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WO2001022995A1 - Procede de preparation de conjugues d'un antigene et d'un composant de liaison muqueux - Google Patents

Procede de preparation de conjugues d'un antigene et d'un composant de liaison muqueux Download PDF

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Publication number
WO2001022995A1
WO2001022995A1 PCT/DK2000/000531 DK0000531W WO0122995A1 WO 2001022995 A1 WO2001022995 A1 WO 2001022995A1 DK 0000531 W DK0000531 W DK 0000531W WO 0122995 A1 WO0122995 A1 WO 0122995A1
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WIPO (PCT)
Prior art keywords
insulin
crosslinker
ctb
antigen
binding component
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/DK2000/000531
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English (en)
Inventor
Karen De Jongh
Jacob Sten Petersen
John Forstrom
Charles R. Petrie
Are Bogsnes
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Novo Nordisk AS
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Novo Nordisk AS
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Priority to AU74048/00A priority Critical patent/AU7404800A/en
Publication of WO2001022995A1 publication Critical patent/WO2001022995A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/0005—Vertebrate antigens
    • A61K39/0008—Antigens related to auto-immune diseases; Preparations to induce self-tolerance
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • 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
    • A61K47/62—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 a protein, peptide or polyamino acid
    • A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
    • A61K47/6415—Toxins or lectins, e.g. clostridial toxins or Pseudomonas exotoxins
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/60—Medicinal preparations containing antigens or antibodies characteristics by the carrier linked to the antigen
    • A61K2039/6031—Proteins
    • A61K2039/6037—Bacterial toxins, e.g. diphteria toxoid [DT], tetanus toxoid [TT]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/62—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier
    • A61K2039/627—Medicinal preparations containing antigens or antibodies characterised by the link between antigen and carrier characterised by the linker

Definitions

  • the present invention relates to a method for preparing a product of conjugates between an antigen and a mucosal binding component, e.g. conjugates between insulin and cholera toxin B subunit (CTB), which product can be used inter a ⁇ a as a means for inducing immunological tolerance and hence suppressing autoimmunity.
  • a mucosal binding component e.g. conjugates between insulin and cholera toxin B subunit (CTB)
  • CTB cholera toxin B subunit
  • the cholera toxin B subunit consists of five identical proteins, each of about 11.6 kDa, that form a doughnut-shaped pentamer into which the toxic A subunit, which is the active agent in inducing diarrhoeal disease, is inserted (Sixma, T.K., Pronk, S.E., Kalk, K.H., Wartna, E.S., van Zanten, B.A.M., Witholt, B. and Hoi, W.G.J. (1991 ) Nature 351 : 371-7).
  • the B subunit pentamer allows the toxin to bind with high affinity to ganglioside G M ⁇ in the gut, and the A subunit is then translocated into the cell where ADP- ribosylation of the G s subunit of adenylate cyclase induces increased cyclic AMP formation and secretion of electrolytes that leads to the life-threatening diarrhoea of cholera.
  • CTB cholera toxin B subunit
  • NOD mouse model of diabetes One of the animal models in which potentiation of oral tolerance by conjugation to CTB has been demonstrated is the NOD mouse model of diabetes. Oral administration of insulin has been shown to delay onset of diabetes in these animals when administered at mg levels (Zhang, Z.J., Davidson, L., Eisenbarth, G. and Weiner, H.L. (1991 ) Proc. Natl. Acad. Sci. 88: 10252-6).
  • the product can be administered in vivo for inducing specific immunological tolerance, inter alia peripheral immunological tolerance.
  • the product can be utilised in vitro as unique research tools, inter alia, for increasing the understanding of specific immunological tolerance.
  • a crosslinker is a compound, which is capable of covalently binding two molecules together. After the reaction, the crosslinker, or a part of the crosslinker, form a part of the linkage between the conjugated molecules.
  • Molecules are referred to as being conjugated if they are covalently bond to each other through one or more crosslinker molecules.
  • An antigen is herein defined as a component capable of invoking immunological tolerance.
  • the antigen may be the target antigen or a fragment or derivative thereof, or a bystander antigen.
  • a target antigen is an antigen for which immunological tolerance is desired.
  • the target antigen will also be the target of an unwanted immunological response (already underway or for which the subject is at risk), and an object will be to decrease, delay, or reduce the risk of the response.
  • a bystander antigen is an antigen, which is antigenically distinct from the target antigen but can substitute for the target antigen in invoking specific immunological tolerance. Usually, a bystander antigen is expressed in the same tissue in the vicinity of the target antigen.
  • Insulin peptide refers to insulin, as well as allelic and synthetic variants, fragments, fusion peptides, conjugates and other derivatives, that contain a region that is homologous (preferably 70% identical, more preferably 80% identical and even more preferably 90% identical at the amino acid level) to at least 10 (preferably 20) consecutive amino acids of insulin, wherein the homologous region of the derivative shares with the insulin an ability to induce tolerance to the target antigen.
  • homologous preferably 70% identical, more preferably 80% identical and even more preferably 90% identical at the amino acid level
  • Insulin as used herein is bovine insulin, porcine insulin, human insulin, including any analogue thereof wherein one or more amino acids have been deleted and/or replaced by other amino acids, including non-codeable amino acids, or human insulin comprising additional amino acids, i.e. more than 51 amino acids; and including any derivative thereof wherein at least one organic substituent is bound to one or more of the amino acids.
  • a mucosal binding component refers to one or more molecules, which either by themselves or in connection with each other are capable of specifically binding to the mucosal cells of the subject treated.
  • mucosal binding component shall thus comprise both a complex of molecules capable of specifically binding to the mucosal cells, for example a multimer of identical subunits or a multimer comprising different subunits, and the individual subunits thereof even though the latter might not by themselves be able to specifically bind to the mucosal cells. If the mucosal binding component is able to bind to the mucosal cells it will also be referred to as a biologically active mucosal binding component. In some instances, the mucosal binding component has the additional characteristic of penetrating or translocating across the mucosal surface.
  • the cholera toxin B subunit refer not only to the intact pentamer subunit, but also to allelic and synthetic variants, fragments, fusion peptides, conjugates and other derivatives, that contain a region that is homologous (preferably 70% identical, more preferably 80% identical and even more preferably 90% identical at the amino acid level) to at least 10 (preferably 30) consecutive amino acids of the CTB subunit, wherein the homologous region of the derivative has mucosal binding activity as well as the monomeric subunits thereof.
  • Crosslinker derivatives of antigen shall refer to antigen derivatised with one or more crosslinker residues.
  • Crosslinker derivatives of antigen and crosslinker derivatives of insulin peptide may for example also be referred to using terms such as derivatised antigen or derivatised insulin peptide, respectively.
  • Crosslinker derivatives of mucosal binding component shall refer to mucosal binding component derivatised with one or more crosslinker residues.
  • Crosslinker derivatives of mucosal binding component and crosslinker derivatives of cholera toxin B subunit may for example also be referred to using terms such as derivatised mucosal binding component or derivatised cholera toxin B subunit, respectively.
  • Activating a crosslinker derivative shall be broadly understood as any treatment of the crosslinker derivative resulting in the crosslinker part of the derivative being converted to a form which can interact with another crosslinker, for example as part of a crosslinker derivative, not treated this way.
  • a crosslinker derivative treated in such a manner may also be referred to as an activated crosslinker derivative of for instance an antigen or an insulin peptide or simply activated antigen or activated insulin peptide.
  • Active immunological tolerance refers to a state in which the tolerance effect(s) are the result of an ongoing biological process; for example, down-regulation of specific effector cells by suppressor cells. Active tolerance can be demonstrated by cell mixing or cell transfer experiments. The following two examples of experimental results (conducted with appropriate controls) are evidence of active immunological tolerance: a) when leucocytes from a tolerized animal are mixed with specific effector cells from a second animal and the activity of the effector cells is diminished; b) when leucocytes from a tolerized animal are transferred to a second animal having a autoimmune disease, and features of the disease are reduced.
  • Invoking immunological tolerance refers to at least one of the following effects: a) a decreased level of a specific immunological response (thought to be mediated at least in part by antigen-receptor effector T lymphocytes, B lymphocytes, antibody, or their equivalents); b) a delay in the onset or progression of a specific immunological response; or c) a reduced risk of the onset or progression of a specific immunological response.
  • immunological tolerance occurs when immunological tolerance is preferably invoked against certain antigens in comparison with others.
  • Sustained immunological tolerance is immunological tolerance that measurably persists for at least 3 weeks.
  • the present invention relates to a method for preparing a product of conjugates between an antigen and a mucosal binding component which method comprises a) reacting the antigen with a first crosslinker thereby producing a mixture of crosslinker derivatives of the antigen, b) isolating the antigen derivatised with a single crosslinker residue, c) activating the isolated crosslinker derivative of the antigen, d) reacting the mucosal binding component with a second crosslinker thereby producing a mixture of crosslinker derivatives of the mucosal binding component, e) reacting the activated crosslinker derivative of the antigen with the mixture of crosslinker derivatives of the mucosal binding component, thereby producing the conjugates between the antigen and the mucosal binding component.
  • the mucosal binding component has G M ⁇ binding activity when present in the product of conjugates between an antigen and said mucosal binding component.
  • the mucosal binding component is the cholera toxin B subunit (CTB).
  • the mucosal binding component is the E. coli heat-labile enterotoxin B subunit (LTB).
  • the mucosal binding component is selected among such as those disclosed in WO 98/47529, which is incorporated herein by reference. An assay for measuring the G M 1 binding activity is also disclosed in WO 98/47529.
  • the antigen is selected from the group of autoantigens (including, but not limited to, insulin peptides, collagen peptides, PLP and myelin basic protein (MBP)), alloantigens (including, but not limited to, HLA class I and II), xenoantigens, or allergens.
  • the antigen may be a polypeptide, polynucleotide, carbohydrate, glycolipid, or other molecule isolated from a biological source, or it may be a chemically synthesized small molecule, polymer, or derivative of a biological material, providing it has the ability of inducing tolerance when conjugated to the mucosal binding component.
  • the antigen is selected among such as those disclosed in WO 98/47529, which is incorporated herein by reference.
  • the antigen is an insulin peptide.
  • the insulin peptide is insulin.
  • the insulin peptide is selected among the precursor form of insulin (comprising an AAK amino acid sequence linking the B chain to the A chain), a single-chain form and a form containing a signal peptide for secretion.
  • the insulin peptide is a recombinant human insulin.
  • the insulin peptide is the A chain of insulin alone, or the B chain of insulin alone.
  • the insulin peptide is selected among the mature B chain residues 1-12, 10-22 or 11-30.
  • the insulin peptide is selected among metabolically inactive forms of insulin, metabolically inactive insulin fragments, and metabolically inactive insulin analogues.
  • metabolically inactive insulin analogues are such as those disclosed in WO 98/47529, which is incorporated herein by reference.
  • the first crosslinker is a bifunctional crosslinker (i.e. with two functional groups), preferably a heterobifunctional crosslinker (i.e. with two different functional groups).
  • the second crosslinker is a bifunctional crosslinker, preferably a heterobifunctional crosslinker.
  • the first and/or the second crosslinker is selected from the non-limiting group of N-succinimidyl(4-iodoacetyl)aminobenzoate (SIAB), N-succinimidyl-3-(2- pyridylthio)propionate (SPDP), N-succinimidyl S-acetylthioacetate (SATA), m- maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), N-g-maleimidobutyryloxy-succinimide ester (GMBS).
  • SIAB N-succinimidyl(4-iodoacetyl)aminobenzoate
  • SPDP N-succinimidyl-3-(2- pyridylthio)propionate
  • SATA N-succinimidyl S-acetylthioacetate
  • MBS m- maleimidobenzoyl-N-hydroxy
  • first and/or the second crosslinker is Traut's Reagent 2-iminothiolane in combination with SPDP.
  • first and/or the second crosslinker is succinimidyl dicarbonyl pentane or disuccinimidyl suberate.
  • first and/or the second crosslinker is selected among such as those disclosed in The 1999/2000 Pierce Products Catalogue (Pierce Chemical Company, 1999, USA) and the Double-AgentsTM Cross-Linking Reagents Selection Guide (Pierce Chemical Company, 1998, USA), which are incorporated herein by reference.
  • the isolated crosslinker derivative of the antigen is substantially free of antigen derivatised with more than one crosslinker. In a further embodiment less than 10 percent (preferably 5 percent) of the isolated crosslinker derivative of the antigen is antigen derivatised with more than one crosslinker.
  • the first crosslinker is a bifunctional crosslinker.
  • the bifunctional crosslinker is a heterobifunctional crosslinker.
  • the second crosslinker is a bifunctional crosslinker.
  • the bifunctional crosslinker is a heterobifunctional crosslinker.
  • the antigen is an insulin peptide.
  • the mucosal binding component as present in the product of conjugates between an antigen and said mucosal binding component has G . binding activity.
  • the present invention relates to a method for preparing a product of conjugates between an insulin peptide and a mucosal binding component which method comprises a) reacting the insulin peptide with a first crosslinker thereby producing a mixture of crosslinker derivatives of the insulin peptide, b) isolating the insulin peptide derivatised with a single crosslinker residue, c) activating the isolated crosslinker derivative of the insulin peptide, d) reacting the mucosal binding component with a second crosslinker thereby producing a mixture of crosslinker derivatives of the mucosal binding component, e) reacting the activated crosslinker derivative of the insulin peptide with the mixture of crosslinker derivatives of the mucosal binding component, thereby producing the conjugates between the insulin peptide and the mucosal binding component.
  • the mucosal binding component as present in the product of conjugates between an antigen and said mucosal binding component has G M . binding activity.
  • mucosal binding component is the cholera toxin B subunit (CTB).
  • the first crosslinker is a bifunctional crosslinker, preferably a heterobifunctional crosslinker.
  • the second crosslinker is a bifunctional crosslinker, preferably a heterobifunctional crosslinker.
  • the first and the second crosslinker is a pyridyl disulphide-containing crosslinker.
  • the first and the second pyridyl disulphide-containing crosslinker is N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP).
  • an insulin peptide derivatised with a pyridyl disulphide-containing crosslinker is activated by converting the pyridyl disulphide group on the derivatised insulin peptide to a thiol group using a reducing agent.
  • the reducing agent is added in an equimolar ratio to the concentration of 2- pyridyl disulphide (SSPY) groups of the derivatised insulin peptide.
  • SSPY 2- pyridyl disulphide
  • the pyridyl disulphide group on the derivatised insulin peptide is converted to a thiol group using Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP).
  • TCEP is added in an equimolar ratio to the concentration of 2-pyridyl disulphide groups of the derivatised insulin peptide. In another special embodiment TCEP is added in a molar ratio of about 1.6:1 to the concentration of derivatised insulin peptide.
  • the derivatised insulin peptide is isolated by preparative reverse-phase HPLC (RP-HPLC).
  • the activated insulin peptide is purified before step e). In a further embodiment the activated insulin peptide is purified by preparative reverse-phase HPLC. In another further embodiment the activated insulin peptide is purified by membrane filtration, such as for instance ultrafiltration or diafiltration. In a further embodiment of the above method the unreacted pyridyl disulphide- containing crosslinker is removed from the pyridyl disulphide-containing crosslinker derivatives of the mucosal binding component before step e).
  • the unreacted pyridyl disulphide-containing crosslinker is removed from the pyridyl disulphide- containing crosslinker derivatives of the mucosal binding component by size exclusion chromatography, such as for instance gel filtration.
  • the unreacted pyridyl disulphide-containing crosslinker is removed from the pyridyl disulphide- containing crosslinker derivatives of the mucosal binding component by use of membrane filtration, such as for instance ultrafiltration.
  • step e) the activated insulin peptide is added in an equimolar amount to the 2-pyridyl disulphide groups of the mixture of pyridyl disulphide protected derivatives of the mucosal binding component.
  • the product of conjugates between the antigen and the mucosal binding component is purified after step e).
  • the product of conjugates between the antigen and the mucosal binding component is purified by size exclusion chromatography, such as for instance gel filtration, membrane filtration, such as for instance ultrafiltration, or ion-exchange chromatography or a combination thereof.
  • the present invention relates to a method for preparing a product of conjugates between an insulin peptide and the cholera toxin B subunit (CTB) which method comprises a) reacting the insulin peptide with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) thereby producing a mixture of pyridyl disulphide-containing crosslinker derivatives of the insulin peptide, b) isolating the insulin peptides derivatised with a single pyridyl disulphide group, c) converting the pyridyl disulphide group on the isolated insulin peptide to a thiol group thereby producing a thiolated insulin peptide, d) reacting the cholera toxin B subunit (CTB) with N-succinimidyl 3-(2-pyridyldithio)- propionate (SPDP) thereby producing a mixture of pyri
  • the insulin peptide is insulin.
  • the isolated insulin peptide in step b), is derivatised with a single pyridyl disulphide group at the amino terminal of the B chain.
  • the molar ratio of SPDP to the insulin peptide is from about 1 :1 to about 2.5:1.
  • the molar ratio of SPDP to the insulin peptide is from about 1 :1 to about 2:1.
  • the molar ratio of SPDP to the insulin peptide is from about 1.1 :1 to 1.5:1.
  • the molar ratio of SPDP to the insulin peptide is 1.13:1.
  • step e) the thiolated insulin peptide is added to the pyridyl disulphide-containing crosslinker derivatives of CTB in a molar ratio of thiolated insulin peptide to pentameric derivatised CTB of about 4:1.
  • the present invention relates to a method for preparing a product of conjugates between an insulin peptide and the cholera toxin B subunit (CTB) which method comprises a) reacting the insulin peptide with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP) thereby producing a mixture of pyridyl disulphide-containing crosslinker derivatives of the insulin peptide, b) isolating the insulin peptides derivatised with a single pyridyl disulphide group by preparative reverse-phase HPLC, c) converting the pyridyl disulphide group on the isolated insulin peptide to a thiol group thereby producing a thiolated insulin peptide using Tris-(2-carboxyethyl)-phosphine hydrochloride (TCEP), d) reacting the cholera toxin B subunit (CTB) with N-succin
  • SPDP N-
  • the present invention relates to a product obtainable by any one of the methods defined above.
  • the present invention relates to a product of conjugates between an antigen and a mucosal binding component wherein the individual conjugate is characterised by consisting of one mucosal binding component conjugated to one or more antigens.
  • the antigen is an insulin peptide.
  • the mucosal binding component as present in the product of conjugates between an antigen and said mucosal binding component has G M . binding activity.
  • the mucosal binding component is the cholera toxin B subunit (CTB).
  • the present invention relates to a product of conjugates between an insulin peptide and the cholera toxin B subunit (CTB) wherein the individual conjugate is characterised by consisting of one cholera toxin B subunit (CTB) conjugated to one or more insulin peptide units.
  • the product of conjugates has an insulin to CTB ratio of from about 1 :1 to about 1 :20.
  • the product of conjugates has an insulin to CTB ratio of from about 1 :2 to about 1 :10, preferably from about 1 :3 to about 1 :5, more preferably of about 1 :4 (the insulin to CTB ratios are based on the pentamer of CTB).
  • the present invention relates to the use of an insulin specific T- cell hybridoma assay for the characterization of the antigen presenting potentiation of a conjugate between an antigen and a mucosal binding component.
  • the present invention relates to a pharmaceutical composition comprising, as an active ingredient, any one of the above-defined products together with a pharmaceutically acceptable carrier or diluent.
  • the composition in unit dosage form comprises from about 0.01 to 100 mg of the product.
  • the present invention relates to a pharmaceutical composition for inducing specific immunological tolerance, the composition comprising, as an active ingredient, any one of the above defined products together with a pharmaceutically acceptable carrier or diluent.
  • the present invention relates to a pharmaceutical composition for treating an autoimmune disease or allergic condition, the composition comprising, as an active ingredient, any one of the above-defined products together with a pharmaceutically acceptable carrier or diluent.
  • the pharmaceutical composition is for oral, nasal, or pulmonary administration.
  • the present invention relates to a method of inducing specific immunological tolerance of a mammal, the method comprising administering to said mammal an effective amount of any one of the above-defined products or any one of the above- defined compositions.
  • the effective amount of the product is in the range of from about 0.00001 to about 10 mg/kg body weight per day.
  • the administration is carried out by the oral, nasal, or pulmonary route.
  • the present invention relates to the use of any one of the above- defined products for the preparation of a medicament.
  • the present invention relates to the use of any one of the above-defined products for the preparation of a medicament for inducing specific immunological tolerance in a mammal.
  • the present invention relates to the use of any one of the above-defined products for the preparation of a medicament for treating an autoimmune disease or allergic reaction in a mammal.
  • the autoimmune disease is selected among such as those disclosed in WO 98/47529, which is incorporated herein by reference.
  • the present invention relates to the use of any of the above defined products or pharmaceutical compositions for inducing tolerance in the context of tissue transplantation.
  • the product can be used for decreasing the risk of rejection in a recipient of a tissue graft transplanted from a donor, by administering the product or composition to a mucosal surface of the recipient. Both xenografts and allografts are included.
  • the product can be used for decreasing the risk of graft-versus-host disease in a recipient of a tissue graft transplanted from a donor, by administering the product or composition to a mucosal surface of the recipient.
  • the antigen is a single HLA antigen, an HLA antigen cocktail, or a cell or cell derivative from one or more individuals.
  • the product can be used for improving transplantation of a tissue graft from a donor into a recipient.
  • the present invention relates to the use of any of the above defined products or pharmaceutical compositions for inducing immunological tolerance to target antigens on the mucosal surface.
  • target antigens preferentially associated with mucosal disorders, exemplified by inflammatory bowel disease, irritable bowel syndrome, ulcerative colitis, Celiac disease, and Chron's disease.
  • the products of the invention may be administered alone or in combination with pharmaceutically acceptable carriers or excipients, in either single or multiple doses.
  • the pharmaceutical compositions according to the invention may be formulated with pharmaceutically acceptable carriers or diluents as well as any other known adjuvants and excipients in accordance with conventional techniques such as those disclosed in Remington: The Science and Practice of Pharmacy, 19 th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995.
  • compositions may be specifically formulated for administration by any suitable route such as the oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), transdermal, intracisternal, intraperitoneal, vaginal, and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intra-dermal) route.
  • suitable route such as the oral, rectal, nasal, pulmonary, topical (including buccal and sublingual), transdermal, intracisternal, intraperitoneal, vaginal, and parenteral (including subcutaneous, intramuscular, intrathecal, intravenous and intra-dermal) route.
  • the pharmaceutical composition is intended for mucosal administration.
  • Selection of a route of administration of a pharmaceutical composition to a mucosal surface will in turn depend, inter alia, on the general condition and age of the subject to be treated, on nature of the clinical condition being treated, on the conjugate chosen, and the ease of administration to a particular surface.
  • the most typical mucosal surfaces used are those of the gastrointestinal tract, the nasal mucosa, the vaginal mucosa, and the airway mucosa.
  • Administration to the gastrointestinal tract may be performed by oral administration, suppositories, intubation, endoscopy, or any other suitable technique.
  • compositions for oral administration include solid dosage forms such as capsules, tablets, dragees, pills, lozenges, powders, and granules. Where appropriate, they can be prepared with coatings such as enteric coatings, or they can be formulated so as to provide controlled release of the active ingredient such as sustained or prolonged release according to methods well known in the art.
  • Liquid dosage forms for oral administration include solutions, emulsions, suspensions, syrups and elixirs, and solid forms suitable for dissolution or suspension in liquid prior to use.
  • Nasal administration typically involves the use of a free flowing liquid, cream, or gel containing an effective concentration in a comfortable volume. Because the nasal mucosa is relatively more quiescent and there is a relative paucity of proteolytic enzymes, an effect may in some instances be obtained using a lower amount of conjugate.
  • the aerosol may either be a finely dispersed liquid, or a powder. Apparatus and methods for forming aerosols are described in Kirk-Othmer, "Encyclopedia of Chemical Technology", 4 th Ed Vol. 1 , Wiley NY USA, pp 670-685, 1991 ; and Newman,
  • a typical oral dosage is in the range of from 0.00001 to about 10 mg/kg body weight per day, preferably from about 0.0001 to about 1 mg/kg body weight per day administered in one or more dosages such as 1 to 3 dosages.
  • the exact dosage will depend upon the expected volume of distribution of the composition before reaching the intended site of action, the degree of degradation and penetration expected for the mode of administration, the frequency of administration, the sex, age, weight and general condition of the subject treated, the nature and severity of the condition treated and any concomitant diseases to be treated and other factors evident to those skilled in the art.
  • a typical unit dosage form for oral administration one or more times per day such as 1 to 3 times per day may contain from about 0.001 to about 1000 mg, preferably from about 0.01 to about 100 mg of the product.
  • Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solution and various organic solvents.
  • solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid, or lower alkyl ethers of cellulose.
  • liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene, or water.
  • the carrier or diluent may include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
  • sustained release material such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.
  • Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules or tablets, each containing a predetermined amount of the active compound, and which may include a suitable excipient. These formulations may be in the form of powder or granules, as a solution or suspension in an aqueous or non-aqueous liquid, or as an oil-in-water or water-in-oil liquid emulsion.
  • the preparation may be tabletted, placed in a hard gelatine capsule in powder or pellet form or it can be in the form of a troche or lozenge.
  • the amount of solid carrier will vary widely but will usually be from about 25 mg to about 1 g.
  • the preparation may be in the form of a syrup, emulsion, soft gelatine capsule, or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.
  • a typical tablet which may be prepared by conventional tabletting techniques, may contain:
  • the products of the invention may be administered in combination with further pharmacologically active substances, including agents that enhance the tolerogenic effect of the conjugate at the mucosal surface.
  • additional active component is a cytokinine, e.g. IL-4, IL-10, or TGF ⁇ . Since the compositions are intended for mucosal administration, it is useful to prepare compositions that are not only stable for the expected shelf life, but also resistant to the pH extremes, enzymes, and other assaults of the mucosal environment.
  • DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine
  • DMPC 1,2-dimyristoyl-sn-glycero-3-phosphocholine
  • IL-2 interleukin-2
  • Antibodies For western blotting experiments the antibodies used were as follows: for insulin detection a monoclonal antibody to human proinsulin was produced at Zymogenetics and a goat anti-mouse antibody coupled to horseradish peroxidase from Rockland Immunochemicals (Gilbertsville, PA) was employed for visualisation; for CTB detection a goat anti-choleragenoid antibody was purchased from List Biological Laboratories (Campbell, CA) and was used with a donkey anti-goat antibody coupled to horseradish peroxidase obtained from Jackson Immunochemicals (West Grove, PA).
  • Antibodies employed in ELISAs were as follows: for the G M . /insulin ELISA a monoclonal antibody against insulin was obtained from Sigma (Missouri, USA) and a goat anti-mouse antibody coupled to horseradish peroxidase from Biosource International (Camarillo, CA) was used as the secondary antibody; the G M .
  • /CTB ELISA employed a goat anti-choleragenoid antibody purchased from List Biological Laboratories (Campbell, CA) and a donkey anti-goat antibody coupled to horseradish peroxidase from Jackson Immunochemicals (West Grove, PA) was used as the secondary antibody; the sandwich ELISA employed the Sigma monoclonal antibody against insulin, a chicken antibody against CTB which was prepared by ZymoGenetics, and a goat anti-chicken antibody coupled to horseradish peroxidase from Promega (Madison, Wl) for visualisation of immunoreactivity.
  • Biacore analysis employed monoclonal antibodies prepared at ZymoGenetics against human proinsulin and human thrombopoietin.
  • the antigen and the first crosslinker should be reacted under conditions, which for a person skilled in the art are appropriate for the specific antigen and crosslinker.
  • the first crosslinker may be added to the antigen in a ratio of crosslinker to antigen of from about 1 :1 to about 2.5:1. A ratio of crosslinker to antigen of about 1.13:1 is preferred.
  • antigen derivatised with a single crosslinker residue may be isolated by use of methods known in the art such as for example size exclusion chromatography, RP-HPLC, membrane filtration, ion-exchange chromatography and other methods known by a person skilled in the art to be suitable for isolation of such a compound.
  • RP-HPLC is preferred for the purpose of the present invention.
  • the derivatised antigen may be worked up using for instance lyofilisation, precipitation, resolubilisation and the like and then, in step c) of a method according to the present invention, activated in a manner suitable for the activation of the chosen first crosslinker.
  • the conditions for the activation should be chosen so that the activation of as many of the crosslinker derivatives as possible, preferably all, is achieved.
  • the resulting activated crosslinker derivative may be isolated by use of any method known by a person skilled in the art to be suitable for isolation of such a compound such as for example size exclusion chromotography, such as for instance gel filtration, membrane filtration, such as for instance gel filtration, and ion-exchange chromatography.
  • the mucosal binding component and the second crosslinker should be reacted under conditions, which for a person skilled in the art are appropriate for the specific mucosal binding component and crosslinker. If the mucosal binding component is of a multimeric structure this should be taken into consideration when choosing the molar ratio of crosslinker to the mucosal binding component. The molar ratio of crosslinker to the mucosal binding component, as the mucosal binding component appears in the product of conjugates, should be chosen to ensure that each biologically active mucosal binding component will be able to be conjugated to at least one antigen residue.
  • the upper limit for the amount of crosslinker conjugated to the mucosal binding component (which eventually will decide the amount of antigen conjugated to the mucosal binding component) will be a result of among others steric considerations, the number of sites on the mucosal binding component available for derivatisation, the nature and potency of the antigen and economic considerations.
  • the resulting reaction mixture may be subjected to such treatments as size exclusion chromatography, such as for instance gel filtration, membrane filtration, such as for instance ultrafiltration, ion-exchange chromatography or the like. Ultrafiltration is preferred for the purpose of the present invention.
  • the derivatised mucosal binding component and the activated antigen should be reacted under conditions, which for a person skilled in the art are appropriate for the specific derivatised mucosal binding component and the specific activated antigen.
  • the mucosal binding component is dissociated into monomeric subunits prior to the conjugation and reassociated after the conjugation.
  • the amount of activated antigen to use should be calculated according to the amount of crosslinker residues on the derivatised mucosal binding component.
  • the molar ratio of activated antigen to multimeric derivatised mucosal binding component should be based on the degree of derivatisation of the derivatised mucosal binding component (i.e. the amount of crosslinker residues on the multimeric derivatised mucosal binding component as described in the previous paragraph) as exemplified in Method B.
  • the pyridine thione released by the conjugation reaction may be removed by use of such treatments as size exclusion chromatography, such as for instance gel filtration, membrane filtration, such as for instance ultrafiltration, ion-exchange chromatography or the like.
  • size exclusion chromatography such as for instance gel filtration, membrane filtration, such as for instance ultrafiltration, ion-exchange chromatography or the like.
  • ultrafiltration is preferred.
  • the method of preparing a product of conjugates between an antigen and a mucosal binding component according to the present invention is exemplified by the following two methods of preparing a product of conjugates between insulin and CTB.
  • the insulin derivatised with 2-pyridyl disulphide (SSPY) at the amino terminus of the B chain is isolated by preparative reverse-phase HPLC (RP- HPLC) using a Waters Delta Prep HPLC at 60 ml/min with a 52 x 250 mm Vydac C4 Column (10-15 ⁇ m, 300 A) and a gradient from 33% acetonitrile/0.1% TFA to 36% acetonitrile/0.1% TFA over 30 minutes.
  • the pool of insulin-SSPY containing the 2-pyridyl disulphide at the amino terminus of the B chain is identified by LC/MS following tryptic digestion.
  • CTB-SSPY SSPY with differing amounts of incorporated cross-linker.
  • Insulin is suspended in 4 mM EDTA to a concentration of 10 mg/ml. 1 M triethanolamine is added to a concentration of triethanolamine of 10 mM and the pH adjusted to 7.5. SPDP is dissolved in ethanol at about 7.5 mg/ml. SPDP is added in a 1.13:1 molar ratio to insulin over a period of 15 to 30 minutes and if necessary the pH is adjusted to 7.5. The reaction mixture is left at room temperature for at least 1 hour. b) Isolation of insulin derivatised with a single crosslinker
  • insulin-SSPY The insulin derivatised with 2-pyridyl disulphide (SSPY) at the amino terminus of the B chain (in the B1 position), referred to as insulin-SSPY, is isolated by preparative reverse- phase HPLC (RP-HPLC) using a C18 substituted Fuji Davison silica (15 ⁇ m, 200 A) Using a a 250 mm column, elution is carried out in a buffer containing 28.1% ethanol/125 mM Na 2 SO 4 /10 mM citric acid, pH3.3/H 2 SO 4 .
  • the pool of insulin-SSPY containing the 2-pyridyl disulphide at the amino terminus of the B chain is identified by LC/MS following tryptic digestion.
  • the eluate from the RP-HPLC is cooled to a temperature of about 4°C under stirring and the resulting precipitate is then resuspended to a final concentration of insulin- SSPY in the aqueous suspension of about 10 mg/ml.
  • the SSPY groups on insulin are converted to thiol groups by addition of TCEP in an molar ratio of TCEP to insulin-SSPY of about 1.6: 1 at a pH of 3.1.
  • the resulting insulin-prop- SH is then subjected to gel filtration on a Pharmacia Sephadex G-25 Medium column using 50 mM NaCI/2 mM citric acid, pH 3.1/NaOH as the eluent.
  • the eluate is immediately cooled to a temperature of about 4°C and stored at that temperature.
  • This procedure is carried out in order to prepare CTB-SSPY with approximately 4 SSPY groups per CTB pentamer.
  • 1 mol/kg triethanolamine, pH 8.0 is added to recombinant CTB (approximately 10 mg/ml in 150 mM NaCI, 22 mM NaH 2 PO 4 , pH 7.4) to a concentration of triethanolamine of 25 mmol/kg and pH is adjusted to 8.0.
  • Freshly made SPDP (7.5 mg/ml in ethanol) is added in a molar ratio of 8.75 SPDP to 1 CTB pentamer over a period of about 15 minutes. The reaction mixture is left at room temperature for at least 1 hour.
  • the pH of the CTB-SSPY from step d) is adjusted to about 3.4 with 1 M HCI which causes the CTB-SSPY to dissociate into monomers. Since the CTB-SSPY is prepared with approximately 4 SSPY groups per CTB pentamer, the insulin-prop-SH from the final gel filtration in step a) is added thereto in a molar ratio of insulin-prop-SH to pentamer CTB- SSPY of 4:1 under gentle stirring at a temperature of between 18 and 25 °C over a period of 0 to 60 minutes.
  • the resulting mixture is kept at room temperature for no longer than about 48 hours and is then subjected to size exclusion chromatography on Pharmacia Superdex 75 under eluation with 50 mM NaCI/10mM citric acid, pH 3.5/NaOH.
  • the pH of the eluate is adjusted to 7.8 with 0.5 M NaOH over a period of about 1 hour, which causes the reassociation of the conjugated product into pentamers.
  • the pH of the resulting mixture is adjusted to 7.8 over a period of 0 to 60 minutes and then the mixture is subjected to size exclusion chromatography an a Pharmacia Superdex 200 under eluation with 50 mM NaCI/10 mM phosphate/pH 7.4. Characterisation and analysis of CTB-insulin
  • Analytical size exclusion chromatography is carried out using a Hewlett Packard HP1050 HPLC equipped with a diode array detector, binary pump system and Chemstation software.
  • a 10 x 300 mm Superdex-200 column (Pharmacia, USA) is used with 50 mM sodium phosphate, pH 7.0 containing 100 mM NaCI as buffer, and a flow rate of 0.6 ml/min.
  • CTB-insulin Three different ELISAs are used to characterise CTB-insulin (CTB conjugated to insulin). Two of these involve binding of conjugate to immobilised ganglioside G M ⁇ , as outlined by Svennerholm and Holmgren (1978), with antibodies against either insulin or CTB used for detection.
  • 96-well ELISA plates are coated with 0.1 ml 5.0 ⁇ g/ml G M ⁇ in 0.1 M sodium carbonate, pH 9.0, at 4°C for 16 hours. Following five washes with 0.2 ml PBS/0.05% Tween-20 (ELISA buffer) unbound sites are blocked by incubating for 10 minutes at room temperature with SuperBlockTM.
  • G M .-bound CTB For detection of G M .-bound CTB a 1 :2,500 dilution of goat anti-CTB antibody and a 1 :10,000 dilution of donkey anti-goat antibody coupled to horseradish-peroxidase are used. Antibodies are diluted in 1 % (w/v) bovine serum albumin in ELISA buffer. Following washing immobilised antibodies are visualised by addition of 0.1 ml development reagent (0.4 mg/ml OPD, 0.024% hydrogen peroxide in 0.1 M citrate, pH 5.0). After 10 minutes at room temperature the reaction is terminated by addition of 0.1 ml 1 M sulphuric acid and absorbance measured at 490 nm.
  • the third ELISA relies on direct capture of CTB-insulin conjugates using antibodies against insulin and detection with antibodies directed against CTB.
  • 96-well plates are coated at 4°C for 16 hours with 0.1 ml of 5.0 ⁇ g/ml anti-insulin monoclonal antibody in 0.1 M sodium carbonate, pH 9.0. Washing, blocking of unbound sites, and application of samples are carried out as outlined above. Plates are then incubated for 1 hour at room temperature with 0.1 ml per well of chicken anti-CTB antibody diluted to 0.5 mg/ml in 1% (w/v) bovine serum albumin in ELISA buffer. Following washing and incubation for 1 hour at 37°C with 0.1 ml 0.5 ⁇ g/ml goat anti-chicken antibody coupled to horseradish peroxidase immobilised conjugate is visualised as outlined above.
  • LC-MS LC-MS
  • the column is equilibrated at 30% B and a linear gradient from 30 to 50% B over 90 minutes is immediately initiated (A: 2% acetonitrile + 0.1% TFA, B: 90% acetonitrile + 0.09% TFA).
  • the outlet from the HPLC UV detector is plumbed directly into a Finnigan LCQ Ion Trap Mass Spectrometer (Thermoquest Corp., San Jose, CA) with no flow splitting, a heated capillary temperature of 200°C, and a sheath gas flow of 80 (arbitrary units).
  • the source voltage is 5.60 kV and the capillary voltage is 43.67 V.
  • Mass spectra from 400-2000 m/z are recorded continuously during the gradient with 3 microscans per full scan.
  • CTB-SSPY is digested with trypsin at 1 :50 (w:w) trypsin:protein in 100 mM Tris, pH 8.5 containing 100 ⁇ M TPCK for 18-24 hrs at 37°O
  • trypsinisation CTB-SSPY is reduced in 6.0 M guanidine/HCI, 150 mM Tris, 0.25 mM EDTA, pH 7.6 with a 25-fold molar excess of dithiothreitol over the number of cysteine residues for 1 hr at 37°O
  • the reduced protein is then alkylated by addition of a 5-fold molar excess of 4-vinylpyridine followed by incubation in the dark at room temperature for 2 hours.
  • the pyridylethylated CTB-SSPY is immediately desalted using
  • Amino acid analysis uses the Waters AccQ-Tag® chemistry according to the manufacturer's instructions. To calculate the concentration, the corrected average pmol for each amino acid is multiplied by the molecular weight of that amino acid. These values are then summed and corrected for the amount of sample hydrolysed and the percentage of the hydrolysed sample injected to the analyser as well as for destruction of Cys, Met and Trp during hydrolysis.
  • CTB T [total moles (a.a.i.) -a.a.] / [a.a.i. - a.a. ⁇ ]
  • a.a. is the moles of amino acid (determined experimentally for the sample)
  • a.a.i. moles of that amino acid in insulin (from the amino acid composition of insulin)
  • a.a.c. moles of that amino acid per CTB T (from the amino acid composition of CTBi).
  • the molar ratio of insulin to CTB T is calculated by dividing moles insulin by moles CTB T for each of the four determinations, and averaging the resulting values. For the purpose of defining the molar insulin ⁇ CTB ! ratio, we assume all the insulin present in the final material is conjugated to CTB.
  • G M ⁇ is carried out using a BIAcore Model 1000 with upgrade and sensor chips coated with G M ⁇ -containing liposomes.
  • the liposomes are prepared by mixing 0.5 mM DMPC and 2% ganglioside G M . in PBS. Following 10 cycles of freeze-thawing in an ethanol / dry ice bath the mixture is passed through a Mini Extruder (Avanti, Alabaster, AL) nine times according to the manufacturer's instructions. Following degassing an HPA Sensor Chip (BIAcore, Inc.,
  • Binding of recombinant CTB (positive control) and CTB-insulin conjugates to flow cells is carried out at a final concentration of 20 ⁇ g/ml in PBS. Following binding of the samples, their ability to bind antibodies against insulin is tested by flowing a monoclonal antibody against proinsulin (20 ⁇ g/ml in PBS) across the flow cell. A monoclonal antibody against thrombopoietin (20 ⁇ g/ml in PBS) is used as a negative control to confirm that binding of the anti-insulin antibody is specific.
  • SDS-PAGE is carried out using 10-20% Tris-Tricine gels and a Novex Xcell II mini-gel box according to the manufacturers instructions with two exceptions.
  • a voltage of 95 V is used during electrophoresis, which is lower than recommended and is necessary to prevent heating of the gel, which causes dissociation of the CTB pentamer (CTB 5 ).
  • CTB 5 CTB pentamer
  • the samples are not heated following addition of sample buffer, again to avoid dissociation of the CTB 5 .
  • 50-200 ng of each sample are loaded on the gel for western blotting and 1 - 5 ⁇ g for coomassie blue staining.
  • the gels are either stained with coomassie blue using the staining solution according to the manufacturers directions, or the samples are electroblotted to nitrocellulose for 2 hours at 400 mA in a Hoefer Mighty Small tank using 25 mM Tris, pH 7.4, 192 mM glycine, 20% methanol as transfer buffer. Unbound sites on the nitrocellulose are blocked with western buffer A (50 mM Tris, 5 mM EDTA, 150 mM NaCI, 0.05% Igepal CA-630, 0.25% gelatine) for 1 hour at room temperature. The membrane is then incubated overnight at 4°C with primary antibody diluted in western buffer A.
  • western buffer A 50 mM Tris, 5 mM EDTA, 150 mM NaCI, 0.05% Igepal CA-630, 0.25% gelatine
  • the mouse monoclonal antibody is used at a 1 :1000 dilution and for CTB detection the goat anti- choleragenoid antibody is used at a 1 :10,000 dilution.
  • the membranes are incubated for 1 hour at room temperature with secondary antibodies in western buffer A at a 1 :4000 dilution for the anti-mouse and a 1 :20,000 dilution for the anti-goat antibodies for insulin and CTB detection respectively.
  • the membranes are then washed three times with western buffer B (50 mM Tris, pH 7.4, 5 mM EDTA, 5% NP-40, 150 mM NaCI, 0.05% Igepal CA-630, 0.4% SDS) for 20 minutes each time, and then incubated with the Amersham ECL reagent according to the manufacturers instructions. Proteins are visualized by exposing the membranes to X-ray film.
  • western buffer B 50 mM Tris, pH 7.4, 5 mM EDTA, 5% NP-40, 150 mM NaCI, 0.05% Igepal CA-630, 0.4% SDS
  • the ability of the various conjugates to activate T cells is determined by monitoring IL-2 production by the insulin specific T cell hybridoma H-11 in the presence of spleen cells derived from NOD mice.
  • NOD mice are killed by cervical dislocation and their spleens removed and cleaned of any remaining pancreas and fat tissue.
  • Each spleen is homogenised into a single cell preparation between two microscope slides and diluted into 50 ml of RPMI 1640 containing 10% FBS and PSN. The cells are washed in 50 ml media, and the red blood cells lysed by addition of 9 volumes of water followed immediately by 1 volume of 10 X PBS. The cells are then washed again in 50 ml media and suspended at
  • the hybridoma T cells are also washed in media and added to the spleen cells at 10 x 10 5 cells / ml.
  • Insulin or CTB-insulin conjugates are diluted into 100 ⁇ l of media and mixed with 100 ⁇ l of cells containing 1 x 10 6 freshly isolated spleen cells and 1 x 10 5 hybridoma T cells. After 24 hours incubation 100 ⁇ l supernatant is removed and assayed for IL-2 by ELISA.
  • FIG. 1 Preparation of insulin-prop-SH.
  • A Purification of insulin-SSPY. Insulin was reacted with a 2-fold molar ratio of SPDP at pH 7.0 for 1 hour at room temperature. Insulin derivatised at the N terminus of the B chain was purified by preparative RP-HPLC on a 52 mm column. LC-MS following reduction and tryptic digestion was used to identify peaks. Some underivatised insulin remained (peak I), as well as insulin derivatised at the N-terminus of the A chain or at lysine 29 of the B chain (peak II), at the N terminus of the B chain (peak III), and at two or three primary amino groups (peak IV).
  • FIG. 2 Preparation of CTB-SSPY.
  • CTB was derivatised with SPDP at various molar ratios and unreacted SPDP was then removed by gel filtration.
  • the number of 2-pyridyl disulphide groups on the CTBi was determined, and the purified CTB-SSPY was analysed by RP-HPLC as outlined in methods. Chromatograms A-D show CTB containing 0, 0.41 , 1.43, or 2.82 2-pyridyl disulphide groups per monomer respectively.
  • FIG. 3 Size exclusion chromatography of CTB-insulin conjugates.
  • CTB-insulin conjugates prepared with CTB-SSPY preparations containing 0.43, 1.43 or 2.82 pyridyl sulphide groups per CTB monomer (panels A - C respectively) were analysed by size exclusion chromatography on a Superdex-200 column as outlined in methods. Fifty ⁇ l of each conjugate was applied to the column, which is equivalent to 31.5, 29.5 and 30.5 ⁇ g CTB for the three conjugates in A-C, respectively. One-minute fractions were collected from 14 to 32 minutes for each run and analysed by three different ELISAs as shown in figure 6. Recombinant CTB elutes from this column at 28 minutes and insulin elutes at 32 minutes.
  • Figure 4 SDS-PAGE analysis of CTB-insulin.
  • CTB-insulin conjugates were electrophoresed on 10-20% tricine gels and either stained with Coomassie Blue (A) or transferred to nitrocellulose and visualised by western blot analysis with antibodies against CTB (B) or insulin (C). The relative migration position of molecular weight markers is indicated.
  • Lanes 1 and 7 contained molecular weight markers while lanes 2, 3, and 4 contained conjugates prepared with CTB-SSPY preparations containing 0.43, 1.43, or 2.82 pyridyl sulphide groups per CTB monomer respectively.
  • Lane 5 contained recombinant CTB while lane 6 contained recombinant insulin.
  • Lanes 1 , 2, and 3 contained conjugates prepared with CTB-SSPY preparations containing 0.43, 1.43, or 2.82 pyridyl sulphide groups per CTB monomer respectively, and lane 5 contained recombinant CTB.
  • Lanes 1 , 2, and 3 contained conjugates prepared with CTB-SSPY preparations containing 0.43, 1.43, or 2.82 pyridyl sulphide groups per CTB monomer respectively.
  • Figure 7 ELISA of fractionated CTB-insulin.
  • FIG. 8 Biacore analysis of CTB-insulin. Binding of CTB and CTB-insulin to a Biacore sensor chip coated with G M ⁇ was carried out as outlined in methods. The arrows indicate the points of addition to the sensor chip of (1) the sample, (2) a buffer wash, (3) antibodies against a non-specific antigen, (4) a buffer wash,
  • G ⁇ were recombinant CTB ( ), conjugate with a molar insulin to CTB1 ratio of 0.33 ( —
  • Insulin ( ⁇ ) or CTB-insulin conjugates with ratios of insulin to CTB1 of 0.33 ( • ), 1.0 ( ⁇ ) or 1.79 ( ⁇ ) at various concentrations were incubated with freshly isolated spleen cells and hybridoma T cells for 24 hours and supernatants were then assayed for IL-2 by ELISA as outlined in "Methods”.
  • Insulin contains three primary amino groups that may be derivatised with SPDP: two are at the NH 2 termini of the A and B chains while the third is the ⁇ -amino group of lysine 29 of the B chain.
  • the lyophilised insulin-SSPY was resuspended at pH 4.0 prior to reduction to the reactive insulin-prop-SH molecule with an equimolar ratio of TCEP to 2-pyridyl disulphide groups.
  • This reducing reagent was chosen since, unlike classic reducing reagents such as dithiothreitol, it is rendered unreactive after reduction of substrate has occurred and hence only stoichiometric reduction may take place.
  • D Amino acid analysis was used to determine the total prolein concentration, the ratio of insulin to CTB, and the concentrations of insulin and CTD in the three conjugates following removal of released pyridine Ihione by size exclusion chromatography.
  • Conjugates were prepared using each of the CTB-SSPY preparations by mixing insulin-prop-SH and CTB-SSPY in ratios designed to minimize the amounts of unreacted components in the final conjugates. Hence, the number of SSPY groups in the purified CTB- SSPY solutions were quantitated and, since each SSPY group may react with one insulin- prop-SH molecule, insulin-prop-SH was added to the CTB-SSPY solution in a 1 :1 molar ratio to SSPY groups (tablel ).
  • Solid insulin-prop-SH was added to the CTB-SSPY solutions and the pyridine thione released in each reaction was removed by size exclusion chromatography.
  • the CTB-insulin conjugates were analysed by SDS-PAGE with Coomassie Blue staining and by western blotting with antibodies against both insulin and CTB (figure 4). In all cases bands larger than CTB 5 were visualized, with apparent molecular weights between approximately 60 and 180 kDa. In each case two major bands were observed, which increased in size as the number of 2-pyridyl disulphide groups on the CTB-SSPY used in the reaction increased. Presumably these two major bands correspond to the two major peaks observed by size exclusion chromatography (figure 3). The bands observed by Coomassie Blue staining were also visualised by western blotting using antibodies against both insulin and CTB, indicating that these bands contain both insulin and CTB, and hence represent conjugates between the two molecules.
  • the total protein concentration of the final conjugates was determined by amino acid analysis. This data was also used to calculate the molar ratio of insulin to CTB monomers in the preparations, and was found to be 0.33, 1.0 and 1.79 for the conjugates prepared using derivatised CTB containing 0.43, 1.43 or 2.82 pyridyl disulphide groups per monomer (table 1 ). Both results were then used to calculate the concentrations of insulin and CTB in each conjugate (table 1).
  • Example 6 Biochemical characterisation of CTB-insulin conjugates - mass spectrometric analysis
  • Mass spectrometric analysis of the preparations was carried out following reverse phase HPLC at acidic pH, which causes dissociation of the CTB pentamer into its monomeric state.
  • the RP-HPLC separated underivatised CTBi from unconjugated insulin-prop-SH, as well as CTBi containing varying amounts of insulin conjugated to it.
  • On-line mass spectrometric analysis of the resolved components allowed confirmation of their identity based on mass.
  • the reverse phase HPLC of the preparation with a molar insulin:CTB ratio of 0.33 revealed primarily a single peak corresponding in mass to underivatised CTBi and multiple peaks of a mass equivalent to CTBi containing one insulin molecule (figure 5A).
  • the multiple peaks observed corresponding in mass to CTB monomer derivatised with a single insulin presumably represent conjugation through multiple primary amines on the
  • the samples were normalised to a constant concentration of CTB.
  • the conjugates with insulin to CTBi ratios of 0.33, 1.0 and 1.79 all contained approximately 0.6 mg CTB / ml, allowing direct comparison of each following dilution to the same extent.
  • Three different ELISAs were used for characterization of the CTB-insulin preparations in this manner.
  • the sandwich ELISA in which antibodies against insulin are bound to the ELISA plate and immobilized conjugate is detected with antibodies against CTB, detects the physical presence of conjugate.
  • the response in this ELISA increased as the molar ratio of insulin:CTB ⁇ in the conjugate increased from 0.33 to 1.0, with a small additional increase observed at a molar insulin ⁇ CTB ! ratio of 1.79 (figure 6A).
  • a different pattern was observed with the ELISAs that detect binding to ganglioside G M1 .
  • Using antibodies against CTB which detect both underivatised CTB as well as CTB-insulin conjugates bound to G M ⁇ , there was a dose response for all three conjugates (figure 6B).
  • each conjugate was also analysed by ELISA following fractionation of similar amounts (based on the concentration of CTB present) by size exclusion chromatography. Analysis of fractions collected from the Superdex-200 column by the sandwich ELISA indicated the presence of conjugate eluting between 17 and 29 minutes (figure 7A). As was observed for the unfractionated conjugate, the response in this ELISA increased as the molar ratio of insulin ⁇ CTB ! in the conjugate increased from 0.33 to 1.0, with no additional increase observed at a molar insulin:CTB ⁇ ratio of 1.79. This trend is consistent with greater insulin antibody immunoreactivity as the amount of insulin in the conjugate increased, up to a point where the assay became saturated. In addition the apparent size of the conjugate, observed as an increase in immunoreactivity at earlier elution times upon size fractionation on Superdex-200, increased as the amount of insulin increased.
  • the assay employed a T cell hybridoma specific for insulin that had been isolated from a NOD mouse, and freshly isolated spleen cells served as a source of antigen presenting cells. Upon addition of insulin or conjugate the T cells are activated and release IL-2. In this model the level of IL-2 is a direct indicator of the ability of the conjugate to activate T cells.
  • Figure 9 shows that insulin alone increases IL-2 production by the T cells in a dose-dependent manner. However, the CTB-insulin conjugates cause a similar degree of activation at doses that are several orders of magnitude lower.

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Abstract

L'invention cocnerne une méthode de préparation d'un produit de conjugués d'un antigène et d'un composant de liaison muqueux, comme, par exemple, des conjugué d'insuline et une sous-unité de la toxine B du choléra (CTB). Ledit produit peut être utilisé, entre autres, comme moyen d'induction de tolérance immunitaire et donc de suppression de l'auto-immunité.
PCT/DK2000/000531 1999-09-30 2000-09-28 Procede de preparation de conjugues d'un antigene et d'un composant de liaison muqueux Ceased WO2001022995A1 (fr)

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Cited By (14)

* Cited by examiner, † Cited by third party
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WO2009077438A1 (fr) * 2007-12-14 2009-06-25 Glaxosmithkline Biologicals S.A. Procédé de préparation de conjugués de protéines
WO2009078796A1 (fr) * 2007-12-19 2009-06-25 Mivac Development Aktiebolag Compositions et procédés pour le traitement de maladies auto-immunes et allergiques
WO2013135359A1 (fr) 2012-03-16 2013-09-19 Merck Patent Gmbh Lipides de ciblage de type acide aminé
US9814780B2 (en) 2010-08-10 2017-11-14 Ecole Polytechnique Federale De Lausanne (Epfl) Compositions for inducing antigen-specific tolerance
US9850296B2 (en) 2010-08-10 2017-12-26 Ecole Polytechnique Federale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US10046056B2 (en) 2014-02-21 2018-08-14 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
CN108912223A (zh) * 2018-03-28 2018-11-30 深圳市新产业生物医学工程股份有限公司 胰岛素免疫原、其制备方法、胰岛素抗体及试剂盒
US10392437B2 (en) 2010-08-10 2019-08-27 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
EP3611270A1 (fr) 2009-05-19 2020-02-19 University Of Miami Kits de présentation d'antigène in vitro, d'évaluation de l'efficacité d'un vaccin, et d'évaluation de l'immunotoxicité d'agents biologiques et de médicaments
US10821157B2 (en) 2014-02-21 2020-11-03 Anokion Sa 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
US11253579B2 (en) 2017-06-16 2022-02-22 The University Of Chicago Compositions and methods for inducing immune tolerance
US12383617B2 (en) 2018-05-09 2025-08-12 The University Of Chicago Compositions and methods concerning immune tolerance

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WO2009077438A1 (fr) * 2007-12-14 2009-06-25 Glaxosmithkline Biologicals S.A. Procédé de préparation de conjugués de protéines
WO2009078796A1 (fr) * 2007-12-19 2009-06-25 Mivac Development Aktiebolag Compositions et procédés pour le traitement de maladies auto-immunes et allergiques
EP3611270A1 (fr) 2009-05-19 2020-02-19 University Of Miami Kits de présentation d'antigène in vitro, d'évaluation de l'efficacité d'un vaccin, et d'évaluation de l'immunotoxicité d'agents biologiques et de médicaments
US10800838B2 (en) 2010-08-10 2020-10-13 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US11246943B2 (en) 2010-08-10 2022-02-15 École Polytechnique Fédérale De Lausanne (Epfl) Antigen-specific tolerance and compositions for induction of same
US9878048B2 (en) 2010-08-10 2018-01-30 Ecole Polytechnique Federale De Lausanne (Epfl) Compositions for generating immune tolerance by targeting erythrocytes
US12060414B2 (en) 2010-08-10 2024-08-13 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US9901645B2 (en) 2010-08-10 2018-02-27 Ecole Polytechnique Fedrale de Lausanne (EPFL) Methods for reducing immune responses
US9901646B2 (en) 2010-08-10 2018-02-27 Ecole Polytechnique Federale De Lausanne (Epfl) Methods for induction of antigen-specific immune tolerance
US11884721B2 (en) 2010-08-10 2024-01-30 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US10919963B2 (en) 2010-08-10 2021-02-16 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US10265415B2 (en) 2010-08-10 2019-04-23 École Polytechnique Fédérale De Lausanne (Epfl) Compositions for inducing antigen-specific tolerance
US10265416B2 (en) 2010-08-10 2019-04-23 École Polytechnique Fédérale de Lausanna (EPFL) Compositions for generation of immune tolerance to specific antigens
US10392437B2 (en) 2010-08-10 2019-08-27 École Polytechnique Fédérale De Lausanne (Epfl) Erythrocyte-binding therapeutics
US10471155B2 (en) 2010-08-10 2019-11-12 École Polytechnique Fédérale De Lausanne (Epfl) Antigen-specific tolerance and compositions for induction of same
US9814780B2 (en) 2010-08-10 2017-11-14 Ecole Polytechnique Federale De Lausanne (Epfl) Compositions for inducing antigen-specific tolerance
US9850296B2 (en) 2010-08-10 2017-12-26 Ecole Polytechnique Federale De Lausanne (Epfl) Erythrocyte-binding therapeutics
WO2013135359A1 (fr) 2012-03-16 2013-09-19 Merck Patent Gmbh Lipides de ciblage de type acide aminé
US9878044B2 (en) 2012-03-16 2018-01-30 Merck Patent Gmbh Targeting aminoacid lipids
US11510988B2 (en) 2012-03-16 2022-11-29 Merck Patent Gmbh Targeting aminoacid lipids
US11654188B2 (en) 2014-02-21 2023-05-23 Ecole Polytechnique Federale De Lausanne (Epfl) Glycotargeting therapeutics
US10953101B2 (en) 2014-02-21 2021-03-23 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US10946079B2 (en) 2014-02-21 2021-03-16 Ecole Polytechnique Federale De Lausanne Glycotargeting therapeutics
US10821157B2 (en) 2014-02-21 2020-11-03 Anokion Sa Glycotargeting therapeutics
US11666638B2 (en) 2014-02-21 2023-06-06 Ecole Polytechnique Federale De Lausanne (Epfl) Glycotargeting therapeutics
US11793882B2 (en) 2014-02-21 2023-10-24 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US11801305B2 (en) 2014-02-21 2023-10-31 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US10046056B2 (en) 2014-02-21 2018-08-14 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US10940209B2 (en) 2014-02-21 2021-03-09 École Polytechnique Fédérale De Lausanne (Epfl) Glycotargeting therapeutics
US11253579B2 (en) 2017-06-16 2022-02-22 The University Of Chicago Compositions and methods for inducing immune tolerance
CN108912223A (zh) * 2018-03-28 2018-11-30 深圳市新产业生物医学工程股份有限公司 胰岛素免疫原、其制备方法、胰岛素抗体及试剂盒
US12383617B2 (en) 2018-05-09 2025-08-12 The University Of Chicago Compositions and methods concerning immune tolerance

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