WO2010047698A1 - Répression de maladies auto-immunes par un inhibiteur de la cytidine désaminase induite par activation - Google Patents

Répression de maladies auto-immunes par un inhibiteur de la cytidine désaminase induite par activation Download PDF

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WO2010047698A1
WO2010047698A1 PCT/US2008/080741 US2008080741W WO2010047698A1 WO 2010047698 A1 WO2010047698 A1 WO 2010047698A1 US 2008080741 W US2008080741 W US 2008080741W WO 2010047698 A1 WO2010047698 A1 WO 2010047698A1
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aid
bxd2
cells
mice
activation
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PCT/US2008/080741
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John D. Mountz
Hui-Chen Hsu
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University Of Alabama At Birmingham
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
    • 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
    • A01K2217/056—Animals comprising random inserted nucleic acids (transgenic) inducing loss of function due to mutation of coding region of the transgene (dominant negative)
    • 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
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • 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 disclosure is generally directed to methods of reducing an autoimmune reaction in an animal.
  • the present disclosure further relates to the suppression of an autoimmune response in an animal by inhibiting an activation-induced cytidine deaminase.
  • BACKGROUND Activation-induced cytidine deaminase plays a key roie in the regulation of B- cell somatic hypermutation (SHM) and class switch recombination (CSR) (Honjo, et al., Immunity 20: 659-668 (2004); Shinkura, et al., Nat. Immunol. 5: 707-712 (2004); Xu, et al., Ann. N. Y. Acad. ScL 1050: 146-162 (2005)).
  • SHM B- cell somatic hypermutation
  • CSR class switch recombination
  • the BXD2 mouse model of generalized autoimmune disease indicates that such animals express dramatically elevated levels of AID in resting B cells isolated directly from the spleen, and that this over-expression occurs in response to extrinsic T-cell factors.
  • IgG autoantibody-producing B cells the spontaneous formation of germinal centers in the spleen, and the development of highly pathogenic multireactive IgG autoantibodies, data indicate excessive T-cell help and dysregulated CSR in BXD2 mice.
  • Activated CD4 + T cells from BXD2 mice produce cytokines and surface molecules that induce high AID expression by B cells, suggesting that the excessive T-cell help directly contributes to the elevated expression of AID in these mice.
  • Multireactive IgM and IgG autoantibodies have been identified in patients with lupus and other autoimmune disease, as well as in models of lupus (Putterman, et al., J Immunol; Thomas Kayden Docket No 222104-2120
  • CSR immunoglobulin class switch recombination
  • SHM and CSR are mediated by AID in the germinal centers of humans and mice.
  • Na ⁇ ve B cells that express surface autoantibodies specific for a particular antigen are activated by T H cells, and their clonal progeny migrate to the dark zone of the germinal center where they become centroblasts.
  • Centroblasts undergo SHM and CSR, and then differentiate into centrocytes.
  • centrocytes interact with antigen presented by follicular dendritic cells.
  • the centrocytes differentiate into plasma or memory B cells.
  • the surface autoantibodies have a reduced affinity for the particular antigen or are autoreactive, the progeny of centrocytes either undergo apoptosis or become anergic.
  • immunoglobulins undergo somatic hypermutation (SHM) and class switch recombination (CSR) in the germinal centers through a process that is dependent on the activity of AID in the B cells, and is independent of RAG1 or RAG2 activity. Upregulation of AID in B cells of BXD2 mice is considered an important pathogenic mechanism.
  • SHM somatic hypermutation
  • CSR class switch recombination
  • embodiments of this disclosure encompass methods for reducing or eliminating autoantibody formation in a host animal by inhibiting the endogenous activation-induced cytidine deaminase (AID) activity in vivo in the host animal by pharmacological or genetic manipulation.
  • AID endogenous activation-induced cytidine deaminase
  • Genetic and pharmacologic inhibition of AID resulted in decreased autoantibody production, and suppression of arthritis and renal disease in the BXD2 mouse model, indicating the important contribution of AID in autoimmune disease.
  • the present study demonstrated that the ability to prevent autoimmune disease and autoantibodies by specifically inhibiting, but not the total deletion of, AID, provides a model comparable to what would be expected for therapeutic inhibition of AID.
  • BXD2 mice could be treated separately with two different competitive inhibitors of cytidine deaminase, tetrahydrourdine (THU), and/or zebularin (ZEB).
  • TNU tetrahydrourdine
  • ZEB zebularin
  • AID-dominant negative gene contained the H56R and E58Q mutations in the AID catalytic domain and the S38A mutation at the phosphorylation site that is essential for interaction of AID with replication protein A (RPA).
  • Sera titers of autoantibodies in animals treated either pharmacologically or genetically manipulated to have a deficiency in AID activity were measured.
  • One aspect of the disclosure therefore, provides methods for inhibiting the generation of an autoantibody in a subject animal, comprising reducing the level of activity of an activation-induced cytidine deaminase in a subject animal, thereby reducing the generation of an autoantibody in the subject animal.
  • the methods may comprise administering to a subject animal an effective amount of a therapeutic composition comprising an activation-induced cytidine deaminase inhibitor compound, whereby the inhibitor reduces the activity of the activation-induced cytidine deaminase in generating somatic hypermutations and/or class switch recombination of an immunoglobulin, thereby reducing the generation of an autoantibody in the subject animal.
  • the methods for inhibiting the generation of an autoantibody further reduces the level of an autoimmune disease in the subject animal.
  • the inhibitor of the activation-induced cytidine deaminase activity is tetrahydrouridine (1-(-D-ribofuranosyl)-4-hydroxytetrahydro-1(1 H)- pyrimidinone).
  • inhibition of an activation-induced cytidine deaminase may inhibit an autoimmune disease.
  • the autoimmune disease may be, but is not limited to, an autoimmune disease selected from the group consisting of: systemic lupus erythematosus, rheumatoid arthritis, spontaneous erosive arthritis, and glomerulonephritis. Thomas Kayden Docket No 222104-2120
  • the method for inhibiting the generation of an autoantibody in a subject animal may comprise reducing the level of activity of an activation-induced cytidine deaminase in a subject animal by delivering to a cell or tissue of the subject animal a heterologous nucleic acid expression construct comprising a nucleic acid region encoding a dominant-negative variant of an activation-induced cytidine deaminase, whereby the variant activation-induced cytidine deaminase in the cell or tissue of the subject animal reduces the activity of the cytidine deaminase in generating somatic hypermutations and/or class switch recombination of an immunoglobulin or plurality of immunoglobulins, thereby reducing the generation of an autoantibody in the subject animal
  • Another aspect of the disclosure provides a transgenic animal comprising a heterologous nucleic acid construct encoding a dominant negative variant of an activation- induced cytidine de
  • the heterologous nucleic acid construct encoding a dominant negative variant of an activation-induced cytidine deaminase may be operably linked to an inducible expression control region
  • Fig 1 is a schematic illustration of an AID-dominant negative construct that was injected directly into BXD2 single-cell Catalytic domain mutations H56R, E58Q, and kinase domain mutation S38A were introduced into wild-type AID, and the AID-dominant negative encoding region was cloned downstream of a chicken- ⁇ -actin CAG promoter The CAGpro- AID-dominant negative was excised and subcloned into the plRES2-AC-GFP1 vector
  • Fig 2 is a digital photograph of an RT-PCR tail RNA analysis of the presence of an expressed AID-dominant negative mRNA in BXD2 AID-dominant negative transgenic mice RT-PCR was carried out using GFP- and AID-IRES-specific primer pairs
  • Fig 3 is a digital photograph of RT-PCR (upper panel) and real-time PCR quantitation of the expression of wild-type-AID carried out using a primer pair that amplifies the untranslated region of wild-type-AID and which had not included in the AID-dominant negative transgene construct RT-PCR and real-time PCR quantitation of the expression of AID-dominant negative was carried out using a primer pair that amplifies the AID-IRES Thomas Kayden Docket No.: 222104-2120
  • Figs. 4A and 4B are graphs illustrating ELISA analysis of the low affinity anti-NP24 (Fig. 4A) and the high affinity anti-NP2 (Fig. 4B) antibody response, respectively, in the indicated groups of mice (all 2 months old) after NP-CGG immunization. Sera were collected 28 days after the immunization ( * P ⁇ 0.05; * P ⁇ 0.01 ; N ⁇ 4 per group).
  • Fig. 5A is a graph illustrating an ELISA analysis of circulating autoantibodies in the sera of B6, wild-type BXD2, and BXD2 AID-dominant negative transgenic mice (* P ⁇ 0.05; ** P ⁇ 0.01; N ⁇ 6 per group).
  • Fig. 5B is a graph illustrating an ELISA analysis of C1q-containing immune complexes in the sera of B6, wild-type BXD2, and BXD2 AID-dominant negative transgenic mice (* P ⁇ 0.05; * P ⁇ 0.01 ; N > 6 per group).
  • Fig. 6 is a series of digital photomicrographs showing hematoxylin/eosin stained joint sections from wildtype-BXD2 and BXD2 AID-dominant negative transgenic mice.
  • Fig. 7 is a series of digital photomicrographs illustrating immunohistochemistry staining of IgM and IgG antibody deposits on the glomerulus of wild-type-BXD2 and BXD2 AID-dominant negative transgenic mice.
  • the objective lens (x10 or x40) used for acquiring each image is indicated.
  • Fig. 8 is an illustration of a flow cytometry analysis of cell viability (upper panels), and percentage of cells undergoing CSR (shown as percentage of GFP + cells) after 5 days of culture (lower panels).
  • Fig. 10 is a graph illustrating that compared to age-matched BXD2 mice, BXD2 AID- dominant negative transgenic mice exhibit a lower number of B cells producing IgG (including lgG2b and lgG2c), as well as less IgM.
  • Fig. 11 shows digital photomicrographs of immunostaining, where IgM-secreting B cells were higher in wild-type BXD2 mice compared to B6 mice, and that the levels were almost 'normal' in BXD2 AID-dominant negative transgenic mice.
  • the objective lens (x10 or x20) used for acquiring each image is indicated.
  • Fig. 12A is a graph illustrating a lower amount of IgG anti-BiP, and both IgM and IgG anti-histone antibody-producing B cells in the spleens of BXD2 AID-dominant negative transgenic mice compared to the spleens of wild-type BXD2 mice.
  • Fig. 12B shows digital photomicrographs of immunostaining, where IgG-secreting B cells were higher in wild-type BXD2 mice compared to B6 mice, and that the levels were Thomas Kayden Docket No.: 222104-2120
  • the objective lens (x10 or x20) used for acquiring each image is indicated.
  • Fig. 13A is a graph illustrating the incidence of arthritis determined by bilateral symmetric erythemia and swelling of either the ankle or fore-limb joint in wild-type BXD2 and BXD2 AID-dominant negative transgenic mice.
  • Fig. 13B is a graph illustrating lymphocyte infiltration, synovial hyperplasia, and bone erosion in 6 month to 8 month old wild-type BXD2 and BXD2 AID-dominant negative transgenic mice.
  • Fig. 13C is a graph illustrating urinary albumin in wild-type BXD2 and BXD2 AID- dominant negative transgenic mice.
  • Fig 14 is a graph illustrating the measurement of spleen size determined by weight (mg) and cell count in wild-type and AID-dominant negative transgenic mice at 6 months to 8 months of age.
  • Fig. 15 is a graph illustrating a FACS analysis of total CD19 + B cells and CD4 T cells in wild-type and AID-dominant negative transgenic mice at 6 months to 8 months of age.
  • Fig. 16 is an illustration of a FACS analysis of percent of Fas + GL-7 + B220 + germinal center B cells in wild-type and AID-dominant negative transgenic mice at 6 months to 8 months of age.
  • Fig. 17 shows graphs illustrating the proliferation, as determined by bromodeoxyuridine (BrdU) incorporation, of CD19 + B cells, and PNA + FaS + germinal center B cells in wild-type and AID-dominant negative transgenic mice at 6 months to 8 months of age.
  • Fig. 18 is a series of digital photomicrographs of an immunohistological analysis (left panel) of Ki67 + cells, TUNEL staining (right panel) of the spleens of wild-type and AID- dominant negative transgenic mice at 6 months to 8 months of age, and a graphical comparison of the results.
  • Fig. 19 shows the quantitation of follicles that contain Ki67 + cells (left panels) and the average number Of TUNEL + cells in each follicle (right panel) in wild-type BXD2 and AID- dominant negative transgenic mice at 6 months to 8 months of age ( * P ⁇ 0.05; *** P ⁇ 0.005; N ⁇ 6 per group).
  • Fig 20 is a graph illustrating the equivalent levels of autoantibodies in the sera of BXD2 mice before treatment with PBS or THU.
  • Fig 21 is a graph illustrating the effects of THU on spleen weight and spleen cell count of BXD2 mice after THU treatment for 3 weeks (*P ⁇ 0.005).
  • Fig. 22 illustrates the results of a FACS analysis of the percent of germinal center B cells and proliferation of B cells in the spleens of BXD2 mice 3 weeks after treatment with THU or vehicle controls. Thomas Kayden Docket No 222104-2120
  • Fig 23 shows digital photomicrographs of the immunohistochemistry staining of K ⁇ 67 in the spleens of BXD2 mice 3 weeks after treatment with THU or vehicle controls
  • Fig 24 shows digital photomicrographs Of TUNEL + apoptotic cells in the spleens of BXD2 mice 3 weeks after treatment with THU or vehicle controls
  • Fig 25 are graphs illustrating the inhibition of autoantibody production in BXD2 mice by treatment with the AID inhibitor THU BXD2 mice were treated with THU (I mg in PBS per mouse, intraperitoneal ⁇ , twice per week) for up to 4 months The graphs illustrate the results of an ELISA assay of serum antibodies in 5 month old animals
  • Fig 26 is a series of photomicrographs of immunohistochemistry staining of IgG deposits (left panel) and hematoxylin/eosin staining of joint disease (right panel) in THU- treated mice, compared to untreated control animals
  • Fig 27 is a graph illustrating a cluster analysis of DNA damage genes of two separates of non-germinal center (N-1 b and N-2a), wild-type germinal center (P-1 and P-2) and AID-dominant negative germinal center (P-3 and P-4)
  • Fig 28 is a graph illustrating a cluster analysis of apoptosis genes of two separates of non-germinal center (N-1b and N-2a), wild-type germinal center (P-1 and P-2) and AID- dominant negative germinal center (P-3 and P-4)
  • Fig 29 is a graph illustrating a quantitative-real-time PCR analysis for the expression of DNA damage sensor genes
  • Fig 30 is a graph illustrating a quantitative-real-time PCR analysis for the expression of DNA damage repair and apopotosis genes
  • Fig 31 A is a graph illustrating the results of a quantitative RT-PCR analysis of DNA damage gene expression in germinal center B cells isolated from BXD2 mice 3 weeks after treatment with THU or vehicle controls
  • Fig 31 B is a graph illustrating the results of a quantitative RT-PCR analysis of DNA repair genes in germinal center B cells isolated from BXD2 mice 3 weeks after treatment with THU or vehicle controls
  • Figs 32A-32C illustrate FACS output images showing increase AID + cells in new emigrant B cells in the PBMCs of autoimmune patients
  • Fig 32A Cells were gated first on CD20 + , and then on IgM + CDIO + Or IgM + CDIO cells
  • Fig 32B A histogram for AID expression for IgM + CDIO + B cells as compared to IgM + CDI O B cells
  • Fig 32C Measurement of rheumatoid factor (RF) and anti ( ⁇ )-DNA antibodies from the same subjects
  • Figs 33A and 33B are digital photographs of a Northern blot (Fig 33A) and immunohistoassays (Fig 33B) illustrating increased AID and spontaneous germinal centers in BXD2 mice Thomas Kayden Docket No.: 222104-2120
  • Fig. 34 is a series of digital images showing the immunohistostaining results illustrating the proximity Of AID + B cells close to germinal centers in the spleens of BXD2 mice.
  • Fig. 35 is a series of digital images of immunohistostained results demonstrating AID + B cells in inflamed tissue of a human autoimmune disease.
  • TUNEL Terminal deoxynucleotidyl transferase dUTP nick end labeling
  • PBMC peripheral blood mononucleocytes
  • AID activation-induced cytidine deaminase
  • SHM somatic hypermutation
  • CSR class switch recombination
  • THU 1-(-D-ribofuranosyl)-4- hydroxytetrahydro-1(1 H)-pyrimidinone
  • RF rheumatoid factor
  • Ig immunoglobulin. Definitions
  • polypeptide refer interchangeably to a polymer in which the monomers are amino acid residues which are joined together through amide bonds, alternatively referred to as a polypeptide.
  • a "single polypeptide” is a continuous peptide that constitutes the protein.
  • the amino acids are alpha-amino acids, either the L-optical isomer or the D-optical isomer can be used, the L- isomers being preferred.
  • unnatural amino acids such as ⁇ -alanine, phenylglycine, and homo-arginine are meant to be included.
  • amino acids which are not gene-encoded can also be used herein, although preferred amino acids are those that are encodable.
  • preferred amino acids are those that are encodable.
  • amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
  • Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, Thomas Kayden Docket No.: 222104-2120
  • Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, and/or methionine methyl sulfonium.
  • Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
  • Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission.
  • Nucleotides may be referred to by their commonly accepted single-letter codes as indicated as follows: Alanine (Ala, A), Arginine (Arg, R), Asparagine (Asn, N), Aspartic Acid (Asp, D), Cysteine (Cys, C), Glutamine (GIn, Q), Glutamic Acid (GIu, E), Glycine (GIy, G), Histidine (His, H), lsoleucine (Me, I), Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M), Phenylalanine (Phe, F), Proline (Pro, P), Serine (Ser, S), Threonine (Thr, T), Tryptophan (Trp, W), Tyrosine (Tyr, Y), and Valine (VaI, V).
  • variant refers to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall (homologous) and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g., substitutions, additions, and/or deletions).
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • Modifications and changes can be made in the structure of the polypeptides of this disclosure and still result in a molecule having similar characteristics as the polypeptide (e.g., a conservative amino acid substitution).
  • certain amino acids can be substituted for other amino acids in a sequence without appreciable loss of activity.
  • certain amino acid sequence substitutions can be made in a polypeptide sequence and nevertheless obtain a polypeptide with like properties. In making such changes, the hydropathic index of amino acids can be considered.
  • hydropathic index on the basis of its hydrophobicity and charge characteristics. Those indices are: isoleucine ( + 4.5); valine ( + 4.2); leucine ( + 3.8); phenylalanine ( + 2.8); cysteine/cysteine ( + 2.5); methionine ( + 1.9); alanine ( + 1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).
  • the relative hydropathic character of the amino acid determines the secondary structure of the resultant polypeptide, which in turn defines the interaction of the polypeptide with other molecules, such as enzymes, substrates, receptors, antibodies, antigens, and the like. It is known in the art that an amino acid can be substituted by another amino acid having a similar hydropathic index and still obtain a functionally equivalent polypeptide. In such changes, the substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity can also be made on the basis of hydrophilicity, particularly where the biologically functional equivalent polypeptide or peptide thereby created is intended for use in immunological embodiments.
  • the following hydrophilicity values have been assigned to amino acid residues: arginine ( + 3.0); lysine ( + 3.0); aspartate ( + 3.0 ⁇ 1); glutamate ( + 3.0 ⁇ 1); serine ( + 0.3); asparagine f ⁇ .2); glutamnine ( + 0.2); glycine (0); proline (-0.5 ⁇ 1); threonine (-0.4); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4).
  • an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent, and in particular, an immunologically equivalent polypeptide.
  • substitution of amino acids whose hydrophilicity values are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like.
  • Exemplary substitutions that take one or more of the foregoing characteristics into consideration are well known to those of skill in the art and include, but are not limited to (original residue: exemplary substitution): (Ala: GIy, Ser), (Arg: Lys), (Asn: GIn, His), (Asp: GIu, Cys, Ser), (GIn: Asn), (GIu: Asp), (GIy: Ala), (His: Asn, GIn), (Me: Leu, VaI), (Leu: lie, VaI), (Lys: Arg), (Met: Leu, Tyr), (Ser: Thr), (Thr: Ser), (Tip: Tyr), (Tyr: Trp, Phe), and (VaI: lie, Leu).
  • Embodiments of this disclosure thus contemplate functional or biological
  • embodiments of the polypeptides can include variants having about 50%, 60%, 70%, 80%, 90%, and 95% sequence identity to the polypeptide of interest.
  • polynucleotide refers to any polyribonucleotide or polydeoxribonucleotide, which may be unmodified RNA or DNA or modified RNA or DNA.
  • polynucleotides as used herein refers to, among others, single-and double-stranded DNA 1 DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • nucleic acid “nucleic acid sequence,” or “oligonucleotide” also encompass a polynucleotide as defined above.
  • polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are “polynucleotides” as that term is intended herein.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein.
  • DNA may be obtained by any method.
  • the DNA includes complementary DNA (cDNA) prepared from mRNA, DNA prepared from genomic DNA, DNA prepared by chemical synthesis, DNA obtained by PCR amplification with RNA or DNA as a template, and DNA constructed by appropriately combining these methods.
  • cDNA complementary DNA
  • phrases for introducing a plasmid into a host are, calcium chloride method, calcium chloride/rubidium chloride method and electroporation method, described in Molecular Cloning, A Laboratory Manual (second edition, Cold Spring Harbor Laboratory, p.1.74 (1989)).
  • Phage vectors can be introduced into host cells by, for example, a method in which the phage DNAs are introduced into grown hosts after in vitro packaging. In vitro packaging can be easily performed with a commercially available in vitro packaging kit (for example, a product from Stratagene or Amersham).
  • vector refers to a DNA molecule serving as a vehicle capable of stably carrying exogeneous genes into host cells.
  • a vector should be replicable, have a system for introducing itself into a host cell, and possess selectable markers.
  • Embodiments of the present disclosure relate to a recombinant vector comprising the DNA encoding the protein used herein.
  • any vector can be used as long as it is capable of retaining replication or self- multiplication in each host cell of prokaryotic and/or eukaryotic cells, including plasmid vectors and phage vectors.
  • the recombinant vector can easily be prepared by ligating the DNA encoding protein with a vector for recombination available in the art (plasmid DNA and bacteriophage DNA) by the usual method.
  • plasmid DNA and bacteriophage DNA a vector for recombination available in the art
  • vectors for recombination are E. co//-derived plasmids such as pBR322, pBR325, pUC12, pUC13, and pUC19, yeast-derived plasmids such as pSH19 and pSH15, and Bacillus subtilis-der ' n/ed plasmids such as pUB110, pTP5, and pC194.
  • phages are a bacteriophage such as ⁇ phage, and an animal or insect virus (pVL1393, Invitrogen) such as a retrovirus, vaccinia virus, and nuclear polyhedrosis virus.
  • expression vector refers to a nucleic acid vector useful for expressing the DNA encoding the protein and for producing the protein.
  • the expression vector is not limited as long as it expresses the gene encoding the protein in various prokaryotic and/or eukaryotic host cells and produces this protein.
  • an expression vector preferably comprises at least a promoter, an initiation codon, the DNA encoding the protein, and a termination codon.
  • the expression vector may also contain, if required, a gene for gene amplification (marker) that is usually used.
  • a gene for gene amplification marker
  • An enhancer sequence, polyadenylation site, and splicing junction that are usually used in the art, such as those derived from SV40 can also be used.
  • a selectable marker usually employed can be used according to the usual method. Examples thereof are resistance genes for antibiotics, such as tetracycline, ampicillin, or kanamycin.
  • genes for gene amplification are dihydrofolate reductase (DHFR) gene, thymidine kinase gene, neomycin resistance gene, glutamate synthase gene, adenosine deaminase gene, ornithine decarboxylase gene, hygromycin-B-phophotransferase gene, aspartate transcarbamylase gene, and the like.
  • DHFR dihydrofolate reductase
  • thymidine kinase gene thymidine kinase gene
  • neomycin resistance gene glutamate synthase gene
  • glutamate synthase gene glutamate synthase gene
  • adenosine deaminase gene adenosine deaminase gene
  • ornithine decarboxylase gene hygromycin-B-phophotransferase gene
  • aspartate transcarbamylase gene and the like
  • An expression vector as intended to be used herein can be prepared by continuously and circularly linking at least the above-mentioned promoter, initiation codon, DNA encoding the protein, termination codon, and terminator region, to an appropriate replicon.
  • appropriate DNA fragments for example, linkers, restriction sites, and so on
  • transformation refers to the introduction of a nucleic acid sequence into the interior of a membrane enclosed space of a living cell, including introduction of the nucleic acid sequence into the cytosol of a cell as well as the interior space of a mitochondria, nucleus or chloroplast.
  • the nucleic acid may be in the form of naked DNA or RNA, associated with various proteins, or the nucleic acid may be incorporated into a vector. Thomas Kayden Docket No.: 222104-2120
  • transformant refers to a host cell prepared by introducing the expression vector mentioned above into host cells.
  • An expression vector can be introduced (transformed (transfected)) into host cells by known methods.
  • Transformation of a host cell with recombinant DNA may be carried out by conventional techniques as are well known to those skilled in the art.
  • the host is prokaryotic, such as E. coli
  • competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCI 2 method by procedures well known in the art.
  • CaCI 2 or RbCI can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.
  • Eukaryotic cells can also be co-transfected with DNA sequences encoding the calcium sensing system of the present disclosure, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene.
  • Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein.
  • a eukaryotic viral vector such as simian virus 40 (SV40) or bovine papilloma virus
  • SV40 simian virus 40
  • bovine papilloma virus bovine papilloma virus
  • host cell as used herein is not limited as long as it is compatible with an expression vector mentioned above and can be transformed.
  • examples thereof are various cells such as wild-type cells or artificially established recombinant cells usually used in technical field (for example, bacteria (Escherichia and Bacillus), yeast (Saccharomyces, Pichia, and such), animal cells, or insect cells).
  • E. coli or animal cells are preferably used. Specific examples are E. coli (DH5 ⁇ , TB1, HB101 , and such), mouse-derived cells (COP, L, C127, Sp2/0, NS-1 , NIH 3T3, and such), rat-derived cells (PC12, PC12h), hamster-derived cells (BHK, CHO 1 and such), monkey-derived cells (COS1 , COS3, COS7, CV1, VeIo, and such), and human-derived cells (HeIa, diploid fibroblast-derived cells, myeloma cells, HepG2, HEK293, and the like).
  • E. coli DH5 ⁇ , TB1, HB101 , and such
  • mouse-derived cells COP, L, C127, Sp2/0, NS-1 , NIH 3T3, and such
  • rat-derived cells PC12, PC12h
  • hamster-derived cells BHK, CHO 1 and such
  • Cultivation of cell lines may be performed by a method known in the art. Cultivation conditions such as temperature, pH of the media, and cultivation time are selected appropriately so that the desired expressed protein is produced in large quantities.
  • codon refers to a specific triplet of mononucleotides in the DNA chain or mRNA that make up an amino acid or termination signal.
  • a preferable initiation codon is, for example, a methionine codon (ATG).
  • codon for example, TAG, TAA, TGA
  • TAG for example, TAG, TAA, TGA
  • TAG for example, TAG, TAA, TGA
  • expression control region and "promoter sequence” as used herein refer to DNA regulatory regions in an operon capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bound at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Within the promoter sequence will be found a transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Various promoters, including inducible promoters may be used to drive the various vectors of the present disclosure.
  • Expression control sequences are operably linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence.
  • expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, and maintenance of the correct reading frame of that gene to permit proper translation of the mRNA, and stop codons.
  • reporter polynucleotide refers to any gene that expresses a detectable gene product, which may be RNA or a reporter polypeptide. Reporter genes include coding sequences for which the transcriptional and/or translational products are readily detectable or selectable.
  • insertion or “addition” as used herein refer to a change in an amino acid or nucleotide sequence resulting in the addition or insertion of one or more amino acid or nucleotide residues, respectively, as compared to the corresponding naturally occurring molecule.
  • deletion or “subtraction” as used herein refer to a change in an amino acid or nucleotide sequence resulting in the deletion or subtraction of one or more amino acid or nucleotide residues, respectively, as compared to the corresponding naturally occurring molecule.
  • substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides, respectively.
  • mutation refers to a heritable change in a DNA sequence relative to a reference “wild-type” DNA sequence. Mutations can occur as a result of a single base change, multiple base changes, or the addition or deletion of more than one nucleotide to a DNA sequence.
  • a “gene mutation” refers to a mutation that occurs entirely within one gene, or its upstream regulatory sequences and can comprise either a point Thomas Kayden Docket No.: 222104-2120
  • mutant refers to a protein that differs in some way from a reference wild-type protein, where the protein may retain biological properties of the reference wild-type (e.g., naturally occurring) protein, or may have biological properties that differ from the reference wild-type protein.
  • biological property of the subject proteins includes, but is not limited to, spectral properties, such as emission maximum, quantum yield, and brightness, and the like; in vivo and/or in vitro stability (e.g., half-life); and the like.
  • Mutants can include single amino acid changes (point mutations), deletions of one or more amino acids (point-deletions), N-terminal truncations, C-terminal truncations, insertions, and the like. Mutants can be generated using standard techniques of molecular biology.
  • the tern "dominant-negative” as used herein refers to a gene product, in particular a polypeptide expressed from an allele of a gene locus, that may prevent a wild-type phenotype being expressed by a cell.
  • a “mutant" strain is not capable of all of the activities of the wild-type strain from which it is derived.
  • a mutant bacterial strain that is defective in its ability to synthesize the amino acid histidine (his strain) requires the presence of exogenous histidine in order to grow.
  • point mutation refers to a change in one, or a small number of base pairs, in a DNA sequence. Point mutations may result from base pair substitutions or from small insertions or deletions.
  • transition refers to a point mutation in which a purine is replaced with a purine or a pyrimidine is replaced with a pyrimidine.
  • conversion refers to a point mutation in which a purine is replaced with a pyrimidine or a pyrimidine with a purine. Generally speaking, transitions are more common than tranversions because the former are not detected by the proofreading enzymes.
  • point mutation can also cause a nonsense mutation resulting from the insertion of a stop codon (amber, ochre, opal).
  • a stop codon amber, ochre, opal.
  • Base pair mutations that generate a translation stop codon causes premature termination of translation of the coded protein.
  • DNA repair mechanism refers to any one of the potential repair mechanisms that exist in both prokaryotes and eukaryotes. For example: postreplication; mismatch repair; nucleotide excision-repair; and photoreactivation or light- dependent repair (not found in mammals). Thomas Kayden Docket No.: 222104-2120
  • host or "organism” as used herein refer to humans, mammals (e.g., cats, dogs, horses, and the like), living cells, and other living organisms.
  • a living organism can be as simple as, for example, a single eukaryotic cell or as complex as a mammal.
  • enzyme activity refers to the ability of the enzyme to catalyze a chemical conversion or reaction.
  • An activity may be, but is not limited to, the deamination (removal of an amine group) from a cytidine moiety.
  • prophylactically effective amount refers to an amount effective to prevent a disease, illness, or sickness.
  • terapéuticaally effective amount refers to an amount effective to treat, cure, or ameliorate a disease, illness, or sickness.
  • pharmaceutically acceptable carrier refers to a diluent, adjuvant, excipient, or vehicle with which a heterodimeric probe of the disclosure is administered and which is approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
  • Such pharmaceutical carriers can be liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, and the like.
  • the pharmaceutical carriers can be saline, gum acacia, gelatin, starch paste, talc, keratin, colloidal silica, urea, and the like.
  • the heterodimeric probe and pharmaceutically acceptable carriers can be sterile.
  • Water is a useful carrier when the heterodimeric probe is administered intravenously.
  • Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
  • Suitable pharmaceutical carriers also include excipients such as glucose, lactose, sucrose, glycerol monostearate, sodium chloride, glycerol, propylene, glycol, water, ethanol, and the like.
  • the present compositions if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
  • compositions advantageously may take the form of solutions, emulsion, sustained-release formulations, or any other form suitable for use.
  • the BXD2 mouse model of generalized autoimmune disease exhibits high levels of autoantibodies with an age-dependent expansion of the autoantibody repertoire, high titers of circulating immune complexes, and the development of glomerulonephritis and spontaneous erosive arthritis (Mountz, et al., Scand. J. Immunol. 61 : 128-138 (2005)).
  • the autoantigens that are commonly recognized by these multireactive autoantibodies include histone, BiP (HSPa ⁇ or GRP78), nitrotyrosine (NT)-modified enolase, and DNA (Hsu, et al., Arthritis Rhem. 54: 343-355 (2006)).
  • Polyreactive autoantibodies isolated from human lupus and rheumatoid arthritis patients exhibited reactivity to lipopolysaccharide. Thomas Kayden Docket No.: 222104-2120
  • the autoantibodies develop in an orderly progression starting with autoantibodies directed against heat-shock proteins (HSPs) including BiP (HSPa5) and HSP47 in mice at 3 months to 4 months of age. Later, the antibodies recognize traditional autoantigen targets including RF, DNA, and histone (Hsu, et al., Arthritis Rheum. 54: 343-355 (2006)). Because of the high levels of somatic hypemnutation and class-switch recombination in pathogenic autoantibodies, the possible role of activation-induced cytidine deaminase (AID), which is an essential enzyme for both processes, was examined in B cells of BXD2 mice.
  • HSPs heat-shock proteins
  • AID activation-induced cytidine deaminase
  • AID was highly expressed in the spleenic germinal centers of BXD2 mice, and that there were increased numbers of spontaneous germinal centers in this mouse strain. Somatic hypermutation and class switch recombination of immunoglobulins are carried out by two separate regions of the 198 amino-acid long AID molecule.
  • the primary sequence of AID contains a nuclear localization signal domain (NLS) and a cytidine deaminase catalytic domain at the N-terminal and a nuclear export motif at the C-terminal site (Shinkura, et al., Nat. Immunol. 5: 707-12 (2004)).
  • AID further contains a conserved cytidine deaminase motif consisting of a histidine and two cysteine residues that coordinate Zn 2+ , together with a glutamate that serves as a proton donor in the deamination reaction (Scott, et al., Exp. Physiol. 84: 791-800 (1999)). Replacement of any of these four amino acids results in a catalytic mutant.
  • AID functions as a homodimer or trimer, and mutant forms of AID can exert dominant negative activity (Papavasiliou, et al., J. Exp. Med. 195: 1193-8 (2002)).
  • An AID variant mutated in two of the deaminase domain residues (H56R/E58Q) was expressed in the constitutively hypermutation human Burkitt lymphoma cancer cell line Ramos. This resulted in a 20-fold decrease in the mutation generating activity of AID, equivalent to that seen in cells lacking AID.
  • the H56R/E58Q mutation exhibited a dominant negative effect that overrides the somatic hypermutation function of AID.
  • Ser38 had been mutated to Ala, the AID was not phosphorylated by protein kinase A (PKA), and they failed to interact with RPA or to mediate deamination of DNA (Basu, et al., Nature 438: 508-11 (2005)).
  • PKA protein kinase A
  • AID class switch recombination activity can be effectively inhibited by treatment of Ramos B cells with a PKA inhibitor, H89, that blocks phosphorylation of AID (Basu, et al., Nature 438: 508-11 (2005)), but not by tetrahydrouridine (THU), a competitive inhibitor of cytidine deaminase that is known to block the somatic hypermutation activity of AID (Matsumoto, et al., Nature 382: 462-6 (1996); Teh, et al., Biochemistry 45: 7825-33 (2006)). Inhibition of AID suppresses autoimmune disease Targeted inhibition of AID can prevent the generation of pathogenic autoantibodies and autoimmune disease.
  • the pathogenic multireactive autoantibodies in BXD2 mice exhibit a 2-3-fold greater level of SHM as compared to the single-reactive non-pathogenic Thomas Kayden Docket No.: 222104-2120
  • Mutations of at least three different sites have been shown to disrupt the function of AID. These include the S38A mutation at the PKA phosphorylation site, the H56R/E58Q mutation at the AID catalytic domain, and the R190X mutation at the CSR domain. Among these, the H56R/E58Q mutation and the R190X mutation can act as dominant-negative mutations that disrupt the SHM and the CSR functions of AID, respectively. It also has been shown that the proper function of AID is highly dependent on the S38 PKA phosphorylation site in mice. Importantly, PKA is not essential for nuclear translocation, but is important for binding of AID to RPA in the nucleus. Defective binding of AID to RPA results in a dramatic suppression of both SHM and CSR.
  • AID-dominant negative transgenic mice Viable founder AID-dominant negative transgenic mice have been produced.
  • a transgenic BXD2 mouse strain expressing a dominant negative form of AID operably linked to the chicken albumin promoter (CAG) was generated, using the construct shown in Fig. 1.
  • This AID-dominant negative transgene contained the H56R/E58Q double mutation in the catalytic domain.
  • a third mutation, S38A, at the phosphorylation site for PKA was included in the AID-dominant negative construct of the transgenic mouse.
  • BXD2 AID-dominant negative transgenic mice Autoantibody levels were significantly suppressed in BXD2 AID-dominant negative transgenic mice at 6 months to 8 months age.
  • BXD2 AID-dominant negative transgenic mice developed minimal arthritis and kidney disease compared to age-matched wild-type BXD2 mice.
  • AID " ' " B6 mice that exhibited enlarged germinal centers during an immune response, there was a decreased number and size of the spontaneous germinal center in BXD2 AID-dominant negative transgenic* mice, and this was associated with an increased number of apoptotic cells in such germinal centers.
  • the AID-dominant negative transgene construct as shown in Fig 1 was inserted directly into BXD2 single-cell embryos
  • the CAG promoter enabled high expression of the AID-dominant negative in all tissues of transgenic mice
  • an AID-specific primer spans the AID-internal ribosomal entry site (IRES), a sequence not found in endogenous mouse AID
  • IRS AID-internal ribosomal entry site
  • Peripheral blood mononuclear cells from approximately 50% of offspring mice carried the AID-dominant negative transgene, as shown in Fig 2
  • There were nearly equivalent levels of wild-type AID expressed in the spleen of wild-type BXD2 see Fig 3, lanes 1 and 2) versus in the AID-dominant negative transgenic BXD2 mice (Fig 3, lanes 3-7)
  • AID-dominant negative transgenic BXD2 mice developed arthritis at the same age (Figs 6 and 13A). Arthritis in the wild-type BXD2 mice was characterized by lymphocytic infiltration, extensive synovial hyperplasia and marginal erosions (Figs. 6 and 13B). The marginal erosions are associated with osteoclast invasion of bone. In BXD2 AID-dominant negative transgenic mice, there is minimal synovial hyperplasia, and the synovial lining was 2 to 3 cells thick. However, there was minimal marginal erosion and no invasion of osteoclasts into bone in the AID-dominant negative BXD2 mouse (Figs. 6 and 13B).
  • BXD2 mice developed significant proteinuria which was significantly decreased in the AID-dominant negative transgenic BXD2 mouse (see Fig. 13C). Histological evaluation showed extensive mesangial proliferation and inflammatory cell infiltration in the glomeruli of wild-type BXD2 mice, which was significantly reduced in the AID-dominant negative transgenic BXD2 mouse (Fig. 7). There were also decreased IgM and IgG glomerular deposits in the AID-dominant negative transgenic BXD2 mouse compared to wild-type BXD2 mice, as shown in Fig. 7.
  • IgM-secreting B cells were somewhat lower in number, as shown in Fig. 11 , and IgG-secreting B cells were higher (shown in Fig. 12), in wild-type BXD2 mice compared to B6 mice.
  • the number of IgG-producing B cells was normalized in Thomas Kayden Docket No.: 222104-2120
  • FaS + GLT + germinal center B cells in the spleens of AID-dominant negative transgenic mice by FACS showed that there was a decreased percentage of germinal center B cells (see Fig. 16) that correlated to a significant decrease in the percentage of BrdlT cycling B cells and cyclining PNA + FaS + germinal center B cells in vivo in the spleen of BXD2 AID-dominant negative transgenic mice (see Fig. 17). Consistent with this, there was a significant decrease in Ki67 staining of follicles in the spleens of BXD2 AID-dominant negative transgenic mice, as shown in Fig. 18.
  • Partial suppression, but not a total deficiency, of AID activity may be associated with a decreased expression of DNA repair genes, and an increase in apoptosis of germinal center B cells in BXD2 mice. While not wishing to be bound by any one theory, if upregulation of DNA repair genes is a consequence of AID-mediated DNA damage, temporal suppression of the AID somatic hypermutation function might be a similar. To test this, 8 month to 10 month old BXD2 mice were treated with either tetrahydrouridine (THU) (1.0 mg/mouse, intraperitoneal ⁇ , twice per week) or with a control vehicle (phosphate buffered saline).
  • TNU tetrahydrouridine
  • THU binds to the catalytic domain of AID and blocks the catalytic activity of AID.
  • all BXD2 mice were pre-screened for sera titers of autoantibodies to ensure that equivalent levels of autoantibodies could be detected from both groups of mice, as shown in Fig. 20.
  • THU exhibited a strong inhibitory effect on sustaining germinal center B cells
  • a long-term treatment of THU to BXD2 mice before the onset of spontaneous germinal centers and autoantibody production was conducted to determine if THU could inhibit AID activity, and thereby prevent the development of autoimmune disease in BXD2 mice, as shown in Fig. 25.
  • THU was administered to BXD2 mice at 4 weeks of age (1.0 mg in PBS per mouse, twice per week), and mice were evaluated at 5 months of age. There was no significant difference in total sera IgM and IgG in vehicle and THU-treated BXD2 mice at 5 months of age. There was, however, a significant decrease in both IgM and IgG anti-histone and anti-BiP in THU-treated 5 month-old BXD2 mice compared to vehicle-treated BXD2 mice (see Fig. 25).
  • THU treatment of wild-type BXD2 mice prevented the development of arthritis, as shown in Fig. 26. Histologically, there was a decrease in synovial hyperplasia and no evidence of marginal erosion in THU-treated wild-type BXD2 mice (Fig. 26). There was a decrease in the IgG immune deposition in the glomerulus of the THU-treated BXD2 mice compared to control vehicle-treated BXD2 mice (Fig. 26). These results indicate that THU specifically inhibits AID, has a profound negative impact on autoantibody production, and suppresses the development of autoimmune disease in BXD2 mice.
  • useful inhibitors such as, but not limited to, zebularine (2-pyrimidone-1- ⁇ -D-riboside; 1-( ⁇ -D-ribofuranosyl)-1 ,2-dihydropyrimidin-2-one; Zeb), 4- hydroxy-3,4-dihydro-1 H-pyridmidin-2-one (HPY), dihydropyrimidine-2,4(1 H,3H)-dione (5,6- dihydrouracil), tetrahedral intermediate of blasticidin (BST), 3,4-dihydro-2p-deoxyuridine-5p monophosphate (DDN), 3,4-dihydrozebularine (DHZ), 1-beta-ribofuranosyl-1 ,3-diazepinone (BRD), and 3-deazacytidine (CTD), and the like, may be considered to have similar effects on the autoimmune system of an animal as does THU (tetrahydrouridine (1-( ⁇ -
  • AID activity is essential for the immunoglobulin gene to undergo somatic hypermutation and class switch recombination, leading to production of T-dependent antibody responses.
  • AID introduces point mutations or S region DNA double strain breaks of the immunoglobulin genes, respectively.
  • AID is up-regulated in T1 and follicular B cell subsets that form the precursors to the germinal center B cells that interact with CD4 T cells.
  • an AID-dominant negative transgenic BXD2 mouse was generated that exhibited somatic hypermutation AID-dominant negative mutations to suppress wild-type-AID somatic hypermutation function and an AID phosphorylation mutation to silent the class switch recombination function of the AID-dominant negative transgenic.
  • the results as described above, and in the following Examples, show that the hyperactivity of AID in germinal center B cells is significant for the induction of autoimmune disease in BXD2 mice. Suppression of the function of, but not the expression levels of, AID prevented the production of pathogenic autoantibodies and ameliorated the development of autoimmune joint and kidney disease.
  • a second possible mechanism is associated with an increase in apoptosis of their germinal centers.
  • Determination of the expression of DNA damage and DNA repair genes in B cells of wild-type and AID-dominant negative transgenic BXD2 mice showed that numerous DNA damage sensor genes, including Atm, Brcai, Gadd45b, Rad51, and Rad54 were dramatically up-regulated in germinal center B cells compared to non-germinal center B cells from both strains of mice. This suggests that there was a robust DNA damage response, most likely induced by AID-induced mutations in germinal center B cells.
  • AID- dominant negative mutations partially suppressed AID-induced damage, but there was residual AID somatic hypermutation and class switch recombination activity existing in germinal center B cells of AID-dominant negative transgenic mice. This is further supported by the higher titers of auto-antibodies and the higher number of autoantibody-producing B cells in BXD2 AID-dominant negative transgenic mice, compared to normal B6 AID " ' " mice.
  • AID-mediated DNA strand breaks might up-regulate numerous DNA repair genes other than the commonly known endonucleases and exonucleases that are involved in repairing DNA strand breaks induced by AID. These include BIm, Fanca, Xrcrf, and Xrcc3.
  • the specificity of AID to up-regulate DNA repair genes in germinal center B cells may be significant since dark-zone germinal center B cells that express AID must undergo dramatic proliferation. Many of the cell cycle genes exhibited many fold higher levels in germinal center versus non-germinal center B cells in BXD2 mice.
  • BXD2 AID-dominant negative transgenic mice germinal centers of BXD2 AID-dominant negative transgenic mice.
  • AID as a mutator, has been long thought to be a major tumor inducer for B-lymphoma development, there is a strong protection mechanism in germinal center B cells of BXD2 mice.
  • the product of this strong repair mechanism in germinal centers of BXD2 mice is the rise of B cells that produce pathogenic autoantibodies.
  • the present disclosure provides data showing the inhibition of the somatic hypermutation function of AID in vivo in BXD2 mice as carried out pharmacologically or by genetic manipulation.
  • the present disclosure therefore, encompasses methods of genetic and pharmacologic inhibition of the activity of AID that can suppress the development of autoimmune disease in BXD2 mice.
  • One aspect of the disclosure provides methods for inhibiting the generation of an autoantibody in a subject animal, comprising reducing the level of activity of an activation-induced cytidine deaminase in a subject animal, thereby reducing the generation of an autoantibody in the subject animal.
  • the methods may comprise administering to a subject animal an effective amount of a therapeutic composition comprising an activation-induced cytidine deaminase inhibitor compound, whereby the inhibitor reduces the activity of the activation-induced cytidine deaminase in generating somatic hypermutations and/or class switch recombination of an immunoglobulin, thereby reducing the generation of an autoantibody in the subject animal.
  • the methods for inhibiting the generation of an autoantibody further reduces the level of an autoimmune disease in the subject animal. Thomas Kayden Docket No.: 222104-2120
  • the effective amount of the composition administered to the subject animal may be a prophylactically effective amount of the composition.
  • the effective amount of the composition administered to the subject animal is a therapeutically effective amount of the composition.
  • the methods comprise administering to a subject animal an effective amount of a therapeutic composition comprising an activation- induced cytidine deaminase inhibitor.
  • useful inhibitors may be, for example, such as, but not limited to, selected from the group consisting of: zebularine (2- pyrimidone-1- ⁇ -D-riboside 1-( ⁇ -D-ribofuranosyl)-1,2-dihydropyrimidin-2-one; ZEB); tetrahydrouridine (1-(-D-ribofuranosyl)-4-hydroxytetrahydro-1(1 H)-pyrimidinone; THU), 4- hydroxy-3,4-dihydro-1 H-pyridmidin-2-one (HPY), dihydropyrimidine-2,4(1 H,3H)-dione (5,6- dihydrouracil), tetrahedral intermediate of blasticidin (BST), 3,4-dihydro-2p-deoxyuridine-5p monophosphate (DDN), 3,4-dihydrozebularine (DHZ), 1-beta-ribofuranosyl-1 ,3-diazepin
  • the inhibitor of the activation-induced cytidine deaminase activity is tetrahydrouridine (1-(-D-ribofuranosyl)-4-hydroxytetrahydro-1 (1H)- pyrimidinone).
  • inhibition of an activation-induced cytidine deaminase may inhibit an autoimmune disease.
  • the autoimmune disease may be, but is not limited to, an autoimmune disease selected from the group consisting of: systemic lupus erythematosus, rheumatoid arthritis, spontaneous erosive arthritis, and glomerulonephritis.
  • the therapeutic composition may comprise the activation-induced cytidine deaminase inhibitor and a pharmaceutically acceptable carrier.
  • the method for inhibiting the generation of an autoantibody in a subject animal may comprise reducing the level of activity of an activation-induced cytidine deaminase in a subject animal by delivering to a cell or tissue of the subject animal a heterologous nucleic acid expression construct comprising a nucleic acid region encoding a dominant-negative variant of an activation-induced cytidine deaminase, whereby the variant activation-induced cytidine deaminase in the cell or tissue of the subject animal reduces the activity of the cytidine deaminase in generating somatic hypermutations and/or class switch recombination of an immunoglobulin or plurality of immunoglobulins, thereby reducing the generation of an autoantibody in the subject animal.
  • transgenic animal comprising a heterologous nucleic acid construct encoding a dominant negative variant of an activation- Thomas Kayden Docket No.: 222104-2120
  • induced cytidine deaminase whereby the variant activation-induced cytidine deaminase, when expressed in a cell or tissue of the transgenic animal, reduces the activity of an endogenous activation-induced cytidine deaminase.
  • the variant activation-induced cytidine deaminase when expressed in a cell or tissue of the transgenic animal, reduces the level of production of an autoantibody in the subject animal.
  • the heterologous nucleic acid construct encoding a dominant negative variant of an activation-induced cytidine deaminase may be operably linked to an inducible expression control region.
  • the AID gene was obtained by TA cloning of the murine AID gene.
  • the full length gene was obtained using the forward primer S'-AGAAAGTCACGCTGGAGACC-S' (SEQ ID NO.: 1) and the reverse primer 5'-ATGTTGCACAGCAAGCTCAG-3' (SEQ ID NO.: 2).
  • the proper function of AID was highly dependent on PKA phosphorylation sites T27 and S38 (mouse), dimerization sites R50 and N51 , catalytic domain sites H56 and E58, and the 3 prime region which can be mutated by introducing a premature stop codon at R190 Thomas Kayden Docket No.: 222104-2120
  • mutations were introduced into the catalytic domain at the amino acid positions: histidine 56 (CAC) to arginine 56 (CGC)/glutamic acid 58 (GAA) to glutamine 58 (CAA) (H56R/E58Q) using the forward primer ⁇ '-ACAAGTCTGGCTGCCGCGTGCAATTGTTGTT-S' (SEQ ID NO.: 3), and the reverse primer 5'-GGCAGCCAGACTTGTTGCGAAGGTGGCC-S' (SEQ ID NO.: 4).
  • the PCR product was inserted into the Sacll and BamH1 restriction enzyme sites in the plRES2-AcGFP1 vector (Clontech), which contains the internal ribosome entry site (IRES) of the encephalomyocarditis virus (ECMV) located between the multiple cloning site (MCS) and the Aequorea coerulescens green fluorescent protein (AcGFPI ) coding region.
  • IRS internal ribosome entry site
  • ECMV encephalomyocarditis virus
  • AcGFPI Aequorea coerulescens green fluorescent protein
  • the chicken ⁇ actin (CAG) promoter was digested using SnaB1 and Xba1 followed by treatment with Klenow enzyme to yield blunt ends.
  • the vector having the AID-dominant negative mutant located between Sacll and BamH1 sites was digested using Nhe1 and Sail followed by treatment with Klenow enzyme to produce blunt ends.
  • the vector was dephosphorylated using ANTARCTICTM (NEB, Inc) phosphatase, and ligated to the blunt ended CAG promoter. Following transformation, colonies were screened for clones having the proper orientation of the CAG promoter using Eco47lll and BamH1 digestion. The final transgene was obtained by digestion of the construct using SnaB1 , Not1 , and Stu1 triple digestion.
  • Example 2 Treatment of mice with THU
  • mice Four-week-old or eight to ten-month-old female BXD2 mice were treated twice per week for 4 months, or 3 weeks, respectively, with control PBS or THU (EMD Chemicals, Inc., Gibbstown, NJ) (1.0 mg/mouse per dose intraperitoneally). Twenty-four hours after the last injection, the mice were sacrificed for the experiments. Thomas Kayden Docket No.: 222104-2120
  • mice were immunized intraperitoneally with 50 ⁇ g of chicken gammaglobulin haptenated with 4-hydroxy-3-nitrophenylacetyl (NP-CGG) (BioSearch Technologies) and adsorbed to 1.3 mg alum (Sigma-Aldrich) in a total volume of 100 ⁇ l PBS. Sera were collected at the indicated times (before immunization, and after days 7, 14, and 21).
  • NP-CGG 4-hydroxy-3-nitrophenylacetyl
  • High affinity anti-NP and total anti-NP lgG2b and lgG2c antibodies were measured by ELISA using a NP-bovine serum albumin conjugate (using either NP 2 -BSA, a low hapten density to detect high affinity anti-NP antibodies, or NP 24 -BSA, a high hapten density to detect both low and high affinity anti-NP antibodies) as the target antigens.
  • lgG2b or lgG2c antibodies bound to the plate were detected with an horse radish peroxidase (HRP)- conjugated goat anti-mouse lgG2c antibody, or an HRP-conjugated goat anti-mouse lgG2b (Southern Biotech). 3,3',5,5'-tetramethylbenzidine (TMB) (Sigma) was used as the substrate.
  • the OD 450 was measured on an Emax Micro-plate reader.
  • RNA samples were coated with 5 ⁇ g/ml of the test autoantigen.
  • BiP Assay Designs, Inc.
  • bovine type Il collagen Chondrex, Inc.
  • the assays were developed with an HRP-labeled isotype-specific goat anti-mouse antibody (Southern Biotechnology Associates) and 3,3',5,5'-tetramethylbenzidine substrate (Sigma-Aldrich).
  • the OD 450 was measured using an Emax Microplate reader.
  • RNA samples from the sorted cells were isolated using the PicoPure RNA isolation kit (Arcturus/Molecular Devices, Sunnyvale, CA). The quality of RNA was confirmed using the Bio-Rad EXPERIONTM method. Each RNA sample was prepared as described above and analyzed using a Mouse Expression Array 430A set (Affimatrix).
  • Transgenic AID specific primers were: transgenic AID (Forward) 5'- CCTCCTGCTCACTGGACTTC-3' (SEQ ID NO.: 7); transgenic AID (Reverse) 5'- CCAAAAGACGGCAATATGGT-3' (SEQ ID NO.: 8) and expected products were a 603 bp fragment for transgenic-IRES AID; 1050 bp for wt AID.
  • wild-type-AID specific primers were designed at the 3' untranslated region of wild-type-AID (Sense: ⁇ '-TTGGGTCGTGAATGATGC-S' (SEQ ID NO.: 9); anti-sense: ⁇ '-CCTGAAAGTGAGCCTTAGAG-S' (SEQ ID NO.: 10) resulting in a 162 bp product.
  • AID-dominant negative specific transcript AID specific primers were designed within the transgene GFP (sense: 5'-
  • Example 8 lmmunohistochemical analysis of tissue sections The tissues were fixed in 10% formaldehyde/PBS and paraffin-embedded. Sections
  • TUNEL staining for the detection of apoptotic cells on tissue sections was carried out using the ApopTag Peroxidase in situ Apoptosis Detection Kit (S7100) according to the manusfacture's instructions (Chemicon/Millipore, Billerica, MA).
  • S7100 ApopTag Peroxidase in situ Apoptosis Detection Kit
  • APC-anti-BrdU antibody was from BD-PharMingen (San Diego, CA).
  • Biotin- conjugated anti-mouse Ly77 (clone GL7) was from eBioscience (San Diego, CA).
  • Biotinylated PNA was from Vector Laboratories (Burlingame, CA).
  • Phycoerythrin (PE) conjugated anti-Fas (clone Jo2)
  • APC- or APC/Cy7-conjugated anti-mouse B220 (clone rheumatoid arthritis3-6B2)
  • PE-Cy5 conjugated CD19 (clone 6D5)
  • PE-Cy5 conjugated streptavidin SA were from Biolegend (San Diego, CA).
  • B cells were enriched from single-cell spleen preparations using a CD19 positive selection column (Miltenyi Biotech). The enriched B cells were then prepared for FACS staining using an APC-anti-B220, PE-anti-mouse Fas, and biotin-PNA detected by a PE-Cy5 conjugated streptavidin. After cell surface staining, cells were either washed with PBS for FACS sorting of different subpopulations of B cells, or washed twice with FACS buffer.
  • mice Female homozygous C57BL/6 and BXD2 recombinant inbred strains of mice were obtained from The Jackson Laboratory (Bar Harbor, ME). The BXD2 female mice, age 20 to 25 days, were super-ovulated with 50 units of human chorionic gonadotropin (hCG), and subsequently with 100 units of pregnant mare serum (PMS) at day 3 and day 1 relative to Thomas Kayden Docket No.: 222104-2120
  • Fig. 2 Shown in Fig. 2 is the result of an RT-PCR tail RNA analysis of the presence of an
  • AID-dominant negative transgene in BXD2 mice was carried out using a GFP and an AID-IRES specific primer pair to determine the expression of AID-dominant negative in transgenic 4 mice.
  • RT-PCR and real-time PCR quantitation of the expression of wild- type-AID was carried out using a primer pair that amplified the untranslated region of wild- type-AID that is not included in the AID-dominant negative transgenic.
  • RT-PCR and realtime PCR quantitation of the expression of AID-dominant negative was carried out using a primer pair that amplified the AID-IRES portion of the AID-dominant negative transgenic (*** P ⁇ 0.0001 ; N > 6 per group).
  • Shown in Figs. 4A and 4B are the results of ELISA analysis of the low affinity anti-
  • Example 12 Decreased autoimmune disease in BXD2 AID-dominant negative transgenic mice
  • Fig. 5A Shown in Fig. 5A is an ELISA analysis of circulating autoantibodies in the sera of the mice (* P ⁇ 0.05; * P ⁇ 0.01 ; N ⁇ 6 per group).
  • Fig. 5B shown is an ELISA analysis of C1q containing immune complexes in the sera ( * P ⁇ 0.05; * P ⁇ 0.01 ; N > 6 per group).
  • FIG. 6 Shown in Fig. 6 is an image of hematoxylin/eosin stained joint sections in wild-type- BXD2 and BXD2 AID-dominant negative transgenic mice, lmmunohistochemistry staining of IgM and IgG antibody deposits on the glomerulus of wild-type-BXD2 and BXD2 AID- dominant negative transgenic mice is shown in Fig. 7.
  • the objective lens used for acquiring each image is indicated.
  • the photomicrographs shown for Figs. 6 and 7 are representative of the results obtained on analysis of tissue sections from at least 6 BXD2 and 6 age- matched BXD2 AID-dominant negative transgenic mice. Thomas Kayden Docket No.: 222104-2120
  • Germinal centers were characterized in wild-type and AID-dominant negative transgenic mice at 6 months to 8 months of age. Shown in Fig. 14 is the measurement of spleen size as determined by weight (mg) (left panel) and cell count (right panel) in wild-type and AID-dominant negative transgenic mice.
  • a FACS analysis of total CD19 + B cells and CD4 T cells in wild-type and AID-dominant negative transgenic BXD2 mice is shown in Fig. 15, whereas a FACS analysis of percent of Fas + GL-7 + B220 + GC B cells in wild-type and AID-dominant negative BXD2 mice is shown in Fig. 16. In Fig.
  • FIG. 17 is graphically illustrated the proliferation of CD19 + B cells and PNA + FaS + GC B cells in wild-type and AID-dominant negative BXD2 mice, as determined by BrdU incorporation.
  • Fig. 18 shows immunohistological analyses of Ki67 + cells in the spleens of wild-type and AID-dominant negative transgenic BXD2 mice.
  • Fig. 19 is shown the quantitation of the results of the number of Ki67+ follicles and the number Of TUNEL+ apoptotic cells per follicle of the spleens of wild-type and AID-dominant negative transgenic BXD2 mice. (* P ⁇ 0.05; *** P ⁇ 0.005; N > 6 per group).
  • Example 14 Effects of THU on germinal center gene expression in BXD2 mice
  • Fig. 20 illustrates the results of an ELISA analysis of IgM and IgG autoantibodies against BiP, histone and DNA in BXD2 mice before vehicle (PBS) or THU treatment.
  • Fig. 21 shows the change in spleen weight and spleen cell count of BXD2 mice from THU treatment.
  • Fig. 22 is shown the results of a FACS analysis of the percent of germinal center B cells and proliferation of B cells in the spleens of BXD2 mice 3 weeks after treatment with THU or vehicle controls.
  • Figs. 23 and 24 are photographs of immunohistochemistry staining of Ki67 (Fig. 23) and TUNEL (Fig.
  • Fig. 27 illustrates a cluster analysis of two separates of non- germinal center (N-1 b and N-2a), wild-type germinal center (P-1 and P-2), and AID-dominant negative germinal center (P-3 and P4).
  • Fig. 28 is shown a cluster analysis of DNA damage and apoptosis genes of two separates of non- germinal center (N-1 b and N-2a), wild-type germinal center (P-1 and P-2), and AID-dominant negative germinal center (P-3 and P4).
  • Figs. 29 and 30 show the quantitative-real-time PCR analysis for the expression of DNA damage (Fig. 29) and DNA repair genes (Fig. 30).
  • AID can be localized to the cytoplasm and the nucleus in CD20 + B cells of autoimmune patients (Figs. 33A and 33B). Expression of AID occurred in approximately one-third of the CD20 + B cells in the germinal centers of Hashimoto's thyroiditis. AID + cells were also detected in ectopic germinal center in rheumatoid arthritis synovium (Fig. 35, right panels). In all situations, AID + cells are located in the germinal center B cell area, but not in the T-cell zone (Fig 35).
  • AID AID in PBMC obtained from 12 rheumatoid arthritis (rheumatoid arthritis), 8 SLE, and 6 non-autoimmune subjects.
  • the B cells were stained with different cell surface markers to enable differentiation of new emigrant B cells (CD20 + , CDIO + , IgM + ) and mature naive B cells (CD20 + , CD10 " , IgM + ).
  • the expression of AID was then determined within either the new emigrant compartment or the mature na ⁇ ve compartment of B cells. The percentage of new emigrant of B cells was higher in both rheumatoid arthritis and SLE patients than in normal controls (Fig. 32A).
  • AID The expression of AID was 3 to 4-fold higher in the newly emigrant B cells from rheumatoid arthritis and SLE patients than the expression of AID in these cells in normal controls (Fig. 32B). In all subjects, AID expression was down-modulated in the mature na ⁇ ve compartment of B cells, although the percentage of AID + B cells remained higher in this population of B cells from autoimmune patients than that from normal controls (Fig. 32C). Rheumatoid arthritis patients had higher RF and anti- DNA, and lupus patients have higher anti-DNA, than normal controls.

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Abstract

La présente invention a pour objet des méthodes de réduction ou d’élimination de la formation d’auto-anticorps chez un animal hôte par inhibition de l’activité de la cytidine désaminase endogène induite par activation (AID). La présente invention concerne également des animaux transgéniques. L’inhibition génétique et pharmacologique de l’AID a entraîné la diminution de la production d’auto-anticorps et la répression de l’arthrite et de la maladie rénale dans le modèle de souris, indiquant la contribution importante de l’AID dans les maladies auto-immunes. La description a pour objet des méthodes qui comprennent l’administration à un animal sujet d’une quantité efficace d’une composition thérapeutique renfermant un composé inhibiteur de la cytidine désaminase induite par activation. Dans un mode de réalisation de la description, l’inhibiteur de l’activité de la cytidine désaminase induite par activation est la tétrahydro-uridine-(1-(-D-ribofuranosyl)- 4-hydroxytétrahydro-1-(1H)-pyrimidinone).
PCT/US2008/080741 2008-10-22 2008-10-22 Répression de maladies auto-immunes par un inhibiteur de la cytidine désaminase induite par activation WO2010047698A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8268800B2 (en) 2007-10-16 2012-09-18 Eisai Inc. Certain compounds, compositions and methods
US8324180B2 (en) 2009-04-06 2012-12-04 Eisai Inc. Compositions and methods for treating cancer
US8329666B2 (en) 2009-04-06 2012-12-11 Eisai Inc. Compositions and methods for treating cancer
US8329665B2 (en) 2009-04-06 2012-12-11 Eisai Inc. Compositions and methods for treating cancer
US8609631B2 (en) 2009-04-06 2013-12-17 Eisai Inc. Compositions and methods for treating cancer

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6723533B2 (en) * 2000-03-10 2004-04-20 Millennium Pharmaceuticals, Inc. 26934, a novel cytidine deaminase-like molecule and uses thereof

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6723533B2 (en) * 2000-03-10 2004-04-20 Millennium Pharmaceuticals, Inc. 26934, a novel cytidine deaminase-like molecule and uses thereof

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HSU, H.C. ET AL.: "INHIBITION OF ACTIVATION-INDUCED CYTIDINE DEAMINASE (AID) PRESERVED SPONTANEOUS GERMINAL CENTERS BUT SUPPRESSED AUTOIMMUNE DISEASE IN BXD2 MICE.", THE FASEB JOURNAL, vol. 22, April 2008 (2008-04-01) *
HSU, H.C. ET AL.: "OVEREXPRESSION OF ACTIVATION-INDUCED CYTIDINE DEAMINASE IN B CELLS IS ASSOCIATED WITH PRODCUTION OF HIGHLY PATHOGENIC AUTOANTIBODIES", THE JOURNAL OF IMMUNOLOGY, vol. 178, no. 8, 2007, pages 5357 - 5365 *
MUTO, T. ET AL.: "NEGATIVE REGULATION OF ACTIVATION-INDUCED CYTIDINE DEAMINASE IN B CELLS.", PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES, vol. 103, no. 8, 2006, pages 2752 - 2757 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8268800B2 (en) 2007-10-16 2012-09-18 Eisai Inc. Certain compounds, compositions and methods
US8618075B2 (en) 2007-10-16 2013-12-31 Eisai Inc. Certain compounds, compositions and methods
US8951987B2 (en) 2007-10-16 2015-02-10 Otsuka Pharmaceuticals Co., Ltd. Certain compounds, compositions and methods
US9567363B2 (en) 2007-10-16 2017-02-14 Otsuka Pharmaceutical Co., Ltd. Certain compounds, compositions and methods
US8324180B2 (en) 2009-04-06 2012-12-04 Eisai Inc. Compositions and methods for treating cancer
US8329666B2 (en) 2009-04-06 2012-12-11 Eisai Inc. Compositions and methods for treating cancer
US8329665B2 (en) 2009-04-06 2012-12-11 Eisai Inc. Compositions and methods for treating cancer
US8609631B2 (en) 2009-04-06 2013-12-17 Eisai Inc. Compositions and methods for treating cancer
US9040501B2 (en) 2009-04-06 2015-05-26 Otsuka Pharmaceutical Co., Ltd. Compositions and methods for treating cancer

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