EP1177298A1 - Variantes de l'autotaxine et leurs utilisations dans le traitement des troubles du metabolisme - Google Patents

Variantes de l'autotaxine et leurs utilisations dans le traitement des troubles du metabolisme

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Publication number
EP1177298A1
EP1177298A1 EP00930429A EP00930429A EP1177298A1 EP 1177298 A1 EP1177298 A1 EP 1177298A1 EP 00930429 A EP00930429 A EP 00930429A EP 00930429 A EP00930429 A EP 00930429A EP 1177298 A1 EP1177298 A1 EP 1177298A1
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European Patent Office
Prior art keywords
autotaxin
amino acid
nucleic acid
seq
polypeptide
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EP00930429A
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German (de)
English (en)
Inventor
James D. Kelly
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Zymogenetics Inc
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Zymogenetics Inc
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Publication of EP1177298A1 publication Critical patent/EP1177298A1/fr
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/465—Hydrolases (3) acting on ester bonds (3.1), e.g. lipases, ribonucleases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy

Definitions

  • the present invention relates generally to a new biologically active polypeptide and to a new form of therapy.
  • the present invention relates to the treatment of metabolic diseases with autotaxin.
  • Lipolysis Alterations in lipolysis are associated with diabetes, and conditions such as obesity and dyslipidemia. These diseases are complicated by elevated levels of circulating free fatty acid in the form of triglycerides, which is an important risk factor for the development of atherosclerosis.
  • the activation of lipolysis increases fatty acids from triglycerols, which are predominantly stored in adipocytes (Arner, Diabetes Reviews 4:450 (1996)).
  • Lipolysis is controlled by hormone-sensitive lipase, which in turn is regulated by a variety of agents including insulin, catecholamines, glucocorticoids, thyroid hormones, growth hormone, and adenosine (Large and Arner, Diabetes & Metabolism 24:409 (1998)).
  • NIDDM Non-insulin dependent diabetes mellitus
  • NIDDM patients and their first-degree relatives demonstrate insulin resistance at the level of skeletal muscle and adipose tissue. This suggests a possible primary role for a defect in the insulin signal transduction cascade that results in stimulation of glucose transport and glycogen synthesis.
  • the signaling defect could involve any protein in the insulin signal transduction pathway, defects in pathways that interface with the insulin signal pathway, or defects in molecules essential for cellular function (DeFronzo, Diabetes Reviews 5:177 (1997)). Although specific genetic defects have been identified in rare syndromes of NIDDM, no specific defect has yet been defined as pathogenic in common forms of human NIDDM.
  • NIDDM is a polygenic disease (DeFronzo, Diabetes Reviews 5:177 (1997); Lowe, “Diabetes Mellitus,” Principles of Molecular Medicine, (Jameson, ed.), pages 433- 442 (Humana Press Inc. 1998)).
  • the present invention provides new human and rat autotaxin proteins and nucleotide sequences encoding the proteins.
  • the present invention also provides new methods for treating metabolic diseases and conditions with autotaxin.
  • Figure 1 shows the insulin-mediated uptake of 2-deoxyglucose by differentiated 3T3 LI cells in the absence or presence of autotaxin (ATX). Glucose uptake is shown as the percent of maximal uptake observed with 1500 pM recombinant human insulin treatment (minus background).
  • Autotaxin is a basic glycoprotein cytokine that was first isolated from the conditioned medium of A2058 human melanoma cells (Stracke et al., J. Biol. Chem. 267:2524 (1992)).
  • Autocrine Motility Factors in Human Cytokines, Vol. II, Aggarwal and Gutterman (eds.), pages 258-285 (Blackwell Science Ltd. 1996); Stracke et al, "Autotaxin, Autocrine Motility Factor,” in Brain Tumor Invasion: Biological, Clinical, and Therapeutic Considerations, Mikkelsen et al. (eds.), pages 343-355 (Wiley-Liss, Inc.
  • autotaxin also has adenosine-5'- triphosphatase (ATPase; EC 3.6.1.3.) and ATP pyrophosphatase (EC 3.6.1.8.) activities (Clair et al, J. Biol. Chem. 272:996 (1997)).
  • Site-directed mutagenesis of the autotaxin phosphodiesterase catalytic site (Y 201 MRPVYPTKTFPN 213 ) has revealed that a phosphorylation of an amino acid at position 210 is required for autotaxin to undergo autophosphorylation, for autotaxin to exhibit phosphodiesterase activity, and for motility-stimulating activities (Lee et al, J. Biol. Chem.
  • Autotaxin has significant identity to a family of proteins that includes a marker of B-cell activation (PC-1), a rat brain nucleotide pyrophosphatase (PD-l ⁇ ), and a rat neural differentiation antigen (gpl30 RB13"6 ) (Goding et al, Immun. Rev. 161:11 (1998); Stracke et al, "Autotaxin, Autocrine Motility Factor," in Brain Tumor Invasion: Biological, Clinical, and Therapeutic Considerations, Mikkelsen et al. (eds.), pages 343-355 (Wiley-Liss, Inc. 1998)). Although PC-1 and gp ⁇ O* 313"6 are predominantly cell surface glycoproteins, autotaxin is secreted, following cleavage near its transmembrane domain and proximal to the somatomedin B domains.
  • PC-1 and gp ⁇ O* 313"6 are predominantly cell surface glycoproteins, autotaxin is secreted,
  • a new form of rat autotaxin was identified while screening for secreted polypeptides that are produced by adipocytes and that affect the metabolism of cultured hepatic cells.
  • This new rat autotaxin has the amino acid sequence of SEQ ID NO:2, and is encoded by the nucleotide sequence of SEQ ID NO:l .
  • Features of the autotaxin include a putative transmembrane domain (amino acid residues 5 to 15 of SEQ ID NO:2) and an RGD binding domain (amino acid residues 122 to 124 of SEQ ID NO:2).
  • this form of rat autotaxin can be distinguished from PD-l ⁇ at least on the basis that the new rat autotaxin comprises the sequence "VPECPAGFVR" (amino acid residues 149 to 158 of SEQ ID NO:2), which PD-l ⁇ lacks, while PD-l ⁇ comprises the sequence "AETGKFRGSKHENKKNLNGSVEPRK” (amino acid residues 591 to 615 of SEQ ID NO:6), which the new rat autotaxin lacks.
  • VPECPAGFVR amino acid residues 149 to 158 of SEQ ID NO:2
  • PD-l ⁇ comprises the sequence "AETGKFRGSKHENKKNLNGSVEPRK” (amino acid residues 591 to 615 of SEQ ID NO:6), which the new rat autotaxin lacks.
  • autotaxin is expressed from a novel promoter, which is located upstream of the previously characterized autotaxin promoter. Accordingly, this form of autotaxin may be expressed in manner that allows a novel form of regulation of the autotaxin gene. Experiments also show that autotaxin can inhibit the differentiation of adipocytes in vitro. See Example 1.
  • human autotaxin polypeptide has the amino acid sequence of SEQ ID NO:9, while an exemplary nucleotide sequence that encodes this amino acid sequence is provided by SEQ ID NO:8.
  • novel human autotaxin include: a putative transmembrane domain (amino acid residues 8 to 26 of SEQ ID NO:9), a putative cleavage site at Ser 44 /Asp 45 of SEQ ID NO:9, an RGD binding domain (amino acid residues 123 to 125 of SEQ ID NO:9), and a putative phosphodiesterase active site (amino acid residues 197 to 209 of SEQ ID NO:9).
  • autotaxin binds various cell types, including cultured murine muscle cells, rat muscle soleus and gastrocnemius, and liver cells.
  • autotaxin binding capacity was observed to increase at least 20-fold following adipocyte differentiation of murine 3T3 LI cells.
  • the effects of autotaxin on adipocyte and liver cells, and the enzymatic activity of autotaxin indicate that autotaxin can be used to treat diseases of metabolism such as obesity, dyslipidemia, and diabetes.
  • autotaxin administration will increase the insulin signaling in adipose tissue by producing substrate for adenosine receptors, which are present in adipocyte cell membranes.
  • Adenosine is known to decrease lipolysis via inhibition of adenylyl cyclase (Large and Arner, Diabetes & Metabolism 24:409 (1998)). Moreover, the enhanced insulin-like signaling will result in an inhibition of lipolysis and lowered serum free fatty acids. Lowered serum free fatty acids may cause decreased hepatic gluconeogenesis, which could result in decreased serum glucose levels, and an increase in insulin sensitivity.
  • the binding of autotaxin to adipocyte and liver cells can provide additional insulin-like signaling, such as the activation of phosphatidylinositol 3 kinase and protein kinase B.
  • This signaling may be a consequence of the binding of autotaxin via its RGD domain to cell-surface integrins.
  • activated integrin associates with insulin receptors and that integrin activation potentiates the insulin-signaling pathway (Schneller et al, EMBO J. 16:5600 (1997); Guilherme et al, J. Biol. Chem. 273:22899 (1998)).
  • RGD domain is present in the new rat autotaxin at amino acid residues 122 to 124 of SEQ ID NO:2.
  • RGD domains are also present in human autotaxin isolated from melanoma cells (Murata et al, J. Biol. Chem. 269:30419 (1994); GenBank accession No. L35594; amino acid residues 127 to 129 of SEQ ID NO:4), human autotaxin isolated from teratocarcinoma cells (Lee et al, Biochem. Biophys. Res. Commun. 218:114 (1996); GenBank accession No.
  • L46720 amino acid residues 127 to 129 of SEQ ID NO:5
  • a new form of human autotaxin described herein amino acid residues 123 to 125 of SEQ ID NO:9
  • rat autotaxin isolated from brain tissue Narita et al, J. Biol. Chem. 269:28235 (1994); GenBank accession No. 1083752 or No. BAA05910; amino acid residues 126 to 128 of SEQ ID NO:6).
  • Autotaxin may provide insulin-like signaling by binding to a cell surface receptor that, when activated, enhances insulin receptor signaling by a variety of means, including phosphorylation of tyrosine residues, incorporation of accessory signaling proteins into larger complexes, and the like.
  • the formation of such large complexes may increase the efficiency of insulin receptor signaling.
  • the present invention provides isolated polypeptides comprising an amino acid sequence that is at least 70%, at least 80%, or at least 90% identical to amino acid residues 32 to 858 of SEQ ID NO:2, wherein the isolated polypeptide specifically binds with an antibody that specifically binds with a polypeptide consisting of the amino acid sequence of SEQ ID NO:2, with the provision that the isolated polypeptide is not an autotaxin selected from the group consisting of human melanoma autotaxin (GenBank accession No. L35594), human teratocarcinoma autotaxin (GenBank accession No. L46720), and rat brain autotaxin (GenBank accession Nos. 1083752 and BAA05910).
  • human melanoma autotaxin GenBank accession No. L35594
  • human teratocarcinoma autotaxin GenBank accession No. L46720
  • rat brain autotaxin GenBank accession Nos. 1083752 and BAA05910
  • the present invention also includes isolated polypeptides comprising either the amino acid sequence of amino acid residues 32 to 858 of SEQ ID NO:2 or the amino acid sequence of amino acid residues 45 to 859 of SEQ ID NO:9, as well as autotaxin polypeptides, comprising amino acid residues 149 to 158 of SEQ ID NO:2.
  • the present invention also provides variant autotaxin polypeptides, wherein the amino acid sequence of the variant polypeptide shares an identity with the amino acid sequence of SEQ ID NO:2 selected from the group consisting of at least 70% identity, at least 80% identity, at least 90% identity, at least 95% identity, or greater than 95% identity, and wherein any difference between the amino acid sequence of the variant polypeptide and the amino acid sequence of SEQ ID NO:2 is due to one or more conservative amino acid substitutions, with the provision that the variant autotaxin polypeptide is not an autotaxin selected from the group consisting of human melanoma autotaxin (GenBank accession No. L35594), human teratocarcinoma autotaxin (GenBank accession No. L46720), and rat brain autotaxin (GenBank accession Nos. 1083752 and BAA05910).
  • the variant autotaxin polypeptide is not an autotaxin selected from the group consisting of human melanoma autotaxin (GenBank accession No. L3559
  • the present invention further provides antibodies and antibody fragments that specifically bind with such polypeptides.
  • exemplary antibodies include polyclonal antibodies, murine monoclonal antibodies, humanized antibodies derived from murine monoclonal antibodies, and human monoclonal antibodies.
  • Illustrative antibody fragments include F(ab') 2 , F(ab) 2 , Fab', Fab, Fv, scFv, and minimal recognition units.
  • An exemplary antibody or antibody fragment binds an autotaxin epitope comprising an amino acid sequence selected from the group consisting of amino acid residues 143 to 158 of SEQ ID NO:2, amino acid residues 149 to 158 of SEQ ID NO:2, and amino acid residues 585 to 595 of SEQ ID NO:2.
  • the present invention also contemplates anti-idiotype antibodies, or anti-idiotype antibody fragments, that specifically bind with an antibody or antibody fragment that binds with an autotaxin polypeptide described herein.
  • the present invention also includes compositions comprising a carrier and a protein, peptide, polypeptide, antibody, or anti-idiotype antibody described herein.
  • the present invention further includes isolated nucleic acid molecules that encode an autotaxin polypeptide, wherein the nucleic acid molecule is either (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3, or (b) a nucleic acid molecule that remains hybridized following stringent wash conditions to a nucleic acid molecule consisting of the nucleotide sequence of SEQ ID NO:l, or the complement of SEQ ID NO:l, with the provision that the isolated nucleic acid molecule does not encode an autotaxin selected from the group consisting of human melanoma autotaxin (GenBank accession No. L35594), human teratocarcinoma autotaxin (GenBank accession No. L46720), and rat brain autotaxin (GenBank accession Nos. 1083752 and BAA05910).
  • the nucleic acid molecule is either (a) a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:3, or (
  • Illustrative nucleic acid molecules include those in which any difference between the amino acid sequence encoded by the nucleic acid molecule and the corresponding amino acid sequence of SEQ ID NO:2 is due to a conservative amino acid substitution.
  • the present invention further contemplates isolated nucleic acid molecules, comprising the nucleotide sequence of either nucleotides 223 to nucleotide 2703 of SEQ ID NO: 1 or nucleotides 237 to 2681 of SEQ ID NO:9.
  • the present invention also includes vectors and expression vectors comprising such nucleic acid molecules.
  • Such expression vectors may comprise a transcription promoter, and a transcription terminator, wherein the promoter is operably linked with the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked with the transcription terminator.
  • the present invention further includes recombinant host cells comprising these vectors and expression vectors.
  • Illustrative host cells include bacterial, yeast, avian, fungal, insect, mammalian, and plant cells.
  • Recombinant host cells comprising such expression vectors can be used to produce autotaxin polypeptides by culturing such recombinant host cells that comprise the expression vector and that produce the autotaxin protein, and, optionally, isolating the autotaxin protein from the cultured recombinant host cells.
  • the present invention also includes methods of treating a subject for a condition associated with elevated serum lipids, comprising the administration of autotaxin or an autotaxin analog, wherein the administration decreases, in the subject, at least one of the level of serum triglyceride or the level of fatty acid bound to albumin in the serum.
  • Examples of such conditions include obesity, dyslipidemia, and diabetes, such as non-insulin dependent diabetes mellitus.
  • the present invention also provides methods of decreasing the serum level of free fatty acid in a subject, comprising the administration of autotaxin or an autotaxin analog, wherein the treatment decreases, in the subject, at least one of the level of serum triglyceride or the level of fatty acid bound to albumin in the serum.
  • the autotaxin can be administered as a pharmaceutical composition that comprises autotaxin polypeptide and a pharmaceutically acceptable carrier.
  • suitable pharmaceutical compositions have a form selected from the group consisting of liquid form, solid form, and aerosol form.
  • the autotaxin can be recombinant autotaxin or natural autotaxin.
  • An illustrative autotaxin analog is an anti-idiotype antibody.
  • a suitable autotaxin polypeptide can further comprise a water-soluble polymer, wherein the water-soluble polymer is conjugated to the autotaxin polypeptide.
  • the water-soluble polymer can be polyethylene glycol.
  • Autotaxin or autotaxin analog can also be administered as a pharmaceutical composition that comprises a nucleic acid molecule encoding an autotaxin or autotaxin analog.
  • the pharmaceutical composition can comprise at least one of an expression vector that comprises the nucleic acid molecule or a recombinant virus that comprises the expression vector.
  • the present invention also includes pharmaceutical compositions, comprising a pharmaceutically acceptable carrier and at least one of autotaxin or an autotaxin analog, as well as pharmaceutical compositions that comprise a pharmaceutically acceptable carrier and at least one of an expression vector, or a recombinant virus that comprises an expression vector.
  • Suitable expression vectors comprise a nucleic acid molecule that encodes autotaxin or an autotaxin analog, a transcription promoter, and a transcription terminator, wherein the promoter is operably linked with the nucleic acid molecule, and wherein the nucleic acid molecule is operably linked with the transcription terminator.
  • the present invention further provides recombinant viruses that comprise such expression vectors.
  • the present invention also provides fusion proteins comprising an autotaxin moiety and an antibody, or antibody fragment.
  • An illustrative antibody fragment is an immunoglobulin heavy chain constant region, such as a human F c fragment.
  • the present invention further includes isolated nucleic acid molecules that encode such fusion proteins.
  • the present invention also provides methods for stimulating glucose uptake by a cell, comprising the administration of autotaxin, or an autotaxin analog, to the cell. If the cell is a cultured cell, then the administration decreases the concentration of glucose in the culture medium. If the autotaxin, or autotaxin analog, is administered to a mammalian subject, then the administration decreases the concentration of glucose in the blood of the subject. Suitable subjects include subjects having diabetes, such as non-insulin dependent diabetes mellitus. An illustrative mammalian subject is a human subject, although, the method can be applied in a veterinary setting as well.
  • the autotaxin, or autotaxin analog can be administered as a composition comprising a polypeptide, a nucleic acid molecule that encodes autotaxin (or an autotaxin analog), or as a virus that comprises such a nucleic acid molecule.
  • insulin is administered prior to the administration of autotaxin (or an autotaxin analog), concomitantly with the administration of autotaxin (or an autotaxin analog), or following the administration of autotaxin (or an autotaxin analog).
  • the present invention further provides compositions that comprise an autotaxin for use as a medicament. Moreover, the present invention provides the use of an autotaxin polypeptide for the manufacture of a medicament for treating a condition associated with an elevated serum glucose level, an elevated serum lipid level, or an elevated serum free fatty acid level.
  • the present invention also includes pharmaceutical compositions comprising a pharmaceutically acceptable carrier and an autotaxin polypeptide that comprises an amino acid sequence selected from the group consisting of (a) amino acid residues 45 to 859 of SEQ ID NO:9, (b) amino acid residues 32 to 858 of SEQ ID NO:2, (c) amino acid residues 49 to 915 of SEQ ID NO:4, (d) amino acid residues 49 to 863 of SEQ ID NO:5, and (e) amino acid residues 36 to 885 of SEQ ID NO:6.
  • Additional pharmaceutical compositions comprise a pharmaceutically acceptable carrier and a polypeptide consisting of an amino acid sequence selected from the amino acid sequences of (a) to (e).
  • compositions comprise a pharmaceutically acceptable carrier and a polypeptide that comprises, or consists, of an amino acid sequence of about 20, about 30, about 60, or about 100 contiguous amino acids of an amino acid sequence selected from the group consisting of (a) amino acid residues 45 to 859 of SEQ ID NO:9, (b) amino acid residues 32 to 858 of SEQ ID NO:2, (c) amino acid residues 49 to 915 of SEQ ID NO:4, (d) amino acid residues 49 to 863 of SEQ ID NO:5, and (e) amino acid residues 36 to 885 of SEQ ID NO:6.
  • the present invention includes pharmaceutical compositions that comprise a pharmaceutically acceptable carrier and at least one of rat brain nucleotide pyrophosphatase (PD-l ⁇ ), or rat neural differentiation antigen (gpnO* 813"6 ).
  • PD-l ⁇ rat brain nucleotide pyrophosphatase
  • gpnO* 813"6 rat neural differentiation antigen
  • the amino acid sequences of these polypeptides are known to those of skill in the art (see, for example, Goding et al, Immun. Rev. 161:11 (1998).
  • pharmaceutical compositions can comprise soluble forms of PD-l ⁇ or gpOO 1 ⁇ 13"6 .
  • nucleic acid or “nucleic acid molecule” refers to polynucleotides, such as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA), oligonucleotides, fragments generated by the polymerase chain reaction (PCR), and fragments generated by any of ligation, scission, endonuclease action, and exonuclease action.
  • Nucleic acid molecules can be composed of monomers that are naturally- occurring nucleotides (such as DNA and RNA), or analogs of naturally-occurring nucleotides (e.g., ⁇ -enantiomeric forms of naturally-occurring nucleotides), or a combination of both.
  • Modified nucleotides can have alterations in sugar moieties and/or in pyrimidine or purine base moieties.
  • Sugar modifications include, for example, replacement of one or more hydroxyl groups with halogens, alkyl groups, amines, and azido groups, or sugars can be functionalized as ethers or esters.
  • the entire sugar moiety can be replaced with sterically and electronically similar structures, such as aza-sugars and carbocyclic sugar analogs.
  • modifications in a base moiety include alkylated purines and pyrimidines, acylated purines or pyrimidines, or other well-known heterocyclic substitutes.
  • Nucleic acid monomers can be linked by phosphodiester bonds or analogs of such linkages.
  • nucleic acid molecule also includes so- called “peptide nucleic acids,” which comprise naturally-occurring or modified nucleic acid bases attached to a polyamide backbone. Nucleic acids can be either single stranded or double stranded.
  • nucleic acid molecule refers to a nucleic acid molecule having a complementary nucleotide sequence and reverse orientation as compared to a reference nucleotide sequence.
  • sequence 5' ATGCACGGG 3' is complementary to 5' CCCGTGCAT 3'.
  • contig denotes a nucleic acid molecule that has a contiguous stretch of identical or complementary sequence to another nucleic acid molecule. Contiguous sequences are said to "overlap" a given stretch of a nucleic acid molecule either in their entirety or along a partial stretch of the nucleic acid molecule.
  • degenerate nucleotide sequence denotes a sequence of nucleotides that includes one or more degenerate codons as compared to a reference nucleic acid molecule that encodes a polypeptide.
  • Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp).
  • structural gene refers to a nucleic acid molecule that is transcribed into messenger RNA (mRNA), which is then translated into a sequence of amino acids characteristic of a specific polypeptide.
  • an "isolated nucleic acid molecule” is a nucleic acid molecule that is not integrated in the genomic DNA of an organism.
  • a DNA molecule that encodes a growth factor that has been separated from the genomic DNA of a cell is an isolated DNA molecule.
  • Another example of an isolated nucleic acid molecule is a chemically-synthesized nucleic acid molecule that is not integrated in the genome of an organism.
  • a nucleic acid molecule that has been isolated from a particular species is smaller than the complete DNA molecule of a chromosome from that species.
  • nucleic acid molecule construct is a nucleic acid molecule, either single- or double-stranded, that has been modified through human intervention to contain segments of nucleic acid combined and juxtaposed in an arrangement not existing in nature.
  • Linear DNA denotes non-circular DNA molecules having free 5' and 3' ends.
  • Linear DNA can be prepared from closed circular DNA molecules, such as plasmids, by enzymatic digestion or physical disruption.
  • cDNA complementary DNA
  • cDNA is a single-stranded DNA molecule that is formed from an mRNA template by the enzyme reverse transcriptase. Typically, a primer complementary to portions of mRNA is employed for the initiation of reverse transcription.
  • cDNA to refer to a double- stranded DNA molecule consisting of such a single-stranded DNA molecule and its complementary DNA strand.
  • the term “cDNA” also refers to a clone of a cDNA molecule synthesized from an RNA template.
  • a “promoter” is a nucleotide sequence that directs the transcription of a structural gene.
  • a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of a structural gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. These promoter elements include RNA polymerase binding sites, TATA sequences, CAAT sequences, differentiation-specific elements (DSEs; McGehee et al, Mol Endocrinol 7:551 (1993)), cyclic AMP response elements (CREs), serum response elements (SREs; Treisman, Seminars in Cancer Biol.
  • DSEs differentiation-specific elements
  • CREs cyclic AMP response elements
  • SREs serum response elements
  • GREs glucocorticoid response elements
  • binding sites for other transcription factors such as CRE/ATF (O'Reilly et al, J. Biol. Chem. 267: 9938 (1992)), AP2 (Ye et al, J. Biol. Chem. 269:25128 (1994)), SP1, cAMP response element binding protein (CREB; Loeken, Gene Expr. 3:253 (1993)) and octamer factors (see, in general, Watson et al, eds., Molecular Biology of the Gene, 4th ed. (The Benjamin/Cummings Publishing Company, Inc. 1987), and Lemaigre and Rousseau, Biochem. J.
  • a promoter contains essential nucleotide sequences for promoter function, including the TATA box and start of transcription. By this definition, a core promoter may or may not have detectable activity in the absence of specific sequences that may enhance the activity or confer tissue specific activity.
  • a “regulatory element” is a nucleotide sequence that modulates the activity of a core promoter.
  • a regulatory element may contain a nucleotide sequence that binds with cellular factors enabling transcription exclusively or preferentially in particular cells, tissues, or organelles. These types of regulatory elements are normally associated with genes that are expressed in a "cell-specific,” “tissue-specific,” or “organelle-specific” manner.
  • the autotaxin regulatory element preferentially induces gene expression in adipocytes and brain tissue.
  • An “enhancer” is a type of regulatory element that can increase the efficiency of transcription, regardless of the distance or orientation of the enhancer relative to the start site of transcription.
  • Heterologous DNA refers to a DNA molecule, or a population of DNA molecules, that does not exist naturally within a given host cell.
  • DNA molecules heterologous to a particular host cell may contain DNA derived from the host cell species (i.e., endogenous DNA) so long as that host DNA is combined with non-host DNA (i.e., exogenous DNA).
  • a DNA molecule containing a non-host DNA segment encoding a polypeptide operably linked to a host DNA segment comprising a transcription promoter is considered to be a heterologous DNA molecule.
  • a heterologous DNA molecule can comprise an endogenous gene operably linked with an exogenous promoter.
  • a DNA molecule comprising a gene derived from a wild-type cell is considered to be heterologous DNA if that DNA molecule is introduced into a mutant cell that lacks the wild-type gene.
  • polypeptide is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides.”
  • a “protein” is a macromolecule comprising one or more polypeptide chains.
  • a protein may also comprise non-peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • a peptide or polypeptide encoded by a non-host DNA molecule is a "heterologous" peptide or polypeptide.
  • an "integrated genetic element” is a segment of DNA that has been incorporated into a chromosome of a host cell after that element is introduced into the cell through human manipulation.
  • integrated genetic elements are most commonly derived from linearized plasmids that are introduced into the cells by electroporation or other techniques. Integrated genetic elements are passed from the original host cell to its progeny.
  • a "cloning vector” is a nucleic acid molecule, such as a plasmid, cosmid, or bacteriophage, which has the capability of replicating autonomously in a host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites that allow insertion of a nucleic acid molecule in a determinable fashion without loss of an essential biological function of the vector, as well as nucleotide sequences encoding a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.
  • Marker genes typically include genes that provide tetracycline resistance or ampicillin resistance.
  • an “expression vector” is a nucleic acid molecule encoding a gene that is expressed in a host cell.
  • an expression vector comprises a transcription promoter, a gene, and a transcription terminator. Gene expression is usually placed under the control of a promoter, and such a gene is said to be “operably linked to” the promoter.
  • a regulatory element and a core promoter are operably linked if the regulatory element modulates the activity of the core promoter.
  • a “recombinant host” is a cell that contains a heterologous nucleic acid molecule, such as a cloning vector or expression vector.
  • a recombinant host is a cell that produces autotaxin from an expression vector. This type of autotaxin is referred to as "recombinant autotaxin.”
  • autotaxin can be produced by a cell that is a "natural source” of autotaxin, and that lacks an expression vector. This type of autotaxin is referred to as "natural autotaxin.”
  • “Integrative transformants” are recombinant host cells, in which heterologous DNA has become integrated into the genomic DNA of the cells.
  • a “fusion protein” is a hybrid protein expressed by a nucleic acid molecule comprising nucleotide sequences of at least two genes.
  • a fusion protein can comprise at least part of an autotaxin polypeptide fused with a polypeptide that binds an affinity matrix.
  • Such a fusion protein provides a means to isolate large quantities of autotaxin using affinity chromatography.
  • Receptor denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand.” This interaction mediates the effect of the ligand on the cell.
  • Receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g., thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
  • Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction. In certain membrane-bound receptors, the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor.
  • the binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell, which in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are often linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
  • secretory signal sequence denotes a DNA sequence that encodes a peptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • secretory peptide a DNA sequence that encodes a peptide that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • isolated polypeptide is a polypeptide that is essentially free from contaminating cellular components, such as carbohydrate, lipid, or other proteinaceous impurities associated with the polypeptide in nature.
  • a preparation of isolated polypeptide contains the polypeptide in a highly purified form, i.e., at least about 80%o pure, at least about 90% pure, at least about 95% pure, greater than 95% pure, or greater than 99% pure.
  • SDS sodium dodecyl sulfate
  • isolated does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • amino-terminal and “carboxyl-terminal” are used herein to denote positions within polypeptides. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a polypeptide is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete polypeptide.
  • expression refers to the biosynthesis of a gene product. For example, in the case of a structural gene, expression involves transcription of the structural gene into mRNA and the translation of mRNA into one or more polypeptides.
  • splice variant is used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene. Splice variants may encode polypeptides having altered amino acid sequence. The term splice variant is also used herein to denote a polypeptide encoded by a splice variant of an mRNA transcribed from a gene.
  • immunomodulator includes cytokines, stem cell growth factors, lymphotoxins, co-stimulatory molecules, hematopoietic factors, and synthetic analogs of these molecules.
  • complement/anti-complement pair denotes non-identical moieties that form a non-covalently associated, stable pair under appropriate conditions.
  • biotin and avidin are prototypical members of a complement/anti-complement pair.
  • Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like.
  • the complement/anti-complement pair preferably has a binding affinity of less than 10 9 M '1 .
  • an "anti-idiotype antibody” is an antibody that binds with the variable region domain of an immunoglobulin.
  • an anti-idiotype antibody binds with the variable region of an anti-autotaxin antibody, and thus, an anti-idiotype antibody mimics an epitope of autotaxin.
  • an “antibody fragment” is a portion of an antibody such as F(ab') 2 , F(ab) 2 , Fab', Fab, and the like. Regardless of structure, an antibody fragment binds with the same antigen that is recognized by the intact antibody. For example, an anti-autotaxin monoclonal antibody fragment binds with an epitope of autotaxin.
  • antibody fragment also includes a synthetic or a genetically engineered polypeptide that binds to a specific antigen, such as polypeptides consisting of the light chain variable region, "Fv” fragments consisting of the variable regions of the heavy and light chains, recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker ("scFv proteins”), and minimal recognition units consisting of the amino acid residues that mimic the hypervariable region.
  • scFv proteins recombinant single chain polypeptide molecules in which light and heavy variable regions are connected by a peptide linker
  • a “chimeric antibody” is a recombinant protein that contains the variable domains and complementary determining regions derived from a rodent antibody, while the remainder of the antibody molecule is derived from a human antibody.
  • Humanized antibodies are recombinant proteins in which murine complementarity determining regions of a monoclonal antibody have been transferred from heavy and light variable chains of the murine immunoglobulin into a human variable domain.
  • a "therapeutic agent” is a molecule or atom, which is conjugated to an antibody moiety to produce a conjugate, which is useful for therapy.
  • therapeutic agents include drugs, toxins, immunomodulators, chelators, boron compounds, photoactive agents or dyes, and radioisotopes.
  • detectable label is a molecule or atom, which can be conjugated to an antibody moiety to produce a molecule useful for diagnosis.
  • detectable labels include chelators, photoactive agents, radioisotopes, fluorescent agents, paramagnetic ions, or other marker moieties.
  • affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • Affinity tags include a poly- histidine tract, protein A (Nilsson et al, EMBO J. 4:1015 (1985); Nilsson et al, Methods Enzymol 198:3 (1991)), glutathione S transferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag (Grussenmeyer et al, Proc.
  • naked antibody is an entire antibody, as opposed to an antibody fragment, which is not conjugated with a therapeutic agent. Naked antibodies include both polyclonal and monoclonal antibodies, as well as certain recombinant antibodies, such as chimeric and humanized antibodies.
  • antibody component includes both an entire antibody and an antibody fragment.
  • An “immunoconjugate” is a conjugate of an antibody component with a therapeutic agent or a detectable label.
  • antibody fusion protein refers to a recombinant molecule that comprises an antibody component and a therapeutic agent.
  • therapeutic agents suitable for such fusion proteins include immunomodulators ("antibody-immunomodulator fusion protein”) and toxins ("antibody -toxin fusion protein”).
  • a “target polypeptide” or a “target peptide” is an amino acid sequence that comprises at least one epitope, and that is expressed on a target cell, such as a tumor cell, or a cell that carries an infectious agent antigen.
  • T cells recognize peptide epitopes presented by a major histocompatibility complex molecule to a target polypeptide or target peptide and typically lyse the target cell or recruit other immune cells to the site of the target cell, thereby killing the target cell.
  • antigenic peptide is a peptide, which will bind a major histocompatibility complex molecule to form an MHC-peptide complex, which is recognized by a T cell, thereby inducing a cytotoxic lymphocyte response upon presentation to the T cell.
  • antigenic peptides are capable of binding to an appropriate major histocompatibility complex molecule and inducing a cytotoxic T cells response, such as cell lysis or specific cytokine release against the target cell, which binds or expresses the antigen.
  • the antigenic peptide can be bound in the context of a class I or class II major histocompatibility complex molecule, on an antigen presenting cell or on a target cell.
  • RNA polymerase II catalyzes the transcription of a structural gene to produce mRNA.
  • a nucleic acid molecule can be designed to contain an RNA polymerase II template in which the RNA transcript has a sequence that is complementary to that of a specific mRNA.
  • the RNA transcript is termed an "anti- sense RNA” and a nucleic acid molecule that encodes the anti-sense RNA is termed an "anti-sense gene.”
  • Anti-sense RNA molecules are capable of binding to mRNA molecules, resulting in an inhibition of mRNA translation.
  • an “anti-sense oligonucleotide specific for autotaxin” or an “autotaxin anti-sense oligonucleotide” is an oligonucleotide having a sequence (a) capable of forming a stable triplex with a portion of the autotaxin gene, or (b) capable of forming a stable duplex with a portion of an mRNA transcript of the autotaxin gene.
  • a "ribozyme” is a nucleic acid molecule that contains a catalytic center.
  • the term includes RNA enzymes, self-splicing RNAs, self-cleaving RNAs, and nucleic acid molecules that perform these catalytic functions.
  • a nucleic acid molecule that encodes a ribozyme is termed a "ribozyme gene.”
  • an “external guide sequence” is a nucleic acid molecule that directs the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, resulting in the cleavage of the mRNA by RNase P.
  • a nucleic acid molecule that encodes an external guide sequence is termed an "external guide sequence gene.”
  • variant autotaxin gene refers to nucleic acid molecules that encode a polypeptide having an amino acid sequence that is a modification of SEQ ID NO:2. Such variants include naturally-occurring polymorphisms of autotaxin genes, as well as synthetic genes that contain conservative amino acid substitutions of the amino acid sequence of SEQ ID NO:2. Additional variant forms of autotaxin genes are nucleic acid molecules that contain insertions or deletions of the nucleotide sequences described herein. A variant autotaxin gene can be identified by determining whether the gene hybridizes with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, or its complement, under stringent conditions.
  • variant autotaxin does not include the following proteins: (1) human autotaxin isolated from melanoma cells (Murata et al, J. Biol. Chem. 269:30419 (1994)), which is encoded by the nucleotide sequence having GenBank accession No. L35594, (2) human autotaxin isolated from teratocarcinoma cells (Lee et al, Biochem. Biophys. Res. Commun. 218:114 (1996)), which is encoded by the nucleotide sequence having GenBank accession No. L46720, and (3) rat autotaxin isolated from brain tissue (Narita et al, J. Biol. Chem. 269:28235 (1994)), which is encoded by the nucleotide sequence having GenBank accession No. 1083752.
  • Variant autotaxin genes can also be identified by sequence comparison.
  • Two amino acid sequences have "100% amino acid sequence identity” if the amino acid residues of the two amino acid sequences are the same when aligned for maximal correspondence.
  • two nucleotide sequences have "100% nucleotide sequence identity” if the nucleotide residues of the two nucleotide sequences are the same when aligned for maximal correspondence. Sequence comparisons can be performed using standard software programs such as those included in the LASERGENE bioinformatics computing suite, which is produced by DNASTAR (Madison, Wisconsin).
  • a variant gene or polypeptide encoded by a variant gene is functionally characterized by at least one of: (1) type I phosphodiesterase activity, (2) ability to bind integrin, (3) ability to inhibit the differentiation of adipocytes, (4) ability to enhance the insulin-mediated uptake of 2- deoxy-glucose by differentiated 3T3 LI cells, and (5) ability to bind specifically to an anti-autotaxin antibody.
  • allelic variant is used herein to denote any of two or more alternative forms of a gene occupying the same chromosomal locus. Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations. Gene mutations can be silent (no change in the encoded polypeptide) or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene.
  • ortholog denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation. "Paralogs" are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, ⁇ - globin, ⁇ -globin, and myoglobin are paralogs of each other.
  • a "functional fragment" of an autotaxin gene refers to a nucleic acid molecule that encodes a portion of an autotaxin polypeptide which has at least one of the biological activities described herein.
  • a functional fragment of an autotaxin gene can comprise a portion of the nucleotide sequence of SEQ ID NO:l, and encode a polypeptide capable of binding integrin or inhibiting the differentiation of adipocytes. Due to the imprecision of standard analytical methods, molecular weights and lengths of polymers are understood to be approximate values. When such a value is expressed as "about” X or “approximately” X, the stated value of X will be understood to be accurate to ⁇ 10%.
  • Nucleic acid molecules encoding a rat autotaxin gene can be obtained by screening a rat cDNA or genomic library using polynucleotide probes based upon SEQ ID NO:l . These techniques are standard and well-established.
  • a nucleic acid molecule that encodes a rat autotaxin gene can be isolated from a rat cDNA library.
  • the first step would be to prepare the cDNA library by isolating RNA from a tissue, such as fat tissue, using methods well-known to those of skill in the art.
  • RNA isolation techniques must provide a method for breaking cells, a means of inhibiting RNase-directed degradation of RNA, and a method of separating RNA from DNA, protein, and polysaccharide contaminants.
  • total RNA can be isolated by freezing tissue in liquid nitrogen, grinding the frozen tissue with a mortar and pestle to lyse the cells, extracting the ground tissue with a solution of phenol/chloroform to remove proteins, and separating RNA from the remaining impurities by selective precipitation with lithium chloride (see, for example, Ausubel et al. (eds.), Short Protocols in Molecular Biology, 3 rd Edition, pages 4-1 to 4-6 (John Wiley & Sons 1995) ["Ausubel (1995)”]; Wu et al, Methods in Gene Biotechnology, pages 33-41 (CRC Press, Inc. 1997) ["Wu (1997)”]).
  • total RNA can be isolated from a suitable tissue by extracting ground tissue with guanidinium isothiocyanate, extracting with organic solvents, and separating RNA from contaminants using differential centrifugation (see, for example, Chirgwin et al, Biochemistry 18:52 (1979); Ausubel (1995) at pages 4-1 to 4-6; Wu (1997) at pages 33-41).
  • poly(A) + RNA In order to construct a cDNA library, poly(A) + RNA must be isolated from a total RNA preparation. Poly(A) + RNA can be isolated from total RNA using the standard technique of oligo(dT)-cellulose chromatography (see, for example, Aviv and Leder, Proc. Nat'l Acad. Sci. USA 69:1408 (1972); Ausubel (1995) at pages 4-11 to 4- 12).
  • Double-stranded cDNA molecules are synthesized from poly(A) + RNA using techniques well-known to those in the art. (see, for example, Wu (1997) at pages 41-46). Moreover, commercially available kits can be used to synthesize double- stranded cDNA molecules. For example, such kits are available from Life Technologies, Inc. (Gaithersburg, MD), CLONTECH Laboratories, Inc. (Palo Alto, CA), Promega Corporation (Madison, WI) and STRATAGENE (La Jolla, CA).
  • a cDNA library can be prepared in a vector derived from bacteriophage, such as a ⁇ gtlO vector. See, for example, Huynh et al, "Constructing and Screening cDNA Libraries in ⁇ gtlO and ⁇ gtl l," in DNA Cloning: A Practical Approach Vol. I, Glover (ed.), page 49 (IRL Press, 1985); Wu (1997) at pages 47-52.
  • double-stranded cDNA molecules can be inserted into a plasmid vector, such as a PBLUESCRIPT vector (STRATAGENE; La Jolla, CA), a LAMDAGEM-4 (Promega Corp.) or other commercially available vectors.
  • a plasmid vector such as a PBLUESCRIPT vector (STRATAGENE; La Jolla, CA), a LAMDAGEM-4 (Promega Corp.) or other commercially available vectors.
  • Suitable cloning vectors also can be obtained from the American Type Culture Collection (Manassas, VA).
  • the cDNA library is inserted into a prokaryotic host, using standard techniques.
  • a cDNA library can be introduced into competent E. coli DH5 cells, which can be obtained, for example, from Life Technologies, Inc. (Gaithersburg, MD).
  • a rat genomic library can be prepared by means well-known in the art
  • Genomic DNA can be isolated by lysing tissue with the detergent Sarkosyl, digesting the lysate with proteinase K, clearing insoluble debris from the lysate by centrifugation, precipitating nucleic acid from the lysate using isopropanol, and purifying resuspended DNA on a cesium chloride density gradient.
  • Genomic DNA fragments that are suitable for the production of a genomic library can be obtained by the random shearing of genomic DNA or by the partial digestion of genomic DNA with restriction endonucleases.
  • Genomic DNA fragments can be inserted into a vector, such as a bacteriophage or cosmid vector, in accordance with conventional techniques, such as the use of restriction enzyme digestion to provide appropriate termini, the use of alkaline phosphatase treatment to avoid undesirable joining of DNA molecules, and ligation with appropriate ligases. Techniques for such manipulation are well-known in the art (see, for example, Ausubel (1995) at pages 5-1 to 5-6; Wu (1997) at pages 307- 327).
  • Nucleic acid molecules that encode a rat autotaxin gene can also be obtained using the polymerase chain reaction (PCR) with oligonucleotide primers having nucleotide sequences that are based upon the nucleotide sequences of the autotaxin gene, as described herein.
  • PCR polymerase chain reaction
  • General methods for screening libraries with PCR are provided by, for example, Yu et al. , "Use of the Polymerase Chain Reaction to Screen Phage Libraries," in Methods in Molecular Biology, Vol. 15: PCR Protocols: Current Methods and Applications, White (ed.), pages 211-215 (Humana Press, Inc. 1993).
  • a library containing cDNA or genomic clones can be screened with one or more polynucleotide probes based upon S ⁇ Q ID NO:l, using standard methods (see, for example, Ausubel (1995) at pages 6-1 to 6-11).
  • Anti-autotaxin antibodies produced as described below, can also be used to isolate DNA sequences that encode rat autotaxin genes from cDNA libraries.
  • the antibodies can be used to screen ⁇ gtl 1 expression libraries, or the antibodies can be used for immunoscreening following hybrid selection and translation (see, for example, Ausubel (1995) at pages 6-12 to 6-16; Margolis et al, "Screening ⁇ expression libraries with antibody and protein probes," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), pages 1-14 (Oxford University Press 1995)).
  • an autotaxin gene can be obtained by synthesizing nucleic acid molecules using mutually priming long oligonucleotides and the nucleotide sequences described herein (see, for example, Ausubel (1995) at pages 8-8 to 8-9).
  • Sources of human autotaxin include A2058 human melanoma cells (ATCC Number: CRL-11147; American Type Culture Collection; Manassas, VA), human teratocarcinoma cell line Ntera2Dl, human neuroblastoma tumor tissues, a neuroblastoma cell line (SMS-KAN), and normal human liver (Stracke et al, J. Biol. Chem. 267:2524 (1992); Lee et al, Biochem. Biophys.
  • nucleic acid molecules of the present invention can also be synthesized with "gene machines” using protocols such as the phosphoramidite method. If chemically-synthesized double stranded DNA is required for an application such as the synthesis of a gene or a gene fragment, then each complementary strand is made separately.
  • the production of short genes (60 to 80 base pairs) is technically straightforward and can be accomplished by synthesizing the complementary strands and then annealing them.
  • Nucleic acid molecules that encode human autotaxin having the amino acid sequence of SEQ ID NO:9 can also be obtained by any of the various methods described above using the nucleic acid and amino acid sequences disclosed herein.
  • the sequence of an autotaxin cDNA or autotaxin genomic fragment can be determined using standard methods. Autotaxin polynucleotide sequences disclosed herein can also be used as probes or primers to clone 5' non-coding regions of an autotaxin gene. In view of the tissue-specific expression observed for autotaxin by northern blotting, this gene region is expected to provide for preferential expression in brain tissue and adipocytes. Promoter elements from an autotaxin gene could thus be used to direct the tissue-specific expression of heterologous genes in, for example, transgenic animals or patients treated with gene therapy. The identification of genomic fragments containing an autotaxin promoter or regulatory element can be achieved using well-established techniques, such as deletion analysis (see, generally, Ausubel (1995)).
  • Cloning of 5' flanking sequences also facilitates production of autotaxin proteins by "gene activation," as disclosed in U.S. Patent No. 5,641,670. Briefly, expression of an endogenous autotaxin gene in a cell is altered by introducing into the autotaxin locus a DNA construct comprising at least a targeting sequence, a regulatory sequence, an exon, and an unpaired splice donor site.
  • the targeting sequence is an autotaxin 5' non-coding sequence that permits homologous recombination of the construct with the endogenous autotaxin locus, whereby the sequences within the construct become operably linked with the endogenous autotaxin coding sequence.
  • an endogenous autotaxin promoter can be replaced or supplemented with other regulatory sequences to provide enhanced, tissue-specific, or otherwise regulated expression.
  • SEQ ID NOs:3 and 10 are degenerate nucleotide sequences that encompass all nucleic acid molecules that encode the autotaxin polypeptides of SEQ ID NOs:2 and 9, respectively. Those skilled in the art will recognize that these degenerate sequences provide all RNA sequences encoding the corresponding amino acid sequences, by substituting U for T.
  • the present invention contemplates autotaxin polypeptide-encoding nucleic acid molecules comprising nucleotide 130 to nucleotide 2703 of SEQ ID NO:l, and their RNA equivalents.
  • the present invention also contemplates autotaxin polypeptide-encoding nucleic acid molecules comprising nucleotide 105 to nucleotide 2681 of SEQ ID NO:8, and their RNA equivalents.
  • Table 1 sets forth the one-letter codes used within SEQ ID NO: 3 to denote degenerate nucleotide positions.
  • “Resolutions” are the nucleotides denoted by a code letter.
  • “Complement” indicates the code for the complementary nucleotide(s). For example, the code Y denotes either C or T, and its complement R denotes A or G, A being complementary to T, and G being complementary to C.
  • degenerate codons used in SEQ ID NOs:3 and 10 encompassing all possible codons for a given amino acid, are set forth in Table 2.
  • degenerate codon representative of all possible codons encoding an amino acid.
  • WSN can, in some circumstances, encode arginine
  • MGN can, in some circumstances, encode serine
  • some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:9. Variant sequences can be readily tested for functionality as described herein.
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2).
  • the amino acid threonine (Thr) may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art.
  • preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species. Therefore, the degenerate codon sequence disclosed in SEQ ID NO: 3 or SEQ ID NO: 10 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
  • the present invention further provides variant polypeptides and nucleic acid molecules that represent counterparts from other species (orthologs). These species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species. Of particular interest are autotaxin polypeptides from mammalian species, including human, mouse, porcine, rat, ovine, bovine, canine, feline, equine, and other primate polypeptides. These autotaxins can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • SEQ ID NO:l represents a single allele of rat autotaxin, and that allelic variation and alternative splicing are expected to occur. Allelic variants of this sequence can be cloned by probing cDNA or genomic libraries from different individuals according to standard procedures. Allelic variants of the nucleotide sequence shown in SEQ ID NO:l or SEQ ID NO:8, including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO:2 or SEQ ID NO:9.
  • cDNA molecules generated from alternatively spliced mRNAs, which retain the properties of the autotaxin polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art.
  • isolated nucleic acid molecules that encode rat autotaxin can hybridize to nucleic acid molecules having the nucleotide sequence of SEQ ID NO:l, or a sequence complementary thereto, under "stringent conditions.”
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • a nucleic acid molecule encoding a variant autotaxin polypeptide can be hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l (or its complement) at 42°C overnight in a solution comprising 50% formamide, 5xSSC (lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution (lOOx Denhardt's solution: 2% (w/v) Ficoll 400, 2% (w/v) polyvinylpyrrolidone, and 2% (w/v) bovine serum albumin), 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA.
  • 5xSSC lxSSC: 0.15 M sodium chloride and 15 mM sodium citrate
  • 50 mM sodium phosphate pH 7.6
  • 5x Denhardt's solution lOOx Den
  • hybridization mixture can be incubated at a higher temperature, such as about 65°C, in a solution that does not contain formamide.
  • premixed hybridization solutions are available (e.g., EXPRESSHYB Hybridization Solution from CLONTECH Laboratories, Inc.), and hybridization can be performed according to the manufacturer's instructions.
  • the nucleic acid molecules can be washed to remove non-hybridized nucleic acid molecules under stringent conditions, or under highly stringent conditions.
  • Typical stringent washing conditions include washing in a solution of 0.5x - 2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 55 - 65°C. That is, nucleic acid molecules encoding a variant autotaxin polypeptide remain hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: l (or its complement) following stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65°C, including 0.5x SSC with 0.1% SDS at 55°C, or 2xSSC with 0.1% SDS at 65°C.
  • One of skill in the art can readily devise equivalent conditions, for example, by substituting SSPE for SSC in the wash solution.
  • Typical highly stringent washing conditions include washing in a solution of O.lx - 0.2x SSC with 0.1% sodium dodecyl sulfate (SDS) at 50 - 65°C.
  • SDS sodium dodecyl sulfate
  • nucleic acid molecules encoding a variant autotaxin polypeptide remain hybridized with a nucleic acid molecule having the nucleotide sequence of SEQ ID NO: 1 (or its complement) under highly stringent washing conditions, in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65°C, including O.lx SSC with 0.1% SDS at 50°C, or 0.2xSSC with 0.1% SDS at 65°C.
  • the present invention also provides isolated autotaxin polypeptides that have a substantially similar sequence identity to the polypeptides of SEQ ID NO:2, SEQ ID NO:9, or their orthologs.
  • substantially similar sequence identity is used herein to denote polypeptides having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the sequences shown in SEQ ID NO:2, or their orthologs.
  • variant autotaxin does not include the following proteins: (1) human autotaxin isolated from melanoma cells (Murata et al, J. Biol. Chem. 269:30419 (1994)), which is encoded by the nucleotide sequence having GenBank accession No.
  • the present invention also contemplates autotaxin variant nucleic acid molecules that can be identified using two criteria: a determination of the similarity between the encoded polypeptide with the amino acid sequence of SEQ ID NO:2, and a hybridization assay, as described above.
  • autotaxin variants include nucleic acid molecules (1) that remain hybridized with a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l (or its complement) following stringent washing conditions, in which the wash stringency is equivalent to 0.5x - 2x SSC with 0.1% SDS at 55 - 65 °C, and (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NO:2.
  • autotaxin variants can be characterized as nucleic acid molecules (1) that remain hybridized with a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l (or its complement) following highly stringent washing conditions, in which the wash stringency is equivalent to O.lx - 0.2x SSC with 0.1% SDS at 50 - 65°C, and (2) that encode a polypeptide having at least 70%, at least 80%, at least 90%, at least 95% or greater than 95% sequence identity to the amino acid sequence of SEQ ID NO:2.
  • Percent sequence identity is determined by conventional methods. See, for example, Altschul et al, Bull. Math. Bio. 48:603 (1986), and Henikoff and Henikoff, Proc. Natl Acad. Sci. USA 59: 10915 (1992). Briefly, two amino acid sequences are aligned to optimize the alignment scores using a gap opening penalty of 10, a gap extension penalty of 1, and the "BLOSUM62" scoring matrix of Henikoff and Henikoff (ibid.) as shown in Table 3 (amino acids are indicated by the standard one- letter codes). The percent identity is then calculated as: ([Total number of identical matches]/ [length of the longer sequence plus the number of gaps introduced into the longer sequence in order to align the two sequences])(100).
  • the "FASTA" similarity search algorithm of Pearson and Lipman is a suitable protein alignment method for examining the level of identity shared by an amino acid sequence disclosed herein and the amino acid sequence of a putative autotaxin variant.
  • the FASTA algorithm is described by Pearson and Lipman, Proc. Nat 'l Acad. Sci. USA 55:2444 (1988), and by Pearson, Meth. Enzymol 183:63 (1990).
  • the ten regions with the highest density of identities are then rescored by comparing the similarity of all paired amino acids using an amino acid substitution matrix, and the ends of the regions are "trimmed" to include only those residues that contribute to the highest score.
  • the trimmed initial regions are examined to determine whether the regions can be joined to form an approximate alignment with gaps.
  • the highest scoring regions of the two amino acid sequences are aligned using a modification of the Needleman-Wunsch-Sellers algorithm (Needleman and Wunsch, J. Mol Biol. 48:444 (1970); Sellers, SIAMJ. Appl. Math. 26:181 (1974)), which allows for amino acid insertions and deletions.
  • FASTA can also be used to determine the sequence identity of nucleic acid molecules using a ratio as disclosed above.
  • the ktup value can range between one to six, preferably from four to six.
  • the present invention includes nucleic acid molecules that encode a polypeptide having a conservative amino acid change, compared with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:9. That is, variants can be obtained that contain one or more amino acid substitutions of SEQ ID NO:2 or SEQ ID NO:9, in which an alkyl amino acid is substituted for an alkyl amino acid in an autotaxin amino acid sequence, an aromatic amino acid is substituted for an aromatic amino acid in an autotaxin amino acid sequence, a sulfur-containing amino acid is substituted for a sulfur-containing amino acid in an autotaxin amino acid sequence, a hydroxy- containing amino acid is substituted for a hydroxy-containing amino acid in an autotaxin amino acid sequence, an acidic amino acid is substituted for an acidic amino acid in an autotaxin amino acid sequence, a basic amino acid is substituted for a basic amino acid in an autotaxin amino acid sequence, or a dibasic monocarboxylic amino acid is substituted for a dibasic monocarboxy
  • a “conservative amino acid substitution” is illustrated by a substitution among amino acids within each of the following groups: (1) glycine, alanine, valine, leucine, and isoleucine, (2) phenylalanine, tyrosine, and tryptophan, (3) serine and threonine, (4) aspartate and glutamate, (5) glutamine and asparagine, and (6) lysine, arginine and histidine.
  • variant autotaxin polypeptides that have an amino acid sequence that differs from SEQ ID NO:2 can be obtained by substituting valine for alanine 93 , valine for alanine 194 , threonine for serine 236 , lysine for arginine 265 , and threonine for serine 289 . Additional variants can be obtained by producing polypeptides having two or more of these amino acid substitutions.
  • the BLOSUM62 table is an amino acid substitution matrix derived from about 2,000 local multiple alignments of protein sequence segments, representing highly conserved regions of more than 500 groups of related proteins (Henikoff and Henikoff, Proc. Nat'/ Acad. Sci. USA 59:10915 (1992)). Accordingly, the BLOSUM62 substitution frequencies can be used to define conservative amino acid substitutions that may be introduced into the amino acid sequences of the present invention. Although it is possible to design amino acid substitutions based solely upon chemical properties (as discussed above), the language "conservative amino acid substitution” preferably refers to a substitution represented by a BLOSUM62 value of greater than -1. For example, an amino acid substitution is conservative if the substitution is characterized by a BLOSUM62 value of 0, 1, 2, or 3.
  • preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 1 (e.g., X, 2 or 3), while more preferred conservative amino acid substitutions are characterized by a BLOSUM62 value of at least 2 (e.g., 2 or 3).
  • Particular variants of rat autotaxin are characterized by having at least
  • Conservative amino acid changes in an autotaxin gene can be introduced by substituting nucleotides for the nucleotides recited in SEQ ID NO:l .
  • Such "conservative amino acid” variants can be obtained, for example, by oligonucleotide- directed mutagenesis, linker-scanning mutagenesis, mutagenesis using the polymerase chain reaction, and the like (see Ausubel (1995) at pages 8-10 to 8-22; and McPherson (ed.), Directed Mutagenesis: A Practical Approach (IRL Press 1991)).
  • the ability of such variants to inhibit the differentiation of adipocytes can be determined using a standard method, such as the assay described herein.
  • a variant autotaxin polypeptide can be identified by the ability to specifically bind anti-autotaxin antibodies.
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, tra «5-3-methylproline, 2,4-methanoproline, cw-4-hydroxyproline, trans-4- hydroxyproline, N-methylglycine, ⁇ // -threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3,3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine.
  • coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2-azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4- fluorophenylalanine).
  • the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al, Biochem. 33:1410 (1994).
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vitro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395 (1993)).
  • a limited number of non-conservative amino acids, amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for autotaxin amino acid residues.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244A08X (1989), Bass et al, Proc. Nat'l Acad. Sci. USA 55:4498 (1991), Coombs and Corey, "Site- Directed Mutagenesis and Protein Engineering,” in Proteins: Analysis and Design, Angeletti (ed.), pages 259-31 1 (Academic Press, Inc. 1998)).
  • sequence analysis can be used to identify autotaxin receptor binding sites
  • location of autotaxin receptor binding domains can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al, Science 255:306 (1992), Smith et al, J. Mol Biol 224:899 (1992), and Wlodaver et al, FEBS Lett. 309:59 (1992).
  • autotaxin labeled with biotin or FITC can be used for expression cloning of autotaxin receptors.
  • variants of the disclosed autotaxin nucleotide and polypeptide sequences can also be generated through DNA shuffling as disclosed by Stemmer, Nature 370:389 (1994), Stemmei, Proc. Nat'l Acad. Sci. USA 9T ⁇ 0141 (1994), and international publication No. WO 97/20078. Briefly, variant DNA molecules are generated by in vitro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNA molecules, such as allelic variants or DNA molecules from different species, to introduce additional variability into the process. Selection or screening for the desired activity, followed by additional iterations of mutagenesis and assay provides for rapid "evolution" of sequences by selecting for desirable mutations while simultaneously selecting against detrimental changes.
  • Mutagenesis methods as disclosed herein can be combined with high- throughput, automated screening methods to detect activity of cloned, mutagenized polypeptides in host cells.
  • Mutagenized DNA molecules that encode biologically active polypeptides, or polypeptides that bind with anti-autotaxin antibodies can be recovered from the host cells and rapidly sequenced using modern equipment. These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • the present invention also includes "functional fragments" of autotaxin polypeptides and nucleic acid molecules encoding such functional fragments.
  • Routine deletion analyses of nucleic acid molecules can be performed to obtain functional fragments of a nucleic acid molecule that encodes an autotaxin polypeptide.
  • DNA molecules having the nucleotide sequence of SEQ ID NO:l or SEQ ID NO: 8 can be digested with Bal3X nuclease to obtain a series of nested deletions. The fragments are then inserted into expression vectors in proper reading frame, and the expressed polypeptides are isolated and tested for biological activity, or for the ability to bind anti-autotaxin antibodies.
  • exonuclease digestion is to use oligonucleotide-directed mutagenesis to introduce deletions or stop codons to specify production of a desired fragment.
  • particular fragments of an autotaxin gene can be synthesized using the polymerase chain reaction.
  • rat autotaxin produced by cultured cells indicates that one form of autotaxin consists of amino acid residues 32 to 858 of SEQ ID NO:2.
  • This example of a functional fragment includes the RGD binding site.
  • Another example of a functional fragment is a polypeptide consisting of the first 500 amino acids of SEQ ID NO:2, which would contain the portion of autotaxin associated with phosphodiesterase activity.
  • a functional fragment of the human autotaxin having the amino acid sequence of SEQ ID NO:9 includes the RGD binding site at amino acids 123 to 125.
  • Another functional fragment of this form of autotaxin comprises amino acids 197 to 209, the putative phosphodiesterase active site.
  • the present invention also contemplates functional fragments of an autotaxin gene that has amino acid changes, compared with the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:9.
  • a variant autotaxin gene can be identified on the basis of structure by determining the level of identity with nucleotide and amino acid sequence of SEQ ID NO:2 or SEQ ID NO:9, as discussed above.
  • An alternative approach to identifying a variant gene on the basis of structure is to determine whether a nucleic acid molecule encoding a potential variant autotaxin gene can hybridize to a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l or SEQ ID NO: 8, as discussed above.
  • the present invention also provides polypeptide fragments or peptides comprising an epitope-bearing portion of an autotaxin polypeptide described herein.
  • Such fragments or peptides may comprise an "immunogenic epitope," which is a part of a protein that elicits an antibody response when the entire protein is used as an immunogen.
  • Immunogenic epitope-bearing peptides can be identified using standard methods (see, for example, Geysen et al, Proc. Nat'l Acad. Sci. USA 81:3998 (1983)).
  • polypeptide fragments or peptides may comprise an "antigenic epitope," which is a region of a protein molecule to which an antibody can specifically bind.
  • Certain epitopes consist of a linear or contiguous stretch of amino acids, and the antigenicity of such an epitope is not disrupted by denaturing agents. It is known in the art that relatively short synthetic peptides that can mimic epitopes of a protein can be used to stimulate the production of antibodies against the protein (see, for example, Sutcliffe et al, Science 219:660 (1983)). Accordingly, antigenic epitope- bearing peptides and polypeptides of the present invention are useful to raise antibodies that bind with the polypeptides described herein.
  • Antigenic epitope-bearing peptides and polypeptides can contain at least four to ten amino acids, at least ten to fifteen amino acids, or about 15 to about 30 amino acids of SEQ ID NO:2 or SEQ ID NO:9. Such epitope-bearing peptides and polypeptides can be produced by fragmenting an autotaxin polypeptide, or by chemical peptide synthesis, as described herein. Moreover, epitopes can be selected by phage display of random peptide libraries (see, for example, Lane and Stephen, Curr. Opin. Immunol. 5:268 (1993), and Cortese et al, Curr. Opin. Biotechnol 7:6X6 (1996)).
  • variant autotaxin gene encodes a polypeptide that is characterized by its biological activity, as described herein, or by the ability to bind specifically to an anti-autotaxin antibody. More specifically, variant autotaxin genes encode polypeptides, which exhibit at least 50%, or even greater than 70, 80, or 90%, of the activity of polypeptide encoded by the autotaxin gene described herein.
  • any autotaxin polypeptide including variants and fusion proteins, one of ordinary skill in the art can readily generate a fully degenerate polynucleotide sequence encoding that variant using the information set forth in Tables 1 and 2 above.
  • the present invention includes a computer-readable medium encoded with a data structure that provides at least one of the following sequences: SEQ ID NO:l, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 10.
  • Suitable forms of computer-readable media include magnetic media and optically-readable media. Examples of magnetic media include a hard or fixed drive, a random access memory (RAM) chip, a floppy disk, digital linear tape (DLT), a disk cache, and a ZIP disk.
  • Optically readable media are exemplified by compact discs (e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD-recordable), and digital versatile/video discs (DVD) (e.g., DVD-ROM, DVD-RAM, and DVD+RW).
  • compact discs e.g., CD-read only memory (ROM), CD-rewritable (RW), and CD-recordable
  • DVD digital versatile/video discs
  • Fusion proteins of autotaxin can be used to express autotaxin in a recombinant host, and to isolate expressed autotaxin. As described below, particular autotaxin fusion proteins also have uses in diagnosis and therapy.
  • One type of fusion protein comprises a peptide that guides an autotaxin polypeptide from a recombinant host cell.
  • a secretory signal sequence also known as a signal peptide, a leader sequence, prepro sequence or pre sequence
  • the secretory signal sequence may be derived from autotaxin, a suitable signal sequence may also be derived from another secreted protein or synthesized de novo.
  • the secretory signal sequence is operably linked to an autotaxin-encoding sequence such that the two sequences are joined in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the nucleotide sequence encoding the polypeptide of interest, although certain secretory signal sequences may be positioned elsewhere in the nucleotide sequence of interest (see, e.g., Welch et ⁇ l, U.S. Patent No. 5,037,743; Holland et ⁇ l, U.S. Patent No. 5,143,830).
  • yeast signal sequence is preferred for expression in yeast cells.
  • suitable yeast signal sequences are those derived from yeast mating phermone ⁇ -factor (encoded by the MF ⁇ l gene), invertase (encoded by the SUC2 gene), or acid phosphatase (encoded by the PH05 gene). See, for example, Romanos et ⁇ l. , "Expression of Cloned Genes in Yeast," in DNA Cloning 2: A Practical Approach, 2 nd Edition, Glover and Hames (eds.), pages 123-167 (Oxford University Press 1995).
  • autotaxin can be expressed as a fusion protein comprising a glutathione S-transferase polypeptide.
  • Glutathione S-transferease fusion proteins are typically soluble, and easily purifiable from E. coli lysates on immobilized glutathione columns.
  • an autotaxin fusion protein comprising a maltose binding protein polypeptide can be isolated with an amylose resin column, while a fusion protein comprising the C-terminal end of a truncated Protein A gene can be purified using IgG-Sepharose.
  • Peptide tags that are useful for isolating heterologous polypeptides expressed by either prokaryotic or eukaryotic cells include polyHistidine tags (which have an affinity for nickel-chelating resin), c-myc tags, calmodulin binding protein (isolated with calmodulin affinity chromatography), substance P, the RYIRS tag (which binds with anti-RYIRS antibodies), the Glu-Glu tag, and the FLAG tag (which binds with anti-FLAG antibodies). See, for example, Luo et al, Arch. Biochem. Biophys. 329:2X5 (1996), Morganti et al, Biotechnol Appl. Biochem. 23:61 (1996), and Zheng et al, Gene 186:55 (1997). Nucleic acid molecules encoding such peptide tags are available, for example, from Sigma-Aldrich Corporation (St. Louis, MO).
  • the present invention also contemplates that the use of the secretory signal sequence contained in the autotaxin polypeptides of the present invention to direct other polypeptides into the secretory pathway.
  • a signal fusion polypeptide may be made wherein a secretory signal sequence derived from amino acid residues 1 to 31 of SEQ ID NO:2 is operably linked to another polypeptide using methods known in the art and disclosed herein.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • Such constructs have numerous applications known in the art.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as a receptor.
  • a normally non-secreted protein such as a receptor.
  • Such fusions may be used in a transgenic animal or in a cultured recombinant host to direct peptides through the secretory pathway.
  • exemplary polypeptides include pharmaceutically active molecules such as Factor Vila, proinsulin, insulin, follicle stimulating hormone, tissue type plasminogen activator, tumor necrosis factor, interleukins (e.g., interleukin-1 (IL-1), IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL- 10, IL-11, IL-12, IL-13, IL-14, and IL-15), colony stimulating factors (e.g., granulocyte-colony stimulating factor (G-CSF) and granulocyte macrophage-colony stimulating factor (GM-CSF)), interferons (e.g., interferons- ⁇ , - ⁇ , - ⁇ , - ⁇ , and -x), the stem cell growth factor designated "SI factor,” erythropoietin, and thrombopoietin.
  • interleukins e.g., interleukin-1 (IL
  • the autotaxin secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway. Fusion proteins comprising an autotaxin secretory signal sequence can be constructed using standard techniques.
  • fusion protein comprises an autotaxin polypeptide and an immunoglobulin heavy chain constant region, typically an F c fragment, which contains two or three constant region domains and a hinge region but lacks the variable region.
  • an immunoglobulin heavy chain constant region typically an F c fragment
  • F c fragment an immunoglobulin heavy chain constant region
  • Chang et al, U.S. Patent No. 5,723,125 describe a fusion protein comprising a human interferon and a human immunoglobulin Fc fragment.
  • the C-terminal of the interferon is linked to the N-terminal of the Fc fragment by a peptide linker moiety.
  • An example of a peptide linker is a peptide comprising primarily a T cell inert sequence, which is immunologically inert.
  • An exemplary peptide linker has the amino acid sequence: GGSGG SGGGG SGGGG S (SEQ ID NO:7).
  • a preferred Fc moiety is a human ⁇ 4 chain, which is stable in solution and has little or no complement activating activity.
  • the present invention contemplates an autotaxin fusion protein that comprises an autotaxin moiety and a human Fc fragment, wherein the C-terminus of the autotaxin moiety is attached to the N-terminus of the Fc fragment via a peptide linker, such as a peptide consisting of the amino acid sequence of SEQ ID NO: 7.
  • the autotaxin moiety can be an autotaxin molecule or a fragment thereof.
  • an autotaxin fusion protein comprises an IgG sequence, an autotaxin moiety covalently joined to the amino terminal end of the IgG sequence, and a signal peptide that is covalently joined to the amino terminal of the autotaxin moiety, wherein the IgG sequence consists of the following elements in the following order: a hinge region, a CH 2 domain, and a CH 3 domain. Accordingly, the IgG sequence lacks a CH, domain.
  • the autotaxin moiety displays an autotaxin activity, as described herein, such as the ability to bind with an autotaxin receptor, or the ability to inhibit the differentiation of adipocytes.
  • Fusion proteins comprising an autotaxin moiety and an Fc moiety can be used, for example, as an in vitro assay tool.
  • the presence of an autotaxin receptor in a biological sample can be detected using an autotaxin-immunoglobulin fusion protein, in which the autotaxin moiety is used to target the cognate receptor, and a macromolecule, such as Protein A or anti-Fc antibody, is used to detect the bound fusion protein-receptor complex.
  • fusion proteins can be used to identify agonists and antagonists that interfere with the binding of autotaxin to its receptor.
  • hybrid autotaxin proteins can be constructed using regions or domains of the inventive autotaxin in combination with those of other insulin-like, or heterologous proteins (see, for example, Picard, Cur. Opin. Biology 5:51 1 (1994)). These methods allow the determination of the biological importance of larger domains or regions in a polypeptide of interest. Such hybrids may alter reaction kinetics, binding, constrict or expand the substrate specificity, or alter tissue and cellular localization of a polypeptide, and can be applied to polypeptides of unknown structure.
  • Fusion proteins can be prepared by methods known to those skilled in the art by preparing each component of the fusion protein and chemically conjugating them. Alternatively, a polynucleotide encoding both components of the fusion protein in the proper reading frame can be generated using known techniques and expressed by the methods described herein. General methods for enzymatic and chemical cleavage of fusion proteins are described, for example, by Ausubel (1995) at pages 16-19 to 16-25.
  • polypeptides of the present invention can be produced in recombinant host cells following conventional techniques.
  • a nucleic acid molecule encoding the polypeptide must be operably linked to regulatory sequences that control transcriptional expression in an expression vector and then, introduced into a host cell.
  • expression vectors can include translational regulatory sequences and a marker gene, which is suitable for selection of cells that carry the expression vector.
  • Expression vectors that are suitable for production of a foreign protein in eukaryotic cells typically contain (1) prokaryotic DNA elements coding for a bacterial replication origin and an antibiotic resistance marker to provide for the growth and selection of the expression vector in a bacterial host; (2) eukaryotic DNA elements that control initiation of transcription, such as a promoter; and (3) DNA elements that control the processing of transcripts, such as a transcription termination/polyadenylation sequence.
  • expression vectors can also include nucleotide sequences encoding a secretory sequence that directs the heterologous polypeptide into the secretory pathway of a host cell.
  • an autotaxin expression vector may comprise an autotaxin gene and a secretory sequence derived from an autotaxin gene or another secreted gene.
  • Autotaxin proteins of the present invention may be expressed in mammalian cells.
  • suitable mammalian host cells include African green monkey kidney cells (Vero; ATCC CRL 1587), human embryonic kidney cells (293- HEK; ATCC CRL 1573), baby hamster kidney cells (BHK-21, BHK-570; ATCC CRL 8544, ATCC CRL 10314), canine kidney cells (MDCK; ATCC CCL 34), Chinese hamster ovary cells (CHO-K1 ; ATCC CCL61 ; CHO DG44 [Chasin et al, Som. Cell. Molec. Genet.
  • rat pituitary cells GHl ; ATCC CCL82
  • HeLa S3 cells ATCC CCL2.2
  • rat hepatoma cells H-4-II-E
  • COS-1 ATCC CRL 1650
  • murine embryonic cells NIH- 3T3; ATCC CRL 1658.
  • Narita et al, J. Biol Chem. 269:28235 (1994) have described the expression of recombinant rat autotaxin in COS-7 and human astrocytoma cell line (U87 cells).
  • the transcriptional and translational regulatory signals may be derived from viral sources, such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression.
  • viral sources such as adenovirus, bovine papilloma virus, simian virus, or the like, in which the regulatory signals are associated with a particular gene which has a high level of expression.
  • Suitable transcriptional and translational regulatory sequences also can be obtained from mammalian genes, such as actin, collagen, myosin, and metallothionein genes.
  • Transcriptional regulatory sequences include a promoter region sufficient to direct the initiation of RNA synthesis.
  • Suitable eukaryotic promoters include the promoter of the mouse metallothionein I gene (Hamer et al, J. Molec. Appl. Genet. 1:213 (1982)), the TK promoter of Herpes virus (McKnight, Cell 31:355 (1982)), the SV40 early promoter (Benoist et al, Nature 290:304 (1981)), the Rous sarcoma virus promoter (Gorman et al, Proc. Nat'l Acad. Sci.
  • a prokaryotic promoter such as the bacteriophage T3 RNA polymerase promoter
  • a prokaryotic promoter can be used to control autotaxin gene expression in mammalian cells if the prokaryotic promoter is regulated by a eukaryotic promoter (Zhou et al, Mol Cell. Biol. 10:4529 (1990), and Kaufman et al, Nucl Acids Res. 7P:4485 (1991)).
  • An expression vector can be introduced into host cells using a variety of standard techniques including calcium phosphate transfection, liposome-mediated transfection, microprojectile-mediated delivery, electroporation, and the like.
  • the transfected cells are selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
  • Techniques for introducing vectors into eukaryotic cells and techniques for selecting such stable transformants using a dominant selectable marker are described, for example, by Ausubel (1995) and by Murray (ed.), Gene Transfer and Expression Protocols (Humana Press 1991).
  • one suitable selectable marker is a gene that provides resistance to the antibiotic neomycin.
  • selection is carried out in the presence of a neomycin-type drug, such as G-418 or the like.
  • Selection systems can also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a suitable amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • drugs resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4, CD8, Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • Autotaxin polypeptides can also be produced by cultured mammalian cells using a viral delivery system.
  • viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno-associated virus (AAV).
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see Becker et al, Meth. Cell Biol 43:161 (1994), and Douglas and Curiel, Science & Medicine 4:44 (1997)).
  • Advantages of the adenovirus system include the accommodation of relatively large DNA inserts, the ability to grow to high-titer, the ability to infect a broad range of mammalian cell types, and flexibility that allows use with a large number of available vectors containing different promoters.
  • Adenovirus vector-infected human 293 cells ATCC Nos. CRL-1573, 45504, 45505
  • Adenovirus vector-infected human 293 cells can be grown as adherent cells or in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al, Cytotechnol. 75:145 (1994)).
  • Autotaxin may also be expressed in other higher eukaryotic cells, such as avian, fungal, insect, yeast, or plant cells.
  • the baculovirus system provides an efficient means to introduce cloned Autotaxin genes into insect cells.
  • Suitable expression vectors are based upon the Autographa calif ornica multiple nuclear polyhedrosis virus (AcMNPV), and contain well-known promoters such as Drosophila heat shock protein (hsp) 70 promoter, Autographa calif ornica nuclear polyhedrosis virus immediate-early gene promoter (/e-7) and the delayed early 39K promoter, baculovirus plO promoter, and the Drosophila metallothionein promoter.
  • hsp Drosophila heat shock protein
  • /e-7 Autographa calif ornica nuclear polyhedrosis virus immediate-early gene promoter
  • the delayed early 39K promoter baculovirus plO promoter
  • baculovirus plO promoter
  • a second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow, et al, J. Virol. 67:4566 (1993)).
  • This system which utilizes transfer vectors, is sold in the BAC-to-BAC kit (Life Technologies, Rockville, MD).
  • This system utilizes a transfer vector, PFASTBAC (Life Technologies) containing a Tn7 transposon to move the DNA encoding the autotaxin polypeptide into a baculovirus genome maintained in E. coli as a large plasmid called a "bacmid.” See, Hill-Perkins and Possee, J. Gen. Virol. 77:971 (1990), Bonning, et al, J. Gen.
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed autotaxin polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al, Proc. Nat'l Acad. Sci. 52:7952 (1985)).
  • a transfer vector containing an autotaxin gene is transformed into E. coli, and screened for bacmids, which contain an interrupted lacZ gene indicative of recombinant baculovirus.
  • the bacmid DNA containing the recombinant baculovirus genome is then isolated using common techniques.
  • the recombinant virus or bacmid is used to transfect host cells.
  • suitable insect host cells include cell lines derived from IPLB-S/ ⁇ l, a Spodoptera frugiperda pupal ovarian cell line, such as 9 (ATCC CRL 1711), S21A ⁇ , and 5 21 (Invitrogen Corporation; San Diego, CA), as well as Drosophila Schneider-2 cells, and the HIGH FIVEO cell line (Invitrogen) derived from Trichoplusia ni (U.S. Patent No. 5,300,435).
  • Commercially available serum-free media can be used to grow and to maintain the cells.
  • Suitable media are Sf900 IITM (Life Technologies) or ESF 921TM (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
  • the cells are typically grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • yeast cells can also be used to express the genes described herein.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris, and Pichia methanolica.
  • Suitable promoters for expression in yeast include promoters from GAL1 (galactose), PGK (phosphoglycerate kinase), ADH (alcohol dehydrogenase), AOX1 (alcohol oxidase), HIS4 (histidinol dehydrogenase), and the like.
  • GAL1 galactose
  • PGK phosphoglycerate kinase
  • ADH alcohol dehydrogenase
  • AOX1 alcohol oxidase
  • HIS4 histidinol dehydrogenase
  • These vectors include Yip-based vectors, such as YIp5, YRp vectors, such as YRpl7, YEp vectors such as YEpl3 and YCp vectors, such as YCpl9.
  • Methods for transforming S. cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311, Kawasaki et al, U.S. Patent No. 4,931,373, Brake, U.S. Patent No. 4,870,008, Welch et al, U.S. Patent No. 5,037,743, and Murray et al, U.S. Patent No.
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine).
  • a suitable vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al. (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media. Additional suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311, Kingsman et al, U.S. Patent No. 4,615,974, and Bitter, U.S. Patent No.
  • Aspergillus cells may be utilized according to the methods of McKnight et al, U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by Sumino et al, U.S. Patent No. 5,162,228. Methods for transforming Neurospora are disclosed by Lambowitz, U.S. Patent No. 4,486,533.
  • Pichia methanolica as host for the production of recombinant proteins is disclosed by Raymond, U.S. Patent No. 5,716,808, Raymond, U.S. Patent No. 5,736,383, Raymond et al, Yeast 74:11-23 (1998), and in international publication Nos. WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
  • DNA molecules for use in transforming P. methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a P.
  • methanolica gene such as a P. methanolica alcohol utilization gene (A UG1 or A UG2).
  • Other useful promoters include those of the dihydroxyacetone synthase (DHAS), formate dehydrogenase (FMD), and catalase (CAT) genes.
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • methanolica ADE2 gene which encodes phosphoribosyl-5- aminoimidazole carboxylase (AIRC; EC 4.1.1.21), and which allows ade2 host cells to grow in the absence of adenine.
  • methanolica ADE2 gene which encodes phosphoribosyl-5- aminoimidazole carboxylase (AIRC; EC 4.1.1.21), and which allows ade2 host cells to grow in the absence of adenine.
  • a UG1 and A UG2 methanol utilization genes
  • host cells deficient in vacuolar protease genes PEP4 and PRB1 are preferred. Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells.
  • methanolica cells can be transformed by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant (t) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Expression vectors can also be introduced into plant protoplasts, intact plant tissues, or isolated plant cells.
  • Methods for introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant tissue with Agrobacterium tumefaciens, microprojectile-mediated delivery, DNA injection, electroporation, and the like. See, for example, Horsch et al, Science 227:1229 (1985), Klein et al, Biotechnology 70:268 (1992), and Miki et al, "Procedures for Introducing Foreign DNA into Plants," in Methods in Plant Molecular Biology and Biotechnology, Glick et al. (eds.), pages 67-88 (CRC Press, 1993).
  • autotaxin genes can be expressed in prokaryotic host cells.
  • Suitable promoters that can be used to express autotaxin polypeptides in a prokaryotic host are well-known to those of skill in the art and include promoters capable of recognizing the T4, T3, Sp6 and T7 polymerases, the P R and P L promoters of bacteriophage lambda, the trp, recA, heat shock, lacUVS, tac, Ipp-lacSpr, phoA, and lacZ promoters of E. coli, promoters of B.
  • subtilis subtilis, the promoters of the bacteriophages of Bacillus, Streptomyces promoters, the int promoter of bacteriophage lambda, the bla promoter of pBR322, and the CAT promoter of the chloramphenicol acetyl transferase gene.
  • Prokaryotic promoters have been reviewed by Glick, J. Ind. Microbiol. 7:277 (1987), Watson et al, Molecular Biology of the Gene, 4th Ed. (Benjamin Cummins 1987), and by Ausubel et al. (1995j.
  • Suitable prokaryotic hosts include E. coli and Bacillus subtilus.
  • Suitable strains of E. coli include BL21(D ⁇ 3), BL21(DE3)pLysS, BL21(DE3)pLysE, DH1, DH4I, DH5, DH5I, DH5IF', DH5IMCR, DH10B, DH10B/p3, DH11S, C600, HB101, JM101, JM105, JM109, JM1 10, K38, RR1, Y1088, Y1089, CSH18, ER1451, and ER1647 (see, for example, Brown (ed.), Molecular Biology Labfax (Academic Press 1991)).
  • Suitable strains of Bacillus subtilus include BR151, YB886, Mil 19, MI 120, and B170 (see, for example, Hardy, "Bacillus Cloning Methods,” in DNA Cloning: A Practical Approach, Glover (ed.) (IRL Press 1985)).
  • the polypeptide When expressing an autotaxin polypeptide in bacteria such as E. coli, the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence. In the former case, the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea. The denatured polypeptide can then be refolded and dimerized by diluting the denaturant, such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the denaturant such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione
  • the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • polypeptides of the present invention can be synthesized by exclusive solid phase synthesis, partial solid phase methods, fragment condensation or classical solution synthesis. These synthesis methods are well-known to those of skill in the art (see, for example, Merrifield, J. Am. Chem. Soc. 55:2149 (1963), Stewart et al, "Solid Phase Peptide Synthesis” (2nd Edition), (Pierce Chemical Co. 1984), Bayer and Rapp, Chem. Pept. Prot.
  • Polypeptides of the present invention can comprise at 15, 20, 25, or 30 contiguous amino acid residues of SEQ ID NO:2.
  • polypeptides can comprise 15, 20, 25, or 30 contiguous amino acids of amino acid residues 135 to 172 of SEQ ID NO:2.
  • the polypeptides comprise 40, 50, 100, or more contiguous residues of SEQ ID NO:2.
  • such polypeptides can comprise 50 contiguous amino acids of amino acid residues 545 to 655 of SEQ ID NO:2.
  • Nucleic acid molecules encoding such peptides and polypeptides are useful as polymerase chain reaction primers and probes.
  • polypeptides of the present invention can be purified to at least about 80% purity, to at least about 90% purity, to at least about 95% purity, or even greater than 95% purity with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents.
  • the polypeptides of the present invention may also be purified to a pharmaceutically pure state, which is greater than 99.9% pure.
  • Certain preparations comprise a purified polypeptide that is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • Fractionation and/or conventional purification methods can be used to obtain preparations of autotaxin purified from natural sources (e.g., adipocytes), and recombinant autotaxin polypeptides and fusion autotaxin polypeptides purified from recombinant host cells.
  • natural sources e.g., adipocytes
  • ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography. Suitable chromatographic media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl- Sepharose FF (Pharmacia), Toyopearl butyl 650 (Toso Haas, Montgomery ville, PA), Octyl-Sepharose (Pharmacia) and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Phenyl- Sepharose FF Pharmacia
  • Toyopearl butyl 650 Toso Haas, Montgomery ville, PA
  • Octyl-Sepharose Pharmacia
  • polyacrylic resins such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica- based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Selection of a particular method for polypeptide isolation and purification is a matter of routine design and is determined in part by the properties of the chosen support (see, for example, Affinity Chromatography: Principles & Methods (Pharmacia LKB Biotechnology 1988), and Doonan, Protein Purification Protocols (The Humana Press 1996)).
  • Example 2 illustrates the use of concanavilin A affinity chromatography to purify autotaxin.
  • autotaxin isolation and purification can be devised by those of skill in the art.
  • anti-autotaxin antibodies obtained as described below, can be used to isolate large quantities of protein by immunoaffinity purification.
  • methods for binding ligands, such as autotaxin, to receptor polypeptides bound to support media are well known in the art.
  • the polypeptides of the present invention can also be isolated by exploitation of particular properties.
  • immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins, including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 5:1 (1985)). Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • IMAC immobilized metal ion adsorption
  • a fusion of the polypeptide of interest and an affinity tag may be constructed to facilitate purification.
  • an affinity tag e.g., maltose-binding protein, an immunoglobulin domain
  • Autotaxin polypeptides or fragments thereof may also be prepared through chemical synthesis, as described below.
  • Autotaxin polypeptides may be monomers or multimers; glycosylated or non-glycosylated; PEGylated or non- PEGylated; and may or may not include an initial methionine amino acid residue.
  • an autotaxin variant can be functionally characterized by its ability to specifically bind with an anti-autotaxin antibody, or by a biological activity that affects metabolism. Illustrative assays that measure such biological activity are described below.
  • a polypeptide produced by an autotaxin variant gene is considered to be an autotaxin agonist if the polypeptide exhibits a biological activity.
  • an autotaxin variant gene product that lacks biological activity may be an autotaxin antagonist.
  • These biologically-inactive autotaxin variants can be initially identified on the basis of hybridization analysis, sequence identity determination, or by the ability to specifically bind anti-autotaxin antibody.
  • An autotaxin antagonist can be further characterized by its ability to inhibit the biological response induced by autotaxin or by an autotaxin agonist. This inhibitory effect may result, for example, from the competitive or non-competitive binding of the antagonist to the autotaxin receptor.
  • Autotaxin its agonists and antagonists are valuable in both in vivo and in vitro uses.
  • autotaxin or an agonist, can be used to inhibit the differentiation of adipocytes to maintain cultures of undifferentiated cells.
  • Antagonists are also useful as research reagents for characterizing sites of interaction between autotaxin and its receptor.
  • pharmaceutical compositions comprising autotaxin antagonists can be used to inhibit autotaxin activity.
  • One general class of autotaxin analogs are agonists or antagonists having an amino acid sequence that is a mutation of the amino acid sequences disclosed herein.
  • Another general class of autotaxin analogs is provided by anti-idiotype antibodies, and fragments thereof, as described below.
  • recombinant antibodies comprising anti-idiotype variable domains can be used as analogs (see, for example, Monfardini et ⁇ l, Proc. Assoc. Am. Physicians 108:420 (1996)). Since the variable domains of anti- idiotype autotaxin antibodies mimic autotaxin, these domains can provide either autotaxin agonist or antagonist activity.
  • Lim and Langer, J. Interferon Res. 75:295 (1993) describe anti-idiotypic interferon- ⁇ antibodies that have the properties of either interferon- ⁇ agonists or antagonists.
  • a third approach to identifying autotaxin analogs is provided by the use of combinatorial 'ibraries.
  • Methods for constructing and screening phage display and other combinatorial libraries are provided, for example, by Kay et al. , Phage Display of Peptides and Proteins (Academic Press 1996), Verdine, U.S. Patent No. 5,783,384, Kay, et. al, U.S. Patent No. 5,747,334, and Kauffman et al, U.S. Patent No. 5,723,323.
  • One method of determining whether an autotaxin variant is functional is to test the ability of the variant to inhibit the differentiation of adipocytes, as described in Example 1 with 7F2 cells.
  • a putative autotaxin variant can be tested for the ability to induce adipocyte differentiation using 3T3-L1 preadipocytes (ATCC No. CL-173; American Type Culture Collection; Manassas, VA; Green, U.S. Patent No. 4,003,789).
  • 3T3-L1 preadipocytes ATCC No. CL-173; American Type Culture Collection; Manassas, VA; Green, U.S. Patent No. 4,003,789
  • Methods for inducing the differentiation of 3T3-L1 cells are described, for example, by Student et al, J. Biol. Chem. 255:4745 (1980). Briefly, 3T3-L1 cells are incubated in Dulbecco's modified Eagle's medium containing 10% fetal calf serum and penicillin/streptomycin (100 units/ml each).
  • differentiation is induced by incubating the cells in medium containing 10% fetal calf serum, dexamethasone (about 1 ⁇ M), insulin (about 160 nM or about 10 ⁇ g/ml), and 3- isobutyl-1-methylxanthine (about 0.5 mM).
  • An experimental group is additionally treated with the putative autotaxin variant to inhibit differentiation. Following two days of treatment, the medium is replaced with fresh medium containing 10% fetal calf serum, and the cells are incubated for three to five days. After this incubation, the cells are treated with Oil Red O stain to determine whether treatment with the putative autotaxin variant inhibited the formation of lipid droplets, which is an indication of adipocyte differentiation.
  • a modified Boyden chamber assay can be used to measure the stimulation of cell motility by autotaxin variants (Stracke et al, J. Biol. Chem. 264:2X544 (1989); Stracke et al, U.S. Patent No. 5,449,753).
  • a phagokinetic track motility assay provides another approach (Siletti et al, Cancer Res. 57:3507 (1991)).
  • autotaxin activity can be identified as the ability to enhance the insulin-mediated uptake of glucose.
  • Example 3 illustrates this type of assay.
  • Type I phosphodiesterase enzymatic activity of autotaxin variants can be measured, for example, using the modification of the method of Razzell, Methods Enzymol 6:236 (1963), as described by Murata et al. J. Biol. Chem. 269:30419 (1994). Briefly, samples are assayed in a 100 ⁇ l volume containing 50 mM Tris-HCl (pH 8.9), and 5 mM ?-nitrophenyl thymidine-5'-monophosphate. After a 30 minute incubation at 37°C, the reactions are terminated by addition of 900 ⁇ l of 0.1 N NaOH, and the amount of product formed is determined by measuring absorbance at 410 nm.
  • hydrolysis of phosphodiester bonds can be detected using a radiolabeled substrate, such as [ ⁇ - 32 P]ATP, [ ⁇ - 32 P]ATP. and [ ⁇ - 2 P]GTP (Clair et al, J. Biol. Chem. 272:996 (1997))
  • a radiolabeled substrate such as [ ⁇ - 32 P]ATP, [ ⁇ - 32 P]ATP. and [ ⁇ - 2 P]GTP (Clair et al, J. Biol. Chem. 272:996 (1997)
  • the activity of autotaxin can be measured by a silicon-based biosensor microphysiometer, which measures the extracellular acidification rate or proton excretion associated with receptor binding and subsequent cellular responses.
  • An exemplary device is the CYTOSENSOR Microphysiometer manufactured by Molecular Devices Corp. (Sunnyvale, CA).
  • a variety of cellular responses, such as cell proliferation, ion transport, energy production, inflammatory response, regulatory and receptor activation, and the like, can be measured by this method (see, for example, McConnell et al, Science 257:1906 (1992), Pitchford et al, Meth. Enzymol. 228:84 (1997), Arimilli et al, J. Immunol. Meth.
  • the microphysiometer can be used for assaying adherent or non-adherent eukaryotic or prokaryotic cells. Since energy metabolism is coupled with the use of cellular ATP, any event that alters cellular ATP levels, such as receptor activation and the initiation of signal transduction, will cause a change in cellular acid section. For example, the studies of the present inventor have indicated that both insulin and autotaxin induce intracellular signaling via STAT or SRE signaling pathways.
  • the microphysiometer directly measures cellular responses to various stimuli, including autotaxin, its agonists, or antagonists.
  • the microphysiometer can be used to measure responses of an autotaxin responsive eukaryotic cell, compared to a control eukaryotic cell that does not respond to autotaxin polypeptide.
  • Autotaxin responsive eukaryotic cells comprise cells into which a receptor for autotaxin has been transfected to create a cell that is responsive to autotaxin, or cells that are naturally responsive to autotaxin (e.g., liver cells, 7F2 cells, 3T3 LI cells, and the like).
  • Autotaxin modulated cellular responses are measured by a change (e.g., an increase or decrease in extracellular acidification) in the response of cells exposed to autotaxin, compared with control cells that have not been exposed to autotaxin.
  • a microphysiometer can be used to identify cells, tissues, or cell lines which respond to an autotaxin stimulated pathway, and which express a functional autotaxin receptor.
  • cells that express a functional autotaxin receptor can be identified by (a) providing test cells, (b) incubating a first portion of the test cells in the absence of autotaxin, (c) incubating a second portion of the test cells in the presence of autotaxin, and (d) detecting a change (e.g., an increase or decrease in extracellular acidification rate, as measured by a microphysiometer) in a cellular response of the second portion of the test cells, as compared to the first portion of the test cells, wherein such a change in cellular response indicates that the test cells express a functional autotaxin receptor.
  • An additional negative control may be included in which a portion of the test cells is incubated with autotaxin and an anti-autotaxin antibody to inhibit the binding of autotaxin with its cognate receptor.
  • the microphysiometer also provides one means to identify autotaxin agonists.
  • agonists of autotaxin can be identified by a method, comprising the steps of (a) providing cells responsive to autotaxin, (b) incubating a first portion of the cells in the absence of a test compound, (c) incubating a second portion of the cells in the presence of a test compound, and (d) detecting a change, for example, an increase or diminution, in a cellular response of the second portion of the cells as compared to the first portion of the cells, wherein such a change in cellular response indicates that the test compound is an autotaxin agonist.
  • An illustrative change in cellular response is a measurable change in extracellular acidification rate, as measured by a microphysiometer.
  • incubating a third portion of the cells in the presence of autotaxin and in the absence of a test compound can be used as a positive control for the autotaxin responsive cells, and as a control to compare the agonist activity of a test compound with that of autotaxin.
  • An additional control may be included in which a portion of the cells is incubated with a test compound (or autotaxin) and an anti- autotaxin antibody to inhibit the binding of the test compound (or autotaxin) with the autotaxin receptor.
  • the microphysiometer also provides a means to identify autotaxin antagonists.
  • autotaxin antagonists can be identified by a method, comprising the steps of (a) providing cells responsive to autotaxin, (b) incubating a first portion of the cells in the presence of autotaxin and in the absence of a test compound, (c) incubating a second portion of the cells in the presence of both autotaxin and the test compound, and (d) comparing the cellular responses of the first and second cell portions, wherein a decreased response by the second portion, compared with the response of the first portion, indicates that the test compound is an autotaxin antagonist.
  • An illustrative change in cellular response is a measurable change extracellular acidification rate, as measured by a microphysiometer.
  • Autotaxin, its analogs, and anti-iodiotype autotaxin antibodies can be used to identify and to isolate autotaxin receptors.
  • proteins and peptides of the present invention can be immobilized on a column and used to bind receptor proteins from membrane preparations that are run over the column (Hermanson et al. (eds.), Immobilized Affinity Ligand Techniques, pages 195-202 (Academic Press 1992)).
  • Radiolabeled or affinity labeled autotaxin polypeptides can also be used to identify or to localize autotaxin receptors in a biological sample (see, for example, Deutscher (ed.), Methods in Enzymol, vol.
  • Antibodies to autotaxin can be obtained, for example, using the product of an autotaxin expression vector or autotaxin isolated from a natural source as an antigen. Particularly useful anti-autotaxin antibodies "bind specifically" with autotaxin. Antibodies are considered to be specifically binding if the antibodies exhibit at least one of the following two properties: (1) antibodies bind to autotaxin with a threshold level of binding activity, and (2) antibodies do not significantly cross-react with polypeptides related to autotaxin.
  • antibodies specifically bind if they bind to an autotaxin polypeptide, peptide or epitope with a binding affinity (K a ) of 10 6 M “1 or greater, preferably 10 7 M “1 or greater, more preferably 10 8 M “1 or greater, and most preferably 10 9 M “1 or greater.
  • K a binding affinity
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, Ann. NY Ac ⁇ d. Sci. 51:660 (1949)).
  • antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect autotaxin, but not known polypeptides using a standard Western blot analysis.
  • Examples of known related polypeptides are orthologs and proteins from the same species that are members of a protein family.
  • specifically-binding anti-autotaxin antibodies bind with the autotaxin described herein, but not with known autotaxin proteins discussed above.
  • Suitable antibodies include antibodies that bind with autotaxin in regions having a low sequence similarity with other autotaxins. Such regions include, for example, amino acid residues 143 to 158 of SEQ ID NO:2, amino acid residues 149 to 158 of SEQ ID NO:2, or amino acid residues 585 to 595 of SEQ ID NO:2.
  • General methods of making anti-autotaxin peptide antibodies are described by Murata et ⁇ l., J. Biol. Chem. 269:30419 (1994), and Narita et ⁇ l, J. Biol. Chem. 269:28235 (1994).
  • Anti-autotaxin antibodies can be produced using antigenic autotaxin epitope-bearing peptides and polypeptides.
  • Antigenic epitope-bearing peptides and polypeptides of the present invention contain a sequence of at least nine, or between 15 to about 30 amino acids contained within SEQ ID NO:2.
  • peptides or polypeptides comprising a larger portion of an amino acid sequence of the invention, containing from 30 to 50 amino acids, or any length up to and including the entire amino acid sequence of a polypeptide of the invention, also are useful for inducing antibodies that bind with autotaxin.
  • amino acid sequence of the epitope-bearing peptide is selected to provide substantial solubility in aqueous solvents (i.e., the sequence includes relatively hydrophilic residues, while hydrophobic residues are preferably avoided). Moreover, amino acid sequences containing proline residues may be also be desirable for antibody production.
  • Polyclonal antibodies to recombinant autotaxin protein or to autotaxin isolated from natural sources can be prepared using methods well-known to those of skill in the art. See, for example, Green et al, "Production of Polyclonal Antisera,” in Immunochemical Protocols (Manson, ed.), pages 1-5 (Humana Press 1992), and Williams et al, "Expression of foreign proteins in E. coli using plasmid vectors and purification of specific polyclonal antibodies, " in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 15 (Oxford University Press 1995).
  • the immunogenicity of an autotaxin polypeptide can be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • an adjuvant such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of autotaxin or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full-length molecule or a portion thereof.
  • polypeptide portion is "hapten-like,” such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH), bovine serum albumin (BSA) or tetanus toxoid
  • an anti- autotaxin antibody of the present invention may also be derived from a subhuman primate antibody.
  • General techniques for raising diagnostically and therapeutically useful antibodies in baboons may be found, for example, in Goldenberg et al, international patent publication No. WO 91/11465, and in Losman et al, Int. J. Cancer 46:3X0 (1990).
  • monoclonal anti-autotaxin antibodies can be generated.
  • Rodent monoclonal antibodies to specific antigens may be obtained by methods known to those skilled in the art (see, for example, Kohler et al, Nature 256:495 (1975), Coligan et al (eds.), Current Protocols in Immunology, Vol. 1, pages 2.5.1-2.6.7 (John Wiley & Sons 1991) ["Coligan”], Picksley et al, "Production of monoclonal antibodies against proteins expressed in E. coli," in DNA Cloning 2: Expression Systems, 2nd Edition, Glover et al. (eds.), page 93 (Oxford University Press 1995)).
  • monoclonal antibodies can be obtained by injecting mice with a composition comprising an autotaxin gene product, verifying the presence of antibody production by removing a serum sample, removing the spleen to obtain B-lymphocytes, fusing the B-lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, selecting positive clones which produce antibodies to the antigen, culturing the clones that produce antibodies to the antigen, and isolating the antibodies from the hybridoma cultures.
  • an anti-autotaxin antibody of the present invention may be derived from a human monoclonal antibody.
  • Human monoclonal antibodies are obtained from transgenic mice that have been engineered to produce specific human antibodies in response to antigenic challenge.
  • elements of the human heavy and light chain locus are introduced into strains of mice derived from embryonic stem cell lines that contain targeted disruptions of the endogenous heavy chain and light chain loci.
  • the transgenic mice can synthesize human antibodies specific for human antigens, and the mice can be used to produce human antibody-secreting hybridomas. Methods for obtaining human antibodies from transgenic mice are described, for example, by Green et al, Nature Genet.
  • Human antibodies can also be obtained using phage display methods (see, for example, Griffiths et al, U.S. patent No.5, 885,793). Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques.
  • isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography (see, for example, Coligan at pages 2.7.1-2.7.12 and pages 2.9.1-2.9.3; Baines et al, "Purification of Immunoglobulin G (IgG),” in Methods in Molecular Biology, Vol. 10, pages 79-104 (The Humana Press, Inc. 1992)).
  • antibody fragments can be obtained, for example, by proteolytic hydrolysis of the antibody.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab') 2 .
  • This fragment can be further cleaved using a thiol reducing agent to produce 3.5S Fab' monovalent fragments.
  • the cleavage reaction can be performed using a blocking group for the sulfhydryl groups that result from cleavage of disulfide linkages.
  • an enzymatic cleavage using pepsin produces two monovalent Fab fragments and an Fc fragment directly.
  • These methods are described, for example, by Goldenberg, U.S. patent No. 4,331,647, Nisonoff et al, Arch Biochem. Biophys. 89:230 (1960), Porter, Biochem. J. 75:119 (1959).
  • Edelman et al. in Methods in Enzymology Vol. 7, page 422 (Academic Press 1967), and by Coligan at pages 2.8.1-2.8.10 and 2.10.-2.10.4.
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • Fv fragments comprise an association of V H and V L chains.
  • This association can be noncovalent, as described by Inbar et al, Proc. Nat'l Acad. Sci. USA 69:2659 (1972).
  • the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde (see, for example, Sandhu, Crit. Rev. Biotech. 72:437 (1992)).
  • the Fv fragments may comprise V H and V L chains, which are connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains which are connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell, such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • a scFV can be obtained by exposing lymphocytes to autotaxin polypeptide in vitro, and selecting antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled autotaxin protein or peptide).
  • Genes encoding polypeptides having potential autotaxin polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli.
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • random peptide display libraries can be used to screen for peptides, which interact with a known target, which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al, U.S. Patent No. 5,223,409, Ladner et al, U.S. Patent No. 4,946,778, Ladner et al, U.S. Patent No. 5,403,484, Ladner et al, U.S. Patent No. 5,571,698, and Kay et al, Phage Display of Peptides and Proteins (Academic Press, Inc.
  • Random peptide display libraries can be screened using the autotaxin sequences disclosed herein to identify proteins, which bind to autotaxin.
  • CDR peptides (“minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells (see, for example, Larrick et al, Methods: A Companion to Methods in Enzymology 2:106 (1991), Courtenay-Luck, "Genetic Manipulation of Monoclonal Antibodies," in Monoclonal Antibodies: Production, Engineering and Clinical Application, Ritter et al.
  • an anti-autotaxin antibody may be derived from a "humanized" monoclonal antibody.
  • Humanized monoclonal antibodies are produced by transferring mouse complementary determining regions from heavy and light variable chains of the mouse immunoglobulin into a human variable domain. Typical residues of human antibodies are then substituted in the framework regions of the murine counterparts.
  • the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of murine constant regions. General techniques for cloning murine immunoglobulin variable domains are described, for example, by Orlandi et al, Proc. Nat'l Acad. Sci. USA 5(5:3833 (1989).
  • Polyclonal anti-idiotype antibodies can be prepared by immunizing animals with anti-autotaxin antibodies or antibody fragments, using standard techniques. See, for example, Green et al, "Production of Polyclonal Antisera,” in Methods In Molecular Biology: Immunochemical Protocols, Manson (ed.), pages 1-12 (Humana Press 1992). Also, see Coligan at pages 2.4.1-2.4.7.
  • monoclonal anti-idiotype antibodies can be prepared using anti-autotaxin antibodies or antibody fragments as immunogens with the techniques, described above.
  • humanized anti-idiotype antibodies or subhuman primate anti-idiotype antibodies can be prepared using the above-described techniques.
  • Nucleic acid molecules can be used to detect the expression of an autotaxin gene in a biological sample.
  • Suitable probe molecules include double- stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NOs: l or 8, or a portion thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NOs: lor 8, or a portion thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • portion refers to at least eight nucleotides to at least 20 or more nucleotides.
  • RNA isolated from a biological sample
  • RNA isolated from a biological sample
  • RNA detection includes northern analysis and dot/slot blot hybridization (see, for example, Ausubel (1995) at pages 4-1 to 4-27, and Wu et ⁇ l (eds.), "Analysis of Gene Expression at the RNA Level," in Methods in Gene Biotechnology, pages 225-239 (CRC Press, Inc. 1997)).
  • Nucleic acid probes can be detectably labeled with radioisotopes such as 32 P or 35 S.
  • autotaxin RNA can be detected with a nonradioactive hybridization method (see, for example, Isaac (ed.), Protocols for Nucleic Acid Analysis by Nonradioactive Probes (Humana Press, Inc. 1993)).
  • nonradioactive detection is achieved by enzymatic conversion of chromogenic or chemiluminescent substrates.
  • Illustrative nonradioactive moieties include biotin, fluorescein, and digoxigenin.
  • oligonucleotide probes are also useful for in vivo diagnosis.
  • l F-labeled oligonucleotides can be administered to a subject and visualized by positron emission tomography (Tavitian et al, Nature Medicine 4:461 (1998)).
  • PCR polymerase chain reaction
  • Standard techniques for performing PCR are well-known (see, generally, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), White (ed.), PCR Protocols: Current Methods and Applications (Humana Press, Inc.
  • RNA is isolated from a biological sample, reverse transcribed to cDNA, and the cDNA is incubated with autotaxin primers (see, for example, Wu et al. (eds.), "Rapid Isolation of Specific cDNAs or Genes by PCR,” in Methods in Gene Biotechnology, pages 15-28 (CRC Press, Inc. 1997)). PCR is then performed and the products are analyzed using standard techniques.
  • RNA is isolated from biological sample using, for example, the guanidinium-thiocyanate cell lysis procedure described above.
  • a solid-phase technique can be used to isolate mRNA from a cell lysate.
  • a reverse transcription reaction can be primed with the isolated RNA using random oligonucleotides, short homopolymers of dT, or autotaxin anti-sense oligomers.
  • Oligo- dT primers offer the advantage that various mRNA nucleotide sequences are amplified that can provide control target sequences, autotaxin sequences are amplified by the polymerase chain reaction using two flanking oligonucleotide primers that are typically 20 bases in length.
  • PCR amplification products can be detected using a variety of approaches.
  • PCR products can be fractionated by gel electrophoresis, and visualized by ethidium bromide staining.
  • fractionated PCR products can be transferred to a membrane, hybridized with a detectably-labeled autotaxin probe, and examined by autoradiography.
  • Additional alternative approaches include the use of digoxigenin-labeled deoxyribonucleic acid triphosphates to provide chemiluminescence detection, and the C-TRAK colorimetric assay.
  • CPT cycling probe technology
  • NASBA nucleic acid sequence-based amplification
  • CATCH cooperative amplification of templates by cross-hybridization
  • LCR ligase chain reaction
  • Autotaxin probes and primers can also be used to detect and to localize autotaxin gene expression in tissue samples.
  • Methods for such in situ hybridization are well-known to those of skill in the art (see, for example, Choo (ed.), In Situ Hybridization Protocols (Humana Press, Inc. 1994), Wu et al. (eds.), "Analysis of Cellular DNA or Abundance of mRNA by Radioactive In Situ Hybridization (RISH),” in Methods in Gene Biotechnology, pages 259-278 (CRC Press, Inc. 1997), and Wu et al.
  • Nucleic acid molecules comprising autotaxin nucleotide sequences can also be used to determine whether a subject's chromosomes contain a mutation in the autotaxin gene. Detectable chromosomal aberrations at the autotaxin gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. Of particular interest are genetic alterations that inactivate the autotaxin gene.
  • Aberrations associated with the autotaxin locus can be detected using nucleic acid molecules of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch analysis, and other genetic analysis techniques known in the art (see, for example, Mathew (ed.), Protocols in Human Molecular Genetics (Humana Press, Inc. 1991), Marian, Chest 705:255 (1995), Coleman and Tsongalis, Molecular Diagnostics (Human Press, Inc.
  • molecular genetic techniques such as restriction fragment length polymorphism analysis, short tandem repeat analysis employing PCR techniques, amplification-refractory mutation system analysis, single-strand conformation polymorphism detection, RNase cleavage methods, denaturing gradient gel electrophoresis, fluorescence-assisted mismatch
  • RNA is isolated from a biological sample, and used to synthesize cDNA. PCR is then used to amplify the autotaxin target sequence and to introduce an RNA polymerase promoter, a translation initiation sequence, and an in- frame ATG triplet. PCR products are transcribed using an RNA polymerase, and the transcripts are translated in vitro with a T7-coupled reticulocyte lysate system.
  • the translation products are then fractionated by SDS-PAGE to determine the lengths of the translation products.
  • the protein truncation test is described, for example, by Dracopoli et al. (eds.), Current Protocols in Human Genetics, pages 9.11.1 - 9.11.18 (John Wiley & Sons 1998).
  • kits for performing a diagnostic assay for autotaxin gene expression or to detect mutations in the autotaxin gene comprise nucleic acid probes, such as double-stranded nucleic acid molecules comprising the nucleotide sequence of SEQ ID NOs: 1 or 8, or a portion thereof, as well as single-stranded nucleic acid molecules having the complement of the nucleotide sequence of SEQ ID NOs:l or 8, or a portion thereof.
  • Probe molecules may be DNA, RNA, oligonucleotides, and the like.
  • Kits may comprise nucleic acid primers for performing PCR. Such a kit can contain all the necessary elements to perform a nucleic acid diagnostic assay described above.
  • a kit will comprise at least one container comprising an autotaxin probe or primer.
  • the kit may also comprise a second container comprising one or more reagents capable of indicating the presence of autotaxin sequences. Examples of such indicator reagents include detectable labels such as radioactive labels, fluorochromes, chemiluminescent agents, and the like.
  • a kit may also comprise a means for conveying to the user that the autotaxin probes and primers are used to detect autotaxin gene expression.
  • written instructions may state that the enclosed nucleic acid molecules can be used to detect either a nucleic acid molecule that encodes autotaxin, or a nucleic acid molecule having a nucleotide sequence that is complementary to an autotaxin-encoding nucleotide sequence.
  • the written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert. 11. Detection of Autotaxin Protein
  • the present invention contemplates the use of anti-autotaxin antibodies to screen biological samples in vitro for the presence of autotaxin.
  • anti-autotaxin antibodies are used in liquid phase.
  • the presence of autotaxin in a biological sample can be tested by mixing the biological sample with a trace amount of labeled autotaxin and an anti-autotaxin antibody under conditions that promote binding between autotaxin and its antibody.
  • Complexes of autotaxin and anti- autotaxin in the sample can be separated from the reaction mixture by contacting the complex with an immobilized protein which binds with the antibody, such as an Fc antibody or St ⁇ phylococcus protein A.
  • the concentration of autotaxin in the biological sample will be inversely proportional to the amount of labeled autotaxin bound to the antibody and directly related to the amount of free labeled autotaxin.
  • in vitro assays can be performed in which anti-autotaxin antibody is bound to a solid-phase carrier.
  • antibody can be attached to a polymer, such as aminodextran, in order to link the antibody to an insoluble support such as a polymer-coated bead, a plate or a tube.
  • polymer such as aminodextran
  • anti-autotaxin antibodies can be used to detect autotaxin in tissue sections prepared from a biopsy specimen. Such immunochemical detection can be used to determine the relative abundance of autotaxin and to determine the distribution of autotaxin in the examined tissue.
  • General immunochemistry techniques are well established (see, for example, Ponder, "Cell Marking Techniques and Their Application " in Mammalian Development: A Practical Approach, Monk (ed.), pages 115-38 (IRL Press 1987), Coligan at pages 5.8.1-5.8.8, Ausubel (1995) at pages 14.6.1 to 14.6.13 (Wiley Interscience 1990), and Manson (ed.), Methods In Molecular Biology, Vol. 10: Immunochemical Protocols (The Humana Press, Inc. 1992)).
  • Immunochemical detection can be performed by contacting a biological sample with an anti-autotaxin antibody, and then contacting the biological sample with a detectably labeled molecule, which binds to the antibody.
  • the detectably labeled molecule can comprise an antibody moiety that binds to anti-autotaxin antibody.
  • the anti-autotaxin antibody can be conjugated with avidin/streptavidin (or biotin) and the detectably labeled molecule can comprise biotin (or avidin/streptavidin). Numerous variations of this basic technique are well-known to those of skill in the art.
  • an anti-autotaxin antibody can be conjugated with a detectable label to form an anti-autotaxin immunoconjugate.
  • Suitable detectable labels include, for example, a radioisotope. a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label or colloidal gold. Methods of making and detecting such detectably-labeled immunoconjugates are well-known to those of ordinary skill in the art, and are described in more detail below.
  • the detectable label can be a radioisotope that is detected by autoradiography. Isotopes that are particularly useful for the purpose of the present invention are 3 H, ,2 T, ⁇ I, 5 S and ,4 C.
  • Anti-autotaxin immunoconjugates can also be labeled with a fluorescent compound.
  • the presence of a fluorescently-labeled antibody is determined by exposing the immunoconjugate to light of the proper wavelength and detecting the resultant fluorescence.
  • Fluorescent labeling compounds include fluorescein isothiocyanate, rhoda- mine, phycoerytherin, phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.
  • anti-autotaxin immunoconjugates can be detectably labeled by coupling an antibody component to a chemiluminescent compound.
  • the presence of the chemiluminescent-tagged immunoconjugate is determined by detecting the presence of luminescence that arises during the course of a chemical reaction.
  • chemiluminescent labeling compounds include luminol, isoluminol, an aromatic acridinium ester, an imidazole, an acridinium salt and an oxalate ester.
  • a bioluminescent compound can be used to label anti-autotaxin immunoconjugates of the present invention.
  • Bioluminescence is a type of chemiluminescence found in biological systems in which a catalytic protein increases the efficiency of the chemiluminescent reaction. The presence of a bioluminescent protein is determined by detecting the presence of luminescence.
  • Bioluminescent compounds that are useful for labeling include luciferin, luciferase and aequorin.
  • anti-autotaxin immunoconjugates can be detectably labeled by linking an anti-autotaxin antibody component to an enzyme.
  • the enzyme moiety reacts with the substrate to produce a chemical moiety, which can be detected, for example, by spectrophotometric, fluorometric or visual means.
  • enzymes that can be used to detectably label polyspecific immunoconjugates include ⁇ -galac- tosidase, glucose oxidase, peroxidase and alkaline phosphatase.
  • kits for performing an immunological diagnostic assay for autotaxin gene expression comprise at least one container comprising an anti-autotaxin antibody, or antibody fragment.
  • a kit may also comprise a second container comprising one or more reagents capable of indicating the presence of autotaxin antibody or antibody fragments.
  • indicator reagents include detectable labels such as a radioactive label, a fluorescent label, a chemiluminescent label, an enzyme label, a bioluminescent label, colloidal gold, and the like.
  • a kit may also comprise a means for conveying to the user that autotaxin antibodies or antibody fragments are used to detect autotaxin protein.
  • written instructions may state that the enclosed antibody or antibody fragment can be used to detect autotaxin.
  • the written material can be applied directly to a container, or the written material can be provided in the form of a packaging insert.
  • the present invention contemplates the use of polypeptides having autotaxin activity, such as autotaxin polypeptides, autotaxin analogs, and autotaxin fusion proteins, to treat metabolic diseases such as obesity, dyslipidemia, and diabetes, especially non-insulin dependent diabetes.
  • a subject e.g., a mammalian subject, such as a human, a domesticated animal, or an animal used in agriculture.
  • the present invention includes the administration of autotaxin for veterinary or human therapeutic uses.
  • dyslipidemia can be characterized by abnormal serum triglyceride levels.
  • a level of 200 to 400 mg triglycerides/dl is considered “borderline high,” while 400 to 1000 mg triglycerides/dl is “high,” and levels greater than 1000 mg triglycerides/dl is considered “very high” (Garg and Grundy, Diabetes Reviews 5:425 (1997).
  • insulin resistance can be inferred in diabetic subjects requiring large daily doses of insulin to control blood glucose levels. Severe insulin resistance may be suspected when the total daily insulin requirement exceeds 1.5 to 2 units per kilogram body weight (Whitelaw and Gilbey, Ann. Clin. Biochem. 55:567 (1998)).
  • the present invention includes the use of polypeptides having autotaxin activity to decrease circulating free fatty acid in the blood of a subject.
  • the effect of such treatment can be observed as at least one of (1) a decrease in the level of serum triglycerides, or (2) a decrease in the amount of free fatty acid bound to albumin in the serum.
  • a subject having elevated serum lipids is suitable for receiving a method of treatment described herein. Those skilled in the art can recognize such a subject in need of an autotaxin treatment of the present invention.
  • autotaxin treatment can be used to increase the sensitivity of cells to insulin.
  • autotaxin can be administered prior to, concomitantly with, or following, insulin treatment to either achieve a greater effect with a standard dose of insulin, or as a means of administering a decreased dosage of insulin.
  • the dosage of administered autotaxin will vary depending upon such factors as the subject's age, weight, height, sex, general medical condition and previous medical history. Typically, it is desirable to provide the recipient with a dosage of autotaxin, which is in the range of from about 1 pg/kg to 10 mg/kg (amount of agent/body weight of subject), although a lower or higher dosage also may be administered as circumstances dictate.
  • Administration of a polypeptide having autotaxin activity to a subject can be intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, intrapleural, intrathecal, by perfusion through a regional catheter, or by direct intralesional injection. When administering therapeutic proteins by injection, the administration may be by continuous infusion or by single or multiple boluses.
  • Additional routes of administration include oral, mucosal-membrane, pulmonary, and transcutaneous.
  • Oral delivery is suitable for polyester microspheres, zein microspheres, proteinoid microspheres, polycyanoacrylate microspheres, and lipid- based systems (see, for example, DiBase and Morrel, "Oral Delivery of Microencapsulated Proteins," in Protein Delivery: Physical Systems, Sanders and Hendren (eds.), pages 255-288 (Plenum Press 1997)).
  • the feasibility of an intranasal delivery is exemplified by such a mode of insulin administration (see, for example, Hinchcliffe and Ilium, Adv. Drug Deliv. Rev. 55:199 (1999)).
  • Dry or liquid particles comprising autotaxin can be prepared and inhaled with the aid of dry-powder dispersers, liquid aerosol generators, or nebulizers (e.g., Pettit and Gombotz, TIBTECH 16:343 (1998); Patton et al, Adv. Drug Deliv. Rev. 35:235 (1999)).
  • This approach is illustrated by the AERX diabetes management system, which is a hand-held electronic inhaler that delivers aerosolized insulin into the lungs.
  • proteins as large as 48,000 kDa have been delivered across skin at therapeutic concentrations with the aid of low-frequency ultrasound, which illustrates the feasibility of trascutaneous administration (Mitragotri et al, Science 269:850 (1995)).
  • Transdermal delivery using electroporation provides another means to administer autotaxin (Potts et al, Pharm. Biotechnol 70:213 (1997)).
  • a pharmaceutical composition comprising autotaxin can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby the therapeutic proteins are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition is said to be a "pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient patient.
  • Sterile phosphate -buffered saline is one example of a pharmaceutically acceptable carrier.
  • Other suitable carriers are well-known to those in the art (see, for example, Gennaro (ed.), Remington's Pharmaceutical Sciences, 19th Edition (Mack Publishing Company 1995)).
  • autotaxin or autotaxin analog or fusion protein
  • a pharmaceutically acceptable carrier are administered to a subject in a therapeutically effective amount.
  • a combination of an autotaxin (or autotaxin analog or fusion protein) and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject. In the present context, an agent is physiologically significant if its presence results in alleviation of a symptom associated with the diseases discussed above.
  • a pharmaceutical composition used to treat elevated serum lipids is physiologically significant if its administration results in a decreased level of serum triglycerides, or in a decrease in the amount of fatty acid bound to albumin in the serum.
  • a pharmaceutical composition comprising autotaxin (or autotaxin analog or fusion protein) can be furnished in liquid form, in an aerosol, or in solid form. Liquid forms, are illustrated by injectable solutions and oral suspensions. Exemplary solid forms include capsules, tablets, and controlled-release forms. The latter form is illustrated by miniosmotic pumps and implants (Bremer et al, Pharm.
  • Liposomes provide one means to deliver autotaxin to a subject intravenously, intraperitoneally, intrathecally, intramuscularly, subcutaneously, or via oral administration, inhalation, or intranasal administration.
  • Liposomes are microscopic vesicles that consist of one or more lipid bilayers surrounding aqueous compartments (see, generally, Bakker-Woudenberg et al, Eur. J. Clin. Microbiol. Infect. Dis.
  • Liposomes are similar in composition to cellular membranes and as a result, liposomes can be administered safely and are biodegradable. Depending on the method of preparation, liposomes may be unilamellar or multilamellar, and liposomes can vary in size with diameters ranging from 0.02 ⁇ m to greater than 10 ⁇ m.
  • a variety of agents can be encapsulated in liposomes: hydrophobic agents partition in the bilayers and hydrophilic agents partition within the inner aqueous space(s) (see, for example, Machy et al, Liposomes In Cell Biology And Pharmacology (John Libbey 1987), and Ostro et al, American J. Hosp. Pharm. 46:1516 (1989)). Moreover, it is possible to control the therapeutic availability of the encapsulated agent by varying liposome size, the number of bilayers, lipid composition, as well as the charge and surface characteristics of the liposomes.
  • Liposomes can adsorb to virtually any type of cell and then slowly release the encapsulated agent.
  • an absorbed liposome may be endocytosed by cells that are phagocytic. Endocytosis is followed by intralysosomal degradation of liposomal lipids and release of the encapsulated agents (Scherphof et al, Ann. N Y. Acad. Sci. 446:368 (1985)).
  • small liposomes (0.1 to 1.0 ⁇ m) are typically taken up by cells of the reticuloendothelial system, located principally in the liver and spleen, whereas liposomes larger than 3.0 ⁇ m are deposited in the lung. This preferential uptake of smaller liposomes by the cells of the reticuloendothelial system has been used to deliver chemotherapeutic agents to macrophages and to tumors of the liver.
  • the reticuloendothelial system can be circumvented by several methods including saturation with large doses of liposome particles, or selective macrophage inactivation by pharmacological means (Claassen et al, Biochim. Biophys. Acta 502:428 (1984)).
  • incorporation of glycolipid- or polyethelene glycol- derivatized phospholipids into liposome membranes has been shown to result in a significantly reduced uptake by the reticuloendothelial system (Allen et al, Biochim. Biophys. Acta 1068:133 (1991); Allen et al, Biochim. Biophys. Acta 1150:9 (1993)).
  • Liposomes can also be prepared to target particular cells or organs by varying phospholipid composition or by inserting receptors or ligands into the liposomes.
  • liposomes prepared with a high content of a nonionic surfactant, have been used to target the liver (Hayakawa et al, Japanese Patent 04- 244,018; Kato et al., Biol. Pharm. Bull. 16:960 (1993)).
  • These formulations were prepared by mixing soybean phospatidylcholine, ⁇ -tocopherol, and ethoxylated hydrogenated castor oil (HCO-60) in methanol, concentrating the mixture under vacuum, and then reconstituting the mixture with water.
  • DPPC dipalmitoylphosphatidylcholine
  • SG soybean-derived sterylglucoside mixture
  • Cho cholesterol
  • various targeting ligands can be bound to the surface of the liposome, such as antibodies, antibody fragments, carbohydrates, vitamins, and transport proteins.
  • liposomes can be modified with branched type galactosyllipid derivatives to target asialoglycoprotein (galactose) receptors, which are exclusively expressed on the surface of liver cells (Kato and Sugiyama, Crit. Rev. Ther. Drug Carrier Syst.
  • Polyaconitylated human serum albumin liposomes provide another approach for targeting liposomes to liver cells (Kamps et al, Proc. Nat'l Acad. Sci. USA 94:11681 (1997)).
  • Geho, et al. U.S. Patent No. 4,603,044 describe a hepatocyte-directed liposome vesicle delivery system, which has specificity for hepatobiliary receptors associated with the specialized metabolic cells of the liver.
  • target cells are prelabeled with biotinylated antibodies specific for a ligand expressed by the target cell (Harasym et al, Adv. Drug Deliv. Rev. 32:99 (1998)). After plasma elimination of free antibody, streptavidin-conjugated liposomes are administered.
  • targeting antibodies are directly attached to liposomes (Harasym et al, Adv. Drug Deliv. Rev. 32:99 (1998)).
  • Polypeptides having autotaxin activity and autotaxin antagonists can be encapsulated within liposomes using standard techniques of protein microencapsulation (see, for example, Anderson et al, Infect. Immun.
  • liposomes may contain a variety of components.
  • liposomes may comprise lipid derivatives of poly(ethylene glycol) (Allen et al, Biochim. Biophys. Acta 7750:9 (1993)).
  • Degradable polymer microspheres have been designed to maintain high systemic levels of therapeutic proteins, and therefore, can provide a means to administer autotaxin.
  • Microspheres are prepared from degradable polymers such as poly(lactide-co-glycolide) (PLG), polyanhydrides, poly (ortho esters), nonbiodegradable ethylvinyl acetate polymers, in which proteins are entrapped in the polymer (Gombotz and Pettit, Bioconjugate Chem.
  • the present invention also contemplates chemically modified autotaxin compositions, in which an autotaxin polypeptide is linked with a polymer.
  • the polymer is water soluble so that the autotaxin conjugate does not precipitate in an aqueous environment, such as a physiological environment.
  • An example of a suitable polymer is one that has been modified to have a single reactive group, such as an active ester for acylation, or an aldehyde for alkylation, In this way, the degree of polymerization can be controlled.
  • An example of a reactive aldehyde is polyethylene glycol propionaldehyde, or mono-(C,-C 10 ) alkoxy, or aryloxy derivatives thereof (see, for example, Harris, et al, U.S. Patent No. 5,252,714).
  • the polymer may be branched or unbranched.
  • a mixture of polymers can be used to produce autotaxin conjugates.
  • Autotaxin conjugates used for therapy can comprise pharmaceutically acceptable water-soluble polymer moieties.
  • Many proteins and peptides have been conjugated with water-soluble polymers, such as PEG, to reduce renal clearance, enhance their systemic circulation half-lives, and reduce immunogenicity (Delgado et al, Crit. Rev. Ther. Drug Carrier Syst. 9:249 (1992), Nieforth et al, Clin. Pharmacol. Ther. 59:636 (1996), and Monkarsh et al, Anal Biochem. 247:434 (1997)).
  • Suitable water-soluble polymers include polyethylene glycol (PEG), monomethoxy-PEG, mono- (C,-C 10 )alkoxy-PEG, aryloxy-PEG, poly-(N -vinyl pyrrolidone)PEG, tresyl monomethoxy PEG, PEG propionaldehyde, bw-succinimidyl carbonate PEG, propylene glycol homopolymers, a polypropylene oxide/ethylene oxide co-polymer, polyoxyethylated polyols (e.g., glycerol), poly vinyl alcohol, dextran, cellulose, or other carbohydrate-based polymers.
  • Suitable PEG may have a molecular weight from about 600 to about 60,000, including, for example, 5,000, 12,000, 20,000 and 25,000.
  • An autotaxin conjugate can also comprise a mixture of such water-soluble polymers.
  • an autotaxin conjugate comprises an autotaxin moiety and a polyalkyl oxide moiety attached to the N-terminus of the autotaxin moiety.
  • PEG is one suitable polyalkyl oxide.
  • autotaxin can be modified with PEG, a process known as "PEGylation.” PEGylation of autotaxin can be carried out by any of the PEGylation reactions known in the art (see, for example, EP 0 154 316, Delgado et al, Crit. Rev. Ther. Drug Carrier Syst 9:249 (1992), Duncan and Spreafico, Clin. Pharmacokinet. 27:290 (1994), and Francis et al, Int J Hematol 65:1 (1998)).
  • PEGylation can be performed by an acylation reaction or by an alkylation reaction with a reactive polyethylene glycol molecule.
  • autotaxin conjugates are formed by condensing activated PEG, in which a terminal hydroxy or amino group of PEG has been replaced by an activated linker (see, for example, Karasiewicz et al, U.S. Patent No. 5,382,657).
  • PE ylation by acylation typically requires reacting an active ester derivative of PEG with an autotaxin polypeptide.
  • An example of an activated PEG ester is PEG esterified to N-hydroxysuccinimide.
  • acylation includes the following types of linkages between autotaxin and a water soluble polymer: amide, carbamate, urethane, and the like.
  • Methods for preparing PEGylated autotaxin by acylation will typically comprise the steps of (a) reacting an autotaxin polypeptide with PEG (such as a reactive ester of an aldehyde derivative of PEG) under conditions whereby one or more PEG groups attach to autotaxin, and (b) obtaining the reaction product(s).
  • PEG such as a reactive ester of an aldehyde derivative of PEG
  • the optimal reaction conditions for acylation reactions will be determined based upon known parameters and desired results. For example, the larger the ratio of PEG: autotaxin, the greater the percentage of polyPEG
  • the product of PEGylation by acylation is typically a polyPEGylated autotaxin product, wherein the lysine ⁇ -amino groups are PEGylated via an acyl linking group.
  • An example of a connecting linkage is an amide.
  • the resulting autotaxin will be at least 95% mono-, di-, or tri-pegylated, although some species with higher degrees of PEGylation may be formed depending upon the reaction conditions.
  • PEGylated species can be separated from unconjugated autotaxin polypeptides using standard purification methods, such as dialysis, ultrafiltration, ion exchange chromatography, affinity chromatography, and the like.
  • PEGylation by alkylation generally involves reacting a terminal aldehyde derivative of PEG with autotaxin in the presence of a reducing agent.
  • PEG groups are preferably attached to the polypeptide via a -CH 2 -NH group.
  • Derivatization via reductive alkylation to produce a monoPEGylated product takes advantage of the differential reactivity of different types of primary amino groups available for derivatization.
  • the reaction is performed at a pH that allows one to take advantage of the pKa differences between the ⁇ -amino groups of the lysine residues and the ⁇ -amino group of the N-terminal residue of the protein.
  • Reductive alkylation to produce a substantially homogenous population of monopolymer autotaxin conjugate molecule can comprise the steps of: (a) reacting an autotaxin polypeptide with a reactive PEG under reductive alkylation conditions at a pH suitable to permit selective modification of the ⁇ -amino group at the amino terminus of the autotaxin, and (b) obtaining the reaction product(s).
  • the reducing agent used for reductive alkylation should be stable in aqueous solution and preferably be able to reduce only the Schiff base formed in the initial process of reductive alkylation. Suitable reducing agents include sodium borohydride, sodium cyanoborohydride, dimethylamine borane, trimethylamine borane, and pyridine borane.
  • the reductive alkylation reaction conditions are those, which permit the selective attachment of the water soluble polymer moiety to the N-terminus of autotaxin.
  • Such reaction conditions generally provide for pKa differences between the lysine amino groups and the ⁇ -amino group at the N-terminus.
  • the pH also affects the ratio of polymer to protein to be used. In general, if the pH is lower, a larger excess of polymer to protein will be desired because the less reactive the N-terminal ⁇ -group, the more polymer is needed to achieve optimal conditions. If the pH is higher, the polyme ⁇ autotaxin need not be as large because more reactive groups are available. Typically, the pH will fall within the range of 3 to 9, or 3 to 6.
  • the molecular weight of the water-soluble polymer is the higher the molecular weight of the polymer, the fewer number of polymer molecules which may be attached to the protein.
  • the typical molecular weight is about 2 kDa to about 100 kDa, about 5 kDa to about 50 kDa, or about 12 kDa to about 25 kDa.
  • the molar ratio of water-soluble polymer to autotaxin will generally be in the range of 1 :1 to 100:1.
  • the molar ratio of water-soluble polymer to autotaxin will be 1 :1 to 20:1 for polyPEGylation, and 1 :1 to 5:1 for monoPEGylation.
  • the biological activities of autotaxin include the ability to bind a cell-surface receptor, the ability to bind integrin through an RGD binding site, and the production of adenosine. In certain cases, it may be desirable to administer a compound that only provides the RGD binding capability.
  • Illustrative synthetic RGD peptides and RGD peptidomimetics include the GRGDSP hexapeptide, the RGDS tetrapeptide, N-(trifluoroacetyl)-O-methyl-ort 7 -(6-guanidino-caproyl)- amino-(L)-tyrosine, N-(trifluoroacetyl)-O-(3-aminopropyl)- ⁇ rt/2 ⁇ -(6-guanidino- caproyl)-amino-(L)-tyrosine, and ⁇ -(2-((5-((aminoiminomethyl)amino)-l - oxopentyl)amino)- 1 -oxoethyl)amino-3-pyridinepropanoic acid, bistrifluoroacetate.( SC56631) (see, for example, Samanen et al, J.
  • an autotaxin antagonist such as an anti-autotaxin antibody, produced as described above.
  • RGD peptides and RGD peptidomimetics can be used to block the binding of high concentrations of endogenously-produced autotaxin to integrin.
  • compositions comprising a peptide or polypeptide described herein.
  • Such compositions can further comprise a carrier.
  • the carrier can be a conventional organic or inorganic carrier. Examples of carriers include water, buffer solution, alcohol, propylene glycol, macrogol, sesame oil, corn oil, and the like.
  • the carrier can be a pharmaceutically acceptable carrier.
  • Autotaxin pharmaceutical compositions can be supplied as a kit comprising a container that comprises autotaxin.
  • Autotaxin can be provided in the form of an injectable composition for single or multiple doses, or as a sterile powder that will be reconstituted before administration.
  • a kit can include a dry- powder disperser, liquid aerosol generator, or nebulizer for administration of autotaxin.
  • a kit can further comprise written information on indications and usage of the pharmaceutical composition. Moreover, such information may include a statement that the autotaxin composition is contraindicated in patients with known hypersensitivity to autotaxin.
  • the present invention includes the use of autotaxin nucleotide sequences to enhance the production of autotaxin in a subject.
  • a therapeutic expression vector can be provided that inhibits autotaxin gene expression, such as an anti-sense molecule, a ribozyme, or an external guide sequence molecule.
  • an expression vector in which a nucleotide sequence encoding an autotaxin gene is operably linked to a core promoter, and optionally a regulatory element, to control gene transcription.
  • a core promoter and optionally a regulatory element, to control gene transcription.
  • Those skilled in the art can direct expression of an autotaxin gene to a particular tissue type.
  • Crystal et al, U.S. Patent No. 5,869,037 teach the use of the aP2 or pi 54 gene regulatory regions to effect adipocyte-specific expression.
  • an autotaxin gene can be delivered using recombinant viral vectors, including for example, adenoviral vectors (e.g., Kass-Eisler et al, Proc. Nat 'l Acad. Sci. USA 90:11498 (1993), Kolls et al, Proc. Nat'l Acad. Sci. USA 97:215 (1994), Li et al, Hum. Gene Ther. 4:403 (1993), Vincent et al, Nat. Genet. 5:130 (1993), and Zabner et al, Cell 75:201 (1993)), adenovirus-associated viral vectors (Flotte et al, Proc. Nat'l Acad. Sci.
  • adenoviral vectors e.g., Kass-Eisler et al, Proc. Nat 'l Acad. Sci. USA 90:11498 (1993), Kolls et al, Proc. Nat'l Acad. Sci. USA 97:215 (1994
  • alphaviruses such as Semliki Forest Virus and Sindbis Virus (Hertz and Huang, J. Vir. 66:851 (1992), Raju and Huang, J. Vir. 65:2501 (1991), and Xiong et al, Science 243:1188 (1989)), herpes viral vectors (e.g., U.S. Patent Nos. 4,769,331, 4,859,587, 5,288,641 and 5,328,688), parvovirus vectors (Koering et al, Hum. Gene Therap. 5:457 (1994)), pox virus vectors (Ozaki et al, Biochem. Biophys. Res. Comm.
  • pox viruses such as canary pox virus or vaccinia virus (Fisher-Hoch et al, Proc. Nat'l Acad. Sci. USA 86:3X1 (1989), and Flexner et al, Ann. N Y. Acad. Sci. 569:86 (1989)), and retroviruses (e.g., Baba et al, J. Neurosurg 79:129 (1993), Ram et al, Cancer Res. 55:83 (1993), Takamiya et al, J. Neurosci.
  • the viral vector itself, or a viral particle which contains the viral vector may be utilized in the methods and compositions described below.
  • adenovirus a double-stranded DNA virus
  • the adenovirus system offers several advantages including: (i) the ability to accommodate relatively large DNA inserts, (ii) the ability to be grown to high-titer, (iii) the ability to infect a broad range of mammalian cell types, and (iv) the ability to be used with many different promoters including ubiquitous, tissue specific, and regulatable promoters.
  • adeno viruses can be administered by intravenous injection, because the viruses are stable in the bloodstream.
  • adenovirus vectors where portions of the adenovirus genome are deleted, inserts are incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential El gene is deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell.
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. Although an adenoviral delivery system with an El gene deletion cannot replicate in the host cells, the host's tissue will express and process an encoded heterologous protein. Host cells will also secrete the heterologous protein if the corresponding gene includes a secretory signal sequence. Secreted proteins will enter the circulation from tissue that expresses the heterologous gene (e.g., the highly vascularized liver).
  • adenoviral vectors containing various deletions of viral genes can be used to reduce or eliminate immune responses to the vector.
  • Such adenoviruses are El-deleted, and in addition, contain deletions of E2A or E4 (Lusky et al, J. Virol 72:2022 (1998); Raper et al, Human Gene Therapy 9:671 (1998)).
  • the deletion of E2b has also been reported to reduce immune responses (Amalfrtano et al, J. Virol. 72:926 (1998)). By deleting the entire adenovirus genome, very large inserts of heterologous DNA can be accommodated.
  • an expression vector comprising an autotaxin gene can be introduced into a subject's cells by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al, Proc. Nat'l Acad. Sci. USA 84:14X3 (1987); Mackey et al, Proc. Nat'l Acad. Sci. USA 55:8027 (1988)).
  • the use of lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • Liposomes can be used to direct transfection to particular cell types, which is particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain. Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters
  • proteins such as antibodies
  • non-peptide molecules can be coupled to liposomes chemically.
  • Electroporation is another alternative mode of administration. For example, Aihara and Miyazaki, Nature Biotechnology 16:861 (1998), have demonstrated the use of in vivo electroporation for gene transfer into muscle.
  • a therapeutic gene may encode an autotaxin anti-sense RNA that inhibits the expression of autotaxin.
  • Suitable sequences for anti-sense molecules can be derived from the nucleotide sequences of autotaxin disclosed herein.
  • an expression vector can be constructed in which a regulatory element is operably linked to a nucleotide sequence that encodes a ribozyme.
  • Ribozymes can be designed to express endonuclease activity that is directed to a certain target sequence in a mRNA molecule (see, for example, Draper and Macejak, U.S. Patent No. 5,496,698, McSwiggen, U.S. Patent No. 5,525,468, Chowrira and McSwiggen, U.S. Patent No. 5,631,359, and Robertson and Goldberg, U.S. Patent No. 5,225,337).
  • ribozymes include nucleotide sequences that bind with autotaxin mRNA.
  • expression vectors can be constructed in which a regulatory element directs the production of RNA transcripts capable of promoting RNase P-mediated cleavage of mRNA molecules that encode an autotaxin gene.
  • an external guide sequence can be constructed for directing the endogenous ribozyme, RNase P, to a particular species of intracellular mRNA, which is subsequently cleaved by the cellular ribozyme (see, for example, Altaian et al, U.S. Patent No. 5,168,053, Yuan et al, Science 265:1269 (1994), Pace et al, international publication No. WO 96/18733, George et al, international publication No.
  • the external guide sequence comprises a ten to fifteen nucleotide sequence complementary to autotaxin mRNA, and a 3'-NCCA nucleotide sequence, wherein N is preferably a purine.
  • the external guide sequence transcripts bind to the targeted mRNA species by the formation of base pairs between the mRNA and the complementary external guide sequences, thus promoting cleavage of mRNA by RNase P at the nucleotide located at the 5'-side of the base-paired region.
  • the dosage of a composition comprising a therapeutic vector having an autotaxin nucleotide acid sequence, such as a recombinant virus will vary depending upon such factors as the subject's age, weight, height, sex, general medical condition and previous medical history.
  • Suitable routes of administration of therapeutic vectors include intravenous injection, intraarterial injection, intraperitoneal injection, intramuscular injection, intratumoral injection, and injection into a cavity that contains a tumor.
  • Horton et al, Proc. Nat 'l Acad. Sci. USA 96:1553 (1999) demonstrated that intramuscular injection of plasmid DNA encoding interferon- ⁇ produces potent antitumor effects on primary and metastatic tumors in a murine model.
  • a composition comprising viral vectors, non-viral vectors, or a combination of viral and non-viral vectors of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby vectors or viruses are combined in a mixture with a pharmaceutically acceptable carrier.
  • a composition such as phosphate-buffered saline is said to be a "pharmaceutically acceptable carrier” if its administration can be tolerated by a recipient subject.
  • suitable carriers are well-known to those in the art (see, for example, Remingto 's Pharmaceutical Sciences, 19th Ed. (Mack Publishing Co. 1995), and Gilman 's the Pharmacological Basis of Therapeutics, 7th Ed. (MacMillan Publishing Co. 1985)).
  • a therapeutic gene expression vector, or a recombinant virus comprising such a vector, and a pharmaceutically acceptable carrier are administered to a subject in a therapeutically effective amount.
  • a combination of an expression vector (or virus) and a pharmaceutically acceptable carrier is said to be administered in a "therapeutically effective amount" if the amount administered is physiologically significant.
  • An agent is physiologically significant if its presence results in a detectable change in the physiology of a recipient subject.
  • an agent is physiologically significant if its presence results in alleviation of a symptom associated with the diseases discussed above.
  • a pharmaceutical composition used to treat elevated serum lipids is physiologically significant if its administration results in a decrease level of serum triglycerides, or in a decrease in the amount of fatty acid bound to albumin in the serum.
  • the therapy is preferably somatic cell gene therapy. That is, the preferred treatment of a human with a therapeutic gene expression vector or a recombinant virus does not entail introducing into cells a nucleic acid molecule that can form part of a human germ line and be passed onto successive generations (i.e., human germ line gene therapy).
  • Transgenic mice can be engineered to over-express the autotaxin gene in all tissues or under the control of a tissue-specific or tissue-preferred regulatory element. These over-producers of autotaxin can be used to characterize the phenotype that results from over-expression, and the transgenic animals can serve as models for human metabolic disease caused by excess autotaxin. Transgenic mice that over- express autotaxin also provide model bioreactors for production of autotaxin in the milk or blood of larger animals.
  • a method for producing a transgenic mouse that expresses an autotaxin gene can begin with adult, fertile males (studs) (B6C3fl, 2-8 months of age (Taconic Farms, Germantown, NY)), vasectomized males (duds) (B6D2fl, 2-8 months, (Taconic Farms)), prepubescent fertile females (donors) (B6C3fl, 4-5 weeks, (Taconic Farms)) and adult fertile females (recipients) (B6D2fl, 2-4 months, (Taconic Farms)).
  • the donors are acclimated for one week and then injected with approximately 8 IU/mouse of Pregnant Mare's Serum gonadotrophin (Sigma Chemical Company; St. Louis, MO) I. P., and 46-47 hours later, 8 IU/mouse of human Chorionic Gonadotropin (hCG (Sigma)) I.P. to induce superovulation.
  • Donors are mated with studs subsequent to hormone injections. Ovulation generally occurs within 13 hours of hCG injection. Copulation is confirmed by the presence of a vaginal plug the morning following mating. Fertilized eggs are collected under a surgical scope. The oviducts are collected and eggs are released into urinanalysis slides containing hyaluronidase (Sigma).
  • Eggs are washed once in hyaluronidase, and twice in Whitten's W640 medium (described, for example, by Menino and O'Claray, Biol. Reprod. 77:159 (1986), and Dienhart and Downs, Zygote 4:129 (1996)) that has been incubated with 5% CO 2 , 5% O , and 90% N at 37°C.
  • the eggs are then stored in a 37°C/5% CO incubator until microinjection.
  • plasmid DNA containing an autotaxin encoding sequence is linearized, gel-purified, and resuspended in 10 mM Tris-HCl (pH 7.4), 0.25 mM EDTA (pH 8.0), at a final concentration of 5-10 nanograms per microliter for microinjection.
  • the autotaxin encoding sequences can encode amino acid residues of SEQ ID NO:2 or SEQ ID NO:9.
  • Plasmid DNA is microinjected into harvested eggs contained in a drop of W640 medium overlaid by warm, CO -equilibrated mineral oil.
  • the DNA is drawn into an injection needle (pulled from a 0.75mm ID, 1mm OD borosilicate glass capillary), and injected into individual eggs. Each egg is penetrated with the injection needle, into one or both of the haploid pronuclei.
  • Picoliters of DNA are injected into the pronuclei, and the injection needle withdrawn without coming into contact with the nucleoli. The procedure is repeated until all the eggs are injected. Successfully microinjected eggs are transferred into an organ tissue-culture dish with pre-gassed W640 medium for storage overnight in a 37°C/5% CO 2 incubator.
  • two-cell embryos are transferred into pseudopregnant recipients.
  • the recipients are identified by the presence of copulation plugs, after copulating with vasectomized duds.
  • Recipients are anesthetized and shaved on the dorsal left side and transferred to a surgical microscope.
  • a small incision is made in the skin and through the muscle wall in the middle of the abdominal area outlined by the ribcage, the saddle, and the hind leg, midway between knee and spleen.
  • the reproductive organs are exteriorized onto a small surgical drape.
  • the fat pad is stretched out over the surgical drape, and a baby serrefine (Roboz, Rockville, MD) is attached to the fat pad and left hanging over the back of the mouse, preventing the organs from sliding back in.
  • a baby serrefine Robot, Rockville, MD
  • the recipients are returned to cages in pairs, and allowed 19-21 days gestation. After birth, 19-21 days postpartum is allowed before weaning.
  • the weanlings are sexed and placed into separate sex cages, and a 0.5 cm biopsy (used for genotyping) is snipped off the tail with clean scissors.
  • Genomic DNA is prepared from the tail snips using, for example, a
  • Genomic DNA is analyzed by PCR using primers designed to amplify an autotaxin gene or a selectable marker gene that was introduced in the same plasmid.
  • animals are back-crossed into an inbred strain by placing a transgenic female with a wild-type male, or a transgenic male with one or two wild-type female(s). As pups are born and weaned, the sexes are separated, and their tails snipped for genotyping.
  • a partial hepatectomy is performed.
  • a surgical prep is made of the upper abdomen directly below the zyphoid process.
  • a small 1.5-2 cm incision is made below the sternum and the left lateral lobe of the liver exteriorized.
  • a tie is made around the lower lobe securing it outside the body cavity.
  • An atraumatic clamp is used to hold the tie while a second loop of absorbable Dexon (American Cyanamid; Wayne, N.J.) is placed proximal to the first tie.
  • a distal cut is made from the Dexon tie and approximately 100 mg of the excised liver tissue is placed in a sterile petri dish.
  • the excised liver section is transferred to a 14 ml polypropylene round bottom tube and snap frozen in liquid nitrogen and then stored on dry ice.
  • the surgical site is closed with suture and wound clips, and the animal's cage placed on a 37°C heating pad for 24 hours post operatively.
  • the animal is checked daily post operatively and the wound clips removed 7-10 days after surgery.
  • the expression level of autotaxin mRNA is examined for each transgenic mouse using an RNA solution hybridization assay or polymerase chain reaction.
  • transgenic mice that over-express autotaxin
  • Such transgenic mice provide useful models for diseases associated with a lack of autotaxin.
  • autotaxin gene expression can be inhibited using anti-sense genes, ribozyme genes, or external guide sequence genes.
  • inhibitory sequences are targeted to autotaxin mRNA.
  • An alternative approach to producing transgenic mice that have little or no autotaxin gene expression is to generate mice having at least one normal autotaxin allele replaced by a nonfunctional autotaxin gene.
  • One method of designing a nonfunctional autotaxin gene is to insert another gene, such as a selectable marker gene, within a nucleic acid molecule that encodes autotaxin. Standard methods for producing these so-called “knockout mice” are known to those skilled in the art (see, for example, Jacob, "Expression and Knockout of Interferons in Transgenic Mice," in Overexpression and Knockout of Cytokines in Transgenic Mice, Jacob (ed.), pages 111- 124 (Academic Press, Ltd. 1994), and Wu et al, "New Strategies for Gene Knockout,” in Methods in Gene Biotechnology, pages 339-365 (CRC Press 1997)).
  • 7F2 is a stromal cell line derived from the bone marrow of p53-deficient mice, as described by
  • ATX cells medium from the transgenic autotaxin- producing cells
  • the cells were plated at 1.1 x 10 4 cells/500 ⁇ l well in collagen-coated 48 well plates, and incubated with ⁇ -MEM containing 10% fetal bovine serum, L-glutamine, and sodium pyruvate.
  • Concentrated (50x) conditioned medium from cells transfected with a rat autotaxin expression vector was tested at a final concentration of O.lx, 0.5x, or 2x. Concentrated medium from cells that did not contain the autotaxin expression vector was included as a control.
  • indomethacin was added to a final concentration of 50 ⁇ M. The cells received fresh medium after three days, and after five days. Oil Red O staining was performed on day 7.
  • the medium was concentrated to 1.5 liters using an AMICON 10,000 spiral cartridge that had been treated with bovine serum albumin.
  • the 1.5 liters was further concentrated to 200 milliliters, and diafiltered into a Concanavilin A equilibration buffer, using an AGT Xampler filter that had a 30,000 molecular weight cut-off.
  • the Concanavilin A equilibration buffer contained: 20mM Tris-HCl (pH 7.4), 0.5 M NaCl, 1 mM Ca ++ , and 1 mM Mg ++ .
  • Con A Sepharose 4B Fifty milliliters of Con A Sepharose 4B (AMERSHAM PHARMACIA BIOTECH, Inc.; Piscataway, NJ) were equilibrated with buffer, 175 milliliters of concentrated medium were added to equilibrated Con A Sepharose 4B, and the mixture was slowly stirred at 4°C for 24 hours. The mixture was then poured into a column (3.2cm x 6.1cm) and washed with three column volumes of equilibration buffer. The elution buffer, which contained 0.5 M ⁇ -D-methylglucoside, was held on the column for 30 minutes, and then, additional elution buffer was added to the column at a flow rate of 3.6 column volumes/hour. Eluate was collected in four tubes, and each was found to contain autotaxin. The autotaxin peak appeared in the third tube, which was equivalent to about one column volume of elution buffer (50 milliliters).
  • 3T3 LI cell differentiation was performed according to the method of Olson et al, Mol. Cell. Biol. 77:2425 (1997). Undifferentiated 3T3 LI cells were plated at a density of 40,000 cells/well on collagen coated 24 well plates, or at 10,000 cells/well on collagen coated 96 well plates (BioCoatTM; BD; Franklin Lakes, NJ) in DMEM supplemented with 10% calf serum (HyClone Laboratories, Inc.; Logan, Utah). The cells were incubated overnight at 8% CO2 and 37°C.
  • cells were differentiated with DMEM supplemented with 10% fetal bovine serum (HyClone), 1 ⁇ g/ml insulin, 1 ⁇ M dexamethasone (Sigma) and 0.5 mM IBMX (ICN Pharmaceuticals, Inc.; Costa Mesa, CA), and the cells were incubated for four days at 37°C and 8% CO2. Media were replaced with DMEM supplemented with 10% fetal bovine serum and 1 ⁇ g/ml insulin, and the cells were incubated another four days at 37°C and 8% CO2. Media were replaced every four days with DMEM (5.6 mM glucose) and 10% fetal bovine serum until the cells were used in the glucose uptake assay.
  • Glucose uptake was measured following a modification of the method described by Walker et al, J. Biol Chem. 264:6581 (1989).
  • DMEM 5.6 mM glucose
  • bovine serum albumin Sigma RIA grade
  • 1 mM sodium pyruvate 1 mM sodium pyruvate
  • 20 mM Hepes buffer 20 mM Hepes buffer.
  • media were replaced with 500 ⁇ l per well DMEM (without glucose) supplemented with 0.25% bovine serum albumin, ImM sodium pyruvate, 20 mM Hepes, and 50 ⁇ M 2 deoxy-D- glucose (Sigma; St. Louis, MO).
  • glucose uptake was observed when cells were incubated with 100 nM autotaxin in the absence of subsequent insulin treatment.

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Abstract

Les altérations de la sensibilité insulinique sont associées au diabète et à des troubles tels que l'obésité et la dyslipidémie. Des niveaux élevés d'acide gras en circulation libre sous forme de triglycérides, qui constituent une facteur de risque important pour l'athérosclérose, viennent compliquer ces troubles. Les troubles du métabolisme tels que l'obésité, la dyslipidémie et le diabète peuvent être traités au moyen de compositions pharmaceutiques renfermant soit des polypeptides d'autotaxine ou des fragments fonctionnels de ces derniers, soit des molécules d'acides nucléiques codant pour lesdits polypeptides ou leurs fragments. Les polypeptides d'autotaxine convenant pour de telles méthodes thérapeutiques renferment par exemples de nouvelles formes d'autotaxine de l'homme et du rat.
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