Classifications

• • 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
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
  • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
  • C07K14/4726—Lectins
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/08—Antiallergic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• the present invention relates to a novel galectin. More specifically, isolated nucleic acid molecules are provided encoding human galectin 11. Galectin 11 polypeptides are also provided, as are vectors, host cells, recombinant methods for producing the same, and antibodies to galectin 11 polypeptides. The invention further relates to screening methods for identifying agonists and antagonists of galectin 11 activity. Also provided are diagnostic methods for detecting cell growth disorders and therapeutic methods for cell growth disorders, including autoimmune diseases, cancer, and inflammatory diseases.
• Lectins are proteins that bind to specific carbohydrate structures and can thus recognize particular glycoconjugates. Barondes et al., J. Biol. Chem. 269(33):20807-20810 (1994). Galectins are members of a family of ⁇ -galactoside-binding lectins with related amino acid sequences (For review see, Barondes et al., Cell 76:597-598 (1994); Barondes et al., J. Biol. Chem. 269(33):20807-20810 (1994)). Although a large number of glycoproteins containing ⁇ -galactoside sugars are produced by the cell, only a few will bind to known galectins in vitro. Such apparent binding specificity suggests a highly specific functional role for the galectins.
• Galectin 1 (conventionally termed LGALS1 for lectin, galactoside-binding, soluble A, but which is also known as: L-14-1, L-14, RL-14.5, galaptin, MGBP, GBP, BHL, CHA, HBP, HPL, HLBP 14, rIML-1) is a homodimer with a subunit molecular mass of 14,500 Daltons. Galectin 1 is expressed abundantly in smooth and skeletal muscle, and to a lesser extent in many other cell types (Couraud et al., J. Biol. Chem. 264. 1310-1316 (1989).
• Galectin 1 is thought to specifically bind laminin, a highly polylactosaminated cellular glycoprotein, as well as the highly polylactosaminated lysosome-associated membrane proteins (LAMPs). Galectin 1 has also been shown to bind specifically to a lactosamine- containing glycolipid found on olfactory neurons and to integrin a 7 b] on skeletal muscle cells.
• LAMPs highly polylactosaminated lysosome-associated membrane proteins
• Galectin 2 was originally found in hepatoma and is a homodimer with a subunit molecular mass of 14,650 Daltons (Gitt et al., J. Biol. Chem. 267. 10601-10606 (1992)).
• Galectin 3 (a.k.a., Mac-2, EPB, CBP-35, CBP-30, and L-29) is abundant in activated macrophages and epithelial cells and is a monomer with an apparent molecular mass between 26,320 and 30,300 Daltons (Cherayil et al., Proc. Natl. Acad. Sci. USA 87: 7324-7326 (1990)).
• Galectin 3 has been observed to bind specifically to laminin, immunoglobulin E and its receptor, and bacterial lipopolysaccharides.
• Galectin 4 has a molecular mass of 36,300 Daltons and contains two carbohydrate-binding domains within a single polypeptide chain (Oda et al., J. Biol. Chem. 268:5929-5939 (1993)).
• Galectins 5 and 6 are discussed in Barondes et al, Cell 76:597-598 (1994).
• Human Galectin 7 has a molecular mass of 15,073 Daltons and is found mainly in stratified squamous epithelium (Madsen et al., J. Biol. Chem.
• Galectin 1 has been shown to either promote or inhibit cell adhesion depending upon the cell type in which it is present. Galectin 1 inhibits cell-matrix interactions in skeletal muscle presumably, by galectin 1 -mediated disruption of laminin-integrin a 7 bj interactions (Cooper et al., J. Cell Biol. 115:1437-1448 (1991)). In several non-skeletal muscle cell types, Galectin 1 promotes cell-matrix adhesion possibly by cross-linking cell surface and substrate glycoconjugates (Zhou et al., Arch. Bioch. Biophys. 300:6-17 (1993); Skrincosky et al., Cancer Res. 53:2667-2675 (1993)).
• Galectin 1 also participates in regulating cell proliferation (Wells et al., Cell 64:91-97 (1991)) and some immune functions (Offner et al., J. Neuroimmunol. 28:177-184 (1990)). Galectin 1 induces the release of tumor necrosis factor from macrophages (Kajikawa et al., Life Sci. 39:1177-1181 (1986). Galectin 1 has also been demonstrated to have therapeutic activity against autoimmune diseases in animal models for experimental myasthenia gravis, and experimental autoimmune encephalomyelitis (Levi et al., Eur. J. Immunol. 13:500-507 (1983); and Offner et al., J. Neuroimmunol. 28: 177-184 (1990), respectively). Additionally, galectin 1 has been shown to regulate immune response by mediating apoptosis of T cells (Perillo et al., Nature 378:736-739 (1995)).
• Galectin 3 promotes the growth of cells cultured under restrictive culture conditions (Yang et al., Proc. Natl. Acad. Sci. USA 93:6737-6742 (June 1996)). Galectin 3 expression in cells confers resistance to apoptosis which indicates that galectin 3 could be a cell death suppresser which interferes in a common pathway of apoptosis. Id. Galectin 3 has also been observed to function in modulating cell-adhesion, as well as in the activation of certain immune cells by cross-linking IgE and IgE receptors.
• HOM-HD-21 a galectin-like antigen designated HOM-HD-21 was found to be highly expressed in a Hodgkin's Disease cDNA library and another galectin, termed PCTA-1, was identified as a specific cell surface marker on human prostate cancer cell lines and patient- derived carcinomas.
• galectins have been observed to be involved in the regulation of immune cell activity, as well as in such diverse processes as cell adhesion, proliferation, inflammation, autoimmunity, and metastasis of tumor cells. Accordingly, there is a need in the art for the identification of novel galectins which can serve as useful tools in the development of therapeutics and diagnostics for regulating immune response, inflammatory disease and cancer.
• the present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding the galectin 11 polypeptide having the amino acid sequence shown in Figure 1 (SEQ ID NO:2), the amino acid sequence encoded by the cDNA clone deposited in a bacterial host as ATCC Deposit Number 209053, on May 16, 1997, and fragments, variants, derivatives, and analogs thereof.
• the present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the galectin 11 polypeptide having the amino acid sequence shown in Figure 6 (SEQ ID NO: 14), referred to herein sometimes as "Galectin- l l ⁇ ” and fragments, variants, derivatives, and analogs thereof.
• the present invention also provides isolated nucleic acid molecules comprising a polynucleotide encoding the galectin 11 polypeptide having the amino acid sequence shown in Figures 6 and 8 (SEQ ID NO: 16), referred to herein sometimes as "Galectin-l l ⁇ ", and fragments, variants, derivatives, and analogs thereof.
• the galectin 11 of Figure 1 (SEQ ID NOS: 1 and 2)
• the galectin 1 l ⁇ of Figure 6 SEQ ID NOS: 1 and 2
• galectin 11 the galectin l l ⁇ of Figures 6-8 (SEQ ID NOS:26 and 27) are often referred to herein collectively as, e.g., "galectin 11.”
• the galectin 11 polynucleotide of Figure 1 (SEQ ID NO:l), the galectin l l ⁇ polynucleotide of Figure 6 (SEQ ID NO:24), and the galectin l l ⁇ polynucleotide of Figure 7 (SEQ ID NO:26) are often referred to herein collectively as, e.g., "galectin 11 polynucleotides.”
• the present invention also relates to recombinant vectors which include the isolated nucleic acid molecules of the invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of galectin 11 polypeptides by recombinant techniques.
• the invention further provides isolated galectin 11 polypeptides, including galectin 11 of SEQ ID NO: 2 and galectin l l ⁇ and ⁇ , having an amino acid sequence encoded by a polynucleotide described herein and antibodies which bind these polypeptides.
• the galectin 11 polypeptide of Figure 1 (SEQ ID NO:2), the galectin l l ⁇ polypeptide of Figure 6 (SEQ ID NO:25), and the galectin l l ⁇ polypeptide of Figure 7 (SEQ ID NO:27) are often referred to herein collectively as, e.g., "galectin 11 polypeptides.”
• the present invention also provides screening methods for identifying compounds capable of enhancing or inhibiting a cellular response, such as, for example, apoptosis, induced by galectin 11.
• these methods involve contacting galectin 11, the candidate compound, and a cell which expresses a galectin 11 ligand, assaying a cellular response resulting from the binding of galectin 11 with the ligand, and comparing the cellular response to a standard, the standard being assayed when contact of galectin 11 and the galectin 11 ligand is made in the absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased cellular response over the standard indicates that the compound is an antagonist.
• a screening assay for agonists and antagonists involves determining the effect a candidate compound has on galectin 11 binding to a ⁇ - galactoside sugar.
• the method involves contacting a ⁇ -galactoside sugar with a galectin 11 polypeptide and a candidate compound and determining whether galectin 11 binding to the ⁇ -galactoside sugar is increased or decreased due to the presence of the candidate compound.
• the invention also provides diagnostic methods useful during diagnosis of disorders associated with elevated, decreased, or otherwise aberrant expression of galectin 11.
• the invention further provides for methods for treating an individual in need of an increased level of galectin 11 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of an isolated galectin 11 polypeptide, fragment, variant, derivative, or analog of the invention, or an agonist thereof.
• the invention provides for methods for treating an individual in need of a decreased level of galectin 11 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a galectin 11 fragment, variant, derivative, analog or antibody of the invention or galectin 11 antagonist.
• Figure 1 shows the nucleotide sequence (SEQ ID NO:l) and deduced amino acid sequence (SEQ ID NO:2) of galectin 11.
• the protein has a deduced molecular mass of about 14.8 kDa.
• the complementary strand of the nucleotide sequence of SEQ ID NO:l is shown in SEQ ID NO: 12.
• Figure 2 shows the regions of similarity between the amino acid sequences of the galectin 11 protein (HJACE54), rat galectin 5 (SEQ ID NO:3), and human galectin 8 (SEQ ID NO:4).
• Identical amino acids shared between the galectins are shaded, while conservative amino acid changes are boxed.
• Figure 3 shows structural and functional features of galectin 11 (SEQ ID NO:2) predicted using the default parameters of the indicated computer programs.
• Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
• the positive peaks indicate locations of the highly antigenic regions of the galectin 11 protein, i.e., regions from which epitope-bearing peptides of the invention can be obtained.
• the domains defined by these graphs are contemplated by the present invention, including for example, amino acid residues 65-70 and 118-124 in Figure 1 (SEQ ID NO:2), which correspond to the shown highly antigenic regions of the galectin 11 polypeptide.
• FIG. 4 Structure of human galectin 11 gene.
• the human galectin 11 gene is located on chromosome 11.
• This figure shows the structure of the region of chromosome 11 containing the galectin 11 gene and discloses the number of nucleotides corresponding to the transcribed (shaded) and untranscribed (open) portions of this region of the chromosome.
• the human galectin 11 gene contains 5 exons.
• the translation initiation site is located on the second exon.
• the nucleotide numbering identified in exons designated by roman numerals correspond to that presented in Figure 1 (SEQ ID NO:l).
• Figure 5 A is a bar graph showing that transfection of Jurkat cells with a galectin 11 expression construct (pEF-Legl l) induces apoptosis of transfected cells. Shaded bars represent % apoptosis of Jurkat cells that have been transfected with the galectin 11 expression construct, whereas open bars represent % apoptosis of Jurkat cells that have been transfected with the pEF control vector. Apoptosis was measured by two-color cytometry using mitoTracker Red.
• Figure 5B is a bar graph showing the survival of GFP positive cells that have been successfully transfected, 4 days after transfection. The survival of the transfected cells was examined after co-transfection with either the control vector (pEFl), or the galectin 11 expression vector (pEF-Legl l). There were about 4 times more surviving GFP positive cells after transfection with pEFl than with pEF-Legl 1.
• Figure 6 shows the nucleotide sequence (SEQ ID NO:24) and deduced amino acid sequence (SEQ ID NO:25) of the complete galectin l l ⁇ cDNA and protein, respectively.
• Figure 7 is a schematic showing the relative positions of the 8 exons which comprise the galectin- 11 gene. Also shown is the difference created by alternative splicing between galectin-l l ⁇ and galectin-l l ⁇ (galectin-l l ⁇ being 7 nucleotides longer at the 5' terminus of exon 2) resulting in divergent N-termini between the variants.
• Figure 8 shows the difference between the polypeptide sequences of galectin-l l ⁇ and galectin- 1 l ⁇ .
• the complete nucleotide and amino acid sequences of galectin- 1 l ⁇ are shown in the sequence listing as SEQ ID NOS: 26 and 27, respectively.
• the present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding a galectin 11 polypeptide having the amino acid sequence shown in Figure 1 (SEQ ID NO:2), Figure 6 (SEQ ID NO:25), or Figures 6 and 8 (SEQ ID NO:27) which were determined by sequencing cloned cDNAs.
• the nucleotide sequence shown in Figure 1 (SEQ ID NO:l) was obtained by sequencing the HJACE54 plasmid which, was deposited on May 16, 1997 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia, and given accession number 209053.
• the galectin 1 1 polypeptides of the present invention share sequence homology with rat galectin 5, chicken galectin 3, and human galectin 8 gene products (see, e.g., Figure 2; SEQ ID NOS: 3-4).
• the invention further provides for fragments, variants, derivatives and analogs of galectin 11 polynucleotides and polypeptides encoded thereby, and antibodies which bind these polypeptides.
• “Functional activity” or “biological activity” refers to galectin 11 polypeptides, fragments, derivatives, variants, and analogs, exhibiting activity similar, but not necessarily identical to, an activity of a galectin 11 polypeptide, including mature forms, as measured in a particular biological assay, with or without dose dependency.
• Such functional activities include, but are not limited to, biological activity (such as, for example, the ability to bind a ⁇ -galactoside sugar, the ability to agglutinate trypsin-treated rabbit erythrocytes and/or to induce apoptosis), antigenicity (ability to bind or compete with a galectin 11 polypeptide for binding to an anti-galectin 11 antibody), immunogenicity (ability to generate antibody which binds to a galectin 11 polypeptide), the ability to form dimers with galectin 11
• Polynucleotide generally refers to any polyribonucleotide or polydeoxyribonucleotide, which may be unmodified RNA or DNA, or modified RNA or DNA.
• Polynucleotides include, without limitation single- and double-stranded DNA, DNA that is a mixture of single- and double- stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
• polynucleotide refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
• the term polynucleotide also includes DNAs or RNAs containing one or more modified bases and DNAs or RNAs with backbones modified for stability or for other reasons.
• Modified bases include, for example, tritylated bases and unusual bases such as inosine.
• polynucleotide embraces chemically, enzymatically or metabolically modified forms of polynucleotides as typically found in nature, as well as the chemical forms of DNA and RNA characteristic of viruses and cells.
• Polynucleotide also embraces relatively short polynucleotides, often referred to as oligonucleotides.
• Polypeptide refers to any peptide or protein comprising two or more amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres.
• Polypeptide refers to both short chains, commonly referred to as peptides, ohgopeptides or oligomers, and to longer chains, generally referred to as proteins. Polypeptides may contain amino acids other than the 20 gene-encoded amino acids.
• Polypeptides include amino acid sequences modified either by natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. Such modifications are well described in basic texts and in more detailed monographs, as well as in a voluminous research literature. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given galectin 11 polypeptide. Also, a given galectin 11 polypeptide may contain many types of modifications.
• Galectin 11 polypeptides may be branched as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched and branched cyclic polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
• Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer- RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
• Variant is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide respectively, but retains functional or biological activity of galectin 11.
• a typical variant of a polynucleotide differs in nucleotide sequence from another, reference polynucleotide. Changes in the nucleotide sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
• a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
• a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
• a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
• a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques or by direct synthesis.
• Antibodies as used herein includes polyclonal and monoclonal antibodies, chimeric, single chain, and humanized antibodies, as well as Fab fragments, including the products of an Fab or other immunoglobulin expression library.
• SEQ ID NO:l The galectin 11 nucleotide sequence identified as SEQ ID NO:l was assembled from partially homologous ("overlapping") sequences obtained from the deposited clone. The overlapping sequences were assembled into a single contiguous sequence of high redundancy resulting in a final sequence identified as SEQ ID NO:l. Therefore, SEQ ID NO: l and the translated SEQ ID NO:2 are sufficiently accurate and otherwise suitable for a variety of uses well known in the art and described further below. For instance, SEQ ID NO: 1 is useful for designing nucleic acid hybridization probes that will detect nucleic acid sequences contained in SEQ ID NO:l or the cDNA contained in the deposited clone.
• polypeptides identified from SEQ ID NO:2 may be used, for example, to generate antibodies which bind specifically to proteins galectin 11.
• nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc.), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
• the present invention provides not only the generated nucleotide sequence identified as SEQ ID NO:l and the predicted translated amino acid sequence identified as SEQ ID NO:2, but also a sample of plasmid DNA containing a human cDNA of galectin 11 deposited with the ATCC.
• the nucleotide sequence of the deposited galectin 11 clone can readily be determined by sequencing the deposited clone in accordance with known methods. The predicted galectin 11 amino acid sequence can then be verified from such deposits. Moreover, the amino acid sequence of the protein encoded by the deposited clone can also be directly determined by peptide sequencing or by expressing the protein in a suitable host cell containing the deposited human galectin 11 cDNA, collecting the protein, and determining its sequence.
• nucleic acid molecule of the present invention encoding a galectin 11 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
• nucleic acid molecule described in Figure 1 SEQ ID NO:l
• SEQ ID NO:l was discovered in a cDNA library derived from Gl phase Jurkat T-cells. This gene was also identified in cDNA libraries generated from human neutrophil and human infant adrenal gland.
• Polynucleotides of the invention can also be obtained from natural sources such as mRNA or genomic DNA using techniques known in the art, or can be chemically synthesized using techniques known in the art.
• the human galectin 11 gene is located on chromosome 11 and contains 5 exons (see, e.g., Figure 4).
• the nucleotide sequence of the galectin 11 cDNA of Figure 1 (SEQ ID NO: l) is 865 nucleotides in length (830 nucleotides discounting the poly A tail of the cDNA) which encodes a predicted open reading frame of 133 amino acid residues.
• the nucleotide sequence of the galectin 11 cDNA of Figure 6 is 1337 nucleotides in length. This is one of two alternatively spliced forms of galectin Hand is referred to as galectin l l ⁇ .
• the other form, galectin l l ⁇ differs only in the loss of 7 nucleotides (nucleotides 136-142 as shown in Figure 6 (SEQ ID NO:24)). See Figure 7.
• the sequence of galectin l l ⁇ is shown in the sequence listing as SEQ ID NO:26.
• the resulting translation products of these splice variants are believed to differ only at the N-terminus.
• the amino acid sequences of galectin l l ⁇ and ⁇ are shown in the sequence listing as SEQ ID NOS:25 and 27, respectively. The differences between the two proteins are highlighted in Figure 8.
• the galectin 11 polypeptide is comprised of two carbohydrate binding domains (CARD domains) separated by a linker sequence.
• the first carbohydrate binding domain consists of the first 121 amino acid residues of galectin-l l ⁇ (SEQ ID NO:25) and the first 142 amino acids of galectin l l ⁇ (SEQ ID NO:27).
• the 29 amino acid residues following the first CARD domain is the linker sequence.
• the last 125 amino acid residues in each protein is the C-terminal CARD domain.
• Preferred polypeptides of the invention comprise either an N-terminal or C-terminal CARD domain. Polynucleotides encoding such polypeptides are also provided.
• allelic variants, orthologs, and/or species homologs are also provided in the present invention. Procedures known in the art can be used to obtain full-length genes, allelic variants, splice variants, full-length coding portions, orthologs, and/or species homologs of genes corresponding to SEQ ID NO: 1-2, 24-25, 26-27, or the deposited clone, using information from the sequences disclosed herein or the clones deposited with the ATCC. For example, allelic variants and/or species homologs may be isolated and identified by making suitable probes or primers from the sequences provided herein and screening a suitable nucleic acid source for allelic variants and/or the desired homologue.
• the predicted galectin 11 polypeptide encoded by the deposited cDNA comprises about 133 amino acid residues, but may be anywhere in the range of 125-150 amino acids.
• nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
• the DNA may be double-stranded or single-stranded.
• Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the complementary or anti-sense strand.
• isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA or RNA, which has been removed from its native environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
• recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
• Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
• Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
• isolated nucleic acid molecules of the invention comprise all or a portion of the coding region of galectin 11, as disclosed in Figure 1 (SEQ ID NO:l) or galectin 1 l ⁇ as disclosed in Figure 6 (SEQ ID NO:24), or galectin 1 l ⁇ as disclosed in SEQ ID NO:26.
• isolated does not refer to genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide/sequences of the present invention.
• Isolated nucleic acid molecules of the present invention include DNA molecules comprising an open reading frame (ORF) or a portion of an ORF shown in Figure 1 or 6 (SEQ ID NO:l, 24, or 26); and DNA molecules which comprise a sequence substantially different from those described above, but which due to the degeneracy of the genetic code, still encode the galectin 11 protein.
• ORF open reading frame
• SEQ ID NO:l, 24, or 26 DNA molecules which comprise a sequence substantially different from those described above, but which due to the degeneracy of the genetic code, still encode the galectin 11 protein.
• the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate variants.
• the invention provides isolated nucleic acid molecules encoding the full length galectin 11 polypeptide depicted in Figure 1 (SEQ ID NO:2), and galectin 11 nucleic acid molecules encoding the galectin 11 polypeptide sequence encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 209053, on May 16, 1997.
• nucleic acid molecules are provided encoding the full length galectin 11 polypeptide lacking the N-terminal methionine.
• the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in Figure 1 (SEQ ID NO:l) or the nucleotide sequence of the galectin 11 cDNA contained in the above-described deposited clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences.
• isolated molecules particularly DNA molecules, have uses which include, but are not limited to, probes for gene mapping by in situ hybridization with chromosomes, and for detecting expression of the galectin 11 gene in human tissue, for instance, by Northern blot analysis.
• the invention further provides a polynucleotide encoding a polypeptide comprising the full-length amino acid sequence shown as SEQ ID NO:25 or 27, with or without an N-terminal methoinine.
• the polynucleotides of the invention are at least 15, at least 30, at least 50, at least 100, at least 125, at least 500, or at least 1000 continuous nucleotides but are less than or equal to 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, 7.5kb, 5 kb, 2.5 kb, 2.0 kb, or 1 kb, in length.
• polynucleotides of the invention comprise a portion of the coding sequences, as disclosed herein, but do not comprise all or a portion of any intron.
• the polynucleotides comprising coding sequences do not contain coding sequences of a genomic fl-inking gene (i.e., 5' or 3' to the galectin 11 gene of interest on chromosome II). In other embodiments, the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
• the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
• a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA, the nucleotide sequence shown in Figures 1 and 6 (SEQ ID NOS:l, 24, and 26), or the complementary strand thereto is intended fragments of at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length.
• fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in Figure 1 (SEQ ID NO:l) or the cDNA shown in Figure 6 (SEQ ID NOS:24 and 26) or the complementary strand thereto.
• DNA fragments comprising 50, 100, 150, 200, 250, 300, 350, 365, 370, 375, 380, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850 contiguous nucleotides of the sequence shown in Figure 1 (SEQ ID NO:l), the strand complementary thereto, or contained in the deposited clone.
• the present invention also encompasses fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in Figure 1 (SEQ ID NO.T) or the complimentary strand thereto.
• the polynucleotide fragments of the invention comprise a sequence which encodes amino acids 1-14, 1-20, 1-40, 1-66, 2-67, 3-8, 3-67, 5-108, 5-128, 10-17, 10-20, 12-16, 13-20, 13-68, 14-67, 23-40, 20-50, 40-108, 41-60, 47-61, 47-108, 47-128, 50-100, 61-80, 65-108, 65-128, 66-108, 76-88, 81-100, 88-108, 88- 128, 95-101, 101-133, 108-120, 114-128, and/or 114-128 of the amino acid sequence depicted in Figure 1 (SEQ ID NO:2).
• polynucleotide fragments of the invention encode a polypeptide which demonstrates a galectin 11 functional activity. Fragments of the invention have numerous uses which include, but are not limited to, diagnostic probes and primers as discussed herein.
• nucleic acid fragments of the present invention include nucleic acid molecules encoding epitope-bearing portions of the galectin 11 protein.
• such nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising amino acid residues from about 65-70 and 118-124 in Figure 1 (SEQ ID NO:2).
• SEQ ID NO:2 The inventors have determined that the above polypeptide fragments are antigenic regions of the galectin 11 protein. Methods for determining other such epitope-bearing portions of the galectin 11 protein are described in detail below.
• the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which hybridizes under stringent hybridization conditions to all or a portion of a galectin 11 polynucleotides (including fragments) described herein, the complementary strand thereof, the cDNA clone contained in ATCC Deposit No. 209053, on May 16, 1997, or fragments thereof.
• stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5X SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 X SSC at 65°C.
• nucleic acid molecules that hybridize to the galectin 11 polynucleotides under lower stringency hybridization conditions.
• Changes in the stringency of hybridization and signal detection are primarily accomplished through the manipulation of formamide concentration (lower percentages of formamide result in lowered stringency); salt conditions, or temperature.
• washes performed following stringent hybridization can be done at higher salt concentrations (e.g. 5X SSC).
• blocking reagents include Denhardt's reagent, BLOTTO, heparin, denatured salmon sperm DNA, and commercially available proprietary formulations.
• the inclusion of specific blocking reagents may require modification of the hybridization conditions described above, due to problems with compatibility.
• a polynucleotide which hybridizes to a portion of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20, still more preferably at least about 30, 50, 60, 75, 100, 150, 175, 200, 250, 300, 350 nt preferable about 30-70 nt, or 80-150 nucleotides, or the entire length of the reference polynucleotide.
• a portion of a polynucleotide of at least "20 nt in length" is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., the deposited cDNA or the nucleotide sequence as depicted in Figure 1 (SEQ ID NO:l).
• the polynucleotide hybridizes to nucleotides 0-20, 0-25, 0-30, 0-50, 51-100, 80-100, 101-200, 201-300, 301-400, 401-450, 451-500, 501-550, 551-600, 601-700, 701-750, 751-780, and/or 780-820 of the nucleotide sequence disclosed in Figure 1 (SEQ ID NO:l).
• the polynucleotide hybridizes to a nucleotide sequence which encodes amino acid residues 1-14, 10-20, 20-50, 50-100, 100-133 of the amino acid sequence depicted in Figure 1 (SEQ ID NO:2).
• the polynucleotide hybridizes to nucleotides 1-20, 1-25, 1-30, 1-50, 51-100, 80-100, 101-200, 201-300, 301-400, 401-450, 451-500, 501-550, 551-600, 601-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951- 1,000, 1,001-1050, 1,051-1,100, 1,101-1,150, 1,151-1,200, 1,201-1,250, and/or 1,251-1,337 of the nucleotide sequence disclosed in SEQ ID NO:24.
• the polynucleotide hybridizes to a nucleotide sequence which encodes amino acid residues 1-14, 10-20, 20-50, 50-100, 100-130, 130-160, 160-210, 210-240 and/or 240-275 of the amino acid sequence depicted in SEQ ID NO:25.
• the polynucleotide hybridizes to nucleotides 1-20, 1-25, 1-30, 1-50, 51-100, 80-100, 101-200, 201-300, 301-400, 401-450, 451-500, 501-550, 551-600, 601-700, 701-750, 751-800, 801-850, 851-900, 901-950, 951- 1,000, 1,001-1050, 1,051-1,100, 1,101-1,150, 1,151-1,200, 1,201-1,250, and/or 1,251-1,330 of the nucleotide sequence disclosed in Figure SEQ ID NO:26.
• the polynucleotide hybridizes to a nucleotide sequence which encodes amino acid residues 1-14, 10-20, 20-50, 50-100, 100-130, 130-160, 160-210, 210-240, 240-270 and/or 270-296 of the amino acid sequence depicted in SEQ ID NO:27.
• These polynucleotides have uses which include, but are not limited to, diagnostic probes and primers, as discussed above and in more detail below.
• nucleic acid molecules of the present invention which encode a galectin
• 11 polypeptide may include, but are not limited to, those encoding the amino acid sequence of the polypeptide, by itself; the coding sequence for the polypeptide and additional sequences, such as those encoding an amino acid leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to, introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
• the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.
• the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
• hexa-histidine provides for convenient purification of the fusion protein.
• the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al., Cell 37:767-778 (1984). As discussed below, other such fusion proteins include the galectin 11 fused to Fc at the N- or C-terminus.
• polynucleotide fragment refers to a short polynucleotide having a nucleic acid sequence which: is a portion of that contained in a deposited clone, or encoding the polypeptide encoded by the cDNA in a deposited clone; is a portion of that shown in SEQ ID NO:l, 24, or 26 or the complementary strand thereto, or is a portion of a polynucleotide sequence encoding the polypeptide of SEQ ID NO:2, 25, or 27.
• the nucleotide fragments of the invention are preferably at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt, at least about 50 nt, at least about 75 nt, or at least about 150 nt in length.
• a fragment "at least 20 nt in length,” for example, is intended to include 20 or more contiguous bases from the cDNA sequence contained in a deposited clone or the nucleotide sequence shown in SEQ ID NO:l, 24, or 26.
• nucleotide fragments include, but are not limited to, as diagnostic probes and primers as discussed herein.
• larger fragments e.g., 50, 150, 500, 600, 2000 nucleotides are prefe ⁇ ed.
• polynucleotide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, a sequence from about nucleotide number 1-48, 49-99, 100-150, 151-201, 202-252, 253-303, 304-354, 355- 405, 406-450, 451-501, and 502 to the end of SEQ ID NO:l, or the complementary strand thereto, or the cDNA contained in the deposited clone.
• “about” includes the particularly recited ranges, and ranges larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
• these fragments encode a polypeptide which has biological activity.
• these polynucleotides can be used as probes or primers as discussed herein.
• Polynucleotides which hybridize to these nucleic acid molecules under stringent hybridization conditions or lower stringency conditions are also encompassed by the invention, as are polypeptides encoded by these polynucleotides.
• the exact formulation, route of administration and dosage of the compounds of the invention to be administrated can be chosen by the individual physician in view of the patient's condition (see e.g., Fingl et al., 1975, in "The Pharmacological Basis of Therapeutics.” Ch. 1 p. 1). Other methods will be known to the skilled artisan and are within the scope of the invention.
• polynucleotide sequences such as EST sequences
• sequence databases Some of these sequences are related to SEQ ID NO: l and may have been publicly available prior to conception of the present invention.
• sequences are related to SEQ ID NO: l and may have been publicly available prior to conception of the present invention.
• polynucleotides are specifically excluded from the scope of the present invention. To list every related sequence would be cumbersome.
• polynucleotides comprising a nucleotide sequence described by the general formula of a-b, where a is any integer between 1 to 851 of SEQ ID NO:l, b is an integer of 15 to 865, where both a and b co ⁇ espond to the positions of nucleotide residues shown in SEQ ID NO:l, and where the b is greater than or equal to a + 14.
• the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode a portion (i.e., fragments), analogs or derivatives of the galectin 11 protein. Variants may occur naturally, such as a natural allelic variant.
• an "allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
• variants include those produced by nucleotide substitutions, deletions or additions which may involve one or more nucleotides
• Particularly prefe ⁇ ed are variants in which the nucleotide sequence encoding 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 15, 20, 25, 30, 35, 40, 50, or, 20-15, 15-10, 10-5 1-5, 1-3, or 1-2 amino acids of a polypeptide of the invention are substituted, deleted, or added in any combination.
• the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.
• Especially prefe ⁇ ed among these are silent substitutions, additions and deletion, which do not alter the properties and activities of the galectin 11 protein or portions thereof. Also especially prefe ⁇ ed in this regard are conservative substitutions.
• nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least 75%, 80%, 85%, or 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% or 98-99% identical to (a) a nucleotide encoding amino acids 1 to 133 of SEQ ID NO:2; (b) a nucleotide encoding amino acids 2 to 133 of SEQ ID NO:2; (c) a nucleotide sequence of the galectin 11 polypeptide encoded by the cDNA contained in ATCC Deposit No.
• nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five nucleotide mismatches per each 100 nucleotides of the reference nucleotide sequence encoding the galectin 11 polypeptide.
• a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
• These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
• the query sequence may be an entire sequence shown of SEQ ID NO:l, the ORF (open reading frame), or any fragment specified as described herein.
• nucleic acid molecule is at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in SEQ ID NOS: 1, 24, and 26 or to the nucleotides sequence of the deposited cDNA clone can be determined conventionally using known computer programs, such as, for example, the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology between two sequences.
• Bestfit program Wiconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711. Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2: 482-489 (1981), to find the best segment of homology
• the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
• a prefe ⁇ ed method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also refe ⁇ ed to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
• the query and subject sequences are both DNA sequences.
• An RNA sequence can be compared by converting U's to T's. The result of said global sequence alignment is in percent identity.
• the percent identity is co ⁇ ected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. A determination of whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment.
• This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to a ⁇ ive at a final percent identity score.
• This corrected score is what is used for the purposes of this embodiment. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.
• a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity.
• the deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end.
• the 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
• a 90 base subject sequence is compared with a 100 base query sequence.
• deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query.
• percent identity calculated by FASTDB is not manually co ⁇ ected.
• bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually co ⁇ ected for. No other manual co ⁇ ections are made for the purposes of this embodiment.
• the galectin 11 variants may contain alterations in the coding regions, non-coding regions, or both.
• polynucleotide variants containing alterations which produce silent substitutions, additions, or deletions, but do not alter the properties or activities of the encoded polypeptide are prefe ⁇ ed.
• Galectin 11 polynucleotide variants can be produced for a variety of reasons, e.g., to optimize codon expression for a particular host (change codons in the human mRNA to those prefe ⁇ ed by a bacterial host such as E. coli).
• Naturally occurring galectin 11 variants are called "allelic variants," and refer to one of several alternate forms of a gene occupying a given locus on a chromosome of an organism.
• allelic variants can vary at either the polynucleotide and/or polypeptide level and are included in the present invention.
• non-naturally occurring variants may be produced by mutagenesis techniques or by direct synthesis.
• variants may be generated to improve or alter the characteristics of the galectin 11 polypeptides. For instance, one or more amino acids can be deleted from the N-terminus or
• the invention further includes galectin 11 polypeptide variants which show substantial biological activity.
• Such variants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as have little effect on activity.
• the present application is directed to nucleic acid molecules at least 75%, 80%, 85%,
• nucleic acid sequences disclosed herein e.g., nucleic acid sequence shown in Figures 1 or 6 (SEQ ID NO:l, 24, or 26), nucleic acid sequence of the deposited cDNA clone, and nucleic acid sequences encoding a polypeptide having the amino acid sequence of an N and/or C terminal deletion disclosed below as m-n of SEQ ID NO:2, 25, or 27), i ⁇ espective of whether they encode a polypeptide having galectin 11 functional activity.
• nucleic acid molecule does not encode a polypeptide having galectin 11 functional activity
• PCR polymerase chain reaction
• nucleic acid molecules of the present invention that do not encode a polypeptide having galectin 11 functional activity include, inter alia, (1) isolating the galectin 11 gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the galectin 11 gene, as described in Verma et al, Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); (3) use in linkage analysis as a marker for chromosome 11; and (4) Northern Blot analysis for detecting galectin 11 mRNA expression in specific tissues.
• FISH in situ hybridization
• nucleic acid molecules having sequences at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence disclosed herein, shown in Figures 1 or 6 (SEQ ID NO:l, 24, or 26), nucleic acid sequence of the deposited cDNA clone, the nucleic acid encoding the polypeptide shown in Figure 1 or 6 (SEQ ID NO:2, 25, or 27), and fragments thereof, which do, in fact, encode a polypeptide having galectin 11 functional activity.
• a polypeptide having galectin 11 functional activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to a functional activity of the galectin 11 protein of the invention (e.g., complete (full-length) galactin 11, and mature galactin 11), as measured in a particular assay.
• galectin 11 protein activity can be measured using a ⁇ -galactoside sugar (e.g., thiodigalactoside or lactose) binding assay, an assay for apoptosis and/or an assay for agglutination of trypsin- treated rabbit erythrocytes, as further described below.
• the first strategy exploits the tolerance of amino acid substitutions by natural selection during the process of evolution. By comparing amino acid sequences in different species, conserved amino acids can be identified. These conserved amino acids are likely important for protein function. In contrast, the amino acid positions where substitutions have been tolerated by natural selection indicates that these positions are not critical for protein function. Thus, positions tolerating amino acid substitution could be modified while still maintaining biological activity of the protein.
• the second strategy uses genetic engineering to introduce amino acid changes at specific positions of a cloned gene to identify regions critical for protein function. For example, site directed mutagenesis or alanme-scanning mutagenesis (introduction of single alanine mutations at every residue in the molecule) can be used. (Cunningham and Wells, Science 244:1081-1085 (1989).) The resulting mutant molecules can then be tested for biological activity.
• tolerated conservative amino acid substitutions involve replacement of the aliphatic or hydrophobic amino acids Ala, Val, Leu and He; replacement of the hydroxyl residues Ser and Thr; replacement of the acidic residues Asp and Glu; replacement of the amide residues Asn and Gin, replacement of the basic residues Lys, Arg, and His; replacement of the aromatic residues Phe, Tyr, and Trp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met, and Gly.
• site directed changes at the amino acid level of galectin 11 of Figure 1 can be made by replacing a particular amino acid with a conservative amino acid.
• conservative mutations include: Ml replaced with A, G, I, L, S, T, or V; S2 replaced with A, G, I, L, T, M, or V; R4 replaced with H, or K; L5 replaced with A, G, I, S, T, M, or V; E6 replaced with D; V7 replaced with A, G, I, L, S, T, or M; S10 replaced with A, G, I, L, T, M, or V; HI 1 replaced with K, or R; A12 replaced with G, I, L, S, T, M, or V; L13 replaced with A, G, I, S, T, M, or V; Q15 replaced with N; G16 replaced with A, I, L, S, T, M, or V; LI 7 replaced with A, G, I, S, T, M, or V
• the resulting constructs can be routinely screened for activities or functions described throughout the specification and known in the art.
• the resulting constructs have an increased and/or a decreased galectin 11 activity or function, while the remaining galectin 11 activities or functions are maintained. More preferably, the resulting constructs have more than one increased and/or decreased galectin 11 activity or function, while the remaining galectin 11 activities or functions are maintained.
• variants of galectin 11 include (i) substitutions with one or more of the non-conserved amino acid residues, where the substituted amino acid residues may or may not be one encoded by the genetic code, or (ii) substitution with one or more of amino acid residues having a substituent group, or (iii) fusion of the mature polypeptide with another compound, such as a compound to increase the stability and/or solubility of the polypeptide (for example, polyethylene glycol), or (iv) fusion of the polypeptide with additional amino acids, such as, for example, an IgG Fc fusion region peptide, or leader or secretory sequence, or a sequence facilitating purification.
• Such variant polypeptides are deemed to be within the scope of those skilled in the art from the teachings herein.
• galectin 11 polypeptide variants containing amino acid substitutions of charged amino acids with other charged or neutral amino acids may produce proteins with improved characteristics, such as less aggregation. Aggregation of pharmaceutical formulations both reduces activity and increases clearance due to the aggregate's immunogenic activity.
• prefe ⁇ ed non-conservative substitutions of galectin 11 of Figure 1 include: Ml replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S2 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P3 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; R4 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L5 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E6 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V7 replaced with D,
• K34 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H35 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; F36 replaced with D,
• E, H, K, R, N, Q, F, W, Y, P, or C L104 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A106 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T107 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S108 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M109 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Nl 10 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; QI 11 replaced with D, E
• Rl 18 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C
• El 19 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C
• L120 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
• R121 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C
• 1122 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
• S123 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
• G124 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C
• the resulting constructs can be routinely screened for activities or functions described throughout the specification and known in the art.
• the resulting constructs have an increased and/or decreased galectin 11 activity or function, while the remaining galectin 11 activities or functions are maintained. More preferably, the resulting constructs have more than one increased and/or decreased galectin 11 activity or function, while the remaining galectin 11 activities or functions are maintained.
• more than one amino acid can be replaced with the substituted amino acids as described above (either conservative or nonconservative).
• the substituted amino acids can occur in the full length, mature, or proprotein form of galectin 11 protein, as well as the N- and C- terminal deletion mutants, having the general formula m-n, [m'-n 1 , m'-n 2 , m'-n 3 , m 2 -n', m 2 -n 2 , m 2 -n 3 ', m 3 -n', m 3 -n 2 and m 3 -n 3 ].
• a further embodiment of the invention relates to a polypeptide which comprises the amino acid sequence of a galectin 11 polypeptide having an amino acid sequence which contains at least one amino acid substitution, but not more than 50 amino acid substitutions, even more preferably, not more than 40 amino acid substitutions, still more preferably, not more than 30 amino acid substitutions, and still even more preferably, not more than 20 amino acid substitutions.
• a polypeptide in order of ever-increasing preference, it is highly preferable for a polypeptide to have an amino acid sequence which comprises the amino acid sequence of a galectin 11 polypeptide, which contains at least one, but not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.
• the number of additions, substitutions, and/or deletions in the amino acid sequence of SEQ ID NOS: 2, 25, or 27 or fragments thereof is 1-5, 5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acid substitutions are preferable.
• the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the polynucleotides and/or recombinant vectors of the invention, and the production of galectin
• Galectin 11 polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
• a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.
• the DNA of the invention is operatively associated with an appropriate heterologous regulatory element (e.g., promoter or enhancer), such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
• an appropriate heterologous regulatory element e.g., promoter or enhancer
• promoter or enhancer such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
• promoter or enhancer such as the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
• Other suitable promoters and enhancers will be
• these constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
• the coding portion of the transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
• the expression vectors will preferably include at least one selectable marker.
• markers include dihydrofolate reductase or G418 neomycin resistance for eukaryotic cell culture and tetracycline, kanamycin or ampicillin resistance genes for culturing in E. coli and other bacteria.
• Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E.
• coli Streptococcus staphylococci, Bacillus subtilis, Streptomyces and Salmonella typhimurium cells
• fungal cells such as yeast cells
• insect cells such as Drosophila S2 and Spodoptera Sf9 cells
• animal cells such as CHO, COS, HeLa, C127, 3T3, BHK, HEK293, and Bowes melanoma cells
• plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
• Such vectors include chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention.
• vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorab
• any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host may be used.
• the appropriate nucleotide sequence may be inserted into an expression vector system by any of a variety of known technique, such as for example, those set forth in Ausubel et al., eds., 1989, Current Protocols in Molecular Biology, Green Publishing Associates, Inc., and John Wiley & Sons, Inc., New York.
• vectors prefe ⁇ ed for use in bacteria include pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
• prefe ⁇ ed eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
• Other suitable vectors will be readily apparent to the skilled artisan.
• the present invention also relates to host cells containing the vector constructs discussed herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoters and/or enhancers) using techniques known in the art.
• the host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
• the host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired.
• Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled.
• different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
• secretion signals may be incorporated into the desired polypeptide using techniques known in the art. These signals may be endogenous to the polypeptide or they may be heterologous signals.
• the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology (1986). It is specifically contemplated that galectin 11 polypeptides may in fact be expressed by a host cell lacking a recombinant vector.
• the polypeptide may be expressed in a modified form, such as a fusion protein (comprising the polypeptide joined via a peptide bond to a heterologous protein sequence (of a different protein)), and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide.
• a fusion protein comprising the polypeptide joined via a peptide bond to a heterologous protein sequence (of a different protein)
• additional heterologous functional regions for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage.
• fusion protein can be made by protein synthetic techniques, e.g., by use of a peptide synthesizer.
• a prefe ⁇ ed fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins.
• EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobulin molecules together with another human protein or part thereof.
• the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232 262).
• human proteins, such as, hIL5- has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, Bennett et al., J. Md. Recog. 8:52-58 (1995) and Johanson et al., J. Biol. Chem. 270(16):9459-9471 (1995).
• the galectin 11 protein can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography ("HPLC") is employed for purification.
• Polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, plant, insect, teleost, avian, and mammalian cells.
• polypeptides of the present invention may be glycosylated or may be non-glycosylated.
• polypeptides of the invention may also include an initial modified methionine residue or may be missing an initial methionine residue, in some cases as a result of host-mediated processes.
• the N-terminal methionine encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells.
• N-terminal methionine on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionine is covalently linked.
• the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., galectin 11 coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with galectin 11 polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous galectin 11 polynucleotides.
• endogenous genetic material e.g., galectin 11 coding sequence
• genetic material e.g., heterologous polynucleotide sequences
• heterologous control regions e.g., promoter and/or enhancer
• endogenous galectin 11 polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit
• heterologous control regions e.g., promoter and/or enhancer
• endogenous galectin 11 polynucleotide sequences via homologous recombination, resulting in the formation of a new transcription unit
• polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller et al., N ⁇ twre, 310:105-111 (1984)).
• a polypeptide co ⁇ esponding to a fragment of a galectin 11 polypeptide can be synthesized by use of a peptide synthesizer.
• nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the galectin 11 polypeptide sequence.
• Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t- butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.
• the amino acid can be D (dextrorotary) or L (levorotary).
• the invention encompasses galectin 11 polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc.
• Additional post-translational modifications encompassed by the invention include, for example, e.g., N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
• the polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
• the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylceliulose, dextran, polyvinyl alcohol and the like.
• the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
• the polymer may be of any molecular weight, and may be branched or unbranched.
• the prefe ⁇ ed molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
• Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of ar-tigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
• the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
• the polyethylene glycol may have a branched structure.
• Branched polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72 (1996); Vorobjev et al., Nucleosides Nucleotides 75:2745-2750 (1999); and Caliceti et al., Bioconjug. Chem. 70:638-646 (1999), the disclosures of each of which are inco ⁇ orated herein by reference.
• polyethylene glycol molecules should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
• attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation of GM-CSF using tresyl chloride).
• polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group. Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
• the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue.
• Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Prefe ⁇ ed for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
• polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
• polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
• One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
• polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (polypeptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
• the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
• Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization of the protein at the N-terminus with a carbonyl group containing polymer is achieved.
• pegylation of the proteins of the invention may be accomplished by any number of means.
• polyethylene glycol may be attached to the protein either directly or by an intervening linker.
• Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249- 304 (1992); Francis et al, Intern. J. ofHematol. 65:1-18 (1998); U.S. Patent No. 4,002,531; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are inco ⁇ orated herein by reference.
• One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO 2 CH CF 3 ).
• MPEG monmethoxy polyethylene glycol
• ClSO 2 CH CF 3 tresylchloride
• polyethylene glycol is directly attached to amine groups of the protein.
• the invention includes protein- polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
• Polyethylene glycol can also be attached to proteins using a number of different intervening linkers.
• U.S. Patent No. 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins.
• Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succinimidylsuccinate, MPEG activated with 1 , 1 '-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate, MPEG-p- nitrophenolcarbonate, and various MPEG-succinate derivatives.
• polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is inco ⁇ orated herein by reference.
• Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.
• the number of polyethylene glycol moieties attached to each protein of the invention i.e., the degree of substitution
• the pegylated proteins of the invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.
• the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20 polyethylene glycol moieties per protein molecule.
• Methods for determining the degree of substitution are discussed, for example, in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).
• the galectin 11 polypeptides of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers and higher multimers). Accordingly, the present invention relates to monomers and multimers of the galectin 11 polypeptides of the invention, their preparation, and compositions (preferably, Therapeutics) containing them.
• the polypeptides of the invention are monomers, dimers, trimers or tetramers.
• the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
• Multimers encompassed by the invention may be homomers or heteromers.
• the term homomer refers to a multimer containing only polypeptides co ⁇ esponding to the amino acid sequence of SEQ ID NO:2, or alternatively SEQ ID NO:25 or 27, or encoded by the cDNA contained in the deposited clone (including fragments, variants, splice variants, and fusion proteins, co ⁇ esponding to these as described herein).
• These homomers may contain galectin 11 polypeptides having identical or different amino acid sequences.
• a homomer of the invention is a multimer containing only galectin 11 polypeptides having an identical amino acid sequence.
• a homomer of the invention is a multimer containing galectin 11 polypeptides having different amino acid sequences.
• the multimer of the invention is a homodimer (e.g., containing galectin 11 polypeptides having identical or different amino acid sequences) or a homotrimer (e.g., containing galectin 11 polypeptides having identical and/or different amino acid sequences).
• the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
• heteromer refers to a multimer containing one or more heterologous polypeptides (i.e., polypeptides of different proteins) in addition to the galectin 11 polypeptides of the invention.
• the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.
• the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer.
• Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
• multimers of the invention such as, for example, homodimers or homotrimers, are formed when polypeptides of the invention contact one another in solution.
• heteromultimers of the invention such as, for example, heterotrimers or heterotetramers, are formed when polypeptides of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.
• multimers of the invention are formed by covalent associations with and/or between the galectin 11 polypeptides of the invention.
• covalent associations may involve one or more amino acid residues contained in the polypeptide sequence (e.g., that recited in SEQ ID NO:2, 25, or 27, or contained in the polypeptide encoded by the clone HJACE54).
• the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences which interact in the native (i.e., naturally occu ⁇ ing) polypeptide.
• the covalent associations are the consequence of chemical or recombinant manipulation.
• covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a galectin 11 fusion protein.
• covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925).
• the covalent associations are between the heterologous sequence contained in a galectin 11 -Fc fusion protein of the invention (as described herein).
• covalent associations of fusion proteins of the invention are between heterologous polypeptide sequence from another protein that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication NO: WO 98/49305, the contents of which are herein inco ⁇ orated by reference in its entirety).
• two or more polypeptides of the invention are joined through peptide linkers. Examples include those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby inco ⁇ orated by reference). Proteins comprising multiple polypeptides of the invention separated by peptide linkers may be produced using conventional recombinant DNA technology.
• Leucine zipper and isoleucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found.
• Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al, Science 240:1759, (1988)), and have since been found in a variety of different proteins.
• leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.
• leucine zipper domains suitable for producing soluble multimeric proteins of the invention are those described in PCT application WO 94/10308, hereby inco ⁇ orated by reference.
• Recombinant fusion proteins comprising a polypeptide of the invention fused to a polypeptide sequence that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric fusion protein is recovered from the culture supernatant using techniques known in the art.
• Trimeric polypeptides of the invention may offer the advantage of enhanced biological activity.
• Prefe ⁇ ed leucine zipper moieties and isoleucine moieties are those that preferentially form trimers.
• One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) and in U.S. patent application Ser. No. 08/446,922, hereby inco ⁇ orated by reference.
• Other peptides derived from naturally occu ⁇ ing trimeric proteins may be employed in preparing trimeric polypeptides of the invention.
• Galectin 11 polynucleotides of the invention are fused to a polynucleotide encoding a "FLAG" polypeptide.
• a galectin 11 -FLAG fusion protein is encompassed by the present invention.
• the FLAG antigenic polypeptide may be fused to an galectin 11 polypeptide of the invention at either or both the amino or the carboxy terminus.
• a galectin 11 -FLAG fusion protein is expressed from a pFLAG-CMV-5a or a pFLAG-CMV-1 expression vector (available from Sigma, St. Louis, MO, USA). See, Andersson, S., et al, J.
• a galectin 11 -FLAG fusion protein is detectable by anti-FLAG monoclonal antibodies (also available from Sigma).
• the multimers of the invention may be generated using chemical techniques known in the art.
• polypeptides desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the sequence of the polypeptides desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• polypeptides of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide and techniques known in the art may be applied to generate multimers containing one or more of these modified polypeptides (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the polypeptide components desired to be contained in the multimer of the invention (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• multimers of the invention may be generated using genetic engineering techniques known in the art.
• polypeptides contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• polynucleotides coding for a homodimer of the invention are generated by li gating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain (or hyrophobic or signal peptide) and which can be inco ⁇ orated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• the invention further provides an isolated galectin 11 polypeptide having the amino acid sequence encoded by the deposited cDNA, the amino acid sequence depicted in Figure 1 (amino acid residues 1-133 of SEQ ID NO:2), the amino acid sequence depicted in Figure 1 less the amino terminal methionine (amino acid residues 2-133 of SEQ ID NO:2), polypeptides which are encoded by a polynucleotide that hybridizes under stringent hybridization conditions to a polynucleotide sequence encoding a polypeptide having the amino acid sequence depicted in Figure 1 (SEQ ID NO:2) and/or contained in the deposited clone, and fragments, variants, derivatives and analogs of these polypeptides.
• polypeptides of the present invention are preferably provided in an isolated form.
• isolated polypeptide is intended a polypeptide removed from its native environment.
• a polypeptide produced and/or contained within a recombinant host cell is considered isolated for pu ⁇ oses of the present invention.
• polypeptides that have been purified, partially or substantially, from a recombinant host cell are polypeptides that have been purified, partially or substantially, from a recombinant host cell.
• a recombinantly produced version of the galectin 11 polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
• the invention further includes variations of the galectin 11 polypeptide which show substantial galectin 11 polypeptide functional activity or which include regions of galectin 11 protein such as the protein portions discussed below. Such mutants include deletions, insertions, inversions, repeats, and type substitutions.
• a fragment, variant, derivative, or analog of the polypeptide of Figure 1 or 6 (SEQ ID NO:2, 25, or 27), or that encoded by the deposited cDNA, include (i) one in which at least one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue(s), and more preferably at least one but less than ten conserved amino acid residues) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification
• the number of additions, substitutions and/or deletions in the amino acid sequence of Figure 1 or 6 (SEQ ID NO:2, 25, or 27) and/or any of the polypeptide fragments described herein is 50, 40, 35, 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1, or 15-20, 15-10, 5-10, 1-5, 1-3, or 1-2.
• Amino acid residues of the galectin 11 polypeptide, fragment, variant, derivative, or analog of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule.
• the resulting mutant molecules are then tested for biological activity or functional activity, such as, receptor binding, ⁇ - galactoside (e.g., thiodigalactoside or lactose) binding, the ability to agglutinate trypsin- treated rabbit erythrocytes, or the ability in vitro or in vivo to induce apoptosis.
• biological activity or functional activity such as, receptor binding, ⁇ - galactoside (e.g., thiodigalactoside or lactose) binding, the ability to agglutinate trypsin- treated rabbit erythrocytes, or the ability in vitro or in vivo to induce apoptosis.
• Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al., Science 255
• the present invention also encompasses polypeptides which are at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99%, or, 97-99% identical to the polypeptides described above.
• polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a galectin 11 polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the galectin 11 polypeptide.
• a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
• These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
• any particular polypeptide is at least 75%, 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in Figure 1 or 6 (SEQ ID NO:2, 25, or 27), the amino acid sequence encoded by deposited cDNA clone, or fragments thereof, can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711.
• the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
• the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence, also refe ⁇ ed to as a global sequence alignment is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
• the percent identity is co ⁇ ected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a co ⁇ esponding subject residue, as a percent of the total bases of the query sequence.
• a determination of whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score.
• This final percent identity score is what is used for the pu ⁇ oses of this embodiment. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the pu ⁇ oses of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity.
• the deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N- terminus.
• the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
• a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query.
• polypeptides of the present invention have uses which include, but are not limited to, molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
• the present invention also encompasses fragments of the above-described polypeptides of the invention.
• these fragments are at least 20, 25, 30, 40, 50, 75, 90, 100, 110, 120, 125, or 130 amino acid residues in length.
• polypeptide fragment refers to an amino acid sequence which is a portion of that contained in SEQ ID NO:2, 25, or 27 or encoded by the cDNA contained in the deposited clone.
• Protein (polypeptide) fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
• Representative examples of polypeptide fragments of the invention include, for example, fragments comprising, or alternatively consisting of, from about amino acid number 1-20, 21-40, 41-60, 61-80, 81-100, 101-120, or 121 to the end of the coding region.
• polypeptide fragments can be about 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, or 130 amino acids in length.
• “about” includes the particularly recited ranges or values, and ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either extreme or at both extremes.
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• polypeptide fragments include the secreted galectin 11 protein as well as the mature form. Further prefe ⁇ ed polypeptide fragments include the secreted galectin 11 protein or the mature form having a continuous series of deleted residues from the amino or the carboxy terminus, or both. For example, any number of amino acids, ranging from 1-60, can be deleted from the amino terminus of either the secreted galectin 11 polypeptide or the mature form.
• any number of amino acids ranging from 1-30, can be deleted from the carboxy terminus of the secreted galectin 11 protein or mature form. Furthermore, any combination of the above amino and carboxy terminus deletions are prefe ⁇ ed. Similarly, polynucleotides encoding these polypeptide fragments are also preferred.
• the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the galectin 11 polypeptide depicted in Figure 1 or 6 (SEQ ID NO:2, 25, or 27) or encoded by the cDNA of the deposited clone.
• N-terminal deletions of the galectin 11 polypeptide can be described by the general formula m to 133, where m is a integer from 1 to 128 co ⁇ esponding to the position of amino acid residue identified in SEQ ID NO:2 and preferably, co ⁇ esponds to one of the N-terminal amino acid residues identified in the N- terminal deletions specified herein.
• N-terminal deletions of the galectin 11 polypeptide of the invention comprise, or alternatively consist of, amino acid residues: S-2 to S-133; P-3 to S-133; R-4 to S-133; L-5 to S-133; E-6 to S-133; V-7 to S-133; P-8 to S-133; C-9 to S-133; S-10 to S-133; H-l l to S-133; A-12 to S-133; L-13 to S-133; P- 14 to S-133; Q-15 to S-133; G-16 to S-133; L-17 to S-133; S-18 to S-133; P-19 to S-133; G- 20 to S-133; Q-21 to S-133; V-22 to S-133; 1-23 to S-133; 1-24 to S-133; V-25 to S-133; R-26 to S-133; G-27 to S-133; L-28 to S-133; V-29 to S-133; L-30 to S-133; Q-31 to S-133
• galectin 11 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities.
• peptides composed of as few as six galectin 11 amino acid residues may often evoke an immune response.
• further embodiments of the invention are directed to C-terminal deletions of the galectin 11 polypeptide described by the general formula 1 to n, where n is an integer from 6 to 132 co ⁇ esponding to the position of amino acid residue identified in SEQ ID NO:2 and preferably, co ⁇ esponds to one of the C-terminal amino acid residues identified in the C- terminal deletions specified herein.
• C terminal deletions of the galectin 11 polypeptide of the invention comprise, or alternatively, consist of, amino acid residues: M-1 to H-132; M-1 to V-131; M-1 to C-130; M-1 to Y-129; M-1 to L-128; M-1 to Q-127; M-1 to V-126; M-1 to S-125; M-1 to G-124; M-1 to S-123; M-1 to 1-122; M-1 to R- 121; M-1 to L-120; M-l to E-l 19; M-1 to R-118; M-1 to L-l 17; M-1 to Q-l 16; M-1 to E- 115; M-1 to L-l 14; M-1 to A-113; M-1 to Q-l 12; M-1 to Q-l 11; M-1 to N-110; M-l to M- 109; M-1 to S-108; M-1 to T-107; M-1 to A-106; M-1 to G-105; M-1 to L-104; M-1 to G-
• polypeptides are also encompassed by the invention.
• any of the above listed N- or C-terminal deletions can be combined to produce a N- and C-terminal deleted galectin 11 polypeptide.
• the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues m-n of SEQ ID NO:2, where n and m are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the polypeptides of the invention comprise, or alternatively, consist of, amino acid residues: M-1 to L-40; M-1 to W-66; P-3 to L-40; L-5 to L-40; L-5 to S-108; L-5 to L-128; P-3 to L-128; L-5 to L-128; L-5 to G-124; C-9 to C-130; L- 13 to L-40; P-14 to L-40; L-40 to S-108; A-47 to S-108; A-47 to L-128; R-65 to S-108; R-65 to L-128; L-88 to S-108; L-88 to L-128; S-108 to L-120; or L-l 14 to L-128 of SEQ ID NO:2.
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• nucleotide sequence encoding a polypeptide consisting of a portion of the complete galectin 11 amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209053, where this portion excludes any integer of amino acid residues from 1 to about 123 amino acids from the amino terminus of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209053, or any integer of amino acid residues from 1 to about 123 amino acids from the carboxy terminus, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209053.
• Polynucleotides encoding all of the above deletion mutant polypeptide forms also are provided.
• the present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the galectin 11 polypeptide sequence set forth herein m-n.
• the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific galectin 11 N- and C-terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• Additional prefe ⁇ ed polypeptide fragments comprise, or alternatively consist of, the amino acid sequence of residues: M-1 to Q-15; S-2 to G-16; P-3 to L-17; R-4 to S-18; L-5 to P-19; E-6 to G-20; V-7 to Q-21; P-8 to V-22; C-9 to 1-23; S-10 to 1-24; H-l l to V-25; A-12 to R-26; L-13 to G-27; P-14 to L-28; Q-15 to V-29; G-16 to L-30; L-17 to Q-31; S-18 to E- 32; P-19 to P-33; G-20 to K-34; Q-21 to H-35; V-22 to F-36; 1-23 to T-37; 1-24 to V-38; V- 25 to S-39; R-26 to L-40; G-27 to R-41; L-28 to D-42; V-29 to Q-43; L-30 to A-44; Q-31 to A-45; E-32 to H-46; P-33 to A
• polypeptide fragments may retain the biological activity of galectin 11 polypeptides of the invention and/or may be useful to generate or screen for antibodies, as described further below.
• Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
• the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the galectin 11 polypeptide depicted in SEQ ID NO:25 or encoded by the cDNA of the deposited clone.
• N-terminal deletions of the galectin 11 polypeptide can be described by the general formula m to 275, where m is a integer from 2 to 270 co ⁇ esponding to the position of amino acid residue identified in SEQ ID NO:25 and preferably, co ⁇ esponds to one of the N-terminal amino acid residues identified in the N- terminal deletions specified herein.
• N-terminal deletions of the galectin 11 polypeptide of the invention comprise, or alternatively consist of, amino acid residues: V-2 to S-275; M-3 to S-275; L-4 to S-275; Q-5 to S-275; G-6 to S-275; V-7 to S-
• C-terminal deletions of the galectin 11 polypeptide described by the general formula 1 to n where n is an integer from 6 to 275 co ⁇ esponding to the position of amino acid residue identified in SEQ ID NO:25 and preferably, co ⁇ esponds to one of the C-terminal amino acid residues identified in the C- terminal deletions specified herein.
• C terminal deletions of the galectin 11 polypeptide of the invention comprise, or alternatively, consist of, amino acid residues: M-1 to H-274; M-1 to V-273; M-1 toC-272; M-1 to Y-271; M-1 to L-270; M-1 to
• polypeptide fragments comprising, or alternatively, consisting of, amino acids described by the general formula m to n, where m and n co ⁇ espond to any one of the amino acid residues specified above for these symbols, respectively.
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the galectin 11 polypeptide depicted in SEQ ID NO:27.
• N- terminal deletions of the galectin 11 polypeptide can be described by the general formula m to 296, where m is an integer from 2 to 291 co ⁇ esponding to the position of amino acid residue identified in SEQ ID NO:27 and preferably, corresponds to one of the N-terminal amino acid residues identified in the N-terminal deletions specified herein.
• N- terminal deletions of the galectin 11 polypeptide of the invention comprise, or alternatively consist of, amino acid residues: S-2 to S-296; F-3 to S-296; F-4 to S-296; S-5 to S-296; C-6 to S-296; S-7 to S-296; G-8 to S-296; G-9 to S-296; S-10 to S-296; L-l l to S-296; C-12 to S-
• S-296 E-l 03 to S-296; E-l 04 to S-296; V-l 05 to S-296; K-106 to S-296; V-l 07 to S-296; S- 108 to S-296; V-l 09 to S-296; N-l 10 to S-296; G-l 11 to S-296; Q-l 12 to S-296; H-l 13 to S- 296; F-l 14 to S-296; L-l 15 to S-296; H-l 16 to S-296; F-l 17 to S-296; R-118 to S-296; Y- 119 to S-296; R-120 to S-296; L-121 to S-296; P-122 to S-296; L-123 to S-296; S-124 to S- 296; H-l 25 to S-296; V-l 26 to S-296; D-127 to S-296; T-128 to S-296; L-l 29 to S-296; G- 130 to S-296; 1-131 to S-2
• FIG. 1 is an integer from 6 to 295 co ⁇ esponding to the position of amino acid residue identified in SEQ ID NO:27 and preferably, co ⁇ esponds to one of the C-terminal amino acid residues identified in the C- terminal deletions specified herein.
• C terminal deletions of the galectin 11 polypeptide of the invention comprise, or alternatively, consist of, amino acid residues: M-1 to H-295; M-1 to V-294; M-1 toC-293; M-1 to Y-292; M-1 to L-291; M-1 to Q-290; M-1 to V-289;M-1 to S-288; M-1 to G-287; M-1 to S-286; M-1 to 1-285; M-1 toR- 284; M-1 to L-283; M-1 to E-282; M-1 to R-281; M-1 to L-280; M-1 to Q-279; M-1 to E- 278; M-1 to L-277; M-1 to A-276; M-1 to Q-275; M-1 to Q-274; M-1 to N-273; M-1 to M- 272; M-1 to S-271; M-1 to T-270; M-1 to A-269; M-1 to G-268; M-1 to L-267; M-1 to G- 266; M-1 to Q-2
• polypeptide fragments comprising, or alternatively, consisting of, amino acids described by the general formula m to n, where m and n co ⁇ espond to any one of the amino acid residues specified above for these symbols, respectively.
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• Figure 2 provides a comparison of the galectin 11 polypeptide with other galectins. Identical amino acids shared between the galectins are shaded, while conservative amino acid changes are boxed. By examining the regions of amino acids shaded and/or boxed, the skilled artisan can readily identify conserved domains between the two polypeptides. The amino acid sequences falling within these conserved, shaded and/or boxed domains are contained in the prefe ⁇ ed polypeptide fragments of the invention. Similar analyses for the full-length galectin- 1 l and ⁇ is deemed to be within the skill of the ordinary artisan given the teachings provided herein.
• polypeptide residue fragments of the invention including, for example, fragments from about amino acid number 1-20, 1-66, 5-108, 5-128, 21-40, 40- 108, 41-60, 47-108, 47-128, 61-80, 65-108, 65-128, 81-100, 88-108, 88-128, 108-120; 114- 128; and 101 to the end of the galectin 11 polypeptide depicted in Figure 1 (SEQ ID NO:2).
• "about” includes the particularly recited ranges larger or smaller by several , 5, 4, 3, 2, or 1 amino acid at either end or at both extremes.
• polypeptide residue fragments of the invention including, for example, fragments from about amino acid number 1-20, 1-66, 5-108, 5-128, 21-40, 40- 108, 41-60, 47-108, 47-128, 61-80, 65-108, 65-128, 81-100, 88-108, 88-128, 108-120; 114-128; 129-150; 145-175; 170-200; 195-225; 220-250; and 245-275 of SEQ ID NO:25.
• polypeptide residue fragments of the invention including, for example, fragments from about amino acid number 1-20, 1-66, 5-108, 5-128, 21-40, 40- 108, 41-60, 47-108, 47-128, 61-80, 65-108, 65-128, 81-100, 88-108, 88-128, 108-120; 114-128; 129-150; 145-175; 170-200; 195-225; 220-250; 245-275; and 270-296 of SEQ ID NO:27.
• Polypeptides comprising such amino acid sequences are provided as well as polynucleotides encoding such polypeptides.
• the polynucleotide fragments of the invention encode a polypeptide which demonstrates a galectin 11 functional activity.
• a polypeptide demonstrating a galectin 11 “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) galectin 11 protein.
• Such functional activities include, but are not limited to, biological activity, antigenicity [ability to bind (or compete with a galectin 11 polypeptide for binding) to an anti-galectin 11 antibody], immunogenicity (ability to generate antibody which binds to a galectin 11 polypeptide), ability to form multimers with galectin 11 polypeptides of the invention.
• fragments characterized by structural or functional attributes of galectin 11 include amino acid residues that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta- sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn- regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., having an antigenic region of three or more contiguous amino acid residues each of which having an antigenic index of greater than or equal to 1.5) of galectin 11.
• Certain prefe ⁇ ed regions are those set out in Figure 3 and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted in Figure 1 (SEQ ID NO:2), such preferred regions include; Garnier-Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Kyte-Doolittle predicted hydrophilic and hydrophobic regions; Eisenberg alpha and beta amphipathic regions; Emini surface-forming regions; and Jameson- Wolf high antigenic index regions, as predicted using the default parameters of these computer programs. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the polynucleotides of the invention encode functional attributes of galectin 11.
• Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of galectin 11.
• the data presented in columns VIII, XII, and XIII of Table I can be used to determine regions of galectin 11 which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, XII, and/or XIII by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response. Certain prefe ⁇ ed regions in these regards are set out in Figure 3, but may, as shown in
• Table I be represented or identified by using tabular representations of the data presented in Figure 3.
• the DNA* STAR computer algorithm used to generate Figure 3 (set on the original default parameters) was used to present the data in Figure 3 in a tabular format (See Table I).
• the tabular format of the data in Figure 3 may be used to easily determine specific boundaries of a prefe ⁇ ed region.
• prefe ⁇ ed regions set out in Figure 3 and in Table I include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in Figure 1.
• prefe ⁇ ed regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions, Chou-Fasman alpha-regions, beta-regions, and turn-regions, Kyte-Doolittle hydrophilic regions, Eisenberg alpha- and beta-amphipathic regions, Ka ⁇ lus-Schulz flexible regions, Emini surface-forming regions and Jameson- Wolf regions of high antigenic index. TABLE I
• Figure 2 provides a comparison of the galectin 11 polypeptide with other galectins. Identical amino acids shared between the galectins are shaded, while conservative amino acid changes are boxed. By examining the regions of amino acids shaded and/or boxed, the skilled artisan can readily identify conserved domains between the two polypeptides. The amino acid sequences falling within these conserved, shaded and/or boxed domains are contained in the prefe ⁇ ed polypeptide fragments of the invention.
• prefe ⁇ ed fragments in this regard are those that comprise regions of galectin 11 that combine several structural features, such as several of the features set out above.
• Other prefe ⁇ ed polypeptide fragments are biologically active galectin 11 fragments.
• Biologically active fragments are those exhibiting activity similar, but not necessarily identical, to an activity of the galectin 11 polypeptide.
• the biological activity of the fragments may include an improved desired activity, or a decreased undesirable activity.
• Polynucleotides encoding these polypeptide fragments are also encompassed by the invention.
• polypeptide residue fragments of the invention including, for example, fragments from about amino acid number 1-20, 1-66, 5-108, 5-128, 21-40, 40- 108, 41-60, 47-108, 47-128, 61-80, 65-108, 65-128, 81-100, 88-108, 88-128, 108-120; 114- 128; and 101 to the end of the galectin 11 polypeptide depicted in Figure 1 (SEQ ID NO:2).
• "about” includes the particularly recited ranges larger or smaller by several, 5, 4, 3, 2, or 1 amino acid at either end or at both extremes.
• galectin 11 polypeptides of the present invention such as, for example, epitope-bearing fragments of galectin 11
• IgG immunoglobulins
• Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric galectin 11 protein or protein fragment alone (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)).
• polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences.
• the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides.
• immunoglobulins IgA, IgE, IgG, IgM
• CHI constant domain of immunoglobulins
• CH2, CH3, or any combination thereof and portions thereof resulting in chimeric polypeptides.
• Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins.
• IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone.
• Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ("HA") tag or flag tag) to aid in detection and purification of the expressed polypeptide.
• an epitope tag e.g., the hemagglutinin ("HA") tag or flag tag
• HA hemagglutinin
• a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al., 1991, Proc. Natl. Acad. Sci. USA 88:8972- 897).
• the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues.
• the tag serves as a matrix binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
• DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., Cu ⁇ . Opinion Biotechnol.
• alteration of polynucleotides co ⁇ esponding to SEQ ID NO:l and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
• DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence.
• polynucleotides of the invention may be altered by being subjected to random mutagenesis by e ⁇ or- prone PCR, random nucleotide insertion or other methods prior to recombination.
• one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
• Polypeptides of the invention include polypeptides encoded by polynucleotides that hybridize (e.g., under stringent hybridization conditions) to the polynucleotide sequence depicted in Figure 1 (SEQ ID NO: l), the complementary strand thereto, and/or the nucleotide sequence contained in the deposited clone.
• the polypeptides of the invention have galectin 11 functional and/or biological activity.
• galectin 11 polypeptides, fragments, variants, derivatives and analogs of the invention can be assayed by various methods.
• various immunoassays known in the art can be used, including, but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA, "sandwich” immunoassays, immunoradiometric assays, and diffusion precipitin reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A as
• galectin 11 polypeptides, fragments, variants, derivatives and analogs of the invention may be determined using, or routinely modifying, assays known in the art.
• lactose binding activity of the expressed galectin 11 polypeptides and fragments, variants, derivatives, or analogs thereof may be assayed by immunodectection of in situ binding activity to asialofetuin (Sigma) immobilized on nitrocellulose (Amersham) (Madsen et al., J. Biol. Chem. 270(11):5823-5829 (1995)).
• nitrocellulose For example, in one assay, thirty ⁇ g of asialofetuin dissolved in 3 ⁇ l of water is spotted on a l-cm2 strip of nitrocellulose. The nitrocellulose pieces are then placed in a 24- well tissue culture plant and incubated overnight in buffer B (58 mM NA 2 HPO 4 , 18 mM KH 2 PO 4 , 75 mM NaCl, 2 mM EDTA, and 3% BSA, pH 7.2) with constant agitation at 22°C.
• buffer B 58 mM NA 2 HPO 4 , 18 mM KH 2 PO 4 , 75 mM NaCl, 2 mM EDTA, and 3% BSA, pH 7.2
• the blocking medium is aspirated and the nitrocellulose pieces are washed three times in buffer A (58 mM Na 2 HPO 4 18 mM KH 2 2PO 4 , 75 mM NaCl, 2 mM EDTA, 4 mM ⁇ -mercaptoethanol and 0.2% BSA, pH 7.2).
• Cell extracts preferably, COS cells
• Cell extracts are prepared containing 1% BSA and either with or without 150 mM lactose (105 ⁇ l of primary extract, 15 ⁇ l of 10% BSA in buffer A and either 30 ⁇ l of 0.75 M lactose in buffer A or 30 ⁇ l of buffer A).
• the immobilized asialofetuin is incubated with the extracts for 2 h and washed 5 times in buffer A.
• the nitrocellulose pieces are then fixed in 2% formalin in PBS (58 mM Na 2 HPO 4 , 18 mM KH 2 PO 4 , 75 mM NaCl, 2 mM EDTA pH 7.2) for 1 h to prevent loss of bound galectin 11.
• PBS 58 mM Na 2 HPO 4 , 18 mM KH 2 PO 4 , 75 mM NaCl, 2 mM EDTA pH 7.2
• Polyclonal serum (generated using techniques known in the art) diluted 1 :100 in PBS for 2h at 22°C.
• the pieces are then washed in PBS and incubated with peroxidase-labeled goat anti-rabbit antibodies (DAKO). Following incubation for 2h at 22°C, the pieces are washed in PBS and the substrate is added. Nitrocellulose pieces are incubated until the color developed and the reaction is stopped by washing in distilled water.
• DAKO peroxidase-labeled goat anti-rabbit antibodies
• galectin 11 polypeptides, fragments, variants, derivatives and analogs of the invention can routinely be assayed using techniques known in the art.
• the ability of the galectin 11 polypeptides, fragments, variants, derivatives and analogs of the invention to induce apoptosis of T-cells may be determined using, or routinely modifying, techniques described herein (see e.g., Example 5) or otherwise known in the art. See e.g., Perillo et al., Nature 378:736-739 (1995); Chinnaiyan et al., Cell 81:505-512 (1995); Boldin et al., J. Biol. Chem. 270:7795-7798 (1995); Kischkel et al., EMBO J. 14:5579-5585 (1995); Chinnaiyan et al., J. Biol. Chem. 271:4961-4965 (1996); the contents of each of which is herein incorporated by reference in its entirety).
• the galectin 11 polynucleotides and polypeptides, and fragments, variants derivatives and analogs thereof; and antibodies, agonists and antagonists thereto; can be tested in vivo for the desired therapeutic or prophylactic activity.
• such compounds can be tested in suitable animal model systems prior to testing in humans.
• animal models include, but are not limited to, rats, mice, chickens, cows, monkeys, rabbits, etc.
• Such testing may also be utilized to routinely determine dosage for delivery to human patients.
• any animal model system known in the art may be used (see, for example, Levi et al., Eur. J. Imun. 13:500-507 (1983); and Offner et al., J.
• EAE allergic encephalomyelitis
• EAE can be induced in certain strains of mice by immunization with myelin in an adjuvant.
• the immunization activates CD4 + T cells specific for myelin basic protein (MBP) and proteolipid (PLP), Bernard et al., J. Immunol. 114:1537- 1540 (1975); Chou et al., J. Immunol. 130:2183-2186 (1983); Kurchroo et al., J. Immunol. 148:3776-3782 (1992).
• the activated T cells enter the central nervous system and their local action causes both the anatomic pathology and clinical signs, e.g., ascending hind limb paresis leading to paralysis, of the disease.
• the galectin 1 has been demonstrated to suppress clinical and histological signs of experimental autoimmune encephalomyelitis in rats (Offner et al, J. Neuroimmunol. 28:177-184 (1990)).
• EAMG experimental autoimmune myasthenia gravis
• AChR purified acetylcholine receptor protein
• galectin 1 has been demonstrated to have a prophylactic and therapeutic action on experimental autoimmune myasthenia gravis in rabbits (Levi et al., Eur. J. Immunol. 13:500-507 (1983)).
• Assays described herein or otherwise known in the art may be applied to routinely determine which galectin 11 polypeptides, fragments, variants, derivatives and analogs of the invention demonstrate galectin 11 functional activity and the optimal concentration at which these compounds demonstrate this activity. These assays may additionally be utilized to identify molecules which enhance (agonists) or suppress (antagonists) galectin 11 functional activity.
• Epitopes The present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2, 25, or 27, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC Deposit No: 209053 or encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO:l, 24, or 26 or contained in ATCC Deposit No: 209053 under stringent hybridization conditions or lower stringency hybridization conditions as defined supra.
• the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:l, 24, or 26), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
• epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
• the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
• An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al., Proc. Natl. Acad. Sci.
• antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
• Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful, for example, to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention.
• Prefe ⁇ ed antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes.
• Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al., Science 219:660-666 (1983)).
• immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al, supra; Chow et al, Proc. Natl. Acad. Sci. USA 82:910-914; and Bittie et al., J. Gen. Virol. 66:2347-2354 (1985).
• Prefe ⁇ ed immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes.
• the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a ca ⁇ ier.
• a carrier protein such as an albumin
• immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
• Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least 7, 9, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 110 or 120 contiguous amino acid residues of the amino acid sequence depicted in Figure 1 or 6 (SEQ ID NO:2, 25, or 27).
• antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids.
• Prefe ⁇ ed polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Additional non-exclusive prefe ⁇ ed antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof.
• Non-limiting examples of antigenic polypeptides or peptides that can be used to generate galectin 11 -specific antibodies include: a polypeptide comprising amino acid residues from about 65-70 and 118-124 in Figure 1 (SEQ ID NO:2). As indicated above, the inventors have determined that the above polypeptide fragments are antigenic regions of the galectin 11 protein.
• the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. See generally, Houghten, R. A., Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This Simultaneous Multiple Peptide Synthesis (SMPS) process is further described in U.S. Patent No. 4,631,211 to Houghten et al. (1986).
• SMPS Simultaneous Multiple Peptide Synthesis
• Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al, supra; Wilson et al., supra, and Bittie et al., J. Gen. Virol., 66:2347-2354 (1985).
• animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
• KLH keyhole limpet hemacyanin
• peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl- N- hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutar aldehyde.
• Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ⁇ g of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
• booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
• the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
• Antibodies Further polypeptides of the invention relate to antibodies and T-cell antigen receptors
• TCR which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:2, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding).
• Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
• antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
• the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
• the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
• Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.
• the antibodies of the invention may be from any animal origin including birds and mammals.
• the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.
• "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
• the antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, et al., J. Immunol. 147:60-69 (1991); U.S. Patent Nos. 4,474,893; 4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol. 148:1547-1553 (1992).
• Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind.
• the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
• Prefe ⁇ ed epitopes of the invention include: amino acids 65-70 and 118-124 of SEQ ID NO:2, as well as polynucleotides that encode these epitopes.
• Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
• Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the co ⁇ esponding epitopes thereof.
• Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
• the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein.
• antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions are also included in the present invention.
• Prefe ⁇ ed binding affinities include those with a dissociation constant or Kd less than 5 X 10 "2 M, 10 "2 M, 5 X 10 '3 M, 10 "3 M, 5 X 10 "4 M, 10 "4 M, 5 X 10 "5 M, 10 “5 M, 5 X 10 “6 M, 10 “ 6 M, 5 X 10 "7 M, 10 7 M, 5 X 10 "8 M, 10 “8 M, 5 X 10 "9 M, 10 “9 M, 5 X 10 "10 M, 10 “10 M, 5 X 10 “ ⁇ M, 10 "11 M, 5 X 10 "12 M, 1( 2 M, 5 X 10 "13 M, 10 "13 M, 5 X 10 '14 M, 10 '14 M, 5 X 10 " 15 M, or 10 ⁇ 15 M.
• the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein.
• the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.
• Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention.
• the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
• antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof.
• the invention features both receptor- specific antibodies and ligand-specific antibodies.
• the invention also features receptor- specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e., signaling) may be determined by techniques described herein or otherwise known in the art.
• receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
• phosphorylation e.g., tyrosine or serine/threonine
• antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
• the invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
• receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
• neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
• antibodies which activate the receptor are also act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor.
• the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein.
• the above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent No. 5,811,097; Deng et al., Blood 92(6): 1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668- 3678 (1998); Ha ⁇ op et al., J. Immunol. 161(4): 1786-1794 (1998); Zhu et al., Cancer Res.
• Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods.
• the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
• the antibodies of the present invention may be used either alone or in combination with other compositions.
• the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
• antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
• the antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
• the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
• the antibodies of the present invention may be generated by any suitable method known in the art.
• Polyclonal antibodies to an antigen-of- interest can be produced by various procedures well known in the art.
• a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
• adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
• Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
• monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties).
• the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
• the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
• mice can be immunized with a polypeptide of the invention or a cell expressing such peptide.
• an immune response e.g., antibodies specific for the antigen are detected in the mouse serum
• the mouse spleen is harvested and splenocytes isolated.
• the splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC. Hybridomas are selected and cloned by limited dilution.
• hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
• Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
• the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen of the invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
• Antibody fragments which recognize specific epitopes may be generated by known techniques.
• Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
• F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
• the antibodies of the present invention can also be generated using various phage display methods known in the art.
• phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
• phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
• Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
• Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
• Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., J. Immunol. Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186 (1995); Kettleborough et al., Eur. J. Immunol.
• the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
• a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
• Methods for producing chimeric antibodies are known in the art. See e.g., Mo ⁇ ison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Gillies et al, (1989) J. Immunol. Methods 125:191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety.
• Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and a framework regions from a human immunoglobulin molecule.
• CDRs complementarity determining regions
• framework residues in the human framework regions will be substituted with the co ⁇ esponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
• These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al, U.S. Patent No.
• Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al, Protein Engineering 7(6):805-814 (1994); Roguska.
• Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
• Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
• the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
• the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
• the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
• the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
• the chimeric mice are then bred to produce homozygous offspring which express human antibodies.
• the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
• Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
• the human immunoglobulin transgenes harbored by the transgenic mice rea ⁇ ange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
• Completely human antibodies which recognize a selected epitope can be generated using a technique refe ⁇ ed to as "guided selection.”
• a selected non-human monoclonal antibody e.g., a mouse antibody
• antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)).
• antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand.
• anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand.
• anti-idiotypic antibodies can be used to bind a polypeptide of the invention and or to bind its ligands/receptors, and thereby block its biological activity.
• polypeptides of the invention and their fragments, variants, derivatives or analogs, or cells expressing them can also be used as immunogens to produce antibodies immunospecific for galectin 11 polypeptide of the invention.
• immunospecific means that the antibodies have substantially greater affinity for the polypeptides of the invention than their affinity for other related polypeptides in the prior art.
• antibody or “monoclonal antibody” (mAb) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab, and F(ab') 2 fragments) which are capable of binding an antigen.
• Fab, Fab', and F (ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl Med. 24:316-325 (1983)).
• Antibodies according to the present invention may be prepared by any of a variety of standard methods using galectin 11 immunogens of the present invention.
• antibodies generated against full-length galectin 11 polypeptides can be obtained by administering the polypeptides or epitope-bearing fragments, variants, derivatives, analogs, or cells, to an animal, preferably a nonhuman, using routine protocols.
• any technique which provides antibodies produced by continuous cell line cultures can be used.
• Examples include the hybridoma technique (Kohler et al, Nature 256:495-497 (1975)), the trioma technique, the human B-cell hybridoma technique (Kozbor et al, Immunology Today 4:72 (1983)) and the EBV-hybridoma technique (Cole et al, MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985). Additionally, techniques for the production of single chain antibodies (U.S. Patent No. 4,946,778) can also be adapted to produce single chain antibodies to polypeptides of the invention. Also, transgenic mice, or other organisms including other mammals, may be used to express humanized antibodies.
• Antibodies of the invention can be used in methods known in the art relating to the localization and activity of the polypeptide sequences of the invention, e.g., for imaging these polypeptides, measuring levels thereof in appropriate physiological samples, etc.
• the antibodies also have use in immunoassays and in therapeutics as agonists and antagonists of galectin 11. Additionally, the antibodies of the invention may be employed to isolate or to identify clones expressing the polypeptide or to purify the polypeptides by affinity chromatography.
• the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof.
• the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:2.
• the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
• a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al, BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
• a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be
• nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.
• the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
• CDRs complementarity determining regions
• one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra.
• the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al, J. Mol. Biol.
• the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
• one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
• Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
• a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
• Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide.
• Techniques for the assembly of functional Fv fragments in E. coli may also be used (Ske ⁇ a et al, Science 242:1038- 1041 (1988)).
• the antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
• an antibody of the invention or fragment, derivative or analog thereof, (e.g., a heavy or light chain of an antibody of the invention or a single chain antibody of the invention), requires construction of an expression vector containing a polynucleotide that encodes the antibody.
• a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
• methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein.
• the invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter.
• Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
• the expression vector is transfe ⁇ ed to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
• the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter.
• vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
• host-expression vector systems may be utilized to express the antibody molecules of the invention.
• Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
• These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
• subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mamm
• bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
• mammalian cells such as Chinese hamster ovary cells (CHO)
• CHO Chinese hamster ovary cells
• a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al, Gene 45:101 (1986); Cockett et al, Bio/Technology 8:2 (1990)).
• a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
• vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
• Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.
• pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
• GST glutathione S-transferase
• fusion proteins are soluble and can easily be purified from lysed cells by adso ⁇ tion and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
• the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
• Autographa califomica nuclear polyhedrosis virus AcNPV
• AcNPV Autographa califomica nuclear polyhedrosis virus
• the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
• a number of viral-based expression systems may be utilized.
• the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
• This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts, (e.g., see Logan & Shenk, Proc. Natl. Acad.
• Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al, Methods in Enzymol. 153:51-544 (1987)).
• a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
• Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the co ⁇ ect modification and processing of the foreign protein expressed.
• eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
• Such mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
• breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D
• normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
• stable expression is prefe ⁇ ed.
• cell lines which stably express the antibody molecule may be engineered.
• host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
• appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
• engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
• the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
• This method may advantageously be used to engineer cell lines which express the antibody molecule.
• Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
• a number of selection systems may be used, including but not limited to the he ⁇ es simplex virus thymidine kinase (Wigler et al, Cell 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al, Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
• antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al, Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al, Proc. Natl Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072 (1981)); neo, which confers resistance to the aminoglycoside G-418 Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87- 95 (1991); Tolstoshev, Ann.
• the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
• vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
• a marker in the vector system expressing antibody is amplifiable
• increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al, Mol. Cell Biol. 3:257 (1983)).
• the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
• the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
• a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)).
• the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
• an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
• chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
• centrifugation e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
• differential solubility e.g., differential solubility, or by any other standard technique for the purification of proteins.
• the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
• the present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention to generate fusion proteins.
• the fusion does not necessarily need to be direct, but may occur through linker sequences.
• the antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the present invention.
• antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.
• Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al, supra, and PCT publication WO 93/21232; EP 439,095; Naramura et al, Immunol. Lett. 39:91-99 (1994); U.S. Patent 5,474,981; Gillies et al, PNAS 89:1428-1432 (1992); Fell et al, J. Immunol 146:2446-2452(1991), which are inco ⁇ orated by reference in their entireties.
• the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
• the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
• the antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
• the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
• Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.
• polypeptides co ⁇ esponding to a polypeptide, polypeptide fragment, or a variant of SEQ ID NO:2 may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art.
• polypeptides co ⁇ esponding to SEQ ID NO:2, 25, or 27 may be fused or conjugated to the above antibody portions to facilitate purification.
• chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins.
• polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dimeric structures may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
• the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
• EP A 232,262 Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
• the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
• human proteins such as hIL-5
• Fc portions for the pu ⁇ ose of high-throughput screening assays to identify antagonists of hIL-5.
• the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification.
• the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
• hexa-histidine provides for convenient purification of the fusion protein.
• peptide tags useful for purification include, but are not limited to, the "HA” tag, which co ⁇ esponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, Cell 37:767 (1984)) and the "flag" tag.
• the present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
• the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions.
• the detectable substance may be coupled or conjugated either directly to the antibody (or fragment thereof) or indirectly, through an intermediate (such as, for example, a linker known in the art) using techniques known in the art. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
• suitable prosthetic group complexes include streptavidin biotin and avidin/biotin;
• suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
• an example of a luminescent material includes luminol;
• examples of bioluminescent materials include luciferase, luciferin, and aequorin;
• suitable radioactive materials include radioisotopes such as iodine ( 131 I, 125 I, 123 I, 12, I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115m In, 113m In, H2 In, In), and technetium
• an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters such as, for example, 213BL
• a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
• Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
• Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6- thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.
• the conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
• the drug moiety may be a protein or polypeptide possessing a desired biological activity.
• proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha, TNF-beta, AIM I (See, International Publication No.
• a thrombotic agent or an anti- angiogenic agent e.g., angiostatin or endostatin
• biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1"), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
• IL-1 interleukin-1
• IL-2 interleukin-2
• IL-6 interleukin-6
• GM-CSF granulocyte macrophage colony stimulating factor
• G-CSF granulocyte colony stimulating factor
• Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
• solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
• Monoclonal Antibodies '84 Biological And Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); "Analysis, Results, And Future Prospective Of The Therapeutic Use Of
• an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is inco ⁇ orated herein by reference in its entirety.
• An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
• the antibodies of the invention may be utilized for immunophenotyping of cell lines and biological samples.
• the translation product of the gene of the present invention may be useful as a cell specific marker, or more specifically as a cellular marker that is differentially expressed at various stages of differentiation and/or maturation of particular cell types.
• Monoclonal antibodies directed against a specific epitope, or combination of epitopes will allow for the screening of cellular populations expressing the marker.
• Various techniques can be utilized using monoclonal antibodies to screen for cellular populations expressing the marker(s), and include magnetic separation using antibody-coated magnetic beads, "panning" with antibody attached to a solid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Patent 5,985,660; and Morrison et al, Cell, 96:737-49 (1999)).
• hematological malignancies i.e. minimal residual disease (MRD) in acute leukemic patients
• GVHD Graft-versus-Host Disease
• these techniques allow for the screening of hematopoietic stem and progenitor cells capable of undergoing proliferation and/or differentiation, as might be found in human umbilical cord blood.
• the antibodies of the invention may be assayed for immunospecific binding by any method known in the art.
• the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
• Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/or protease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g., 1-4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
• a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1% sodium deoxy
• the ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g., western blot analysis.
• One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding of the antibody to an antigen and decrease the background (e.g., pre- clearing the cell lysate with sepharose beads).
• immunoprecipitation protocols see, e.g., Ausubel et al, eds, 1994, Cu ⁇ ent Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New York at 10.16.1.
• Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3% BSA or non-fat milk), washing the membrane in washing buffer (e.g., PBS-Tween 20), blocking the membrane with primary antibody (the antibody of interest) diluted in blocking buffer, washing the membrane in washing buffer, blocking the membrane with a secondary antibody (which recognizes the primary antibody, e.g., an anti- human antibody) conjugated to an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) or radioactive molecule (e.g., 32 P or 125 I) diluted in blocking buffer, washing the membrane in wash buffer, and detecting the presence of the antigen.
• ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
• a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
• a detectable compound such as an enzymatic substrate (e.g., horseradish peroxidase or alkaline phosphatase)
• a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well
• ELISAs see, e.g., Ausubel et al, eds, 1994, Cu ⁇ ent Protocols in Molecular Biology, Vol. 1 , John Wiley & Sons, Inc., New York at 11.2.1.
• the binding affinity of an antibody to an antigen and the off-rate of an antibody- antigen interaction can be determined by competitive binding assays.
• a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3 H or 125 I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
• labeled antigen e.g., 3 H or 125 I
• the affinity of the antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassay s.
• the antigen is incubated with antibody of interest conjugated to a labeled compound (e.g., 3 H or 125 I) in the presence of increasing amounts of an unlabeled second antibody.
• a labeled compound e.g., 3 H or 125 I
• suitable radioactive materials include, but are not limited to, radioisotopes such as iodine ( 131 I, 125 I, 123 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115 ⁇ Tn, 113m In, 112 In, H 1 In), and technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( ,8 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, ,40 La, ,75 Yb,
• Therapeutic Uses is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions.
• Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein).
• the antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with abe ⁇ ant expression and/or activity of a polypeptide of the invention, including, but not limited to, any one or more of the diseases, disorders, or conditions described herein.
• the treatment and/or prevention of diseases, disorders, or conditions associated with abe ⁇ ant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases, disorders or conditions.
• Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
• a summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below.
• the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
• lymphokines or hematopoietic growth factors such as, e.g., IL-2, IL-3 and IL-7
• the antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
• Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10 "2 M, 10 "2 M, 5 X 10 "3 M, 10 “3 M, 5 X 10 “4 M, lO “4 M, 5 X 10 '5 M, 10 "5 M, 5 X 10 "6 M, 10 “6 M, 5 X 10 "7 M, 10 “7 M, 5 X 10 “8 M, 10 “8 M, 5 X 10 "9 M, 10 '9 M, 5 X 10 "10 M, 10 “10 M, 5 X 10 "1 1 M, 10 "11 M, 5 X 10 "12 M, 10 “12 M, 5 X 10 "13 M, 10 " 13 M, 5 X 10 "14 M, 10 “14 M, 5 X 10 "15 M, and 10 "15 M.
• nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit or prevent a disease or disorder associated with abe ⁇ ant expression and/or activity of a polypeptide of the invention, by way of gene therapy.
• Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid.
• the nucleic acids produce their encoded protein that mediates a therapeutic effect.
• the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host.
• nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue- specific.
• nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression of the antibody encoding nucleic acids (Koller and Smithies, Proc. Natl.
• the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, of the antibody.
• Delivery of the nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
• the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product.
• This can be accomplished by any of numerous methods known in the art, e.g., by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g., by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No.
• microparticle bombardment e.g., a gene gun; Biolistic, Dupont
• coating lipids or cell-surface receptors or transfecting agents, encapsulation in liposomes, microparticles, or microcapsules, or by administering them in linkage to a peptide which is known to enter the nucleus, by administering it in linkage to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)) (which can be used to target cell types specifically expressing the receptors), etc.
• nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes, allowing the nucleic acid to avoid lysosomal degradation.
• the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635; WO92/20316; WO93/14188, WO 93/20221).
• the nucleic acid can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al, Nature 342:435-438 (1989)).
• viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used.
• a retroviral vector can be used (see Miller et al, Meth. Enzymol 217:581-599 (1993)). These retroviral vectors contain the components necessary for the co ⁇ ect packaging of the viral genome and integration into the host cell DNA.
• the nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient.
• retroviral vectors More detail about retroviral vectors can be found in Boesen et al, Biotherapy 6:291-302 (1994), which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
• Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al, J. Clin. Invest. 93:644-651 (1994); Kiem et al, Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson, Cu ⁇ . Opin. in Genetics and Devel 3:110-114 (1993).
• Adenoviruses are other viral vectors that can be used in gene therapy. Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson, Cu ⁇ ent Opinion in Genetics and Development 3:499-503 (1993) present a review of adenovirus-based gene therapy.
• Adeno-associated virus has also been proposed for use in gene therapy (Walsh et al, Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Patent No. 5,436,146).
• Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
• the method of transfer includes the transfer of a selectable marker to the cells.
• the cells are then placed under selection to isolate those cells that have taken up and are expressing the transfe ⁇ ed gene. Those cells are then delivered to a patient.
• the nucleic acid is introduced into a cell prior to administration in vivo of the resulting recombinant cell.
• introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc.
• Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol. 217:599-618 (1993); Cohen et al, Meth. Enzymol.
• the technique should provide for the stable transfer of the nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
• the resulting recombinant cells can be delivered to a patient by various methods known in the art.
• Recombinant blood cells e.g., hematopoietic stem or progenitor cells
• Cells into which a nucleic acid can be introduced for pu ⁇ oses of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, Blymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g., as obtained from bone ma ⁇ ow, umbilical cord blood, peripheral blood, fetal liver, etc.
• the cell used for gene therapy is autologous to the patient.
• nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
• stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment of the present invention (see e.g. PCT Publication WO
• the nucleic acid to be introduced for pu ⁇ oses of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the appropriate inducer of transcription. Demonstration of Therapeutic or Prophylactic Activity
• the compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans.
• in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample.
• the effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays.
• in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
• autoimmune diseases which include, but are not limited to, lupus erythematosus (SLE), rheumatoid arthritis (RA), insulin-dependent diabetes, multiple sclerosis (MS), giant cell arteritis, polyarteritis nodosa, myasthenia gravis, scleroderma, and graft versus host disease; graft rejection; mammalian cancers which include, but are not limited, to, melanoma, renal astrocytoma, Hodgkin's disease, breast, ovarian, prostate, bone, liver, lung, pancreatic, and spleenic cancers; inflammatory diseases; asthma; and allergeic diseases) express significantly altered (e.g., enhanced or decreased) levels of the galectin 11 polypeptide and mRNA encoding the galectin 11 polypeptide when compared to a co ⁇ esponding "standard" ma
• the invention provides a diagnostic method useful during diagnosis, which involves assaying the expression level of the gene encoding the galectin 11 polypeptide in mammalian cells or body fluid and comparing the gene expression level with a standard galectin 11 gene expression level, whereby an increase or decrease in the gene expression level over the standard is indicative of the disease.
• the present invention is useful as a prognostic indicator, whereby patients exhibiting altered galectin 11 gene expression will experience a worse clinical outcome relative to patients expressing the gene at a normal level.
• the expression level of the gene encoding the galectin 11 polypeptide is intended qualitatively or quantitatively measuring or estimating the level of the galectin 11 polypeptide or the level of the mRNA encoding the galectin 11 polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute polypeptide or mRNA level) or relatively (e.g., by comparing to the galectin 11 polypeptide level or mRNA level in a second biological sample).
• Nucleic acids for diagnosis may be obtained from a biological sample of a subject, such as from blood, urine, saliva, tissue biopsy or autopsy material, using techniques known in the art.
• the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR or other amplification techniques prior to analysis.
• RNA or cDNA may also be used in similar fashion. Deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to labeled galectin 11 nucleotide sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase digestion or by differences in melting temperatures.
• DNA sequence differences may also be detected by alterations in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents, or by direct DNA sequencing (see, e.g., Myers et al, Science 230:1242 (1985)). Sequence changes at specific locations may also be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (see Cotton et al, Proc. Natl. Acad. Sci. USA 85:4397-4401 (1985)).
• an a ⁇ ay of oligonucleotides probes comprising galectin 11 polynucleotide sequences or fragments thereof, can be constructed to conduct efficient screening of e.g., genetic mutations.
• Array technology methods are well known and have general applicability and can be used to address a variety of questions in molecular genetics including gene expression, genetic linkage, and genetic variability (see for example, Chee et al, Science 274:610-613 (1996)).
• the diagnostic assays offer a process for diagnosing or determining a susceptibility to specific diseases through detection of mutation in the galectin 11 gene by the methods described.
• RNAse protection the polymerase chain reaction (PCR), reverse transcription in combination with the polymerase chain reaction (RT-PCR) (Makino et al, Technique 2:295-301 (1990), reverse transcription in combination with the ligase chain reaction (RT-LCR) and other hybridization methods.
• galectin 11 polypeptide levels in a biological sample can be by any techniques known in the art, which include, but are not limited to, radioimmunoassays, competitive-binding assays, Western Blot analysis and enzyme linked immimosorbent assays (ELISAs) and other antibody-based techniques.
• galectin 11 polypeptide expression in tissues can be studied with classical immunohistological methods (Jalkanen et al, J. Cell Biol 101:976-985 (1985); Jalkanen et al, J. Cell. Biol. 105:3087-3096 (1987)).
• Suitable labels are known in the art and include enzyme labels, such as, Glucose oxidase, and radioisotopes, such as iodine ( 131 I, 125 I, 123 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( 115m In, U3m In, H2 In, m In), and technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), ,53 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, ,75 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rh
• the present invention relates to a diagnostic kit for a disease or susceptibility to a disease which comprises:
• a galectin 11 polynucleotide preferably the nucleotide sequence of SEQ ID NO:l, 24, or 26, or a fragment thereof ;
• b a nucleotide sequence complementary to that of (a);
• galectin 11 polypeptide of the invention preferably the polypeptide of SEQ ID NO:2, 25, or 27 or a fragment thereof;
• an antibody to a galectin 11 polypeptide of the invention preferably to the polypeptide of SEQ ID NO: 2, 25, or 27.
• kits may comprise a substantial component.
• the invention also provides a diagnostic method useful during diagnosis of a disorder, involving measuring the expression level of polynucleotides of the present invention in cells or body fluid from an individual and comparing the measured gene expression level with a standard level of polynucleotide expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of a disorder.
• the invention includes a kit for analyzing samples for the presence of proliferative and/or cancerous polynucleotides derived from a test subject.
• the kit includes at least one polynucleotide probe containing a nucleotide sequence that will specifically hybridize with a polynucleotide of the present invention and a suitable container.
• the kit includes two polynucleotide probes defining an internal region of the polynucleotide of the present invention, where each probe has one strand containing a 31'mer-end internal to the region.
• the probes may be useful as primers for polymerase chain reaction amplification.
• the present invention is useful as a prognostic indicator, whereby patients exhibiting enhanced or depressed polynucleotide of the present invention expression will experience a worse clinical outcome relative to patients expressing the gene at a level nearer the standard level
• measuring the expression level of polynucleotide of the present invention is intended qualitatively or quantitatively measuring or estimating the level of the polypeptide of the present invention or the level of the mRNA encoding the polypeptide in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the polypeptide level or mRNA level in a second biological sample).
• the polypeptide level or mRNA level in the first biological sample is measured or estimated and compared to a standard polypeptide level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder.
• a standard polypeptide level or mRNA level is known, it can be used repeatedly as a standard for comparison.
• biological sample any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains the polypeptide of the present invention or mRNA.
• biological samples include body fluids (such as semen, lymph, sera, plasma, urine, synovial fluid and spinal fluid) which contain the polypeptide of the present invention, and other tissue sources found to express the polypeptide of the present invention. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the prefe ⁇ ed source.
• the method(s) provided above may preferrably be applied in a diagnostic method and/or kits in which polynucleotides and/or polypeptides are attached to a solid support.
• the support may be a "gene chip” or a "biological chip” as described in US Patents 5,837,832, 5,874,219, and 5,856,174.
• a gene chip with polynucleotides of the present invention attached may be used to identify polymo ⁇ hisms between the polynucleotide sequences, with polynucleotides isolated from a test subject. The knowledge of such polymo ⁇ hisms (i.e.
• galectin 11 expression is responsible for many biological functions, including many pathologies. Accordingly, it is desirous to find compounds and drugs which enhance galectin 11 activity or, alternatively, suppress galectin 11 activity.
• the invention also provides a method of screening compounds to identify those which enhance or suppress galectin 11 activity.
• An agonist is a compound which increases the natural biological functions of galectin 11 or which functions in a manner similar to galectin 11, while antagonists decrease or eliminate such functions.
• embodiments of the invention are directed to assays designed to identify compounds that interact with (e.g., bind to) galectin 11 polypeptides of the invention, compounds that interfere or enhance the interaction of galectin 11 with its cognate ligands, and to compounds which modulate the galectin 11 gene (i.e., modulate the level of galectin 11 gene expression) or modulate the level of galectin 11 functional or biological activity.
• Assays may also be used to identify compounds which bind galectin 11 gene regulatory sequences (e.g., promoter sequences) and which may modulate galectin 11 gene expression. See e.g., Platt, J. Biol. Chem.
• polypeptides of the invention may be used to assess the binding of small molecule substrates and ligands in, for example, cells, cell-free preparations, chemical libraries, and natural product mixtures.
• substrates and ligands may be natural substrates and ligands or may be structural or functional mimetics. See Coligan et al, Current Protocols in Immunology 1(2): Chapter 5 (1991).
• peptides such as, for example soluble peptides, including but not limited to, those found: in random peptide libraries (see, e.g., Lam et al, Nature 354:84-86 (1991)), and combinatorial chemistry-derived molecular libraries made of D- and L- configuration amino acid; phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries (see e.g., Songyang et al, Cell 72:767-778 (1993)); antibodies (including but not limited to, monoclonal, humanized, anti-idiotypic, chimeric or single chain antibodies, and Fab, F(ab')2, and FAB expression library fragments, and epitope-binding fragments thereof); and small organic or inorganic molecules.
• peptides such as, for example soluble peptides, including but not limited to, those found: in random peptide libraries (see, e.g., Lam et al, Nature
• Numerous experimental methods may be used to select and detect compounds that bind galectin 11 polypeptides of the invention and thereby modulate galectin 11 expression or activity, including, but not limited to, protein affinity chromotography, affinity blotting, immunoprecipitation, cross-linking, and library based methods such as protein probing, phage display, the two-hybrid system (Fields and Song, Nature 340:245-246 (1989)), and modified versions of the two-hybrid system (Gyuris et al, Cell 75:791-803 (1993); Zervos et al, Cell 72:223-232 (1993)). See generally, Phizicky et al, Microbiol Rev. 59:94-123 (1995).
• the principle behind assays that identify compounds that bind to galectin 11 polypeptides of the invention involves preparing a reaction mixture of galectin 11 polypeptide and test compound under conditions that allow the two components to interact and bind, thus forming a complex which can be detected in the reaction mixture and purified using techniques known in the art. Accordingly, the assays may simply test binding of a candidate compound to galectin 11.
• the assays may simply comprise the steps of combining a candidate compound with a solution containing a galectin 11 polypeptide to form a mixture, and determining the ability of galectin 11 contained in this mixture to bind galectin 11 cognate ligands (e.g., compounds containing a ⁇ galactoside sugar and/or molecules expressed on the surface of T-cells), to agglutinate trypsin-treated rabbit erythrocytes, or to induce apoptosis of T-cells, and comparing this ability with that observed for the galectin 11 polypeptide in the same or similar solution under the same or similar conditions, but absent the candidate compound.
• galectin 11 cognate ligands e.g., compounds containing a ⁇ galactoside sugar and/or molecules expressed on the surface of T-cells
• the ability of the candidate molecule to interfere with binding of galectin 11 to the cognate ligand is reflected in decreased binding of the labeled galectin 11 to the cognate ligand relative to that in the absence of candidate molecule.
• Molecules which interfere with the ability of galectin 11 to elicit cellular responses (e.g., apoptosis) resulting from galectin 11 binding to its cognate ligand are antagonists.
• Molecules that enhance galectin 11 induced cellular responses when mixed with galectin 11 , or which are able to induce a similar cellular response in the absence of galectin 11, are agonists.
• the galectin 11 polynucleotides, polypeptides, and antibodies of the invention may also be used to configure assays for detecting the effect of added compounds on the production of galectin 11 mRNA and protein in cells.
• an ELISA may be constructed for measuring secreted or cell associated levels of galectin 11 protein using monoclonal and polyclonal antibodies by standard methods known in the art, and this can be used to discover agents which may inhibit or enhance the production of galectin 11 (also called antagonist or agonist, respectively) from suitably manipulated cells or tissues. Standard methods for conducting screening assays are well understood in the art.
• galectin 11 antagonists include antibodies or, in some cases, oligonucleotides or proteins which are closely related to the galectin 11 or its cognate ligand, e.g., a fragment of galectin 11 or galectin 11 ligand, or small molecules which bind to the cognate ligand, but do not elicit a response, so that the activity of the galectin 11 is prevented.
• the present invention relates to a screening kit for identifying agonists, antagonists, ligands, receptors, substrates, enzymes, etc. for galectin 11 polypeptides; or compounds which decrease or enhance the production of galectin 11, which comprises: (a) a galectin 11 polypeptide of the invention, such as, for example, that of SEQ ID NO:
• a cell expressing a galectin 11 ligand such as, for example, a T-cell
• Compounds identified via assays such as those described herein, may be useful, for example, in elaborating the biological function of the galectin 11 gene product and for regulating cell growth, cell proliferation and differentiation, and apoptosis.
• antibodies against galectin 11 and galectin 11 polypeptides, fragments, derivatives, variants or analogs of the invention may be employed to suppress galectin 11 activity to treat abnormalities resulting from elevated galectin 11.
• a pharmaceutically acceptable carrier e.g., as described herein
• their administration to treat or prevent growth regulatory and immunomodulatory disorders including, but not limited to, autoimmune diseases, cancer, and inflammatory diseases, are also encompassed by the invention.
• galectin 11 shares significant homology with other galectins. Additionally, as disclosed herein, galectin 11, like galectin 1 induces apoptosis of T-cell lines. Further, as discussed above, galectin 1 has been demonstrated to play a role in regulating cell proliferation and some immune functions (e.g., therapeutic activity against autoimmune diseases in experimental myasthenia gravis and experimental autoimmune encephalomyelitis animal model systems).
• galectin 11 like galectin 1, is active in modulating growth regulatory activities (e.g., cell differentiation and/or cell proliferation) , immunomodulatory activity, cell-cell and cell-substrate interactions, and apoptosis.
• Apoptosis or programmed cell death, is a physiological mechanism involved in the deletion of peripheral T lymphocytes of the immune system, and its dysregulation can lead to a number of different pathogenic processes.
• Diseases associated with increased cell survival, or the inhibition of apoptosis, that could be treated or detected by galectin 11 polynucleotides or polypeptides, as well as antagonists or agonists of galectin 11, include cancers (such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma,
• galectin 11 polynucleotides, polypeptides, and/or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above.
• galectin 11 polynucleotides or polypeptides, or agonists or antagonists of galectin 11, include, but are not limited to, progression, and or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sar
• galectin 11 polynucleotides or polypeptides include AIDS; neurodegenerative disorders (such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease); autoimmune disorders (such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary ci ⁇ hosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune-related glomerulonephritis and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft v.
• neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease
• autoimmune disorders such as, multiple sclerosis, S
• ischemic injury such as that caused by myocardial infarction, stroke and reperfusion injury
• liver injury e.g., hepatitis related liver injury, ischemia/reperfusion injury, cholestosis (bile duct injury) and liver cancer
• toxin-induced liver disease such as that caused by alcohol
• septic shock e.g., septic shock, cachexia and anorexia.
• Any method which neutralizes or enhances galectin 11 activity can be used to modulate growth regulatory activities (e.g., cell proliferation), immunomodulatory activity, cell-cell and cell-substrate interactions, and apoptosis.
• Galectin 11 polypeptides or polynucleotides may be useful in treating deficiencies or disorders of the immune system, by activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells.
• Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
• the etiology of these immune deficiencies or disorders may be genetic, somatic, such as cancer or some autoimmune disorders, acquired (e.g., by chemotherapy or toxins), or infectious.
• galectin 11 polynucleotides or polypeptides can be used as a marker or detector of a particular immune system disease or disorder.
• Galectin 11 polynucleotides or polypeptides may be useful in treating or detecting deficiencies or disorders of hematopoietic cells.
• galectin 11 polypeptides or polynucleotides or agonists or antagonists of galectin 11 could be used to increase differentiation and proliferation of hematopoietic cells, including the pluripotent stem cells, in an effort to treat those disorders associated with a decrease in certain (or many) types hematopoietic cells.
• immunologic deficiency syndromes include, but are not limited to: blood protein disorders (e.g.
• agammaglobulinemia agammaglobulinemia, dysgammaglobulinemia), ataxia telangiectasia, common variable immunodeficiency, Digeorge Syndrome, HIV infection, HTLV-BLV infection, leukocyte adhesion deficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction, severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder, anemia, thrombocytopenia, or hemoglobinuria.
• SIDs severe combined immunodeficiency
• galectin 11 polypeptides or polynucleotides can also be used to modulate hemostatic (the stopping of bleeding) or thrombolytic activity (clot formation).
• galectin 11 polynucleotides or polypeptides could be used to treat blood coagulation disorders (e.g., afibrinogenemia, factor deficiencies), blood platelet disorders (e.g. thrombocytopenia), or wounds resulting from trauma, surgery, or other causes.
• galectin 11 polynucleotides or polypeptides, or agonists or antagonists of galectin 11, that can decrease hemostatic or thrombolytic activity could be used to inhibit or dissolve clotting. These molecules could be important in the treatment of heart attacks (infarction), strokes, or scarring.
• Galectin 11 polynucleotides or polypeptides may also be useful in treating or detecting autoimmune disorders.
• galectin 11 induces apoptosis of T-cell lines (see Example 5, Figures 5A and 5B).
• Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue.
• the administration of galectin 11 polypeptides or polynucleotides that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells may be an effective therapy in preventing autoimmune disorders.
• autoimmune disorders that can be treated or detected include, but are not limited to: Addison's Disease, hemolytic anemia, antiphospholipid syndrome, rheumatoid arthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis, Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, Myasthenia Gravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus, Polyendocrinopathies, Pu ⁇ ura, Reiter's Disease, Stiff-Man Syndrome, Autoimmune Thyroiditis, Systemic Lupus Erythematosus, Autoimmune Pulmonary Inflammation, Guillain-Ba ⁇ e Syndrome, insulin dependent diabetes mellitis, and autoimmune
• allergic reactions and conditions such as asthma (particularly allergic asthma) or other respiratory problems, may also be treated by galectin 11 polypeptides or polynucleotides, or agonists or antagonists of galectin 11.
• these molecules can be used to treat anaphylaxis, hypersensitivity to an antigenic molecule, or blood group incompatibility.
• Galectin 11 polynucleotides or polypeptides may also be used to treat and/or prevent organ rejection or graft- versus-host disease (GVHD).
• Organ rejection occurs by host immune cell destruction of the transplanted tissue through an immune response.
• an immune response is also involved in GVHD, but, in this case, the foreign transplanted immune cells destroy the host tissues.
• galectin 11 polypeptides or polynucleotides, or agonists or antagonists of galectin 11, that inhibits an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing organ rejection or GVHD.
• galectin 11 polypeptides or polynucleotides may also be used to modulate inflammation.
• galectin 11 polypeptides or polynucleotides, or agonists or antagonists of galectin 11 may inhibit the proliferation and differentiation of cells involved in an inflammatory response.
• These molecules can be used to treat inflammatory conditions, both chronic and acute conditions, including inflammation associated with infection (e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)), ischemia-reperfusion injury, endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g., TNF or IL-1).
• infection e.g., septic shock, sepsis, or systemic inflammatory response syndrome (SIRS)
• ischemia-reperfusion injury e.g., endotoxin lethality, arthritis, complement-mediated hyperacute rejection, nephritis, cytokine or chemokine induced lung injury, inflammatory bowel disease, Crohn's disease, or resulting from over production of cytokines (e.g.,
• Galectin 11 polypeptides or polynucleotides can be used to treat or detect hype ⁇ roliferative disorders, including neoplasms.
• Galectin 11 polypeptides or polynucleotides, or agonists or antagonists of galectin 11 may inhibit the proliferation of the disorder through direct or indirect interactions.
• galectin 11 polypeptides or polynucleotides, or agonists or antagonists of galectin 11 may proliferate other cells which can inhibit the hype ⁇ roliferative disorder.
• hype ⁇ roliferative disorders can be treated.
• This immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
• decreasing an immune response may also be a method of treating hype ⁇ roliferative disorders, such as a chemotherapeutic agent.
• Examples of hype ⁇ roliferative disorders that can be treated or detected by galectin 11 polynucleotides or polypeptides include, but are not limited to, neoplasms located in the: colon, abdomen, bone, breast, digestive system, liver, pancreas, prostate, peritoneum, endocrine glands (adrenal, parathyroid, pituitary, testicles, ovary, thymus, thyroid), eye, head and neck, nervous (central and peripheral), lymphatic system, pelvic, skin, soft tissue, spleen, thoracic, and urogenital
• hype ⁇ roliferative disorders can also be treated or detected by galectin 11 polynucleotides or polypeptides, or agonists or antagonists of galectin 11.
• Examples of such hype ⁇ roliferative disorders include, but are not limited to: hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias, pu ⁇ ura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia, Gaucher's Disease, histiocytosis, and any other hype ⁇ roliferative disease, besides neoplasia, located in an organ system listed above.
• One prefe ⁇ ed embodiment utilizes polynucleotides of the present invention to inhibit abe ⁇ ant cellular division, by gene therapy using the present invention, and/or protein fusions or fragments thereof.
• the present invention provides a method for treating cell proliferative disorders by inserting into an abnormally proliferating cell a polynucleotide of the present invention, wherein said polynucleotide represses said expression.
• polynucleotides of the present invention is a DNA construct comprising a recombinant expression vector effective in expressing a DNA sequence encoding said polynucleotides.
• the DNA construct encoding the poynucleotides of the present invention is inserted into cells to be treated utilizing a retrovirus, or more prefe ⁇ ably an adenoviral vector (See G J. Nabel, et.
• the viral vector is defective and will not transform non-proliferating cells, only proliferating cells.
• the polynucleotides of the present invention inserted into proliferating cells either alone, or in combination with or fused to other polynucleotides can then be modulated via an external stimulus (i.e. magnetic, specific small molecule, chemical, or drug administration, etc.), which acts upon the promoter upstream of said polynucleotides to induce expression of the encoded protein product.
• an external stimulus i.e. magnetic, specific small molecule, chemical, or drug administration, etc.
• the beneficial therapeutic affect of the present invention may be expressly modulated (i.e. to increase, decrease, or inhibit expression of the present invention) based upon said external stimulus.
• Polynucleotides of the present invention may be useful in repressing expression of oncogenic genes or antigens.
• repressing expression of the oncogenic genes is intended the suppression of the transcription of the gene, the degradation of the gene transcript (pre- message RNA), the inhibition of splicing, the destruction of the messenger RNA, the prevention of the post-translational modifications of the protein, the destruction of the protein, or the inhibition of the normal function of the protein.
• polynucleotides of the present invention may be administered by any method known to those of skill in the art including, but not limited to transfection, electroporation, microinjection of cells, or in vehicles such as liposomes, lipofectin, or as naked polynucleotides, or any other method described throughout the specification.
• the polynucleotide of the present invention may be delivered by known gene delivery systems such as, but not limited to, retroviral vectors (Gilboa, J. Virology 44:845 (1982); Hocke, Nature 320:275 (1986); Wilson, et al, Proc. Natl. Acad. Sci. U.S.A.
• the polynucleotides of the present invention may be delivered directly to cell proliferative disorder/disease sites in internal organs, body cavities and the like by use of imaging devices used to guide an injecting needle directly to the disease site.
• the polynucleotides of the present invention may also be administered to disease sites at the time of surgical intervention.
• cell proliferative disease any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which is characterized by single or multiple local abnormal proliferations of cells, groups of cells, or tissues, whether benign or malignant. Any amount of the polynucleotides of the present invention may be administered as long as it has a biologically inhibiting effect on the proliferation of the treated cells.
• biologically inhibiting is meant partial or total growth inhibition as well as decreases in the rate of proliferation or growth of the cells.
• the biologically inhibitory dose may be determined by assessing the effects of the polynucleotides of the present invention on target malignant or abnormally proliferating cell growth in tissue culture, tumor growth in animals and cell cultures, or any other method known to one of ordinary skill in the art.
• the present invention is further directed to antibody-based therapies which involve administering of anti-polypeptides and anti-polynucleotide antibodies to a mammalian, preferably human, patient for treating one or more of the described disorders.
• Methods for producing anti-polypeptides and anti-polynucleotide antibodies polyclonal and monoclonal antibodies are described in detail elsewhere herein. Such antibodies may be provided in pharmaceutically acceptable compositions as known in the art or as described herein.
• a summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g. as mediated by complement (CDC) or by effector cells (ADCC). Some of these approaches are described in more detail below.
• the antibodies, fragments and derivatives of the present invention are useful for treating a subject having or developing cell proliferative and/or differentiation disorders as described herein.
• Such treatment comprises administering a single or multiple doses of the antibody, or a fragment, derivative, or a conjugate thereof.
• the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors, for example, which serve to increase the number or activity of effector cells which interact with the antibodies.
• Prefe ⁇ ed binding affinities include those with a dissociation constant or Kd less than 5X10 "6 M, 10 "6 M, 5X10 “7 M, 10 “7 M, 5X10 “8 M, 10 “8 M, 5X10 “9 M, 10- 9 M ⁇ 5X10- 10 M, 10 "10 M, 5X10- U M, 10 "n M, 5X10 "12 M, 10 "12 M, 5X10 "13 M, 10 " 13 M, 5X10 "14 M, 10 "14 M, 5X10 "15 M, and 10 "15 M.
• polypeptides of the present invention are useful in inhibiting the angiogenesis of proliferative cells or tissues, either alone, as a protein fusion, or in combination with other polypeptides directly or indirectly, as described elsewhere herein.
• said anti-angiogenesis effect may be achieved indirectly, for example, through the inhibition of hematopoietic, tumor-specific cells, such as tumor- associated macrophages (See Joseph IB, et al. J Natl Cancer Inst, 90(21): 1648-53 (1998), which is hereby inco ⁇ orated by reference).
• Antibodies directed to polypeptides or polynucleotides of the present invention may also result in inhibition of angiogenesis directly, or indirectly (See Witte L, et al, Cancer Metastasis Rev. 17(2):155-61 (1998), which is hereby inco ⁇ orated by reference)).
• Polypeptides including protein fusions, of the present invention, or fragments thereof may be useful in inhibiting proliferative cells or tissues through the induction of apoptosis.
• Said polypeptides may act either directly, or indirectly to induce apoptosis of proliferative cells and tissues, for example in the activation of a death-domain receptor, such as tumor necrosis factor (TNF) receptor- 1, CD95 (Fas/APO-1), TNF-receptor-related apoptosis- mediated protein (TRAMP) and TNF-related apoptosis-inducing ligand (TRAIL) receptor- 1 and -2 (See Schulze-Osthoff K, etal, Eur J Biochem 254(3):439-59 (1998), which is hereby inco ⁇ orated by reference).
• TNF tumor necrosis factor
• TRAMP TNF-receptor-related apoptosis- mediated protein
• TRAIL TNF-related apoptos
• said polypeptides may induce apoptosis through other mechanisms, such as in the activation of other proteins which will activate apoptosis, or through stimulating the expression of said proteins, either alone or in combination with small molecule drugs or adjuviants, such as apoptonin, galectins, thioredoxins, antiinflammatory proteins (See for example, Mutat Res 400(l-2):447-55 (1998), Med Hypotheses.50(5):423-33 (1998), Chem Biol Interact. Apr 24; 111-112:23-34 (1998), J Mol Med.76(6):402-12 (1998), Int J Tissue React;20(l):3-15 (1998), which are all hereby inco ⁇ orated by reference).
• small molecule drugs or adjuviants such as apoptonin, galectins, thioredoxins, antiinflammatory proteins
• Polypeptides, including protein fusions to, or fragments thereof, of the present invention are useful in inhibiting the metastasis of proliferative cells or tissues. Inhibition may occur as a direct result of administering polypeptides, or antibodies directed to said polypeptides as described elsewere herein, or indirectly, such as activating the expression of proteins known to inhibit metastasis, for example alpha 4 integrins, (See, e.g., Cu ⁇ Top Microbiol Immunol 1998;231 :125-41, which is hereby inco ⁇ orated by reference). Such thereapeutic affects of the present invention may be achieved either alone, or in combination with small molecule drugs or adjuvants.
• the invention provides a method of delivering compositions containing the polypeptides of the invention (e.g., compositions containing polypeptides or polypeptide antibodes associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells expressing the polypeptide of the present invention.
• compositions containing the polypeptides of the invention e.g., compositions containing polypeptides or polypeptide antibodes associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs
• Polypeptides or polypeptide antibodes of the invention may be associated with with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
• Polypeptides, protein fusions to, or fragments thereof, of the present invention are useful in enhancing the immunogenicity and/or antigenicity of proliferating cells or tissues, either directly, such as would occur if the polypeptides of the present invention 'vaccinated' the immune response to respond to proliferative antigens and immunogens, or indirectly, such as in activating the expression of proteins known to enhance the immune response (e.g. chemokines), to said antigens and immunogens.
• Galectin 11 polypeptides or polynucleotides can be used to treat or detect infectious agents.
• infectious diseases may be treated.
• the immune response may be increased by either enhancing an existing immune response, or by initiating a new immune response.
• galectin 11 polypeptides or polynucleotides may also directly inhibit the infectious agent, without necessarily eliciting an immune response.
• viruses are one example of an infectious agent that can cause disease or symptoms that can be treated or detected by galectin 11 polynucleotides or polypeptides.
• viruses include, but are not limited to, the following DNA and RNA viral families: Arbovirus, Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV, Flaviviridae, Hepadnaviridae (Hepatitis), He ⁇ esviridae (such as, Cytomegalo virus, He ⁇ es Simplex, He ⁇ es Zoster), Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g., Influenza A, Influenza B, and parainfluenza), Papiloma virus, Papovaviridae,
• Viruses falling within these families can cause a variety of diseases or symptoms, including, but not limited to: arthritis, bronchiollitis, respiratory syncytial virus, encephalitis, eye infections (e.g., conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, Chronic Active, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valley fever, yellow fever, meningitis, opportunistic infections (e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemo ⁇ hagic fever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts), and viremia.
• arthritis bronchiollitis
• respiratory syncytial virus e.g., respiratory syncytial virus
• Galectin 11 polypeptides or polynucleotides, or agonists or antagonists of the invention can be used to treat or detect any of these symptoms or diseases.
• polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat: meningitis, Dengue, EBV, and/or hepatitis (e.g., hepatitis B).
• polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat patients nonresponsive to one or more other commercially available hepatitis vaccines.
• polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat AIDS.
• bacterial or fungal agents that can cause disease or symptoms and that can be treated or detected by galectin 11 polynucleotides or polypeptides and/or agonist or antagonists of the present invention include, but are not limited to, the following Gram- Negative and Gram-positive bacterial families and fungi: Actinomycetales (e.g., Corynebacterium, Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis, Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Bo ⁇ elia (e.g., Bo ⁇ elia burgdorferi), Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatoc
• Enterobacteriaceae Klebsiella, Salmonella (e.g., Salmonella typhi, and Salmonella paratyphi), Se ⁇ atia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Mycobacterium leprae, Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea, Menigococcal), Meisseria meningitidis, Pasteurellacea Infections (e.g., Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B), Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, Shigella spp
• bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections (e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gono ⁇ hea, meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually
• Galectin 11 polypeptides or polynucleotides, or agonists or antagonists of the invention can be used to treat or detect any of these symptoms or diseases.
• polynucleotides, polypeptides, agonists or antagonists of the invention are used to treat: tetanus, Diptheria, botulism, and/or meningitis type B.
• parasitic agents causing disease or symptoms that can be treated or detected by galectin 11 polynucleotides or polypeptides, agonists or antagonists include, but not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas and Sporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodium malariae and Plasmodium ovale).
• These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease (e.g., dysentery, giardiasis), liver disease, lung disease, opportunistic infections (e.g., AIDS related), malaria, pregnancy complications, and toxoplasmosis.
• Galectin 11 polypeptides or polynucleotides, or agonists or antagonists can be used to treat or detect any of these symptoms or diseases.
• polynucleotides, polypeptides, or agonists or antagonists of the invention are used to treat malaria.
• treatment using a polypeptide or polynucleotide and/or agonist or antagonist of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy).
• the polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
• Galectin 11 polynucleotides or polypeptides can be used to differentiate, proliferate, and attract cells, leading to the regeneration of tissues.
• the regeneration of tissues could be used to repair, replace, or protect tissue damaged by congenital defects, trauma (wounds, burns, incisions, or ulcers), age, disease (e.g. osteoporosis, osteocarthritis, periodontal disease, liver failure), surgery, including cosmetic plastic surgery, fibrosis, reperfusion injury, or systemic cytokine damage.
• Tissues that could be regenerated using the present invention include organs (e.g., pancreas, liver, intestine, kidney, skin, endothelium), muscle (smooth, skeletal or cardiac), vascular (including vascular endothelium), nervous, hematopoietic, and skeletal (bone, cartilage, tendon, and ligament) tissue.
• organs e.g., pancreas, liver, intestine, kidney, skin, endothelium
• muscle smooth, skeletal or cardiac
• vascular including vascular endothelium
• nervous hematopoietic
• hematopoietic skeletal tissue
• skeletal bone, cartilage, tendon, and ligament
• galectin 11 polynucleotides or polypeptides may increase regeneration of tissues difficult to heal. For example, increased tendon/ligament regeneration would quicken recovery time after damage.
• Galectin 11 polynucleotides or polypeptides, or agonists or antagonists of the present invention could also be used prophylactically in an effort to avoid damage.
• Specific diseases that could be treated include of tendinitis, ca ⁇ al tunnel syndrome, and other tendon or ligament defects.
• a further example of tissue regeneration of non-healing wounds includes pressure ulcers, ulcers associated with vascular insufficiency, surgical, and traumatic wounds.
• nerve and brain tissue could also be regenerated by using galectin 11 polynucleotides or polypeptides, or agonists or antagonists of the present invention, to proliferate and differentiate nerve cells.
• Diseases that could be treated using this method include central and peripheral nervous system diseases, neuropathies, or mechanical and traumatic disorders (e.g., spinal cord disorders, head trauma, cerebrovascular disease, and stoke).
• diseases associated with peripheral nerve injuries could all be treated using the galectin 11 polynucleotides or polypeptides or agonists or antagonists of galectin 11.
• Galectin 11 polynucleotides or polypeptides, or agonists or antagonists of the present invention may have chemotaxis activity.
• a chemotaxic molecule attracts or mobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells, mast cells, eosinophils, epithelial and/or endothelial cells) to a particular site in the body, such as inflammation, infection, or site of hype ⁇ roliferation.
• the mobilized cells can then fight off and/or heal the particular trauma or abnormality.
• Galectin 11 polynucleotides or polypeptides, or agonists or antagonists of the present invention may increase chemotaxic activity of particular cells. These chemotactic molecules can then be used to treat inflammation, infection, hype ⁇ roliferative disorders, or any immune system disorder by increasing the number of cells targeted to a particular location in the body. For example, chemotaxic molecules can be used to treat wounds and other trauma to tissues by attracting immune cells to the injured location. Chemotactic molecules of the present invention can also attract fibroblasts, which can be used to treat wounds.
• galectin 11 polynucleotides or polypeptides, or agonists or antagonists of the present invention may inhibit chemotactic activity. These molecules could also be used to treat disorders. Thus, galectin 11 polynucleotides or polypeptides, or agonists or antagonists of the present invention, could be used as an inhibitor of chemotaxis.
• Nervous system disorders which can be treated with the galectin 11 compositions of the invention (e.g., galectin 11 polypeptides, polynucleotides, and/or agonists or antagonists), include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.
• the galectin 11 compositions of the invention include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination.
• Nervous system lesions which may be treated in a patient (including human and non-human mammalian patients) according to the invention, include but are not limited to, the following lesions of either the central (including spinal cord, brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, he ⁇ es z
• the galectin 11 polypeptides, polynucleotides, or agonists or antagonists of the present invention are used to protect neural cells from the damaging effects of cerebral hypoxia.
• the galectin 11 compositions of the invention are used to treat or prevent neural cell injury associated with cerebral hypoxia.
• the galectin 11 polypeptides, polynucleotides, or agonists or antagonists of the invention are used to treat or prevent neural cell injury associated with cerebral ischemia.
• the galectin 11 polypeptides, polynucleotides, or agonists or antagonists of the present invention are used to treat or prevent neural cell injury associated with cerebral infarction.
• the galectin 11 polypeptides, polynucleotides, or agonists or antagonists of the present invention are used to treat or prevent neural cell injury associated with a stroke.
• the galectin 11 polypeptides, polynucleotides, or agonists or antagonists of the present invention are used to treat or prevent neural cell injury associated with a heart attack.
• compositions of the invention which are useful for treating or preventing a nervous system disorder may be selected by testing for biological activity in promoting the survival or differentiation of neurons.
• galectin 11 compositions of the invention which elicit any of the following effects may be useful according to the invention: (1) increased survival time of neurons in culture; (2) increased sprouting of neurons in culture or in vivo; (3) increased production of a neuron-associated molecule in culture or in vivo, e.g., choline acetyltransferase or acetylcholinesterase with respect to motor neurons; or (4) decreased symptoms of neuron dysfunction in vivo.
• Such effects may be measured by any method known in the art.
• increased survival of neurons may routinely be measured using a method set forth herein or otherwise known in the art, such as, for example, the method set forth in Arakawa et al. (J. Neurosci. 10:3507-3515 (1990)); increased sprouting of neurons may be detected by methods known in the art, such as, for example, the methods set forth in Pestronk et al. (Exp. Neurol 70:65-82 (1980)) or Brown et al. (Ann. Rev. Neurosci.
• neuron-associated molecules may be measured by bioassay, enzymatic assay, antibody binding, Northern blot assay, etc., using techniques known in the art and depending on the molecule to be measured; and motor neuron dysfunction may be measured by assessing the physical manifestation of motor neuron disorder, e.g., weakness, motor neuron conduction velocity, or functional disability.
• motor neuron disorders that may be treated according to the invention include, but are not limited to, disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar palsy, primary lateral sclerosis, infantile and juvenile muscular atrophy, progressive bulbar paralysis of childhood (Fazio- Londe syndrome), poliomyelitis and the post polio syndrome, and Hereditary Motorsensory Neuropathy (Charcot-Marie-Tooth Disease).
• disorders such as infarction, infection, exposure to toxin, trauma, surgical damage, degenerative disease or malignancy that may affect motor neurons as well as other components of the nervous system, as well as disorders that selectively affect neurons such as amyotrophic lateral sclerosis, and including, but not limited to, progressive spinal muscular atrophy, progressive bulbar
• epilepsy such as generalized epilepsy which includes infantile spasms, absence epilepsy, myoclonic epilepsy which includes MERRF Syndrome, tonic-clonic epilepsy, partial epilepsy such as complex partial epilepsy, frontal lobe epilepsy and temporal lobe epilepsy, post-traumatic epilepsy, status epilepticus such as Epilepsia Partialis Continua, Hallervorden-Spatz Syndrome, hydrocephalus such as Dandy-Walker Syndrome and normal pressure hydrocephalus, hypothalamic diseases such as hypothalamic neoplasms, cerebral malaria, nar
• Bacterial meningtitis which includes Haemophilus Meningtitis, Listeria Meningtitis, Meningococcal Meningtitis such as Waterhouse-Friderichsen Syndrome, Pneumococcal Meningtitis and meningeal tuberculosis, fungal meningitis such as Cryptococcal Meningtitis, subdural effusion, meningoencephalitis such as uvemeningoencephalitic syndrome, myelitis such as transverse myelitis, neurosyphilis such as tabes dorsalis, poliomyelitis which includes bulbar poliomyelitis and postpoliomyelitis syndrome, prion diseases (such as Creutzfeldt-Jakob Syndrome, Bovine Spongiform Encephalopathy, Gerstmann-Straussler Syndrome, Kuru, Scrapie) cerebral toxoplasmosis, central nervous system neoplasms such as brain neoplasms that include cerebellear neoplasms such
• Postpoliomyelitis Syndrome Muscular Dystrophy, Myasthenia Gravis, Myotonia Afrophica, Myotonia Confenita, Nemaline Myopathy, Familial Periodic Paralysis, Multiplex Paramyloclonus, Tropical Spastic Paraparesis and Stiff-Man Syndrome, peripheral nervous system diseases such as acrodynia, amyloid neuropathies, autonomic nervous system diseases such as Adie's Syndrome, Ba ⁇ e-Lieou Syndrome, Familial Dysautonomia, Homer's Syndrome, Reflex Sympathetic Dystrophy and Shy-Drager Syndrome, Cranial Nerve Diseases such as Acoustic Nerve Diseases such as Acoustic Neuroma which includes Neurofibromatosis 2, Facial Nerve Diseases such as Facial Neuralgia,Melkersson-Rosenthal Syndrome, ocular motility disorders which includes amblyopia, nystagmus, oculomotor nerve paralysis, ophthalmoplegia such as Duane'
• galectin 11 polynucleotides or polypeptides as well as agonists or antagonists of galectin 11, for therapeutic pu ⁇ oses, for example, to stimulate epithelial cell proliferation and basal keratinocytes for the pu ⁇ ose of wound healing, and to stimulate hair follicle production and healing of dermal wounds.
• Galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11, may be clinically useful in stimulating wound healing including surgical wounds, excisional wounds, deep wounds involving damage of the dermis and epidermis, eye tissue wounds, dental tissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers, cubitus ulcers, arterial ulcers, venous stasis ulcers, bums resulting from heat exposure or chemicals, and other abnormal wound healing conditions such as uremia, malnutrition, vitamin deficiencies and complications associted with systemic treatment with steroids, radiation therapy and antineoplastic drugs and antimetabolites.
• Galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11, could be used to promote dermal reestablishment subsequent to dermal loss
• Galectin 11 polynucleotides or polypeptides could be used to increase the adherence of skin grafts to a wound bed and to stimulate re-epithelialization from the wound bed.
• galectin 11 polynucleotides or polypeptides, agonists or antagonists of galectin 11, could be used to increase adherence to a wound bed: autografts, artificial skin, allografts, autodermic graft, autoepdermic grafts, avacular grafts, Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft, delayed graft, dermic graft, epidermic graft, fascia graft, full thickness graft, heterologous graft, xenograft, homologous graft, hype ⁇ lastic graft, lamellar graft, mesh graft, mucosal graft, Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft, penetrating graft, split skin graft, thick split graft.
• galectin 11 polynucleotides or polypeptides will also produce changes in hepatocyte proliferation, and epithelial cell proliferation in the lung, breast, pancreas, stomach, small intesting, and large intestine.
• Galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11 could promote proliferation of epithelial cells such as sebocytes, hair follicles, hepatocytes, type II pneumocytes, mucin-producing goblet cells, and other epithelial cells and their progenitors contained within the skin, lung, liver, and gastrointestinal tract.
• Galectin 11 polynucleotides or polypeptides, agonists or antagonists of galectin 11 may promote proliferation of endothelial cells, keratinocytes, and basal keratinocytes.
• Galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11, could also be used to reduce the side effects of gut toxicity that result from radiation, chemotherapy treatments or viral infections.
• Galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11, may have a cytoprotective effect on the small intestine mucosa.
• Galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11 may also stimulate healing of mucositis (mouth ulcers) that result from chemotherapy and viral infections.
• Galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11, could further be used in full regeneration of skin in full and partial thickness skin defects, including bums, (i.e., repopulation of hair follicles, sweat glands, and sebaceous glands), treatment of other skin defects such as psoriasis.
• Galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11, could be used to treat epidermolysis bullosa, a defect in adherence of the epidermis to the underlying dermis which results in frequent, open and painful blisters by accelerating reepithelialization of these lesions.
• Galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11, could also be used to treat gastric and doudenal ulcers and help heal by scar formation of the mucosal lining and regeneration of glandular mucosa and duodenal mucosal lining more rapidly.
• Inflamamatory bowel diseases such as Crohn's disease and ulcerative colitis, are diseases which result in destruction of the mucosal surface of the small or large intestine, respectively.
• galectin 11 polynucleotides or polypeptides could be used to promote the resurfacing of the mucosal surface to aid more rapid healing and to prevent progression of inflammatory bowel disease.
• Treatment with galectin 11 polynucleotides or polypeptides, agonists or antagonists of galectin 11, is expected to have a significant effect on the production of mucus throughout the gastrointestinal tract and could be used to protect the intestinal mucosa from injurious substances that are ingested or following surgery.
• Galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11 could be used to treat diseases associate with the under expression of galectin 11.
• galectin 11 polynucleotides or polypeptides could be used to prevent and heal damage to the lungs due to various pathological states.
• a growth factor such as galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11, which could stimulate proliferation and differentiation and promote the repair of alveoli and brochiolar epithelium to prevent or treat acute or chronic lung damage.
• galectin 11 polynucleotides or polypeptides agonists or antagonists of galectin 11.
• galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11 could be used to stimulate the proliferation of and differentiation of type II pneumocytes, which may help treat or prevent disease such as hyaline membrane diseases, such as infant respiratory distress syndrome and bronchopulmonary displasia, in premature infants.
• Galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11, could stimulate the proliferation and differentiation of hepatocytes and, thus, could be used to alleviate or treat liver diseases and pathologies such as fulminant liver failure caused by ci ⁇ hosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).
• liver diseases and pathologies such as fulminant liver failure caused by ci ⁇ hosis, liver damage caused by viral hepatitis and toxic substances (i.e., acetaminophen, carbon tetraholoride and other hepatotoxins known in the art).
• galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11, could be used treat or prevent the onset of diabetes mellitus.
• galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11, could be used to maintain the islet function so as to alleviate, delay or prevent permanent manifestation of the disease.
• galectin 11 polynucleotides or polypeptides, as well as agonists or antagonists of galectin 11 could be used as an auxiliary in islet cell transplantation to improve or promote islet cell function.
• Galectin 11 polynucleotides or polypeptides, or agonists or antagonists of galectin 11 , encoding galectin 11 may be used to treat cardiovascular disorders, including peripheral artery disease, such as limb ischemia.
• Cardiovascular disorders include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome.
• Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextiocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septal defects.
• Cardiovascular disorders also include heart disease, such as a ⁇ hythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular syphilis, and cardiovascular tuberculosis.
• heart disease such as a ⁇ hythmias, carcinoi
• a ⁇ hythmias include sinus a ⁇ hythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation.
• Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
• Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
• Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Keams Syndrome, myocardial reperfusion injury, and myocarditis.
• Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
• coronary disease such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
• Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease, Klippel- Trenaunay- Weber Syndrome, Sturge- Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemo ⁇ hoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CREST syndrome, retinal vein
• Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, mptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
• Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
• Cerebrovascular disorders include carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemo ⁇ hage, epidural hematoma, subdural hematoma, subaraxhnoid hemo ⁇ hage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.
• Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms.
• Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
• Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia.
• Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-Henoch pu ⁇ ura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
• Galectin 11 polynucleotides or polypeptides, or agonists or antagonists of galectin 11, are especially effective for the treatment of critical limb ischemia and coronary disease.
• Galectin 11 polypeptides may be administered using any method known in the art, including, but not limited to, direct needle injection at the delivery site, intravenous injection, topical administration, catheter infusion, biolistic injectors, particle accelerators, gelfoam sponge depots, other commercially available depot materials, osmotic pumps, oral or suppositorial solid pharmaceutical formulations, decanting or topical applications during surgery, aerosol delivery. Such methods are known in the art.
• Galectin 11 polypeptides may be administered as part of a Therapeutic, described in more detail below. Methods of delivering galectin 11 polynucleotides are described in more detail herein.
• angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases.
• a number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g., reviews by Moses et al, Biotech. 9:630-634 (1991); Folkman et al, N Engl J. Med., 333:1757-1763 (1995); Auerbach et al, J. Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp . 175-203 (1985); Patz, Am. J. Opthalmol 94:7X5-743 (1982); and Folkman et al, Science 221:719-725 (1983).
• the present invention provides for treatment of diseases or disorders associated with neovascularization by administration of the polynucleotides and/or polypeptides of the invention, as well as agonists or antagonists of the present invention.
• Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides, or agonists or antagonists of the invention include, but are not limited to, malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al, Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).
• the present invention provides a method of treating an angiogenesis-related disease and/or disorder, comprising administering to an individual in need thereof a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the invention.
• a polynucleotide, polypeptide, antagonist and/or agonist of the invention may be utilized in a variety of additional methods in order to therapeutically treat a cancer or tumor.
• Cancers which may be treated with polynucleotides, polypeptides, antagonists and/or agonists of the present invention include, but are not limited to solid tumors, including prostate, lung, breast, ovarian, stomach, pancreas, larynx, esophagus, testes, liver, parotid, biliary tract, colon, rectum, cervix, uterus, endometrium, kidney, bladder, thyroid cancer; primary tumors and metastases; melanomas; glioblastoma; Kaposi's sarcoma; leiomyosarcoma; non- small cell lung cancer; colorectal cancer; advanced malignancies; and blood bom tumors such as leukemias.
• polynucleotides, polypeptides, antagonists and/or agonists of the present invention may be delivered topically, in order to treat cancers such as skin cancer, head and neck tumors, breast tumors, and Kaposi's sarcoma.
• polynucleotides, polypeptides, antagonists and/or agonists of the present invention may be utilized to treat superficial forms of bladder cancer by, for example, intravesical administration.
• Polynucleotides, polypeptides, antagonists and/or agonists of the present invention may be delivered directly into the tumor, or near the tumor site, via injection or a catheter.
• the appropriate mode of administration will vary according to the cancer to be treated. Other modes of delivery are discussed herein.
• Polynucleotides, polypeptides, antagonists and/or agonists of the present invention may be useful in treating other disorders, besides cancers, which involve angiogenesis.
• disorders include, but are not limited to: benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas; artheroscleric plaques; ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, comeal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, uvietis and Pterygia (abnormal blood vessel growth) of the eye; rheumatoid arthritis; psoriasis; delayed wound healing; endometriosis; vasculogenesis; granulations; hypertrophic scars (keloids); nonunion
• methods for treating hypertrophic scars and keloids comprising the step of administering a polynucleotide, polypeptide, antagonist and/or agonist of the invention to a hypertrophic scar or keloid.
• polypeptides, antagonists and/or agonists of the present invention are injected directly into a hypertrophic scar or keloid, in order to prevent the progression of these lesions.
• This therapy is of particular value in the prophylactic treatment of conditions which are known to result in the development of hypertrophic scars and keloids (e.g., bums), and is preferably initiated after the proliferative phase has had time to progress (approximately 14 days after the initial injury), but before hypertrophic scar or keloid development.
• the present invention also provides methods for treating neovascular diseases of the eye, including for example, comeal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
• neovascular diseases of the eye including for example, comeal neovascularization, neovascular glaucoma, proliferative diabetic retinopathy, retrolental fibroplasia and macular degeneration.
• Ocular disorders associated with neovascularization which can be treated with the polynucleotides and polypeptides of the present invention (including agonists and/or antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, comeal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g., reviews by Waltman et al, Am. J. Ophthal 55:704-710 (1978) and Gartner et al, Surv. Ophthal. 22:291-312 (1978).
• neovascular diseases of the eye such as comeal neovascularization (including comeal graft neovascularization)
• a therapeutically effective amount of a compound (as described above) to the cornea such that the formation of blood vessels is inhibited.
• the cornea is a tissue which normally lacks blood vessels.
• capillaries may extend into the cornea from the pericorneal vascular plexus of the limbus.
• the cornea becomes vascularized, it also becomes clouded, resulting in a decline in the patient's visual acuity. Nisual loss may become complete if the cornea completely opacitates.
• comeal infections e.g., trachoma, he ⁇ es simplex keratitis, leishmaniasis and onchocerciasis
• immunological processes e.g., graft rejection and Stevens- Johnson's syndrome
• alkali bums e.g., trauma, inflammation (of any cause), toxic and nutritional deficiency states, and as a complication of wearing contact lenses.
• saline may be prepared for topical administration in saline (combined with any of the preservatives and antimicrobial agents commonly used in ocular preparations), and administered in eyedrop form.
• the solution or suspension may be prepared in its pure form and administered several times daily.
• anti-angiogenic compositions prepared as described above, may also be administered directly to the cornea.
• the anti-angiogenic composition is prepared with a muco-adhesive polymer which binds to cornea.
• the anti-angiogenic factors or anti-angiogenic compositions may be utilized as an adjunct to conventional steroid therapy.
• Topical therapy may also be useful prophylactically in comeal lesions which are known to have a high probability of inducing an angiogenic response (such as chemical bums).
• the treatment likely in combination with steroids, may be instituted immediately to help prevent subsequent complications.
• the compounds described above may be injected directly into the comeal stroma by an ophthalmologist under microscopic guidance.
• the prefe ⁇ ed site of injection may vary with the mo ⁇ hology of the individual lesion, but the goal of the administration would be to place the composition at the advancing front of the vasculature (i.e., interspersed between the blood vessels and the normal cornea). In most cases this would involve perilimbic comeal injection to "protect" the cornea from the advancing blood vessels.
• This method may also be utilized shortly after a comeal insult in order to prophylactically prevent comeal neovascularization.
• the material could be injected in the perilimbic cornea interspersed between the comeal lesion and its undesired potential limbic blood supply.
• Such methods may also be utilized in a similar fashion to prevent capillary invasion of transplanted corneas. In a sustained-release form injections might only be required 2-3 times per year.
• a steroid could also be added to the injection solution to reduce inflammation resulting from the injection itself.
• methods for treating neovascular glaucoma, comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the present invention to the eye, such that the formation of blood vessels is inhibited.
• the compound may be administered topically to the eye in order to treat early forms of neovascular glaucoma.
• the compound may be implanted by injection into the region of the anterior chamber angle.
• the compound may also be placed in any location such that the compound is continuously released into the aqueous humor.
• proliferative diabetic retinopathy comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist to the eyes, such that the formation of blood vessels is inhibited.
• proliferative diabetic retinopathy may be treated by injection into the aqueous humor or the vitreous, in order to increase the local concentration of the polynucleotide, polypeptide, antagonist and/or agonist in the retina.
• this treatment should be initiated prior to the acquisition of severe disease requiring photocoagulation.
• methods for treating retrolental fibroplasia comprising the step of administering to a patient a therapeutically effective amount of a polynucleotide, polypeptide, antagonist and/or agonist of the present invention to the eye, such that the formation of blood vessels is inhibited.
• the compound may be administered topically, via intravitreous injection and/or via intraocular implants.
• disorders which can be treated with the polynucleotides, polypeptides, agonists and/or agonists of the present invention include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osier-Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
• disorders and/or states, which can be treated with be treated with the the polynucleotides, polypeptides, agonists and/or agonists of the present invention include, but are not limited to, solid tumors, blood bom tumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benign tumors, for example hemangiomas, acoustic neuromas, neurofibromas, trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis, ocular angiogenic diseases, for example, diabetic retinopathy, retinopathy of prematurity, macular degeneration, comeal graft rejection, neovascular glaucoma, retrolental fibroplasia, rubeosis, retinoblastoma, and uvietis, delayed wound healing, endometriosis, vascluogenesis, granulations, hypertrophic scars (kel).
• an amount of the compound sufficient to block embryo implantation is administered before or after intercourse and fertilization have occurred, thus providing an effective method of birth control, possibly a "morning after" method.
• Polynucleotides, polypeptides, agonists and/or agonists of the present invention may also be used in controlling menstruation or administered as either a peritoneal lavage fluid or for peritoneal implantation in the treatment of endometriosis.
• Polynucleotides, polypeptides, agonists and/or agonists of the present invention may be inco ⁇ orated into surgical sutures in order to prevent stitch granulomas.
• compositions in the form of, for example, a spray or film
• a compositions may be utilized to coat or spray an area prior to removal of a tumor, in order to isolate normal surrounding tissues from malignant tissue, and/or to prevent the spread of disease to su ⁇ ounding tissues.
• compositions e.g., in the form of a spray
• surgical meshes which have been coated with anti- angiogenic compositions of the present invention may be utilized in any procedure wherein a surgical mesh might be utilized.
• a surgical mesh laden with an anti- angiogenic composition may be utilized during abdominal cancer resection surgery (e.g., subsequent to colon resection) in order to provide support to the structure, and to release an amount of the anti-angiogenic factor.
• methods for treating tumor excision sites, comprising administering a polynucleotide, polypeptide, agonist and/or agonist of the present invention to the resection margins of a tumor subsequent to excision, such that the local recu ⁇ ence of cancer and the formation of new blood vessels at the site is inhibited.
• the anti-angiogenic compound is administered directly to the tumor excision site (e.g., applied by swabbing, brushing or otherwise coating the resection margins of the tumor with the anti-angiogenic compound).
• the anti-angiogenic compounds may be inco ⁇ orated into known surgical pastes prior to administration.
• the anti- angiogenic compounds are applied after hepatic resections for malignancy, and after neurosurgical operations.
• polynucleotides, polypeptides, agonists and/or agonists of the present invention may be administered to the resection margin of a wide variety of tumors, including for example, breast, colon, brain and hepatic tumors.
• anti-angiogenic compounds may be administered to the site of a neurological tumor subsequent to excision, such that the formation of new blood vessels at the site are inhibited.
• polynucleotides, polypeptides, agonists and/or agonists of the present invention may also be administered along with other anti-angiogenic factors.
• anti-angiogenic factors include: Anti-Invasive Factor, retinoic acid and derivatives thereof, paclitaxel, Suramin, Tissue Inhibitor of Metalloproteinase-1, Tissue Inhibitor of Metalloproteinase-2, Plasminogen Activator Inhibitor- 1, Plasminogen Activator Inhibitor-2, and various forms of the lighter "d group" transition metals.
• Lighter "d group” transition metals include, for example, vanadium, molybdenum, tungsten, titanium, niobium, and tantalum species. Such transition metal species may form transition metal complexes. Suitable complexes of the above-mentioned transition metal species include oxo transition metal complexes.
• vanadium complexes include oxo vanadium complexes such as vanadate and vanadyl complexes.
• Suitable vanadate complexes include metavanadate and orthovanadate complexes such as, for example, ammonium metavanadate, sodium metavanadate, and sodium orthovanadate.
• Suitable vanadyl complexes include, for example, vanadyl acetylacetonate and vanadyl sulfate including vanadyl sulfate hydrates such as vanadyl sulfate mono- and trihydrates.
• Representative examples of tungsten and molybdenum complexes also include oxo complexes.
• Suitable oxo tungsten complexes include tungstate and tungsten oxide complexes.
• Suitable tungstate complexes include ammonium tungstate, calcium tungstate, sodium tungstate dihydrate, and tungstic acid.
• Suitable tungsten oxides include tungsten (IN) oxide and tungsten (VI) oxide.
• Suitable oxo molybdenum complexes include molybdate, molybdenum oxide, and molybdenyl complexes.
• Suitable molybdate complexes include ammonium molybdate and its hydrates, sodium molybdate and its hydrates, and potassium molybdate and its hydrates.
• Suitable molybdenum oxides include molybdenum (VI) oxide, molybdenum (VI) oxide, and molybdic acid.
• Suitable molybdenyl complexes include, for example, molybdenyl acetylacetonate.
• Other suitable tungsten and molybdenum complexes include hydroxo derivatives derived from, for example, glycerol, tartaric acid, and sugars.
• anti-angiogenic factors include platelet factor 4; protamine sulphate; sulphated chitin derivatives (prepared from queen crab shells), (Murata et al, Cancer Res.
• SP- PG Sulphated Polysaccharide Peptidoglycan Complex
• the function of this compound may be enhanced by the presence of steroids such as estrogen, and tamoxifen citrate
• Staurosporine modulators of matrix metabolism, including for example, proline analogs, cishydroxyproline, d,L-3,4-dehydroproline, Thiaproline, alpha,alpha-dipyridyl, aminopropionitrile fumarate; 4-propyl-5-(4-pyridinyl)-2(3H)- oxazolone; Methotrexate; Mitoxantrone; Heparin; Interferons; 2 Macroglobulin-serum; ChIMP-3 (Pavloff et al, J.
• the present invention is directed to a method for enhancing apoptosis, cell proliferation, cell differentiation, or other cell growth activity regulated by galectin 11, which involves administering to an individual in need of an increased level of galectin 11 functional or biological activity, a therapeutically effective amount of galectin 11 polypeptide, fragment, variant, derivative, or analog, or an agonist capable of increasing galectin 11 mediated cellular responses.
• galectin 11 mediated signaling is increased to treat a disease wherein decreased apoptosis is exhibited.
• galectin 11 Given the activities modulated by galectin 11, it is readily apparent that a substantially altered (increased or decreased) level of expression of galectin 11 in an individual compared to the standard or "normal” level produces pathological conditions such as those described above. It will also be appreciated by one of ordinary skill that the galectin 11 polypeptides of the invention will exert its modulating activities on any of its target cells. Therefore, it will be appreciated that conditions caused by a decrease in the standard or normal level of galectin 11 activity in an individual, can be treated by administration of galectin 11 protein or an agonist thereof.
• the invention encompasses methods of administering galectin 11 polypeptides or polynucleotides (including fragments, variants, derivatives and analogs, and agonists and antagonists as described herein) to elevate galectin 11 associated biological activity.
• galectin 11 polypeptides or polynucleotides including fragments, variants, derivatives and analogs, and agonists and antagonists as described herein
• any method which elevates galectin 11 concentration and/or activity can be used to stimulate hematopoiesis.
• the galectin 11 polypeptide and nucleotide sequences described herein may be used to stimulate hematopoiesis.
• galectin 11 polypeptides and polynucleotides are used in erythropoietin therapy, which is directed toward supplementing the oxygen carrying capacity of blood.
• Galectin 11 treatment within the scope of the invention includes, but is not limited, to patients generally requiring blood transfusions, such as, for example, trauma victims, surgical patients, dialysis patients, and patients with a variety of blood composition-affecting disorders, such as hemophilia, cystic fibrosis, pregnancy, menstrual disorders, early anemia of prematurity, spinal cord injury, space flight, aging, various neoplastic disease states, and the like.
• Examples of patient conditions that require supplementation of the oxygen carrying capacity of blood include but are not limited to: treatment of blood disorders characterized by low or defective red blood cell production, anemia associated with chronic renal failure, stimulation of reticulocyte response, development of fe ⁇ okinetic effects (such as plasma iron turnover effects and ma ⁇ ow transit time effects), erythrocyte mass changes, stimulation of hemoglobin C synthesis, and increasing levels of hematocrit in vertebrates.
• the invention also provides for treatment to enhance the oxygen-carrying capacity of an individual, such as for example, an individual encountering hypoxic environmental conditions.
• the invention also encompasses combining the galectin 11 polypeptides and polynucleotides described herein with other proposed or conventional hematopoietic therapies.
• galectin 11 can be combined with compounds that singly exhibit erythropoietic stimulatory effects, such as erythropoietin, testosterone, progenitor cell stimulators, insulin-like growth factor, prostaglandins, serotonin, cyclic AMP, prolactin, and triiodothyzonine.
• aplastic anemia such as methenolene, stanozolol, and nandrolone
• iron- deficiency anemia such as iron preparations
• malignant anemia such as vitamin B12 and/or folic acid
• hemolytic anemia such as adrenocortical steroids, e.g., corticoids.
• erythropoietin Compounds that enhance the effects of or synergize with erythropoietin are also useful as adjuvants herein, and include but are not limited to, adrenergic agonists, thyroid hormones, androgens, hepatic erythropoietic factors, erythrotropins, and erythrogenins, See for e.g., Dunn, "Current Concepts in Erythropoiesis", John Wiley and Sons (Chichester, England, 1983); Weiland et al, 1982, Blut, 44:173-175; Kalmani, 1982, Kidney Int., 22:383- 391; Shahidi, 1973, New Eng. J.
• Methods for stimulating hematopoiesis comprise administering a hematopoietically effective amount (i.e, an amount which effects the formation of blood cells) of a pharmaceutical composition containing galectin 11 to a patient.
• a hematopoietically effective amount i.e, an amount which effects the formation of blood cells
• the galectin 11 is administered to the patient by any suitable technique, including but not limited to, parenteral, sublingual, topical, intrapulmonary and intranasal, and those techniques further discussed herein.
• the pharmaceutical composition optionally contains one or more members of the group consisting of erythropoietin, testosterone, progenitor cell stimulators, insulin-like growth factor, prostaglandins, serotonin, cyclic AMP, prolactin, triiodothyzonine, methenolene, stanozolol, and nandrolone, iron preparations, vitamin B12, folic acid and/or adrenocortical steroids.
• the galectin 11 and cotreatment drug(s) are suitably delivered by separate or by the same administration route, and at the same or at different times, depending, e.g., on dosing, the clinical condition of the patient, etc.
• galectin 11 For treating abnormal conditions related to an under-expression of galectin 11 and its activity, or in which elevated or decreased levels of galectin 11 are desired, several approaches are available.
• One approach comprises administering to an individual in need of an increased level of galectin 11 in the body, a therapeutically effective amount of an isolated galectin 11 polypeptide, fragment, variant, derivative or analog of the invention, or a compound which activates galectin 11, i.e., an agonist as described above, optionally in combination with a pharmaceutically acceptable carrier.
• gene therapy may be employed to effect the endogenous production of galectin 11 by the relevant cells in the subject.
• a polynucleotide of the invention may be engineered for expression in a replication defective retroviral vector using techniques known in the art.
• the retroviral expression construct may then be isolated and introduced into a packaging cell transduced with a retroviral plasmid vector containing RNA encoding a polypeptide of the present invention such that the packaging cell now produces infectious viral particles containing the gene of interest.
• These producer cells may be administered to a subject for engineering cells in vivo and expression of the polypeptide in vivo.
• treatment can be administered, for example, in the form of gene replacement therapy.
• one or more copies of a galectin 11 nucleotide sequence of the invention that directs the production of a galectin 11 gene product exhibiting normal function may be inserted into the appropriate cells within a patient or animal subject, using vectors which include, but are not limited to, adenovirus, adeno-associated vims, retrovirus and he ⁇ esvirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes and gene activated matrices.
• vectors which include, but are not limited to, adenovirus, adeno-associated vims, retrovirus and he ⁇ esvirus vectors, in addition to other particles that introduce DNA into cells, such as liposomes and gene activated matrices.
• galectin 11 gene is expressed in neutiophils
• gene replacement techniques should be capable of delivering galectin 11 gene sequence to these cells within patients, or, alternatively, should involve direct administration of such galectin 11 polynucleotide sequences to the site of the cells in which the galectin 11 gene sequences are to be expressed.
• targeted homologous recombination can be utilized to co ⁇ ect the defective endogenous galectin 11 gene and/or regulatory sequences thereof (e.g., promoter and enhancer sequences), or alternatively, to "turn on" other dormant galectin 11 activity in the appropriate tissue or cell type.
• Additional methods which may be utilized to increase the overall level of galectin 11 expression and/or galectin 11 activity include the introduction of appropriate galectin 11- expressing cells, preferably autologous cells, into a patient at positions and in numbers which are sufficient to ameliorate the symptoms of abnormalities in cells growth regulation. Such cells may be either recombinant or non-recombinant.
• Such cells may be either recombinant or non-recombinant.
• normal cells which express the galectin 11 gene.
• Cell-based gene therapy techniques are well known to those skilled in the art, see, e.g., Anderson et al, U.S. Patent No. 5,399,349; and Mulligan & Wilson, U.S. Patent No. 5,460,959.
• a further aspect of the invention is related to a method for treating an individual in need of a decreased level of galectin 11 activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a galectin 11 polypeptide, fragment, variant, derivative or analog of the invention which acts as a galectin 11 antagonist, optionally, in combination with a pharmaceutically acceptable carrier.
• galectin 11 activity is decreased to treat a disease wherein increased apoptosis or other cell growth activity regulated by galectin 11 is exhibited.
• one embodiment of the invention comprises administering to a subject an inhibitor compound (antagonist), such as for example, an antibody or fragment, variant, derivative or analog of the invention, along with a pharmaceutically acceptable carrier in an amount effective to suppress (i.e. lower) galectin 11 activity.
• an inhibitor compound such as for example, an antibody or fragment, variant, derivative or analog of the invention
• galectin 11 activity can be reduced or inhibited by decreasing the level of galectin 11 gene expression.
• antagonists according to the present invention are nucleic acids co ⁇ esponding to the sequences contained in SEQ ID NO:l, or the complementary strand thereof, and/or to nucleotide sequences contained in the deposited clone 209053. In one embodiment, this is accomplished through the use of antisense sequences, either internally generated, by the organism, or separately administered (see, for example, O'Connor, J. Neurochem. (1991) 56:560 in Otigodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
• Antisense technology can be used to control gene expression through antisense DNA or RNA or through triple-helix formation.
• Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
• Triple helix formation is discussed in, for instance, Lee et al, Nucleic Acids Research 10-1573 (1979); Cooney et al, Science 241:456 (1988); and Dervan et al, Science 251:1360 (1991).
• the methods are based on binding of a polynucleotide to a complementary DNA or RNA.
• c-myc and c-myb antisense RNA constructs to inhibit the growth of the non-lymphocytic leukemia cell line HL-60 and other cell lines was previously described. (Wickstrom et al. (1988); Anfossi et al. (1989)). These experiments were performed in vitro by incubating cells with the oligoribonucleotide. A similar procedure for in vivo use is described in WO 91/15580. Briefly, a pair of oligonucleotides for a given antisense RNA is produced as follows: A sequence complimentary to the first 15 bases of the open reading frame is flanked by an EcoRI site on the 5 end and a Hindlll site on the 3 end.
• the pair of oligonucleotides is heated at 90°C for one minute and then annealed in 2X ligation buffer (20mM TRIS HCl pH 7.5, lOmM MgC12, 10MM dithiothreitol (DTT) and 0.2 mM ATP) and then ligated to the EcoRl Hind III site of the retroviral vector PMV7 (WO 91/15580).
• the 5' coding portion of a polynucleotide that encodes galectin 11 polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
• a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription thereby preventing transcription and the production of the galectin 11 polypeptide.
• the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into polypeptide.
• the galectin 11 antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence.
• a vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
• RNA antisense nucleic acid
• Such a vector would contain a sequence encoding the galectin 11 antisense nucleic acid.
• Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
• Such vectors can be constructed by recombinant DNA technology methods standard in the art. Vectors can be plasmid, viral, or others know in the art, used for replication and expression in vertebrate cells.
• Expression of the sequence encoding galectin 11, or fragments thereof can be by any promoter known in the art to act in vertebrate, preferably human cells.
• Such promoters can be inducible or constitutive.
• Such promoters include, but are not limited to, the SV40 early promoter region (Bemoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma vims (Yamamoto et al, Cell 22:787-797 (1980), the he ⁇ es thymidine promoter (Wagner et al, Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981)), the regulatory sequences of the metallothionein gene (Brinster et al, Nature 296:39-42 (1982)), etc.
• the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA transcript of a galectin 11 gene.
• absolute complementarity although preferred, is not required.
• the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid.
• the larger the hybridizing nucleic acid the more base mismatches with a galectin 11 RNA it may contain and still form a stable duplex (or triplex as the case may be).
• One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
• Oligonucleotides that are complementary to the 5' end of the message should work most efficiently at inhibiting translation.
• sequences complementary to the 3' untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner, R., 1994, Nature 372:333-335.
• oligonucleotides complementary to either the 5'- or 3'- non- translated, non-coding regions of galectin 11 shown in Figures 1 could be used in an antisense approach to inhibit translation of endogenous galectin 11 mRNA.
• Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
• Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention.
• antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
• the polynucleotides of the present invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
• the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
• the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al, 1989, Proc. Natl. Acad. Sci. U.S.A.
• the oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
• the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine,
• the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
• the antisense oligonucleotide is an a-anomeric oligonucleotide.
• An a-anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual b-units, the strands run parallel to each other (Gautier et al, 1987, Nucl. Acids Res. 15:6625-6641).
• the oligonucleotide is a 2'-0- methylribonucleotide (Inoue et al, 1987, Nucl. Acids Res. 15:6131-6148), or a chimeric
• RNA-DNA analogue (Inoue et al, 1987, FEBS Lett. 215:327-330).
• Polynucleotides of the invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
• phosphorothioate oligonucleotides may be synthesized by the method of Stein et al. (1988, Nucl. Acids Res. 16:3209), methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85:7448-7451), etc.
• galectin 11 antagonists also include catalytic RNA, or a ribozyme (See, e.g., PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al, Science 247:1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy galectin 1 1 mRNAs, the use of hammerhead ribozymes is prefe ⁇ ed.
• Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
• the construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within the nucleotide sequence of galectin 11 ( Figure 1; SEQ ID NO:l).
• the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the galectin 11 mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
• DNA constmcts encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA.
• the ribozymes of the invention can be composed of modified oligonucleotides (e.g. for improved stability, targeting, etc.) and should be delivered to cells which express galectin 11 in vivo.
• DNA constmcts encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA.
• a prefe ⁇ ed method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous galectin 11 messages and inhibit translation. Since ribozymes, unlike antisense molecules are catalytic, a lower intracellular concentration is required for efficiency.
• Endogenous galectin 11 gene expression can also be reduced by inactivating or "knocking out” the galectin 11 gene or its promoter using targeted homologous recombination (e.g., see Smithies et al, Nature 317:330-234 (1985); Thomas et al, Cell 51:503-512 (1987); Thompson et al, Cell 5:313-321 (1989); each of which is inco ⁇ orated by reference herein in its entirety).
• targeted homologous recombination e.g., see Smithies et al, Nature 317:330-234 (1985); Thomas et al, Cell 51:503-512 (1987); Thompson et al, Cell 5:313-321 (1989); each of which is inco ⁇ orated by reference herein in its entirety.
• Such approach can be adapted for use in humans provided the recombinant DNA constmcts are directly administered or targeted to the required site in vivo using appropriate viral vectors.
• endogenous galectin 11 gene expression can be reduced by targeted deoxyribonucleotide sequences complementary to the regulatory region of the galectin 11 gene (i.e., the galectin 11 promoter and/or enhancers) to form triple helical structures that prevent transcription of the galectin 11 gene in target cells in the body, see generally, Helene et al, Ann, N.Y. Acad. Sci. 660:27-36 (1992); Helene, C, Anticancer Dmg Des., 6(6):569-584 (1991); and Maher, L.J., Bioassays 14(12):807-815 (1992)).
• targeted deoxyribonucleotide sequences complementary to the regulatory region of the galectin 11 gene i.e., the galectin 11 promoter and/or enhancers
• the activity of galectin 11 can be reduced using a "dominant negative".
• constmcts which encode defective galectin 11 such as, for example, mutants lacking all or a portion of region of galectin 11 that binds ⁇ - galactosides, can be used in gene therapy approaches to diminish the activity of galectin 11 on appropriate target cells.
• nucleotide sequences that direct host cell expression of galectin 11 in which all or a portion of the region of galectin 1 1 that binds ⁇ -galactoside is altered or missing can be introduced into neutrophil cells, or other cells or tissue which express galectin 11 (either by in vivo or ex vivo gene therapy methods as for example, described herein).
• targeted homologous recombination can be utilized to introduce such deletions or mutations into the subjects endogenous galectin 11 gene in neutiophils or other cells expressing galectin 11.
• Antagonist/agonist compounds may be employed to inhibit the cell growth and proliferation effects of the polypeptides of the present invention on neoplastic cells and tissues, i.e. stimulation of angiogenesis of tumors, and, therefore, retard or prevent abnormal cellular growth and proliferation, for example, in tumor formation or growth.
• the antagonist/agonist may also be employed to prevent hyper-vascular diseases, and prevent the proliferation of epithelial lens cells after extracapsular cataract surgery. Prevention of the mitogenic activity of the polypeptides of the present invention may also be desirous in cases such as restenosis after balloon angioplasty.
• the antagonist/agonist may also be employed to prevent the growth of scar tissue during wound healing.
• the antagonist/agonist may also be employed to treat the diseases described herein.
• the invention provides a method of treating disorders or diseases, including but not limited to the disorders or diseases listed throughout this application, associated with overexpression of a polynucleotide of the present invention by administering to a patient (a) an antisense molecule directed to the polynucleotide of the present invention, and/or (b) a ribozyme directed to the polynucleotide of the present invention.
• the invention further provides a method of treating an individual in need of an increased level of galectin 11 activity comprising administering to such an individual a pharmaceutical composition comprising an effective amount of an isolated galectin 11 polypeptide or fragment, variant, derivative, or analog of the invention, such as for example, the full length form of the galectin 11, effective to increase the galectin 11 activity level in such an individual.
• the invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention.
• the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side-effects).
• the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
• the dosage range required depends on the choice of peptide, the route of administration, the nature of the formulation, the nature of the subject's condition, and the judgment of the attending practitioner.
• the total pharmaceutically effective amount of galectin 11 polypeptide administered parenterally per dose will be in the range of about 1 ⁇ g/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans this dose is in the range of 0.1-100 mg/kg of subject, or between about 0.01 and 1 mg/kg/day.
• the galectin 11 polypeptide is typically administered at a dose rate of about 1 ⁇ g/kg/hour to about 50 ⁇ g kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump.
• An intravenous bag solution may also be employed. Wide variations in the needed dosage, however, are to be expected in view of the variety of compounds available and the differing efficiencies of various routes of administration. For example, oral administration would be expected to require higher dosages than administration by intravenous injection. Variations in these dosage levels can be adjusted using standard empirical routines for optimization, as is well understood in the art.
• compositions containing the galectin 11 polypeptides and polynucleotides of the invention may be routinely formulated in combination with a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier is meant a nontoxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
• pharmaceutically acceptable means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly humans.
• Nonlimiting examples of suitable pharmaceutical carriers according to this embodiment are provided in "Remington's Pharmaceutical Sciences” by E.W. Martin, and include sterile liquids, such as water, saline, buffered saline, glycerol, ethanol, and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Formulation should suit the mode of administration, and is well within the skill of the art. For example, water is a prefe ⁇ ed carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can be employed as liquid carriers, particularly for injectable solutions.
• the invention additionally relates to pharmaceutical packs and kits comprising one or more containers filled with one or more of the ingredients of the aforementioned compositions of the invention.
• Polypeptides and other compounds of the present invention may be administered alone or in conjunction with other compounds, such as therapeutic compounds.
• the pharmaceutical composition of the invention may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
• Prefe ⁇ ed forms of systemic administration of the pharmaceutical compositions include parenteral injection, typically by intravenous injection. Other injection routes, such as subcutaneous, intramuscular, intrasternal, intraarticular or intraperitoneal, can be used.
• transmucosal and transdermal administration using penetrants such as bile salts or fusidic acids or other detergents.
• penetrants such as bile salts or fusidic acids or other detergents.
• oral administration may also be possible.
• Administration of these compounds may also be topical and/or localized, in the form of salves, pastes, gels and the like.
• Polypeptides used in treatment can also be generated endogenously in the subject, in treatment modalities often refe ⁇ ed to as "gene therapy” as described above.
• cells from a subject may be engineered with a polynucleotide, such as a DNA or RNA, to encode a polypeptide ex vivo, and for example, by the use of a retroviral plasmid vector. The cells are then introduced into the subject.
• Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
• a compound of the invention e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid as part of a retroviral or other vector, etc.
• Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
• the compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by abso ⁇ tion through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
• Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
• a protein, including an antibody, of the invention care must be taken to use materials to which the protein does not absorb.
• the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, Science 249:1527-1533 (1990); Treat et al, in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353- 365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)
• the compound or composition can be delivered in a controlled release system.
• a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng.
• polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Dmg Bioavailability, Dmg Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al, Science 228:190 (1985); During et al, Ann. Neurol.
• a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115- 138 (1984)).
• the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No.
• a nucleic acid can be introduced intracellularly and inco ⁇ orated within host cell DNA for expression, by homologous recombination.
• compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable ca ⁇ ier.
• pharmaceutically acceptable means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• ca ⁇ ier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like.
• Water is a prefe ⁇ ed ca ⁇ ier when the pharmaceutical composition is administered intravenously.
• Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.
• Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like.
• the composition if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
• compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained-release formulations and the like.
• the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
• Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
• Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
• the formulation should suit the mode of administration.
• the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
• compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection.
• the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent.
• composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
• an ampoule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
• the compounds of the invention can be formulated as neutral or salt forms.
• Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.
• the amount of the compound of the invention which will be effective in the treatment, inhibition and prevention of a disease or disorder associated with abe ⁇ ant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques.
• in vitro assays may optionally be employed to help identify optimal dosage ranges.
• the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.
• the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
• the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight.
• human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
• the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
• the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
• Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
• Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic pu ⁇ oses to detect, diagnose, or monitor diseases and/or disorders associated with the abe ⁇ ant expression and/or activity of a polypeptide of the invention.
• the invention provides for the detection of abe ⁇ ant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of abe ⁇ ant expression.
• the invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
• a diagnostic assay for diagnosing a disorder comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
• the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior
• galectin 11 polypeptide levels in a biological sample can occur using antibody-based techniques. For example, galectin 11 polypeptide expression in tissues can be studied with classical immunohistological methods (Jalkanen, M., et al, J. Cell. Biol. 101:976-985 (1985); Jalkanen, M., et al, J. Cell . Biol 705:3087-3096 (1987)).
• antibody-based methods useful for detecting galectin 11 polypeptide gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
• Suitable antibody assay labels are known in the art and include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( 131 1, 125 I, I23 1, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( H5m In, 113m In, H2 In, 1 H In), and technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, 177 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47
• diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with abe ⁇ ant expression of the polypeptide of interest.
• Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for
• 131 L 112j nj 99m TC) ( 131 Tj 12 ⁇ 123 ⁇ 121 ⁇ carbon (14 ⁇ sulfi] ⁇ (35 ⁇ ⁇ . ⁇ ( 3 H)? ⁇ (HSmj ⁇ H3 ⁇ Tn, 112 In, H 1 In), and technetium ( 99 Tc, 99m Tc), thallium ( 201 Ti), gallium ( 68 Ga, 67 Ga), palladium ( 103 Pd), molybdenum ( 99 Mo), xenon ( 133 Xe), fluorine ( 18 F), 153 Sm, ,77 Lu, 159 Gd, 149 Pm, 140 La, 175 Yb, 166 Ho, 90 Y, 47 Sc, 186 Re, 188 Re, 142 Pr, 105 Rh, 97 Ru), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for cell proliferation disorder.
• the size of the subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
• the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99m Tc.
• the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain galectin 11 protein.
• In vivo tumor imaging is described in S.W. Burchiel et al, "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A.
• galectin 11 ligand any galectin 11 polypeptide whose presence can be detected, can be administered.
• galectin 11 polypeptides labeled with a radio-opaque or other appropriate compound can be administered and visualized in vivo, as discussed, above for labeled antibodies. Further such galectin 11 polypeptides can be utilized for in vitro diagnostic procedures.
• the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
• monitoring of the disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
• Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label.
• Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
• CT computed tomography
• PET position emission tomography
• MRI magnetic resonance imaging
• sonography sonography
• the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instmment (Thurston et al, U.S. Patent No. 5,441,050).
• the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instmment.
• the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography.
• the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
• MRI magnetic resonance imaging
• kits comprises an antibody of the invention, preferably a purified antibody, in one or more containers.
• the kits of the present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit.
• the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest.
• kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
• a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate.
• the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides.
• a kit may include a control antibody that does not react with the polypeptide of interest.
• a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody.
• a kit includes means for detecting the binding of said antibody to the antigen (e.g., the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry).
• the kit may include a recombinantly produced or chemically synthesized polypeptide antigen.
• the polypeptide antigen of the kit may also be attached to a solid support.
• the detecting means of the above-described kit includes a solid support to which said polypeptide antigen is attached.
• a kit may also include a non-attached reporter-labeled anti-human antibody.
• binding of the antibody to the polypeptide antigen can be detected by binding of the said reporter-labeled antibody.
• the invention includes a diagnostic kit for use in screening semm containing antigens of the polypeptide of the invention.
• the diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody.
• the antibody is attached to a solid support.
• the antibody may be a monoclonal antibody.
• the detecting means of the kit may include a second, labeled monoclonal antibody. Alternatively, or in addition, the detecting means may include a labeled, competing antigen.
• test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods of the present invention.
• the reagent After binding with specific antigen antibody to the reagent and removing unbound semm components by washing, the reagent is reacted with reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support.
• the reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined.
• the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
• the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96- well plate or filter material. These attachment methods generally include non-specific adso ⁇ tion of the protein to the support or covalent attachment of the protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
• the invention provides an assay system or kit for carrying out this diagnostic method.
• the kit generally includes a support with surface- bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
• the nucleic acid molecules of the present invention are also valuable for chromosome identification.
• the sequence is specifically targeted to and can hybridize with a particular location on human chromosome 11.
• the mapping of DNAs to chromosomes according to the present invention is an important first step in co ⁇ elating those sequences with genes associated with disease.
• the galectin 11 gene has been mapped to a precise chromosomal location, the physical position of the sequence on the chromosome can be co ⁇ elated with genetic map data.
• genetic map data are found, for example, in V. McKusick, Mendelian Inheritance In Man, available on-line through Johns Hopkins University, Welch Medical Library. The relationship between genes and diseases that have been mapped to the same chromosomal region are then identified through linkage analysis (coinheritance of physically adjacent genes).
• Example 1 Expression and Purification of Galectin 11 in E. coli
• the DNA sequence encoding the galectin 11 protein in the deposited cDNA clone is amplified using PCR oligonucleotide primers specific to the amino terminal sequences of the galectin 11 protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and 3' sequences respectively.
• the 5' galectin 11 oligonucleotide primer has the sequence 5' cgc CCATGG ATGAGCCCCAGGCTGGAGGTG 3' (SEQ ID NO:23) containing the underlined Ncol restriction site and nucleotides 49 to 69 of the galectin 11 nucleotide sequence depicted in Figure 1 (SEQ ID NO: 1).
• the 3' galectin 11 primer has the sequence 5' cgc AAGCTT TCAGGAGTGGACACAGTAG 3' (SEQ ID NO:6) containing the underlined Hindlll restriction site followed by nucleotides complementary to position 431 to 451 of the galectin 11 nucleotide sequence depicted in Figure 1 (SEQ ID NO:l).
• the restriction sites are convenient to restriction enzyme sites in the bacterial expression vector pQE60 which are used for bacterial expression in these examples. (Qiagen,
• pQE60 encodes ampicillin antibiotic resistance (“ Ampr”) and contains a bacterial origin of replication (“ori”), an IPTG inducible promoter, a ribosome binding site (“RBS”), a 6-His tag and restriction enzyme sites.
• the amplified galectin 11 DNA and the pQE60 vector is digested with Ncol and
• E. coli strain M15/rep4 containing multiple copies of the plasmid pREP4, which expresses lac repressor and confers kanamycin resistance ("Kanr"), is used in carrying out the example described herein.
• Kanr kanamycin resistance
• Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis.
• Clones containing the desired constmcts are grown overnight ( O/N ) in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
• the O/N culture is used to inoculate a large culture, at a dilution of approximately 1:100 to 1:250.
• the cells are grown to an optical density at 600nm ("OD600") of between 0.4 and 0.6.
• Isopropyl-B-D-thiogalactopyranoside (IPTG ) is then added to a final concentration of 1 mM to induce transcription from lac repressor sensitive promoters, by inactivating the lacl repressor.
• Cells subsequently are incubated further for 3 to 4 hours. Cells then are harvested by centrifugation and dismpted, by standard methods.
• Inclusion bodies are purified from the dismpted cells using routine collection techniques, and protein is solubilized from the inclusion bodies into 8M urea.
• the 8M urea solution containing the solubilized polypeptide is passed over a PD-10 column in 2X phosphate-buffered saline ("PBS"), thereby removing the urea, exchanging the buffer and refolding the protein.
• PBS 2X phosphate-buffered saline
• the polypeptide is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered.
• the sterile filtered protein preparation was stored in 2X PBS at a concentration of 95 ⁇ /ml.
• Example 2 Cloning and Expression of Galectin 11 protein in a Baculovirus Expression System
• the cDNA sequence encoding the full length galectin 11 protein in the deposited clone is amplified using PCR oligonucleotide primers co ⁇ esponding to the 5' and 3' sequences of the gene:
• the 5' galectin 11 oligonucleotide primer has the sequence 5' cgc CCC GGG GCCT ATGAGCCCCAGGCTGGAGG 3' (SEQ ID NO:7) containing the underlined Smal restriction site and nucleotides 49 to 66 of the galectin 11 nucleotide sequence depicted in Figure 1 (SEQ ID NO: 1).
• the 3' Galectin 11 primer has the sequence 5' cgc GGT ACC TCAGGAGTGGACACAGTAG 3' (SEQ ID NO:8) containing the underlined Asp718 restriction site followed by nucleotides complementary to position 432 to 450 of the galectin 11 nucleotide sequence depicted in Figure 1 (SEQ ID NO:l).
• the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean,” BIO 101 Inc., La Jolla, Ca.). The fragment then is digested with Xbal and again is purified on a 1% agarose gel. This fragment is designated herein F2.
• the vector pA2-GP is used to express the galectin 11 protein in the baculovims expression system, using standard methods, as described in Summers et al, A MANUAL OF METHODS FOR BACULOVIRUS VECTORS AND INSECT CELL CULTURE PROCEDURES, Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
• This expression vector contains the strong polyhedrin promoter of the Autographa californica nuclear polyhedrosis vims (AcMNPV) followed by convenient restriction sites.
• the signal peptide of AcMNPV gp67, including the N-terminal methionine, is located just upstream of a BamHI site.
• the polyadenylation site of the simian vims 40 (“SV40") is used for efficient polyadenylation.
• SV40 simian vims 40
• the beta-galactosidase gene from E. coli is inserted in the same orientation as the polyhedrin promoter and is followed by the polyadenylation signal of the polyhedrin gene.
• the polyhedrin sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable vims that express the cloned polynucleotide.
• baculovims vectors could be used in place of pA2-GP, such as pAc373, pVL941 and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required.
• Such vectors are described in Luckow et al, Virology 170: 31-39, among others.
• the plasmid is digested with the restriction enzyme Smal and Asp718 and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
• the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated herein V2 .
• Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase.
• E. coli HB101 cells are transformed with ligation mix and spread on culture plates.
• Bacteria are identified that contain the plasmid with the human galectin 11 gene by digesting DNA from individual colonies using Xbal and then analyzing the digestion product by gel electrophoresis. The sequence of the cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBacgalectin 11.
• 5 ⁇ g of the plasmid pBacgalectin 11 is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovims DNA ("BaculoGold baculovims DNA", Pharmingen, San Diego, CA.), using the lipofection method described by Feigner et al, Proc. Natl. Acad. Sci. USA 84: 7413-7417 (1987).
• l ⁇ g of BaculoGold vims DNA and 5 ⁇ g of the plasmid pBacgalectin 1 1 are mixed in a sterile well of a microtiter plate containing 50 ⁇ l of serum-free Grace's medium (Life Technologies Inc., Gaithersburg, MD).
• plaque assay After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, cited above. An agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal- expressing clones, which produce blue-stained plaques. (A detailed description of a "plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10).
• the vims is added to the cells. After appropriate incubation, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant vimses is then resuspended in an Eppendorf tube containing 200 ⁇ l of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovims is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supematants of these culture dishes are harvested and then they are stored at 4°C.
• V-galectin 11 A clone containing properly inserted hESSB I, II and III is identified by DNA analysis including restriction mapping and sequencing. This is designated herein as V-galectin 11. Sf9 cells are grown in Grace's medium supplemented with 10% heat-inactivated FBS.
• the cells are infected with the recombinant baculovims V-galectin 11 at a multiplicity of infection ("MOI") of about 2 (about 1 to about 3).
• MOI multiplicity of infection
• the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Gaithersburg).
• 5 ⁇ Ci of 35S-methionine and 5 ⁇ Ci 35S-cysteine available from Amersham
• the cells are further incubated for 16 hours and then they are harvested by centrifugation, lysed and the labeled proteins are visualized by SDS-PAGE and autoradiography.
• vectors used for the transient expression of the galectin 11 polypeptide gene sequence in mammalian cells should carry the SV40 origin of replication. This allows the replication of the vector to high copy numbers in cells (e.g. COS cells) which express the T antigen required for the initiation of viral DNA synthesis. Any other mammalian cell line can also be utilized for this pu ⁇ ose.
• a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the long terminal repeats (LTRs) from Retro vimses, e.g. RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular signals can also be used (e.g. human actin promoter).
• Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and ⁇ BC12MI (ATCC 67109).
• Mammalian host cells that could be used include, human Hela, 283, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, African green monkey cells, quail QC1-3 cells, mouse L cells and Chinese hamster ovary cells.
• the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome. The co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation of the transfected cells.
• the transfected gene can also be amplified to express large amounts of the encoded protein.
• the DHFR dihydrofolate reductase
• GS glutamine synthase
• Another useful selection marker is the enzyme glutamine synthase (GS) (Mu ⁇ hy et al, Biochem J. 227:277- 279 (1991); Bebbington et al, Bio/Technology 10:169-175 (1992)).
• GS glutamine synthase
• the mammalian cells are grown in selective medium and the cells with the highest resistance are selected.
• These cell lines contain the amplified gene(s) integrated into a chromosome.
• Chinese hamster ovary (CHO) cells are often used for the production of proteins.
• the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Vims (Cullen et al, Molecular and Cellular Biology, 438-4470 (March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al, Cell 41:521-530 (1985)). Multiple cloning sites, e.g. with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest.
• the vectors contain in addition the 3 intron, the polyadenylation and termination signal of the rat preproinsulin gene.
• the expression plasmid, pgalectin 11, is made by cloning a cDNA encoding galectin 11 into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
• the expression vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cells; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells; (3) an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal a ⁇ anged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
• a DNA fragment encoding the galectin 11 protein and an HA tag fused in frame to its 3' end is cloned into the polylinker region of the vector so that recombinant protein expression is directed by the CMV promoter.
• the HA tag co ⁇ esponds to an epitope derived from the influenza hemagglutinin protein described by Wilson et al, Cell 37: 767 (1984).
• the fusion of the HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
• the plasmid construction strategy is as follows.
• the galectin 11 cDNA of the deposited clone is amplified using primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of galectin 11 in E. coli.
• primers that contain convenient restriction sites, much as described above regarding the construction of expression vectors for expression of galectin 11 in E. coli.
• one of the primers contains a hemagglutinin tag ("HA tag") as described above.
• Suitable primers include the following, which are used in this example.
• the 5' galectin 11 primer has the sequence 5' cgc CCC GGG gcc ate ATG GCCTATC ATGAGCCCCAGGCTGGAGG 3' (S ⁇ Q ID NO:9) containing the underlined Smal restriction enzyme site followed by nucleotide sequence 49 to 66 of Figure 1 (S ⁇ Q ID NO:l).
• the 3' galectin 11 primer has the sequence 5' cgc GGT ACC
• TCAGGAGTGGACACAGTAG 3' (S ⁇ Q ID NO: 8) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 432 to 450 of the galectin 11 nucleotide sequence depicted in Figure 1 (S ⁇ Q ID NO:l).
• the PCR amplified DNA fragment and the vector, pcDNAI/Amp are digested with Hindlll and Xhol and then ligated.
• the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 11099 North To ⁇ ey Pines Road, La Jolla, CA 92037), and the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the galectin 11 -encoding fragment.
• COS cells are transfected with an expression vector, as described above, using DEAE-DEXTRAN, as described, for instance, in
• galectin 11 HA fusion protein is detected by radiolabelling and immunoprecipitation, using methods described in, for example Harlow et al, ANTIBODIES: A LABORATORY MANUAL, 2nd Ed.; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing 35S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and the lysed with detergent-containing RIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 1% NP-40, 0.5% DOC, 50 mM TRIS, pH 7.5, as described by Wilson et al.
• Proteins are precipitated from the cell lysate and from the culture media using an HA-specific monoclonal antibody. The precipitated proteins then are analyzed by SDS-PAGE gels and autoradiography. An expression product of the expected size is seen in the cell lysate, which is not seen in negative controls.
• the vector pCl is used for the expression of galectin 11 protein.
• Plasmid pCl is a derivative of the plasmid pSV2-dhfr [ATCC Accession No. 37146]. Both plasmids contain the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate.
• a selective medium alpha minus MEM, Life Technologies
• MTX methotrexate
• DHFR target enzyme
• a second gene is linked to the DHFR gene it is usually co-amplified and over-expressed. It is state of the art to develop cell lines carrying more than 1,000 copies of the genes. Subsequently, when the methotrexate is withdrawn, cell lines contain the amplified gene integrated into the chromosome(s).
• Plasmid pCl contains for the expression of the gene of interest a strong promoter of the long terminal repeat (LTR) of the Rouse Sarcoma Vims (Cullen, et al, Molecular and Cellular Biology, March 1985:438-4470) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart et al, Cell 41:521-530, 1985).
• LTR long terminal repeat
• CMV cytomegalovirus
• Downstream of the promoter are the following single restriction enzyme cleavage sites that allow the integration of the genes: BamHI, Pvull, and Nrul Behind these cloning sites the plasmid contains translational stop codons in all three reading frames followed by the 3 intron and the polyadenylation site of the rat preproinsulin gene.
• Other high efficient promoters can also be used for the expression, e.g., the human -actin promoter, the SV40 early or late promoters or the long terminal repeats from other retrovimses, e.g., H1N and HTLVI.
• H1N and HTLVI For the polyadenylation of the mR A other signals, e.g., from the human growth hormone or globin genes can be used as well.
• Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
• the plasmid pCl is digested with the restriction enzyme BamHI and then dephosphorylated using calf intestinal phosphatase by procedures known in the art.
• the vector is then isolated from a 1% agarose gel.
• the D ⁇ A sequence encoding galectin 11, ATCC Deposit No. 209053 is amplified using PCR oligonucleotide primers co ⁇ esponding to the 5' and 3' sequences of the gene:
• the 5' Galectin 11 primer has the sequence 5' cgc CCC GGG gcc ate ATG GCCTATC ATGAGCCCCAGGCTGGAGG 3' (SEQ ID NO:9) containing the underlined Smal restriction enzyme site followed by nucleotide sequence 49-66 of Figure 1 (SEQ ID NO:l).
• Inserted into an expression vector, as described below, the 5' end of the amplified fragment encoding human galectin 11 provides an efficient signal peptide.
• An efficient signal for initiation of translation in eukaryotic cells, as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987) is appropriately located in the vector portion of the construct.
• the 3' Galectin 11 primer has the sequence 5' cgc GGT ACC TCAGGAGTGGACACAGTAG 3' (SEQ ID NO:8) containing the Asp718 restriction followed by nucleotides complementary to nucleotides 432-450 of the galectin 11 nucleotide sequence depicted in Figure 1 (SEQ ID NO:l).
• the amplified fragments are isolated from a 1% agarose gel as described above and then digested with the endonucleases Smal and Asp718 and then purified again on a 1% agarose gel.
• the isolated fragment and the dephosphorylated vector are then ligated with T4 DNA ligase.
• E. coli HB101 cells are then transformed and bacteria identified that contained the plasmid pCl inserted in the co ⁇ ect orientation using the restriction enzyme Smal. The sequence of the inserted gene is confirmed by DNA sequencing.
• 5 ⁇ g of the expression plasmid Cl are cotransfected with 0.5 ⁇ g of the plasmid pSVneo using the lipofecting method (Feigner et al, supra).
• the plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
• the cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 days.
• single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM). Clones growing at the highest concentrations of methotrexate are then transfe ⁇ ed to new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 ⁇ M, 2 ⁇ M, 5 ⁇ M). The same procedure is repeated until clones grow at a concentration of 100 ⁇ M.
• Northern blot analysis is carried out to examine galectin 11 gene expression in human tissues, using methods described by, among others, Sambrook et al, cited above.
• a cDNA probe containing the entire nucleotide sequence encoding galectin 11 protein (SEQ ID NO: l) is labeled with 32P using the r ⁇ b ' prime DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN- 100 column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT 1200-1. The purified labeled probe is then used to examine various human tissues for galectin 11 mRNA.
• MTN Multiple Tissue Northern
• H human tissues
• IM human immune system tissues
• Example 5 Galectin 11 induced apoptosis in transfected cells This example presents data demonstrating that transfection of a constitutive galectin
• a T cell is a type of lymphocyte, or "white blood cell", that mediates the cellular immune response to foreign macromolecule, termed antigens. While T cells are necessary for normal mammalian immune responses, in some instances it is desirable to inhibit their activation: for example, in some autoimmune diseases, the T cells of a subject respond to "self-antigens", i.e., macromolecule produced by the subject, rather than foreign-made macromolecule, and damage the cells and tissues of the subject. Autoimmune T cell responses are found in subjects having systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), insulin-dependent diabetes, myasthenia gravis, and multiple sclerosis (MS) and contribute to the pathophysiology of each.
• SLE systemic lupus erythematosus
• RA rheumatoid arthritis
• MS multiple sclerosis
• T cells also cause graft rejection and graft versus host disease (GVHD).
• Graft rejection is caused by an immune response against the transplanted tissues (the graft), which are recognized as “foreign” by T cells of the recipient (host).
• Graft versus host disease is caused by engrafted T cells, which recognize host-made macromolecule as "foreign.”
• the DNA sequence encoding the galectin 11 protein in the deposited cDNA clone was amplified using PCR oligonucleotide primers specific to the amino terminal sequences of the galectin 11 protein and to vector sequences 3' to the gene.
• the 5' galectin 11 oligonucleotide primer had the sequence 5' CGCCGCCACCATGAGCCCCAGGC 3' (SEQ ID NO: 10) containing nucleotides 49 to 61 of the galectin 11 nucleotide sequence in Figure 1 (SEQ ID NO:l).
• the 3' galectin 11 primer has the sequence 5' GGAATCTAGATCAGGAGTGGAC 3' (SEQ ID NO: 11) containing the underlined Xbal restriction site followed by nucleotide sequence complementary to position 439 to 450 of the galectin 11 nucleotide sequence in Figure 1 (SEQ ID NO: 1).
• the amplified galectin 11 fragments were isolated from a 1% agarose gel as described above, digested with the endonuclease Xbal, purified again on a 1% agarose gel, and ligated into the multiple cloning site of restricted pEFl using T4 DNA ligase.
• the pEFl vector was generated by replacing the CMV promoter on pIRESlneo
• This vector also contains a bovine growth hormone poly A signal and a ampicilin resistance gene, a ribosome binding site ("RBS”), a 6-His tag and restriction enzyme sites. This vector was digested with EcoRI, BamHI, and phosphatase using techniques known in the art.
• Insertion of the isolated galectin 11 fragment into the restricted pEFl vector placed the galectin 11 polypeptide coding region downstream of and operably associated with the vector's constitutive elongation factor- 1 promoter and in-frame with an initiating AUG appropriately positioned for translation of galectin 11.
• E. coli cells were then transformed with the ligation reaction and those cells containing the desired constmct (pEFLegl l) were identified using techniques known in the art. Cells containing the pEFLegl l expression constmct were then cultured under known conditions favoring high yield and the expression constmct was isolated from the bacterial cell culture using techniques known in the art.
• apoptosis For detection of apoptosis, techniques known in the art were used to cotransfect human Jurkat T-cells with the pEFLegl 1 expression constmct together with a marker plasmid encoding green fluorescent protein (GFP). The transfected cells were then stained with MitoTracker Red (Molecular Probes) to determine the transmembrane potentials of mitochondria, and analyzed by two-color flow cytometry. Transfected populations were identified by emission of green fluorescence due to the expression of GFP. Apoptotic cells exhibit dismpted mitochondrial transmembrane potential and thus have lower red fluorescence emission because of their reduced ability to sequester the dye MitoTracker Red.
• MitoTracker Red Molecular Probes
• Galectin 11 expression vector pEFl-Legl 1. There were about 4 times more surviving GFP positive cells after transfection with pEFl than with pEFl-Legl 1 ( Figure 5B).
• oligonucleotide primers of about 15-25 nucleotides are derived from the desired 5' and 3' positions of a polynucleotide of SEQ ID NO:l. The 5' and 3' positions of the primers are determined based on the desired galectin 11 polynucleotide fragment. An initiation and stop codon are added to the 5' and 3' primers respectively, if necessary, to express the galectin 11 polypeptide fragment encoded by the polynucleotide fragment. Prefe ⁇ ed galectin 11 polynucleotide fragments are those encoding the N-terminal and C-terminal deletion mutants disclosed above in the "Galectin 11 Polypeptide and Fragments" section of the Specification.
• Additional nucleotides containing restriction sites to facilitate cloning of the galectin 11 polynucleotide fragment in a desired vector may also be added to the 5' and 3' primer sequences.
• the galectin 11 polynucleotide fragment is amplified from genomic DNA or from the deposited cDNA clone using the appropriate PCR oligonucleotide primers and conditions discussed herein or known in the art.
• the galectin 11 polypeptide fragments encoded by the galectin 11 polynucleotide fragments of the present invention may be expressed and purified in the same general manner as the full length polypeptides, although routine modifications may be necessary due to the differences in chemical and physical properties between a particular fragment and full length polypeptide.
• the polynucleotide encoding the galectin 11 polypeptide fragment L-5 to L-128 is amplified and cloned as follows: A 5' primer is generated comprising a restriction enzyme site followed by an initiation codon in frame with the polynucleotide sequence encoding the N-terminal portion of the polypeptide fragment beginning with L-5. A complementary 3' primer is generated comprising a restriction enzyme site followed by a stop codon in frame with the polynucleotide sequence encoding C-terminal portion of the galectin 11 polypeptide fragment ending with L-128.
• the amplified polynucleotide fragment and the expression vector are digested with restriction enzymes which recognize the sites in the primers.
• the digested polynucleotides are then ligated together.
• the galectin 11 polynucleotide fragment is inserted into the restricted expression vector, preferably in a manner which places the galectin 11 polypeptide fragment coding region downstream from the promoter.
• the ligation mixture is transformed into competent E. coli cells using standard procedures and as described in the Examples herein. Plasmid DNA is isolated from resistant colonies and the identity of the cloned DNA confirmed by restriction analysis, PCR and DNA sequencing.
• Galectin 11 polypeptides are preferably fused to other proteins. These fusion proteins can be used for a variety of applications. For example, fusion of galectin 11 polypeptides to His-tag, HA-tag, protein A, IgG domains, and maltose binding protein facilitates purification. (See Example 5; see also EP A 394,827; Traunecker, et al, Nature 331:84-86 (1988).) Similarly, fusion to IgG-1, IgG-3, and albumin increases the haiflife time in vivo.
• Nuclear localization signals fused to galectin 11 polypeptides can target the protein to a specific subcellular localization, while covalent heterodimer or homodimers can increase or decrease the activity of a fusion protein. Fusion proteins can also create chimeric molecules having more than one function. Finally, fusion proteins can increase solubility and/or stability of the fused protein compared to the non-fused protein. All of the types of fusion proteins described above can be made by modifying the following protocol, which outlines the fusion of a polypeptide to an IgG molecule, or the protocol described in Example 3.
• the human Fc portion of the IgG molecule can be PCR amplified, using primers that span the 5' and 3' ends of the sequence described below. These primers also should have convenient restriction enzyme sites that will facilitate cloning into an expression vector, preferably a mammalian expression vector.
• the human Fc portion can be ligated into the BamHI cloning site. Note that the 3' BamHI site should be destroyed.
• the vector containing the human Fc portion is re-restricted with BamHI, linearizing the vector, and galectin 11 polynucleotide, isolated by the PCR protocol described in Example 1, is ligated into this BamHI site. Note that the polynucleotide is cloned without a stop codon, otherwise a fusion protein will not be produced.
• pC4 does not need a second signal peptide.
• the vector can be modified to include a heterologous signal sequence. (See, e.g., WO 96/34891.)
• the antibodies of the present invention can be prepared by a variety of methods. (See, Cu ⁇ ent Protocols, Chapter 2.) As one example of such methods, cells expressing galectin 11 are administered to an animal to induce the production of sera containing polyclonal antibodies. In a prefe ⁇ ed method, a preparation of galectin 11 protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity. Monoclonal antibodies specific for galectin 11 protein are prepared using hybridoma technology. (Kohler et al, Nature 256:495 (1975); Kohler et al, Eur. J. Immunol.
• an animal preferably a mouse
• galectin 11 polypeptide or, more preferably, with a secreted galectin 11 polypeptide-expressing cell.
• Such polypeptide-expressing cells are cultured in any suitable tissue culture medium, preferably in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56°C), and supplemented with about 10 g/1 of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ⁇ g/ml of streptomycin.
• tissue culture medium preferably in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56°C), and supplemented with about 10 g/1 of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ⁇ g/ml of streptomycin.
• the splenocytes of such mice are extracted and fused with a suitable myeloma cell line.
• a suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the ATCC.
• SP2O parent myeloma cell line
• the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 80:225-232 (1981)).
• the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the galectin 11 polypeptide.
• additional antibodies capable of binding to galectin 11 polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies.
• Such a method makes use of the fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody.
• protein specific antibodies are used to immunize an animal, preferably a mouse.
• the splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the galectin 11 protein- specific antibody can be blocked by galectin 11.
• Such antibodies comprise anti-idiotypic antibodies to the galectin 11 protein-specific antibody and are used to immunize an animal to induce formation of further galectin 11 protein-specific antibodies.
• an antibody is "humanized".
• Such antibodies can be produced using genetic constmcts derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed herein. (See, for review, Mo ⁇ ison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Cabilly et al, U.S. Patent No.
• Naturally occurring V-genes isolated from human PBLs are constmcted into a library of antibody fragments which contain reactivities against Galectin 11 to which the donor may or may not have been exposed (see e.g., U.S. Patent 5,885,793 inco ⁇ orated herein by reference in its entirety).
• a library of scFvs is constmcted from the RNA of human PBLs as described in PCT publication WO 92/01047.
• To rescue phage displaying antibody fragments approximately 109 E. coli harboring the phagemid are used to inoculate 50 ml of 2xTY containing 1% glucose and 100 ⁇ g/ml of ampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking.
• M13 delta gene III is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious Ml 3 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage mo ⁇ hogenesis. The culture is incubated for 1 hour at 37° C without shaking and then for a further hour at 37°C with shaking. Cells are spun down (IEC-Cenfra 8,400 r.p.m.
• Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 ⁇ g/ml or 10 ⁇ g/ml of a polypeptide of the present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37°C and then washed 3 times in PBS. Approximately 1013 TU of phage is applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and 10 times with PBS.
• Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of 1.0M Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TGI by incubating eluted phage with bacteria for 30 minutes at 37°C. The E. coli are then plated on TYE plates containing 1% glucose and 100 ⁇ g/ml ampicillin. The resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection.
• Clones positive in ELISA are further characterized by PCR fmge ⁇ rinting (see, e.g., PCT publication WO 92/01047) and then by sequencing. These ELISA positive clones may also be further characterized by techniques known in the art, such as, for example, epitope mapping, binding affinity, receptor signal transduction, ability to block or competitively inhibit antibody/antigen binding, and competitive agonistic or antagonistic activity.
• the following protocol produces a supernatant containing galectin 11 polypeptide to be tested. This supernatant can then be used in the Screening Assays described in Examples 14-21.
• dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution (lmgml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516F Biowhittaker) for a working solution of 50ug/ml.
• PBS w/o calcium or magnesium 17-516F Biowhittaker
• PBS Phosphate Buffered Saline
• the transfection should be performed by tag-teaming the following tasks.
• tags on time is cut in half, and the cells do not spend too much time on PBS.
• person A aspirates off the media from four 24-well plates of cells, and then person B rinses each well with .5-lml PBS.
• Person A then aspirates off PBS rinse, and person B, using a 12-channel pipetter with tips on every other channel, adds the 200ul of DNA/Lipofectamine/Optimem I complex to the odd wells first, then to the even wells, to each row on the 24-well plates. Incubate at 37 degree C for 6 hours.
• Adjust osmolarity to 327 mOsm) with 2mm glutamine and lx penstrep. (BSA (81-068-3 Bayer) lOOgm dissolved in IL DMEM for a 10% BSA stock solution). Filter the media and collect 50 ul for endotoxin assay in 15ml polystyrene conical. The transfection reaction is terminated, preferably by tag-teaming, at the end of the incubation period. Person A aspirates off the transfection media, while person B adds 1.5ml appropriate media to each well Incubate at 37 degree C for 45 or 72 hours depending on the media used: 1%BSA for 45 hours or CHO-5 for 72 hours.

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