in embodiment E53 wherein the glucocorticoid receptor agonist has a logP of about 3.5 to about 4.5.
The invention provides as embodiment E55 a nanoparticle as defined in embodiment E53 wherein the glucocorticoid receptor agonist is betamethasone-17-valerate. The invention provides as embodiment E56 a nanoparticle as defined in embodiment E53 wherein the glucocorticoid receptor agonist is betamethasone-17,21 -dipropionate.
The invention provides as embodiment E57 a process for preparing a nanoparticle according to any one of embodiments E1 to E56, wherein: (i) the antigen is solubilized in an aqueous solution (w^ at a concentration of at least 0.5 mg/mL (preferably at least 5mg/ml_); (ii) the antigen solution from step (i) is homogenised with an organic phase (o) that contains the glucocorticoid and polymer to form an w^o emulsion; (iii) the w^o emulsion is added to a second aqueous solution (w2) containing an emulsifier with an HLB value greater than about 10 and homogenised to form a
Figure imgf000009_0001
double emulsion; (iv) solvent is removed to form the nanoparticle; and (v) a targeting ligand is conjugated to the surface of the nanoparticle.
The invention provides as embodiment E58 a process for preparing a nanoparticle as defined in embodiment E57 wherein step (v) is performed at a pH between about 5 and about 7.
The invention provides as embodiment E59 a process for preparing a nanoparticle as defined in embodiment E58 wherein step (v) is performed at a pH between about 5.5 and about 6.5.
General definitions
"About" or "approximately," when used in connection with a measurable numerical variable, refers to the indicated value of the variable and to all values of the variable that are within the experimental error of the indicated value (e.g. within the 95% confidence interval for the mean) or within 10 percent of the indicated value, whichever is greater. Numeric ranges are inclusive of the numbers defining the range.
References to "treatment" include references to curative, palliative and prophylactic treatment. The term treatment encompasses any objective or subjective improvement in a subject with respect to a relevant condition or disease.
The term "prophylactic treatment," as used herein to describe the present invention, means that the nanoparticle or pharmaceutical composition thereof is administered to a subject to inhibit or stop the relevant condition from occurring in a subject, particularly in a subject or member of a population that is significantly predisposed to the relevant condition.
The term "palliative treatment," as used herein to describe the present invention, means that the nanoparticle or pharmaceutical composition thereof is administered to a subject to remedy signs and/or symptoms of a condition, without necessarily modifying the progression of, or underlying etiology of, the relevant condition.
The term "curative treatment," as used herein to describe the present invention, means that the nanoparticle or pharmaceutical composition thereof is administered to a subject for the purpose of bringing the disease or disorder into complete remission, or that the disease or disorder is undetectable after such treatment.
Whilst the present invention has been defined in terms of a "nanoparticle", it is understood that the invention will be practiced by making and using a population of such nanoparticles. Any reference to "nanoparticle" is therefore understood to apply equally to a "population of nanoparticles" wherein each member of the population has the characteristics of the single nanoparticle. The nanoparticles within such a population may be heterogeneous in nature, e.g. having a range of sizes or targeting ligand density. Where the nanoparticle is defined by a numerical parameter given as a range, it is understood that this parameter applies, in the case of a population of nanoparticles, to the distribution within the population as a whole, unless the context requires a different interpretation
Nanoparticle with polymeric matrix
The nanoparticle comprises a polymeric matrix that is capable of encapsulating the antigen and the glucocorticoid receptor agonist and onto which a targeting ligand for DEC- 205 is covalently attached. The size of the nanoparticle is preferably from about 20 to about 500 nm in diameter, most preferably from about 60 to about 300 nm in diameter. Within a population of the nanoparticles, the mean size of the population is preferably from about 100 to about 150 nm. The polymers used are preferably biocompatible (e.g. poly(ethylene glycol, PEG) and may optionally be biodegradable (e.g. poly(lactic-co-glycolic acid), PLGA). Block co-polymers such as PEG-PLGA may also be used. A sub-population of the polymer chains must be functionalised with a reactive group in order to facilitate attachment of the targeting ligand for DEC-205 to the exterior of the particles. Functionalisation is preferably effected on a more hydrophilic part of the polymer chains in order to encourage the reactive groups to locate on the exterior of the nanoparticles when fabricated. The term "polymeric matrix" means the three-dimensional fabric of the nanoparticle which consists of an entanglement of polymer chains from which the nanoparticle is constructed. The chains are not covalently attached to each other but held together by non- covalent interactions such as van der Waals forces, hydrogen bonds and hydrophobic effects.
By "encapsulating" it is meant that the antigen and glucocorticoid receptor agonist are held within the polymer matrix by non-covalent means. Typically, the antigen will initially be held within small droplets of aqueous phase that form in holes or channels within the polymer matrix during fabrication of the nanoparticles and will remain in these holes or channels when solvent is removed. The glucocorticoid receptor agonist, being more lipophilic, will be distributed throughout the nanoparticle embedded in spaces between the polymer chains. Thus, the antigen and glucocorticoid agonist, not being covalently bound, are free to diffuse out of the polymer matrix when it is suspended in solution and/or as the polymer matrix is degraded in vivo.
The term "biocompatible" as used herein means something that is biologically inert or non-reactive with intracellular and extra cellular biological molecules, and non-toxic. Biocompatible polymers for use in the present invention have preferably been approved by regulatory authorities for use in human beings.
Preferably, the polymer from which the nanoparticle is constructed is a block copolymer of poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG) or a mixture of a PLGA-PEG co-polymer and PLGA. Such polymers are commercially available and may be purchased, for example, from Akina Inc. (West Lafayette, Indiana) as part of their PolySciTech® range.
The present invention therefore also provides a nanoparticle comprising a polymeric matrix and a covalently-bound surface-orientated targeting ligand for DEC-205, said polymeric matrix encapsulating an antigen which is the subject of an undesirable immune response and a glucocorticoid receptor agonist and said polymeric matrix comprising (and more preferably consisting of) a block co-polymer of PLGA-PEG or a mixture of a PLGA and a block co-polymer of PLGA-PEG.
In some embodiments, the nanoparticles are fabricated from a PLGA-PEG block copolymer. Preferably, the ratio of lactic acid to glycolic acid is about 1 :1 and the average molecular weight of the PLGA and PEG in each chain is from about 15,000 to about 50,000 daltons (e.g. about 20,000 daltons) for the PLGA and from about 2,500 to about 20,000 daltons (e.g. about 5000 daltons) for the PEG.
In some embodiments, the nanoparticles are fabricated from a mixture of 80% PLGA- PEG block co-polymer and 20% PLGA. Preferably, the ratio of lactic acid to glycolic acid is 1 :1 and the average molecular weight of the PLGA and PEG in each chain is from about 15,000 to about 50,000 daltons (e.g. about 20,000 daltons) for the PLGA and from about 2,500 to about 20,000 daltons (e.g. about 5000 daltons) for the PEG.
In these embodiments, it is preferred that a proportion of the PEG chains have a moiety, preferably a terminal moiety, that facilitates attachment of the DEC-205 targeting ligand. In one embodiment, at least 50% of the PEG chains have a moiety (preferably a terminal moiety) that facilitates attachment of the DEC-205 targeting ligand. In another embodiment, about 50% of the PEG chains have a moiety (preferably a terminal moiety) that facilitates attachment of the DEC-205 targeting ligand. In some embodiments, the amount of PEG chains with a moiety (preferably a terminal moiety) that facilitates attachment of the DEC-205 targeting ligand is at least about 55% or 60% or 65% or 70% of 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99%.
In these embodiments it is also preferred that the PLGA and PLGA-PEG polymer chains are neutral, i.e. do not contain any group that is charged at pH 7. Preferably, such neutral polymer chains terminate in a methoxy group, i.e. a methyl ester in the case of PLGA, a methyl ether in the case of PEG.
Targeting ligand for DEC-205
The DEC-205 targeting ligand is surface-oriented, i.e. attached to the surface of the nanoparticle and is covalently attached to the polymeric matrix from which the nanoparticle is constructed. Surface-oriented in this context means that the binding region of the target ligand is solvent accessible and is not sterically or otherwise hindered by the rest of the nanoparticle from binding to its DEC-205 target
Preferably, the DEC-205 targeting ligand is exclusively attached to the surface of the nanoparticle and does not form part of the interior of the nanoparticle or is only minimally internalised. However, in some embodiments a proportion of the targeting ligand may be located inside the polymeric matrix of the nanoparticle as long as sufficient targeting ligand is located on the surface to facilitate targeting of the nanoparticles to DEC-205-expressing cells. Preferably, the percentage of DEC-205 targeting ligand that is surface-orientated is at least about 50% or 55% or 60% or 65% or 70% of 75% or 80% or 85% or 90% or 95% or 96% or 97% or 98% or 99%.
DEC-205 is a surface protein expressed primarily by cells of the immune system, particularly by dendritic cells, which facilitates internalisation of nanoparticles that bind to it. The full length sequence of human DEC-205 may be found under GenBank accession number NP_002340. The targeting ligand must be specific for the DEC-205 protein expressed by the mammal undergoing treatment. For the treatment of humans, the DEC-205 targeting ligand must target human DEC-205. In some embodiments, the targeting ligand binds to DEC-205 with high affinity, for instance with a binding affinity (Kd) of less than about 1000 nM or with a Kd of less than about 1 00 nM or with a Kd of less than about 10 nM or with a Kd of less that about 1 nM. In some embodiments, the targeting ligand binds to the DEC- 205 with high avidity as a result of the co-operative binding of targeting ligands on the nanoparticle surface. The affinity and binding properties of a targeting ligand according to the invention for
DEC-205 may be determined using in vitro assays (biochemical or immunological based assays) known in the art for antigen-binding domain, including but not limited to enzyme- linked immu nosorbent assay (ELISA) assay, surface plasmon resonance (SPR) assay, Bio- Layer Interferometry, or immunoprecipitation assays. The term "binding affinity" (Kd) as used herein, is intended to refer to the dissociation rate of a particular antigen-antibody interaction. The Kd is the ratio of the rate of dissociation, also called the "off-rate (koff)", to the association rate, or "on-rate (kon)". Thus, Kd equals koff/kon and is expressed as a molar concentration (M). It follows that the smaller the Kd , the stronger the affinity of binding . Therefore, a Kd of 1 μ indicates weak binding affinity compared to a KD of 1 nM. Kd values for antibodies can be determined using methods well established in the art. One method for determining the Kd of an antibody is by using surface plasmon resonance (SPR), typically using a biosensor system such as a BIACORE® system. BIAcore kinetic analysis comprises analyzing the binding and dissociation of an antigen from chips with immobilized molecules (e.g. molecules comprising epitope binding domains), on their surface. Another method for determining the Kd of an antibody is by using Bio-Layer Interferometry, typically using OCTET® technology (Octet QKe system, ForteBio).
Any ligand that binds specifically to DEC-205 may be used as the targeting ligand . In particular, the targeting ligand may be an antibody that binds to DEC-205 or an antigen binding portion thereof, such as a full-length antibody for DEC-205 or a fragment of a full- length antibody (Fab) in which some of the non-binding domains have been deleted e.g. a fragment of a full-length antibody containing a portion of the hinge region (Fab'). Other antibody-related entities that may be utilised as the targeting ligand include single-chain antibodies (scFv), diabodies (db), small immunoproteins (SIP), Vhh domains (Vhh) and other similar immunoproteins known to those skilled in the art. An antibody is an immunoglobulin molecule capable of specific binding to a target or antigen, such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen-binding site, located in the variable region of the immunoglobulin molecule.
As used herein, unless otherwise indicated by context, the term is intended to encompass not only intact polyclonal or monoclonal antibodies comprising two identical full- length heavy chain polypeptides and two identical light chain polypeptides, but also fragments thereof (such as Fab, Fab', F(ab')2, Fv), single chain (ScFv) and domain antibodies (dAbs), including shark and camelid antibodies, and fusion proteins comprising an antibody portion, multivalent antibodies, multispecific antibodies (e.g. bispecific antibodies so long as they exhibit the desired biological activity) and antibody fragments as described herein, and any other modified configuration of the immunoglobulin molecule that comprises an antigen recognition site, for example without limitation, minibodies, maxibodies, monobodies, peptibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NAR and bis- scFv. Antigen-binding portions may be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact antibodies. Antigen-binding portions include, inter alia, Fab, Fab', F(ab')2, Fv, dAb, and complementarity determining region (CDR) fragments, single-chain antibodies (scFv), chimeric antibodies, diabodies and polypeptides that contain at least a portion of an Ig that is sufficient to confer specific antigen binding to the polypeptide.
An immunoglobulin (Ig) is a heteromultimeric molecule. In a naturally occurring Ig, each multimer is composed primarily of identical pairs of polypeptide chains, each pair having one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa).
The amino-terminal portion of each chain includes a variable region, of about 100 to 1 10 or more amino acids primarily responsible for antigen recognition. The carboxy-terminal portion of each chain defines a constant region primarily responsible for effector function.
Variable domains exhibit the same general structure of relatively conserved framework regions (FR) joined by 3 hypervariable regions, also called complementarity determining regions or CDRs. The CDRs from the 2 chains of each pair are aligned by the framework regions, enabling binding to a specific epitope. From N-terminus to C-terminus, both light and heavy chains comprise the domains FR1 , CDR1 , FR2, CDR2, FR3, CDR3 and FR4.
If the targeting ligand is an antibody, it is preferably a human antibody or a humanized antibody. The term "human antibody" includes all antibodies that have one or more variable and constant regions derived from human Ig sequences. In some embodiments of the present invention, all of the variable and constant domains of the antibody are derived from human Ig sequences (a fully human antibody). Fully human antibodies are expected to minimize the immunogenic and allergic responses intrinsic to mouse or mouse-derivatized monoclonal antibodies (Mabs) and thus to increase the efficacy and safety of the administered nanoparticles. A humanized antibody is an antibody that is derived from a non-human species, in which certain amino acids have been mutated so as to avoid or abrogate an immune response in humans. Alternatively, a humanized antibody may be produced by fusing the constant domains from a human antibody to the variable domains of a non-human species.
Some antibodies for DEC-205 are commercially available. Others may be generated and characterised by methods well known to the skilled person in the art. Antibodies may be generated using the full-length protein or just the extracellular domain thereof. Examples of commercially available antibodies to DEC-205 include anti-human CD205 purified monoclonal antibody No. 14-2059 sold by Affymetrix eBioscience (San Diego, California) and purified anti-human/mouse/rat CD205 antibody No. 359202 sold by Biolegend (San Diego, California). In some embodiments of the invention, the targeting ligand is an antibody that is specific for a protein comprising the amino acid sequence of SEQ ID NO: 29. In some embodiments of the invention the targeting ligand is an antibody that is specific for a protein comprising the amino acid sequence of SEQ ID NO: 30.
Other non-antibody based targeting ligands can also be used if they show specific binding to DEC-205. Such molecules include aptamers and other similar oligonucleotides, peptides, small organic molecule ligands and other various non-covalent and covalent binding ligands with specificity towards DEC-205.
The preferred targeting ligand for DEC-205 is a fragment of a full-length monoclonal antibody (Fab) which is easier to prepare and conjugate than a full length antibody. The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigen. Further, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. Specific DEC-205 targeting ligands that may be used in the nanoparticles of the invention incorporate the NLDC binding sequences set out in Table 2. In one embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 4. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 5. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 6. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 10. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 1 1 . In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 12. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO:6. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 10, SEQ ID NO: 1 1 and SEQ ID NO: 12. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 10, SEQ ID NO: 1 1 and SEQ ID NO: 12. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 26. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 27. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 26 and SEQ ID NO: 27. In another embodiment, the targeting ligand is an antibody fragment comprising SEQ ID NO: 28.
In the case of an antibody-based DEC-205 targeting ligand, conventional protein engineering may be used to introduce functionality into the ligand to facilitate covalent attachment to the nanoparticle. In one embodiment of the invention, a cysteine may be introduced in the antibody or antibody fragment providing the targeting ligand with a free nucleophilic thiol (-SH) group capable of being conjugated to the nanoparticle in various ways. For instance, the thiol group can be directly added by nucleophilic addition to a maleimide group borne on the polymer chains from which the nanoparticle is fabricated or can be further functionalised with a small linker which itself bears a reactive functional group (e.g. a strained alkyne) tailored to participate in a conjugation reaction with a moiety attached to the polymer (e.g. in the case of a strained alkyne on the targeting ligand, an azide) . Such a cysteine may be incorporated at any position on the antibody peptide chain or chains that is accessible for conjugation and does not interfere with the binding of the targeting ligand to DEC-205 such as, for example, at the C-terminus of one of the peptide chains, particularly the C-terminus of the heavy chain. In other embodiments, a unique "handle" suitable for bioconjugation may be engineered into the targeting ligand, such as an enzyme substrate sequence (e.g. a transglutaminase substrate), so that attachment of the targeting ligand may be accomplished by enzymatic reaction. The density of the targeting ligand on the surface of the nanoparticle has been found to be an important factor in optimising the targeting of the nanoparticles to relevant cells of the immune system. In the case where the targeting ligand is an antibody fragment (Fab), particularly one having a molecular weight of about 50 kD, the density, as measured by reverse phase high pressure liquid chromatography (RP-HPLC), typically ranges from about 0.05 nmol to about 67 nmol targeting ligand per mg of nanoparticle polymer matrix, preferably from about 0.1 nmol to about 26 nmol targeting ligand per mg of nanoparticle polymer matrix. The optimal density was found to be from about 1 nmol to about 20 nmol targeting ligand per mg of nanoparticle polymer matrix. The actual mass will differ according to the molecular weight of the targeting ligand. Although during the conjugation process a certain amount of targeting ligand may be adsorbed onto the surface of the nanoparticle, only covalently-bound targeting ligand has been found to play a significant role in achieving specific and targeted immune cell delivery of the nanoparticles.
Figure 14 shows the results of experiments carried out using the procedure of Example 9 to measure in vitro the amount of nanoparticle uptake by DEC-205 expressing cells using various kinds of nanoparticle at various target ligand densities. In these experiments, a model nanoparticle was constructed using PLGA-PEG and a DEC-205 targeting ligand was conjugated to the surface using maleimide chemistry (see Example 3). Nanoparticles with different targeting ligand density were prepared by varying the ratio of targeting ligand:nanoparticle polymer and the resulting target ligand density was measured using a reverse phase HPLC method. As shown in Figure 15, there is a direct correlation between the ratio of targeting ligand:nanoparticle polymer used in the conjugation reaction and the resulting target ligand density on the nanoparticle measured in μg targeting ligand/mg nanoparticle polymer. The results obtained by measuring the uptake of targeted nanoparticles by DEC-205 expressing cells (Figure 14, DEC-205-specific Fab-NP) show that a targeting ligand density of about 2 nmol/mg nanoparticle polymer is needed in order to achieve optimal cellular uptake. From about 0.05 nmol target ligand/mg nanoparticle polymer to about 2 nmol target ligand/mg nanoparticle polymer, there is an increase in cellular binding/uptake of approximately 2.5-3.0-fold. From about 2 nmol to about 12 nmol targeting ligand/mg nanoparticle polymer, there is a plateau where an increase in targeting ligand density does not appear to change the binding capability of the nanoparticle. Surprisingly, cellular uptake declines slightly above a target ligand density of about 12 nmol/mg nanoparticle polymer. Several control nanoparticles were also tested. Nanoparticles bearing a non-specific targeting ligand (Figure 14, Non-specific Fab-NP) or no targeting ligand at all (Figure 14, Cys-NP) did not achieve an acceptable level of cellular uptake. Nanoparticles that did not have any covalently bound targeting ligand but had adsorbed targeting ligand on the surface (Figure 14, DEC-205-specific Fab/NF-NP) also exhibited a very low level of cellular uptake. The results show that the use of a covalently bound targeting ligand that is specific to DEC-205 at a target ligand density of from about 0.5 nmol to about 26 nmol or more preferably from about 1 nmol to about 20 nmol/mg nanoparticle polymer, leads to the optimal level of uptake by the target cells of the immune system and an optimal biological response.
Attachment of targeting ligand to the polymeric matrix
A subpopulation of the polymer chains from which the polymeric matrix is constructed must be functionalised with a surface-orientated reactive moiety onto which the targeting ligand for DEC-205 may be attached. The reactive moiety may be present at any position on the polymer chain which forms part of the surface of the fabricated particle but is typically at the terminus of the chain. It may be bound directly to the polymeric chain or by means of a short linker. The targeting ligand is typically attached after fabrication of the nanoparticle (in which case it will be exclusively located on the exterior of the nanoparticle) but, in certain instances, where the targeting ligand is sufficiently robust to withstand the conditions used during fabrication of the nanoparticle, it may be attached to the polymer chains which make up the polymeric matrix prior to nanoparticle fabrication (in which case a certain proportion of the targeting ligand may be found in the interior of the nanoparticle). In the latter case, the targeting ligand is preferably attached to a more hydrophilic region of the polymer (e.g. the PEG part of a PEG-PLGA block co-polymer) in order to favour its location on the exterior of the nanoparticles when fabricated.
The nature of the reactive moiety will depend on the kind of conjugation chemistry that is employed in order to generate the covalent link between the polymeric chain and the targeting ligand. The targeting ligand can be coupled directly to the reactive moiety on the nanoparticle or through a bifunctional linker. The reactive moiety may comprise a number of different functional groups such as an amino group, a carboxylic acid group, a sulfhydryl group, a hydroxyl group, a hydroxylamine group, a hydrazine group, a tetrazine group or an azide group. It may be desirable to protect the chosen functional group during the fabrication of the nanoparticle and deprotect it shortly before coupling in order to prevent any unwanted reactions between the reactive moiety and other components of the nanoparticle such as the antigen. In relation to the choice of suitable protecting group methodology, reference may be made to the standard textbook in the field, Greene's Protective Groups in Organic Synthesis, 5th edition, 2014 (Wiley). Preferably, a bioorthogonal approach is selected such that the reactive moiety does not interact with the encapsulated protein without requiring protection but allows for direct conjugation with the target ligand functionality.
For example, if the link between the nanoparticle and the targeting ligand is to be constructed by forming an amide bond then the polymeric chain should bear a carboxylic acid or amine moiety, or a protected variant of either which can be deprotected prior to conjugation. Alternatively, a maleimide, haloacetamide or methanesulfonylheterocycle moiety may be attached to the polymer chain and the conjugation of the targeting ligand achieved by means of the nucleophilic addition of a thiol group borne by the targeting ligand.
In a preferred embodiment, the polymer chain bears an azide moiety and the conjugation is achieved by means of the cycloaddition of an alkyne group borne on the targeting ligand or via reaction with a phosphine moiety through a Staudinger Ligation. Such a cycloaddition will typically be a strain-promoted alkyne-azide cycloaddition (SpAAC) using a reagent such as dibenzocyclooctyne (DBCO) or bicycle[6.1 .Ojnonyne (BCN). A copper- catalyzed alkyne-azide cycloaddition may also be used. Alternatively, an inverse electron demand Diels-Alder reaction (iEDDA) may be used, such as the tetrazine/trans-cyclooct-5-enol (TCO) conjugation.
If a bifunctional linker is employed in the conjugation methodology, it may contain a cleavage element such as a disulphide bond, pH cleavable moiety or protease labile group which would enable facile release of the targeting group within the target cell. An additional route to targeting ligand conjugation is the pre-fabrication route whereby the targeting ligand is conjugated to the polymer chain prior to nanoparticle fabrication. This route is acceptable for targeting ligands, such as, but not limited to, organic small molecules, peptides, oligonucleotides or aptamers, that are robust and resistant to the organic solvent and high stress conditions of the nanoparticle emulsion process. Such targeting ligands should have a covalent bond to the polymer chain, preferably connected at the terminal position of a hydrophilic region of the polymer (e.g. a PEG chain), so that the targeting ligand will be primarily surface-oriented after nanoparticle fabrication.
It is important to ensure that nanoparticles do not agglomerate during the conjugation process as this can lead to particles having an inappropriate size distribution which can lead to reduced efficacy. In particular, it has been found that the use of a pH below about 7, preferably from about 5 to about 7, most preferably from about 5.5 to about 6.5, prevents agglomeration during conjugation. As shown in Preparative Example 8 and Figures 12-13, conjugation of a DEC-205 targeting ligand to nanoparticles using maleimide chemistry using a process in which the nanoparticles and targeting ligand were incubated together on an orbital shaker for 2 hours in aqueous buffer at pH 7 resulted in agglomerated nanoparticles with a size distribution having a maximum diameter at around 800 nm which is too large to achieve an optimal biological response in vivo (Figure 12). Attempts to prevent agglomeration using a lower reaction concentration, a reduced reaction temperature and sonication were each largely unsuccessful. Surprisingly, however, the use of a buffer with an acidic pH (in this case pH 6) prevented agglomeration, resulting in conjugated nanoparticles with an average diameter of about 200 nm, similar to the diameter of the non-conjugated nanoparticle used as starting material (Figure 13).
Antigen
The antigen is the subject of an undesirable immune response. Such an undesirable immune response may be associated with an autoimmune disease or may be the result of introducing an exogenous protein into the body, e.g. as part of an enzyme replacement therapy. Antigens according to the invention include, but are not limited to, those listed in Table 1 below, and fragments thereof. Where the antigen is a naturally occurring protein which is associated with an autoimmune disease or an exogenous protein used in replacement therapy that is the subject of an undesirable immune response, the antigen to be used in accordance with the present invention is preferably the whole protein. In other embodiments, however, the antigen may be a fragment of the protein which contains the epitopes giving rise to the unwanted immune response.
The encapsulated antigen preferably comprises from about 0.25 to about 5% by weight of the total dry mass of the nanoparticle.
For the treatment of an unwanted immune response to factor VIII, the preferred antigen is the full-length human sequence of factor VIII commercially available as Advate® (GenBank accession number AAA52484, SEQ ID NO: 24) or the B-domain deleted human sequence commercially available as Refacto® (SEQ ID NO: 25).
Glucocorticoid receptor agonist The encapsulated glucocorticoid receptor agonist preferably comprises from about 1 to about 20% by weight of the total dry mass of the nanoparticle. The glucocorticoid receptor agonist is a compound which, when delivered to cells, acts as an agonist at the glucocorticoid receptor. As such, the glucocorticoid receptor agonist may be a compound which acts directly as an agonist at the glucocorticoid receptor itself or a prodrug which has little or no agonist activity itself but which is hydrolysed to an active compound in vivo, for example, by hydrolytic cleavage (e.g. by esterase enzymes in the case of an ester prodrug). Further information on the use of prodrugs may be found in 'Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T Higuchi and W Stella) and 'Bioreversible Carriers in Drug Design', Pergamon Press, 1987 (ed. E B Roche, American Pharmaceutical Association). Prodrugs in accordance with the invention can, for example, be produced by replacing appropriate functionalities present in the glucocorticoid receptor agonist with certain moieties known to those skilled in the art as 'pro-moieties' as described, for example, in "Design of Prodrugs" by H Bundgaard (Elsevier, 1985). In particular, where the glucocorticoid receptor agonist contains a hydroxyl group, it may be possible to esterify the hydroxyl group to make an ester prodrug using a pharmaceutically acceptable acid, for example a methanoate, ethanoate, propanoate (propionate), butanoate or pentanoate (valerate) ester. Where the glucocorticoid receptor agonist contains 2 hydroxyl groups it may be possible to esterify either one or both in order to make prodrugs. The use of ester prodrugs may be particularly advantageous in adjusting the physicochemical properties of the glucocorticoid receptor agonist such as its lipophilicity.
The glucocorticoid receptor agonist must have a degree of lipophilicity that is conducive to its ready incorporation and retention between the chains of the polymer from which the nanoparticle is constructed but also allows for a reasonable rate of release when the nanoparticle is administered to a patient. Lipophilicity is usually measured in terms of logP which is the logarithm of the ratio of an unionized compound's solubility in 1 -octanol to its solubility in water. A higher logP therefore correlates with a higher lipophilicity and an increased affinity between the glucocorticoid receptor agonist and the polymeric matrix. It has been found, particularly in the case where the polymeric matrix is fabricated using a block co-polymer of poly(lactic-co-glycolic acid) (PLGA) and poly(ethylene glycol) (PEG), or a mixture of PLGA-PEG and PLGA, that the glucocorticoid receptor agonist should preferably have a logP from about 3 to about 7, most preferably from about 3.5 to about 4.5. Particularly preferred glucocorticoid receptor agonists that fall within this range are betamethasone-17-valerate (logP = 4.185) and betamethasone-17,21-dipropionate (4.185).
The present invention therefore also provides a nanoparticle comprising a polymeric matrix and a covalently-bound surface-orientated targeting ligand for DEC-205, said polymeric matrix encapsulating an antigen which is the subject of an undesirable immune response and a glucocorticoid receptor agonist, said polymeric matrix comprising (and more preferably consisting of) a block co-polymer of PLGA-PEG or a mixture of PLGA and a block co-polymer of PLGA-PEG, and said glucocorticoid receptor agonist having a logP in the range 3 to 7 (and more preferably being betamethasone-17-valerate or betamethasone- 17,21 -dipropionate).
Other preferred glucocorticoid receptor agonists (calculated logP in brackets) include dexamethasone 17-propionate (3.12), dexamethasone 21 -propionate (3.12), budesonide (3.186), dexamethasone isonicotinate (3.195), 21 -0-(1-ethoxy)ethyl desoxymetasone (3.648), clobetasol propionate (3.678), dexamethasone 17-, 21 -dipropionate (4.185), flumethasone pivalate (4.267), mometasone furoate (4.268), beclomethasone dipropionate (4.492), and fluticasone propionate (4.605).
Other glucocorticoid receptor agonists that can be used with the nanoparticles of the present invention include: dexamethasone, dexamethasone acetate, dexamethasone palmitate, fluticasone furoate, hydrocortisone, prednisone, mifepristone, desoximetasone-21 - cinnamate, desoxymetasone, fludrocortisone and Z-guggulsterone.
Release profile for antigen and glucocorticoid receptor agonist
The antigen and glucocorticoid receptor agonist are slowly release from the matrix by diffusion and, if the polymeric matrix is biodegradable, by degradation of the polymer.
Drug release from nanoparticles can be measured in vitro using a dialysis method in which the amount of drug retained within a nanoparticle is measured to determine the cumulative amount of drug released over time. At select time points, a sample is removed from the dialysis unit and then lyophilized. The lyophilized samples are dissolved in DMSO, and the amount of glucocorticoid receptor agonist encapsulated is measured by reverse phase HPLC. The results demonstrate that drug release from particles varies from one glucocorticoid receptor agonist to another, a compound with a lower logP typically having faster rate of drug release.
Other optional features
The polymeric chains from which the nanoparticle is fabricated can also bear other moieties in addition to the attachment means for the DEC 205 targeting ligand. For instance, in some cases it may be desired to attach charged groups such as carboxyl groups or amino groups in order to fine-tune the surface properties of the nanoparticles and modify their pharmacokinetic and pharmacodynamics properties.
In other cases, it may be desirable to attach a label such as a fluorophore (e.g. FKR648) in order to be able to trace the movement of the particles in vitro and in vivo.
Indications
There are a number of diseases associated with an undesirable immune response which may be treated with the nanoparticles of the invention. In each case, a protein antigen relevant to the disease is encapsulated in the nanoparticle. A list of relevant diseases, along with examples of the disease-relevant protein antigens that could be utilised and, where appropriate, references thereto, is included below in Table 1 .
Table 1 - Diseases and disease-relevant antigens
Source of undesirable Disease-relevant protein Reference for protein antigen immune tesponse antigen
Haemophilia A Factor VIII
Refacto®
Advate®
Haemophilia B Factor IX
Benefix®
Celiac disease Gliadin Journal of Biological Chemistry,
1967, 242, 445-450
Recombinant gliadin
Hordein Carlsberg Research
Communications, 1979, 44, 431-438
Secalin Journal of Experimental Botany,
1982, 33, 261-268
Pompe's disease Recombinant alglucosidase
alpha
Lumizyme®
Myozyme® Fabry disease Recombinant algalsidase
beta
Fabryzyme®
Mucopolysaccharidoses Recombinant laronidase
Aldurazymet®
Idursulfase
Elaprase®
Arylsulfatase B
Naglazyme®
Gene-therapy Proteins associated with Gene Therapy, 1999, 6, 1574- adeno-associated virus 1583
Proteins associated with
retrovirus
Proteins associated with Nature Medicine, 2001, 7, 33- lentivirus 40
Type 1 diabetes Purified pro-insulin FEBS Letters, 1997, 402, 124- 130
Recombinant pro-insulin
Peptide fragments of pro- insulin
Recombinant glutamate Diabetes, 2001, 50, 1749-1754 decarboxylate-65 and 67
Recombinant insulinoma- Diabetes, 2001, 50, 1749-1754 associated protein 2 (IA-2
and IA-213)
Recombinant islet-specific Journal of Biological Chemistry, glucose-6-phosphatase 2004, 279, 13976-13983
Recombinant ZnT8 Proceedings of the National
Academy of Sciences, 2007, 104, 17040-17045.
Recombinant chromagranin Regulatory Peptides, 1995, 56: A and other pancreatic beta- 71-88
cell associated antigens
Primary biliary cirrhosis Recombinant E2 Hepatology, 2001, 34, 243-248 components of pyruvate dehydrogenase complexes
Multiple sclerosis Recombinant, purified, or Brain, 1999, 122, 2047-2056 peptide fragments of Myelin
oligodendrocyte glycoprotein
(MOG)
Recombinant, purified, or Brain, 1999, 122, 2047-2056 peptide fragments of myelin
basic protein
Recombinant, purified, or American journal of peptide fragments of neuroradiology, 1999, 20, 965- proteolipid protein (13) 976
neuromyelitis optica Recombinant aquaporin-4 Journal of the neurological sciences, 2010, 291, 52-56 myasthenia gravis Recombinant acetylcholine The Journal of immunology, receptor 1991, 146, 2245-2248
Muscle specific kinase Journal of neuroimmunology,
2006, 175, 107-117
Grave's disease Recombinant TSH receptor Endocrinology, 1994, 134, 549- 554
ANCA-associated Recombinant European journal of vasculitides myeloperoxidase biochemistry, 1991, 197, 605- 614
Recombinant desmoglein 1 Clinical and Developmental or 3 Immunology 2009
Recombinant proteinase 3 FEBS letters, 1996, 390, 265- 270
The treatment of haemophilia A is a preferred embodiment. The treatment of multiple sclerosis is another preferred embodiment.
The invention therefore provides a nanoparticle, as defined in any of the preceding embodiments, for use as a medicament. The invention also provides a nanoparticle, as defined in any of the preceding embodiments, for use in the treatment of an autoimmune disease (such one or more of the diseases listed in Table 1 above, notably haemophilia A). The invention also provides a method of treating an autoimmune disease (such one or more of the diseases listed in Table 1 above, notably haemophilia A), in a mammal, including administering to said mammal an effective amount of a nanoparticle as defined in any of the preceding embodiments. The invention also provides a pharmaceutical composition comprising a nanoparticle as defined in any of the preceding embodiments, for use in treating an autoimmune disease.
Formulation and administration
Following fabrication, the nanoparticles may be stored by suspending them in an aqueous medium containing a cryoprotectant and freezing the suspension (typically at - 80°C). A suitable cryoprotectant is a sugar or polyol. Preferably, the nanoparticles are suspended in an aqueous solution of at least 10 % (weight/volume) sucrose or trehalose, at a concentration of 1 -20 mg/mL (preferably at a concentration of about 5 mg/mL), and the suspension is frozen. The frozen suspension may be stored without further treatment or lyophilized. The nanoparticles are conveniently administered to a patient by as a suspension by sub-cutaneous injection or by intravenous injection/infusion. Administration by intravenous injection or infusion is preferred. Suitable devices for intravenous administration include needle-based injectors and infusion techniques. Suitable devices for subcutaneous injection include needle-based injectors. The invention therefore also provides a pharmaceutical composition suitable for subcutaneous or intravenous administration comprising a nanoparticle, as defined in any of the preceding embodiments, and a pharmaceutically acceptable excipient.
Pharmaceutical compositions suitable for subcutaneous or intravenous administration are typically aqueous solutions which may contain excipients such as salts, amino acids, carbohydrates and buffering agents. The nanoparticles of the invention may be administered in an isotonic aqueous buffer including, but not limited to, a phosphate buffer, an acetate buffer, a citrate buffer, a tartrate buffer or a bicarbonate buffer. Suitable carbohydrates include anhydrous and hydrated forms of lactose, mannose, sucrose, glucose and fructose. In one embodiment, a freeze-dried formulation of the nanoparticles is reconstituted with sterile water for injection and then diluted with 0.9% aqueous sodium chloride or 5% dextrose solution prior to intravenous infusion. A volume of 10-200 ml is typically used. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and freeze drying that yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile filtered solution thereof. The proper fluidity of a solution can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. .
The composition may be administered once, but may also be administered multiple times. For example, the composition may be administered from once daily to once every six months or longer. The administering may be on a schedule such as three times daily, twice daily, once daily, once every two days, once every three days, once weekly, once every two weeks, once every month, once every two months, once every three months and once every six months.
The composition may be administered once, at least twice or for at least the period of time until the disease is treated, palliated or cured. The composition generally may be administered for as long as the disease is present.
The compositions of the invention may include a therapeutically effective amount or a prophylactically effective amount of the nanoparticles of the invention. In preparing the composition, the therapeutically effective amount of the nanoparticles present in the composition can be determined, for example, by taking into account the desired dose volumes and mode(s) of administration, the nature and severity of the condition to be treated, and the age and size of the subject.
Exemplary, non-limiting dose ranges for administration of the pharmaceutical compositions of the present invention to a subject are from about 0.01 mg/kg to about 200 mg/kg (expressed in terms of milligrams (mg) of nanoparticle administered per kilogram (kg) of subject weight), from about 0.1 mg/kg to about 100 mg/kg, from about 1 .0 mg/kg to about 50 mg/kg, from about 5.0 mg/kg to about 20 mg/kg, or about 15 mg/kg. For purposes of the present invention, an average human subject weighs about 70 kg. Ranges intermediate to any of the dosages cited herein, e.g., about 0.02 mg/kg - 199 mg/kg, are also intended to be part of this invention. For example, ranges of values using a combination of any of the recited values as upper and/or lower limits are intended to be included.
Dosage regimens can also be adjusted to provide the optimum desired response (e.g., a therapeutic or prophylactic response) by administering several divided doses to a subject over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the nanoparticle and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an antibody for the treatment of sensitivity in individuals. Unit dosage forms of the nanoparticles of the inventions include freeze-dried compositions for reconstitution and dilution and liquid compositions.
In the case of a liquid composition of the present invention, a unit dosage per vial may contain from 1 to 1000 milliliters (mis) of different concentrations of the nanoparticle. In other embodiments, a unit dosage per vial may contain about 1 ml, 2 ml, 3 ml, 4 ml, 5 ml, 6 ml, 7 ml, 8 ml, 9 ml, 10 ml, 15 ml, 20 ml, 30 ml, 40 ml, 50 ml or 100 ml of different concentrations of the nanoparticles. If necessary, these preparations can be adjusted to a desired concentration by adding a sterile diluent to each vial. The liquid compositions of the present invention can also be prepared as unit dosage forms in sterile bags or containers, which are suitable for connection to an intravenous administration line or catheter.
The pharmaceutical composition may be packaged in a variety of ways depending upon the method used for administering the drug. Generally, an article for distribution includes a container having deposited therein the pharmaceutical formulation in an appropriate form. Suitable containers are well-known to those skilled in the art and include materials such as vials, bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and the like. The container may also include a tamper-proof assemblage to prevent indiscreet access to the contents of the package. In addition, the container has deposited thereon a label that describes the contents of the container. The label may also include appropriate warnings.
Sequence list
Nucleotide and amino acid sequences relevant to the invention are set out in the following Table.
Table 2 - Sequence list
SE Description Sequence Fab-NLDC-145 VH ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGCGT nucleotide with GCACTCCGAG GTGAAGCTGT TGGAATCTGG AGGAGGTTTG GTACAGCCGG
GGGGTTCTCT GAGACTCTCC TGTGCAGCTT CTGGATTCAC CTTCAATGAT
secretory leader
TTCTACATGA ACTGGATCCG CCAGCCTCCA GGGCAGGCAC CTGAGTGGTT
GGGTGTTATT AGAAACAAAG GTAATGGTTA CACAACAGAG GTCAATACAT
CTGTGAAGGG GCGGTTCACC ATCTCCAGAG ATAATACCCA AAACATCCTC
TATCTTCAAA TGAACAGCCT GAGAGCTGAG GACACCGCCA TTTACTACTG
TGCAAGAGGC GGTCCTTATT ACTACAGTGG TGACGACGCC CCTTACTGGG
GCCAAGGAGT CATGGTCACA GTCTCCTCA
Fab-NLDC-145 VH MGWSCIILFL VATATGVHSE VKLLESGGGL VQPGGSLRLS CAASGFTFND with secretory FYMNWIRQPP GQAPEWLGVI RNKGNGYTTE VNTSVKGRFT ISRDNTQNIL
YLQMNSLRAE DTAIYYCARG GPYYYSGDDA PYWGQGVMVT VSS
leader
Fab-NLDC-145 VH MGWSCIILFL VATATGVHS
secretory leader
Fab-NLDC-145 GFTFNDFYMN
CDRH1
Fab-NLDC-145 VIRNKGNGYT TEVNTSVKG
CDRH2
Fab-NLDC-145 GGPYYYSGDD APY
CDRH3
Fab-NLDC-145 VL ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGCGT nucleotide with GCACTCCGAC ATCCAGATGA CCCAGTCTCC ATCCTCCCTG TCTGCATCTG
TAGGAGACAG AGTCACCATC ACTTGCCATG CCAGTCAGAA CATCAAGGGT
secretory leader
TGGTTAGCCT GGTATCAGCA GAAACCAGGG AAAGCCCCTA AGCTCCTGAT
CTATAAGGCA TCTAGCCTGC AATCAGGGGT CCCATCAAGG TTCAGTGGCA
GTGGATCTGG GACAGATTTC ACTCTCACCA TCAGCAGTCT GCAACCTGAA
GATTTTGCAA CTTACTACTG TCAGCATTAT CAAAGCTTTC CGTGGACCTT
CGGTCAAGGC ACCAAGGTGG AAATCAAA
Fab-NLDC-145 VL MGWSCIILFL VATATGVHSD IQMTQSPSSL SASVGDRVTI TCHASQNIKG with secretory WLAWYQQKPG KAPKLLIYKA SSLQSGVPSR FSGSGSGTDF TLTISSLQPE
DFATYYCQHY QSFPWTFGQG TKVEIK
leader
Fab-NLDC-145 VL MGWSCIILFL VATATGVHS
secretory leader
Fab-NLDC-145 HASQNIKGWL A CDRL1
Fab-NLDC-145 KASSLQS
CDRL2
Fab-NLDC-145 QHYQSFPWT
CDRL3
huNLDC-145 VH EVKLLESGGG LVQPGGSLRL SCAASGFTFN DFYMNWIRQP PGQAPEWLGV without secretory IRNKGNGYTT EVNTSVKGRF TISRDNTQNI LYLQMNSLPA EDTAIYYCAR
GGPYYYSGDD APYWGQGVMV TVSS
leader huNLDC-145 JH WGQGVMVTVS S
CH1 ASTKGPSVFP LAPSSKSTSG GTAALGCLVK DYFPEPVTVS WNSGALTSGV
HTFPAVLQSS GLYSLSSWT VPSSSLGTQT YICNVNHKPS NTKVDKKV
HINGE EPKSCDKTHT GPPPC
Fab-NLDC-145 VH GAGGTGAAGC TGTTGGAATC TGGAGGAGGT TTGGTACAGC CGGGGGGTTC nucleotide without TCTGAGACTC TCCTGTGCAG CTTCTGGATT CACCTTCAAT GATTTCTACA
TGAACTGGAT CCGCCAGCCT CCAGGGCAGG CACCTGAGTG GTTGGGTGTT
secretory leader
ATTAGAAACA AAGGTAATGG TTACACAACA GAGGTCAATA CATCTGTGAA
GGGGCGGTTC ACCATCTCCA GAGATAATAC CCAAAACATC CTCTATCTTC
AAATGAACAG CCTGAGAGCT GAGGACACCG CCATTTACTA CTGTGCAAGA
GGCGGTCCTT ATTACTACAG TGGTGACGAC GCCCCTTACT GGGGCCAAGG
AGTCATGGTC ACAGTCTCCT CA
Fab-NLDC-145 VL ATGGGATGGA GCTGTATCAT CCTCTTCTTG GTAGCAACAG CTACAGGCGT nucleotide without GCACTCCGAC ATCCAGATGA CCCAGTCTCC ATCCTCCCTG TCTGCATCTG
TAGGAGACAG AGTCACCATC ACTTGCCATG CCAGTCAGAA CATCAAGGGT
secretory leader
TGGTTAGCCT GGTATCAGCA GAAACCAGGG AAAGCCCCTA AGCTCCTGAT
CTATAAGGCA TCTAGCCTGC AATCAGGGGT CCCATCAAGG TTCAGTGGCA
GTGGATCTGG GACAGATTTC ACTCTCACCA TCAGCAGTCT GCAACCTGAA
GATTTTGCAA CTTACTACTG TCAGCATTAT CAAAGCTTTC CGTGGACCTT
CGGTCAAGGC ACCAAGGTGG AAATCAAA
huNLDC-145 HC MGWSCIILFL VATATGVHSE VKLLESGGGL VQPGGSLRLS CAASGFTFND with secretory FYMNWIRQPP GQAPEWLGVI RNKGNGYTTE VTTSVKGRFT ISRDNTQNIL
YLQMNSLRAE DTAIYYCARG GPYYYSGDDA PYWGQGVMVT VSSASTKGPS
leader
VFPLAPSSKS TSGGTAALGC LVKDYFPEPV TVSWNSGALT SGVHTFPAVL
QSSGLYSLSS WTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSCDKTH
TGPPPC huNLDC-145 VL DIQMTQSPSS LSASVGDRVT ITCHASQNIK GWLAWYQQKP GKAPKLLIYK
ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQH YQSFPWTFGQ
GTKVEIK huNLDC-145 JK FGQGTKVEIK
CL RTVAAPSVFI FPPSDEQLKS GTASWCLLN NFYPREAKVQ WKVDNALQSG
NSQESVTEQD SKDSTYSLSS TLTLSKADYE KHKVYACEVT HQGLSSPVTK
SFNRGEC huNLDC-145 LC DIQMTQSPSF LSTSLGNSIT ITCHASQNIK GWLAWYQQKS GNAPQLLIYK
ASSLQSGVPS RFSGSGSGTD YIFTISNLQP EDIATYYCQH YQSFPWTFGG
GTKLELKRTV AAPSVFIFPP SDEQLKSGTA SWCLLNNFY PREAKVQWKV
DNALQSGNSQ ESVTEQDSKD STYSLSSTLT LSKADYEKHK VYACEVTHQG
LSSPVTKSFN RGEC
Human factor VIII MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP
(HC followed by LC PRVPKSFPFN TSWYKKTLF VEFTVHLFNI AKPRPPWMGL LGPTIQAEVY
DTWITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG
in italics)
GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE
GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM
HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH
RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE
EPQLRMKNNE EAEDYDDDLT DSEMDWRFD DDNSPSFIQI RSVAKKHPKT
WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY
TDETFKTREA IQHESGILGP LLYGEVGDTL LI I FKNQASR PYNIYPHGIT
DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR
YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE
NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL
HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS
MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDI SAYLL
SKNNAIEPRS FSQNSRHPST RQKQFNATTI PENDIEKTDP WFAHRTPMPK
IQNVSSSDLL MLLRQSPTPH GLSLSDLQEA KYETFSDDPS PGAIDSNNSL
SEMTHFRPQL HHSGDMVFTP ESGLQLRLNE KLGTTAATEL KKLDFKVSST
SNNLISTIPS DNLAAGTDNT SSLGPPSMPV HYDSQLDTTL FGKKSSPLTE
SGGPLSLSEE NNDSKLLESG LMNSQESSWG KNVSSTESGR LFKGKPAHGP
ALLTKDNALF KVSISLLKTN KTSNNSATNR KTHIDGPSLL IENSPSVWQN
ILESDTEFKK VTPLIHDRML MDKNATALRL NHMSNKTTSS KNMEMVQQKK
EGPIPPDAQN PDMSFFKMLF LPESARWIQR THGKNSLNSG QGPSPKQLVS
LGPEKSVEGQ NFLSEKNKW VGKGEFTKDV GLKEMVFPSS RNLFLTNLDN LHENNTHNQE KKIQEEIEKK ETLIQENWL PQIHTVTGTK NFMKNLFLLS
TRQNVEGSYE GAYAPVLQDF RSLNDSTNRT KKHTAHFSKK GEEENLEGLG
NQTKQIVEKY ACTTRISPNT SQQNFVTQRS KPALKQFRLP LEETELEKRI
IVDDTSTQWS KNMKHLTPST LTQIDYNEKE KGAITQSPLS DCLTRSHSIP
QANRSPLPIA KVSSFPSIRP IYLTRVLFQD NSSHLPAASY RKKDSGVQES
SHFLQGAKKN NLSLAILTLE MTGDQREVGS LGTSATNSVT YKKVENTVLP
KPDLPKTSGK VELLPKVHIY QKDLFPTETS NGSPGHLDLV EGSLLQGTEG
AIKWNEANRP GKVPFLRVAT ESSAKTPSKL LDPLAWDNHY GTQI PKEEWK
SQEKSPEKTA FKKKDTILSL NACESNHAIA AINEGQNKPE IEVTWAKQGR
TERLCSQNPP VLKRHQREIT RTTLQSDQEE IDYDDTISVE MKKEDFDIYD
EDENQSPRSF QKKTRHYFIA AVERLWDYGM SSSPHVLRNR AQSGSVPQFK
KVVFQEFTDG SFTQPLYRGE LNEHLGLLGP YIRAEVEDNI MVTFRNQASR
PYSFYSSLIS YEEDQRQGAE PRKNFVKPNE TKTYFWKVQH HMAPTKDEFD
CKAWAYFSDV DLEKDVHSGL IGPLLVCHTN TLNPAHGRQV TVQEFALFFT
IFDETKSWYF TENMERNCRA PCNIQMEDPT FKENYRFHAI NGYIMDTLPG
LVMAQDQRIR WYLLSMGSNE NIHSIHFSGH VFTVRKKEEY KMALYNLYPG
VFETVEMLPS KAGIWRVECL IGEHLHAGMS TLFLVYSNKC QTPLGMASGH
IRDFQITASG QYGQWAPKLA RLHYSGSINA WSTKEPFSWI KVDLLAPMII
HGIKTQGARQ KFSSLYISQF IIMYSLDGKK WQTYRGNSTG TLMVFFGNVD
SSGIKHNIFN PPIIARYIRL HPTHYSIRST LRMELMGCDL NSCSMPLGME
SKAISDAQIT ASSYFTNMFA TWSPSKARLH LQGRSNAWRP QVNNPKEWLQ
VDFQKTMKVT GVTTQGVKSL LTSMYVKEFL ISSSQDGHQW TLFFQNGKVK
VFQGNQDSFT PVVNSLDPPL LTRYLRIHPQ SWVHQIALRM EVLGCEAQDL Y
B-domain deleted ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSWYKKTL factor VIII (HC FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTWITLKN MASHPVSLHA
VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD
followed by LC in
PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA
italics)
VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR
KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL
MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL
TDSEMDWRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL
APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG
PLLYGEVGDT LLI I FKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD
FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RDLASGLIGP
LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN IQRFLPNPAG
VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS
VFFSGYTFKH KMVYEDTLTL FPFSGETVFM SMENPGLWIL GCHNSDFRNR
GMTALLKVSS CDKNTGDYYE DSYEDI SAYL LSKNNAIEPR SFSQNPPVLK
REQREITRTTLQSDQEEIDY DDTISVEMKK EDFDIYDEDE NQSPRSFQKK TRHYFIAAVE RLWDYGMSSS PHVLRNRAQS GSVPQFKKVV FQEFTDGSFT QPLYRGELNE HLGLLGPYIR AEVEDNIMVT FRNQASRPYS FYSSLISYEE DQRQGAEPRK NFVKPNETKT YFWKVQHHMA PTKDEFDCKA WAYFSDVDLE KDVHSGLIGP LLVCHTNTLN PAHGRQVTVQ EFALFLTIFD ETKSWYFTEN MERNCRAPCN IQMEDPTFKE NYRFHAINGY IMDTLPGLVM AQDQRIRWYL LSMGSNENIH SIHFSGHVFT VRKKEEYKMA LYNLYPGVFE TVEMLPSKAG IWRVECLIGE HLHAGMSTLF LVYSNKCQTP LGMASGHIRD FQITASGQYG QWAPKLARLH YSGSINAWST KEPFSWIKVD LLAPMIIHGI KTQGARQKFS SLYISQFIIM YSLDGKKWQT YRGNSTGTLM VFFGNVDSSG IKHNIFNPPI IARYIRLHPT HYSIRSTLRM ELMGCDLNSC SMPLGMESKA ISDAQITASS YFTNMFATWS PSKARLHLQG RSNAWRPQVN NPKEWLQVDF QKTMKVTGVT TQGVKSLLTS MYVKEFLISS SQDGHQWTLF FQNGKVKVFQ GNQDSFTPVV NSLDPPLLTR YLRIHPQSWV HQIALRMEVL GCEAQDLY I Fab-NLDC-145 VH EVKLLESGGG LVQPGGSLRL SCAASGFTFN DFYMNWIRQP PGQAPEWLGV without leader IRNKGNGYTT EVNTSVKGRF TISRDNTQNI LYLQMNSLRA EDTAIYYCAR
GGPYYYSGDD APYWGQGVMV TVSS
I Fab-NLDC-145 DIQMTQSPSS LSASVGDRVT ITCHASQNIK GWLAWYQQKP GKAPKLLIYK without leader ASSLQSGVPS RFSGSGSGTD FTLTISSLQP EDFATYYCQH YQSFPWTFGQ
GTKVEIK
I Full NLDC-145 Fab EVKLLESGGG LVQPGGSLRL SCAASGFTFN DFYMNWIRQP PGQAPEWLGV HC and LC without IRNKGNGYTT EVTTSVKGRF TISRDNTQNI LYLQMNSLRA EDTAIYYCAR
GGPYYYSGDD APYWGQGVMV TVSSASTKGP SVFPLAPSSK STSGGTAALG
secretory leader
CLVKDYFPEP VTVSWNSGAL TSGVHTFPAV LQSSGLYSLS SWTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTGPPPCDIQ MTQSPSFLST SLGNSITITC HASQNIKGWL AWYQQKSGNA PQLLIYKASS LQSGVPSRFS GSGSGTDYIF TISNLQPEDI ATYYCQHYQS FPWTFGGGTK LELKRTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNA LQSGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C
I Truncated human METDTLLLWV LLLWVPGSTG SGPAANDPFT IVHGNTGKCI KPVYGWIVAD DEC205 DCDETEDKLW KWVSQHRLFH LHSQKCLGLD ITKSVNELRM FSCDSSAMLW
WKCEHHSLYG AARYRLALKD GHGTAI SNAS DVWKKGGSEE SLCDQPYHEI
extracellular
YTRDGNSYGR PCEFPFLIDG TWHHDCILDE DHSGPWCATT LNYEYDRKWG
domain fused to a
ICLKPENGIE GRMDGGGGSG GGGSGGPSVF LFPPKPKDTL MISRTPEVTC
monomeric human
VWDVSHEDP EVKFNWYVDG VEVHNAKTKP REEQYNSTYR WSVLTVLHQ
lgG1 Fc domain
DWLNGKEYKC KVSNKALPAP IEKTISKAKG QPREPQVYTL PPSREEMTKN QVNLTCLVKG FYPSDIAVEW ESNGQPENNY KTTPPVLDSD GSFFLNSTLT VDKSRWQQGN VFSCSVLHEA LHSHYTQKSL SLSPK Full length human MRTGWATPRR PAGLLMLLFW FFDLAEPSGR AANDPFTIVH GNTGKCIKPV
DEC205 Genbank YGWIVADDCD ETEDKLWKWV SQHRLFHLHS QKCLGLDITK SVNELRMFSC accession number DSSAMLWWKC EHHSLYGAAR YRLALKDGHG TAISNASDVW KKGGSEESLC
DQPYHEIYTR DGNSYGRPCE FPFLIDGTWH HDCILDEDHS GPWCATTLNY
NP_002340 amino
EYDRKWGICL KPENGCEDNW EKNEQFGSCY QFNTQTALSW KEAYVSCQNQ
acid
GADLLSINSA AELTYLKEKE GIAKIFWIGL NQLYSARGWE WSDHKPLNFL
NWDPDRPSAP TIGGSSCARM DAESGLWQSF SCEAQLPYVC RKPLNNTVEL
TDVWTYSDTR CDAGWLPNNG FCYLLVNESN SWDKAHAKCK AFSSDLISIH
SLADVEVWT KLHNEDIKEE VWIGLKNINI PTLFQWSDGT EVTLTYWDEN
EPNVPYNKTP NCVSYLGELG QWKVQSCEEK LKYVCKRKGE KLNDASSDKM
CPPDEGWKRH GETCYKIYED EVPFGTNCNL TITSRFEQEY LNDLMKKYDK
SLRKYFWTGL RDVDSCGEYN WATVGGRRRA VTFSNWNFLE PASPGGCVAM
STGKSVGKWE VKDCRSFKAL SICKKMSGPL GPEEASPKPD DPCPEGWQSF
PASLSCYKVF HAERIVRKRN WEEAERFCQA LGAHLSSFSH VDEIKEFLHF
LTDQFSGQHW LWIGLNKRSP DLQGSWQWSD RTPVSTIIMP NEFQQDYDIR
DCAAVKVFHR PWRRGWHFYD DREFIYLRPF ACDTKLEWVC QIPKGRTPKT
PDWYNPDPAG IHGPPLIIEG SEYWFVADLH LNYEEAVLYC ASNHSFLATI
TSFVGLKAIK NKIANISGDG QKWWIRISEW PIDDHFTYSR YPWHRFPVTF
GEECLYMSAK TWLIDLGKPT DCSTKLPFIC EKYNVSSLEK YSPDSAAKVQ
CSEQWIPFQN KCFLKIKPVS LTFSQASDTC HSYGGTLPSV LSQIEQDFIT
SLLPDMEATL WIGLRWTAYE KINKWTDNRE LTYSNFHPLL VSGRLRI PEN
FFEEESRYHC ALILNLQKSP FTGTWNFTSC SERHFVSLCQ KYSEVKSRQT
LQNASETVKY LNNLYKI I PK TLTWHSAKRE CLKSNMQLVS ITDPYQQAFL
SVQALLHNSS LWIGLFSQDD ELNFGWSDGK RLHFSRWAET NGQLEDCWL
DTDGFWKTVD CNDNQPGAIC YYSGNETEKE VKPVDSVKCP SPVLNTPWIP
FQNCCYNFI I TKNRHMATTQ DEVHTKCQKL NPKSHILSIR DEKENNFVLE
QLLYFNYMAS WVMLGITYRN KSLMWFDKTP LSYTHWRAGR PTIKNEKFLA
GLSTDGFWDI QTFKVIEEAV YFHQHSILAC KIEMVDYKEE YNTTLPQFMP
YEDGIYSVIQ KKVTWYEALN MCSQSGGHLA SVHNQNGQLF LEDIVKRDGF
PLWVGLSSHD GSESSFEWSD GSTFDYI PWK GQTSPGNCVL LDPKGTWKHE
KCNSVKDGAI CYKPTKSKKL SRLTYSSRCP AAKENGSRWI QYKGHCYKSD
QALHSFSEAK KLCSKHDHSA TIVSIKDEDE NKFVSRLMRE NNNITMRVWL
GLSQHSVDQS WSWLDGSEVT FVKWENKSKS GVGRCSMLIA SNETWKKVEC
EHGFGRWCK VPLGPDYTAI AIIVATLSIL VLMGGLIWFL FQRHRLHLAG
FSSVRYAQGV NEDEIMLPSF HD
Myelin EVG YR3PF SRWHLYR G K
oligodendrocyte
glycoprotein (MOG)
35-55 B-domain deleted ATRRYYLGAV ELSWDYMQSD LGELPVDARF PPRVPKSFPF NTSWYKKTL factor VIII FVEFTDHLFN IAKPRPPWMG LLGPTIQAEV YDTWITLKN MASHPVSLHA
VGVSYWKASE GAEYDDQTSQ REKEDDKVFP GGSHTYVWQV LKENGPMASD
Heavy chain
PLCLTYSYLS HVDLVKDLNS GLIGALLVCR EGSLAKEKTQ TLHKFILLFA
VFDEGKSWHS ETKNSLMQDR DAASARAWPK MHTVNGYVNR SLPGLIGCHR
KSVYWHVIGM GTTPEVHSIF LEGHTFLVRN HRQASLEISP ITFLTAQTLL
MDLGQFLLFC HISSHQHDGM EAYVKVDSCP EEPQLRMKNN EEAEDYDDDL
TDSEMDWRF DDDNSPSFIQ IRSVAKKHPK TWVHYIAAEE EDWDYAPLVL
APDDRSYKSQ YLNNGPQRIG RKYKKVRFMA YTDETFKTRE AIQHESGILG
PLLYGEVGDT LLI I FKNQAS RPYNIYPHGI TDVRPLYSRR LPKGVKHLKD
FPILPGEIFK YKWTVTVEDG PTKSDPRCLT RYYSSFVNME RDLASGLIGP
LLICYKESVD QRGNQIMSDK RNVILFSVFD ENRSWYLTEN IQRFLPNPAG
VQLEDPEFQA SNIMHSINGY VFDSLQLSVC LHEVAYWYIL SIGAQTDFLS
VFFSGYTFKH KMVYEDTLTL FPFSGETVFM SMENPGLWIL GCHNSDFRNR
GMTALLKVSS CDKNTGDYYE DSYEDI SAYL LSKNNAIEPR SFSQNPPVLK
RHQR
B-domain deleted EITRTTLQSD QEEIDYDDTI SVEMKKEDFD IYDEDENQSP RSFQKKTRHY factor VIII FIAAVERLWD YGMSSSPHVL RNRAQSGSVP QFKKWFQEF TDGSFTQPLY
RGELNEHLGL LGPYIPAEVE DNIMVTFRNQ ASRPYSFYSS LI SYEEDQRQ
Light Chain
GAEPRKNFVK PNETKTYFWK VQHHMAPTKD EFDCKAWAYF SDVDLEKDVH
SGLIGPLLVC HTNTLNPAHG RQVTVQEFAL FLTI FDETKS WYFTENMERN
CPAPCNIQME DPTFKENYRF HAINGYIMDT LPGLVMAQDQ RIRWYLLSMG
SNENIHSIHF SGHVFTVRKK EEYKMALYNL YPGVFETVEM LPSKAGIWRV
ECLIGEHLHA GMSTLFLVYS NKCQTPLGMA SGHIRDFQIT ASGQYGQWAP
KLARLHYSGS INAWSTKEPF SWIKVDLLAP MIIHGIKTQG ARQKFSSLYI
SQFIIMYSLD GKKWQTYRGN STGTLMVFFG NVDSSGIKHN IFNPPIIARY
IRLHPTHYSI RSTLRMELMG CDLNSCSMPL GMESKAISDA QITASSYFTN
MFATWSPSKA RLHLQGRSNA WRPQVNNPKE WLQVDFQKTM KVTGVTTQGV
KSLLTSMYVK EFLISSSQDG HQWTLFFQNG KVKVFQGNQD SFTPWNSLD
PPLLTRYLRI HPQSWVHQIA LRMEVLGCEA QDLY
Human factor VIII MQIELSTCFF LCLLRFCFSA TRRYYLGAVE LSWDYMQSDL GELPVDARFP
Heavy chain PRVPKSFPFN TSWYKKTLF VEFTVHLFNI AKPRPPWMGL LGPTIQAEVY
DTWITLKNM ASHPVSLHAV GVSYWKASEG AEYDDQTSQR EKEDDKVFPG
GSHTYVWQVL KENGPMASDP LCLTYSYLSH VDLVKDLNSG LIGALLVCRE
GSLAKEKTQT LHKFILLFAV FDEGKSWHSE TKNSLMQDRD AASARAWPKM
HTVNGYVNRS LPGLIGCHRK SVYWHVIGMG TTPEVHSIFL EGHTFLVRNH
RQASLEISPI TFLTAQTLLM DLGQFLLFCH ISSHQHDGME AYVKVDSCPE
EPQLRMKNNE EAEDYDDDLT DSEMDWRFD DDNSPSFIQI RSVAKKHPKT
WVHYIAAEEE DWDYAPLVLA PDDRSYKSQY LNNGPQRIGR KYKKVRFMAY
TDETFKTREA IQHESGILGP LLYGEVGDTL LI I FKNQASR PYNIYPHGIT DVRPLYSRRL PKGVKHLKDF PILPGEIFKY KWTVTVEDGP TKSDPRCLTR
YYSSFVNMER DLASGLIGPL LICYKESVDQ RGNQIMSDKR NVILFSVFDE
NRSWYLTENI QRFLPNPAGV QLEDPEFQAS NIMHSINGYV FDSLQLSVCL
HEVAYWYILS IGAQTDFLSV FFSGYTFKHK MVYEDTLTLF PFSGETVFMS
MENPGLWILG CHNSDFRNRG MTALLKVSSC DKNTGDYYED SYEDI SAYLL
SKNNAIEPRS FSQNSRHPST RQKQFNATTI PENDIEKTDP WFAHRTPMPK
IQNVSSSDLL MLLRQSPTPH GLSLSDLQEA KYETFSDDPS PGAIDSNNSL
SEMTHFRPQL HHSGDMVFTP ESGLQLRLNE KLGTTAATEL KKLDFKVSST
SNNLISTIPS DNLAAGTDNT SSLGPPSMPV HYDSQLDTTL FGKKSSPLTE
SGGPLSLSEE NNDSKLLESG LMNSQESSWG KNVSSTESGR LFKGKPAHGP
ALLTKDNALF KVSISLLKTN KTSNNSATNR KTHIDGPSLL IENSPSVWQN
ILESDTEFKK VTPLIHDRML MDKNATALRL NHMSNKTTSS KNMEMVQQKK
EGPIPPDAQN PDMSFFKMLF LPESARWIQR THGKNSLNSG QGPSPKQLVS
LGPEKSVEGQ NFLSEKNKW VGKGEFTKDV GLKEMVFPSS RNLFLTNLDN
LHENNTHNQE KKIQEEIEKK ETLIQENWL PQIHTVTGTK NFMKNLFLLS
TRQNVEGSYE GAYAPVLQDF RSLNDSTNRT KKHTAHFSKK GEEENLEGLG
NQTKQIVEKY ACTTRISPNT SQQNFVTQRS KPALKQFRLP LEETELEKRI
IVDDTSTQWS KNMKHLTPST LTQIDYNEKE KGAITQSPLS DCLTRSHSIP
QANRSPLPIA KVSSFPSIRP IYLTRVLFQD NSSHLPAASY RKKDSGVQES
SHFLQGAKKN NLSLAILTLE MTGDQREVGS LGTSATNSVT YKKVENTVLP
KPDLPKTSGK VELLPKVHIY QKDLFPTETS NGSPGHLDLV EGSLLQGTEG
AIKWNEANRP GKVPFLRVAT ESSAKTPSKL LDPLAWDNHY GTQI PKEEWK
SQEKSPEKTA FKKKDTILSL NACESNHAIA AINEGQNKPE IEVTWAKQGR
TERLCSQNPP VLKRHQR
Human factor VIII EITRTTLQSD QEEIDYDDTI SVEMKKEDFD IYDEDENQSP RSFQKKTRHY
Light chain FIAAVERLWD YGMSSSPHVL RNRAQSGSVP QFKKWFQEF TDGSFTQPLY
RGELNEHLGL LGPYIPAEVE DNIMVTFRNQ ASRPYSFYSS LI SYEEDQRQ
GAEPRKNFVK PNETKTYFWK VQHHMAPTKD EFDCKAWAYF SDVDLEKDVH
SGLIGPLLVC HTNTLNPAHG RQVTVQEFAL FFTI FDETKS WYFTENMERN
CPAPCNIQME DPTFKENYRF HAINGYIMDT LPGLVMAQDQ RIRWYLLSMG
SNENIHSIHF SGHVFTVRKK EEYKMALYNL YPGVFETVEM LPSKAGIWRV
ECLIGEHLHA GMSTLFLVYS NKCQTPLGMA SGHIRDFQIT ASGQYGQWAP
KLARLHYSGS INAWSTKEPF SWIKVDLLAP MIIHGIKTQG ARQKFSSLYI
SQFIIMYSLD GKKWQTYRGN STGTLMVFFG NVDSSGIKHN IFNPPIIARY
IRLHPTHYSI RSTLRMELMG CDLNSCSMPL GMESKAISDA QITASSYFTN
MFATWSPSKA RLHLQGRSNA WRPQVNNPKE WLQVDFQKTM KVTGVTTQGV
KSLLTSMYVK EFLISSSQDG HQWTLFFQNG KVKVFQGNQD SFTPWNSLD
PPLLTRYLRI HPQSWVHQIA LRMEVLGCEA QDLY
Figures Figure 1 shows that dendritic cells expressing DEC-205 selectively recognise and internalise nanoparticles with a covalently bound DEC-205 targeting ligand in vitro. The graph plots geometric mean fluorescent intensity (MFI; a measure of nanoparticle uptake) against time of incubation (minutes) for non-functionalised nanoparticles (NF-NPS), DEC- 205-targeted nanoparticles (NLDC - targeting ligand densities of 1 :5 and 1 :1 used) and nanoparticles functionalised with a ligand that does not target DEC-205 (8.8).
Figure 2 shows that dendritic cells expressing DEC-205 selectively recognise and internalise nanoparticles with a covalently bound DEC-205 targeting ligand in vivo. There is significant DEC-205-targeted nanoparticle uptake by CD8a+ dendritic cells compared to control Fab-conjugated and non-functionalized (untargeted) nanoparticles.
Figure 3 shows that CD8 + dendritic cells isolated from mouse spleen have the highest level of DEC-205 expression in comparison to B cells (B220+), neutrophils, macrophages, myeloid dendritic cells (CD1 1 b+ DC) and monocytes.
Figure 4 shows that free glucocorticoid receptor agonists suppress the activation of JAWSM dendritic cells (measured in terms of TNFa release) when stimulated with LPS.
Figure 5 shows that nanoparticle encapsulated glucocorticoid receptor agonists suppress the activation of JAWSM dendritic cells (measured in terms of TNFa release) when stimulated with LPS.
Figure 6 shows that free glucocorticoid receptor agonists suppress the activation of human whole blood cells (measured in terms of TNFa release) when stimulated with LPS
Figure 7 shows that nanoparticle encapsulated glucocorticoid receptor agonists suppress the activation of human whole blood cells (measured in terms of TNFa release) when stimulated with LPS.
Figure 8 shows that nanoparticles co-encapsulating ovalbumin and betamethasone- 17-valerate (PF575) cause the greatest amount of Foxp3+ T regulatory cell induction in vitro as compared with nanoparticles encapsulating ovalbumin alone (PF556) and nanoparticles encapsulating ovalbumin alone + free beta-methasone-17-valerate in solution (PF556 + FD).
Figure 9 shows that nanoparticles co-encapsulating a protein antigen and a glucocorticoid receptor agonist reduce cognate T cell expansion in vivo. Figure 10 shows the size distribution curve for a batch of DEC-205 targeted nanoparticles encapsulating B-domain deleted Factor VIM and betamethasone dipropionate Figure 1 1 shows the selective delivery of DEC-205 targeted nanoparticles encapsulating factor VI II and betamethasone dipropionate to JAWSI I dendritic cells in vitro
Figures 12-13 show that nanoparticle agglomeration during the targeting ligand conjugation process can be prevented by controlling the pH during conjugation Figure 14 shows that nanoparticle uptake by DEC-205-expressing cells varies with targeting ligand density
Figure 1 5 shows that targeting ligand density can be controlled by adjusting the ratio of targeting ligand to nanoparticle in the conjugation reaction
Figure 16 shows that the treatment of mice with nanoparticles containing the peptide MOG (relevant to multiple sclerosis) prior to immune challenge with MOG limited the production of MOG-specific antibodies.
General preparative methods
The nanoparticles of the invention are preferably prepared by first fabricating the polymeric matrix in the presence of the antigen and glucocorticoid receptor agonist in order to form particles encapsulating both these agents. The targeting ligand for DEC-205 is then attached to the exterior of the particles so formed.
Specifically, the nanoparticles may be prepared using a double-emulsion process in which a first aqueous phase containing the antigen (referred to as "w ') is emulsified with an immiscible organic phase containing the polymer and the glucocorticoid receptor agonist (referred to as "o") to form a first water-in-oil emulsion. Formation of this w^o emulsion is achieved by coarse mixing with a rotor-stator, followed by homogenization on a homogenizer. This first emulsion w-i/o is then further emulsified into a second aqueous phase (referred to as "w2") to form a water-in-oil-in-water double emulsion
Figure imgf000038_0001
Formation of a
Figure imgf000038_0002
double emulsion is also achieved by coarse mixing with a rotor- stator, followed by homogenization on a homogenizer. The solvent is then removed to leave discrete polymeric particles containing antigen and glucocorticoid receptor agonist. During this process, the protein is retained within aqueous droplets that fill pores in the polymeric matrix and the glucocorticoid receptor agonist becomes lodged directly between the polymeric chains, being lipophilic.
The first aqueous phase (w^ containing the antigen is generally an aqueous buffer in which is suitable for solubilising and stabilising the antigen. For example, factor VII I or B- domain deleted factor VIII may be formulated in an aqueous buffer containing 19 millimolar histidine, 3.4 millimolar calcium chloride, 250 millimolar arginine (pH 7.1). The antigen is present at a concentration of at least 0.5 mg/mL, preferably at a concentration of 5 mg/mL or greater. With PLGA and PLGA-PEG polymers, sodium chloride should be excluded from the aqueous phase in order to prevent the nanoparticles from becoming too porous.
The organic phase (o) preferably has a polymer concentration of 10-100 mg/mL. An organic solvent such as dichloromethane, chloroform, or ethyl acetate is typically used and the glucocorticoid content is 5-30 % (weight/weight) with respect to the initial polymer content. The organic phase may optionally contain an emulsifying agent with a low hydrophobic-lipophilic (HLB) value (typically less than 10).
The volume of the first aqueous phase is typically 1 to 20 times less than the volume of the organic phase (preferably 5 to 10 times less).
Generally, the double emulsion needs to be stabilised at the interface between the organic phase and the second aqueous phase (w2) and the second aqueous phase therefore contains an stabilising emulsifier with a high HLB value, typically greater than about 10. Preferably, polyvinyl alcohol) is used as the stabilising agent, more preferably polyvinyl alcohol) with a molecular weight of 30-70 kDa and 87-90% hydrolysed. The amount of stabliliser required is typically 1 -10% (weight/volume).
The second aqueous phase generally has a volume 2-10 times greater than the w^o solution.
Emulsion formation is achieved in each case by high-energy mixing, e.g. using a rotor-stator or sonicator followed by high-pressure homogenisation (e.g. using a Microfluidics (Westwood, MA) processor).
Solvent may be removed in order to generate the particles by means of evaporation or by particle wash through a tangential flow filtration device.
Examples
Embodiments of the present invention are illustrated by the following Examples. It is to be understood, however, that the embodiments of the invention are not limited to the specific details of these Examples, as other variations thereof will be known, or apparent in light of the instant disclosure, to one of ordinary skill in the art. Example 1 - Fabrication and characterization of nanoparticles containing the antigen ovalbumin and various glucocorticoid receptor agonists
A 100 mg amount of glucocorticoid receptor agonist (see Table 3 below) and 1 g of polymer (see Table 3 below) was dissolved in 20 ml_ of dichloromethane. To this solution, 2 mL of 5 mg/mL aqueous endotoxin free ovalbumin (Hyglos, Germany) was added, and then mixed with a rotor-stator. The entire volume of this coarse emulsion was then homogenized on a Microfluidics instrument for 5 complete cycles through an F20Y processor at a pressure of 18000 psi. The resulting emulsion was then added to 100 mL of 1 % aqueous polyvinyl alcohol), mixed under a rotor stator, and then homogenized for 5 cycles on the Microfluidics instrument with F20Y processor at 5000 psi for 4 passes. This emulsion was transferred to an open beaker with 100 mL water, and then continuously stirred for approximately 18 hours to evaporate off the organic solvent. Subsequently, the particles were pelleted by centrifugation at 35000 rcf for at least 10 minutes at 4 °C. The supernatant was removed and the particle pellet was resuspended with water and washed. The wash and centrifugation step was repeated 1 to 3 more times. After the final wash, the nanoparticles were resuspended at 5 mg/mL or less in 10% (w/v) sucrose, frozen, and then lyophilized.
The total yield of nanoparticle was determined by gravimetric measurement of an aliquot in water. The encapsulation of glucocorticoid receptor agonist was measured by using reverse phase high performance liquid chromatography (RP-HPLC) with an absorbance detector. Specifically, nanoparticles were dissolved in 100% dimethyl sulfoxide (DMSO) or dimethylformamide (DMF), and then analysed by RP-HPLC using a gradient protocol consisting of 0.1 % trifluoroacetic acid (TFA) in acetonitrile and 0.1 % TFA in water. Glucocorticoid receptor agonist absorbance was detected at 239 nm. For measurement of ovalbumin content, an SDS-PAGE was performed with Coomassie or silver stain, followed by a densitometric image analysis of the gel to quantify protein content. In formulations that contain a fluorescent tracer, the amount of fluourescence from a dissolved sample can be measured with a fluorimeter, such as a Safire2 (Tecan, Switzerland), and compared to a standard curve. The size and charge of the nanoparticle is measured by dynamic light scattering, using a Malvern nanosizer.
For measurements of drug release in buffer, the dialysis method was used.
Table 3 - Nanoparticles containing various glucocorticoid receptor agonists and ovalbumin Experiment Polymer Glucocorticoid Glucocorticoid Ovalbumin number receptor agonist receptor loading by agonist loading SDS-PAGE (Mg/mg) by (ug/mg) HPLC
1 80% PLGA-PEG, 20% Betamethasone 64.64 ± 7.42 5.07
PLGA-FKR6481 dipropionate
2 PLGA-PEG Beclamethasone 72.49 ± 7.1 1 5.63
dipropionate
3 80% PLGA-PEG, 20% Dexamathasone 230.63 ± 3.67 5.3
PLGA-FKR6481 pa Imitate
4 PLGA-PEG Dexamethasone 1 1 .58 ± 0.17 1 1 .86
dipropionate
5 PLGA-PEG Betamethasone 39.19 ± 3.40 5.24
valerate
1 . FKR648 is a fluorophore allowing visualisation of the nanoparticles
Example 2 - Fabrication and characterization of nanoparticles containing the antigen
Factor VIII and betamethasone dipropionate A 1 g polymer mixture of 50% (weight/weight) PLGA(20K)-PEG-maleimide, 30%
(weight/weight) PLGA(20K)-PEG, and 20% (weight/weight) PLGA-FKR648 was dissolved in 20 mL of dichloromethane with 100 mg of betamethasone dipropionate. To this solution, a 2 mL aqueous solution of FVIM at 10 mg/ml was added drop-wise. The resulting emulsion was mixed with a rotor-stator at 8000 rpm, and then transferred to a Microfluidics homogenizer (M-1 10P) and processed for 5 passes at 18000 psi through an F20Y chamber. Next, the resulting mixture was added to 100 mL of deionized water containing 1 % PVA, mixed with a rotor-stator at 5500 rpm, and then transferred to a Microfluidics homogenizer and processed for four passes at 5000 psi through an F20Y chamber.
The particulate suspension was stirred overnight in an open beaker to remove organic solvent, and then centrifuged at 30000 rcf for 25 min. The supernatant was decanted, and the pellet was washed with water. This centrifugation and wash step was repeated twice. The particles were finally suspended in a 10% (w/v) sucrose solution, frozen, and then lyophilized. Particle size was measured by dynamic light scattering, using a Malvern Zetasizer (Nano ZS model). The encapsulation efficiency of FVIII was measured with a BCA assay, and the betamethasone dipropionate loading was measured by reverse phase HPLC.
Table 4 - Nanoparticles containing a glucocorticoid receptor agonist and factor VIII
Figure imgf000042_0001
1 . FKR648 is a fluorophore allowing visualisation of the nanoparticles
Example 3 - Conjugation of a DEC-205 targeting Iigand using maleimide chemistry
A DEC-205 targeting Iigand comprised of Fab protein with a single C-terminal cysteine (~50 kDa MW) was buffer exchanged into phosphate buffer, pH 6.5. A reducing agent, tris(2-carboxyethyl)phosphine (TCEP), was used to reduce the C-terminal cysteine to give a free thiol (from any disulfide bound residual free cysteine or glutathione from protein expression). After reduction and buffer exchange into reaction buffer (20 mM phosphate, 150 mM NaCI, 1 mM EDTA, pH 7.0), an Ellman's assay was used to confirm the reduction of the Fab cysteine to a single free thiol. An appropriate aliquot of maleimide-functionalized PEG-PLGA nanoparticles (Mal-
NP, 15 mg) was added to a 100 kD MWCO centrifuge filter unit, CFU, and centrifuged (2100 rpm, 4°C) for at least 90 minutes and until the final volume was less than 750 μΙ_. The concentrated Mal-NP solution was removed from the CFU. The filter membrane was washed with reaction buffer (20 mM phosphate, 150 mM NaCI, 1 mM EDTA, pH 7.0) to recover residual Mal-NP and pooled with the solution. The final volume was brought to 750 μΙ_ with reaction buffer, for a theoretical concentration of 20 mg/mL of Mal-NP. The size and charge of the nanoparticles were measured using a Malvern Zetasizer instrument.
For conjugation of targeting Iigand to Mal-NP, a concentrated 2x solution of targeting Iigand with a single free thiol was added in equal volume to the 20 mg/mL (2x) solution of Mal-NP. The amount of accessible maleimide on the surface of the nanoparticle was assayed using a thiol-containing fluorophore reagent. The concentration of the targeting ligand solution will be dependent on the molar ratio targeted. As an example, a 1 :20 (targeting ligand:Mal-NP) molar ratio was tested for conjugation. The amounts needed in the example reaction are shown in Table 5 below:
Table 5 - Conjugation using maleimide chemistry
Figure imgf000043_0001
The conjugation reaction was incubated on an orbital shaker (250 rpm) at ambient temperature (approx. 22°C) for 2 hours. The reaction was quenched with 0.4 mM Cysteine to quench any free maleimides. The targeting ligand-conjugated nanoparticles, TL-NP, were centrifuged to filter out all unreacted/un-associated targeting ligand using 100 kD MWCO CFUs. Three centrifugations were completed, each for 1 .5 hours (2100 rpm, 4°C) with subsequent addition of fresh storage buffer (20 mM histidine, pH 5.8, 8.5% sucrose) to the retentate solution. The TL-NPs in the retentate were analyzed for size and charge upon completion of each centrifugation. Each filtrate wash was collected for analysis (Reverse- phase HPLC method to determine targeting ligand conjugated-PEG-PLGA polymer).
Analysis of the TL-NPs included target ligand density by reverse-phase high performance liquid chromatography (HPLC) with a C4 column using in-line detectors such as UV-VIS, Fluorescence and Evaporative Light-Scattering Detection (ELSD) or Charged Aerosol Detection (CAD). This RP-HPLC method measures both the conjugated and free targeting ligand associated with the nanoparticle. The amount of free targeting ligand in solution not associated with TL-NP was measured by RP-HPLC analysis of the final centrifugation filtrate. Particle size and size distribution was measured by dynamic light scattering (DLS) and zeta potential and zeta potential distribution by Laser Doppler Micro- electrophoresis. Nanoparticle concentration was measured using either a freeze-dried weight, an HPLC assay of the polymer components or a plate based assay using a modified Dragendorff reagent for PEG quantitation. Nanoparticle morphology was analyzed using cryo-transmission electron microscopy (cryo-TEM).
Example 4 - Generation of recombinant human DEC-205 protein containing monomeric human IgG Fc
Recombinant human DEC-205 extracellular domain (ECD) proteins were constructed from synthetic fragments ordered from Integrated DNA Technologies (IDT; Coralville, IA) and cloned using conventional molecular biological techniques. Complementary deoxyribonucleic acid (cDNA) was generated for truncated human and mouse DEC-205 ECD fragments based on full length sequence (accession numbers NP_002340; NP_038853). The ECD fragment was then cloned into the mammalian expression vector pSMED2 containing the canonical Factor Xa cleavage sequence (IEGRMD) and a human immunoglobulin gammal (lgG1) Fc domain that has been modified to prevent dimerization (J. Biol. Chem., 2013, 288(23), 16529-16537). The vector was sequence-confirmed and transiently transfected into Freestyle 293 human embryonic kidney cells (HEK) (Life Technologies, Grand Island, NY) according to the manufacturer's method and expressed over 120 hours. Soluble protein of interest was purified using standard protein A chromatographic techniques (Protein A FF, GE Healthcare, Piscataway NJ) followed by gel filtration size exclusion chromatography (Superdex200, GE Healthcare, Piscataway NJ). Purified protein was characterized for purity and activity by binding ELISA using commercially-available anti-human DEC-205 monoclonal antibodies (eBiosciences, San Diego, CA).
Example 5 - Use of ELISA to measure binding of anti-DEC-205 proteins to DEC-205- Fc Protein Constructs
Human or mouse DEC-205-Fc protein or a negative control (non-specific protein-Fc fusion) was coated overnight at 4°C on 96 well Nunc Maxisorp plates (Thermo Fisher Scientific, Madison, CT) at a concentration of 1 μg/mL in PBS. Plates were washed three times using PBS + 0.05% TWEEN-20 and blocked for 1 hour at room temperature in 3% milk/PBS. Samples prepared in block buffer were added to the blocked plates for 1 hour at room temperature. Plates were washed three times as before, prior to the addition of secondary antibody (either anti-human IgG-HRP 1 :4000 (Southern Biotech, Birmingham, AL) or anti penta-histidine 1 :2000 (Qiagen, Valencia, CA). Plates were incubated for a further 1 hour at room temperature and washed three times as before. Signal was developed using TMB (SurModics, Eden Prairie, MN), the reaction stopped with H2S04, and the absorbance read at 450 nm on an EnVision plate reader (Perkin Elmer, Waltham, MA).
Example 6 - Generation of anti-DEC-205 NLDC-145 Fab
Rat-anti-murine DEC-205 hybridoma NLDC-145 was obtained from American Type
Culture Collection (Kraal et al., J, Exp. Med., 1986, 163, 981 -997). The complementary deoxyribonucleic acid (cDNA) sequence encoding the immunoglobulin gamma (IgG) variable regions (VH and VL) for NLDC-145 was determined using conventional molecular biological techniques and the manufacturer's protocols unless otherwise noted. Briefly, one million NLDC-145 hybridoma cells were homogenized for total RNA isolation with QIAGEN RNAeasy Mini kit (Valencia, CA). First strand cDNA was then produced using Superscript III RT kit (LifeTechnologies, Grand Island, NY). Double stranded cDNAs for variable regions (VH and VL) of NLDC-145 were generated by PCR using primers from the rat IgG heavy chain (lgG1 , 2a, 2b) and light chain (kappa or lambda) constant regions, as described below. PCR cycling conditions: 1 cycle at 95°C for 1 min; 25 cycles at 95°C for 1 min, 63°C for 1 min and 72 °C for 1 min. The resulting RT-PCR products were cloned into TOPO-Blunt cloning vector (LifeTechnologies, Grand Island, NY) and sequenced by conventional methods. Following sequence confirmation of the rat-anti-DEC-205 variable regions, a human chimeric Fab was generated as follows: rat constant domains CL1 and CH1 were replaced with human constant domains CL1 and CH1 . The rat hinge region was also replaced with a modified human hinge region. Mutations for site-specific maleimide conjugation were made to the lower hinge region positions C226G, C229P, P230C (EU numbering). Additionally, a single point mutation was introduced in the VH position 62 (N62T; Kabat). Expression vectors for the chimeric NLDC-145 Fab VH and VL were then synthesized (Blue Heron, Bothell, WA) and sequence-confirmed.
Example 7 - Purification of engineered Fab constructs
Following transient transfection, conditioned media (CM) was clarified by ultrafiltration and loaded onto a 46 mL CaptureSelect KappaXL column (GE Healthcare, Piscataway, NJ) previously equilibrated with PBS. The CM was loaded at 25-30 mL/min overnight at 4°C. The column was then washed with 10 column volumes (CV) of PBS pH 7.2. The protein of interest was step-eluted with 20 mM citric acid, pH 3.5 for 5 CVs, then immediately neutralized with 5% of 1 .0 M Tris-HCI, pH 8.0. The captured pools were loaded onto a 320 mL Superdex75 gel filtration column (GE Healthcare, Piscataway, NJ) to remove high molecular weight (HMW) species. The peaks were pooled based on the analytical size exclusion (SEC) using a TSK G3000 SEC column (Tosoh Bioscience, King of Prussia, PA) and filtered through a 0.22μηι PES membrane. The final pool was analyzed by OD280 (NanoDrop, Thermo Fisher Scientific, Madison, CT), SDS-PAGE (LifeTechnologies, Grand Island, NY), analytical SEC, mass spectrometry and endotoxin level by Limulus Amebocyte Lysate (LAL) assay (Charles River, Wilmington, MA). Less than 1 % high HMW species was observed by analytical SEC.
Example 8 - Conjugation of targeting ligand to nanoparticles and prevention of agglomeration
Targeting ligand (DEC-205 specific Fab bearing a C-terminal cysteine) was reduced using 2x molar excess of reducing agent (tris (2-carboxyethyl)phosphine (TCEP). The reduction step was allowed to proceed at ambient temperature for 30 minutes. After reduction, the excess TCEP was removed while the reduced targeting ligand was buffer exchanged to reaction buffer and concentrated using 10 kD MWCO centrifugal filter units (CFU) to 4 mg/ml. The control reaction buffer was 20 mM sodium phosphate, 150 mM sodium chloride, 1 mM EDTA, pH 7.0. The final concentration of the targeting ligand was > 40 mg/ml.
An equal volume of 20 mg/ml nanoparticle suspension (bearing maleimide functional groups on the polymer matrix) was added to 4 mg/ml DEC-205-specific Fab for all reactions (1 :5 molar ratio of targeting ligand to nanoparticle). The reactions were incubated on an orbital shaker at 250 rpm at room temperature for 2 hours. The reactions were quenched using 0.4 mM cysteine. The size of the nanoparticles was measured prior to purification, to check the integrity of the nanoparticles following conjugation.
Several conditions were tested by varying the procedure described above. These conditions included lowering the concentrations of both nanoparticle and targeting ligand (an equal volume of 10 mg/ml nanoparticle suspension was added to 2 mg/ml DEC-205-specific Fab), performing the reaction at 4°C, using sonication during the reaction, and changing the reaction pH to 6.0 from 7.0. The conditions tested are set out in Table 6 below:
Table 6 - Conjugation of nanoparticle and targeting ligand
Reaction pH Reaction Incubation conditions temperature
Control 7 Ambient (~22°C) 2 h, Rotator, 250 rpm
Diluted reaction 7 Ambient (~22°C) 2 h, Rotator, 250 rpm mixture
Cooled reaction 7 4°C 2 h, Rotator, 250 rpm mixture
Sonicated reaction 7 Ice bath (~0°C) 2 h, sonicated, low mixture energy
Low pH reaction 6 Ambient (~22°C) 2 h, Rotator, 250 rpm mixture
Size measurements were made at the start of each reaction, after 2 h, and post quenching. Figures 12 and 13 below show the size distribution of samples at t = 0 (before conjugation) and t = 2 hours (after conjugation). Figure 12 shows the results of the control reaction. Figure 13 shows the results of reducing the pH of the reaction. The only experiment in which nanoparticle population size and shape was not significantly affected during the conjugation reaction was the one conducted at a reaction pH of 6.0.
Example 9 - In vitro assay determining DEC-205 targeting Fluorescently-labelled, functionalized nanoparticles were fabricated using the
Figure imgf000047_0001
double emulsion method (see Example 1 ) with 50% maleimide-PEG-PLGA (MW = 5k:20k Da; 50:50 lactic acid:glycolic acid), 20% FKR648-PLGA (20k Da), and 30% PEG-PLGA (5k:20k Da; 50:50 lactic acid:glycolic acid) (polymers sourced from PolySciTech). These maleimide surface-functioned nanoparticles were conjugated to either a DEC-205 targeting ligand (clone NLDC-145 Fab fragments) or a control targeting ligand without DEC-205 specificity (clone 8.8 Fab fragments) at a 1 :1 and 1 :5 targeting ligand/maleimide ratio (see Example 3). Nanoparticles conjugated to DEC-205 targeting ligands (denoted by NLDC) were compared to nanoparticles conjugated to control targeting ligands (denoted by Fab) in a JAWSII uptake assay. To perform the assay, 200,000 cells of a JAWSII mouse dendritic cell line are added to a 96 well plate. Nanoparticles are added at a final concentration of 0.2 mg/mL (based on Dragondorff assay concentrations), with the assay performed simultaneously at 37°C and on ice. At completion of the time course, the 37°C plate is chilled in an ice bath, and washed 3x with ice-cold buffer (PBS/2mM EDTA/0.5% FBS/0.02%NaN3), with cells fixed in 2% formaldehyde for 20min before washing and reading on an LSR Fortessa cytometer. Upon acquisition and data analysis, line plots were constructed from cytometer geometric mean fluorescent intensity values using GraphPad software. Nanoparticles were incubated with JAWSII cells at 37deg for the indicated length of time (T=0, 5, 15, 30, 60, 120, 180 and 240 minutes), whereupon cells were washed and assessed for nanoparticle internalization by detection of the FKR-648 fluorophore by fluorescent-activated cell sorting. As shown in Figure 1 , DEC-205-targeted nanoparticles (NLDC:NP) exhibited increased internalization rates in comparison to nanoparticles with a ligand that is not specific for DEC-205 (Fab:NP) and nanoparticle without any targeting ligand (NF-NPS). Furthermore, nanoparticles with a higher density of attached targeting ligand (1 :1 NLDC:NP) were recognised and internalised faster than nanoparticles with a lower density of attached targeting ligand (1 :5 NLDC:NP).
Example 10 - In vivo assay determining DEC-205 targeting
DEC-205 targeted nanoparticles target CD8a+DEC205+ CD1 1 c+ dendritic cells in comparison to untargeted nanoparticles. C57BL/6 mice were injected with 0.5mg of either DEC-205 targeted (NLDC145 1 :1 PF554,) or untargeted (PF129, PF130, 8.8 1 :1 PF554 and PF541 ) nanoparticles containing 20% FKR648-PLGA as a fluorescent label. At T=5, 60, 300 and 1440 minutes, two mice per time-point were sacrificed and engagement of nanoparticles by CD8a+DEC205+ dendritic cells in the spleen was assessed. Upon sacrifice of mice, spleens were removed and stored at room temperature in PBS/2mM EDTA / 0.5% FBS in Miltenyi GentleMACS C-tubes until further processing. Spleens were reduced to single-cell suspensions using an OctoMACS, with red blood cell lysis subsequently performed on both blood and spleen single-cell suspension samples. Lymphocytes were washed extensively in fluorescent activated cell sorting (FACS) buffer (PBS/2mM EDTA/0.5% FBS), with conventional dendritic cells identified by FACS using CD8a-AF488+ and CD1 1 c-BV421 + antibodies. Upon gating on CD8a+ conventional dendritic cells, the association of DEC-205 targeted or untargeted nanoparticles was determined by FKR648 signal. Figure 2 shows the engagement of DEC-205 targeted nanoparticles (denoted NLDC) by spleen CD8 + dendritic cells in comparison to untargeted nanoparticles, with each number representing the percent of cells having engaged a nanoparticle. CD8a+ dendritic cells are known to have the highest level of DEC-205 expression in comparison to B cells (B220+), neutrophils, macrophages, myeloid dendritic cells (CD1 1 b+ DC) and monocytes (see Figure 3). Of note, no untargeted nanoparticles were found to be associated with CD8a+ dendritic cells.
Example 1 1 - In vitro assay determining functional tolerogenic activity of nanoparticles
Tolerogenic dendritic cells are characterized by their resistance to producing inflammatory cytokines in response to a strong inflammatory stimulus. The potency of a therapeutic in maintaining a tolerogenic state can be ranked by its ability to inhibit the production of inflammatory cytokines by dendritic cells stimulated by lipopolysaccharide (LPS). To rank the ability of PLGA nanoparticles encapsulating various glucocorticoids to maintain a tolerogenic functionality upon LPS stimulation, JAWSII cells were pre-incubated with either nanoparticles or soluble glucocorticoids, with TNFa release assessed upon LPS stimulation. JAWSII cells were seeded the morning of the assay into 384-well plates. In parallel, nanoparticle preparations were normalized to 10μΜ of drug, with soluble glucocorticoids normalized to the same concentration. Cells were pre-incubated for 2 hours with either DMSO vehicle, nanoparticles, or soluble glucocorticoids, whereupon LPS was added at a final concentration of 100 ng/ml for an additional 18 hours at 37°C, 5% C02. At the end of the incubation period, supernatants were removed and stored, and TNFa concentrations were measured by a mouse TNFa R&D Systems ELISA. The results are shown in Figure 4 (soluble glucocorticoid receptor agonist) and Figure 5 (nanoparticle encapsulated glucocorticoid receptor agonist). The IC50 (in units of M) of soluble dexamethasone was observed to be 2.41 -2.61 E-09, betamethasone diproprionate to be between 7.8-8.01 E-10, and betamethasone valerate to be 5.17-7.02E-10; while nanoparticle-encapsulated dexamethasone exhibited an IC50 of 2.61 -3.74E-09, betamethasone diproprionate of 9.42-9.88E-10, and betamethasone valerate of 1 .72-1 .89E- 10.
Another method of assessing the potency of glucocorticoid-encapsulated nanoparticles is to co-culture either nanoparticles or soluble compound with human whole blood. Heparinized human whole blood from two different donors was diluted 1 :10 final with complete RPMI / 10%FBS. Diluted blood was aliquoted to a 96-well plate, with soluble glucocorticoids or nanoparticle-encapsulated glucocorticoids subsequently added and pre- incubated for 5 minutes. LPS was added at a concentration of 0.2ng/ml, with samples incubated for 18 hours, with TNFa levels assessed by ELISA (R&D Systems Quantkine) after 18hr incubation. The results are shown in Figure 6 (soluble glucocorticoid receptor agonist) and Figure 7 (nanoparticle encapsulated glucocorticoid receptor agonist). Soluble dexamethasone, betamethasone diproprionate, beclomethasone diproprionate, and betamethasone valerate exhibited IC50s of 3.5E-9, 2.0E-9, 4.3E-9 and 3.4E-10 M respectively. PLGA nanoparticle encapsulated dexamethasone, betamethasone diproprionate, beclomethasone diproprionate and betamethasone valerate exhibited IC50s of 4.7E-10, 7.2E-10, 8.0E-10 and 3.4E-1 1 M. Notably, PLGA-encapsulated rapamycin did not impact TNFa release in this assay. Furthermore, rapamycin delivered in a soluble manner was found to increase TNFa production in comparison to vehicle. Example 12 - In vitro assay demonstrating increased potency of nanoparticle- delivered glucocorticoid receptor agonist versus soluble compound and DEC-205 targeted nanoparticle-delivered glucocorticoid receptor agonist versus untargeted nanoparticles
Co-delivery of nanoparticle-encapsulated ovalbumin with betamethasone-17 valerate increases the generation of CD4+OT-II+ inducible T regulatory cells in vitro (Figure 8). At all doses tested, nanoparticle encapsulating ovalbumin (OVA) and beta methasone-17 valerate (PF575, Figure 8) resulted in an approximate 2 fold increase in the percentage of FoxP3+ T cells observed compared with nanoparticle encapsulating ovalbumin alone (PF556, Figure 8). Similarly, nanoparticle encapsulating ovalbumin in the presence of free solubilised beta methasone-17 valerate (PF556 + FD, Figure 8) also resulted in an approximate 2 fold increase in the percentage of FoxP3+ T cells observed compared with nanoparticle encapsulating ovalbumin alone (PF556, Figure 8). At expected therapeutic levels (103-205 nM) a greater induction of Foxp3+ T regulatory cells was observed with nanoparticle encapsulating ovalbumin and beta methasone-17 valerate (PF575, Figure 8) as compared with nanoparticle encapsulating ovalbumin in the presence of free solubilised beta methasone-17 valerate (PF556 + FD, Figure 8), indicating a significant advantage to co- encapsulating both protein antigen together with betamethasone-17 valerate. These results are consistent with the capacity of glucocorticoids to induce tolerance by decreasing the co- stimulatory signal strength provided by dendritic cells, manifesting in an increased conversion of CD4+ T cells intra regulatory T cells at sites of inflammation. To perform this experiment, bone marrow derived dendritic cells (0.5x106/well) were incubated with titrating doses of nanoparticles encapsulating ovalbumin (PF-556), nanoparticles encapsulating ovalbumin and free solubilised betamethasone 17-valerate (PF-556+FD) and nanoparticles encapsulating both albumin and betamethasone 17-valerate for 4 hours followed by overnight maturation with LPS (50ng/ml). The following day, dendritic cells were washed, counted and re-plated at 2x104 cells/well in a 96 well plate. Violet labelled splenic CD4+ OT- II T-cells (1x105cells/well) were co-cultured with the matured BM-dendritic cells in the presence of interleukin-2 (5ng/ml), and as a control, titrating doses of whole ovalbumin were normalized to encapsulated ovalbumin (nM) was included separately. On day 6, cells were harvested, and stained for Foxp3 expression and analysed in conjunction with Violet dilution. The data is presented as the replicate average for each NP/whole OVA dilution point.
Example 13 - In vivo assay measuring the functional tolerogenic activity of nanoparticles Nanoparticles co-encapsulating protein antigen and a glucocorticoid receptor agonist suppress cognate T cell expansion in vivo (Figure 9). CD45.1 + congenically marked C57BL/6 mice (strain B6.S J L-Pfprca Pepc VBoyJ; Jackson Laboratory) were adoptively transferred with labelled 2x10E6 CD4+CD45.2+ OT II Ova-specific T cells on Day 0. To obtain labelled OT II T cells, spleens of B6.129S6-f?ag2imiFwaTg(TcraTcrb)425Cbn (Taconic) were harvested and reduced to single cell suspension using GentleMACS. After erythrocyte lysis, cells were counted and labelled with the proliferation tracking dye CellTracker Violet (Invitrogen) at a concentration of 5μΜ. Cells were then washed, the frequency of OT II cells determined by FACS, and mice injected intravenously in the contralateral tail vein using insulin syringes with cells in cold PBS. The next day, PLGA nanoparticles loaded with either 300μg of ovalbumin only or ovalbumin together with 0.61 mgs per kg of rapamycin, 0.29 mgs per kg of betamethasone diproprionate or 0.21 mgs per kg beclomethasone diproprionate were injected i.v. in the contralateral tail vein. After 72 hours (Day 4), mice were euthanized, spleens harvested and reduced to single-cell suspensions by GentleMACS, and erythrocytes lysed. After counting, the expansion of OT2 T cells in spleens were assessed and quantified by FACS by using anti-CD45.2, anti-CD4, anti-CD3 and anti-CD45 antibodies and AquaLiveDead viability stain (Invitrogen). All three nanoparticles incorporating ovalbumin in combination with an immunosuppressant achieved reduced cognate T cell expansion in comparison with nanoparticles incorporating ovalbumin alone.
Example 14 - DEC-205 targeted nanoparticles co-encapsulating Factor VIII and a glucocorticoid receptor agonist efficiently target DEC-205-expressing cells in vitro
DEC-205 targeted nanoparticles (PF584) loaded with B-domain deleted Factor VIII and betamethasone diproprionate were formulated using the double emulsion method and a polymer blend of 50% PLGA-PEG-Maleimide, 20% PLGA-FKR648, and 30% PLGA-PEG. FKR648 is a fluorescent label.
The B-domain deleted factor VIII was reformulated to ~20 fold higher concentration (based on the commercial formulation) to enable the desired loading of BDD-FVIII in the nanoparticles. To enhance solubility and stability, B-domain deleted Factor VIII was formulated at 10-12 mg/mL concentration in a sodium chloride-free medium consisting of 19mM histidine, 3.4mM calcium chloride and 250mM arginine (pH 7.1). This formulation was used as the inner aqueous phase (w^ during the encapsulation process.
The characteristics of the nanoparticles are shown in Table 7 below. A plot of size distribution is shown in Figure 10. Table 7 - Characterisation of maleimide functionalised nanoparticles encapsulating factor VIII and betamethasone dipropionate.
Figure imgf000052_0001
B-domain deleted Factor VIII was loaded at 1 .5% w/w loading, or 15.76 μg/mg loading as determined by BCA assay. Betamethasone diproprionate was loaded at 9.4% w/w loading, or 94.29 +/- 4.2 μg/mg. PF584 was conjugated either to NLDC-145 Fab (NT162), 8.8 Fab (NT161), or to free cysteine (Cys, NT163), with a targeting ligand density of 215.85 μg/mg for NLDC-145 and 176.32 g/mg for 8.8. To assess the ability of either DEC-205 targeted or untargeted PF584 to target DEC-205 expressed on the surface of JAWSII cells, an in vitro uptake assay was performed. Nanoparticles at a concentration of 0.2 mg/ml were added to 100,000 JAWSII cells in a 96 well plate, and either kept on ice or at 37 degrees Celsius for the duration of the binding assay. At 15 and 240 minutes, cells were washed three times with cold PBS and fixed with 2% formaldehyde for 20 minutes. Samples were run on an LSR Fortessa, and the geographic mean fluorescent intensity (GMFI) of FKR648 by JAWSII cells was determined. Samples were graphed using geometric mean fluorescent intensity (see Figure 1 1 for 37°C data). DEC-205 nanoparticles demonstrate a strikingly greater engagement of JAWSII cells in comparison to 8.8 and Cys controls both at 4 and 37 degrees, indicating efficient targeting of FVIII/betamethasone diproprionate co- encapsulated nanoparticles.
Example 15 - DEC-205 targeted particles containing myelin oligodendrocyte glycoprotein (MOG) and betamethasone dipropionate attenuated MOG-specific antibodies in mice challenged with Complete Freud's Adjuvant (CFAVMOG Anti-DEC-205 nanoparticles containing MOG35-55 peptide and betamethasone dipropionate (NT191 , NT192, NT193, NT196) were formulated as described in Table 8. These particles were administered i.v. two times, a week apart, to C57BL/6 female mice (day -14 and day - 7), at a dose of approximately 0.04 mpk MOG peptide and 1 .2 mpk betamethasone dipropionate. One week after the last dose (day 0), mice were challenged with CFA/MOG35-55. Plasma from days 10, 17, and 24 after challenge with CFA/MOG were analyzed by ELISA for the amount of MOG35-55 antibodies. Treatment with DEC205 targeted particles that contained MOG35-55 peptide and betamethasone dipropionate led to reduced MOG-specific antibodies, relative to mice that did not receive any nanoparticles, and with greater effect than non-targeted particles observed on day 17 (Fig. 16). Treatment with non-targeted particles was eventually able to achieve similar reductions by day 24. Moreover administration of nanoparticles containing betamethasone dipropionate and an irrelevant protein (ovalbumin, OVA) had less of an impact on reducing MOG-specific antibody titers.
Table 8 - Summary of nanoparticle characteristics used in studies of MOG/CFA challenge
Figure imgf000053_0001
The 10 treatment protocols illustrated in Fig. 16, from left to right are: (1) CFA challenge alone without MOG; (2) MOG immunization alone; (3) pre-treatment with DEC205-targeted (NLDC) nanoparticles containing MOG and betamethasone dipropionate before MOG immunization; (4) pre-treatment with Fab8 targeted nanoparticles containing MOG and betamethasone dipropionate before MOG immunization; (5) pre-treatment with untargeted nanoparticles containing MOG and betamethasone dipropionate before MOG immuniazation; (6) pre-treatment with DEC205-targeted nanoparticles containing MOG before MOG immunization; (7) pre-treatment with DEC205-targeted nanoparticles containing OVA and betamethasone dipropionate before MOG immunization.

Claims

Claims
1 . A nanoparticle comprising a polymeric matrix and a covalently-bound surface- orientated targeting ligand for DEC-205, said polymeric matrix encapsulating an antigen which is the subject of an undesirable immune response and a glucocorticoid receptor agonist.
2. A nanoparticle according to claim 1 , wherein said polymeric matrix comprises a block co-polymer of poly(lactic-co-glycolic acid) and poly(ethylene glycol) or a mixture of poly(lactic-co-glycolic acid) and a block co-polymer of poly(lactic-co-glycolic acid) and poly(ethylene glycol).
3. A nanoparticle according to claim 2, wherein said polymeric matrix consists of a block co-polymer of poly(lactic-co-glycolic acid) and poly(ethylene glycol) or a mixture of poly(lactic-co-glycolic acid) and a block co-polymer of poly(lactic-co-glycolic acid) and poly(ethylene glycol).
4. A nanoparticle according to claim 2 or claim 3, wherein the ratio of lactic acid to glycolic acid is about 1 :1 and the average molecular weight of the poly(lactic-co-glycolic acid) and poly(ethylene glycol) in each chain is from 15,000 to 50,000 daltons (e.g. about 20,000 daltons) for the poly(lactic-co-glycolic acid) and from 2,500 to 20,000 daltons (e.g. about 5000 daltons) for the poly(ethylene glycol).
5. A nanoparticle according to any one of claims 2 to 4, wherein the targeting ligand is attached to the poly(ethylene glycol) chains.
6. A nanoparticle according to any one of claims 1 to 5, wherein the DEC-205 targeting ligand is a fragment of a full-length monoclonal antibody (Fab) for DEC-205.
7. A nanoparticle according to any one of claim 6, wherein the DEC-205 targeting ligand comprises the amino acid sequence SEQ ID NO: 28.
8. A nanoparticle according to any one of claims 1 to 7, wherein the targeting ligand density on the surface of the nanoparticle is from about 1 nmol to about 20 nmol targeting ligand per mg of nanoparticle polymer matrix.
9. A nanoparticle according to any one of claims 1 to 8, wherein the targeting ligand is attached to the polymer matrix by means of bonds formed by the cycloaddition of an azide moiety on the polymer matrix with an alkyne group borne on the targeting ligand.
10. A nanoparticle according to any one of claims 1 to 9, wherein the encapsulated glucocorticoid receptor agonist comprises from about 1 to about 20% by weight of the total dry mass of the nanoparticle.
1 1 . A nanoparticle according to any one of claims 1 to 10, wherein the encapsulated glucocorticoid receptor agonist has a logP of from about 3 to about 7.
12. A nanoparticle according to claim 1 1 , wherein the encapsulated glucocorticoid receptor agonist is betamethasone-17-valerate or betamethasone-17,21-dipropionate.
13. A nanoparticle according to any one of claims 1 to 12, wherein the encapsulated antigen comprises from about 0.25 to about 5% by weight of the total dry mass of the nanoparticle.
14. A nanoparticle according to any one of claims 1 to 13, wherein the encapsulated antigen is human factor VIII comprising the amino acid sequence of SEQ ID NO: 24, or a fragment thereof, such as a beta-domain deleted sequence comprising the amino acid sequence of SEQ ID NO: 25.
15. A process for preparing nanoparticles according to any one of claims 1 to 14, wherein:
(i) the antigen is solubilized in an aqueous solution (w at a concentration of at least 0.5 mg/mL, preferably at least 5mg/ml;
(ii) the antigen solution from step (i) is homogenised with an organic phase (o) that contains the glucocorticoid and polymer to form an w^o emulsion;
(iii) the w^o emulsion is added to a second aqueous solution (w2) containing an emulsifier with an HLB value greater than about 10 and homogenised to form a
Figure imgf000056_0001
double emulsion;
(iv) solvent is removed to form the nanoparticles; and (v) a targeting ligand is conjugated to the surface of the nanoparticles.
16. The process according to claim 15, wherein step (v) is performed at a pH of from about 5 to about 7.
17. A pharmaceutical composition suitable for intravenous administration comprising nanoparticles according to any one of claims 1 to 13, and a pharmaceutically acceptable excipient.
18. A pharmaceutical composition suitable for intravenous administration comprising nanoparticles according to claim 14, and a pharmaceutically acceptable excipient.
19. A nanoparticle according to any one of claims 1 to 13 or a pharmaceutical composition as defined in claim 17, for use in the treatment of an autoimmune disease or other disease associated with an undesirable immune response.
20. A nanoparticle according to claim any one of claims 1 to 14, or a pharmaceutical composition according to claim 17 or claim 18, for use in the treatment of haemophilia A.
21 . A nanoparticle according to any one of claims 1 to 13, or a pharmaceutical composition according to claim 17, for use in the treatment of multiple sclerosis.
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Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10265415B2 (en) 2010-08-10 2019-04-23 École Polytechnique Fédérale De Lausanne (Epfl) Compositions for inducing antigen-specific tolerance
EA032386B1 (en) * 2018-03-19 2019-05-31 Общество С Ограниченной Ответственностью "Марлин Биотех" Method for delivery of nuclear acids to the heart by means of plga nanoparticles
US10392437B2 (en) 2010-08-10 2019-08-27 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US10800838B2 (en) 2010-08-10 2020-10-13 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US10821157B2 (en) 2014-02-21 2020-11-03 Anokion Sa Glycotargeting therapeutics
US10940209B2 (en) 2014-02-21 2021-03-09 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US10946079B2 (en) 2014-02-21 2021-03-16 Ecole Polytechnique Federale De Lausanne Glycotargeting therapeutics
US10953101B2 (en) 2014-02-21 2021-03-23 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
WO2021108637A1 (en) * 2019-11-26 2021-06-03 Cedars-Sinai Medical Center Compositions and methods for treating diseases and conditions by depletion of mitochondrial or genomic dna from circulation
CN113030281A (en) * 2019-12-24 2021-06-25 重庆华邦制药有限公司 Method for separating compound D and/or J
US11253579B2 (en) 2017-06-16 2022-02-22 The University Of Chicago Compositions and methods for inducing immune tolerance

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020187131A1 (en) * 1995-01-31 2002-12-12 Daniel Hawiger Enhanced antigen delivery and modulation of the immune response therefrom
US20140328854A1 (en) * 2013-05-03 2014-11-06 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers and therapeutic macromolecules for reduced or enhanced pharmacodynamic effects

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20020187131A1 (en) * 1995-01-31 2002-12-12 Daniel Hawiger Enhanced antigen delivery and modulation of the immune response therefrom
US20140328854A1 (en) * 2013-05-03 2014-11-06 Selecta Biosciences, Inc. Tolerogenic synthetic nanocarriers and therapeutic macromolecules for reduced or enhanced pharmacodynamic effects

Non-Patent Citations (16)

* Cited by examiner, † Cited by third party
Title
"Bailliere's Clinical Haematology", vol. 11, 1998, pages: 287 - 303
"Bioreversible Carriers in Drug Design", 1987, PERGAMON PRESS
"Textbook of Haemophilia", 2011, JOHN WILEY AND SONS
BLOOD, vol. 119, 2012, pages 1335 - 1344
H BUNDGAARD: "Design of Prodrugs", 1985, ELSEVIER
HAEMOPHILIA: THE OFFICIAL JOURNAL OF THE WORLD FEDERATION OF HAEMOPHILIA, vol. 13, no. 2, 2007, pages 10 - 15
J. BIOL. CHEM., vol. 288, no. 23, 2013, pages 16529 - 16537
JOURNAL OF THROMBOSIS AND HAEMOSTASIS, vol. 7, 2009, pages 787 - 794
KRAAL ET AL., J, EXP. MED., vol. 163, 1986, pages 981 - 997
MALDONADO R A ET AL: "Polymeric synthetic nanoparticles for the induction of antigen-specific immunological tolerance", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA 20150113 NATIONAL ACADEMY OF SCIENCES USA, vol. 112, no. 2, 13 January 2015 (2015-01-13), pages E156 - E165, XP002764343, ISSN: 0027-8424 *
NEW ENGLAND JOURNAL OF MEDICINE, vol. 318, 1988, pages 947 - 950
SALUJA SANDEEP S ET AL: "Targeting human dendritic cells via DEC-205 using PLGA nanoparticles leads to enhanced cross-presentation of a melanoma-associated antigen", INTERNATIONAL JOURNAL OF NANOMEDICINE, vol. 9, 2014, pages 5231 - 5246, XP002764344 *
T HIGUCHI; W STELLA: "Pro-drugs as Novel Delivery Systems", vol. 14, ACS SYMPOSIUM SERIES
THE LANCET, vol. 339, 1992, pages 594 - 598
THROMBOSIS AND HAEMOSTASIS, vol. 110, 2013, pages 1114 - 1120
THROMBOSIS AND HAEMOSTASIS, vol. 79, 1998, pages 762 - 766

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