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Title:  Truncated CRAF1 inhibits CD40 signaling

United States Patent:  6,822,070

Issued:  November 23, 2004

Inventors:  Baltimore; David (508 Union Wharf, Boston, MA 02109); Cheng; Genhong (827 Leverin Ave., Apt. 304, Los Angeles, CA 90024); Ye; Zheng-Sheng (1233 York Ave., New York, NY 10021); Lederman; Seth (533 W. 112th St., Apt. 8C, New York, NY 10025); Cleary; Aileen (60 Haven Ave., New York, NY 10032)

Appl. No.:  813323

Filed:  March 10, 1997

Abstract

Overexpression of a CRAF1 (CD40 receptor-associated factor 1) gene truncated by 323 to about 414 amino acids at the amino inhibits CD40-mediated cell activation, and is used to treat conditions characterized by an unwanted level of CD40-mediated intracellular signaling.

BACKGROUND OF THE INVENTION

CD40 (1) is a receptor on B cells that interacts with the helper T cell surface protein CD40L (CD40 ligand, also known as T-BAM, gp39, or TRAP) (2-4). CD40L is found particularly on lymphoid follicle CD4+ T lymphocytes, where it delivers a contact-dependent signal that stimulates B cell survival, growth, and differentiation (2-4). Signaling through CD40 rescues B cells from apoptosis induced by Fas (CD95) or by cross-linking of the immunoglobulin M (IgM) complex (5); it also induces B cells to differentiate and to undergo Ig isotype switching (3) and to express CD80 (B7 or BB-1) (6). The crucial role of CD40L-CD40 interaction is illustrated by humans with defects in CD40L, who manifest a serious immune deficiency syndrome, the X-linked hyper-IgM syndrome (HIGMX-1) characterized by an absence of IgG, IgA, and IgE, elevated IgM, and no lymphoid follicles (7). The essential roles of CD40L and CD40 in the phenotype of HIGMX-1 syndrome has been confirmed by targeted disruption of either CD40L (8) or CD40 (9) in mice. In addition to B cells, CD40 is also expressed by follicular dendritic cells (10), dendritic cells (11), activated macrophages (12), epithelial cells (including thymic epithelium) (13), and a variety of tumor cells.

Stimulation of CD40 causes the tyrosine phosphorylation of multiple substrates including Src family kinases such as p53-p56lyn, activates multiple serine-threonine-specific protein kinases, and induces the phosphorylation of phospholipase C-.gamma.2 and of phosphoinositide-3' kinase (14).

In mice the CD40 cytoplasmic tail is necessary for signaling (15). Proteins which interact with the cytoplasmic tail of CD40 have been described (H. M. Hu, et al., J. Biol. Chem. 269: 30069 (1994); and G. Mosialos, et al., Cell 80:389 (1995)). These proteins are the same as CRAF1.

SUMMARY OF THE INVENTION

This invention provides a protein comprising CRAF1 truncated by from about 323 to about 414 amino acid residues at the amino terminus, or a variant thereof capable of inhibiting CD40-mediated cell activation.

This invention provides a method of inhibiting activation by CD40 ligand of cells bearing CD40 on the cell surface, comprising providing the cells with an agent capable of inhibiting CD40-mediated intracellular signaling, the agent being present in an amount effective to inhibit activation of the cells.

This invention provides a method of providing a subject with an amount of a protein comprising CRAF1 truncated by from about 323 to about 414 amino acid residues at the amino terminus, or a variant thereof effective to inhibit activation by CD40 ligand of cells bearing CD40 on the cell surface in the subject, comprising: introducing into CD40-bearing cells of the subject, a nucleic acid sequence encoding the protein under conditions such that the cells express in the subject an activation inhibiting effective amount of the protein.

This invention provides a method of treating a condition characterized by an aberrant or unwanted level of CD40-mediated intracellular signaling, in a subject, comprising providing the subject with a therapeutically effective amount of an agent capable of inhibiting CD40-mediated intracellular signaling in cells bearing CD40 on the cell surface.

This invention provides a nucleic acid molecule encoding a protein comprising CRAF1 truncated by from about 323 to about 414 amino acid residues at the amino terminus, or a variant thereof capable of inhibiting CD40-mediated cell activation.

This invention provides a method of identifying an agent capable of inhibiting CD40-mediated intracellular signaling in a cell expressing CD40 on the cell surface, comprising providing the cell with the agent under conditions permitting activation of the cell in the absence of the agent, and determining decreased or absent activation, thereby identifying an agent capable of inhibiting CD40-mediated intracellular signaling in a cell expressing CD40 on the cell surface.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides a protein comprising CRAF1 truncated by from about 323 to about 414 amino acid residues at the amino terminus, or a variant thereof capable of inhibiting CD40-mediated cell activation. In an embodiment the variant comprises a conservative amino acid substitution.

Variants can differ from naturally occurring CD40 or CD40 ligand in amino acid sequence or in ways that do not involve sequence, or both. Variants in amino acid sequence are produced when one or more amino acids in naturally occurring CD40 or CD40 ligand is substituted with a different natural amino acid, an amino acid derivative or non-native amino acid. When a nucleic acid molecule encoding the protein is expressed in a cell, one naturally occurring amino acid will generally be substituted for another. Conservative substitutions typically include the substitution of one amino acid for another with similar characteristics such as substitutions within the following groups: valine, glycine; glycine, alanine; valine, isoleucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. The non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid.

Other conservative substitutions can be taken from Table 1, and yet others are described by Dayhoff in the Atlas of Protein Sequence and Structure (1988).

                             TABLE 1
               Conservative Amino Acid Replacements
    For Amino Acid     Code   Replace with any of
    Alanine              A    D-Ala, Gly, beta-ALa, L-Cys, D-Cys
    Arginine             R    D-Arg, Lys, homo-Arg, D-homo-
                              Arg, Met, D-Met, Ile, D-Ile,
                              Orn, D-Orn
    Asparagine           N    D-Asn, Asp, D-Asp, Glu, D-Glu,
                              Gln, D-Gln
    Aspartic Acid        D    D-Asp, D-Asn, Asn, Glu, D-Glu,
                              Gln, D-Gln
    Cysteine             C    D-Cys, S-Me-Cys, Met, D-Met, Thr,
                              D-Thr
    Glutamine            Q    D-Gln, Asn, D-Asn, Glu, D-Glu, Asp,
                              D-Asp
    Glutamic Acid        E    D-Glu, D-Asp, Asp, Asn, D-Asn,
                              Gln, D-Gln
    Glycine              G    Ala, D-Ala, Pro, D-Pro, Beta-Ala, Acp
    Isoleucine           I    D-Ile, Val, D-Val, Leu, D-Leu,
                              Met, D-Met
    Leucine              L    D-Leu, Val, D-Val, Met, D-Met
    Lysine               K    D-Lys, Arg, D-Arg, homo-Arg, D-
                              homo-Arg, Met, D-Met, Ile, D-
                              Ile, Orn, D-Orn
    Methionine           M    D-Met, S-Me-Cys, Ile, D-Ile,
                              Leu, D-Leu, Val, D-Val, Norleu
    Phenylalanine        F    D-Phe, Tyr, D-Thr, L-Dopa, His, D-
                              His, Trp, D-Trp, Trans 3,4 or
                              5-phenylproline, cis 3,4 or 5
                              phenylproline
    Proline              P    D-Pro, L-I-thioazolidine-4-
                              carboxylic acid, D- or L-1-
                              oxazolidine-4-carboxylic acid
    Serine               S    D-Ser, Thr, D-Thr, allo-Thr,
                              Met, D-Met, Met(O), D-Met(O),
                              Val, D-Val
    Threonine            T    D-Thr, Ser, D-Ser, allo-Thr,
                              Met, D-Met, Met(O) D-Met(O),
                              Val, D-Val
    Tyrosine             Y    D-Tyr,Phe, D-Phe, L-Dopa,
                              His,D-His
    Valine               V    D-Val, Leu,D-Leu,Ile,D-Ile,
                              Met, D-Met

Other variants within the invention are those with modifications which increase peptide stability. Such variants may contain, for example, one or more non-peptide bonds (which replace the peptide bonds) in the peptide sequence. Also included are: variants that include residues other than naturally occurring L-amino acids, such as D-amino acids or non-naturally occurring or synthetic amino acids such as beta or gamma amino acids and cyclic variants. Incorporation of D- instead of L-amino acids into the polypeptide may increase its resistance to proteases. See, e.g., U.S. Pat. No. 5,219,990.

The protein of this invention may also be modified by various changes such as insertions, deletions and substitutions, either conservative or non-conservative where such changes might provide for certain advantages in their use.

In other embodiments, variants with amino acid substitutions which are less conservative may also result in desired derivatives, e.g., by causing changes in charge, conformation and other biological properties. Such substitutions would include for example, substitution of hydrophilic residue for a hydrophobic residue, substitution of a cysteine or proline for another residue, substitution of a residue having a small side chain for a residue having a bulky side chain or substitution of a residue having a net positive charge for a residue having a net negative charge. When the result of a given substitution cannot be predicted with certainty, the derivatives may be readily assayed according to the methods disclosed herein to determine the presence or absence of the desired characteristics.

Variants within the scope of the invention include proteins and peptides with amino acid sequences having at least eighty percent homology with the COOH-terminal domain of CRAF1 (corresponding roughly to residues 415-567 (SEQ ID NO:12)) or with C26 (residues 324-567 of CRAF1 (SEQ ID NO:11)). More preferably the sequence homology is at least ninety percent, or at least ninety-five percent.

Just as it is possible to replace substituents of the scaffold, it is also possible to substitute functional groups which decorate the scaffold with groups characterized by similar features. These substitutions will initially be conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group. Non-sequence modifications may include, for example, in vivo or in vitro chemical derivatization of portions of the protein of this invention, as well as changes in acetylation, methylation, phosphorylation, carboxylation or glycosylation.

In a further embodiment the protein is modified by chemical modifications in which activity is preserved. For example, the proteins may be amidated, sulfated, singly or multiply halogenated, alkylated, carboxylated, or phosphorylated. The protein may also be singly or multiply acylated, such as with an acetyl group, with a farnesyl moiety, or with a fatty acid, which may be saturated, monounsaturated or polyunsaturated. The fatty acid may also be singly or multiply fluorinated. The invention also includes methionine analogs of the protein, for example the methionine sulfone and methionine sulfoxide analogs. The invention also includes salts of the proteins, such as ammonium salts, including alkyl or aryl ammonium salts, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, dihydrogen phosphate, thiosulfate, carbonate, bicarbonate, benzoate, sulfonate, thiosulfonate, mesylate, ethyl sulfonate and benzensulfonate salts.

In specific embodiments the CRAF1 is mouse or human CRAF1.

This invention provides a method of inhibiting activation by CD40 ligand of cells bearing CD40 on the cell surface, comprising providing the cells with an agent capable of inhibiting CD40-mediated intracellular signaling, the agent being present in an amount effective to inhibit activation of the cells. In an embodiment the agent is a protein comprising CRAF1 truncated by from about 323 to about 414 amino acid residues at the amino terminus, or a variant thereof.

In an embodiment of the method of inhibiting activation by CD40 ligand of cells bearing CD40 on the cell surface, the cells are provided with the protein of this invention by introducing into the cells a nucleic acid sequence encoding the protein under conditions such that the cells express an amount of the protein effective to inhibit activation of the cells. The nucleic acid may be DNA (including cDNA) or RNA. It may be single or double stranded, linear or circular. It may be in the form of a vector such as a plasmid or a viral vector. Preferably the nucleic acid sequence is operably linked to a transcriptional control sequence recognized by the host cell.

In another embodiment the agent is a small molecule. As used herein a small molecule is a compound capable of entering the cell. Preferably it has a molecular weight between 20 Da and 1x106 Da, preferably from 50 Da to 2 kDa.

In an embodiment the agent is modified from a lead inhibitory agent. In an embodiment the agent specifically binds to CD40 intracellular domain.

In embodiments of the methods described herein, the CD40-bearing cells are selected from the group consisting of B cells, fibroblasts, endothelial cells, epithelial cells, T cells, basophils, macrophages, Reed-Steinberg cells, dendritic cells, renal cells, and smooth muscle cells.

In a more specific embodiment the B cells are resting B cells, primed B cells, myeloma cells, lymphocytic leukemia B cells, or B lymphoma cells. In another specific embodiment the epithelial cells are keratinocytes. In another embodiment the fibroblasts are synovial membrane fibroblasts, dermal fibroblasts, pulmonary fibroblasts, or liver fibroblasts. In another specific embodiment the renal cells are selected from the group consisting of glomerular endothelial cells, mesangial cells, distal tubule cells, proximal tubule cells, parietal epithelial cells (e.g., crescent parietal epithelial cells), visceral epithelial cells, cells of a Henle limb, and interstitial inflammatory cells. In another embodiment the smooth muscle cells are smooth muscle cells of the bladder, vascular smooth muscle cells, aortic smooth muscle cells, coronary smooth muscle cells, pulmonary smooth muscle cells, or gastrointestinal smooth muscle cells. In a more specific embodiment the gastrointestinal smooth muscle cells are esophageal smooth muscle cells, stomachic smooth muscle cells, smooth muscle cells of the small intestine, or smooth muscle cells of the large intestine.

This invention provides a method of providing a subject with an amount of the protein of this invention effective to inhibit activation by CD40 ligand of cells bearing CD40 on the cell surface in the subject, comprising: introducing into CD40-bearing cells of the subject, a nucleic acid sequence encoding the protein of this invention, under conditions such that the cells express in the subject an activation inhibiting effective amount of the protein.

In an embodiment of this invention the introducing of the nucleic acid into cells of the subject comprises: a) treating cells of the subject ex vivo to insert the nucleic acid sequence into the cells; and b) introducing the cells from step a) into the subject.

The subject which can be treated by the above-described methods is an animal. Preferably the animal is a mammal. Subjects specifically intended for treatment with the method of the invention include humans, as well as nonhuman primates, sheep, horses, cattle, goats, pigs, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats and mice, as well as the organs, tumors, and cells derived or originating from these hosts.

This invention provides a method of treating a condition characterized by an unwanted level of CD40-mediated intracellular signaling, in a subject, comprising providing the subject with an amount of an agent capable of inhibiting CD40-mediated intracellular signaling in cells bearing CD40 on the cell surface.

In an embodiment the agent is a protein comprising CRAF1 truncated by from about 323 to about 414 amino acid residues at the amino terminus, or a variant thereof capable of inhibiting CD40-mediated cell activation. In an embodiment the protein is provided by introducing into CD40-bearing cells of the subject, a nucleic acid sequence encoding the protein, under conditions such that the cells express in the subject an activation inhibiting effective amount of the protein.

In an embodiment of this invention the agent is a small molecule. In an embodiment the molecule is modified from a lead inhibitory agent. In an embodiment the agent specifically binds to CD40 intracellular domain.

In an embodiment the condition is organ rejection in a subject receiving transplant organs. Examples of suitable transplant organs include a kidney, heart or liver, as well as others known to those of skill in the art. In another embodiment the condition is an immune response in a subject receiving gene therapy. One difficulty encountered in gene therapy is an immune response by the patient to the gene therapy vector and the proteins it expresses. Because the protein of this invention inhibits the immune response, gene therapy with the protein of this invention does not trigger an immune response. Its immunosuppressant effect also makes it useful as an adjunct to other forms of gene therapy. For example, at the same time that a vector being administered to provide a gene therapy patient with a desired gene product, the patient is also administered a vector which provides the protein of this invention.

In another embodiment the condition is a CD40-dependent immune response. In a specific embodiment the CD40-dependent immune response is an autoimmune response in a subject suffering from an autoimmune disease, including but not limited to rheumatoid arthritis, Myasthenia gravis, systemic lupus erythematosus, Graves' disease, idiopathic thrombocytopenia purpura, hemolytic anemia, diabetes mellitus, a drug-induced autoimmune disease such as drug-induced lupus, psoriasis, or hyper IgE syndrome.

In another embodiment the condition is an allergic response, including but not limited to hay fever or a penicillin allergy.

In an embodiment of this invention the immune response comprises induction of CD23, CD80 upregulation, or rescue from CD95-mediated apoptosis. Because CD40, which is expressed by many tumors, is involved in rescuing cells from apoptosis, inhibitors of CD40-mediated activity are useful as adjunctive agents in chemotherapy.

In an embodiment of this invention the immune response is autoimmune manifestations of an infectious disease. In more specific embodiments the autoimmune manifestations are derived from Reiter's syndrome, spondyloarthritis, Lyme disease, HIV infections, syphilis or tuberculosis.

In an embodiment the condition is dependent on CD40 ligand-induced activation of fibroblast cells, for example arthritis, scleroderma, and fibrosis. In more specific embodiments the arthritis is rheumatoid arthritis, non-rheumatoid inflammatory arthritis, arthritis associated with Lyme disease, or osteoarthritis. In another specific embodiment the fibrosis is pulmonary fibrosis, hypersensitivity pulmonary fibrosis, or a pneumoconiosis. Examples of pulmonary fibrosis include pulmonary fibrosis secondary to adult respiratory distress syndrome, drug-induced pulmonary fibrosis, idiopathic pulmonary fibrosis, or hypersensitivity pneumonitis. Examples of pneumoconiosis include asbestosis, siliconosis, or Farmer's lung. In another specific embodiment the fibrosis is a fibrotic disease of the liver or lung, including fibrotic disease of the lung caused by rheumatoid arthritis or scleroderma, and fibrotic diseases of the liver selected from the group consisting of: Hepatitis-C; Hepatitis-B; cirrhosis; cirrhosis of the liver secondary to a toxic insult; cirrhosis of the liver secondary to drugs; cirrhosis of the liver secondary to a viral infection; and cirrhosis of the liver secondary to an autoimmune disease. In a specific embodiment the toxic insult is alcohol consumption. In another specific embodiment the viral infection is Hepatitis B, Hepatitis C, or hepatitis non-B non-C. In another specific embodiment the autoimmune disease is primary biliary cirrhosis, or Lupoid hepatitis.

In an embodiment of this method the condition is dependent on CD40 ligand-induced activation of endothelial cells. In specific embodiments the condition dependent on CD40 ligand-induced activation of endothelial cells is selected from the group consisting of atherosclerosis, reperfusion injury, allograft rejection, organ rejection, and chronic inflammatory autoimmune diseases. In a more specific embodiment the atherosclerosis is accelerated atherosclerosis associated with organ transplantation. In another specific embodiment the chronic inflammatory autoimmune disease is vasculitis, rheumatoid arthritis, scleroderma, or multiple sclerosis.

In an embodiment the condition is dependent on CD40 ligand-induced activation of epithelial cells. In a specific embodiment the epithelial cells are keratinocytes, and the condition is psoriasis. In another specific embodiment the condition is an inflammatory kidney disease, including inflammatory kidney disease not initiated by autoantibody deposition in kidney and kidney disease which is initiated by autoantibody deposition. In specific embodiments the kidney disease is selected from the group consisting of: membranous glomerulonephritis; minimal change disease/acute tubular necrosis; pauci-immune glomerulonephritis; focal segmental glomerulosclerosis; interstitial nephritis; antitissue antibody-induced glomerular injury; circulating immune-complex disease; a glomerulopathy associated with a multisystem disease; and drug-induced glomerular disease. In an embodiment the antitissue antibody-induced glomerular injury is anti-basement membrane antibody disease. In another embodiment the circulating immune-complex disease is selected from the group consisting of: infective endocarditis; leprosy; syphilis; hepatitis B; malaria; and a disease associated with an endogenous antigen. In a more specific embodiment the endogenous antigen is DNA, thyroglobulin, an autologous immunoglobulin, erythrocyte stroma, a renal tubule antigen, a tumor-specific antigen, or a tumor-associated antigen. In another embodiment the glomerulopathy associated with a multisystem disease is selected from the group consisting of: diabetic nephropathy; systemic lupus erythematosus; Goodpasture's disease; Henoch-Schonlein purpura; polyarteritis; Wegener's granulomatosis; cryoimmunoglobulinemia; multiple myeloma; Waldenstrom's macroglobulinemia; and amyloidosis. In an embodiment the pauci-immune glomerulonephritis is ANCA+ pauci-immune glomerulonephritis, or Wegener's granulomatosis. In an embodiment the interstitial nephritis is drug-induced interstitial nephritis. In another embodiment the kidney disease affects renal tubules, including but not limited to: a kidney disease associated with a toxin; a neoplasia; hypersensitivity nephropathy; Sjogren's syndrome; and AIDS.

In an embodiment the condition is a smooth muscle cell-dependent disease. Examples include vascular diseases such as atherosclerosis; gastrointestinal diseases such as esophageal dysmotility, inflammatory bowel disease, and scleroderma; and bladder diseases.

In an embodiment of this method, the condition is associated with Epstein-Barr virus. Examples of Epstein-Barr virus-associated conditions include mononucleosis, B cell tumors (particularly in immunosuppressed individuals such as chemotherapy patients and those with acquired immune deficiency syndrome (AIDS)), Burkitt's lymphoma, and nasopharyngeal carcinoma. Epstein-Barr virus (EBV) transforms cells using latent infection membrane protein 1 (LMP1). LMP1 binds to CRAF1 (also known as LAP1)(33).

This invention provides a nucleic acid molecule encoding the protein of this invention. The nucleic acid may be DNA (including cDNA) or RNA. It may be single or double stranded, linear or circular. It may be in the form of a vector, such as a plasmid or viral vector, which comprises the nucleic acid molecule operably linked to a transcriptional control sequence recognized by a host cell transformed with the vector.

This invention provides a method of identifying an agent capable of inhibiting CD40-mediated intracellular signaling in a cell expressing CD40 on the cell surface, comprising providing the cell with the agent under conditions permitting activation of the cell in the absence of the agent, and determining decreased or absent activation, thereby identifying an agent capable of inhibiting CD40-mediated intracellular signaling in a cell expressing CD40 on the cell surface. In an embodiment the activation comprises up-regulation of CD23. In an embodiment the conditions permitting activation of the cell comprises contacting the cell with CD40 ligand or portion thereof effective to activate the cell.

Claim 1 of 2 Claims

What is claimed is:

1. An isolated protein consisting of amino acids 416-568 of SEQ ID NO:2.


____________________________________________
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