WO2011029126A1 - Procedes et reactifs pour produire des anticorps contre des autoantigenes - Google Patents

Procedes et reactifs pour produire des anticorps contre des autoantigenes Download PDF

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Publication number
WO2011029126A1
WO2011029126A1 PCT/AU2010/000653 AU2010000653W WO2011029126A1 WO 2011029126 A1 WO2011029126 A1 WO 2011029126A1 AU 2010000653 W AU2010000653 W AU 2010000653W WO 2011029126 A1 WO2011029126 A1 WO 2011029126A1
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protein
antibody
autoantigen
aire
human animal
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PCT/AU2010/000653
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English (en)
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Ian Keith Campbell
Ian Peter Wicks
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Walter And Eliza Hall Institute Of Medical Research
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A01K2217/054—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function
    • A01K2217/058—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function due to expression of inhibitory nucleic acid, e.g. siRNA, antisense
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/0306—Animal model for genetic diseases
    • A01K2267/0325—Animal model for autoimmune diseases

Definitions

  • the present invention relates to methods for producing antibodies and proteins comprising autoantigen binding domains.
  • Antibodies and proteins comprising antigen binding domains are now widely used as research reagents, diagnostic/prognostic reagents and therapeutic agents. This broad ranging applicability arises from the ability of antibodies and proteins comprising antigen binding domains thereof to bind to an antigen with a high degree of specificity and affinity. Accordingly, antibodies and proteins comprising antigen binding domains thereof are able to specifically detect an antigen in a sample and permit quantification or to bind specifically to a cell expressing the antigen, e-g-, to kill the cell or to deliver a therapeutic payload.
  • antibodies were produced by immunizing an animal subject with an antigen or an epitope thereof to which the subject would raise an immune response.
  • immune cells e.g., spleen cells or lymphocytes
  • transformed cells e.g., myeloma cells
  • a difficulty that arises with traditional methods for antibody production is that it is difficult to raise antibodies against antigens expressed in the subject (autoantigens, also known as self-antigens) or proteins having a high degree of structural similarity thereto (i.e., structurally conserved with self antigens; Griffiths et al, 1993).
  • This difficulty results from tolerance mechanisms that prevent antigen-mediated expansion of B-cell clones with self specificities. After antibody gene rearrangement, B-cells may display antibodies with self-reactivity, however tolerance mechanisms can lead to their deletion.
  • T cell tolerance prevents the production of T helper cells having specificity for self-antigens, which are required for B cell maturation and antibody production.
  • TSAs tissue-specific antigens
  • TRAs tissue-specific antigens
  • peripheral tissue antigens TSAs
  • TSAs tissue-specific antigens
  • TSAs are normally present only in specialized peripheral organs.
  • these TSAs are presented by mTECs or dendritic cells to differentiating thymocytes as self antigens and self-reactive T cells are deleted or functionally inactivated.
  • any antibodies capable of binding to self antigens generally do so with low affinity, and are not produced in large amounts.
  • phage-display technology to screen libraries of antibody fragments to identify fragments capable of binding to the antigen of interest.
  • these libraries are made from rearranged heavy and light chain variable regions from non-immunized human donors. Because the phage displayed libraries are not subjected to tolerance mechanisms, they contain antibody fragments capable of binding to self antigens. However, these antibody fragments generally bind with low affinity, as also occurs in vivo. In vivo, genes encoding low affinity binding antibodies undergo somatic hypermutation to generate genes encoding high-affinity antibodies. This process does not naturally occur with phage displayed libraries, which instead require often lengthy in vitro affinity maturation processes to improve binding specificity. Furthermore, such in vitro processes may result in an antibody that, while having a high affinity for the antigen of interest is also immunogenic and thus of limited therapeutic value.
  • the present inventors have found that administering an autoantigen to a subject having reduced expression of AIRE caused the subject to produce antibodies against the autoantigen. In contrast, subjects having normal levels of AIRE rarely produced antibodies against the autoantigen. For example, using mutant mice deficient in AIRE protein expression the inventors produced antibodies against autoantigens from mice and from different species. These findings establish that subjects having reduced AIRE expression are useful in methods for producing antibodies, for example, antibodies against a self antigen or a protein having structural conservation with a self antigen.
  • the present invention provides a method for producing an antibody that binds to an autoantigen, the method comprising immunizing a non-human animal having reduced AIRE protein activity with one or more autoantigen(s) such that an antibody against the autoantigen is produced.
  • the non-human animal has reduced AIRE protein production.
  • the non-human animal is genetically-modified to reduce production of the AIRE protein.
  • Exemplary genetically-modified animals comprise a genetic modification within an AIRE-encoding gene that reduces expression of said gene. Such animals can be heterozygous or homozygous for the genetic modification.
  • Preferred autoantigens are capable of eliciting an autoimmune response in the non-human animal.
  • Such autoantigens can be from the same or a different species as the immunized non-human animal.
  • the autoantigen is from a human.
  • Exemplary autoantigens are selected from the group consisting of a tissue, a tissue homogenate, a mixture of cells, an isolated protein and an isolated peptide.
  • the autoantigen is collagen type II (e.g., mouse type II collagen) or an immunogenic fragment thereof, e.g., a cyanogen bromide fragment, such as CB1 1.
  • the autoantigen is a granulocyte macrophage colony stimulating factor (GM-CSF), e.g., GM-CSF alpha chain or an immunogenic fragment thereof.
  • GM-CSF granulocyte macrophage colony stimulating factor
  • the method comprises immunizing the non-human animal with the autoantigen once.
  • the method does not comprise additional steps of immunizing the animal with the autoantigen.
  • the non- human animal is not given a booster following immunization.
  • the method comprises isolating the antibody and/or a cell producing the antibody no more than 50 days after immunizing the non-human animal.
  • the method comprises isolating the antibody and/or a cell producing the antibody no more than 45 days or 44, 43, 42 or, 40 days or 39, 38, 37, 36 or 35 days or 34, 33, 32, 31 or 30 days after immunizing the non-human animal.
  • the method comprises isolating the antibody and/or a cell producing the antibody about 40 days after immunizing the non-human animal.
  • the method comprises isolating the antibody and/or a cell producing the antibody about 30 days after immunizing the non-human animal.
  • a method as described herein according to any embodiment or example of the invention additionally comprises isolating the antibody or a plurality of the antibodies that bind(s) to the autoantigen.
  • the non-human animal is a mammal, such as a rodent.
  • the non-human animal is murine, e.g., a mouse.
  • the present invention also provides an isolated antibody or plurality thereof produced by a method as described herein.
  • the present invention also provides a method for isolating a cell that produces an antibody that binds to an autoantigen, the method comprising performing the method of the present invention and isolating a cell expressing an antibody that binds to the autoantigen.
  • the method comprises, immortalizing the cell.
  • the method comprises fusing the cell with a cancer cell, such as a myeloma cell to produce a hybridoma.
  • the method comprises expressing one or more genes in the cell to thereby immortalize the cell.
  • the invention further provides a recombinant method for producing an antibody producing cell or a cell that produces a protein comprising an autoantigen binding domain.
  • the present invention provides a method for producing a cell that produces an antibody that binds to an autoantigen or that produces a protein comprising an autoantigen binding domain, the method comprising: .
  • the present invention also produces a cell isolated or produced by a method according to any embodiment or example of the present invention.
  • the present invention additionally provides an isolated cell producing an antibody that binds to an autoantigen, wherein the cell comprises a genetic modification that reduces production of the AIRE protein.
  • the. cell may be derived from an animal comprising a genetic modification in its genome that reduces AIRE protein expression.
  • the present invention also provides an isolated hybridoma derived from a cell of the present invention, the hybridoma producing the antibody.
  • the present invention also provides a method for producing a protein comprising an autoantigen binding domain of an antibody, the method comprising performing a method of the invention to produce a cell capable of expressing the protein and culturing the cell to produce the protein. Also provided is a protein comprising an autoantigen binding domain produced by this method of the invention.
  • the present invention further provides a method for producing a library of cells or particles displaying one or more proteins comprising an autoantigen binding domain of an antibody, the method comprising:
  • nucleic acid encoding a polypeptide that facilitates display of the autoantigen binding domains in/on the cells or particles
  • the proteins are displayed on the surface of the cells or particles.
  • the autoantigen binding domains are displayed on the surface of a virus and the expression constructs comprise the nucleic acid encoding the protein comprising the autoantigen binding domain linked to a nucleic acid encoding a viral coat protein or part thereof.
  • the present invention also provides a library of nucleic acids encoding antibodies and/or proteins comprising autoantigen binding domains from (e.g., obtained from) a non-human-animal having reduced AIRE protein activity.
  • the present invention additionally provides a library of expression constructs comprising a nucleic acid from the library described in the previous paragraph operably linked to a promoter.
  • the present invention also provides a library of cells or particles comprising cells or particles displaying one or more proteins comprising an autoantigen binding domain of an antibody from a non-human animal having reduced AIRE protein activity.
  • the invention also provides a library of proteins comprising an autoantigen binding domain from a non-human animal having reduced Autoimmune Regulator (AIRE) protein activity.
  • AIRE Autoimmune Regulator
  • the one or more proteins are displayed on the surface of the cells or particles.
  • a library according to the invention is produced from a non- human animal that was immunized with an autoantigen.
  • the present invention additionally provides a method for isolating an autoantigen binding domain, the method comprising contacting a library of the, and/or produced by a method of the, invention with the autoantigen and isolating a cell or particle that displays a protein comprising an autoantigen binding domain that binds the autoantigen.
  • the method additionally comprises, humanizing the autoantigen binding domain.
  • the method comprises isolating nucleic acid encoding the protein comprising the autoantigen binding domain.
  • the nucleic acid encodes the autoantogen binding domain.
  • the nucleic acid is introduced into a cell such that the autoantigen binding domain is expressed.
  • the present invention additionally provides an isolated autoantigen binding domain produced by a method of the present invention.
  • Also provided is a method for producing an antibody that binds to an autoantigen or a protein comprising an autoantigen binding domain comprising performing a method of the invention to produce an autoantigen binding domain, and producing an antibody or a protein comprising the autoantigen binding domain.
  • the present invention also encompasses the product of this process.
  • the antibodies and proteins of the present invention also facilitate diagnostic, prognostic, therapeutic and prophylactic methods.
  • the present invention also provides a method for diagnosing or prognosing a condition associated with production of a self-antigen in a subject, the method comprising contacting a sample from the subject with an antibody, autoantigen binding domain, or the protein of the invention such that the antibody, domain or protein forms a complex with the self- antigen and detecting the complex, wherein detection of the complex is diagnostic or prognostic of the condition.
  • the present invention also provides a method for localising or detecting a self- antigen in a subject, said method comprising:
  • the present invention also provides a method for treating or preventing a condition associated with production of a self-antigen in a subject, the method comprising administering to the subject an antibody, autoantigen binding domain, or the protein of the invention, wherein the antibody, domain or protein binds to the self- antigen, and treats or prevents the condition.
  • the ability to produce antibodies that bind a self-antigen also enables the production of animal models of autoimmune conditions, preferably human autoimmune conditions.
  • the present invention provides a method for producing a non- human animal model of an autoimmune condition, the method comprising performing a method of the invention to produce an antibody that binds to an autoantigen, wherein the antibody also binds to a self-antigen in the non-human animal and induces the autoimmune condition.
  • the present invention provides a method for producing a non- human animal model of an autoimmune condition, the method comprising administering the' antibody, the autoantigen binding domain or the protein of the invention or nucleic acid encoding the antibody, domain or protein to a non-human animal or expressing theantibody, domain or protein in a non-human animal, wherein the antibody, domain or protein binds to a self-antigen in the non-human animal and induces the autoimmune condition.
  • the present invention also provides a method for identifying a compound for treating or preventing an autoimmune condition, the method comprising:
  • the method comprises isolating or producing the identified compound.
  • the present invention additionally provides a genetically-modified non-human animal having reduced AIRE protein activity, the animal genetically-modified to express one or more of the following:
  • the present invention also provides a non-human animal having reduced AIRE protein activity, wherein the animal is immunized with an autoantigen.
  • the genetically-modified non-human animal is genetically- modified to reduce expression of the AIRE protein.
  • the present invention also provides a composition comprising one or more of the following and a pharmaceutically acceptable carrier:
  • kits comprising one or more of the following:
  • the present inventors have also shown that activated T cells are involved in the progression of autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Accordingly, by targeting such cells or reducing the activity of such cells, APECED is treated.
  • the present invention provides a method for treating or preventing APECED, the method comprising administering a compound that binds to and/or reduces the activity of a costimulatory molecule on activated T cells, and treats or prevents APECED.
  • the compound binds to the costimulatory molecule and prevents the molecule binding to a ligand or receptor thereof.
  • the compound kills or induces death of a cell to which it binds.
  • Figure 2 A is a graphical representation showing enhanced collagen-induced arthritis in AIRE' ' mice following immunization with chick type II collagen (CII).
  • CII chick type II collagen
  • Figure 2B includes copies of photomicrographs showing frontal sections from nonarthritic AIRE +/+ (i), nonarthritic AIRE 1' (ii), arthritic AIRE + + (iii), and arthritic AIRE ' ' (iv) mouse metacarpophalangeal (MCP)/ metatarsophalangeal (MTP) joints, 50 days after immunization. (Hematoxylin and eosin stained; original magnification x 100).
  • Figure 2C is a graphical representation showing results of histologic assessment of joints from AIRE +/+ , AIRE + ' and AIRE 1' mice. For each group, >155 MCP/MTP joints from 12 mice were assessed. Bars show the mean and SEM. P values were determined by Mann- Whitney 1 -tailed test.
  • Figure 3A is a graphical representation showing normal response of AIRE 1' T cells and dendritic cells (DCs) to type II collagen (CII).
  • Figure 3B is a graphical representation showing limiting dilution of CD4 T cells in an antigen recall assay.
  • CD4 T cells from chick Cll-immunized AIRE' ' and wild-type mice were cultured with chick CII and Cll-primed wild-type antigen presenting cells (APCs) for up to 72 hours. Control cultures received no CII.
  • Figure 3C is a graphical representation showing interleukin-17 (IL-17) concentrations (after 24 hours) and IL-4 concentrations (after 48 hours) in the group of culture supernatants with the highest concentration of T cells shown in Figure 3B, cultured with and without CII. Bars show the mean ⁇ SEM.
  • IL-17 interleukin-17
  • Figure 3D includes copies of photographic representations showing results of enzyme-linked immunospot assay for IFNy production. Representative wells from AIRE' ' and wild-type mouse CD4 T cells cultured for 48 hours with wild-type APCs and either no stimulus (i), 100 ⁇ g/ml of chick CII (ii), or 2 ⁇ g/ml of concanavalin A (iii) are shown.
  • Figure 3E includes graphical representations showing 3H-thymidine incorporation in DCs from ovalbumin (OVA)-injected or phosphate buffered saline.
  • OVA ovalbumin
  • PBS phosphate buffered saline.
  • OVA-specific class II major histocompatibility complex (MHC)-restricted transgenic T cells (OTII) or OVA-specific class I MHC-restricted transgenic T cells (OT-I) (n 6 wells).
  • Results are representative of experiments performed 2 or more times. Bars show the mean ⁇ SEM.
  • Figure 4A is a graphical representation showing enhanced antibody response to murine type II collagen (CII) in AIRE 1' mice.
  • Figure 4B is a graphical representation showing serum anti-murine CII IgM concentrations in wild-type and AIRE " " mice. Bars show the mean and SEM arbitrary units/ml. j
  • Figure 4D includes copies of images of immunofluorescent staining using peanut agglutinin to detect germinal centers in splenic B cell follicles from wild-type and AIRE' ' mice 14 days after CII immunization (original magnification x 40).
  • FIG. 5B is a graphical representation showing T cell help to B cells for anti- murine type II collagen (anti-CII) antibody production.
  • AIRE' ' or wild-type CD4 + CD25 " T cells were cotransferred with wild-type B220 + cells into Rag! ' ' ' mice, which were then immunized with chick CII/Freund's complete adjuvant.
  • AIRE' ' and wild-type littermate (WT) mice were immunised by intradermal injection at the base of the tail with 100 ⁇ of an emulsion containing mouse type II collagen (100 ⁇ g) and complete Freund's adjuvant. Arthritis, seen as redness and swelling of the paws, was evaluated for up to 41 days post injection. 1 ) 1
  • AIRK and wild-type littermate (WT) mice were immunised by intradermal injection at the base of the tail with 100 ⁇ of an emulsion containing mouse type II collagen (100 ⁇ g) and complete Freund's adjuvant.
  • AIRE 1' and wild-type littermate (WT) mice were immunised by intradermal injection at the base of the tail with 100 ⁇ of an emulsion containing mouse type II collagen (100 ⁇ g) and complete Freund's adjuvant. Mice were bled 41 days later and the serum anti-mouse type II collagen antibody levels were determined.
  • Figure 7A is a graphical representation showing the concentration of anti type II collagen immunoglobulin G (IgG) per ml of serum from mice immunized once with an autoantigen (type II collagen). Results from AIRE " " (solid bars) and wild type (open bars) are shown. Results from various time points are depicted as indicated on the X- axis.
  • IgG immunoglobulin G
  • Figure 7B is a graphical representation showing the concentration of anti type II collagen immunoglobulin M (IgM) per ml of serum from mice immunized once with an autoantigen (type II collagen). Results from AIRE " " (solid bars) and wild type (open bars) are shown. Results from various time points are depicted as indicated on the X- axis.
  • IgM anti type II collagen immunoglobulin M
  • SEQ ID NO: 1 is a cDNA sequence encoding AIRE protein from Mus musculus.
  • SEQ ID NO: 2 is an amino acid sequence of AIRE protein from Mus musculus.
  • SEQ ID NO: 3 is a cDNA sequence encoding AIRE protein from Rattus norvegicus.
  • SEQ ID NO: 4 is an amino acid sequence of AIRE protein from Rattus norvegicus.
  • SEQ ID NO: 5 is a cDNA sequence encoding AIRE protein from Pan troglodytes.
  • SEQ ID NO: 6 is an amino acid sequence of AIRE protein from Pan troglodytes.
  • SEQ ID NO: 7 is a cDNA sequence encoding AIRE protein from Homo sapiens.
  • SEQ ID NO: 8 is an amino acid sequence of AIRE protein from.Homo sapiens/
  • SEQ ID NO: 9 is an amino acid sequence of a peptide derived from mouse oligodendrocyte glycoprotein (MOG) designated MOG35-55.
  • SEQ ID NO: 10 is an amino acid sequence of a peptide derived from human interphotoreceptor retinoid binding protein (IRBP) designated IRBP1-20. Detailed Description of the Preferred Embodiments
  • the term "autoimmune regulator protein” or "AIRE protein” shall be understood to mean a protein capable of regulating expression or presentation of TS As in the thymus, preferably in mTECs, that preferably comprises a sequence at least about 65% identical to a sequence set forth in one or more of SEQ ID NOs: 2, 4, 6 or 8.
  • the AIRE protein is encoded by a gene comprising a sequence at least about 65% identicial to a sequence set forth in any one of SEQ ID NOs: 1, 3, 5 or 7, preferably SEQ ID NO: 1.
  • the degree of sequence identity is at least about 68% or 69% or 75% or 80% or 85% or 88% or 90% or 95% or 99%.
  • the AIRE protein comprises a nuclear import signal and a caspase- recruitment domain and a SAND domain and a plant homeodomain (PHD) zinc finger domain (preferably 2 PHD domains) and one or more nuclear receptor binding motif(s) and a proline rich region.
  • PHD plant homeodomain
  • the AIRE protein comprises a sequence set forth in SEQ ID NO: 2 or is a homolog or ortholog thereof.
  • the term "genetically-modified to reduce AIRE protein expression levels” encompasses non-human animals that have been modified to express a nucleic acid that reduces translation of mRNA encoding AIRE protein or that induces degradation or cleavage of said mRNA or that have been genetically-modified to disrupt, interrupt or remove one or more coding regions of an AIRE-encoding gene to thereby reduce expression of said gene. This term encompasses animals that are modified to reduce AIRE protein expression to the level that it is undetectable or such that AIRE protein is not expressed.
  • a subject having a "reduced” level of AIRE protein activity shall be understood to have a lower level of activity (and/or production) compared to a subject of the same species or genus.
  • the level. of activity is less than the mean level of activity in a population of subjects of the same species.
  • the level of activity is less than the level in an isogenic or near isogenic subject.
  • an isogenic or near isogenic subject will be understood to be a subject isogenic or near isogenic other than the genetic modification to reduce AIRE protein production.
  • the term "reduced" activity will be understood to include undetectable activity or no activity.
  • non-human animal shall be taken to mean any animal that endogenously expresses an AIRE protein other than a human (i.e., Homo sapiens).
  • Preferred animals are those in which antibodies are routinely produced, such as mice, rats, chickens, goats, rabbits, donkeys, amongst other.
  • the subject is one that is readily genetically-modified, such as a rodent, e.g., a mouse or a rat.
  • the non-human animal is a mammal, such as a rodent, preferably murine, such as a mouse.
  • protein comprising an autoantigen binding domain shall be taken to mean protein comprising a region of an antibody that is capable of binding (preferably, specifically binding) to an autoantigen.
  • An autoantigen binding domain of an antibody includes a single variable region, e.g., from a light chain or a heavy chain (e.g., a domain antibody).
  • the autoantigen binding domain need not be a series of contiguous amino acids, or even amino acids in a single polypeptide chain.
  • a Fv region from an antibody is produced from two different polypeptide chains (i.e., a heavy chain variable region (VH) and a light chain variable region (VL)) that interact to form an antigen binding site.
  • VH heavy chain variable region
  • VL light chain variable region
  • protein comprising an autoantigen binding domain includes fragments directly derived from an antibody as well as proteins corresponding to such a fragment produced using recombinant means or proteins comprising such fragments.
  • the VH is not linked to a heavy chain constant domain (CH) 1 and/or the VL is not linked to a light chain constant domain (CL).
  • Exemplary proteins comprising autoantigen binding domains include a Fab fragment, a Fab' fragment, a F(ab') fragment, a single chain Fv (scFv), a diabody, a triabody, a tetrabody or higher order complex, or any of the foregoing linked to a constant region or domain thereof, e.g., CH2 or CH3 domain or a chimeric antibody.
  • a "Fab fragment” consists essentially of a monovalent antigen-binding fragment of an antibody, and can be produced by digestion of a whole antibody with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain- or can be produced using recombinant means.
  • a "Fab' fragment” of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody treated in this manner. A Fab' fragment can also be produced by recombinant means.
  • An "F(ab')2 fragment” of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • An "Fab 2 " fragment is a recombinant fragment comprising two Fab fragments linked using, for example a leucine zipper or a CH3 domain.
  • a “single chain Fv” or “scFv” is a recombinant molecule containing the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable, flexible polypeptide linker.
  • the present invention encompasses any of the foregoing proteins additionally comprising a Fc region or a CH2 region or a CH3 region or combinations thereof.
  • the term "protein” shall be taken to include a single polypeptide chain, i.e., a series of contiguous amino acids linked by peptide bonds or a series of polypeptide chains covalently or non-covalently linked to one another (i.e., a polypeptide complex).
  • the series of polypeptide chains can be covalently linked using a suitable chemical or a disulphide bond.
  • non-covalent bonds include hydrogen bonds, ionic bonds, Van der Waals forces, and hydrophobic interactions.
  • a non-covalent bond contemplated by the present invention is the interaction between a VH and a VL, e.g., in some forms of diabody or a triabody or a tetrabody. . It will be understood from the foregoing that a "polypeptide chain" is a series of contiguous amino acids linked by peptide bonds.
  • antigen shall be understood to mean any composition of matter against which a subject can raise an antibody response.
  • exemplary antigens include proteins, peptides, polypeptides, carbohydrates, phosphate groups, phosphor- peptides or polypeptides, glyscpsylated peptides, etc, as well as mixtures thereof (e.g., in tissues, tissue homogenates or cells).
  • an antibody is raised against an epitope or antigenic determinant in the antigen.
  • epitope or antigenic determinant is not limited to peptides or polypeptides.
  • antibodies are capable of binding to carbohydrates or glycosylated peptides or polypeptides, phosphates or phospo-peptides or polypeptides amongst other epitopes.
  • An epitope or peptide or polypeptide comprising same can be administered to an animal to generate antibodies against the epitope.
  • the term "epitope” or "antigenic determinant” also encompasses a peptide or polypeptide comprising the epitope.
  • Such a peptide or polypeptide can be a fragment of the antigen in which the epitope occurs in nature or recombinant form of the fragment, or a different peptide or polypeptide, e.g., a carrier polypeptide linked to the epitope.
  • autoantigen shall be understood to mean an antigen that is capable of eliciting an antibody response in a subject against an antigen expressed by the subject (i.e., a "self-antigen”).
  • the autoantigen can be a self-antigen produced by the subject or an epitope thereof, or can be an antigen from another subject or epitope thereof or a synthetic antigen. It will be apparent to -the skilled artisan from the foregoing that an autoantigen need not be the same or even highly similar to the self-antigen against which an antibody response is elicited.
  • the autoantigen may have an epitope similar to or that forms a similar structure to an epitope in the self-antigen against the autoimmune response is raised.
  • the autoantigen is from the subject being immunized, e.g., the subject is a mouse and the autoantigen is from a mouse.
  • the autoantigen is from a subject different to the subject being immunized, e.g., from a different species and/or genus and/or family and/or order and/or class and/or phylum and/or kingdom.
  • the subject being immunized is a mouse and the autoantigen is from a human.
  • autoantigen also encompasses synthetic or non-naturally occurring antigens.
  • autoantigen also encompasses a nucleic acid encoding an antigen that is administered to a subject such that the subject expresses the antigen e.g., a DNA vaccine. In such a case, the subject raises an antibody response against the encoded autoantigen.
  • autoantigen also includes a plurality of previously isolated autoantigens or a mixture of autoantigens derived directly from a tissue or a mixture of cells or a tissue sample or a tissue homogenate. For example, this term includes a mixture of cells or a tissue sample or a tissue homogenate from a joint or from cartilage or from a pancreas (e.g., pancreatic beta cells) or from neuronal tissue.
  • an antibody or protein comprising an autoantigen binding domain may be identified or isolated using the cells or tissue or homogenate or using one or more isolated autoantigens that occur or are suspected to occur in the cells or tissue.
  • self-antigen will be understood to mean an antigen expressed by a non-human animal having reduced AIRE protein activity (in constrast to an autoantigen, which does not necessarily have to be expressed by the non-human animal).
  • a self ⁇ antigen is a type of autoantigen.
  • the self-antigen is endogenous to the subject, however this term also encompasses antigens that a subject is genetically modified to express.
  • the term "associated" when discussing the association of a condition with an antigen shall be taken to mean an antigen is. causative of a condition (e.g., a mutant protein), or an antigen expressed by an agent that is causative of a condition (e.g., a cancer cell), or an antigen that is overexpressed or overproduced or underexpressed or underproduced in a condition.
  • the term "immunize” and grammatical equivalents thereof shall be understood to mean the administration of an autoantigen to a subject in such a manner that the subject raises an antibody response against the autoantigen.
  • This term does not mean that the antigen is administered solus (i.e., without other components), for example, the antigen may be administered with an adjuvant to increase the immune response by the subject or in the form of a vaccine.
  • the term "immunizing the non-human animal with the autoantigen once" shall be understood to mean that the autoantigen is administered to a subject at a single point in time, i.e., without repeating administration at a later point of time.
  • the subject may receive multiple immunizations of the autoantigen at a single point in time.
  • the terms "expression”, “expressed” or “express” shall be taken to mean transcription of a nucleic acid to produce a RNA and translation of the RNA to produce a peptide, polypeptide or protein. Reducing expression may be achieved by, for example, reducing transcription, which in turn reduces translation, or by reducing levels of RNA available for translation or by reducing translation.
  • expression construct is to be taken in its broadest context and includes a promoter that operably linked to a nucleic acid encoding an antibody or protein comprising an autoantigen binding domain such that the promoter can confer expression on the nucleic acid.
  • the expression construct is an expression vector.
  • expression vector refers to a nucleic acid comprising an expression construct and comprising sequences that permit maintenance or replication of the vector in a cell.
  • Exemplary expression vectors include a plasmid, bacteriophage, phagemid, cosmid, virus sub-genomic or genomic fragment, or other nucleic acid capable of maintaining and or replicating heterologous DNA in an expressible format.
  • operably linked in the context of a promoter operably linked to a nucleic acid means positioning said nucleic acid such that its expression is controlled by the promoter. Promoters are generally positioned 5' (upstream) to the nucleic acid upon which they confer expression. To construct heterologous promoter/structural gene combinations, it is generally preferred to position the promoter at a distance from the gene transcription start site that is approximately the same as the distance between that promoter and the gene it controls in its natural setting, i.e., the gene from which the promoter is derived. As is known in the art, some variation in this distance can be accommodated without loss of promoter function.
  • the preferred positioning of a regulatory sequence element with respect to a heterologous gene to be placed under its control is defined by the positioning of the element in its natural setting, i.e., the gene from which it is derived. Again, as is known in the art, some variation in this distance can also occur.
  • promoter is to be taken in its broadest context and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiator element, which is required for accurate transcription initiation, with or without additional regulatory elements (i.e., upstream activating sequences, transcription factor binding sites, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue specific manner.
  • promoter is also used to describe a recombinant, synthetic or fusion molecule, or derivative which confers, activates or enhances the expression of a nucleic acid molecule to which it is operably linked.
  • Preferred promoters can contain additional copies of one or more specific regulatory elements to further enhance expression and/or alter the spatial expression and/or temporal expression of said nucleic acid molecule.
  • the term "display” and grammatical equivalents shall be understood to mean that an antibody or protein comprising .an autoantigen binding domain thereof is bound to or immobilized in or on a cell or particle in such a manner that it forms a conformation suitable for binding to an antigen that is present in/on or contacted to the antibody or protein.
  • an antibody or protein is displayed on the surface of a cell or particle.
  • particle means a composition of matter capable of displaying an antibody or protein comprising an autoantigen binding domain and capable of directly or indirectly linking nucleic acid encoding the antibody or domain to the encoded protein.
  • exemplary particles include phage and other viruses and ribosomes.
  • the term "obtaining nucleic acids... from a non-human animal” shall be understood to include merely indicating a source of the nucleic acids as opposed to requiring any specific method steps to obtain the nucleic acids.
  • the nucleic acids may be provided by a third party.
  • This term also includes providing or isolating the nucleic acids.
  • the term includes isolating the nucleic acids from the non-human animal or from cells isolated from the non-human animal.
  • this term includes isolating cells from a non-human animal (e.g., antibody producing cells) and isolating the nucleic acids from those cells.
  • the term "derived from” shall be taken to indicate that a specified integer is obtained from a particular source albeit not necessarily directly from that source. Accordingly, this term does not require any specific method steps to, obtaining the integer, merely that it is obtained from the specified source by some means.
  • autoimmune response means that an autoantigen induces antibody production against a self-antigen produced by the non-human animal immunized with the autoantigen.
  • the subject produces B cells that proliferate and express and secrete antibodies that bind to the self-antigen.
  • the subject may also produce T helper cells that recognize the self-antigen (and preferably, the autoantigen) and induce antibody production.
  • autoimmune condition means a condition arising from reaction of a subject's immune system to one or more antigens, preferably one or more self-antigens.
  • the autoimmune condition is associated with the production by a subject of antibodies that bind to the self-antigen(s).
  • preventing include administering a therapeutically effective amount of an inhibitor(s) and/or agent(s) described herein sufficient to stop or hinder the development of at least one symptom of a specified disease or condition.
  • treating include administering a therapeutically effective amount of a compound described herein sufficient to reduce or eliminate at least one symptom of a specified disease or condition.
  • diagnosis includes any primary diagnosis of a clinical state or diagnosis of recurrent disease.
  • Prognosis refers to the likely outcome or course of a disease, including the chance of recovery or recurrence.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • the present invention contemplates the use of non-human animals naturally having reduced AIRE protein activity or induced (e.g., genetically-modified) to have reduced AIRE protein activity.
  • the animal can be modified to produce a molecule that reduces AIRE production, e.g., by expressing a nucleic acid that reduces AIRE gene transcription and/or translation of the transcribed mRNA.
  • the animal can be genetically-modified to mutate an AIRE- encoding gene to reduce AIRE protein production.
  • Animals can also be modified to reduce AIRE activity, e.g., by mutating a region of an AIRE-encoding gene required for activity, e.g., a SAND domain. Accordingly, any embodiment herein directed to reducing AIRE expression shall be taken to apply mutatis mutandis to reducing AIRE activity.
  • nucleic acids include a catalytic nucleic acid (e.g., a ribozyme), interfering RNA, siRNA or microRNA.
  • catalytic nucleic acid/nucleic acid refers to a RNA or RNA-containing molecule (also known as a "ribozyme”) which specifically recognizes a distinct substrate and catalyses the chemical modification of this substrate, preferably cleavage of the substrate.
  • the nucleic acid bases in the catalytic nucleic acid can be bases A, C, G, and U.
  • the catalytic nucleic acid contains an antisense sequence for specific recognition of a target nucleic acid (i.e., a mRNA encoding an AIRE protein), and a nucleic acid cleaving enzymatic activity (also referred to herein as the "catalytic domain").
  • a target nucleic acid i.e., a mRNA encoding an AIRE protein
  • a nucleic acid cleaving enzymatic activity also referred to herein as the "catalytic domain”
  • Exemplary ribozymes useful in this invention are a hammerhead ribozyme (Haseloff and Gerlach, 1988) and a hairpin ribozyme (Klein et ah, 1998).
  • DNA encoding the ribozymes of the present invention can be chemically synthesized using methods known in the art. Such DNA is then operably linked to a promoter and used to produce a transgenic animal by methods known in the art and/or described herein.
  • Catalytic nucleic acids useful in the invention should be capable of "hybridizing" a target nucleic acid molecule (for example an mRNA encoded by a nucleic acid of the present invention) under physiological conditions, namely those conditions within a cell in a non-human animal.
  • a target nucleic acid molecule for example an mRNA encoded by a nucleic acid of the present invention
  • RNA interference is useful for specifically inhibiting the production of a particular transcript or protein, i.e., an AIRE-encoding mRNA or an AIRE protein.
  • a particular transcript or protein i.e., an AIRE-encoding mRNA or an AIRE protein.
  • dsRNA duplex RNA
  • This technology relies on the presence of dsRNA molecules that contain a sequence that is substantially identical to the mRNA of the gene of interest or part thereof, in this case an mRNA encoded by a nucleic acid of the present invention.
  • the dsRNA can be produced from a single promoter in a recombinant vector or host cell, where the sense and anti-sense sequences are flanked by an unrelated sequence which enables the sense and antisense sequences to hybridize to one another to form the dsR A molecule with the unrelated sequence forming a loop structure.
  • the design and production of suitable dsRNA molecules for the present invention is well within the capacity of a person skilled in the art, particularly considering Waterhouse et al. (1998), W099/32619, WO99/53050, WO99/49029, and WO01/34815.
  • a DNA is introduced into a subject that directs ⁇ the synthesis of an at least partly double stranded RNA product(s) with homology to mRNA encoding an AIRE protein.
  • the DNA therefore comprises both sense and antisense sequences that, when transcribed into RNA, can hybridize to form a double stranded RNA region.
  • the sense and antisense sequences are separated by a spacer region that comprises an intron which, when transcribed into RNA, is spliced out. This arrangement has been shown to result in a higher efficiency of gene silencing.
  • the double stranded region may comprise one or two RNA molecules, transcribed from either one DNA region or two.
  • the presence of the double stranded molecule is generally considered to trigger a response from an endogenous mammalian system that destroys both the double stranded RNA and also the homologous RNA transcript from the target mammalian gene, efficiently reducing or eliminating the activity of the target gene.
  • the length of the sense and antisense sequences that hybridize should each be at least 19 contiguous nucleotides, preferably at least 30 or 50 nucleotides, and more preferably at least 100, 200, 500 or 1000 nucleotides.
  • the full-length sequence corresponding to the entire gene transcript may be used. The lengths are most preferably 100-2000 nucleotides.
  • the degree of identity of the sense and antisense sequences to the targeted transcript should be at least 85%, preferably at least 90% and more preferably 95-100%.
  • the RNA molecule may of course comprise unrelated sequences which may function to stabilize the molecule.
  • the RNA molecule may be expressed under the control of a RNA polymerase II or RNA polymerase III promoter. Examples of the latter include tRNA or snRNA promoters.
  • Preferred small interfering RNA (“siRNA”) molecules comprise a nucleotide sequence that is identical to about 19-21 contiguous nucleotides of the target mRNA.
  • the siRNA sequence commences with the dinucleotide AA, comprises a GC- content of about 30-70% (preferably, 30-60%, more preferably 40-60% and more preferably about 45%-55%), and does not have a high percentage identity to any nucleotide sequence other than the target in the genome of the mammal in which it is to be introduced, for example as determined by standard BLAST search.
  • microRNA small interfering RNA
  • MicroR A regulation is a specialized branch of the RNA silencing pathway that evolved towards gene regulation, diverging from conventional RNAi.
  • MicroRNAs are a specific class of small RNAs that are encoded in gene-like elements organized in a characteristic inverted repeat. When transcribed, microRNA genes give rise to stem- looped precursor RNAs from which the microRNAs are subsequently processed. MicroRNAs are typically about 21 nucleotides in length. The released miRNAs exert sequence-specific gene repression (see, for example, Millar and Waterhouse, 2005; Pasquinelli et al, 2005).
  • Nucleic acids encoding molecules that reduce AIRE-protein production are used to produce transgenic animals.
  • an expression construct as described herein according to any embodiment is injected into the pronucleus of a fertilized oocyte and the oocyte then implanted into a uterus of a pseudopregnant female recipient. Any resulting offspring born are then screened to determine those carrying and preferably expressing the introduced nucleic acid, i.e., "founder animals". Founder animals are then bred to produce offspring, which may be heterozygous or homozygous for the introduced nucleic acid.
  • Such methods for producing a genetically-modified cell and/or animal are known in the art and/or described in US 10/820,777 and WO93/14200. Methods for producing genetically- modified cows using microinjection are described, for example, in Krimpenfort et al. ( 1991 ) and/or Hyttinen et al. ( 1994).
  • a genetically-modified non-human mammal is produced by introducing an expression construct or expression vector as described herein according to any embodiment into a somatic cell, e.g., a fibroblast of a non-human animal, resulting in a genetically-modified non-human animal cell.
  • Methods for introducing a nucleic acid into a somatic cell include, for example, electroporation, microinjection, transfection mediated by DEAE-dextran, transfection mediated by calcium phosphate, transfection mediated by liposomes such as by using Lipofectamine (Invitrogen) and/or cellfectin (Invitrogen), transduction by Adenoviuses, Herpesviruses, Togaviruses or Retroviruses and microparticle bombardment such as by using DNA- coated tungsten or gold particles (Agacetus Inc., WI, USA).
  • the genetically-modified non-human animal cell or the nucleus thereof is then injected into or fused with an enucleated mature oocyte from the same species of non-human animal as the somatic cell to produce a monocell embryo.
  • This monocell embryo is then maintained for a time and under conditions sufficient for a multi-cell embryo to form, e.g., a blastocyst to form, and the multi-cell embryo is then administered to or implanted into a uterus of a non-human animal and maintained under conditions for a non-human animal to develop and be born.
  • an expression construct is included in a retrovirus particle, preferably containing the envelope protein from vesicular somatic protein.
  • the retrovirus particle is injected between the zona pellucida and the membrane of an oocyte of a non-human animal at a period when the nuclear membrane is absent, e.g., during metaphase II (Mil) of the second meiosis.
  • the infected oocyte is then fertilized with a sperm cell, and incubated for a time and under conditions sufficient for an embryo, e.g., a blastocyst to form.
  • the embryo is then administered to, e.g., implanted in a uterus of a female non-human animal and a genetically-modified non-human animal permitted to develop and be born.
  • a suitable retrovirus-mediated method for producing a genetically- modified non-human mammal, e.g., a cow or bull, is described in Chan et al. (1998).
  • the genetically modified non-human animals described herein can be produced by methods other than the specific methods taught herein, e.g., sperm-mediated transgenesis (Lavitrano et al, 1989), linker based sperm-mediated gene transfer technology (LB-SMGT; US7,067,308), or embryonic stem cell-mediated transgenesis (e.g., as described in the context of rats in Li et al, 2008).
  • non-human animals (or cells) having reduced AIRE activity can be produced by mutating a gene encoding an AIRE protein, e.g., using gene-targeting (knock-in or knock-out, syn. homologous recombination or targeted disruption) technology.
  • Such animals can have a mutation introduced into a region of an AIRE protein required for activity, e.g., mutations in the CARD domain and/or SAND domain and/or plant homeodomain(s) have been shown to reduce AIRE protein activity (Mathis and Benoist, 2009).
  • an AIRE-encoding gene is mutated to reduce or prevent production of a functional AIRE protein, i.e., the gene is disrupted or knocked out or to remove a region of the protein required for activity (e.g., as domain discussed above).
  • an AIRE-encoding gene is mutated to reduce or prevent production of AIRE protein, i.e., the gene is disrupted or knocked out or to remove a region of the protein required for expression (e.g., transcription and/or translation) and/or the entire gene is removed.
  • Methods for producing knock-in and knock-out animals are known in the art and described, for example, in Nagy et al, 2002, and Tymms and Kola, 2001.
  • One of two configurations of constructs is generally used for a. vector for mutating a gene using homologous recombination, i.e., an insertion construct or a replacement construct.
  • An insertion construct comprises a region of homology to the target gene cloned as a single continuous sequence.
  • the insertion construct additionally comprises a heterologous nucleic acid that is to be inserted into the target gene positioned adjacent to and, if required, in-frame with the region of homology.
  • the insertion vector is then linearised, e.g., by cleavage of a unique restriction site. Homologous recombination introduces the insertion construct sequences and any adjacent nucleic acid into the homologous site of the target gene, interrupting normal target-gene structure by adding an additional sequence, for example, a LoxP site.
  • Such a vector is useful for, for example, introducing one or more LoxP sites into a region of interest to flank a nucleic acid region or for introducing a mutation that alters the sequence of the nucleic acid of the invention (e.g., by introducing a stop codon).
  • Such a vector is also useful for mutating a region of a gene that encodes a region of the protein required for activity, e.g., to reduce AIRE protein activity.
  • a replacement construct is more commonly used to knock-out a gene of interest or to introduce a mutation to a gene of interest.
  • This form of construct contains two regions of homology to the target gene located on either side of a heterologous nucleic acid (for example, encoding one or more positive selectable markers, such as, for example, a fluorescent protein (e.g. enhanced green fluorescent protein), ⁇ -galactosidase, an antibiotic resistance protein (e.g. neomycin resistance or zeocin resistance) or a fusion protein (e.g. ⁇ -galactosidase-neomycin resistance fusion protein, ⁇ -geo) and/or a mutation to be introduced.
  • a fluorescent protein e.g. enhanced green fluorescent protein
  • ⁇ -galactosidase an antibiotic resistance protein
  • a fusion protein e.g. ⁇ -galactosidase-neomycin resistance fusion protein, ⁇ -geo
  • homologous recombination occurs by at least two recombination events (or a double cross-over event) that leads to the replacement of target-gene sequences with the replacement-construct sequences. More specifically, each region of homology in the vector induces at least one recombination event that leads to the heterologous nucleic acid in the vector replacing the nucleic acid located between the regions of homology in the target gene.
  • an effective targeting vector comprises a nucleic acid comprising a nucleotide sequence that is effective for homologous recombination with the nucleic acid.
  • a replacement targeting vector comprises at least two regions of nucleic acid that are substantially identical to a genomic sequence of the nucleic acid or a region thereof.
  • some degree of non-identity does not significantly adversely affect the gene targeting capability of a construct of the invention.
  • a higher the degree of identity between the regions of homology in the vector and the gene increases the likelihood of effective homologous recombination.
  • a region of a vector homologous to a target nucleic acid comprises a nucleotide sequence that is at least about 80% identical to the target sequence, more preferably, 90% or 95% identical.
  • each region of homology comprises at least about 1500bp that is substantially identical to a target sequence, more preferably, 2000bp and even more preferably, at least about 3000bp. Guidelines for the selection and use of sequences are described for example in Bollag et al. (1989).
  • Suitable sites for gene targeting will also be apparent to the skilled artisan.
  • a wild-type gene is mutated and/or disrupted by inserting a heterologous nucleic acid into all or a portion of the region of the gene that encodes a polypeptide.
  • a- targeting construct is designed to recombine with a particular portion within an enhancer, promoter, coding region, start codon, noncoding sequence, intron/s or exon/s of the gene (preferably, a region required for expression of a functional expression product).
  • Suitable targeting constructs of the invention are prepared using standard molecular biology techniques known to those of skill in the art. For example, techniques useful for the preparation of suitable vectors are described by Sambrook et al. (2001).
  • An appropriate vector includes, for example, an insertion vector such as the insertion vector described by Capecchi (1989); or a vector based on a promoter trap strategy or a polyadenylation trap, or "tag-and-exchange" strategy described by Bradley et al. (1992); or Askew et al. (1999).
  • an insertion vector such as the insertion vector described by Capecchi (1989); or a vector based on a promoter trap strategy or a polyadenylation trap, or "tag-and-exchange" strategy described by Bradley et al. (1992); or Askew et al. (1999).
  • any of a number of appropriate vectors known in the art can be used as the basis of a suitable targeting vector.
  • any vector that is capable of accommodating the recombinant nucleic acid required for homologous recombination and to disrupt the target gene is useful.
  • pBR322, pACY164, pKK223-3, pUC8, pKG, pUC19, pLG339, pR290, pKClOl or other plasmid vectors can be used.
  • a viral vector such as the lambda gtl l vector system is useful as the backbone (e.g. cassette) for a targeting construct.
  • the targeting vector comprises one or more recombination sites (i.e., recombinase binding sites), such as, for example, a LoxP site (which is a recognition site of the PI recombination enzyme Cre) or a fit site (which is a recognition site of the yeast recombinase flp).
  • recombination sites i.e., recombinase binding sites
  • LoxP site which is a recognition site of the PI recombination enzyme Cre
  • a fit site which is a recognition site of the yeast recombinase flp
  • loxP sites in the same orientation near each other in a nucleic acid, it is possible to excise the nucleic acid intervening the LoxP sites by expressing the enzyme, Cre in the cell thereby, leaving a single loxP site in the original DNA and the remaining loxP sites in a circular piece of DNA containing the intervening sequence. Accordingly, loxP sites or fit sites that are inserted flanking a region gene or the entire gene are useful for the removal of the intervening sequence.
  • Conditional silencing or knocking out a gene means that that the silencing of the gene is dependent upon an external stimulus that may be spatially and/or temporally controlled.
  • the gene is flanked by two or more loxP sites (i.e., it is "floxed") and upon expression of Cre recombinase the gene is removed.
  • the expression of Cre may be spatially controlled (e.g., under the control of a tissue or cell specific promoter) and/or may be temporally controlled (e.g. under control of a promoter that is expressed at a certain stage of development or that is inducible, e.g., a tet inducible or repressible promoter).
  • the present invention also provides a genetically-modified non-human animal comprising one or more (preferably at least two) recombinase binding sites positioned within or flanking an AIRE-encoding gene or a region thereof, e.g., within its genome.
  • Recombination sites are also useful for removing one or more selectable markers and or producing a new selectable marker following integration into the genome of a cell or organism (e.g., as exemplified herein).
  • a suitable cell for the production of a knockout animal is, for example, an embryonic stem cell.
  • An embryonic stem cell is a pluripotent cell isolated from the inner cell mass of mammalian blastocyst.
  • ES cells can be cultured in vitro under appropriate culture conditions in an undifferentiated state and retain the ability to resume normal in vivo development as a result of being combined with blastocyst and introduced into the uterus of a pseudopregnant foster mother.
  • stem cells and stem cell lines are known in the art, such as, for example, AB-1, HM-1, D3.
  • a suitable stem cell or stem cell line will depend upon the type of genetically-modified animal to be produced. For example, should a genetically-modified mouse be desired a mouse ES cell line is used. Furthermore, should an inbred strain of genetically-modified mouse be preferred, an ES cell line derived from the same strain of mouse that is to be used is preferred. Following transfection, a cell is maintained under conditions sufficient for homologous recombination to occur while maintaining the pluripotency of the ES cell.
  • an ES cell is selected that has homologously recombined to introduce the targeting vector into its genome (as opposed to random integration).
  • a method used for eliminating cells in which the construct integrated into the genome randomly, thus further enriching for homologous recombinants, is known as positive-negative selection. Such methods are described, for example, in US 5,464,764.
  • a cell is screened using, for example, PCR or Southern blotting to determine a targeting construct that has integrated into the correct region of the genome rather than randomly integrated. Methods for such screening are known in the art, and described, for example, in Nagy et al (2002) and/or Tymms and Kola (2001).
  • the cell is preferably grown to form an ES cell colony using methods known in the art. One or more cells from the colony are then used to produce a chimeric animal.
  • An example of a method used to generate chimeras involves injecting a genetically modified ES cell into the blastocoel cavity of a developing embryo.
  • a genetically modified ES cell For example, should the targeted ES cell be of mouse origin, an ES cell is injected into the blastocoel cavity of a 3.5 -day-old mouse embryo.
  • the injected embryo is surgically implanted into the uterus of a foster mother, for example, a pseudopregant female.
  • a resultant offspring is a chimera as its tissues is derived from both the host embryo and from the ES cell. Should the ES cell populate the germ line, the chimera can pass an altered gene to offspring, resulting in a new mouse strain in which all cells contain an altered gene.
  • the present invention clearly contemplates both heterozygous and homozygous knockout non-human mammals.
  • the knockout mammals described herein can be produced by methods other than the embryonic stem cell method described above, for example by the pronuclear injection of recombinant genes into the pronuclei of a one-cell embryo or other gene targeting methods which do not rely on the use of a transfected ES cell.
  • suitable methods for the production of a knockout animal using nuclear transfer are known in the art and/or described, for example, in Kolber-Simonds et al. (2004); or reviewed in Gong and Rong (2003).
  • Additional methods known in the art include the use of zinc finger nucleases to remove an entire gene or a region there required to expression or to encode an active protein, e.g., as described in Geurts et al. (2009).
  • the present invention contemplates a genetically-modified non-human animal that comprises a further genetic modification.
  • Such animals are produced by introduing a further genetic modification (e.g., as described herein) or by crossing the genetically- modified anial having reduced AIRE protein activity with a genetically-modified animal having the desired modification.
  • Examplary genetically-modified animals useful in such crosses are mice having a humanized immunoglobulin locus (e.g., as discussed herein) or cattle having humanized immunoglobulin locus (e.g., as described in Kuroiwa et al, 2009). Such modifications could also be introduced into a genetically-modified anial having reduced AIRE protein activity, e.g., using methods described herein.
  • Other genetically-modified animals include transgenic non-human animals expressing scFv or Fab or an antigen of interest and/or a T cell receptor specific to an antigen of interest.
  • the present invention is useful for producing antibodies against any autoantigen.
  • the only requirement is that the autoantigen is capable of eliciting production of antibodies that recognize an antigen produced by the subject, i.e., a self-antigen.
  • an autoantigen can be from or endogenous to the subject to be immunized, e.g., directly isolated from the subject or a related subject or produced using synthetic or recombinant means.
  • the autoantigen can also be from another subject (e.g., of a different species, etc) or an artificial autoantigen, provided that the autoantigen is capable of eliciting an immune response against an antigen produced by the subject.
  • the autoantigen is structurally related to a self-antigen, or comprises an epitope structurally related to an epitope of a self-antigen.
  • the term "structurally-related" includes related at the primary structural level (e.g., amino acid sequence) or related at the secondary or tertiary or quaternary level.
  • the autoantigen has at least about 70% or 80% or 85% or 90% or 95% sequence identity to at least a region of a self- antigen of the subject to be immunized.
  • the autoantigen is an antigen associated with a condition against which antibodies are desired for therapeutic, prophylactic, diagnostic and/or prognostic applications.
  • the autoantigen is expressed on a cancer cell in humans and is structurally similar to an antigen in the subject to be immunized.
  • Exemplary antigens include carcinoembryonic antigen, CD20, VEGF, amongst others.
  • the autoantigen is associated with an autoimmune disease, e.g., tumor necrosis factor.
  • the autoantigen is capable of inducing production of antibodies that induce an autoimmune condition in a non-human animal.
  • the autoantigen is collagen, preferably collagen type II (which induces arthritis or an arthritis-like condition); other collagens such as collagen type IX, collagen type V, collagen type XXVII, collagen type XVIII, collagen type IV,aggrecan I; pancreas- specific protein disulphide isomerise A2 (which induces diabetes or a diabetes-like condition); interphotoreceptor retinoid binding protein (IRBP; which induces autoimmune uveitis or an autoimmune uveitis-like ' disease); protein lipoprotein (which induces multiple sclerosis or a multiple-sclerosis-like condition); myelin basic protein (which induces multiple sclerosis or a multiple-sclerosis-like condition and Experimental Autoimmune Encephalomyelitis (EAE)); myelin oligodendrocyte glycoprotein (which induces multiple sclerosis or a multiple-associated a
  • ICA69 islet cell autoantigen
  • cytochrome P450 1A2 CYP1A2; involved in hepatitis and APS 1
  • Tph and Fabp2 tryptophan hydroxylase and fatty acid- binding protein 2
  • Tgn thyroglobulin
  • salivary proteins 1 and 2 Sptl and 2; involved in Sjogren's syndrome
  • Mater maternal effect gene
  • Preferred autoantigens include mouse Type II collagen and GM-CSF receptor.
  • the autoantigen is encoded by an AIRE-regulated gene or induces an immune response against a protein encoded by an AIRE-regulated gene.
  • exemplary autoantigens/proteins include Procollagen, type IX, al, Adipocyte, Clq and collagen domain containing, Procollagen, type V, a3, Procollagen, type XXVII, al, Procollagen, type XVIII, al, Macrophage receptor with collagenous structure, Procollagen, type IX, a3, Procollagen, type IV, a5, Aggrecan 1, Procollagen, type IV, al, Procollagen lysine, 2-oxoglutarate 5-dioxygenase 2, Procollagen, type II, al, Insulin II, promyeloperoxidase, myelin oligodendrocyte glycoprotein, myelin basic protein, proteolipid protein, interphotoreceptor retinoid-binding protein, insulin, IGF2, BAFF, peripheral tissue antigens (PTAs), peripheral tissue
  • NT_039268 Neutrophilic granule protein, Glutamic acid decarboxylase 1 gene (GAD67 protein), Glutamic acid decarboxylase 2 gene (GAD65 protein), alpha casein, intestinal trefoil factor, C defensin-related cryptdin peptide, salivary proteins 1 and 2, gamma casein, major urinary proteins 1, 3 and 4, gastric inhibitory polypeptide, oxytocin, amelogenin X chromosome, neuropeptide Y, preproneuropeptide y, alpha 1 microglobulin, spermine binding protein, hemoglobin y beta-like embryonic chain, neurotoxin homologue, cryptidin related sequence 2, lactotransferrin, serine protease (BSSP), prostaglandin D, purkinje cell protein 4, glucose-dependent insulinotropic polypeptide, mast cell protease-2, SI 00 calcium binding protein A9, aldose reductase, preproinsulin II, inter-alpha
  • the autoantigen is a fragment of any of the autoantigens mentioned above.
  • Particular examples include fragments of GAD67 or GAD65, the Al- 13 and the B9-23 peptides of insulin and the Bl 1-25 peptide of IGF2, fragments of GM- CSF receptor a chain and fragments of collagen, for example, fragments of Type II collagen.
  • Particular examples of fragments of collagen are those fragments generated by cyanogen bromide cleavage, for example the CB11 peptide from collagen (e.g., as described in Seyer et al, 1989).
  • Further examples include fragments of myelin oligodendrocyte glycoprotein, for example, the peptide
  • MEVGWYRSPFSRVVHLYRNGK (mouse MOG35-55; SEQ ID NO: 9), and fragments of IRBP, for example, the peptide GPTHLFQPSLVLDMAKVLLD (human IRBPl-20; SEQ ID NO: 10).
  • Preferred fragments of an antigen are immunogenic fragments, preferably fragments comprising a B cell epitope.
  • a preferred B-cell epitope will be capable of eliciting the production of antibodies when administered to a subject.
  • shorter B cell epitopes are preferred, to facilitate peptide synthesis.
  • the length of the B cell epitope will not exceed about 30 amino acids in length. More preferably, the B cell epitope sequence consists of about 25 amino acid residues or less, and more preferably less than 20 amino acid residues, and even more preferably about 5- 20 amino acid residues in length derived from the sequence of the full-length protein.
  • the present invention also encompasses the use of tissues and parts thereof, tissue homogenates and mixtures of cells as autoantigens.
  • the non-human animal immunized with the autoantigen does not produce detectable antibodies to the autoantigen prior to immunization.
  • an "antibody” is generally considered to be a protein that comprises a variable region made up of a plurality of polypeptide chains, e.g., a light chain variable region (VL) and a heavy chain variable region (VH).
  • An antibody also generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallisable (Fc).
  • Antibodies can bind specifically to one or a few closely related antigens.
  • antibodies comprise a four-chain structure as their basic unit. Full-length antibodies comprise two heavy chains (-50-70 kD) covalently linked and two light chains (-23 kD each).
  • a light chain generally comprises a variable region and a constant domain and in mammals is either a ⁇ light chain or a ⁇ light chain.
  • a heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s).
  • Heavy chains of mammals are of one of the following types ⁇ , ⁇ , ⁇ , ⁇ , or ⁇ .
  • Each light chain is also covalently linked to one of the heavy chains.
  • the two heavy chains and the heavy and light chains are held together by inter-chain disulfide bonds and by non- covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies.
  • Each chain has an N-terminal variable region (VH or VL wherein each are -1 10 amino acids in length) and one or more constant domains at the C- terminus.
  • the constant domain of the light chain (CL which is -110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (CH which is about 330-440 amino acids in length).
  • the light chain variable region is aligned with the variable region of the heavy chain.
  • the antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region can be identified between the CHI and Cm constant domains.
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgA) and IgA 2 ) or subclass.
  • the term "antibody” also encompasses humanized antibodies, primatized antibodies and de-immunized antibodies.
  • antibody variable domain refers to the portions of the light and heavy chains of antibody molecules that include amino acid sequences of complementarity determining regions (CDRs; i.e., CDRI, CDR2, and CDR3), and Framework Regions (FRs).
  • CDRs complementarity determining regions
  • FRs Framework Regions
  • VH refers to the variable domain of the heavy chain.
  • VL refers to the variable domain of the light chain.
  • CDRs and FRs may be defined according to Kabat ( 1987 and 1991 ) or Chothia and Lesk ( 1987).
  • the heavy chain constant region can be selected from any of the five isotypes: alpha, delta, epsilon, gamma or mu. Further, heavy chains of various subclasses (such as the IgG subclasses of heavy chains) are responsible for different effector functions and thus, by choosing the desired heavy chain constant region, antibodies with desired effector function can be produced.
  • Preferred heavy chain constant regions are gamma 1 (IgGl), gamma 2 (IgG2), gamma 3 (IgG3) and gamma 4 (IgG4).
  • Light chain constant regions can be of the kappa or lambda type, preferably of the kappa type.
  • CDRs complementarity determining regions
  • CDRl complementarity determining regions
  • CDR2 complementarity determining regions
  • CDR3 refers to the amino acid residues of ah antibody variable domain the presence of which are necessary for antigen binding.
  • Each variable domain typically has three CDR regions identified as CDRl, CDR2 and CDR3.
  • Each complementarity determining region may comprise amino acid residues from a "complementarity determining region” as defined by Kabat et al. (1987 and 1991) and/or those residues from a "hypervariable loop" Chothia and Lesk (1987).
  • Framework regions are those variable domain residues other than the CDR residues.
  • an autoantigen or a cell or particle expressing and displaying same is administered to a non-human animal, e.g., in the form of an injectable composition.
  • Injection may be intranasal, intramuscular, subcutaneous, intravenous, intradermal, intraperitoneal, or by other known route.
  • intravenous injection it is desirable to include one or more fluid and nutrient replenishers.
  • Means for preparing and characterizing antibodies are known in the art. (See, e.g., ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, 1988).
  • Autoantigens for generating antibodies can be covalently coupled to an immunogenic carrier protein, such as diphtheria toxoid (DT), keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or the nuclear protein of influenza virus (NP), using any of several conjugation chemistries known in the art.
  • an immunogenic carrier protein such as diphtheria toxoid (DT), keyhole limpet hemocyanin (KLH), tetanus toxoid (TT) or the nuclear protein of influenza virus (NP), using any of several conjugation chemistries known in the art.
  • DT diphtheria toxoid
  • KLH keyhole limpet hemocyanin
  • TT tetanus toxoid
  • NP nuclear protein of influenza virus
  • conjugate molecules so produced may be purified and employed in immunogenic compositions to elicit, upon administration to a host, an immune response to the protein and/or peptide which is potentiated in comparison to the protein or peptide alone.
  • Such conjugates fall within the scope of the term "autoantigen”.
  • the efficacy of the autoantigen in producing an antibody is established by injecting an animal as described herein, for example, a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat or pig, and then monitoring the immune response to the autoantigen. Both primary and secondary immune responses are monitored.
  • the antibody titer is determined using any conventional immunoassay, such as, for example, ELISA, or radio-immunoassay.
  • polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster injection, may be given, if required to achieve a desired antibody titer. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal is bled and the serum isolated and stored, and/or the animal is used to generate monoclonal antibodies (Mabs).
  • Mabs monoclonal antibodies
  • Monoclonal antibodies are preferred.
  • the term "monoclonal antibody” refers to a homogeneous antibody population capable of binding to the same antigen(s) and, preferably, to the same epitopic determinant within the antigen(s). This term is not intended to be limited as regards to the source of the antibody or the, manner in which it is made.
  • any one of a number of known techniques may be used, such as, for example, the procedure exemplified in US4, 196,265 or ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, 1988, incorporated herein by reference.
  • a suitable animal as described herein is immunized with an effective amount of an autoantigen under conditions sufficient to stimulate antibody producing cells.
  • Rodents such as rabbits, mice and rats are preferred animals, however, the use of sheep or frog cells is also possible.
  • the use of rats may provide certain advantages, but mice or rabbits are preferred, with the BALB/c or C57/bl6 mouse being preferred as the most routinely used animal and one that generally gives a higher percentage of stable fusions.
  • an animal e.g., a mouse genetically-engineered to express human immunoglobulin proteins, and preferably not express murine immunoglobulin proteins
  • an antibody is immunized to produce an antibody.
  • Such mice are known in the art and commercially available.
  • Regeneron, Inc. have produced the VeloclmmuneTM mouse in which human variable regions have been homologously recombined or knocked-in to the mouse genome to replace endogenous mouse variable region encoding genes.
  • Such mice are described, for example, in WO2002/066630.
  • mice have produced strains of mice in which the endogenous mouse immunoglobulin loci are inactivated or "knocked-out" and human immunoglobulin loci introduced using yeast artificial chromosomes. Examples of these mice are described or reviewed in Lonberg et al. (1994); Lonberg (1994); Tomizuka et al. (2000) and Jakobovits et al. (2007).
  • B lymphocytes B cells
  • somatic cells with the potential for producing antibodies are selected for use in the monoclonal antibody generating protocol.
  • B cells may be obtained from biopsies of spleens, tonsils or lymph nodes, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of antibody-producing cells that are in the dividing plasmablast stage, and the latter because peripheral blood is easily accessible.
  • Spleen lymphocytes are obtained by homogenizing the spleen with a syringe.
  • the B cells from the immunized animal are then fused with cells of an immortal myeloma cell, generally derived from the same species as the animal that was immunized with the autoantigen.
  • myeloma cells Any one of a number of myeloma cells may be used and these are known to those of skill in the art (e.g. murine P3-X63/Ag8, X63-Ag8.653, NSl/1 .Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-1 1, MPC 11 -X45-GTG 1.7 and S194/5XX0).
  • murine P3-X63/Ag8, X63-Ag8.653, NSl/1 .Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-1 1, MPC 11 -X45-GTG 1.7 and S194/5XX0 e.g. murine P3-X63/Ag8, X63-Ag8.653, NSl/1 .Ag 4 1, Sp210-Agl4, FO, NSO/U, MPC-1 1, MPC 11 -X45-GTG 1.7 and S194/5XX
  • somatic cells are mixed with myeloma cells in the presence of an agent or agents (chemical or electrical) that promote the fusion of cell membranes.
  • Fusion methods using Se . ndai virus have . been described by Kohler and Milstein, (1975); and Kohler and Milstein, (1976).
  • Methods using polyethylene glycol (PEG), such as 37% (v/v) PEG, are described in detail by Gefter et al, (1977). The use of electrically induced fusion methods is also appropriate.
  • Hybrids are amplified by culture in a selective medium comprising an agent that blocks the de novo synthesis of nucleotides in the tissue culture media.
  • agents are aminopterin, methotrexate and azaserine.
  • the amplified hybridomas are subjected to a functional selection for antibody specificity and/or titer, such as, for example, by immunoassay (e.g. radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and the like).
  • immunoassay e.g. radioimmunoassay, enzyme immunoassay, cytotoxicity assay, plaque assay, dot immunoassay, and the like.
  • the selected hybridomas are serially diluted and cloned into individual antibody- producing cell lines, which clones can then be propagated for an extended period, e.g., indefinitely to provide monoclonal antibodies.
  • the cell lines may be exploited for monoclonal antibody production in various manners. For example, a sample of the hybridoma is injected, usually in the peritoneal cavity, into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors secreting the specific monoclonal antibody produced by the fused cell hybrid. The body fluids of the animal, such as serum or ascites fluid, can then be tapped to provide monoclonal antibodies in high concentration.
  • the individual cell lines could also be cultured in vitro, where the monoclonal antibodies are naturally secreted into the culture medium from which they are readily obtained in high concentrations.
  • Monoclonal antibodies produced by either means may be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC or affinity chromatography.
  • ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to produce cell lines secreting monoclonal antibodies (mAbs) against an autoantigen.
  • This technology comprises infecting splenocytes from immunized non- human animals according to the invention with replication-incompetent retrovirus comprising the oncogenes v-abl and c-myc.
  • Splenocytes are transplanted into naive mice which then develop ascites fluid containing cell lines producing monoclonal antibodies (mAbs) against an autoantigen.
  • the monoclonal antibodies are purified from ascites using protein G or protein A, e.g., bound to a solid matrix, depending on the isotype of the mAb.
  • the ABL-MYC technology is described generically in detail in Largaespada (1990); Largaespada et al, (1992); and Largaespada et al, (1996).
  • the antibody specifically binds to an autoantigen (and preferably, a self-antigen).
  • an autoantigen and preferably, a self-antigen.
  • the term "specifically binds" shall be taken to mean an antibody (or protein comprising an autoantigen binding domain) reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity with a particular antigen or antigens or cell expressing same than it does with alternative antigens or cells.
  • an antibody or protein that specifically binds to an antigen binds that antigen(s) with greater affinity, avidity, more readily, and/or with greater duration than it binds to other antigens.
  • binding does not necessarily require exclusive binding or non-detectable binding of another antigen, this is meant by the term “selective binding”.
  • reference to binding means specific binding, and each term shall be understood to provide explicit support for the other term
  • Antibodies or autoantigen binding regions produced by performing a method of the invention can also be humanized.
  • humanized antibody shall be understood to refer to a chimeric molecule, generally prepared using recombinant techniques, having an antigen binding site derived from an antibody from a non-human animal (i.e., having reduced AIRE- protein activity) and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human antibody.
  • the antigen-binding site preferably comprises the complementarity determining regions (CDRs) from the non-human antibody grafted onto appropriate framework regions in the variable domains of a human antibody and the remaining regions from a human antibody.
  • Antigen binding sites may be wild type or modified by one or more amino acid substitutions.
  • Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from the non- human antibody.
  • Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
  • Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence.
  • the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • an example of the present invention provides a method for producing a humanized antibody or protein comprising a humanized autoantigen binding domain, the method comprising:
  • the method additionally comprises mutating or otherwise altering one or more amino acids in a CDR or a FR to improve binding affinity of the antibody or protein for an autoantigen.
  • the present invention also contemplates primatized antibodies (e.g., as described in WO2007/070979) and de-immunized antibodies (e.g., as described in WO2004/108158).
  • the present invention contemplates a wide variety of proteins comprising autoantigen binding domains.
  • the following description is examples of some such proteins and is not intended to limit the present invention.
  • the following description focuses on proteins produced using recombinant techniques, however as discussed above, the present invention clearly contemplates other techniques (e.g., enzymatic digestion of antibodies).
  • Antibodies produced using the methods of the present invention can be modified, e.g., to reduce their immunogenicity.
  • An example of such a modified antibody is a chimeric antibody (i.e., an antibody produced according to the methods of the invention is chimerized).
  • 'chimeric antibody refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species (e.g., murine, such as mouse) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species (e.g., primate, such as human) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (US4,816,567 and Morrison et al. 1984).
  • a particular species e.g., murine, such as mouse
  • primate such as human
  • chimeric antibodies utilize rodent or rabbit variable regions (in the case of the present invention from an antibody against an autoantigen) and human constant regions, in order to produce an antibody with predominantly human domains.
  • a chimeric antibody comprises a variable region from a mouse antibody as described herein according to any embodiment fused to a human constant region.
  • the production of such chimeric antibodies is known in the art, and may be achieved by standard means (as described, e.g., in Morrison (1985); US5, 807,715; US4,816,567 and US4,816397).
  • an example of the present invention provides a method for producing a chimeric antibody, the method comprising: (i) performing a method as described herein according to any embodiment to produce an antibody that binds to an autoantigen or an autoantigen binding domain;
  • Exemplary proteins comprising an autoantigen binding domains include diabodies, triabodies, tetrabodies and higher order protein complexes such as those described in WO98/044001 and WO94/007921.
  • the term "diabody” shall be taken to mean a protein comprising two associated polypeptide chains, each polypeptide chain comprising the structure VL- X-VH or VH-X-VL, wherein V L is an antibody light chain variable region, V H is an antibody heavy chain variable region, X is a linker comprising insufficient residues to permit the VH and V L in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the VH of one polypeptide chain binds to a VL of the other polypeptide chain to form an antigen binding site, i.e., to form a Fv molecule capable of specifically binding to one or more antigens.
  • the VL and VH can be the same in each polypeptide chain or the VL and VH can be different in each polypeptide chain so as to form a bispecific diabody (i.e., comprising two Fvs having different specificity).
  • the term "triabody” shall be taken to mean a protein comprising three associated polypeptide chains, each polypeptide chain comprising the structure VL- X-VH or VH-X-VL, wherein V L is an antibody light chain variable region, VH is an antibody heavy chain variable region, X is a linker comprising insufficient residues to permit the VH and V L in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the VH of one polypeptide chain is associated with the VL of another polypeptide chain to thereby form a trimeric protein (a triabody).
  • a VH of a first polypeptide chain is associated with the VL of a second polypeptide chain
  • the VH of the second polypeptide chain is associated with the VL of a third polypeptide chain
  • the VH of the third polypeptide is associated with the V L of the first polypeptide chain.
  • the VL and VH associate so as to form an antigen binding site, i.e., a Fv capable of specifically binding to one or more antigens.
  • the VL and VH can be the same in each polypeptide chain (i.e., to produce a monospecific triabody) or two of the VL and two of the VH can be the same and the third of each different in the third polypeptide chain to produce a bispecific protein or the VL and VH can be different in each polypeptide chain so as to form a trivalent protein.
  • tetrabody shall be taken to mean a protein comprising four associated polypeptide chains, each polypeptide chain comprising the structure VL- X-VH or VH-X-VL, wherein VL is an antibody light chain variable region, VH is an antibody heavy chain variable region, X is a linker comprising insufficient residues to permit the V H and VL in a single polypeptide chain to associate (or form an Fv) or is absent, and wherein the VH of one polypeptide chain is associated with the VL of another polypeptide chain to thereby form a tetrameric protein (a tetrabody).
  • the VL and VH associate so as to form an antigen binding site, i.e., a Fv capable of specifically binding to one or more antigens.
  • a Fv capable of specifically binding to one or more antigens.
  • the VH of a first polypeptide chain is associated with the VL of a second polypeptide chain
  • the VH of the second polypeptide chain is associated with the VL of a third polypeptide chain
  • the VH of the third polypeptide chain is associated with the VL of a fourth polypeptide chain
  • the V H of the fourth polypeptide chain is associated with the VL of the first polypeptide chain.
  • the VL and VH can be the same in each polypeptide chain (i.e., to produce a monospecific tetrabody) or the VL and VH can be of one type in two polypeptide chains and a different type in the other two polypeptide chains to produce a bispecific tetrabody or the V L and VH can be different in each polypeptide chain so as to form a tetraspecific tetrabody.
  • these proteins comprise a polypeptide chain in which a VH and a VL are linked directly or using a linker that is of insufficient length to permit the VH and VL to associate.
  • the VH and VL can be positioned in any order, i.e., VL-VH or VH-VL.
  • the V H and VL are readily obtained, e.g., by isolating nucleic acid encoding these polypeptide chains from a cell expressing an antibody comprising one or more variable region(s) of interest (including an antibody or a chimeric antibody or a humanized antibody or a human antibody).
  • Proteins comprising VH and VL associate to form diabodies, triabodies and/or tetrabodies depending on the length of the linker (if present) and/or the order of the VH and VL domains.
  • the linker comprises 12 or fewer amino acids.
  • X is a linker
  • a linker having 3-12 residues generally results in formation of diabodies
  • a linker having 1 or 2 residues or where a linker is absent generally results in formation of triabodies.
  • Exemplary publications describing diabodies, triabodies and/or tetrabodies include WO94/07921 ; WO98/44001 ; Hollinger et al. (1993); Hollinger and Hudson (2005); and references cited therein.
  • scFvs comprise VH and V L regions in a single polypeptide chain.
  • the polypeptide chain further comprises a polypeptide linker between the V H and VL which enables the scFv to form the desired structure for antigen binding (i.e., for the VH and VL of the single polypeptide chain to associate with one another to form a Fv).
  • the linker comprises in excess of 12 amino acid residues with (Gly 4 Ser) 3 being one of the more favoured linkers for a scFv.
  • a minibody comprises the VH and V L domains of an immunoglobulin fused to the CH2 and/or CH3 domain of an antibody.
  • the minibody comprises a hinge region between the VH and a VL, sometimes this conformation is referred to as a Flex Minibody (Hu et al, 1996).
  • a minibody does not comprise a CHI or a CL.
  • the VH and VL domains are fused to the hinge region and the CH3 domain of an immunoglobulin. Each of the regions may be derived from the same immunoglobulin.
  • the VH and VL domains can be derived from one immunoglobulin and the hinge and CH /CH3 from another, or the hinge and CH2/CH3 can also be derived from different antibodies.
  • Exemplary minibodies and methods for their production are described, for example, in WO94/09817.
  • the present invention also provides display libraries, methods and reagents for producing display libraries, methods for screening display libraries and products of such screens.
  • display libraries are produced by obtaining nucleic acid encoding autoantigen binding domains from a non-human animal having reduced AIRE-protein activity that has been immunized with an autoantigen, introducing the nucleic acid into a suitable expression construct, and expressing the autoantigen binding domains such that they are displayed in/on cells or particles (preferably, on the surface of the cells or particles).
  • These libraries are useful for screening to identify or isolate a protein comprising an autoantigen binding domain.
  • the display library is an in vitro display library (i.e., the autoantigen binding domains are displayed using in vitro display wherein the expressed domain is linked to the nucleic acid from which it was expressed such that said domain is presented in the absence of a host cell).
  • expression libraries produced by in vitro display technologies are not limited by transformation or transfection efficiencies. Examples of methods of in vitro display include ribosome display, covalent display and mRNA display.
  • the in vitro display library is a ribosome display library.
  • a ribosome display library directly links mRNA encoded by the expression library to the protein that it encodes.
  • Means for producing a ribosome display library comprise placing nucleic acid encoding the autoantigen binding domain in operable connection with an appropriate promoter sequence and ribosome binding sequence.
  • Preferred promoter sequences are the bacteriophage T3 and T7 promoters.
  • the nucleic acid is placed in operable connection with a spacer sequence and a modified terminator sequence with the terminator codon removed.
  • spacer sequence shall be understood to mean a series of nucleic acids that encode a peptide that is fused to the peptide.
  • the spacer sequence is incorporated into the gene construct, as the peptide encoded by the spacer sequence remains within the ribosomal tunnel following translation, while allowing the autoantigen binding domain to freely fold and interact with another protein or a nucleic acid.
  • a preferred spacer sequence is, for example, a nucleic acid that encodes amino acids 21 1-299 of gene III of filamentous phage Ml 3 mpl9.
  • the display library is transcribed and translated in vitro using methods known in the art and/or described for example, in Ausubel et al. (1987) and Sambrook et al. (2001).
  • Examples of commercially available systems for in vitro transcription and translation include, for example, the TNT in vitro transcription and translation systems from Promega. Cooling the expression reactions on ice generally terminates translation.
  • the ribosome complexes are stabilized against dissociation from the peptide and/or its encoding mRNA by the addition of reagents such as, for example, magnesium acetate or chloroamphenicol.
  • Such in vitro display libraries are screened by a variety of methods, as described herein.
  • the display library of the present invention is a ribosome inactivation display library.
  • a nucleic acid is operably linked to a nucleic acid encoding a first spacer sequence. It is preferred that this spacer sequence is a glycine/serine rich sequence that allows a autoantigen binding domain encoded therefrom to freely fold and interact with a target antigen.
  • the first spacer sequence is linked to a nucleic acid that encodes a toxin that Inactivates a ribosome.
  • the toxin comprises the ricin A chain, which inactivates eukaryotic ribosomes and stalls the ribosome on the translation complex without release of the mRNA or the encoded peptide.
  • the nucleic acid encoding the toxin is linked to another nucleic acid that encodes a second spacer sequence.
  • the second spacer is an anchor to occupy the tunnel of the ribosome, and allow both the peptide and the toxin to correctly fold and become active. Examples of such spacer sequences are sequences derived from gene III of Ml 3 bacteriophage.
  • Ribosome inactivation display libraries are generally transcribed and translated in vitro, using a system such as the rabbit reticulocyte lysate system available from Promega. Upon translation of the mRNA encoding the toxin and correct folding of this protein, the ribosome is inactivated while still bound to both the encoded polypeptide and the mRNA from which it was translated.
  • the display library is an mRNA display library.
  • a nucleic acid is operably linked to a nucleic acid encoding a spacer sequence, such as a glycine/serine rich sequence that allows an autoantigen binding domain encoded by the expression library of the present invention to freely fold and interact with a target antigen.
  • the nucleic acid encoding the spacer sequence is operably linked to a transcription terminator.
  • mRNA display libraries are generally transcribed in vitro using methods known in the art, such as, for example, the HeLaScribe Nuclear Extract In Vitro Transcription System available from Promega.
  • Encoded mRNA is subsequently covalently linked to a DNA oligonucleotide that is covalently linked to a molecule that binds to a ribosome, such as, for example, puromycin, using techniques known in the art and are described in, for example, Roberts and Szostak (1997).
  • the oligonucleotide is covalently linked to a psoralen moiety, whereby the oligonucleotide is photo-crosslinked to a mRNA encoded by the expression library of the present invention.
  • the mRNA transcribed from the expression library is then translated using methods known in the art.
  • the ribosome When the ribosome reaches the junction of the mRNA and the oligonucleotide the ribosome stalls and the puromycin moiety enters the phosphotransferase site of the ribosome and thus covalently links the encoded polypeptide to the mRNA from which it was expressed.
  • the display library is a covalent display library.
  • a nucleic acid encoding an autoantigen binding domain is operably linked to a second nucleic acid that encodes a protein that interacts with the DNA from which it was encoded.
  • a protein that interacts with the DNA from which it interacts include, but are not limited to, the E. coli bacteriophage P2 viral A protein (P2A) and equivalent proteins isolated from phage 186, HP1 and PSP3.
  • P2A E. coli bacteriophage P2 viral A protein
  • HP1 and PSP3 HP1 and PSP3.
  • a covalent display gene construct is transcribed and translated in vitro, using a system such as the rabbit reticulocyte lysate system available from Promega.
  • the P2A protein nicks the nucleic acid to which it binds and forms a covalent bond therewith. Accordingly, a nucleic acid fragment is covalently linked to the peptide that it encodes.
  • the display library is a phage display library wherein the expressed autoantigen binding domains are displayed on the surface of a bacteriophage, as described, for example, in US5,821,047; US6,248,516 and US6, 190,908.
  • the basic principle described relates to the fusion of a first nucleic acid comprising a sequence encoding an autoantigen binding domain to a second nucleic acid comprising a sequence encoding a phage coat protein, such as, for example a phage coat proteins selected from the group, Ml 3 protein-3, Ml 3 protein-7, or Ml 3, protein-8.
  • coat protein also encompasses fragments or parts there that are capable of incorporating into a viral coat or capsid and displaying the autoantigen binding domain, without disrupting production of viral particles. These sequences are then inserted into an appropriate vector, i.e., one that is able to replicate in bacterial cells. Suitable host cells, such as, for example E. coli, are then transformed with the recombinant vector. Said host cells are also infected with a helper phage particle encoding an unmodified form of the coat protein to which a nucleic acid fragment is operably linked.
  • Transformed, infected host cells are cultured under conditions suitable for forming recombinant phagemid particles comprising more than one copy of the fusion protein on the surface of the particle.
  • This system has been shown to be effective in the generation of virus particles such as, ⁇ phage, T4 phage, Ml 3 phage, T7 phage and baculovirus.
  • virus particles such as, ⁇ phage, T4 phage, Ml 3 phage, T7 phage and baculovirus.
  • Such phage display particles are then screened to identify a displayed domain having a conformation sufficient for binding to a target antigen.
  • viral display libraries include a retroviral display library wherein the expressed peptides or protein domains are displayed on the surface of a retroviral particle, e.g., as described in US6,297,004
  • the present invention also contemplates bacterial display libraries, e.g., as described in US5,516,637; yeast display libraries, e.g., as described in US6,423,538 or a mammalian display library, e.g., as described in Strenglin et al. (1988).
  • a display library of the present invention is screened using affinity purification.
  • Affinity purification techniques are known in the art and are described in, for example, Scopes (1994).
  • Methods of affinity purification typically involve contacting the autoantigen binding domains displayed by the library with a target autoantigen and, following washing, eluting those domains that remain bound to the autoantigen.
  • the autoantigen is preferably bound to another molecule to allow for ease of purification, such as, for example, a molecule selected from the group consisting of protein A, protein G, Sepharose, agarose, biotin, glutathione S-transferase (GST), and FLAG epitope.
  • the target protein or nucleic acid is isolated simply through centrifugation, or through binding to another molecule, eg. streptavidin, or binding of a specific antibody, eg. anti-FLAG antibodies, or anti-GST antibodies.
  • the. display library of the present invention is expressed so as to allow identification of a bound peptide using FACS analysis.
  • the screening of libraries using FACS analysis is described in US645563.
  • an in vitro display library is screened by FACS sorting.
  • In vitro display proteins are covalently linked to a particle or bead suitable for FACS sorting, such as, for example, glass, polymers such as for example polystyrene, latex or cross-linked dextrans such as Sepharose, cellulose, nylon, teflon, amongst others.
  • the displayed library bound to particles or beads is added to a target autoantigen that has been labelled with a labelling moiety, such as for example, a fluorescent molecule, or a molecule which is detected by a second fluorescent molecule.
  • a labelling moiety such as for example, a fluorescent molecule, or a molecule which is detected by a second fluorescent molecule.
  • the beads are then washed and subjected to sorting by FACS, which allows the beads with bound fluorescent target autoantigen, to be separated from the beads that have not bound to a fluorescent target protein or nucleic acid.
  • the library is screened using a biosensor-based assay, such as, for example, Biacore sensor chip technology (Biacore AB, UK).
  • Biacore sensor chip is a glass surface coated with a thin layer of gold modified with carboxymethylated dextran, to which the target protein or nucleic acid is covalently attached.
  • the libraries of the present invention are then exposed to the Biacore sensor chip comprising the autoantigen.
  • An antibody or protein comprising an autoantigen binding domain produced according to the present invention can be isolated using any of a variety of techniques, for example, hydroxyl apatite chromatography, gel electrophoresis, dialysis, affinity chromatography, with affinity chromatography being the preferred purification technique.
  • Protein A affinity chromatography can be used to purify antibodies that are based on human ⁇ , ⁇ 2, or ⁇ 4 heavy chains (Lindmark et al., 1983). Protein G is recommended for all mouse isotypes and for human ⁇ 3 (Guss et al, 1986).
  • an autoantigen is used to isolate antibodies or proteins comprising an autoantigen binding domain that bind thereto.
  • the matrix to which the affinity ligand is attached is most often agarose, but other matrices are available.
  • Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose.
  • Other techniques for protein purification such as fractionation on an ion-exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSETM chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.
  • the mixture comprising the antibody or protein of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography.
  • An antibody or protein comprising an autoantigen binding domain can be produced using recombinant techniques.
  • DNA encoding an antibody or protein of the invention is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • a hybridoma cell can serve as a preferred source of such DNA.
  • the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells that do not otherwise produce the antibody or protein.
  • the nucleic acid encoding it is preferably isolated and inserted into an expression construct, preferably an expression vector.
  • DNA encoding the antibody is readily isolated or synthesized using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to DNAs encoding the heavy and light chains of the antibody).
  • the vector components generally include, but are not limited to, one or more of the following: a signal sequence, a sequence encoding an antibody of the present invention or fragment thereof (e.g., derived from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence.
  • exemplary signal sequences include prokaryotic secretion signals (e.g., alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, a factor leader, or acid phosphatase leader) or mammalian secretion signals, (e.g., herpes simplex gD signal).
  • prokaryotic secretion signals e.g., alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II
  • yeast secretion signals e.g., invertase leader, a factor leader, or acid phosphatase leader
  • mammalian secretion signals e.g., herpes simplex gD signal
  • Exemplary promoters include those active in prokryotes (e.g., phoA promoter , ⁇ -lactamase and lactose promoter systems, alkaline phosphatase, a tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter), and those active in mammalian cells (e.g., cytomegalovirus immediate early promoter (CMV), the human elongation factor 1-a promoter (EF1), the small nuclear RNA promoters (Ula and Ulb), a-myosin heavy chain promoter, Simian virus 40 promoter (SV40), Rous sarcoma virus promoter (RSV), Adenovirus major late promoter, ⁇ -actin promoter; hybrid regulatory element comprising a CMV enhancer/ ⁇ -actin promoter or an immunoglobulin promoter or active fragment thereof.
  • CMV cytomegalovirus immediate early promoter
  • EF1 human elongation factor 1-a promoter
  • Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryote, yeast, or higher eukaryote cells.
  • Suitable prokaryotes for this purpose include eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B. subtilis and B.
  • Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus
  • Salmonella e.g., Salmonella typhimurium
  • Serratia e.g., Serrati
  • E. coli 294 ATCC 31,446
  • E. coli B E. coli X 1776
  • E. coli W3110 ATCC 27,325
  • eukaryotic microbes such as filamentous fungi or yeast' are suitable cloning or expression hosts for antibody-encoding vectors.
  • Saccharomyces cerevisiae or common baker's yeast, is the most commonly used among lower eukaryotic host microorganisms.
  • a number of other genera, species, and strains are commonly available and useful herein, such as Schizosaccharomyces pombe; Pichia pastoris (EP 183,070); and filamentous fungi such as, e.g., Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.
  • Suitable host cells for the expression of glycosylated antibody are derived from multicellular organisms.
  • invertebrate cells include plant and insect cells.
  • Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori have been identified.
  • Examples of useful mammalian host cell lines are monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture, Graham et al. (1977) ; baby hamster kidney cells (BHK, ATCC CCL 10); Chinese hamster ovary cells (CHO, Urlaub et al.
  • mice Sertoli cells (TM4, Mather (1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3 A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRJ cells (Mather et al, 1982); MRC 5 cells; FS4 cells; and PER.C6TM (Crucell NV).
  • the host cells used to produce the antibody of this invention may be cultured in a variety of media.
  • Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells.
  • any of the media described in Ham et al. (1979), Barnes et al. (1980), US4,767,704; US4,657,866; US4,927,762; US4,560,655; US5,122,469; WO 90/03430; WO 87/00195 may be used as culture media for the host cells.
  • the present invention also provides derivatives of an antibody or a proteins comprising autoantigen binding domain as described herein according to any embodiment, e.g., a conjugate (immunoconjugate) comprising an antibody or protein of the present invention conjugated to a distinct moiety, e.g., a therapeutic agent which is directly or indirectly bound to the antibody.
  • a conjugate immunoconjugate
  • a distinct moiety e.g., a therapeutic agent which is directly or indirectly bound to the antibody.
  • moieties include, but are not limited to, a cytotoxin, a radioisotope (e.g., iodine-131, yttrium-90 or indium-I l l), an immunomodulatory agent, an anti-angiogenic agent, an anti-neo vascularization and/or other vascularization .agent, a toxin, an anti-proliferative agent, a pro-apoptotic agent, a chemotherapeutic agent and a therapeutic nucleic acid.
  • a cytotoxin e.g., iodine-131, yttrium-90 or indium-I l l
  • an immunomodulatory agent e.g., an anti-angiogenic agent, an anti-neo vascularization and/or other vascularization .agent, a toxin, an anti-proliferative agent, a pro-apoptotic agent, a chemotherapeutic agent and a therapeutic nucle
  • a cytotoxin includes any agent that is detrimental to (e.g., kills) cells.
  • kills include any agent that is detrimental to (e.g., kills) cells.
  • these classes of drugs which are known in the art, and their mechanisms of action, see Goodman et al. (1990). Additional techniques relevant to the preparation of antibody immunotoxins are provided in for instance US5,194,594.
  • Exemplary toxins include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin and the tricothecenes. See, for example, W093/21232.
  • Suitable therapeutic agents for forming derivatives of antibodies or proteins of the present invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabin, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (such as mechlorethamine, thio
  • radionuclides are available for the production of radioconjugated antibodies or proteins. Examples include, but are not limited to, 212 Bi, 131 I, 90 Y, and
  • the antibody or protein may be conjugated to a "receptor” (such as streptavidin) for utilization in pretargeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a "ligand” (e.g., avidin) that is conjugated to a therapeutic agent (e.g., a radionucleotide).
  • a "receptor” such as streptavidin
  • a "ligand” e.g., avidin
  • a therapeutic agent e.g., a radionucleotide
  • the antibodies or proteins of the present invention can be further modified to contain additional nonproteinaceous moieties that are known in the art and readily available.
  • the moieties suitable for derivatization of the antibody are water soluble polymers.
  • water soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran or polyvinyl alcohol.
  • an antibody or protein as described herein comprises one or more detectable labels to facilitate detection and/or isolation.
  • the antibody or protein comprises a fluorescent label such as, for example, fluorescein (FITC), 5, 6-carboxy methyl fluorescein, Texas red, nitrobenz-2-oxa- 1,3- diazol-4-yl (NBD), coumarin, dahsyl chloride, rhodamine, 4'-6-diamidino-2- phenylinodole (DAPI), and the cyanine dyes Cy3, Cy3.5, Cy5, Cy5.5 and Cy7, fluorescein (5-carboxyfluorescein-N-hydroxysuccinimide ester), rhodamine (5,6- tetramethyl rhodamine).
  • FITC fluorescein
  • NBD nitrobenz-2-oxa- 1,3- diazol-4-yl
  • DAPI nitrobenz-2-oxa- 1,3- diazol-4-yl
  • the absorption and emission maxima, respectively, for these fluors are: FITC (490 nm; 520 nm), Cy3 (554 nm; 568 nm), Cy3.5 (581 nm; 588 nm), Cy5 (652 nm: 672 nm), Cy5.5 (682 nm; 703 nm) and Cy7 (755 nm; 778 nm).
  • the antibody or protein is labelled with, for example, a fluorescent semiconductor nanocrystal (as described, for example, in US6,306,610).
  • the antibody or protein is labelled with, for example, a magnetic or paramagnetic compound, such as, iron, steel, nickel, cobalt, rare earth materials, neodymium-iron-boron, ferrous-chromium-cobalt, nickel-ferrous, cobalt- platinum, or strontium ferrite.
  • a magnetic or paramagnetic compound such as, iron, steel, nickel, cobalt, rare earth materials, neodymium-iron-boron, ferrous-chromium-cobalt, nickel-ferrous, cobalt- platinum, or strontium ferrite.
  • the proteins and/or antibodies of the present invention are useful for parenteral, topical, oral, or local administration, aerosol administration, or transdermal administration for prophylactic or for therapeutic treatment.
  • the pharmaceutical compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
  • unit dosage forms suitable for oral administration include powder, tablets, pills, capsules and lozenges or by parenteral administration.
  • the pharmaceutical compositions of this invention when administered orally, should be protected from digestion. This is typically accomplished either by complexing the antibodies or proteins with a composition to render it resistant to acidic and enzymatic hydrolysis or by packaging the compound in an appropriately resistant carrier such as a liposome. Means of protecting proteins from digestion are known in the art.
  • an effective amount preferably a therapeutically or prophylactically effective amount of an antibody or protein of the invention will be formulated into a composition for administration to a subject.
  • a therapeutically effective amount refers to an amount sufficient to provide, promote, induce, and/or enhance treatment or other therapeutic effect in a subject.
  • a prophylactically effective amount refers to an amount sufficient to prevent or reduce onset or progression of a disease or a symptom thereof.
  • concentration of antibodies and/or proteins of the present invention in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs.
  • a therapeutically effective amount may be about 1 ⁇ g/kg to 15 mg/kg (e.g. 0.1-20 mg/kg) of protein and/or antibody, whether, for example, by one or more separate administrations, or by continuous infusion.
  • a typical daily dosage might range from about 1 ⁇ g/kg to 100 mg/kg or more.
  • An exemplary dosage of the antibody or protein to be administered to a patient is in the range of about 0.1 to about 10 mg/kg of patient weight.
  • An exemplary dosing regimen comprises administering an initial loading dose of about 4mg/kg, followed by a weekly maintenance dose of about 2 mg/kg of the protein. Other dosage regimens may be useful. The progress of this therapy is easily monitored by conventional techniques and assays.
  • the antibody or protein of the invention is formulated at a concentrated dose that is diluted to a therapeutically effective dose prior to administration to a subject.
  • compositions of this invention are preferably useful for parenteral administration, e.g., formulated for injection via the intravenous, intramuscular, sub-cutaneous, transdermal, or other such routes, including peristaltic administration and direct instillation into a tumour or disease site (intracavity administration).
  • the compositions for administration will commonly comprise a solution of the proteins and/or antibodies of the present invention dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
  • a pharmaceutically acceptable carrier preferably an aqueous carrier.
  • aqueous carriers can be used, e.g., buffered saline and the like.
  • Other exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin.
  • Nonaqueous vehicles such as mixed oils and ethyl oleate may also be used. Liposomes may also be used as carriers.
  • the vehicles may contain minor amounts of additives that enhance isotonicity and chemical stability, e.g., buffers and preservatives.
  • the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
  • WO2002/080967 describes compositions and methods for administering aerosolized compositions comprising proteins for the treatment of, e.g., asthma, which are also suitable for administration of protein of the present invention.
  • Suitable dosages of compounds of the present invention will vary depending on the specific protein, the condition to be diagnosed/treated/prevented and/or the subject being treated. It is within the ability of a skilled physician to determine a suitable dosage, e.g., by commencing with a sub-optimal dosage and incrementally modifying the dosage to determine an optimal or useful dosage. Alternatively, to determine an appropriate dosage for treatment/prophylaxis, data from cell culture assays or animal studies are used, wherein a suitable dose is within a range of circulating concentrations that include the ED 5 o of the active compound with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. A therapeutically/prophylactically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma maybe measured, for example, by high performance liquid chromatography.
  • a protein or antibody produced according to the invention may be combined in a pharmaceutical combination formulation, or dosing regimen as combination therapy, with a second compound.
  • the second compound of the pharmaceutical combination formulation or dosing regimen preferably has complementary activities to the protein of the combination such that they do not adversely affect each other.
  • the second compound may be, for example, a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agents anti-hormonal agent, and/or cardioprotectant.
  • a chemotherapeutic agent for example, a chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agents anti-hormonal agent, and/or cardioprotectant.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • Slow release capsules or compositions may also be used. Slow release formulations are generally designed to give a constant drug level over an extended period and may be used to deliver compounds of the present invention.
  • the present invention also provides a method of treating or preventing a condition in a subject, the method comprising administering a therapeutically effective amount of a protein or antibody of the invention to a. subject in need thereof.
  • the term "subject” shall be taken to mean any animal including humans, preferably a mammal.
  • exemplary subjects include but are not limited to humans, primates, livestock (e.g. sheep, cows, horses, donkeys, pigs), companion animals (e.g. dogs, cats), laboratory test animals (e.g. mice, rabbits, rats, guinea pigs, hamsters), captive wild animals (e.g. fox, deer).
  • livestock e.g. sheep, cows, horses, donkeys, pigs
  • companion animals e.g. dogs, cats
  • laboratory test animals e.g. mice, rabbits, rats, guinea pigs, hamsters
  • captive wild animals e.g. fox, deer.
  • the mammal is a human or primate. More preferably the mammal is a human.
  • a "condition” is a disruption of or interference with normal function, and is not to be limited to any specific condition, and will include diseases or disorders.
  • the condition is a cancer or an autoimmune disorder. '
  • Exemplary cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoid malignancies. More particular examples of such cancers include squamous cell cancer (e.g. epithelial squamous cell cancer), lung cancer including small-cell lung , cancer, non-small cell lung cancer, adenocarcinoma of the lung and squamous carcinoma of the lung, cancer of the peritoneum, hepatocellular cancer, gastric or stomach cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectal cancer, endometrial or uterine carcinoma, salivary gland carcinoma, kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head and neck cancer.
  • squamous cell cancer e.
  • Autoimmune conditions include autoimmune diseases and hypersensitivity responses (e.g. Type I: anaphylaxis, hives, food allergies, asthma; Type II: autoimmune haemolytic anaemia, blood transfusion reactions; Type III: serum sickness, necrotizing vasculitis, glomerulonephritis, rheumatoid arthritis, lupus; Type IV: contact dermatitis, graft rejection).
  • Type I anaphylaxis, hives, food allergies, asthma
  • Type II autoimmune haemolytic anaemia, blood transfusion reactions
  • Type III serum sickness, necrotizing vasculitis, glomerulonephritis, rheumatoid arthritis, lupus
  • Type IV contact dermatitis, graft rejection
  • Autoimmune diseases include rheumatologic disorders (such as, for example, rheumatoid arthritis, Sjogren's syndrome, scleroderma, lupus such as SLE and lupus nephritis, polymyositis/dermatomyositis, cryoglobulinemia, anti-phospholipid antibody syndrome, and psoriatic arthritis), osteoarthritis, autoimmune gastrointestinal and liver disorders (such as, for example, inflammatory bowel diseases (e.g., ulcerative colitis and Crohn's disease), autoimmune gastritis and pernicious anemia, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, and celiac disease), vasculitis (such as, for example, ANCA-associated vasculitis, including Churg-Strauss vasculitis, Wegener's granulomatosis, and polyarteriitis) autoimmune neurological disorders (such as,
  • More preferred such diseases include, for example, rheumatoid arthritis, ulcerative colitis, ANCA-associated vasculitis, lupus, multiple sclerosis, Sjogren's syndrome, Graves' disease, IDDM, pernicious anemia, thyroiditis, and glomerulonephritis.
  • the condition is an inflammatory condition. Inflammation is a protective response of body tissues to irritation or injury- and can be acute or chronic.
  • inflammatory conditions include diseases involving neutrophils, monocytes, mast cells, basophils, eosinophils, macrophages where cytokine release, histamine release, oxidative burst, phagocytosis, release of other granule enzymes, and chemotaxis occur.
  • Hypersensitivity responses can also be regarded as inflammatory diseases (acute or chronic) since they often involve complement activation and recruitment/infiltration of various leukocytes such as neutrophils, mast cells, basophils, etc.
  • compositions of the present invention will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically/prophylactically effective.
  • Formulations are easily administered in a variety of manners, e.g., by ingestion or injection or inhalation.
  • an antibody or protein of the invention is administered by injection.
  • the combination therapy may be administered as a simultaneous or sequential regimen.
  • the combination may be administered in two or more administrations.
  • the combined administration includes coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities.
  • the present invention additionally provides a method for treating or preventing APECED.
  • APECED is a monogenic autoimmune syndrome, generally caused by mutations in AIRE protein.
  • the most common symptoms of APECED are chronic mucocutaneous candidiasis, hypoparathyroidism and/or Addison's disease.
  • the range of clinical features is broad arid variable and includes several endocrine and/or autoimmune conditions, such as premature gonadal failure, hypothyroidism, hypophysitis, pernicious anemia, type 1 diabetes, autoimmune hepatitis, gastritis, alopecia and/or vitiligo.
  • a method of treating APECED comprises administering to a subject suffering from APECED or at risk of developing APECED a compound that binds to and/or reduces the activity of a costimulatory molecule on activated T cells and treats APECED.
  • a subject "at risk of developing APECED” is a subject carrying a mutation in an AIRE gene associated with the development of APECED, who is yet to develop the disease or a symptom thereof.
  • Exemplary compounds reduce the expression of an AIRE protein.
  • the compound is a nucleic acid compound, e.g., as described herein.
  • nucleic acid compounds that reduce expression of T cell costimulatory molecules include siR A that reduce expression of CD28 (e.g., Xu et al, 2005), lentiviral delivered shRNA that reduce expression of CD40 (Zhang et al, 2009), siRNA or shRNA that reduce expression of ICOS (commercially available from Santa Cruz Biotechnology, Inc, CA, USA).
  • Additional compounds useful in a method of treating APECED include those that bind to the costimulatory molecule and prevent the molecule binding to a ligand or receptor thereof and/or that kill or induce death of a cell to which it binds.
  • Such compounds are known in the art and include, for example, anti-CD 137 monoclonal antibodies (Sun et al, 2002), ICOS-Ig fusion protein (US 20080279851), anti-OX40 monoclonal antibodies (e.g., as described in Weinberg et al, 2002), anti-CD40 monoclonal antibodies, (e:g., BG9588, Boumpass et al, 2002; or IDEC-131, Kalinian et al, 2002), or CTLA-4-Ig fusion protein (abatacept, Mihara et al, 2000).
  • compositions comprising such compounds in addition to methods of treatment are described herein and shall be taken to apply mutatis mutandis to the present embodiment of the invention.
  • the present invention provides methods for diagnosing or prognosing a condition.
  • the method comprises detecting an antigen (e.g., a self-antigen) or determining the amount of an antigen (e.g., a self-antigen) in a sample. Accordingly, in some embodiments, the method comprises determining the level of an antigen in a sample from a subject and comparing that level to the level in a normal and/or healthy subject, wherein an increased or reduced level in the sample from the subject is diagnostic or prognostic of the condition.
  • an antigen e.g., a self-antigen
  • the method comprises determining the level of an antigen in a sample from a subject and comparing that level to the level in a normal and/or healthy subject, wherein an increased or reduced level in the sample from the subject is diagnostic or prognostic of the condition.
  • the antibodies and/or proteins of the invention have utility in applications such as cell sorting (e.g., flow cytometry, fluorescence activated cell sorting), for diagnostic or research purposes.
  • a sample is contacted with a protein of the invention for a time and under conditions sufficient for it to bind to an antigen and form a complex and the complex is then detected or the level of complex is determined.
  • the antibodies and/or proteins can be labelled or unlabeled.
  • the antibodies and/or proteins can be directly labelled, e.g., using a method described herein. When unlabeled, the antibodies and/or proteins can be detected using suitable means, for example, an agglutination assay.
  • Unlabeled antibodies or fragments can also be used in combination with another (i.e., one or more) suitable reagent which can be used to detect a protein, such as a labelled antibody (e.g., a second antibody) reactive with the protein or other suitable reagent (e.g., labelled protein A).
  • a suitable reagent which can be used to detect a protein, such as a labelled antibody (e.g., a second antibody) reactive with the protein or other suitable reagent (e.g., labelled protein A).
  • an antibody or protein of the invention is used in an immunoassay.
  • an immunoassay selected from the group consisting of, immunohistochemistry, immunofluorescence, enzyme linked immunosorbent assay (ELISA), fluorescence linked immunosorbent assay (FLISA) Western blotting, radioimmunoassay (RIA), a biosensor assay, a protein chip assay and an immunostaining assay (e.g. immunofluorescence).
  • Standard solid-phase ELISA or FLISA formats are useful in determining the concentration of an antigen in or from a variety of samples.
  • such an assay involves immobilizing a biological sample onto a solid matrix, such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide).
  • a solid matrix such as, for example a polystyrene or polycarbonate microwell or dipstick, a membrane, or a glass support (e.g. a glass slide).
  • An antibody or protein of the invention that specifically binds to an antigen of interest is brought into direct contact with the immobilized sample, and binds to its target antigen present in the sample.
  • the antibody or protein of the invention is generally labelled with a detectable reporter molecule, such as for example, a fluorescent label (e.g. FITC or Texas Red) or a fluorescent semiconductor nanocrystal (as described in US 6,306,610) in the case of a FLISA or an enzyme (e.g.
  • HRP horseradish peroxidase
  • AP alkaline phosphatase
  • ⁇ - galactosidase a labelled secondary antibody that binds to the antibody or protein of the invention.
  • a substrate such as for example hydrogen peroxide, TMB, or toluidine, or 5-bromo-4-chloro-3-indol-beta-D- galaotopyranoside (x-gal) in the case of an enzymatic label.
  • Such ELISA or FLISA based systems are particularly suitable for quantification of the amount of an antigen in a sample, by calibrating the detection system against known amounts of the antigen, such as for example, an isolated and/or recombinant protein or immunogenic fragment thereof or epitope thereof.
  • an ELISA or FLISA comprises immobilizing an antibody protein of the invention or another antibody that binds to an antigen of interest on a solid matrix, such as, for example, a membrane, a polystyrene or polycarbonate microwell, a polystyrene or polycarbonate dipstick or a glass, support.
  • a sample is then brought into physical relation with said antibody or protein, and the antigen to which the protein or antibody binds is bound or 'captured'.
  • the bound protein is then detected using a labelled antibody or protein of the invention that binds to a different epitope in the antigen.
  • a third labelled antibody can be used that binds the second (detecting) antibody.
  • Biosensor devices generally employ an electrode surface in combination with current or impedance measuring elements to be integrated into a device in combination with the assay substrate (such as that described in US5,567,301).
  • An antibody or protein of the invention that specifically binds to an autoantigen is incorporated onto the surface of a biosensor device and a sample contacted to the device.
  • a change in the detected current or impedance by the biosensor device indicates protein binding to the antibody or protein.
  • Some forms of biosensors known in the art also rely on surface plasmon resonance to detect protein interactions, whereby a change in the surface plasmon resonance surface of reflection is indicative of an antigen binding to the antibody or protein (US5,485,277 and US5,492,840).
  • Biosensors are of particular use in high throughput analysis due to the ease of adapting such systems to micro- or nano-scales. Furthermore, such systems are conveniently adapted to incorporate several detection reagents, allowing for multiplexing of diagnostic reagents in a single biosensor unit. This permits the simultaneous detection of several proteins or peptides in a small amount of body fluids.
  • an antibody or protein of the invention is conjugated to a detectable label, which can be any molecule or agent that can emit a signal that is detectable by imaging.
  • the detectable label may be a protein, a radioisotope, a fluorophore, a visible light emitting fluorophore, infrared light emitting fluorophore, a metal, a ferromagnetic substance, an electromagnetic emitting substance a substance with a specific MR spectroscopic signature, an X-ray absorbing or reflecting substance, or a sound altering substance.
  • the antibody or protein of the present invention can be administered either systemically or locally to the tumour, organ, or tissue to be imaged, prior to the imaging procedure.
  • the antibody or protein is administered in one or more doses effective to achieve the desired optical image of a tumour, tissue, or organ.
  • doses may vary widely, depending upon the particular antibody or protein employed, the tumour, tissue, or organ subjected to the imaging procedure, the imaging . equipment being used, and the like.
  • the antibody or protein of the invention is used as in vivo optical imaging agents of tissues and organs in various biomedical applications including, but not limited to, imaging of tumours, tomographic imaging of organs, monitoring of organ functions, coronary angiography, fluorescence endoscopy, laser guided surgery,, photoacoustic and sonofluorescence methods, and the like.
  • Exemplary diseases, e.g., cancers, in which an antibody or protein of the invention is useful for imaging are described herein and shall be taken to apply mutatis mutandis to the present embodiment of the invention.
  • the antibody or protein of the invention is useful for laser- assisted surgery. In yet another example, the antibody or protein of the invention is useful in the diagnosis of atherosclerotic plaques and blood clots.
  • imaging methods include magnetic resonance imaging (MRI), MR spectroscopy, radiography, CT, ultrasound, planar gamma camera imaging, single-photon emission computed tomography (SPECT), positron emission tomography (PET), other nuclear medicine-based imaging, optical imaging using visible light, optical imaging using luciferase, optical imaging using a fluorophore, other optical imaging, imaging using near infrared light, or imaging using infrared light.
  • MRI magnetic resonance imaging
  • MR spectroscopy radiography
  • CT computed tomography
  • PET positron emission tomography
  • other nuclear medicine-based imaging nuclear medicine-based imaging
  • optical imaging using visible light optical imaging using luciferase
  • optical imaging using a fluorophore other optical imaging, imaging using near infrared light, or imaging using infrared light.
  • Certain examples of the methods of the present invention further include imaging a tissue during a surgical procedure on a subject.
  • optical imaging is one imaging modality that has gained widespread acceptance in particular areas of medicine. Examples include optical labeling of cellular components, and angiography such as fluorescein angiography and iridocyanine green angiography.
  • optical imaging agents include, for example, fluorescein, a fluorescein derivative, indocyanine green, Oregon green, a derivative of Oregon green derivative, rhodamine green, a derivative of rhodamine green, an eosin, an erytlirosin, Texas red, a derivative of Texas red, malachite green, nanogold sulfosuccinimidyl ester, cascade blue, a coumarin derivative, a naphthalene, a pyridyloxazole derivative, cascade yellow dye, dapoxyl dye.
  • Gamma camera imaging is contemplated as a method of imaging that can be utilized for measuring a signal derived from the detectable label.
  • One of ordinary skill in the art would be familiar with techniques for application of gamma camera imaging.
  • measuring a signal can involve use of gamma-camera imaging of an l u In or 99m Tc conjugate, in particular 11 'in- octreotide or 99m Tc-somatostatin analogue.
  • CT Computerized tomography
  • a computer is programmed to display two- dimensional slices from any angle and at any depth. The slices may be combined to build three-dimensional representations.
  • contrast agents aid in assessing the vascularity of a soft tissue lesion.
  • the use of contrast agents may aid the delineation of the relationship of a tumor and adjacent vascular structures.
  • CT contrast agents include, for example, iodinated contrast media. Examples of these agents include iothalamate, iohexol, diatrizoate, iopamidol, ethiodol, and iopanoate. Gadolinium agents have also been reported to be of use as a CT contrast agent, for example, gadopentate.
  • Magnetic resonance imaging is an imaging modality that uses a high- strength magnet and radio-frequency signals to produce images.
  • the sample to be imaged is placed in a strong static magnetic field and excited with a pulse of radio frequency (RF) radiation to produce a net magnetization in the sample.
  • RF radio frequency
  • Various magnetic field gradients and other RF pulses then act to code spatial information into the recorded signals.
  • By collecting and analysing these signals it is possible to compute a three- dimensional image which, like a CT image, is normally displayed in two-dimensional slices.
  • the slices may be combined to build three-dimensional representations.
  • Contrast agents used in MRI or MR spectroscopy imaging differ from those used in other imaging techniques.
  • MRI contrast agents include gadolinium chelates, manganese chelates, chromium chelates, and iron particles.
  • a protein of the invention is conjugated to a compound comprising a chelate of a paramagnetic metal selected from the group consisting of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, molybdenum, ruthenium, cerium, indium, praseodymium, neodymium, profnethium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, and ytterbium.
  • a paramagnetic metal selected from the group consisting of scandium, titanium, vanadium, chromium, manganese, iron, cobalt, nickel, copper, moly
  • imaging agents useful for the present invention is halocarbon-based nanoparticle such as PFOB or other fluorine-based MRI agents.
  • CT and MRI provide anatomical information that aid in distinguishing tissue boundaries and vascular structure.
  • Imaging modalities that provide information pertaining to information at the cellular level, such as cellular viability, include positron emission tomography (PET) and single- photon emission computed tomography (SPECT).
  • PET positron emission tomography
  • SPECT single- photon emission computed tomography
  • PET a patient ingests or is injected with a radioactive substance that emits positrons, which can be monitored as the substance moves through the body.
  • SPECT single-photon emission computed tomography
  • the major difference between the two is that instead of a positron-emitting substance, SPECT uses a radioactive tracer that emits high-energy photons.
  • SPECT is valuable for diagnosing multiple illnesses including coronary artery disease, and already some 2.5 million SPECT heart studies are done in the United States each year.
  • a protein of the invention is commonly labelled with positron-emitters such as n C, 13 N, 15 0, 18 F, 82 Rb, 62 Cu, and 68 Ga. Proteins of the invention are labelled with positron emitters such as 99mTc, 201 T1, and 67 Ga, l u In for SPECT.
  • Non-invasive fluorescence imaging of animals and humans can also provide in vivo diagnostic information and be used in a wide variety of clinical specialties. For instance, techniques have been developed including simple observations following UV excitation of fluorophores up to sophisticated spectroscopic imaging using advanced equipment (see, e.g., Andersson-Engels et al, 1997).
  • fluorescence e.g., from fluorophores or fluorescent proteins
  • specific devices or methods known in the art for the in vivo detection of fluorescence include, but are not limited to, in vivo near- infrared fluorescence (see, e.g., Frangioni, 2003), the MaestroTM in vivo fluorescence imaging system (Cambridge Research & Instrumentation, Inc.; Woburn, MA), in vivo fluorescence imaging using a flying-spot scanner (see, e.g., Ramanujam et al, 2001), and the like.
  • Other methods or devices for detecting an optical response include, without limitation, visual inspection, CCD cameras, video cameras, photographic film, laser- scanning devices, fluorometers, photodiodes, quantum counters, epifluorescence microscopes, scanning microscopes, flow cytometers, fluorescence microplate readers, or signal amplification using photomultiplier tubes.
  • an imaging agent is tested using an in vitro or in vivo assay prior to use in humans, e.g., using a model described herein.
  • sample should be understood as a reference to any sample of biological material derived from an animal such as, but not limited to, a body fluid (e.g., blood or synovial fluid or cerebrospinal fluid), cellular material (e.g. tissue aspirate), tissue biopsy specimens or surgical specimens.
  • body fluid e.g., blood or synovial fluid or cerebrospinal fluid
  • cellular material e.g. tissue aspirate
  • tissue biopsy specimens or surgical specimens e.g. tissue aspirate
  • sample includes extracts and/or derivatives and/or fractions of said sample, e.g., serum, plasma, peripheral blood mononuclear cells (PBMC), a buffy coat fraction.
  • PBMC peripheral blood mononuclear cells
  • the biological sample which is used according to the method of the present invention may be used directly or may require some form of treatment prior to use.
  • a biopsy or surgical sample may require homogenization or other form of cellular dispersion prior to use.
  • a reagent such as a buffer
  • such an assay may require the use of a suitable control, e.g. a normal or healthy individual or a typical population, e.g., for quantification.
  • a suitable control e.g. a normal or healthy individual or a typical population, e.g., for quantification.
  • abnormal individual shall be taken to mean that the subject is selected on the basis that they do not have an abnormal level of the antigen to be detected or quantified.
  • a "healthy subject” is one that has not been diagnosed as suffering from the condition being diagnosed or prognosed. .
  • a suitable control sample is a control data set. comprising measurements of the marker being assayed for a typical population of subjects known not to suffer from the condition being diagnosed or prognosed.
  • a reference sample is not included in an assay. Instead, a suitable reference sample is derived from an established data set previously generated from a typical population. Data derived from processing, analysing and/or assaying a test sample is then compared to data obtained for the sample population.
  • the methods of the present invention for inducing an antibody response (or producing an antibody) against an autoantigen that is a self-antigen are also useful for inducing an autoimmune response (e.g., to produce a model of an autoimmune condition).
  • a model of an autoimmune response may be produced by administering the autoantigen to the subject to thereby induce an autoimmune antibody response.
  • the method comprises administering antibodies or proteins of the invention that bind to the self-antigen to a subject to thereby induce an autoimmune antibody response.
  • Exemplary self antigens associated with an autoimmune condition are selected from the group consisting of collagen, preferably collagen type II (which induces arthritis or an arthritis-like condition); other collagens such as collagen type IX, collagen type V, collagen type XXVII, collagen type XVIII, collagen type IV, collagen type IX; aggrecan I; pancreas-specific protein disulphide isomerise A2 (which induces diabetes or a diabetes-like condition); interphotoreceptor retinoid binding protein (IRBP, which induces autoimmune uveitis or an autoimmune uveitis-like disease); the human IRBP peptide GPTHLFQPSLVLDMAKVLLD (IRBPl-20; SEQ ID NO: 10) (which induces autoimmune uveitis or an autoimmune uveitis-like disease); protein lipoprotein (which induces multiple sclerosis or a multiple-sclerosis-like condition); myelin basic protein (which induces multiple sclerosis or a multiple-sclerosis-like condition);
  • ICA69 involved in diabetes
  • CYP1A2 involved in hepatitis and APSl
  • Tph and Fabp2 both involved in APECED and APSl
  • Tgn involved in autoimmune thyroiditis and APSl
  • Sptl & 2 involved in Sjogren's syndrome
  • Mater involved in ovarian failure
  • CB11 peptide from collagen which induces arthritis or an arthritis-like condition
  • the self-antigen is collagen type II and the autoimmune condition is arthritis or an arthritis-like condition.
  • the present invention also provides methods for identifying or isolating a compound for treating or preventing an autoimmune condition.
  • Suitable compounds ' for screening include, for example, nucleic acids, antibodies, peptides or small molecules.
  • this method comprises administering the compound to a non-human animal having an autoimmune condition produced by performing a method of the invention and determining the effect of the compound on one or more symptoms of the autoimmune condition.
  • exemplary symptoms include :
  • arthritis or an arthritis-like condition swelling and/or of the joints, inflammation in joints as assessed using histochemistry, presence of inflammatory cells in a joint (e.g., assessed using ELISA or ELISPOT assays), and/or immune response against collagen or other connective tissue protein;
  • diabetes or a diabetes-like condition reduced blood glucose levels or reduced plasma insulin levels, reduced glucose tolerance, fewer pancreatic beta islet cells and/or an immune response against a pancreatic beta islet cell antigen (e.g., GAD65); and
  • This invention also provides for the provision of information concerning the identified or isolated compound. Accordingly, the screening methods are further modified by:
  • providing the compound shall be taken to include any chemical or recombinant synthetic means for producing said compound or alternatively, the provision of a compound that has been previously synthesized by any person or means. This clearly includes isolating the compound.
  • the compound or the name or structure of the compound is provided with an indication as to its use e.g., as determined by a screen described herein.
  • the screening assays can be further modified by:
  • the synthesized compound or the name or structure of the compound is provided with an indication as to its use e.g., as determined by a screen described herein.
  • the compound is identified or isolated from a library of compounds, each of which or a subset of which may be separated from other members (i.e., physically isolated).
  • a compound is isolated from the library by its identification, which then permits a skilled person to produce that compound in isolation, e.g., in the absence of other members of the library.
  • AIRE 1' mice were derived as previously described using B6 embryonic stem cells (Hubert et al, 2008). Littermate B6 mice were used as wild-type controls.
  • Medullary TECs (CD45 " MHCII high Ly51 ,0W ) were isolated from the thymi of 6-8- week-old mice essentially as previously described (Hubert et al., 2008; Gray et al, 2002). R A was extracted using an RNeasy MicroKit (Qiagen, Chatsworth, CA) and was subjected to 2 rounds of amplification using a Megascript T7 kit (Ambion, Austin, TX) prior to hybridization to GeneChip Mouse Genome 430 2.0 (Affymetrix, Santa Clara, CA). For analysis, probe intensities were normalized and summarized using the robust multichip algorithm (Irrizary et al, 2003). Differential expression was assessed using empirical Bayes-moderated /-statistics from the LIMMA package (Smyth, 2004).
  • RNA was reverse-transcribed using Superscript III (Invitrogen, San Diego, CA), and PCR was performed on an ABI 7900 instrument using prevalidated TaqMan gene-specific assays (Applied Biosystems, Foster City, CA). Gene expression was normalized to that of Pgkl, with analysis of change in cycle threshold.
  • CIA suboptimal CIA was induced in mice by a single immunization with chick collagen type II (CII) (Sigma, St. Louis, MO) in complete Freund's adjuvant (CFA), as previously described (Campbell et al, 2000). Limbs were assessed for redness and swelling, and a clinical score (maximum of 12) was assigned.
  • CII chick collagen type II
  • CFA complete Freund's adjuvant
  • mice were killed, paws were removed, and hematoxylin and eosin-stained sections were histologically scored by an evaluator who was blinded with regard to experimental group, using a scale of 0-3, where 0 - normal, 1 - mild inflammation and minimal joint tissue disruption, 2 - moderate inflammation and tissue destruction with joint features remaining intact, and 3 - severe inflammation and tissue destruction with marked perturbation of joint architecture.
  • inguinal lymph node (LN) cells (2 x 10 s per well) from mice immunized with CII were evaluated essentially as previously described (Campbell et al, 2000), using either chick CII (Sigma) or murine CII (Chondrex, Redmond, WA) as the antigenic stimulus.
  • cytokines interferon- ⁇ [IFNy], interleukin-4 [IL-4], and IL-17
  • ELISA enzyme-linked immunosorbent assay
  • Serum anti-CII antibody levels were determined by ELISAs essentially as previously described (Campbell et al, 2000). Both murine and chick CII were used in separate assays as the target antigens.
  • Immobilon-P membrane plates (Multiscreen-IP; Millipore, Bedford, MA) that had , been pretreated with ethanol were coated with 10 ⁇ g/ml of rat anti-mouse IFNy (clone R4-6A2; BD PharMingen) in phosphate buffered saline (PBS), then washed and blocked with RPMI medium containing 10% fetal calf serum.
  • PBS phosphate buffered saline
  • Purified CD4 T cells (2.5 x 10 5 ; pooled from draining LNs/spleen) of AIRE' ' and wild-type mice immunized with chick CII were cultured with irradiated (20 Gy) splenic APCs (5.0 x 10 5 ) prepared from naive wild-type mice. Cells were cultured with and without 100 ⁇ g/ml of chick CII for 48 hours at 37°C. Wells containing 2 ⁇ g/ml of concanavalin A served as a positive control and an indication of equal cell numbers.
  • Wild-type and AIRE 1' mice were intravenously injected with 3 mg of OVA (grade V [-98% purity]) (Sigma) or PBS; splenic dendritic cells (DCs) were isolated 16 hours later, essentially as previously described (Vremec et al, 1997), and the pooled cells of 2 mice were cultured with OVA-specific TCR-transgenic T cells (1 x 10 4 OT-I or OT-II cells/well), and 3H-thymidine incorporation was measured.
  • OVA grade V [-98% purity]
  • DCs splenic dendritic cells
  • NP-KLH The T cell-dependent (4-hydroxy-3-nitrophenyl)acetyl-keyhole limpet hemocyanin (NP-KLH) antigen antibody response was induced and measured essentially as previously described (Tarlinton et al, 2003).
  • AIRE 1' and wild-type mice were immunized on days 0 and 35 by intraperitoneal injection with 100 ⁇ g of NP-KLH (prepared by coupling the hapten NP-OSu [Biosearch Technologies, Novato, CA] with the carrier protein KLH [95% purity; Calbiochem]) precipitated in alum.
  • NP-KLH prepared by coupling the hapten NP-OSu [Biosearch Technologies, Novato, CA] with the carrier protein KLH [95% purity; Calbiochem]
  • levels of high- affinity and total NP-specific IgG were determined by ELISA using plates coated with 10 ⁇ g/ml of either NP-2-bovine serum albumin [BSA] or
  • CD4 T cells were purified with an autoMACS magnetic cell sorter (Miltenyi Biotec, Sunnyvale, CA). Single-cell suspensions of erythrocyte-depleted mouse splenocytes were stained with fluorescein isothiocyanate (FITC)-conjugated anti-CD4 monoclonal antibody (mAb) (Caltag, South San Francisco, CA) followed by anti-FITC microbeads. Cell purity was -95% by flow cytometry.
  • FITC fluorescein isothiocyanate
  • mAb monoclonal antibody
  • CD4 + CD25 " T cells were prepared from the spleens of naive AIRE 1' or wild-type mice by sequential sorting on an autoMACS system.
  • CD25 " cells were negatively selected from erythrocyte depleted splenocytes using FITC-conjugated anti-CD25 mAb (clone PC61/F7) and anti-FITC microbeads.
  • CD4 " CD25 " cells were then positively , selected using phycoerythrin (PE)-conjugated anti-CD4 mAb and anti-PE microbeads.
  • PE phycoerythrin
  • B cells were positively selected from the spleens of separate naive wild-type mice using PE-conjugated anti-B220 mAb (clone RA36B2; BD PharMingen) and anti-PE microbeads.
  • RagI ' mice were injected intraperitoneally with a mixture containing either AIRE 1' or wild-type CD4 + CD25 " cells (5 x ' lO 6 ) together with wild-type B220 + cells (2 x 10 7 ). Cell purity was >94% for CD4 " CD25 ' cells and >98% for B220 + cells. Evaluation of Germinal Center Formation
  • PNA biotinylated peanut agglutinin
  • Alexa 594-conjugated streptavidin Molecular Probes, Eugene, OR
  • the percentages of PNA-B220 + live cells were determined by flow cytometry Using FITC-labeled PNA and PE-conjugated anti-B220 mAb. For each analysis, 3 niice per genotype were examined at 7, 14, and 21 days after immunization with CII,
  • Medullary TECs were isolated from AIRE 1' and wild-type mice for gene expression analysis. Gene products that are structurally associated with CII in articular cartilage were strongly regulated by AIRE in mTECs (Table 1). These included type IX collagen ⁇ CoWal and Col9a3) and the noncollagenous proteoglycan aggrecan (Agcl). Several of these products, including CII (Co al), meet the criteria for TRAs as defined by Gardner et al (2008).
  • AIRE 1' mice spontaneously develop lymphocytic infiltrates of nonlymphoid organs and associated tissue-specific autoantibodies.
  • Targets include the retina, salivary glands, ovaries, lung, stomach, and lacrimal glands, but synovial joints do not appear to be targets for spontaneous autoimmunity.
  • AIRE 1' mice might harbor more T cells with potential reactivity against TRAs, e.g., TRAs in cartilage, such as CII, AIRE 1' , AIRE*' ' , and AIRE '* (wild-type) mice were given a single immunization with chick CII in CFA, a protocol that typically results in only mild CIA in B6 mice (Campbell et al, 2000). Chick and mouse CII share up to 94% sequence homology (Luross et al, 2001). A weak response was seen in wild-type B6 mice, with 56% of mice showing mild disease by day 50 (Figure 2A).
  • TRAs in cartilage such as CII, AIRE 1' , AIRE*' ' , and AIRE '* (wild-type) mice were given a single immunization with chick CII in CFA, a protocol that typically results in only mild CIA in B6 mice (Campbell et al, 2000). Chick and mouse
  • AIRE 1' mice showed significantly greater disease incidence (96%; P _ 0.01), with earlier onset of disease (mean ⁇ SEM day of onset 25.8 ⁇ 1.6 versus 35.8 ⁇ 2.7 in wild-type mice; P ⁇ 0.01).
  • Disease was also more severe in AIRE' ' mice, with significantly higher clinical scores (mean ⁇ SEM 4.4 ⁇ 0.6 versus 1.0 ⁇ 0.3 in wild-type mice; P ⁇ 0.001) (Figure 2A).
  • AIRE is reported to have a gene-dose effect on the expression of organ-specific autoimmunity (Liston et al, 2004), and so this was investigated in CIA.
  • Mice that were heterozygous for AIRE (AIRE* ' ' ' ) showed a trend toward an intermediate clinical response between those found in AIRE' ' and wild-type mice ( Figure 2A). This difference was statistically significant at the histologic level ( Figure 2C).
  • CII is found in the vitreous humor of the eye and some older AIRE' ' B6 mice develop retinal inflammation (Hubert et al, 2009), but no acceleration of eye disease was apparent in mice immunized with CII (results not shown).
  • CII is a highly conserved molecule, and cross-reactive cellular and humoral immune responses to CII are necessary for CIA (Campbell et al, 2000).
  • Immunization of DBA/1 mice with heterologous CII results in a weak cross-reacting T cell proliferative response to murine CII (Michaelsson et al, 1992), and so this was investigated.
  • AIRE' ' T cells showed a relatively minor proliferative response to murine CII, similar to that of wild-type cells (data not shown).
  • AIRE' ' CD4 T cells showed a relatively minor proliferative response to murine CII, similar to that of wild-type cells (data not shown).
  • increasing numbers of AIRE' ' or wild-type CD4 T cells were cultured with CII- primed wild-type APCs, and cytokine production (IFNy, IL-17, and IL-4) ( Figures 3B and C) and cell proliferation (data not shown) were evaluated. There was no difference in the response of the 2 genotypes.
  • DCs express AIRE, and in its absence, there may be enhanced T cell activation by these cells (Ramsey et al, 2006) that might not be apparent from whole lymph node cultures.
  • OVA-transgenic T cell system was used. Purified splenic DCs from OVA- injected AIRE' ' and wild-type mice were cultured with OVA-specific TCR-transgenic T cells. No difference was observed between AIRE' ' and wild-type DC antigen presentation to either CD4 (OT-II) or CD8 (OT-I) T cells ( Figure 3E).
  • mice with heterologous CII elicits a T cell-dependent antibody response.
  • the generation of complement-fixing antibodies (IgG2a, IgG2b, and IgG2c) to CII is critical for the development of CIA (Watson et al, 1985), and these appear shortly before arthritis develops (Wooley et al, 1988).
  • Naive AIRE 1' mice had no detectable autoantibodies against CII (data not shown), but AIRE' ' mice immunized with CII/CFA had a greater anti-CII total IgG response, whether murine CII ( Figure 4A) or chick CII (data not shown) was used as the detecting antigen.
  • Higher antibody titers were generally obtained in arthritic mice than in nonarthritic mice ( Figure 4A).
  • the percentage of splenic PNA+B220+ cells was also comparable between the genotypes (mean ⁇ SEM 4.8 ⁇ 0.6% for AIRE' ' mice versus 3.2 ⁇ 0.6% for wild-type mice) (P ⁇ 0.19). Similar results were obtained 7 and 21 days after CII immunization (data not shown).
  • mice were immunised by intradermal injection at the base of the tail with 100 ⁇ of an emulsion containing mouse type II collagen (100 g) and CFA. Arthritis, seen as redness and swelling of the paws, was evaluated for up to 41 days post injection.
  • An emulsion was formed by mixing equal volumes of antigen solution (e.g. 2 mg/ml mouse type II collagen in 10 mM acetic acid) and Freund's complete adjuvant (CFA; containing 5 mg/ml heat-killed Mycobacterium tuberculosis). Mice were shaved at the base of the tail and then immunised by intra-dermal injection of emulsion (100 ⁇ total, given over 2 or more sites). The following variations of the injection regime are contemplated: other adjuvants (e.g. Freund's incomplete adjuvant, Alum), other injection sites (e.g. subcutaneous, intra-muscular, intra-peritoneal). The mice were monitored for a serum antibody response over the subsequent 30 days. No boost injection was given. At day 30 (or thereabouts) the mice were killed and the spleens removed for generation of hybridomas and monoclonal antibodies.
  • CFA Freund's complete adjuvant
  • the CB11 fragment of Collagen type II ( ⁇ g/ml in 50mM Tris, 150mM NaCl) or mouse Collagen type II protein (Chondrex, Australia) ( ⁇ g/ml in lOmM acetic acid) were spotted at 2 ⁇ 1 volumes onto nitrocellulose membrane and allowed to dry for one hour at RT. Non-specific sites were then blocked by gentle rocking in blocking buffer overnight at 4°C. After incubation, membranes were washed three times with tris buffered saline-Tween (TBS-T) and allowed to dry for one hour, following which; 2 ⁇ 1 of hybridoma fusion supernatants were spotted onto the corresponding sample dots and left for at least one hour at room temperature (RT).
  • TBS-T tris buffered saline-Tween
  • the membranes were then washed three times with TBS-T and incubated with goat anti-mouse IgG total secondary antibody conjugated with horse-radish peroxidase (HRP) (in TBS-T + 5% skim milk) for 45 minutes at RT. After incubation, the membranes were washed three times with TBS-T and incubated with ECL reagent (GE Healthcare, Buckinghamshire, UK) for five minutes, sandwiched between plastic bags and exposed to film in the dark.
  • HRP horse-radish peroxidase
  • the CB11 fragment or mouse Collagen type II protein was diluted to ⁇ g/ml in MT-PBS, added at 70 ⁇ 1 ⁇ 11 of a 96-well NUNC-Immuno plate (Thermo Fisher Scientific, Roskilde, Denmark), sealed and incubated overnight at 4°C. Following incubation, plates were washed three times with ELISA wash buffer and blocked with ELISA block buffer for one hour at RT. Plates were washed three times and 70 ⁇ 1 of hybridoma fusion supernatants were added to the appropriate wells, sealed and incubated for two hours at RT.
  • Plates were washed three times and 70 ⁇ 1 of goat amouse HRP secondary antibody diluted in ELISA was buffer was added to the appropriate wells, sealed and incubated for two hours at RT. Plates were washed with ELISA wash buffer and ⁇ of TMB substrate solution was added to the appropriate wells and incubated for 20 minutes at RT. The colour reaction was stopped by adding 50 ⁇ 1 ⁇ 11 of 0.5M H 2 S0 4 . Plates were read at 450nm on a Multiskan Ascent microplate photometer (Thermo Scientific, Pathtech party Ltd., Australia) and data analysed using Multiskan Ascent® software (Thermo Scientific, USA).
  • the hybridomas described in Example 4 are sub-cloned at least twice, resulting in between 50 to 100 sub-clone supernatants each round. These supernatants are subsequently screened by dot-blot and ELISA essentially as described in Example 4 and cells producing monoclonal antibodies against the autoantigen grown out for the next round of sub-cloning. After the sub-cloning process, a panel of monoclonal antibodies specific for both native type II collagen and the CB11 fragment of type II collagen is obtained. The antibodies are injected into mice either alone or in combination with each other to observe onset of collagen antibody-induced arthritis compared with a commercial cocktail of anti-collagen antibodies.

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Abstract

L'invention concerne un procédé permettant de produire un anticorps qui se lie à un autoantigène, le procédé consistant à immuniser un animal non humain présentant une activité protéique du régulateur auto-immun (AIRE) réduite avec l'autoantigène, de sorte qu'un anticorps contre l'autoantigène est produit. L'invention concerne également des anticorps contre des autoantigènes, des protéines comprenant des domaines de liaison d'autoantigènes, des bibliothèques d'acides nucléiques codant les anticorps et/ou les protéines, et des bibliothèques de cellules et particules affichant les anticorps et/ou les protéines.
PCT/AU2010/000653 2009-09-14 2010-05-27 Procedes et reactifs pour produire des anticorps contre des autoantigenes WO2011029126A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014091034A1 (fr) * 2012-12-14 2014-06-19 Universitätsmedizin Der Johannes Gutenberg-Universität Mainz Nouveaux antigènes associés à une tumeur indépendante du cmh
US11001804B2 (en) * 2017-08-30 2021-05-11 Wayne State University Methods for the production of therapeutic, diagnostic, or research antibodies

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WO1999015559A1 (fr) * 1997-09-23 1999-04-01 Finnish Immunotechnology Ltd. Nouveau gene sans apeced et ses utilisations
WO2002076406A2 (fr) * 2001-03-27 2002-10-03 Gershwin M Eric Anticorps contre autoantigenes de cirrhose biliaire primaire et methodes de preparation et d'utilisations associees
WO2005062959A2 (fr) * 2003-12-22 2005-07-14 The University Of Tennessee Research Foundation Recepteurs isoles de lymphocytes t specifique aux auto-antigenes humains en complexe avec des molecules de complexe majeur d'histocompatibilite et leurs procedes de fabrication et d'utilisation

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WO1999015559A1 (fr) * 1997-09-23 1999-04-01 Finnish Immunotechnology Ltd. Nouveau gene sans apeced et ses utilisations
WO2002076406A2 (fr) * 2001-03-27 2002-10-03 Gershwin M Eric Anticorps contre autoantigenes de cirrhose biliaire primaire et methodes de preparation et d'utilisations associees
WO2005062959A2 (fr) * 2003-12-22 2005-07-14 The University Of Tennessee Research Foundation Recepteurs isoles de lymphocytes t specifique aux auto-antigenes humains en complexe avec des molecules de complexe majeur d'histocompatibilite et leurs procedes de fabrication et d'utilisation

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Title
ANDERSON, M.S. ET AL.: "Projection of an Immunological Self Shadow Within the Thymus by the Aire.", PROTEIN. SCIENCE., vol. 298, no. 5597, 2002, pages 1395 - 1401 *
CAMPBELL, I.K. ET AL.: "Autoimmune Regulator Controls T Cell Help for Pathogenetic Autoantibody Production in Collagen-Induced Arthritis.", ARTHRITIS & RHEUMATISM., vol. 60, no. 6, 2009, pages 1683 - 1693 *
GAVANESCU, I. ET AL.: "B cells are required for Aire-deficient mice to develop multi- organ autoinflammation: A therapeutic approach for APECED patients.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE USA., vol. 105, no. 35, 2008, pages 13009 - 13014 *
HUBERT, F-X. ET AL.: "Aire-Deficient C57BL/6 Mice Mimicking the Common Human 13-Base Pair Deletion Mutation Present with Only a Mild Autoimmune Phenotype.", JOURNAL OF IMMUNOLOGY, vol. 182, no. 6, March 2009 (2009-03-01), pages 3902 - 3918 *
MISHARIN, A.V. ET AL.: "Studies in Mice for the Autoimmune Regulator (Aire) and Transgenic for the Thyrotropin Receptor Reveal a Role for Aire in Tolerance for Thyroid Autoantigens.", ENDOCRINOLOGY., vol. 150, no. 6, June 2009 (2009-06-01), pages 2948 - 2956 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014091034A1 (fr) * 2012-12-14 2014-06-19 Universitätsmedizin Der Johannes Gutenberg-Universität Mainz Nouveaux antigènes associés à une tumeur indépendante du cmh
US9861688B2 (en) 2012-12-14 2018-01-09 Universitaetsmedizin Der Johannes Gutenberg-Universitaet Mainz T cell receptors with MHC independent binding to GM-CSF receptor alpha chain
AU2013357239B2 (en) * 2012-12-14 2019-10-17 Biontech Rna Pharmaceuticals Gmbh Novel MHC-independent tumor-associated antigens
US10987413B2 (en) 2012-12-14 2021-04-27 Biontech Rna Pharmaceuticals Gmbh MHC-independent tumor-associated antigens
US11001804B2 (en) * 2017-08-30 2021-05-11 Wayne State University Methods for the production of therapeutic, diagnostic, or research antibodies

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