WO2003046127A2 - Molecules du marqueur endothelial tumoral 5 et utilisation associee - Google Patents

Molecules du marqueur endothelial tumoral 5 et utilisation associee Download PDF

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Publication number
WO2003046127A2
WO2003046127A2 PCT/US2002/035662 US0235662W WO03046127A2 WO 2003046127 A2 WO2003046127 A2 WO 2003046127A2 US 0235662 W US0235662 W US 0235662W WO 03046127 A2 WO03046127 A2 WO 03046127A2
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polypeptide
tem5
amino acid
nucleic acid
seq
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PCT/US2002/035662
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Todd Juan
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Amgen Inc.
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid

Definitions

  • the - present invention relates to Tumor Endothelial Marker 5 (TEM5) polypeptides and nucleic acid molecules encoding the same.
  • TEM5 Tumor Endothelial Marker 5
  • the invention also relates to selective binding agents, vectors, host cells, and methods for producing TEM5 polypeptides.
  • the invention further relates to pharmaceutical compositions and methods for the diagnosis, treatment, amelioration, or prevention of diseases, disorders, and conditions associated with TEM5 polypeptides.
  • the present invention relates to novel TEM5 nucleic acid molecules and encoded polypeptides.
  • the invention provides for an isolated nucleic acid molecule comprising a nucleotide sequence:
  • the invention also provides for an isolated nucleic acid molecule comprising: (a) a nucleotide sequence encoding a polypeptide that is at least about 70 percent identical to the polypeptide as set forth in SEQ ID NO: 2, wherein the encoded polypeptide has TEM5 activity;
  • nucleotide sequence encoding an allelic variant or splice variant of the nucleotide sequence as set forth in SEQ ID NO: 1 or the nucleotide sequence of (a);
  • nucleotide sequence that hybridizes under at least moderately stringent conditions to the complement of the nucleotide sequence of any of (a) - (d), wherein the nucleic acid molecule encodes a polypeptide having TEM5 activity; or (f) a nucleotide sequence complementary to the nucleotide sequence of any of (a) - (e).
  • the invention further provides for an isolated nucleic acid molecule comprising a nucleotide sequence: (a) encoding a polypeptide as set forth in SEQ ID NO: 2 with at least one conservative amino acid substitution, wherein the encoded polypeptide has TEM5 activity;
  • the present invention provides for an isolated polypeptide comprising the amino acid sequence set forth in SEQ ID NO: 2.
  • the invention also provides for an isolated polypeptide comprising: (a) an amino acid sequence for an ortholog of SEQ ID NO: 2;
  • polypeptide (e) with at least one modification that is an amino acid substitution, amino acid insertion, amino acid deletion, C-terminal truncation, or N-terminal truncation; wherein the polypeptide has TEM5 activity.
  • fusion polypeptides comprising TEM5 amino acid sequences.
  • the present invention also provides for an expression vector comprising the isolated nucleic acid molecules as set forth herein, recombinant host cells comprising the recombinant nucleic acid molecules as set forth herein, and a method of producing a TEM5 polypeptide comprising culturing the host cells and optionally isolating the polypeptide so produced.
  • a transgenic non-human animal comprising a nucleic acid molecule encoding a TEM5 polypeptide is also encompassed by the invention.
  • the TEM5 nucleic acid molecules are introduced into the animal in a manner that allows expression and increased levels of a TEM5 polypeptide, which may include increased circulating levels.
  • the TEM5 nucleic acid molecules are introduced into the animal in a manner that prevents expression of endogenous TEM5 polypeptide (i.e., generates a transgenic animal possessing a TEM5 polypeptide gene knockout).
  • the transgenic non-human animal is preferably a mammal, and more preferably a rodent, such as a rat or a mouse.
  • selective binding agents such as antibodies and peptides capable of specifically binding the TEM5 polypeptides of the invention.
  • Such antibodies and peptides may be agonistic or antagonistic.
  • compositions comprising the nucleotides, polypeptides, or selective binding agents of the invention and one or more pharmaceutically acceptable formulation agents are also encompassed by the invention.
  • the pharmaceutical compositions are used to provide therapeutically effective amounts of the nucleotides or polypeptides of the present invention.
  • the invention is also directed to methods of using the polypeptides, nucleic acid molecules, and selective binding agents.
  • TEM5 polypeptides and nucleic acid molecules of the present invention may be used to treat, prevent, ameliorate, and/or detect diseases and disorders, including those recited herein.
  • the present invention also provides a method of assaying test molecules to identify a test molecule that binds to a TEM5 polypeptide.
  • the method comprises contacting a TEM5 polypeptide with a test molecule to determine the extent of binding of the test molecule to the polypeptide.
  • the method further comprises determining whether such test molecules are agonists or antagonists of a TEM5 polypeptide.
  • the present invention further provides a method of testing the impact of molecules on the expression of TEM5 polypeptide or on the activity of TEM5 polypeptide. Methods of regulating expression and modulating (i.e., increasing or decreasing) levels of a TEM5 polypeptide are also encompassed by the invention.
  • One method comprises administering to an animal a nucleic acid molecule encoding a TEM5 polypeptide.
  • a nucleic acid molecule comprising elements that regulate or modulate the expression of a TEM5 polypeptide may be administered. Examples of these methods include gene therapy, cell therapy, and anti-sense therapy as further described herein.
  • TEM5 polypeptides can be used for identifying ligands thereof.
  • Various forms of "expression cloning" have been used for cloning ligands for receptors (See, e.g., Davis et al, 1996, Cell, 87:1161-69). These and other TEM5 ligand cloning experiments are described in greater detail herein. Isolation of the TEM5 ligand(s) allows for the identification or development of novel agonists and/or antagonists of the TEM5 signaling pathway.
  • Such agonists and antagonists include TEM5 ligand(s), anti-TEM5 ligand antibodies and derivatives thereof, small molecules, or antisense oligonucleotides, any of which can be used for potentially treating one or more diseases or disorders, including those recited herein.
  • Figures 1A-1H show the nucleotide sequence of the human TEM5 gene (SEQ ID NO: 1) and the deduced amino acid sequence of human TEM5 polypeptide (SEQ ID NO:
  • Figures 2A-2C show an amino acid sequence alignment of human TEM5 polypeptide (upper sequence; SEQ ID NO: 2) and human TEM5 ⁇ polypeptide (lower sequence; SEQ ID NO: 3);
  • Figure 3 illustrates the locations of several conserved domains possessed by the human TEM5 polypeptide (SEQ ID NO: 2), as indicated following a BLAST analysis of the amino acid sequences against the conserveed Domain Database.
  • TEM5 gene or "TEM5 nucleic acid molecule” or “TEM5 polynucleotide” refer to a nucleic acid molecule comprising or consisting of a nucleotide sequence as set forth in SEQ ID NO: 1, a nucleotide sequence encoding the polypeptide as set forth in SEQ ID NO: 2, and nucleic acid molecules as defined herein.
  • TEM5 polypeptide allelic variant refers to one of several possible naturally occurring alternate forms of a gene occupying a given locus on a chromosome of an organism or a population of organisms.
  • TEM5 polypeptide splice variant refers to a nucleic acid molecule, usually RNA, which is generated by alternative processing of intron sequences in an RNA transcript of TEM5 polypeptide amino acid sequence as set forth in SEQ ID NO: 2.
  • isolated nucleic acid molecule refers to a nucleic acid molecule of the invention that (1) has been separated from at least about 50 percent of proteins, lipids, carbohydrates, or other materials with which it is naturally found when total nucleic acid is isolated from the source cells, (2) is not linked to all or a portion of a polynucleotide to which the "isolated nucleic acid molecule" is linked in nature, (3) is operably linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature as part of a larger polynucleotide sequence.
  • the isolated nucleic acid molecule of the present invention is substantially free from any other contaminating nucleic acid molecule(s) or other contaminants that are found in its natural environment that would interfere with its use in polypeptide production or its therapeutic, diagnostic, prophylactic or research use.
  • nucleic acid sequence refers to a DNA or RNA sequence.
  • the term encompasses molecules formed from any of the known base analogs of DNA and RNA such as, but not limited to 4-acetylcytosine, 8- hydroxy-N6-methyladenosine, aziridinyl-cytosine, pseudoisocytosine, 5- (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-bromouracil, 5- carboxymethylaminomethyl-2-thiouracil, 5-carboxy-methylaminomethyluracil, dihydrouracil, inosine, N6-iso-pentenyladenine, 1-methyladenine, 1- methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethyl-guanine, 2- methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6- methyladenine, 7-
  • vector is used to refer to any molecule (e.g., nucleic acid, plasmid, or virus) used to transfer coding information to a host cell.
  • molecule e.g., nucleic acid, plasmid, or virus
  • expression vector refers to a vector that is suitable for transformation of a host cell and contains nucleic acid sequences that direct and/or control the expression of inserted heterologous nucleic acid sequences. Expression includes, but is not limited to, processes such as transcription, translation, and RNA splicing, if introns are present.
  • flanking sequence operably linked is used herein to refer to an arrangement of flanking sequences wherein the flanking sequences so described are configured or assembled so as to perform their usual function.
  • a flanking sequence operably linked to a coding sequence may be capable of effecting the replication, transcription and/or translation of the coding sequence.
  • a coding sequence is operably linked to a promoter when the promoter is capable of directing transcription of that coding sequence.
  • a flanking sequence need not be contiguous with the coding sequence, so long as it functions correctly.
  • intervening untranslated yet transcribed sequences can be present between a promoter sequence and the coding sequence and the promoter sequence can still be considered “operably linked" to the coding sequence.
  • host cell is used to refer to a cell which has been transformed, or is capable of being transformed with a nucleic acid sequence and then of expressing a selected gene of interest.
  • the term includes the progeny of the parent cell, whether or not the progeny is identical in morphology or in genetic make-up to the original parent, so long as the selected gene is present.
  • TEM5 polypeptide refers to a polypeptide comprising the amino acid sequence of SEQ ID NO: 2 and related polypeptides.
  • Related polypeptides include TEM5 polypeptide fragments, TEM5 polypeptide orthologs, TEM5 polypeptide variants, and TEM5 polypeptide derivatives, which possess at least one activity of the polypeptide as set forth in SEQ ID NO: 2.
  • TEM5 polypeptides may be mature polypeptides, as defined herein, and may or may not have an amino-terminal methionine residue, depending on the method by which they are prepared.
  • TEM5 polypeptide fragment refers to a polypeptide that comprises a truncation at the amino-terminus (with or without a leader sequence) and/or a truncation at the carboxyl-terminus of the polypeptide as set forth in SEQ ID NO: 2.
  • the term “TEM5 polypeptide fragment” also refers to amino-terminal and/or carboxyl-terminal truncations of TEM5 polypeptide orthologs, TEM5 polypeptide derivatives, or TEM5 polypeptide variants, or to amino-terminal andor carboxyl- terminal truncations of the polypeptides encoded by TEM5 polypeptide allelic variants or TEM5 polypeptide splice variants.
  • TEM5 polypeptide fragments may result from alternative RNA splicing or from in vivo protease activity. Membrane- bound forms of a TEM5 polypeptide are also contemplated by the present invention. In preferred embodiments, truncations and/or deletions comprise about 10 amino acids, or about 20 amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or more than about 100 amino acids. The polypeptide fragments so produced will comprise about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids, or more than about 200 amino acids. Such TEM5 polypeptide fragments may optionally comprise an amino-terminal methionine residue. It will be appreciated that such fragments can be used, for example, to generate antibodies to TEM5 polypeptides.
  • TEM5 polypeptide ortholog refers to a polypeptide from another species that corresponds to TEM5 polypeptide amino acid sequence as set forth in SEQ ID NO: 2.
  • mouse and human TEM5 polypeptides are considered orthologs of each other.
  • TEM5 polypeptide variants refers to TEM5 polypeptides comprising amino acid sequences having one or more amino acid sequence substitutions, deletions (such as internal deletions and/or TEM5 polypeptide fragments), and/or additions (such as internal additions and/or TEM5 fusion polypeptides) as compared to the TEM5 polypeptide amino acid sequence set forth in SEQ ID NO: 2 (with or without a leader sequence).
  • Variants may be naturally occurring (e.g., TEM5 polypeptide allelic variants, TEM5 polypeptide orthologs, and TEM5 polypeptide splice variants) or artificially constructed.
  • Such TEM5 polypeptide variants may be prepared from the corresponding nucleic acid molecules having a DNA sequence that varies accordingly from the DNA sequence as set forth in SEQ ID NO: 1.
  • the variants have from 1 to 3, or from 1 to 5, or from 1 to 10, or from 1 to 15, or from 1 to 20, or from 1 to 25, or from 1 to 50, or from 1 to 75, or from 1 to 100, or more than 100 amino acid substitutions, insertions, additions and or deletions, wherein the substitutions may be conservative, or non-conservative, or any combination thereof.
  • TEM5 polypeptide derivatives refers to the polypeptide as set forth in SEQ ID NO: 2, TEM5 polypeptide fragments, TEM5 polypeptide orthologs, or TEM5 polypeptide variants, as defined herein, that have been chemically modified.
  • TEM5 polypeptide derivatives also refers to the polypeptides encoded by TEM5 polypeptide allelic variants or TEM5 polypeptide splice variants, as defined herein, that have been chemically modified.
  • mature TEM5 polypeptide refers to a TEM5 polypeptide lacking a leader sequence.
  • a mature TEM5 polypeptide may also include other modifications such as proteolytic processing of the amino-terminus (with or without a leader sequence) and/or the carboxyl-terminus, cleavage of a smaller polypeptide from a larger precursor, N-linked and/or O-linked glycosylation, and the like.
  • TEM5 fusion polypeptide refers to a fusion of one or more amino acids (such as a heterologous protein or peptide) at the amino- or carboxyl-terminus of the polypeptide as set forth in SEQ ID NO: 2, TEM5 polypeptide fragments, TEM5 polypeptide orthologs, TEM5 polypeptide variants, or TEM5 derivatives, as defined herein.
  • TEM5 fusion polypeptide also refers to a fusion of one or more amino acids at the amino- or carboxyl-terminus of the polypeptide encoded by TEM5 polypeptide allelic variants or TEM5 polypeptide splice variants, as defined herein.
  • biologically active TEM5 polypeptides refers to TEM5 polypeptides having at least one activity characteristic of the polypeptide comprising the amino acid sequence of SEQ ID NO: 2.
  • a TEM5 polypeptide may be active as an immunogen; that is, the TEM5 polypeptide contains at least one epitope to which antibodies may be raised.
  • isolated polypeptide refers to a polypeptide of the present invention that (1) has been separated from at least about 50 percent of polynucleotides, lipids, carbohydrates, or other materials with which it is naturally found when isolated from the source cell, (2) is not linked (by covalent or noncovalent interaction) to all or a portion of a polypeptide to which the "isolated polypeptide” is linked in nature, (3) is operably linked (by covalent or noncovalent interaction) to a polypeptide with which it is not linked in nature, or (4) does not occur in nature.
  • the isolated polypeptide is substantially free from any other contaminating polypeptides or other contaminants that are found in its natural environment that would interfere with its therapeutic, diagnostic, prophylactic or research use.
  • identity refers to a relationship between the sequences of two or more polypeptide molecules or two or more nucleic acid molecules, as determined by comparing the sequences.
  • identity also means the degree of sequence relatedness between nucleic acid molecules or polypeptides, as the case may be, as determined by the match between strings of two or more nucleotide or two or more amino acid sequences.
  • Identity measures the percent of identical matches between the smaller of two or more sequences with gap alignments (if any) addressed by a particular mathematical model or computer program (i.e., "algorithms”).
  • similarity is a related concept, but in contrast to “identity,” “similarity” refers to a measure of relatedness that includes both identical matches and conservative substitution matches. If two polypeptide sequences have, for example, 10/20 identical amino acids, and the remainder are all non-conservative substitutions, then the percent identity and similarity would both be 50%. If in the same example, there are five more positions where there are conservative substitutions, then the percent identity remains 50%, but the percent similarity would be 75% (15/20). Therefore, in cases where there are conservative substitutions, the percent similarity between two polypeptides will be higher than the percent identity between those two polypeptides.
  • naturally occurring or “native” when used in connection with biological materials refers to materials which are found in nature and are not manipulated by man.
  • non-naturally occurring or “non-native” as used herein refers to a material that is not found in nature or that has been structurally modified or synthesized by man.
  • naturally occurring or “native” refers to the bases adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U).
  • the terms “naturally occurring” and “native” refer to the 20 amino acids alanine (A), cysteine (C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y).
  • ⁇ ективное amount and “therapeutically effective amount” each refer to the amount of a TEM5 polypeptide or TEM5 nucleic acid molecule used to support an observable level of one or more biological activities of the TEM5 polypeptides as set forth herein.
  • pharmaceutically acceptable carrier or “physiologically acceptable carrier” as used herein refers to one or more formulation materials suitable for accomplishing or enhancing the delivery of the TEM5 polypeptide, TEM5 nucleic acid molecule, or TEM5 selective binding agent as a pharmaceutical composition.
  • antigen refers to a molecule or a portion of a molecule capable of being bound by a selective binding agent, such as an antibody, and additionally capable of being used in an animal to produce antibodies capable of binding to an epitope of that antigen.
  • a selective binding agent such as an antibody
  • An antigen may have one or more epitopes.
  • selective binding agent refers to a molecule or molecules having specificity for a TEM5 polypeptide.
  • specific and specificity refer to the ability of the selective binding agents to bind to human TEM5 polypeptides and not to bind to human non-TEM5 polypeptides. It will be appreciated, however, that the selective binding agents may also bind orthologs of the polypeptide as set forth in SEQ ID NO: 2, that is, interspecies versions thereof, such as mouse and rat TEM5 polypeptides.
  • transduction is used to refer to the transfer of genes from one bacterium to another, usually by a phage. “Transduction” also refers to the acquisition and transfer of eukaryotic cellular sequences by retro viruses.
  • transfection is used to refer to the uptake of foreign or exogenous DNA by a cell, and a cell has been "transfected" when the exogenous DNA has been introduced inside the cell membrane.
  • transfection techniques are well known in the art and are disclosed herein. See, e.g., Graham et al, 1973, Virology 52:456; Sambrook et al, Molecular Cloning, A Laboratory Manual (Cold Spring Harbor Laboratories, 1989); Davis et al, Basic Methods in Molecular Biology (Elsevier, 1986); and Chu et al, 1981, Gene 13:197.
  • Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells.
  • transformation refers to a change in a cell's genetic characteristics, and a cell has been transformed when it has been modified to contain a new DNA.
  • a cell is transformed where it is genetically modified from its native state.
  • the transforming DNA may recombine with that of the cell by physically integrating into a chromosome of the cell, may be maintained transiently as an episomal element without being replicated, or may replicate independently as a plasmid.
  • a cell is considered to have been stably transformed when the DNA is replicated with the division of the cell.
  • nucleic acid molecules include allelic or splice variants of the nucleic acid molecule of SEQ ID NO: 1, and include sequences which are complementary to any of the above nucleotide sequences.
  • Related nucleic acid molecules also include a nucleotide sequence encoding a polypeptide comprising or consisting essentially of a substitution, modification, addition and/or deletion of one or more amino acid residues compared to the polypeptide as set forth in SEQ ID NO: 2.
  • Such related TEM5 polypeptides may comprise, for example, an addition and/or a deletion of one or more N-linked or 0-linked glycosylation sites or an addition and/or a deletion of one or more cysteine residues.
  • nucleic acid molecules also include fragments of TEM5 nucleic acid molecules which encode a polypeptide of at least about 25 contiguous amino acids, or about 50 amino acids, or about 75 amino acids, or about 100 amino acids, or about 150 amino acids, or about 200 amino acids, or more than about 200 amino acid residues of the TEM5 polypeptide of SEQ ID NO: 2.
  • related TEM5 nucleic acid molecules also include those molecules which comprise nucleotide sequences which hybridize under moderately or highly stringent conditions as defined herein with the fully complementary sequence of the TEM5 nucleic acid molecule of SEQ ID NO: 1, or of a molecule encoding a polypeptide, which polypeptide comprises the amino acid sequence as shown in SEQ ID NO: 2, or of a nucleic acid fragment as defined herein, or of a nucleic acid fragment encoding a polypeptide as defined herein.
  • Hybridization probes may be prepared using the TEM5 sequences provided herein to screen cDNA, genomic or synthetic DNA libraries for related sequences. Regions of the DNA and/or amino acid sequence of TEM5 polypeptide that exhibit significant identity to known sequences are readily determined using sequence alignment algorithms as described herein and those regions may be used to design probes for screening.
  • highly stringent conditions refers to those conditions that are designed to permit hybridization of DNA strands whose sequences are highly complementary, and to exclude hybridization of significantly mismatched DNAs.
  • Hybridization stringency is principally determined by temperature, ionic strength, and the concentration of denaturing agents such as formamide.
  • Examples of "highly stringent conditions” for hybridization and washing are 0.015 M sodium chloride, 0.0015 M sodium citrate at 65-68°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C.
  • Examples are 0.1% bovine serum albumin, 0.1% polyvinyl- pyrrolidone, 0.1% sodium pyrophosphate, 0.1% sodium dodecylsulfate, NaDodS0 4 , (SDS), ficoll, Denhardt's solution, sonicated salmon sperm DNA (or another non- complementary DNA), and dextran sulfate, although other suitable agents can also be used.
  • concentration and types of these additives can be changed without substantially affecting the stringency of the hybridization conditions.
  • Hybridization experiments are usually carried out at pH 6.8-7.4; however, at typical ionic strength conditions, the rate of hybridization is nearly independent of pH. See Anderson et al., Nucleic Acid Hybridisation: A Practical Approach Ch. 4 (IRL Press Limited).
  • T m (°C) 81.5 + 16.6(log[Na+]) + 0.41(%G+C) - 600/N - 0.72(%formamide)
  • N is the length of the duplex formed
  • [Na+] is the molar concentration of the sodium ion in the hybridization or washing solution
  • %G+C is the percentage of (guanine+cytosine) bases in the hybrid.
  • the melting temperature is reduced by approximately 1°C for each 1% mismatch.
  • moderately stringent conditions refers to conditions under which a DNA duplex with a greater degree of base pair mismatching than could occur under
  • “highly stringent conditions” is able to form.
  • typical “moderately stringent conditions” are 0.015 M sodium chloride, 0.0015 M sodium citrate at 50- 65°C or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 20% formamide at 37-50°C.
  • “moderately stringent conditions” of 50°C in 0.015 M sodium ion will allow about a 21% mismatch.
  • Tm 2°C per A-T base pair + 4°C per G-C base pair *The sodium ion concentration in 6X salt sodium citrate (SSC) is 1M. See Suggs et al, Developmental Biology Using Purified Genes 683 (Brown and Fox, eds., 1981).
  • Fligh stringency washing conditions for oligonucleotides are usually at a temperature of 0-5°C below the Tm of the oligonucleotide in 6X SSC, 0.1% SDS.
  • nucleic acid molecules comprise or consist of a nucleotide sequence that is at least about 70 percent identical to the nucleotide sequence as shown in SEQ ID NO: 1.
  • nucleotide sequences are about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percent identical to the nucleotide sequence as shown in SEQ ID NO: 1.
  • Related nucleic acid molecules encode polypeptides possessing at least one activity of the polypeptide set forth in SEQ ID NO: 2.
  • Differences in the nucleic acid sequence may result in conservative and/or non-conservative modifications of the amino acid sequence relative to the amino acid sequence of SEQ ID NO: 2.
  • amino acid sequence of SEQ ID NO: 2 (and the corresponding modifications to the encoding nucleotides) will produce a polypeptide having functional and chemical characteristics similar to those of TEM5 polypeptides.
  • substantial modifications in the functional and or chemical characteristics of TEM5 polypeptides may be accomplished by selecting substitutions in the amino acid sequence of SEQ ID NO: 2 that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain.
  • a “conservative amino acid substitution” may involve a substitution of a native amino acid residue with a nonnative residue such that there is little or no effect on the polarity or charge of the amino acid residue at that position.
  • any native residue in the polypeptide may also be substituted with alanine, as has been previously described for "alanine scanning mutagenesis.”
  • Conservative amino acid substitutions also encompass non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in biological systems. These include peptidomimetics, and other reversed or inverted forms of amino acid moieties.
  • Naturally occurring residues may be divided into classes based on common side chain properties:
  • non-conservative substitutions may involve the exchange of a member of one of these classes for a member from another class.
  • substituted residues may be introduced into regions of the human TEM5 polypeptide that are homologous with non-human TEM5 polypeptides, or into the non-homologous regions of the molecule.
  • hydropathic index of amino acids may be considered. Each amino acid has been assigned a hydropathic index on the basis of its hydrophobicity and charge characteristics.
  • the hydropathic indices are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (- 0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (- 3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (- 4.5).
  • hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte et al, 1982, /. Mol. Biol. 157:105-31). It is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are within ⁇ 2 is preferred, those within ⁇ 1 are particularly preferred, and those within ⁇ 0.5 are even more particularly preferred.
  • hydrophilicity values have been assigned to these amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ⁇ 1); glutamate (+3.0 ⁇ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ⁇ 1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); and tryptophan (-3.4).
  • Desired amino acid substitutions can be determined by those skilled in the art at the time such substitutions are desired.
  • amino acid substitutions can be used to identify important residues of the TEM5 polypeptide, or to increase or decrease the affinity of the TEM5 polypeptides described herein.
  • Exemplary amino acid substitutions are set forth in Table I.
  • Leu ⁇ orleucine lie, lie Val, Met, Ala, Phe
  • a skilled artisan will be able to determine suitable variants of the polypeptide as set forth in SEQ ID NO: 2 using well-known techniques. For identifying suitable areas of the molecule that may be changed without destroying biological activity, one skilled in the art may target areas not believed to be important for activity. For example, when similar polypeptides with similar activities from the same species or from other species are known, one skilled in the art may compare the amino acid sequence of a TEM5 polypeptide to such similar polypeptides. With such a comparison, one can identify residues and portions of the molecules that are conserved among similar polypeptides.
  • One skilled in the art can also analyze the three-dimensional structure and amino acid sequence in relation to that structure in similar polypeptides. In view of such information, one skilled in the art may predict the alignment of amino acid residues of TEM5 polypeptide with respect to its three dimensional structure. One skilled in the art may choose not to make radical changes to amino acid residues predicted to be on the surface of the protein, since such residues may be involved in important interactions with other molecules. Moreover, one skilled in the art may generate test variants containing a single amino acid substitution at each amino acid residue. The variants could be screened using activity assays known to those with skill in the art. Such variants could be used to gather information about suitable variants.
  • Additional methods of predicting secondary structure include “threading” (Jones, 1997, Curr. Opin. Struct. Biol. 7:377-87; Sippl et al, 1996, Structure 4:15- 19), “profile analysis” (Bowie et al, 1991, Science, 253:164-70; Gribskov et al, 1990, Methods Enzymol. 183:146-59; Gribskov et al, 1987, Proc. Nat. Acad. Sci. U.S.A. 84:4355-58), and “evolutionary linkage” (See Holm et al, supra, and Brenner et al, supra).
  • TEM5 polypeptide variants include glycosylation variants wherein the number and/or type of glycosylation sites have been altered compared to the amino acid sequence set forth in SEQ ID NO: 2.
  • TEM5 polypeptide variants comprise a greater or a lesser number of N-linked glycosylation sites than the amino acid sequence set forth in SEQ ID NO: 2.
  • An N-linked glycosylation site is characterized by the sequence: Asn-X-Ser or Asn-X-Thr, wherein the amino acid residue designated as X may be any amino acid residue except proline. The substitution of amino acid residues to create this sequence provides a potential new site for the addition of an N-linked carbohydrate chain.
  • TEM5 variants include cysteine variants, wherein one or more cysteine residues are deleted or substituted with another amino acid (e.g., serine) as compared to the amino acid sequence set forth in SEQ ID NO: 2.
  • cysteine variants are useful when TEM5 polypeptides must be refolded into a biologically active conformation such as after the isolation of insoluble inclusion bodies. Cysteine variants generally have fewer cysteine residues than the native protein, and typically have an even number to minimize interactions resulting from unpaired cysteines.
  • TEM5 polypeptide variants comprise an amino acid sequence as set forth in SEQ ID NO: 2 with at least one amino acid insertion and wherein the polypeptide has TEM5 activity, or an amino acid sequence encoding a polypeptide as set forth in SEQ ID NO: 2 with at least one amino acid deletion and wherein the polypeptide has TEM5 activity.
  • TEM5 polypeptide variants also comprise an amino acid sequence as set forth in SEQ ID NO: 2 wherein the polypeptide has a carboxyl- and/or amino-terminal truncation and further wherein the polypeptide has TEM5 activity.
  • TEM5 polypeptide variants further comprise an amino acid sequence as set forth in SEQ ID NO: 2 with at least one modification that is an amino acid substitution, amino acid insertion, amino acid deletion, carboxyl- terminal truncation, or amino-terminal truncation, and wherein the polypeptide has TEM5 activity.
  • TEM5 polypeptide variants comprise an amino acid sequence that is at least about 70 percent identical to the amino acid sequence as set forth in SEQ ID NO: 2.
  • TEM5 polypeptide variants comprise an amino acid sequence that is at least about 75 percent, or about 80 percent, or about 85 percent, or about 90 percent, or about 95, 96, 97, 98, or 99 percent identical to the amino acid sequence as set forth in SEQ ID NO: 2.
  • TEM5 polypeptide variants possess at least one activity of the polypeptide set forth in SEQ ID NO: 2.
  • polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or other TEM5 polypeptide, may be fused to a homologous polypeptide to form a homodimer or to a heterologous polypeptide to form a heterodimer.
  • Heterologous peptides and polypeptides include, but are not limited to: an epitope to allow for the detection and/or isolation of a TEM5 fusion polypeptide; a transmembrane receptor protein or a portion thereof, such as an extracellular domain or a transmembrane and intracellular domain; a ligand or a portion thereof which binds to a transmembrane receptor protein; an enzyme or portion thereof which is catalytically active; a polypeptide or peptide which promotes oligomerization, such as a leucine zipper domain; a polypeptide or peptide which increases stability, such as an immunoglobulin constant region; and a polypeptide which has a therapeutic activity different from the polypeptide comprising the amino acid sequence as set forth in SEQ ID NO: 2, or other TEM5 polypeptide.
  • Fusions can be made either at the amino-terminus or at the carboxyl-terminus of the polypeptide comprising the amino acid sequence set forth in SEQ ED NO: 2, or other TEM5 polypeptide. Fusions may be direct with no linker or adapter molecule or may be through a linker or adapter molecule.
  • a linker or adapter molecule may be one or more amino acid residues, typically from about 20 to about 50 amino acid residues.
  • a linker or adapter molecule may also be designed with a cleavage site for a DNA restriction endonuclease or for a protease to allow for the separation of the fused moieties. It will be appreciated that once constructed, the fusion polypeptides can be derivatized according to the methods described herein.
  • the polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or other TEM5 polypeptide is fused to one or more domains of an Fc region of human IgG.
  • Antibodies comprise two functionally independent parts, a variable domain known as "Fab,” that binds an antigen, and a constant domain known as "Fc,” that is involved in effector functions such as complement activation and attack by phagocytic cells.
  • Fab variable domain known as "Fab”
  • Fc constant domain
  • An Fc has a long serum half- life, whereas an Fab is short-lived.
  • an Fc domain When constructed together with a therapeutic protein, an Fc domain can provide longer half- life or incorporate such functions as Fc receptor binding, protein A binding, complement fixation, and perhaps even placental transfer. Id. Table II summarizes the use of certain Fc fusions known in the art.
  • a human IgG hinge, CH2, and CH3 region may be fused at either the amino-terminus or carboxyl-tenninus of the TEM5 polypeptides using methods known to the skilled artisan.
  • a human IgG hinge, CH2, and CH3 region may be fused at either the amino-terminus or carboxyl-terminus of a TEM5 polypeptide fragment (e.g., the predicted extracellular portion of TEM5 polypeptide).
  • the resulting TEM5 fusion polypeptide may be purified by use of a Protein A affinity column.
  • Peptides and proteins fused to an Fc region have been found to exhibit a substantially greater half-life in vivo than the unfused counterpart.
  • a fusion to an Fc region allows for dimerization/multimerization of the fusion polypeptide.
  • the Fc region may be a naturally occurring Fc region, or may be altered to improve certain qualities, such as therapeutic qualities, circulation time, or reduced aggregation.
  • Useful modifications of protein therapeutic agents by fusion with the "Fc" domain of an antibody are discussed in detail in U.S. Patent App. No. 09/428,082 (International Pub. No. WO 99/25044), which is hereby incorporated by reference in its entirety.
  • That patent application discusses linkage to a "vehicle” such as polyethylene gycol (PEG), dextran, or an Fc region.
  • a "vehicle” such as polyethylene gycol (PEG), dextran, or an Fc region.
  • PEG polyethylene gycol
  • Fc region an Fc region.
  • Identity and similarity of related nucleic acid molecules and polypeptides are readily calculated by known methods. Such methods include, but are not limited to those described in Computational Molecular Biology (A.M. Lesk, ed., Oxford University Press 1988); Biocomputing: Informatics and Genome Projects (D.W. Smith, ed., Academic Press 1993); Computer Analysis of Sequence Data (Part 1, A.M. Griffin and H.G. Griffin, eds., Humana Press 1994); G. von Heijne, Sequence Analysis in Molecular Biology (Academic Press 1987); Sequence Analysis Primer (M. Gribskov and J. Devereux, e
  • Preferred methods to determine identity and/or similarity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are described in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include, but are not limited to, the GCG program package, including GAP (Devereux et al, 1984, Nucleic Acids Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, and FASTA (Altschul et al, 1990, /. Mol. Biol. 215:403-10).
  • GCG program package including GAP (Devereux et al, 1984, Nucleic Acids Res. 12:387; Genetics Computer Group, University of Wisconsin, Madison, WI), BLASTP, BLASTN, and FASTA (Altschul et al, 1990, /. Mol. Biol. 215:403-10).
  • the BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources (Altschul et al, BLAST Manual (NCB NLM NIH, Bethesda, MD); Altschul et al, 1990, supra).
  • NCBI National Center for Biotechnology Information
  • NCB NLM NIH Bethesda
  • MD Altschul et al, 1990, supra.
  • the well-known Smith Waterman algorithm may also be used to determine identity.
  • Certain alignment schemes for aligning two amino acid sequences may result in the matching of only a short region of the two sequences, and this small aligned region may have very high sequence identity even though there is no significant relationship between the two full-length sequences. Accordingly, in a preferred embodiment, the selected alignment method (GAP program) will result in an alignment that spans at least 50 contiguous amino acids of the claimed polypeptide.
  • GAP Genetics Computer Group, University of Wisconsin, Madison, WI
  • two polypeptides for which the percent sequence identity is to be determined are aligned for optimal matching of their respective amino acids (the "matched span," as determined by the algorithm).
  • a gap opening penalty (which is calculated as 3X the average diagonal; the "average diagonal” is the average of the diagonal of the comparison matrix being used; the “diagonal” is the score or number assigned to each perfect amino acid match by the particular comparison matrix) and a gap extension penalty (which is usually 0.1X the gap opening penalty), as well as a comparison matrix such as PAM 250 or BLOSUM 62 are used in conjunction with the algorithm.
  • Preferred parameters for polypeptide sequence comparison include the following:
  • the GAP program is useful with the above parameters.
  • the aforementioned parameters are the default parameters for polypeptide comparisons (along with no penalty for end gaps) using the GAP algorithm.
  • Preferred parameters for nucleic acid molecule sequence comparison include the following:
  • the GAP program is also useful with the above parameters.
  • the aforementioned parameters are the default parameters for nucleic acid molecule comparisons.
  • Other exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices, and thresholds of similarity may be used, including those set forth in the Program Manual, Wisconsin Package, Version 9, September, 1997.
  • the particular choices to be made will be apparent to those of skill in the art and will depend on the specific comparison to be made, such as DNA-to-DNA, protein-to- protein, protein-to-DNA; and additionally, whether the comparison is between given pairs of sequences (in which case GAP or BestFit are generally preferred) or between one sequence and a large database of sequences (in which case FASTA or BLASTA are preferred).
  • Nucleic Acid Molecules are the default parameters for nucleic acid molecule comparisons.
  • Other exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices, and thresholds of similarity may be used, including those set forth in the Program Manual, Wisconsin Package, Version 9,
  • nucleic acid molecules encoding a polypeptide comprising the amino acid sequence of a TEM5 polypeptide can readily be obtained in a variety of ways including, without limitation, chemical synthesis, cDNA or genomic library screening, expression library screening, and/or PCR amplification of cDNA.
  • a gene encoding the amino acid sequence of a TEM5 polypeptide may be used as a probe to identify orthologs or related genes from the same species.
  • the probes or primers may be used to screen cDNA libraries from various tissue sources believed to express the TEM5 polypeptide.
  • part or all of a nucleic acid molecule having the sequence as set forth in SEQ ID NO: 1 may be used to screen a genomic library to identify and isolate a gene encoding the amino acid sequence of a TEM5 polypeptide.
  • conditions of moderate or high stringency will be employed for screening to minimize the number of false positives obtained from the screening.
  • Nucleic acid molecules encoding the amino acid sequence of TEM5 polypeptides may also be identified by expression cloning which employs the detection of positive clones based upon a property of the expressed protein.
  • nucleic acid libraries are screened by the binding an antibody or other binding partner (e.g., receptor or ligand) to cloned proteins that are expressed and displayed on a host cell surface.
  • the antibody or binding partner is modified with a detectable label to identify those cells expressing the desired clone.
  • Recombinant expression techniques conducted in accordance with the descriptions set forth below may be followed to produce these polynucleotides and to express the encoded polypeptides.
  • a nucleic acid sequence that encodes the amino acid sequence of a TEM5 polypeptide into an appropriate vector, one skilled in the art can readily produce large quantities of the desired nucleotide sequence. The sequences can then be used to generate detection probes or amplification primers.
  • a polynucleotide encoding the amino acid sequence of a TEM5 polypeptide can be inserted into an expression vector. By introducing the expression vector into an appropriate host, the encoded TEM5 polypeptide may be produced in large amounts.
  • PCR polymerase chain reaction
  • cDNA is prepared from poly(A)+RNA or total RNA using the enzyme reverse transcriptase.
  • Two primers typically complementary to two separate regions of cDNA encoding the amino acid sequence of a TEM5 polypeptide, are then added to the cDNA along with a polymerase such as Taq polymerase, and the polymerase amplifies the cDNA region between the two primers.
  • a polymerase such as Taq polymerase
  • Another means of preparing a nucleic acid molecule encoding the amino acid sequence of a TEM5 polypeptide is chemical synthesis using methods well known to the skilled artisan such as those described by Engels et al, 1989, Angew. Chem. Intl. Ed. 28:716-34. These methods include, inter alia, the phosphotriester, phosphoramidite, and H-phosphonate methods for nucleic acid synthesis.
  • a preferred method for such chemical synthesis is polymer-supported synthesis using standard phosphoramidite chemistry.
  • the DNA encoding the amino acid sequence of a TEM5 polypeptide will be several hundred nucleotides in length.
  • Nucleic acids larger than about 100 nucleotides can be synthesized as several fragments using these methods. The fragments can then be ligated together to form the full-length nucleotide sequence of a TEM5 gene.
  • the DNA fragment encoding the amino-terminus of the polypeptide will have an ATG, which encodes a methionine residue. This methionine may or may not be present on the mature form of the TEM5 polypeptide, depending on whether the polypeptide produced in the host cell is designed to be secreted from that cell. Other methods known to the skilled artisan may be used as well.
  • nucleic acid variants contain codons which have been altered for optimal expression of a TEM5 polypeptide in a given host cell. Particular codon alterations will depend upon the TEM5 polypeptide and host cell selected for expression. Such "codon optimization” can be carried out by a variety of methods, for example, by selecting codons which are preferred for use in highly expressed genes in a given host cell. Computer algorithms which incorporate codon frequency tables such as "Eco_high.Cod" for codon preference of highly expressed bacterial genes may be used and are provided by the University of Wisconsin Package Version 9.0 (Genetics Computer Group, Madison, WI).
  • Codon frequency tables include “Celegans_high.cod,” “Celegans_low.cod,” “Drosophilajhigh.cod,” “Human iigh.cod,” “Maize_.high.cod,” and “Yeast_high.cod.”
  • nucleic acid molecules encoding TEM5 polypeptide variants may be produced using site directed mutagenesis, PCR amplification, or other appropriate methods, where the primer(s) have the desired point mutations (see Sambrook et al, supra, and Ausubel et al, supra, for descriptions of mutagenesis techniques). Chemical synthesis using methods described by Engels et al, supra, may also be used to prepare such variants. Other methods known to the skilled artisan may be used as well.
  • a nucleic acid molecule encoding the amino acid sequence of a TEM5 polypeptide is inserted into an appropriate expression vector using standard ligation techniques.
  • the vector is typically selected to be functional in the particular host cell employed (i.e., the vector is compatible with the host cell machinery such that amplification of the gene and/or expression of the gene can occur).
  • a nucleic acid molecule encoding the amino acid sequence of a TEM5 polypeptide may be amplified/expressed in prokaryotic, yeast, insect (baculovirus systems) and/or eukaryotic host cells. Selection of the host cell will depend in part on whether a TEM5 polypeptide is to be post-translationally modified (e.g., glycosylated and/or phosphorylated). If so, yeast, insect, or mammalian host cells are preferable.
  • yeast, insect, or mammalian host cells are preferable.
  • expression vectors used in any of the host cells will contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
  • sequences collectively referred to as “flanking sequences” in certain embodiments will typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcriptional termination sequence, a complete intron sequence containing a donor and acceptor splice site, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a polylinker region for inserting the nucleic acid encoding the polypeptide to be expressed, and a selectable marker element.
  • a promoter one or more enhancer sequences
  • an origin of replication a transcriptional termination sequence
  • a complete intron sequence containing a donor and acceptor splice site a sequence encoding a leader sequence for polypeptide secreti
  • the vector may contain a "tag"-encoding sequence, le., an oligonucleotide molecule located at the 5' or 3' end of the TEM5 polypeptide coding sequence; the oligonucleotide sequence encodes polyHis (such as hexaHis), or another "tag” such as FLAG, FIA (hemaglutinin influenza virus), or myc for which commercially available antibodies exist.
  • This tag is typically fused to the polypeptide upon expression of the polypeptide, and can serve as a means for affinity purification of the TEM5 polypeptide from the host cell. Affinity purification can be accomplished, for example, by column chromatography using antibodies against the tag as an affinity matrix.
  • the tag can subsequently be removed from the purified TEM5 polypeptide by various means such as using certain peptidases for cleavage.
  • Flanking sequences may be homologous (i.e., from the same species and/or strain as the host cell), heterologous (i.e., from a species other than the host cell species or strain), hybrid (i.e., a combination of flanking sequences from more than one source), or synthetic, or the flanking sequences may be native sequences that normally function to regulate TEM5 polypeptide expression.
  • the source of a flanking sequence may be any prokaryotic or eukaryotic organism, any vertebrate or invertebrate organism, or any plant, provided that the flanking sequence is functional in, and can be activated by, the host cell machinery.
  • Flanking sequences useful in the vectors of this invention may be obtained by any of several methods well known in the art.
  • flanking sequences useful herein - other than the TEM5 gene flanking sequences - will have been previously identified by mapping and/or by restriction endonuclease digestion and can thus be isolated from the proper tissue source using the appropriate restriction endonucleases.
  • the full nucleotide sequence of a flanking sequence may be known.
  • the flanking sequence may be synthesized using the methods described herein for nucleic acid synthesis or cloning.
  • flanking sequence may be obtained using PCR and/or by screening a genomic library with a suitable oligonucleotide and/or flanking sequence fragment from the same or another species.
  • flanking sequence may be not known, a fragment of DNA containing a flanking sequence may be isolated from a larger piece of DNA that may contain, for example, a coding sequence or even another gene or genes. Isolation may be accomplished by restriction endonuclease digestion to produce the proper DNA fragment followed by isolation using agarose gel purification, Qiagen ® column chromatography (Chatsworth, CA), or other methods known to the skilled artisan. The selection of suitable enzymes to accomplish this purpose will be readily apparent to one of ordinary skill in the art.
  • An origin of replication is typically a part of those prokaryotic expression vectors purchased commercially, and the origin aids in the amplification of the vector in a host cell. Amplification of the vector to a certain copy number can, in some cases, be important for the optimal expression of a TEM5 polypeptide. If the vector of choice does not contain an origin of replication site, one may be chemically synthesized based on a known sequence, and ligated into the vector.
  • the origin of replication from the plasmid pBR322 (New England Biolabs, Beverly, MA) is suitable for most gram-negative bacteria and various origins (e.g., SV40, polyoma, adenovirus, vesicular stomatitus virus (VSV), or papillomaviruses such as HPV or BPV) are useful for cloning vectors in mammalian cells.
  • origin of replication component is not needed for mammalian expression vectors (for example, the SV40 origin is often used only because it contains the early promoter).
  • a transcription termination sequence is typically located 3' of the end of a polypeptide coding region and serves to terminate transcription.
  • a transcription termination sequence in prokaryotic cells is a G-C rich fragment followed by a poly-T sequence. While the sequence is easily cloned from a library or even purchased commercially as part of a vector, it can also be readily synthesized using methods for nucleic acid synthesis such as those described herein.
  • a selectable marker gene element encodes a protein necessary for the survival and growth of a host cell grown in a selective culture medium.
  • Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, tetracycline, or kanamycin for prokaryotic host cells; (b) complement auxotrophic deficiencies of the cell; or (c) supply critical nutrients not available from complex media.
  • Preferred selectable markers are the kanamycin resistance gene, the ampicillin resistance gene, and the tetracycline resistance gene.
  • a neomycin resistance gene may also be used for selection in prokaryotic and eukaryotic host cells.
  • Other selection genes may be used to amplify the gene that will be expressed.
  • Amplification is the process wherein genes that are in greater demand for the production of a protein critical for growth are reiterated in tandem within the chromosomes of successive generations of recombinant cells.
  • suitable selectable markers for mammalian cells include dihydrofolate reductase (DHFR) and thymidine kinase.
  • DHFR dihydrofolate reductase
  • the mammalian cell transformants are placed under selection pressure wherein only the transformants are uniquely adapted to survive by virtue of the selection gene present in the vector. Selection pressure is imposed by culturing the transformed cells under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to the amplification of both the selection gene and the DNA that encodes a TEM5 polypeptide. As a result, increased quantities of TEM5 polypeptide are synthesized from the amplified DNA.
  • a ribosome binding site is usually necessary for translation initiation of mRNA and is characterized by a Shine-Dalgarno sequence (prokaryotes) or a Kozak sequence (eukaryotes).
  • the element is typically located 3' to the promoter and 5' to the coding sequence of a TEM5 polypeptide to be expressed.
  • the Shine-Dalgarno sequence is varied but is typically a polypurine (i.e., having a high A-G content).
  • Many Shine-Dalgarno sequences have been identified, each of which can be readily synthesized using methods set forth herein and used in a prokaryotic vector.
  • a leader, or signal, sequence may be used to direct a TEM5 polypeptide out of the host cell.
  • a nucleotide sequence encoding the signal sequence is positioned in the coding region of a TEM5 nucleic acid molecule, or directly at the 5' end of a TEM5 polypeptide coding region.
  • Many signal sequences have been identified, and any of those that are functional in the selected host cell may be used in conjunction with a TEM5 nucleic acid molecule. Therefore, a signal sequence may be homologous (naturally occurring) or heterologous to the TEM5 nucleic acid molecule. Additionally, a signal sequence may be chemically synthesized using methods described herein.
  • the signal sequence may be a component of the vector, or it may be a part of a TEM5 nucleic acid molecule that is inserted into the vector.
  • nucleotide sequence encoding a native TEM5 polypeptide signal sequence joined to a TEM5 polypeptide coding region or a nucleotide sequence encoding a heterologous signal sequence joined to a TEM5 polypeptide coding region.
  • the heterologous signal sequence selected should be one that is recognized and processed, i.e., cleaved by a signal peptidase, by the host cell.
  • the signal sequence is substituted by a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, or heat-stable enterotoxin II leaders.
  • a prokaryotic signal sequence selected, for example, from the group of the alkaline phosphatase, penicillinase, or heat-stable enterotoxin II leaders.
  • yeast secretion the native TEM5 polypeptide signal sequence may be substituted by the yeast invertase, alpha factor, or acid phosphatase leaders.
  • the native signal sequence is satisfactory, although other mammalian signal sequences may be suitable.
  • the final protein product may have, in the -1 position (relative to the first amino acid of the mature protein) one or more additional amino acids incident to expression, which may not have been totally removed.
  • the final protein product may have one or two amino acid residues found in the peptidase cleavage site, attached to the amino-terminus.
  • use of some enzyme cleavage sites may result in a slightly truncated form of the desired TEM5 polypeptide, if the enzyme cuts at such area within the mature polypeptide.
  • transcription of a nucleic acid molecule is increased by the presence of one or more introns in the vector; this is particularly true where a polypeptide is produced in eukaryotic host cells, especially mammalian host cells.
  • the introns used may be naturally occurring within the TEM5 gene especially where the gene used is a full-length genomic sequence or a fragment thereof. Where the intron is not naturally occurring within the gene (as for most cDNAs), the intron may be obtained from another source.
  • the position of the intron with respect to flanking sequences and the TEM5 gene is generally important, as the intron must be transcribed to be effective.
  • the preferred position for the intron is 3' to the transcription start site and 5' to the poly-A transcription termination sequence.
  • the intron or introns will be located on one side or the other (i.e., 5' or 3') of the cDNA such that it does not interrupt the coding sequence.
  • Any intron from any source including viral, prokaryotic and eukaryotic (plant or animal) organisms, may be used to practice this invention, provided that it is compatible with the host cell into which it is inserted.
  • synthetic introns may be used to practice this invention, provided that it is compatible with the host cell into which it is inserted.
  • more than one intron may be used in the vector.
  • the expression and cloning vectors of the present invention will typically contain a promoter that is recognized by the host organism and operably linked to the molecule encoding the TEM5 polypeptide. Promoters are untranscribed sequences located upstream (i.e., 5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcription of the structural gene. Promoters are conventionally grouped into one of two classes: inducible promoters and constitutive promoters. Inducible promoters initiate increased levels of transcription from DNA under their control in response to some change in culture conditions, such as the presence or absence of a nutrient or a change in temperature.
  • Constitutive promoters initiate continual gene product production; that is, there is little or no control over gene expression.
  • a large number of promoters, recognized by a variety of potential host cells, are well known.
  • a suitable promoter is operably linked to the DNA encoding TEM5 polypeptide by removing the promoter from the source DNA by restriction enzyme digestion and inserting the desired promoter sequence into the vector.
  • the native TEM5 promoter sequence may be used to direct amplification and/or expression of a TEM5 nucleic acid molecule.
  • a heterologous promoter is preferred, however, if it permits greater transcription and higher yields of the expressed protein as compared to the native promoter, and if it is compatible with the host cell system that has been selected for use.
  • Promoters suitable for use with prokaryotic hosts include the beta-lactamase and lactose promoter systems; alkaline phosphatase; a tryptophan (trp) promoter system; and hybrid promoters such as the tac promoter. Other known bacterial promoters are also suitable. Their sequences have been published, thereby enabling one skilled in the art to ligate them to the desired DNA sequence, using linkers or adapters as needed to supply any useful restriction sites.
  • Suitable promoters for use with yeast hosts are also well known in the art.
  • Yeast enhancers are advantageously used with yeast promoters.
  • Suitable promoters for use with mammalian host cells are well known and include, but are not limited to, those obtained from the genomes of viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably Simian Virus 40 (SV40).
  • viruses such as polyoma virus, fowlpox virus, adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, retroviruses, hepatitis-B virus and most preferably Simian Virus 40 (SV40).
  • Other suitable mammalian promoters
  • Additional promoters which may be of interest in controlling TEM5 gene expression include, but are not limited to: the SV40 early promoter region (Bernoist and Chambon, 1981, Nature 290:304-10); the CMV promoter; the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto, et al, 1980, Cell 22:787-97); the herpes thymidine kinase promoter (Wagner et al, 1981, Proc. Natl. Acad. Sci. U.S.A.
  • elastase I gene control region which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38:639-46; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol.
  • beta-globin gene control region which is active in myeloid cells (Mogram et al, 1985, Nature 315:338-40; Kollias et al, 1986, Cell 46:89-94); the myelin basic protein gene control region which is active in oligodendrocyte cells in the brain (Readhead et al, 1987, Cell 48:703-12); the myosin light chain-2 gene control region which is active in skeletal muscle (Sani, 1985, Nature 314:283-86); and the gonadotropic releasing hormone gene control region which is active in the hypothalamus (Mason et al, 1986, Science 234:1372-78).
  • Enhancers are cis-acting elements of DNA, usually about 10-300 bp in length, that act on the promoter to increase transcription. Enhancers are relatively orientation and position independent. They have been found 5' and 3' to the transcription unit.
  • enhancer sequences available from mammalian genes are known (e.g., globin, elastase, albumin, alpha-feto-protein and insulin). Typically, however, an enhancer from a virus will be used.
  • the SV40 enhancer, the cytomegalovirus early promoter enhancer, the polyoma enhancer, and adenovirus enhancers are exemplary enhancing elements for the activation of eukaryotic promoters. While an enhancer may be spliced into the vector at a position 5' or 3' to a TEM5 nucleic acid molecule, it is typically located at a site 5' from the promoter.
  • Expression vectors of the invention may be constructed from a starting vector such as a commercially available vector. Such vectors may or may not contain all of the desired flanking sequences. Where one or more of the flanking sequences described herein are not already present in the vector, they may be individually obtained and ligated into the vector. Methods used for obtaining each of the flanking sequences are well known to one skilled in the art.
  • Preferred vectors for practicing this invention are those that are compatible with bacterial, insect, and mammalian host cells.
  • Such vectors include, inter alia, pCRII, pCR3, and pcDNA3.1 (Invitrogen, Carlsbad, CA), pBSII (Stratagene, La Jolla, CA), pET15 (Novagen, Madison, WI), pGEX (Pharmacia Biotech, Piscataway, NJ), pEGFP-N2 (Clontech, Palo Alto, CA), pETL (BlueBacII, Invitrogen), pDSR- alpha (International Pub. No. WO 90/14363) and pFastBacDual (Gibco-BRL, Grand Island, NY).
  • vectors include, but are not limited to, cosmids, plasmids, or modified viruses, but it will be appreciated that the vector system must be compatible with the selected host cell.
  • Such vectors include, but are not limited to plasmids such as Bluescript plasmid derivatives (a high copy number ColEl -based phagemid; Stratagene Cloning Systems; La Jolla CA), PCR cloning plasmids designed for cloning Taq-amplified PCR products (e.g., TOPOTM TA Cloning ® Kit, , ®
  • PCR2.1 plasmid derivatives include Invitrogen, and mammalian, yeast or virus vectors such as a baculovirus expression system (pBacPAK plasmid derivatives; Clontech; Palo Alto, CA).
  • pBacPAK plasmid derivatives include Clontech; Palo Alto, CA.
  • the completed vector may be inserted into a suitable host cell for amplification and/or polypeptide expression.
  • the transformation of an expression vector for a TEM5 polypeptide into a selected host cell may be accomplished by well known methods including methods such as transfection, infection, calcium chloride, electroporation, microinjection, lipofection, DEAE-dextran method, or other known techniques. The method selected will in part be a function of the type of host cell to be used. These methods and other suitable methods are well known to the skilled artisan, and are set forth, for example, in Sambrook et al, supra.
  • Host cells may be prokaryotic host cells (such as E. coli) or eukaryotic host cells (such as a yeast, insect, or vertebrate cell).
  • the host cell when cultured under appropriate conditions, synthesizes a TEM5 polypeptide that can subsequently be collected from the culture medium (if the host cell secretes it into the medium) or directly from the host cell producing it (if it is not secreted).
  • the selection of an appropriate host cell will depend upon various factors, such as desired expression levels, polypeptide modifications that are desirable or necessary for activity (such as glycosylation or phosphorylation) and ease of folding into a biologically active molecule.
  • suitable host cells are known in the art and many are available from the American Type Culture Collection (ATCC), Manassas, VA. Examples include, but are not limited to, mammalian cells, such as Chinese hamster ovary cells (CHO), CHO DHFR(-) cells (Urlaub et al, 1980, Proc. Natl. Acad. Sci. U.S.A. 97:4216-20), human embryonic kidney (HEK) 293 or 293T cells, or 3T3 cells.
  • CHO Chinese hamster ovary cells
  • CHO DHFR(-) cells Urlaub et al, 1980, Proc. Natl. Acad. Sci. U.S.A. 97:4216-20
  • HEK human embryonic kidney
  • suitable mammalian cell lines are the monkey COS-1 and COS-7 cell lines, and the CV-1 cell line.
  • Further exemplary mammalian host cells include primate cell lines and rodent cell lines, including transformed cell lines. Normal diploid cells, cell strains derived from in vitro culture of primary tissue, as well as primary explants, are also suitable.
  • Candidate cells may be genotypically deficient in the selection gene, or may contain a dominantly acting selection gene.
  • Other suitable mammalian cell lines include but are not limited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3 lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines. Each of these cell lines is known by and available to those skilled in the art of protein expression.
  • bacterial cells e.g., HB101, DH5 ⁇ , DH10, and MCI 061 are well-known as host cells in the field of biotechnology.
  • Various strains of ⁇ . subtilis, Pseudomonas spp., other Bacillus spp., Streptomyces spp., and the like may also be employed in this method.
  • yeast cells include, for example, Saccharomyces cerivisae and Pichia pastoris.
  • insect cell systems may be utilized in the methods of the present invention. Such systems are described, for example, in Kitts et al, 1993, Biotechniques, 14:810-17; Lucklow, 1993, Curr. Opin. Biotechnol 4:564-72; and Lucklow et al, 1993, /. Virol, 67:4566-79.
  • Preferred insect cells are Sf-9 and Hi5 (Invitrogen).
  • transgenic animals to express glycosylated TEM5 polypeptides.
  • a transgenic milk-producing animal a cow or goat, for example
  • plants to produce TEM5 polypeptides, however, in general, the glycosylation occurring in plants is different from that produced in mammalian cells, and may result in a glycosylated product which is not suitable for human therapeutic use.
  • Host cells comprising a TEM5 polypeptide expression vector may be cultured using standard media well known to the skilled artisan.
  • the media will usually contain all nutrients necessary for the growth and survival of the cells.
  • Suitable media for culturing E. coli cells include, for example, Luria Broth (LB) and/or Terrific Broth (TB).
  • Suitable media for culturing eukaryotic cells include Roswell Park Memorial Institute medium 1640 (RPMI 1640), Minimal Essential Medium (MEM) and/or Dulbecco's Modified Eagle Medium (DMEM), all of which may be supplemented with serum and/or growth factors as necessary for the particular cell line being cultured.
  • a suitable medium for insect cultures is Grace's medium supplemented with yeastolate, lactalbumin hydrolysate, and/or fetal calf serum as necessary.
  • an antibiotic or other compound useful for selective growth of transfected or transformed cells is added as a supplement to the media.
  • the compound to be used will be dictated by the selectable marker element present on the plasmid with which the host cell was transformed.
  • the selectable marker element is kanamycin resistance
  • the compound added to the culture medium will be kanamycin.
  • Other compounds for selective growth include ampicillin, tetracycline, and neomycin.
  • the amount of a TEM5 polypeptide produced by a host cell can be evaluated using standard methods known in the art. Such methods include, without limitation, Western blot analysis, SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, High Performance Liquid Chromatography (HPLC) separation, immunoprecipitation, and/or activity assays such as DNA binding gel shift assays.
  • Western blot analysis SDS-polyacrylamide gel electrophoresis, non-denaturing gel electrophoresis, High Performance Liquid Chromatography (HPLC) separation, immunoprecipitation, and/or activity assays such as DNA binding gel shift assays.
  • HPLC High Performance Liquid Chromatography
  • TEM5 polypeptide has been designed to be secreted from the host cells, the majority of polypeptide may be found in the cell culture medium. If however, the TEM5 polypeptide is not secreted from the host cells, it will be present in the cytoplasm and/or the nucleus (for eukaryotic host cells) or in the cytosol (for gram- negative bacteria host cells).
  • the intracellular material can be extracted from the host cell using any standard technique known to the skilled artisan.
  • the host cells can be lysed to release the contents of the periplasm cytoplasm by French press, homogenization, and/or sonication followed by centrifugation. If a TEM5 polypeptide has formed inclusion bodies in the cytosol, the inclusion bodies can often bind to the inner and/or outer cellular membranes and thus will be found primarily in the pellet material after centrifugation.
  • the pellet material can then be treated at pH extremes or with a chaotropic agent such as a detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies.
  • a chaotropic agent such as a detergent, guanidine, guanidine derivatives, urea, or urea derivatives in the presence of a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies.
  • a reducing agent such as dithiothreitol at alkaline pH or tris carboxyethyl phosphine at acid pH to release, break apart, and solubilize the inclusion bodies.
  • a TEM5 polypeptide may not be biologically active upon isolation.
  • Various methods for "refolding" or converting the polypeptide to its tertiary structure and generating disulfide linkages can be used to restore biological activity. Such methods include exposing the solubilized polypeptide to a pH usually above 7 and in the presence of a particular concentration of a chaotrope. The selection of chaotrope is very similar to the choices used for inclusion body solubilization, but usually the chaotrope is used at a lower concentration and is not necessarily the same as chaotropes used for the solubilization.
  • the refolding/oxidation solution will also contain a reducing agent or the reducing agent plus its oxidized form in a specific ratio to generate a particular redox potential allowing for disulfide shuffling to occur in the formation of the protein's cysteine bridges.
  • Some of the commonly used redox couples include cysteine/cystamine, glutathione (GSH)/dithiobis GSH, cupric chloride, dithiothreitol(DTT)/dithiane DTT, and 2-2-mercaptoethanol(bME)/dithio-b(ME).
  • a cosolvent may be used or may be needed to increase the efficiency of the refolding, and the more common reagents used for this purpose include glycerol, polyethylene glycol of various molecular weights, arginine and the like.
  • polypeptide will be found primarily in the supernatant after centrifugation of the cell homogenate.
  • the polypeptide may be further isolated from the supernatant using methods such as those described herein.
  • TEM5 polypeptide from solution can be accomplished using a variety of techniques. If the polypeptide has been synthesized such that it contains a tag such as Hexahistidine (TEM5 polypeptide/hexaHis) or other small peptide such as FLAG (Eastman Kodak Co., New Haven, CT) or myc (Invitrogen) at either its carboxyl- or amino-terminus, it may be purified in a one-step process by passing the solution through an affinity column where the column matrix has a high affinity for the tag. For example, polyhistidine binds with great affinity and specificity to nickel.
  • a tag such as Hexahistidine (TEM5 polypeptide/hexaHis) or other small peptide such as FLAG (Eastman Kodak Co., New Haven, CT) or myc (Invitrogen) at either its carboxyl- or amino-terminus
  • FLAG Eastman Kodak Co., New Haven, CT
  • myc Invitrogen
  • an affinity column of nickel (such as the Qiagen ® nickel columns) can be used for purification of TEM5 polypeptide/polyHis. See, e.g., Current Protocols in
  • TEM5 polypeptides may be purified through the use of a monoclonal antibody that is capable of specifically recognizing and binding to a TEM5 polypeptide.
  • Suitable procedures for purification include, without limitation, affinity chromatography, immunoaffinity chromatography, ion exchange chromatography, molecular sieve chromatography, HPLC, electrophoresis (including native gel electrophoresis) followed by gel elution, and preparative isoelectric focusing ("Isoprime” machine/technique, Hoefer Scientific, San Francisco, CA).
  • two or more purification techniques may be combined to achieve increased purity.
  • TEM5 polypeptides may also be prepared by chemical synthesis methods
  • Such polypeptides may be synthesized with or without a methionine on the amino-terminus.
  • Chemically synthesized TEM5 polypeptides may be oxidized using methods set forth in these references to form disulfide bridges.
  • Chemically synthesized TEM5 polypeptides are expected to have comparable biological activity to the corresponding TEM5 polypeptides produced recombinantly or purified from natural sources, and thus may be used interchangeably with a recombinant or natural TEM5 polypeptide.
  • Another means of obtaining TEM5 polypeptide is via purification from biological samples such as source tissues and/or fluids in which the TEM5 polypeptide is naturally found. Such purification can be conducted using methods for protein purification as described herein. The presence of the TEM5 polypeptide during purification may be monitored, for example, using an antibody prepared against recombinantly produced TEM5 polypeptide or peptide fragments thereof.
  • the procedure involves generating a heterogeneous pool of oligonucleotides, each having a 5' randomized sequence, a central preselected sequence, and a 3' randomized sequence.
  • the resulting heterogeneous pool is introduced into a population of cells that do not exhibit the desired biological function.
  • Subpopulations of the cells are then screened for those that exhibit a predetermined biological function. From that subpopulation, oligonucleotides capable of carrying out the desired biological function are isolated.
  • U.S. Patent Nos. 5,763,192; 5,814,476; 5,723,323; and 5,817,483 describe processes for producing peptides or polypeptides. This is done by producing stochastic genes or fragments thereof, and then introducing these genes into host cells which produce one or more proteins encoded by the stochastic genes. The host cells are then screened to identify those clones producing peptides or polypeptides having the desired activity.
  • RAGE-GD Random Activation of Gene Expression for Gene Discovery
  • the process involves the activation of endogenous gene expression or over-expression of a gene by in situ recombination methods. For example, expression of an endogenous gene is activated or increased by integrating a regulatory sequence into the target cell that is capable of activating expression of the gene by non-homologous or illegitimate recombination.
  • the target DNA is first subjected to radiation, and a genetic promoter inserted. The promoter eventually locates a break at the front of a gene, initiating transcription of the gene. This results in expression of the desired peptide or polypeptide.
  • nucleic acid and polypeptide molecules described herein may be produced by recombinant and other means.
  • selective binding agent refers to a molecule that has specificity for one or more TEM5 polypeptides.
  • Suitable selective binding agents include, but are not limited to, antibodies and derivatives thereof, polypeptides, and small molecules. Suitable selective binding agents may be prepared using methods known in the art.
  • An exemplary TEM5 polypeptide selective binding agent of the present invention is capable of binding a certain portion of the TEM5 polypeptide thereby inhibiting the binding of the polypeptide to a TEM5 polypeptide receptor.
  • the antibodies may be polyclonal including monospecific polyclonal; monoclonal (MAbs); recombinant; chimeric; humanized, such as complementarity-determining region (CDR)-grafted; human; single chain; and/or bispecific; as well as fragments; variants; or derivatives thereof.
  • Antibody fragments include those portions of the antibody that bind to an epitope on the TEM5 polypeptide. Examples of such fragments include
  • Fab and F(ab') fragments generated by enzymatic cleavage of full-length antibodies.
  • Other binding fragments include those generated by recombinant DNA techniques, such as the expression of recombinant plasmids containing nucleic acid sequences encoding antibody variable regions.
  • Polyclonal antibodies directed toward a TEM5 polypeptide generally are produced in animals (e.g., rabbits or mice) by means of multiple subcutaneous or intraperitoneal injections of TEM5 polypeptide and an adjuvant.
  • a TEM5 polypeptide may be conjugated to a carrier protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum, albumin, bovine thyroglobulin, or soybean trypsin inhibitor.
  • a carrier protein that is immunogenic in the species to be immunized
  • aggregating agents such as alum are used to enhance the immune response. After immunization, the animals are bled and the serum is assayed for anti-TEM5 antibody titer.
  • Monoclonal antibodies directed toward TEM5 polypeptides are produced using any method that provides for the production of antibody molecules by continuous cell lines in culture.
  • suitable methods for preparing monoclonal antibodies include the hybridoma methods of Kohler et al, 1975, Nature 256:495-97 and the human B-cell hybridoma method (Kozbor, 1984, /. Immunol. 133:3001; Brön et al, Monoclonal Antibody Production Techniques and Applications 51-63 (Marcel Dekker, Inc., 1987).
  • Also provided by the invention are hybridoma cell lines that produce monoclonal antibodies reactive with TEM5 polypeptides.
  • Monoclonal antibodies of the invention may be modified for use as therapeutics.
  • One embodiment is a "chimeric" antibody in which a portion of the heavy (H) and/or light (L) chain is identical with or homologous to a corresponding sequence in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is/are identical with or homologous to a corresponding sequence in antibodies derived from another species or belonging to another antibody class or subclass.
  • fragments of such antibodies so long as they exhibit the desired biological activity. See U.S. Patent No. 4,816,567; Morrison et al, 1985, Proc. Natl. Acad. Sci. 81:6851-55.
  • a monoclonal antibody of the invention is a "humanized" antibody.
  • Methods for humanizing non-human antibodies are well known in the art. See U.S. Patent Nos. 5,585,089 and 5,693,762.
  • a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Humanization can be performed, for example, using methods described in the art (Jones et al, 1986, Nature 321:522-25; Riechmann et al, 1998, Nature 332:323-27; Verhoeyen et al, 1988, Science 239:1534-36), by substituting at least a portion of a rodent complementarity-determining region for the corresponding regions of a human antibody.
  • human antibodies that bind TEM5 polypeptides.
  • transgenic animals e.g., mice
  • a TEM5 polypeptide antigen i.e., having at least 6 contiguous amino acids
  • a carrier i.e., having at least 6 contiguous amino acids
  • such transgenic animals are produced by incapacitating the endogenous loci encoding the heavy and light immunoglobulin chains therein, and inserting loci encoding human heavy and light chain proteins into the genome thereof.
  • Partially modified animals that is animals having less than the full complement of modifications, are then cross-bred to obtain an animal having all of the desired immune system modifications.
  • these transgenic animals produce antibodies with human (rather than, e.g., murine) amino acid sequences, including variable regions that are immunospecific for these antigens. See International Pub. Nos. WO 96/33735 and WO 94/02602. Additional methods are described in U.S. Patent No. 5,545,807, International Pub. Nos.
  • Human antibodies can also be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.
  • human antibodies can also be produced from phage-display libraries (Hoogenboom et al, 1991, / Mol. Biol. 227:381; Marks et al, 1991, /. Mol. Biol. 222:581). These processes mimic immune selection through the display of antibody repertoires on the surface of filamentous bacteriophage, and subsequent selection of phage by their binding to an antigen of choice.
  • phage-display libraries Hoogenboom et al, 1991, / Mol. Biol. 227:381; Marks et al, 1991, /. Mol. Biol. 222:581).
  • phage-display libraries Hoogenboom et al, 1991, / Mol. Biol. 227:381; Marks et al, 1991, /. Mol. Biol. 222:581).
  • phage-display libraries Hoogenboom et al, 1991, / Mol. Biol. 227:381; Marks et al, 1991,
  • Nucleic acids encoding the antibodies are introduced into host cells and expressed using materials and procedures described herein.
  • the antibodies are produced in mammalian host cells, such as CHO cells.
  • Monoclonal (e.g., human) antibodies may be produced by the expression of recombinant DNA in host cells or by expression in hybridoma cells as described herein.
  • the anti-TEM5 antibodies of the invention may be employed in any known assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Sola, Monoclonal Antibodies: A Manual of Techniques 147-158 (CRC Press, Inc., 1987)) for the detection and quantitation of
  • TEM5 polypeptides The antibodies will bind TEM5 polypeptides with an affinity that is appropriate for the assay method being employed.
  • anti-TEM5 antibodies may be labeled with a detectable moiety.
  • the detectable moiety can be any one that is capable of producing, either directly or indirectly, a detectable signal.
  • the detectable moiety may be a radioisotope, such as 3 H, 14 C, 32 P, 35 S, 125 1, 99 Tc, ⁇ u ln, or 67 Ga; a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin; or an enzyme, such as alkaline phosphatase, ⁇ -galactosidase, or horseradish peroxidase (Bayer, et al, 1990, Meth. Enz. 184:138- 63).
  • a labeled standard e.g., a TEM5 polypeptide, or an immunologically reactive portion thereof
  • analyte an TEM5 polypeptide
  • the amount of a TEM5 polypeptide in the test sample is inversely proportional to the amount of standard that becomes bound to the antibodies.
  • the antibodies typically are insolubilized before or after the competition, so that the standard and analyte that are bound to the antibodies may conveniently be separated from the standard and analyte that remain unbound.
  • Sandwich assays typically involve the use of two antibodies, each capable of binding to a different immunogenic portion, or epitope, of the protein to be detected and or quantitated.
  • the test sample analyte is typically bound by a first antibody that is immobilized on a solid support, and thereafter a second antibody binds to the analyte, thus forming an insoluble three-part complex.
  • the second antibody may itself be labeled with a detectable moiety (direct sandwich assays) or may be measured using an anti- immunoglobulin antibody that is labeled with a detectable moiety (indirect sandwich assays).
  • sandwich assay is an enzyme-linked immunosorbent assay (ELISA), in which case the detectable moiety is an enzyme.
  • the selective binding agents are also useful for in vivo imaging.
  • An antibody labeled with a detectable moiety may be administered to an animal, preferably into the bloodstream, and the presence and location of the labeled antibody in the host assayed.
  • the antibody may be labeled with any moiety that is detectable in an animal, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.
  • Selective binding agents of the invention may be used as therapeutics. These therapeutic agents are generally agonists or antagonists, in that they either enhance or reduce, respectively, at least one of the biological activities of a TEM5 polypeptide.
  • antagonist antibodies of the invention are antibodies or binding fragments thereof which are capable of specifically binding to a TEM5 polypeptide and which are capable of inhibiting or eliminating the functional activity of a TEM5 polypeptide in vivo or in vitro.
  • the selective binding agent e.g., an antagonist antibody, will inhibit the functional activity of a TEM5 polypeptide by at least about 50%, and preferably by at least about 80%.
  • the selective binding agent may be an anti-TEM5 polypeptide antibody that is capable of interacting with a TEM5 polypeptide binding partner (a ligand or receptor) thereby inhibiting or eliminating TEM5 polypeptide activity in vitro or in vivo.
  • a TEM5 polypeptide binding partner a ligand or receptor
  • Selective binding agents including agonist and antagonist anti-TEM5 polypeptide antibodies, are identified by screening assays that are well known in the art.
  • the invention also relates to a kit comprising TEM5 selective binding agents (such as antibodies) and other reagents useful for detecting TEM5 polypeptide levels in biological samples.
  • TEM5 selective binding agents such as antibodies
  • Such reagents may include a detectable label, blocking serum, positive and negative control samples, and detection reagents.
  • DNA microarray technology can be utilized in accordance with the present invention.
  • DNA microarrays are miniature, high-density arrays of nucleic acids positioned on a solid support, such as glass. Each cell or element within the array contains numerous copies of a single nucleic acid species that acts as a target for hybridization with a complementary nucleic acid sequence (e.g., mRNA).
  • mRNA is first extracted from a cell or tissue sample and then converted enzymatically to fluorescently labeled cDNA. This material is hybridized to the microarray and unbound cDNA is removed by washing.
  • the expression of discrete genes represented on the array is then visualized by quantitating the amount of labeled cDNA that is specifically bound to each target nucleic acid molecule. In this way, the expression of thousands of genes can be quantitated in a high throughput, parallel manner from a single sample of biological material.
  • This high throughput expression profiling has a broad range of applications with respect to the TEM5 molecules of the invention, including, but not limited to: the identification and validation of TEM5 disease-related genes as targets for therapeutics; molecular toxicology of related TEM5 molecules and inhibitors thereof; stratification of populations and generation of surrogate markers for clinical trials; and enhancing related TEM5 polypeptide small molecule drug discovery by aiding in the identification of selective compounds in high throughput screens.
  • TEM5 polypeptide derivatives may be prepared by one skilled in the art, given the disclosures described herein.
  • TEM5 polypeptide derivatives are modified in a manner that is different - either in the type or location of the molecules naturally attached to the polypeptide.
  • Derivatives may include molecules formed by the deletion of one or more naturally-attached chemical groups.
  • the polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or other TEM5 polypeptide may be modified by the covalent attachment of one or more polymers.
  • the polymer selected is typically water-soluble so that the protein to which it is attached does not precipitate in an aqueous environment, such as a physiological environment. Included within the scope of suitable polymers is a mixture of polymers.
  • the polymer will be pharmaceutically acceptable.
  • the polymers each may be of any molecular weight and may be branched or unbranched.
  • the polymers each typically have an average molecular weight of between about 2 kDa to about 100 kDa (the term "about” indicating that in preparations of a water-soluble polymer, some molecules will weigh more, some less, than the stated molecular weight).
  • the average molecular weight of each polymer is preferably between about 5 kDa and about 50 kDa, more preferably between about 12 kDa and about 40 kDa and most preferably between about 20 kDa and about 35 kDa.
  • Suitable water-soluble polymers or mixtures thereof include, but are not limited to, N-linked or 0-linked carbohydrates, sugars, phosphates, polyethylene glycol (PEG) (including the forms of PEG that have been used to derivatize proteins, including mono-(C 1 -C 1 o), alkoxy-, or aryloxy-polyethylene glycol), monomethoxy- polyethylene glycol, dextran (such as low molecular weight dextran of, for example, about 6 kD), cellulose, or other carbohydrate based polymers, poly-(N-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, polypropylene oxide/ethylene oxide co-polymers, polyoxyethylated polyols (e.g., glycerol), and polyvinyl alcohol.
  • bifunctional crosslinking molecules that may be used to prepare covalently attached TEM5 polypeptide multimers.
  • chemical derivatization may be performed under any suitable condition used to react a protein with an activated polymer molecule.
  • Methods for preparing chemical derivatives of polypeptides will generally comprise the steps of: (a) reacting the polypeptide with the activated polymer molecule (such as a reactive ester or aldehyde derivative of the polymer molecule) under conditions whereby the polypeptide comprising the amino acid sequence of SEQ ID NO: 2, or other TEM5 polypeptide, becomes attached to one or more polymer molecules, and (b) obtaining the reaction products.
  • the optimal reaction conditions will be determined based on known parameters and the desired result.
  • the TEM5 polypeptide derivative may have a single polymer molecule moiety at the amino-terminus.
  • the pegylation of a polypeptide may be specifically carried out using any of the pegylation reactions known in the art. Such reactions are described, for example, in the following references: Francis et al, 1992, Focus on Growth Factors 3:4-10; European Patent Nos. 0154316 and 0401384; and U.S. Patent No. 4,179,337.
  • pegylation may be carried out via an acylation reaction or an alkylation reaction with a reactive polyethylene glycol molecule (or an analogous reactive water- soluble polymer) as described herein.
  • a selected polymer should have a single reactive ester group.
  • a selected polymer should have a single reactive aldehyde group.
  • a reactive aldehyde is, for example, polyethylene glycol propionaldehyde, which is water stable, or mono Ci- o alkoxy or aryloxy derivatives thereof (see U.S. Patent No. 5,252,714).
  • TEM5 polypeptides may be chemically coupled to biotin.
  • the biotin/TEM5 polypeptide molecules are then allowed to bind to avidin, resulting in tetravalent avidin/biotin/TEM5 polypeptide molecules.
  • TEM5 polypeptides may also be covalently coupled to dinitrophenol (DNP) or trinitrophenol (TNP) and the resulting conjugates precipitated with anti-DNP or anti-TNP-IgM to form decameric conjugates with a valency of 10.
  • DNP dinitrophenol
  • TNP trinitrophenol
  • conditions that may be alleviated or modulated by the administration of the present TEM5 polypeptide derivatives include those described herein for TEM5 polypeptides.
  • the TEM5 polypeptide derivatives disclosed herein may have additional activities, enhanced or reduced biological activity, or other characteristics, such as increased or decreased half -life, as compared to the non-derivatized molecules.
  • non-human animals such as mice, rats, or other rodents; rabbits, goats, sheep, or other farm animals, in which the genes encoding native TEM5 polypeptide have been disrupted (i.e., "knocked out") such that the level of expression of TEM5 polypeptide is significantly decreased or completely abolished.
  • Such animals may be prepared using techniques and methods such as those described in U.S. Patent No. 5,557,032.
  • the present invention further includes non-human animals such as mice, rats, or other rodents; rabbits, goats, sheep, or other farm animals, in which either the native form of a TEM5 gene for that animal or a heterologous TEM5 gene is over- expressed by the animal, thereby creating a "transgenic" animal.
  • non-human animals such as mice, rats, or other rodents; rabbits, goats, sheep, or other farm animals, in which either the native form of a TEM5 gene for that animal or a heterologous TEM5 gene is over- expressed by the animal, thereby creating a "transgenic" animal.
  • Such transgenic animals may be prepared using well known methods such as those described in U.S. Patent No 5,489,743 and International Pub. No. WO 94/28122.
  • the present invention further includes non-human animals in which the promoter for one or more of the TEM5 polypeptides of the present invention is either activated or inactivated (e.g., by using homologous recombination methods) to alter the level of expression of one or more of the native TEM5 polypeptides.
  • non-human animals may be used for drug candidate screening.
  • the impact of a drug candidate on the animal may be measured.
  • drug candidates may decrease or increase the expression of the TEM5 gene.
  • the amount of TEM5 polypeptide that is produced may be measured after the exposure of the animal to the drug candidate.
  • one may detect the actual impact of the drug candidate on the animal. For example, over-expression of a particular gene may result in, or be associated with, a disease or pathological condition. In such cases, one may test a drug candidate's ability to decrease expression of the gene or its ability to prevent or inhibit a pathological condition.
  • the production of a particular metabolic product such as a fragment of a polypeptide, may result in, or be associated with, a disease or pathological condition.
  • a drug candidate may test a drug candidate's ability to decrease the production of such a metabolic product or its ability to prevent or inhibit a pathological condition.
  • TEM5 Polypeptide Activity it may be desirable to identify molecules that are modulators, i.e., agonists or antagonists, of the activity of TEM5 polypeptide.
  • Natural or synthetic molecules that modulate TEM5 polypeptide may be identified using one or more screening assays, such as those described herein. Such molecules may be administered either in an ex vivo manner or in an in vivo manner by injection, or by oral delivery, implantation device, or the like.
  • Test molecule refers to a molecule that is under evaluation for the ability to modulate (i.e., increase or decrease) the activity of a TEM5 polypeptide. Most commonly, a test molecule will interact directly with a TEM5 polypeptide. However, it is also contemplated that a test molecule may also modulate TEM5 polypeptide activity indirectly, such as by affecting TEM5 gene expression, or by binding to a TEM5 polypeptide binding partner (e.g., receptor or ligand).
  • a TEM5 polypeptide binding partner e.g., receptor or ligand
  • a test molecule will bind to a TEM5 polypeptide with an affinity constant of at least about IO "6 M, preferably about 10 "8 M, more preferably about IO "9 M, and even more preferably about 10 "10 M.
  • Methods for identifying compounds that interact with TEM5 polypeptides are encompassed by the present invention.
  • a TEM5 polypeptide is incubated with a test molecule under conditions that permit the interaction of the test molecule with a TEM5 polypeptide, and the extent of the interaction is measured.
  • the test molecule can be screened in a substantially purified form or in a crude mixture.
  • a TEM5 polypeptide agonist or antagonist may be a protein, peptide, carbohydrate, lipid, or small molecular weight molecule that interacts with TEM5 polypeptide to regulate its activity.
  • Molecules which regulate TEM5 polypeptide expression include nucleic acids which are complementary to nucleic acids encoding a TEM5 polypeptide, or are complementary to nucleic acids sequences which direct or control the expression of TEM5 polypeptide, and which act as anti-sense regulators of expression.
  • test molecule Once a test molecule has been identified as interacting with a TEM5 polypeptide, the molecule may be further evaluated for its ability to increase or decrease TEM5 polypeptide activity.
  • the measurement of the interaction of a test molecule with TEM5 polypeptide may be carried out in several formats, including cell-based binding assays, membrane binding assays, solution-phase assays, and immunoassays.
  • a test molecule is incubated with a TEM5 polypeptide for a specified period of time, and TEM5 polypeptide activity is determined by one or more assays for measuring biological activity.
  • test molecules with TEM5 polypeptides may also be assayed directly using polyclonal or monoclonal antibodies in an immunoassay.
  • modified forms of TEM5 polypeptides containing epitope tags as described herein may be used in solution and immunoassays.
  • TEM5 polypeptides display biological activity through an interaction with a binding partner (e.g., a receptor or a ligand)
  • a variety of in vitro assays may be used to measure the binding of a TEM5 polypeptide to the corresponding binding partner (such as a selective binding agent, receptor, or ligand). These assays may be used to screen test molecules for their ability to increase or decrease the rate and/or the extent of binding of a TEM5 polypeptide to its binding partner.
  • a TEM5 polypeptide is immobilized in the wells of a microtiter plate.
  • Radiolabeled TEM5 polypeptide binding partner for example, iodinated TEM5 polypeptide binding partner
  • a test molecule can then be added either one at a time (in either order) or simultaneously to the wells. After incubation, the wells can be washed and counted for radioactivity, using a scintillation counter, to determine the extent to which the binding partner bound to the TEM5 polypeptide.
  • a molecule will be tested over a range of concentrations, and a series of control wells lacking one or more elements of the test assays can be used for accuracy in the evaluation of the results.
  • An alternative to this method involves reversing the "positions" of the proteins, i.e., immobilizing TEM5 polypeptide binding partner to the microtiter plate wells, incubating with the test molecule and radiolabeled TEM5 polypeptide, and determining the extent of TEM5 polypeptide binding. See, e.g., Current Protocols in Molecular Biology, chap. 18 (Ausubel et al, eds., Green Publishers Inc. and Wiley and Sons 1995).
  • a TEM5 polypeptide or its binding partner may be conjugated to biotin, and the presence of biotinylated protein can then be detected using streptavidin linked to an enzyme, such as horse radish peroxidase (HRP) or alkaline phosphatase (AP), which can be detected colorometrically, or by fluorescent tagging of streptavidin.
  • HRP horse radish peroxidase
  • AP alkaline phosphatase
  • An antibody directed to a TEM5 polypeptide or to a TEM5 polypeptide binding partner, and which is conjugated to biotin may also be used for purposes of detection following incubation of the complex with enzyme- linked streptavidin linked to AP or HRP.
  • a TEM5 polypeptide or a TEM5 polypeptide binding partner can also be immobilized by attachment to agarose beads, acrylic beads, or other types of such inert solid phase substrates.
  • the substrate-protein complex can be placed in a solution containing the complementary protein and the test compound. After incubation, the beads can be precipitated by centrifugation, and the amount of binding between a TEM5 polypeptide and its binding partner can be assessed using the methods described herein.
  • the substrate-protein complex can be immobilized in a column with the test molecule and complementary protein passing through the column. The formation of a complex between a TEM5 polypeptide and its binding partner can then be assessed using any of the techniques described herein (e.g., radiolabelling or antibody binding) .
  • a surface plasmon resonance detector system such as the BIAcore assay system (Pharmacia, Piscataway, NJ).
  • the BIAcore system is utilized as specified by the manufacturer.
  • This assay essentially involves the covalent binding of either TEM5 polypeptide or a TEM5 polypeptide binding partner to a dextran-coated sensor chip that is located in a detector.
  • the test compound and the other complementary protein can then be injected, either simultaneously or sequentially, into the chamber containing the sensor chip.
  • the amount of complementary protein that binds can be assessed based on the change in molecular mass that is physically associated with the dextran-coated side of the sensor chip, with the change in molecular mass being measured by the detector system.
  • test compounds it may be desirable to evaluate two or more test compounds together for their ability to increase or decrease the formation of a complex between a
  • TEM5 polypeptide and a TEM5 polypeptide binding partner a TEM5 polypeptide binding partner.
  • the assays set forth herein can be readily modified by adding such additional test compound(s) either simultaneously with, or subsequent to, the first test compound. The remainder of the steps in the assay are as set forth herein.
  • In vitro assays such as those described herein may be used advantageously to screen large numbers of compounds for an effect on the formation of a complex between a TEM5 polypeptide and TEM5 polypeptide binding partner.
  • the assays may be automated to screen compounds generated in phage display, synthetic peptide, and chemical synthesis libraries.
  • Compounds which increase or decrease the formation of a complex between a TEM5 polypeptide and a TEM5 polypeptide binding partner may also be screened in cell culture using cells and cell lines expressing either TEM5 polypeptide or TEM5 polypeptide binding partner.
  • Cells and cell lines may be obtained from any mammal, but preferably will be from human or other primate, canine, or rodent sources.
  • the binding of a TEM5 polypeptide to cells expressing TEM5 polypeptide binding partner at the surface is evaluated in the presence or absence of test molecules, and the extent of binding may be determined by, for example, flow cytometry using a biotinylated antibody to a TEM5 polypeptide binding partner.
  • Cell culture assays can be used advantageously to further evaluate compounds that score positive in protein binding assays described herein.
  • Cell cultures can also be used to screen the impact of a drug candidate.
  • drug candidates may decrease or increase the expression of the TEM5 gene.
  • the amount of TEM5 polypeptide or a TEM5 polypeptide fragment that is produced may be measured after exposure of the cell culture to the drug candidate.
  • one may detect the actual impact of the drug candidate on the cell culture.
  • the over-expression of a particular gene may have a particular impact on the cell culture. In such cases, one may test a drug candidate's ability to increase or decrease the expression of the gene or its ability to prevent or inhibit a particular impact on the cell culture.
  • the production of a particular metabolic product such as a fragment of a polypeptide, may result in, or be associated with, a disease or pathological condition.
  • a drug candidate may test a drug candidate's ability to decrease the production of such a metabolic product in a cell culture.
  • the tat protein sequence (from HJV) can be used to internalize proteins into a cell. See, e.g., Falwell et al, 1994, Proc. Natl. Acad. Sci. USA. 91:664-68.
  • an 11 amino acid sequence (Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 4) of the HJV tat protein (termed the "protein transduction domain," or TAT PDT) has been described as mediating delivery across the cytoplasmic membrane and the nuclear membrane of a cell. See Schwarze et al, 1999, Science 285:1569-72; and Nagahara et al, 1998, Nat. Med. 4:1449-52.
  • FITC-constructs (FITC- labeled G-G-G-G- Y-G-R-K-K-R-R-Q-R-R-R; SEQ ID NO: 5), which penetrate tissues following intraperitoneal administration, are prepared, and the binding of such constructs to cells is detected by fluorescence-activated cell sorting (FACS) analysis.
  • FACS fluorescence-activated cell sorting
  • Cells treated with a t t- ⁇ -gal fusion protein will demonstrate ⁇ -gal activity.
  • expression of such a construct can be detected in a number of tissues, including liver, kidney, lung, heart, and brain tissue. It is believed that such constructs undergo some degree of unfolding in order to enter the cell, and as such, may require a refolding following entry into the cell.
  • the tat protein sequence may be used to internalize a desired polypeptide into a cell.
  • a TEM5 antagonist such as an anti-TEM5 selective binding agent, small molecule, soluble receptor, or antisense oligonucleotide
  • TEM5 molecule refers to both TEM5 nucleic acid molecules and TEM5 polypeptides as defined herein.
  • the TEM5 protein itself may also be internally administered to a cell using these procedures. See also, Straus, 1999, Science 285:1466-67.
  • nucleic acids encoding a TEM5 polypeptide can be used as a probe to identify cells described herein by screening the nucleic acids of the cells with such a probe.
  • anti-TEM5 polypeptide antibodies to test for the presence of TEM5 polypeptide in cells, and thus, determine if such cells are of the types described herein.
  • TEM5 Polypeptide compositions and Administration Therapeutic compositions are within the scope of the present invention.
  • Such TEM5 polypeptide pharmaceutical compositions may comprise a therapeutically effective amount of a TEM5 polypeptide or a TEM5 nucleic acid molecule in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
  • Pharmaceutical compositions may comprise a therapeutically effective amount of one or more TEM5 polypeptide selective binding agents in admixture with a pharmaceutically or physiologically acceptable formulation agent selected for suitability with the mode of administration.
  • Acceptable formulation materials preferably are nontoxic to recipients at the dosages and concentrations employed.
  • the pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition.
  • Suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine, or lysine), antimicrobials, antioxidants (such as ascorbic acid, sodium sulfite, or sodium hydrogen-sulfite), buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates, or other organic acids), bulking agents (such as mannitol or glycine), chelating agents (such as ethylenediamine tetraacetic acid (EDTA)), complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin, or hydroxypropyl- beta-cyclodextrin), fillers, monosaccharides, disaccharides, and other carbohydrates (such as glucose, mannose, or dextrins), proteins (such as serum albumin, gelatin, or immunoglobulins), coloring, flavoring and diluting agents, emulsifying agents
  • the optimal pharmaceutical composition will be determined by a skilled artisan depending upon, for example, the intended route of administration, delivery format, and desired dosage. See, e.g., Remington's Pharmaceutical Sciences, supra.
  • compositions may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the TEM5 molecule.
  • the primary vehicle or carrier in a pharmaceutical composition may be either aqueous or non-aqueous in nature.
  • a suitable vehicle or carrier for injection may be water, physiological saline solution, or artificial cerebrospinal fluid, possibly supplemented with other materials common in compositions for parenteral administration.
  • Neutral buffered saline or saline mixed with serum albumin are further exemplary vehicles.
  • Other exemplary pharmaceutical compositions comprise Tris buffer of about pH 7.0-8.5, or acetate buffer of about pH 4.0-5.5, which may further include sorbitol or a suitable substitute.
  • TEM5 polypeptide compositions may be prepared for storage by mixing the selected composition having the desired degree of purity with optional formulation agents (Remington's Pharmaceutical Sciences, supra) in the form of a lyophilized cake or an aqueous solution. Further, the TEM5 polypeptide product may be formulated as a lyophilizate using appropriate excipients such as sucrose.
  • the TEM5 polypeptide pharmaceutical compositions can be selected for parenteral delivery. Alternatively, the compositions may be selected for inhalation or for delivery through the digestive tract, such as orally.
  • the preparation of such pharmaceutically acceptable compositions is within the skill of the art.
  • the formulation components are present in concentrations that are acceptable to the site of administration. For example, buffers are used to maintain the composition at physiological pH or at a slightly lower pH, typically within a pH range of from about 5 to about 8.
  • the therapeutic compositions for use in this invention may be in the form of a pyrogen-free, parenterally acceptable, aqueous solution comprising the desired TEM5 molecule in a pharmaceutically acceptable vehicle.
  • a particularly suitable vehicle for parenteral injection is sterile distilled water in which a TEM5 molecule is formulated as a sterile, isotonic solution, properly preserved.
  • Yet another preparation can involve the formulation of the desired molecule with an agent, such as injectable microspheres, bio-erodible particles, polymeric compounds (such as polylactic acid or polyglycolic acid), beads, or liposomes, that provides for the controlled or sustained release of the product which may then be delivered via a depot injection.
  • Hyaluronic acid may also be used, and this may have the effect of promoting sustained duration in the circulation.
  • Other suitable means for the introduction of the desired molecule include implantable drug delivery devices.
  • a pharmaceutical composition may be formulated for inhalation.
  • TEM5 polypeptide may be formulated as a dry powder for inhalation.
  • TEM5 polypeptide or nucleic acid molecule inhalation solutions may also be formulated with a propellant for aerosol delivery.
  • solutions may be nebulized. Pulmonary administration is further described in International Pub. No. WO 94/20069, which describes the pulmonary delivery of chemically modified proteins. It is also contemplated that certain formulations may be administered orally.
  • TEM5 polypeptides that are administered in this fashion can be formulated with or without those carriers customarily used in the compounding of solid dosage forms such as tablets and capsules.
  • a capsule may be designed to release the active portion of the formulation at the point in the gastrointestinal tract when bioavailability is maximized and pre-systemic degradation is minimized.
  • Additional agents can be included to facilitate absorption of the TEM5 polypeptide. Diluents, flavorings, low melting point waxes, vegetable oils, lubricants, suspending agents, tablet disintegrating agents, and binders may also be employed.
  • Another pharmaceutical composition may involve an effective quantity of
  • TEM5 polypeptides in a mixture with non-toxic excipients that are suitable for the manufacture of tablets.
  • Suitable excipients include, but are not limited to, inert diluents, such as calcium carbonate, sodium carbonate or bicarbonate, lactose, or calcium phosphate; or binding agents, such as starch, gelatin, or acacia; or lubricating agents such as magnesium stearate, stearic acid, or talc.
  • Additional TEM5 polypeptide pharmaceutical compositions will be evident to those skilled in the art, including formulations involving TEM5 polypeptides in sustained- or controlled-delivery formulations.
  • Techniques for formulating a variety of other sustained- or controlled-delivery means such as liposome carriers, bio- erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. See, e.g., International Pub. No. WO 93/15722, which describes the controlled release of porous polymeric microparticles for the delivery of pharmaceutical compositions.
  • sustained-release preparations include semipermeable polymer matrices in the form of shaped articles, e.g. films, or microcapsules.
  • Sustained release matrices may include polyesters, hydrogels, polylactides (U.S. Patent No. 3,773,919 and European Patent No. 058481), copolymers of L-glutamic acid and gamma ethyl-L-glutamate (Sidman et al, 1983, Biopolymers 22:547-56), poly(2-hydroxyethyl-methacrylate) (Langer et al, 1981, /. Biomed. Mater. Res. 15:167-277 and Langer, 1982, Chem. Tech.
  • Sustained-release compositions may also include liposomes, which can be prepared by any of several methods known in the art. See, e.g., Eppstein et al, 1985, Proc. Natl. Acad. Sci. USA 82:3688-92; and European Patent Nos. 036676, 088046, and 143949.
  • the pharmaceutical composition may be stored in sterile vials as a solution, suspension, gel, emulsion, solid, or as a dehydrated or lyophilized powder.
  • Such formulations may be stored either in a ready-to-use form or in a form (e.g. , lyophilized) requiring reconstitution prior to administration.
  • kits for producing a single-dose administration unit may each contain both a first container having a dried protein and a second container having an aqueous formulation. Also included within the scope of this invention are kits containing single and multi-chambered pre-filled syringes (e.g., liquid syringes and lyosyringes).
  • a TEM5 pharmaceutical composition to be employed therapeutically will depend, for example, upon the therapeutic context and objectives.
  • a typical dosage may range from about 0.1 ⁇ g/kg to up to about 100 mg/kg or more, depending on the factors mentioned above. In other embodiments, the dosage may range from 0.1 ⁇ g/kg up to about 100 mg/kg; or 1 ⁇ g/kg up to about 100 mg/kg; or 5 ⁇ g/kg up to about 100 mg/kg.
  • the frequency of dosing will depend upon the pharmacokinetic parameters of the TEM5 molecule in the formulation being used. Typically, a clinician will administer the composition until a dosage is reached that achieves the desired effect.
  • the composition may therefore be administered as a single dose, as two or more doses (which may or may not contain the same amount of the desired molecule) over time, or as a continuous infusion via an implantation device or catheter. Further refinement of the appropriate dosage is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them. Appropriate dosages may be ascertained through use of appropriate dose-response data.
  • the route of administration of the pharmaceutical composition is in accord with known methods, e.g., orally; through injection by intravenous, intraperitoneal, intracerebral (intraparenchymal), intracerebroventricular, intramuscular, intraocular, intraarterial, intraportal, or intralesional routes; by sustained release systems; or by implantation devices.
  • the compositions may be administered by bolus injection or continuously by infusion, or by implantation device.
  • the composition may be administered locally via implantation of a membrane, sponge, or other appropriate material onto which the desired molecule has been absorbed or encapsulated.
  • the device may be implanted into any suitable tissue or organ, and delivery of the desired molecule may be via diffusion, timed-release bolus, or continuous administration.
  • TEM5 polypeptide pharmaceutical compositions it may be desirable to use TEM5 polypeptide pharmaceutical compositions in an ex vivo manner.
  • cells, tissues, or organs that have been removed from the patient are exposed to TEM5 polypeptide pharmaceutical compositions after which the cells, tissues, or organs are subsequently implanted back into the patient.
  • a TEM5 polypeptide can be delivered by implanting certain cells that have been genetically engineered, using methods such as those described herein, to express and secrete the TEM5 polypeptide.
  • Such cells may be animal or human cells, and may be autologous, heterologous, or xenogeneic.
  • the cells may be immortalized.
  • the cells may be encapsulated to avoid infiltration of surrounding tissues.
  • the encapsulation materials are typically biocompatible, semi-permeable polymeric enclosures or membranes that allow the release of the protein product(s) but prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissues.
  • Additional embodiments of the present invention relate to cells and methods (e.g., homologous recombination and/or other recombinant production methods) for both the in vitro production of therapeutic polypeptides and for the production and delivery of therapeutic polypeptides by gene therapy or cell therapy.
  • Homologous and other recombination methods may be used to modify a cell that contains a normally transcriptionally-silent TEM5 gene, or an under-expressed gene, and thereby produce a cell which expresses therapeutically efficacious amounts of TEM5 polypeptides.
  • Homologous recombination is a technique originally developed for targeting genes to induce or correct mutations in transcriptionally active genes. Kucherlapati, 1989, Prog, in Nucl. Acid Res. & Mol. Biol. 36:301.
  • the basic technique was developed as a method for introducing specific mutations into specific regions of the mammalian genome (Thomas et al, 1986, Cell 44:419-28; Thomas and Capecchi, 1987, Cell 51:503-12; Doetschman et al, 1988, Proc. Natl. Acad. Sci. U.S.A. 85:8583-87) or to correct specific mutations within defective genes (Doetschman et al, 1987, Nature 330:576-78).
  • the DNA sequence to be inserted into the genome can be directed to a specific region of the gene of interest by attaching it to targeting DNA.
  • the targeting DNA is a nucleotide sequence that is complementary (homologous) to a region of the genomic DNA. Small pieces of targeting DNA that are complementary to a specific region of the genome are put in contact with the parental strand during the DNA replication process. It is a general property of DNA that has been inserted into a cell to hybridize, and therefore, recombine with other pieces of endogenous DNA through shared homologous regions.
  • a promoter/enhancer element, a suppressor, or an exogenous transcription modulatory element is inserted in the genome of the intended host cell in proximity and orientation sufficient to influence the transcription of DNA encoding the desired TEM5 polypeptide.
  • the control element controls a portion of the DNA present in the host cell genome.
  • the expression of the desired TEM5 polypeptide may be achieved not by transfection of DNA that encodes the TEM5 gene itself, but rather by the use of targeting DNA (containing regions of homology with the endogenous gene of interest) coupled with DNA regulatory segments that provide the endogenous gene sequence with recognizable signals for transcription of a TEM5 gene.
  • the expression of a desired targeted gene in a cell is altered via homologous recombination into the cellular genome at a preselected site, by the introduction of DNA that includes at least a regulatory sequence, an exon, and a splice donor site.
  • DNA that includes at least a regulatory sequence, an exon, and a splice donor site.
  • These components are introduced into the chromosomal (genomic) DNA in such a manner that this, in effect, results in the production of a new transcription unit (in which the regulatory sequence, the exon, and the splice donor site present in the DNA construct are operatively linked to the endogenous gene).
  • the expression of the desired endogenous gene is altered.
  • Altered gene expression encompasses activating (or causing to be expressed) a gene which is normally silent (unexpressed) in the cell as obtained, as well as increasing the expression of a gene which is not expressed at physiologically significant levels in the cell as obtained.
  • the embodiments further encompass changing the pattern of regulation or induction such that it is different from the pattern of regulation or induction that occurs in the cell as obtained, and reducing (including eliminating) the expression of a gene which is expressed in the cell as obtained.
  • homologous recombination can be used to increase, or cause, TEM5 polypeptide production from a cell's endogenous TEM5 gene involves first using homologous recombination to place a recombination sequence from a site- specific recombination system (e.g., Cre/loxP, FLP/FRT) (Sauer, 1994, Curr. Opin. Biotechnol, 5:521-27; Sauer, 1993, Methods Enzymol, 225:890-900) upstream of (i.e., 5' to) the cell's endogenous genomic TEM5 polypeptide coding region.
  • site-specific recombination system e.g., Cre/loxP, FLP/FRT
  • a plasmid containing a recombination site homologous to the site that was placed just upstream of the genomic TEM5 polypeptide coding region is introduced into the modified cell line along with the appropriate recombinase enzyme.
  • This recombinase causes the plasmid to integrate, via the plasmid' s recombination site, into the recombination site located just upstream of the genomic TEM5 polypeptide coding region in the cell line (Baubonis and Sauer, 1993, Nucleic Acids Res. 21:2025-29; O'Gorman et al, 1991, Science 251:1351-55).
  • flanking sequences known to increase transcription e.g., enhancer/promoter, intron, translational enhancer
  • if properly positioned in this plasmid would integrate in such a manner as to create a new or modified transcriptional unit resulting in de novo or increased TEM5 polypeptide production from the cell's endogenous TEM5 gene.
  • a further method to use the cell line in which the site specific recombination sequence had been placed just upstream of the cell's endogenous genomic TEM5 polypeptide coding region is to use homologous recombination to introduce a second recombination site elsewhere in the cell line's genome.
  • the appropriate recombinase enzyme is then introduced into the two-recombination-site cell line, causing a recombination event (deletion, inversion, and translocation) (Sauer, 1994, Curr. Opin.
  • An additional approach for increasing, or causing, the expression of TEM5 polypeptide from a cell's endogenous TEM5 gene involves increasing, or causing, the expression of a gene or genes (e.g., transcription factors) and/or decreasing the expression of a gene or genes (e.g., transcriptional repressors) in a manner which results in de novo or increased TEM5 polypeptide production from the cell's endogenous TEM5 gene.
  • This method includes the introduction of a non-naturally occurring polypeptide (e.g., a polypeptide comprising a site specific DNA binding domain fused to a transcriptional factor domain) into the cell such that de novo or increased TEM5 polypeptide production from the cell's endogenous TEM5 gene results.
  • the present invention further relates to DNA constructs useful in the method of altering expression of a target gene.
  • the exemplary DNA constructs comprise: (a) one or more targeting sequences, (b) a regulatory sequence, (c) an exon, and (d) an unpaired splice-donor site.
  • the targeting sequence in the DNA construct directs the integration of elements (a) - (d) into a target gene in a cell such that the elements (b) - (d) are operatively linked to sequences of the endogenous target gene.
  • the DNA constructs comprise: (a) one or more targeting sequences, (b) a regulatory sequence, (c) an exon, (d) a splice-donor site, (e) an intron, and (f) a splice-acceptor site, wherein the targeting sequence directs the integration of elements (a) - (f) such that the elements of (b) - (f) are operatively linked to the endogenous gene.
  • the targeting sequence is homologous to the preselected site in the cellular chromosomal DNA with which homologous recombination is to occur.
  • the exon is generally 3' of the regulatory sequence and the splice-donor site is 3' of the exon.
  • sequence of a particular gene is known, such as the nucleic acid sequence of TEM5 polypeptide presented herein
  • a piece of DNA that is complementary to a selected region of the gene can be synthesized or otherwise obtained, such as by appropriate restriction of the native DNA at specific recognition sites bounding the region of interest.
  • This piece serves as a targeting sequence upon insertion into the cell and will hybridize to its homologous region within the genome. If this hybridization occurs during DNA replication, this piece of DNA, and any additional sequence attached thereto, will act as an Okazaki fragment and will be incorporated into the newly synthesized daughter strand of DNA.
  • the present invention therefore, includes nucleotides encoding a TEM5 polypeptide, which nucleotides may be used as targeting sequences.
  • TEM5 polypeptide cell therapy e.g., the implantation of cells producing TEM5 polypeptides
  • This embodiment involves implanting cells capable of synthesizing and secreting a biologically active form of TEM5 polypeptide.
  • Such TEM5 polypeptide-producing cells can be cells that are natural producers of TEM5 polypeptides or may be recombinant cells whose ability to produce TEM5 polypeptides has been augmented by transformation with a gene encoding the desired TEM5 polypeptide or with a gene augmenting the expression of TEM5 polypeptide.
  • Such a modification may be accomplished by means of a vector suitable for delivering the gene as well as promoting its expression and secretion.
  • the natural cells producing TEM5 polypeptide be of human origin and produce human TEM5 polypeptide.
  • the recombinant cells producing TEM5 polypeptide be transformed with an expression vector containing a gene encoding a human TEM5 polypeptide.
  • Implanted cells may be encapsulated to avoid the infiltration of surrounding tissue.
  • Human or non-human animal cells may be implanted in patients in biocompatible, semipermeable polymeric enclosures or membranes that allow the release of TEM5 polypeptide, but that prevent the destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissue.
  • the patient's own cells, transformed to produce TEM5 polypeptides ex vivo may be implanted directly into the patient without such encapsulation.
  • the devices provide for the delivery of the molecules from living cells to specific sites within a recipient.
  • U.S. Patent Nos. 4,892,538; 5,011,472; and 5,106,627 A system for encapsulating living cells is described in International Pub. No. WO 91/10425 (Aebischer et al). See also, International Pub. No. WO 91/10470 (Aebischer et al); Winn et al, 1991, Exper. Neurol. 113:322-29; Aebischer et al, 1991, Exper. Neurol. 111:269-75; and Tresco et al, 1992, ASAIO 38:17-23.
  • TEM5 gene therapy technique is to use the TEM5 gene (either genomic DNA, cDNA, and/or synthetic DNA) encoding a TEM5 polypeptide that may be operably linked to a constitutive or inducible promoter to form a "gene therapy DNA construct."
  • the promoter may be homologous or heterologous to the endogenous TEM5 gene, provided that it is active in the cell or tissue type into which the construct will be inserted.
  • Other components of the gene therapy DNA construct may optionally include DNA molecules designed for site-specific integration (e.g., endogenous sequences useful for homologous recombination), tissue-specific promoters, enhancers or silencers, DNA molecules capable of providing a selective advantage over the parent cell, DNA molecules useful as labels to identify transformed cells, negative selection systems, cell specific binding agents (as, for example, for cell targeting), cell-specific internalization factors, transcription factors enhancing expression from a vector, and factors enabling vector production.
  • DNA molecules designed for site-specific integration e.g., endogenous sequences useful for homologous recombination
  • tissue-specific promoters e.g., enhancers or silencers
  • DNA molecules capable of providing a selective advantage over the parent cell DNA molecules useful as labels to identify transformed cells
  • negative selection systems e.g., cell specific binding agents (as, for example, for cell targeting), cell-specific internalization factors, transcription factors enhancing expression from a vector, and factors enabling vector production.
  • a gene therapy DNA construct can then be introduced into cells (either ex vivo or in vivo) using viral or non- viral vectors.
  • One means for introducing the gene therapy DNA construct is by means of viral vectors as described herein.
  • Certain vectors, such as retroviral vectors will deliver the DNA construct to the chromosomal DNA of the cells, and the gene can integrate into the chromosomal DNA.
  • Other vectors will function as episomes, and the gene therapy DNA construct will remain in the cytoplasm.
  • regulatory elements can be included for the controlled expression of the TEM5 gene in the target cell. Such elements are turned on in response to an appropriate effector. In this way, a therapeutic polypeptide can be expressed when desired.
  • One conventional control means involves the use of small molecule dimerizers or rapalogs to dimerize chimeric proteins which contain a small molecule-binding domain and a domain capable of initiating a biological process, such as a DNA-binding protein or transcriptional activation protein (see International Pub. Nos. WO 96/41865, WO 97/31898, and WO 97/31899).
  • the dimerization of the proteins can be used to initiate transcription of the transgene.
  • An alternative regulation technology uses a method of storing proteins expressed from the gene of interest inside the cell as an aggregate or cluster.
  • the gene of interest is expressed as a fusion protein that includes a conditional aggregation domain that results in the retention of the aggregated protein in the endoplasmic reticulum.
  • the stored proteins are stable and inactive inside the cell.
  • the proteins can be released, however, by administering a drug (e.g., small molecule ligand) that removes the conditional aggregation domain and thereby specifically breaks apart the aggregates or clusters so that the proteins may be secreted from the cell.
  • a drug e.g., small molecule ligand
  • Other suitable control means or gene switches include, but are not limited to, the systems described herein.
  • Mifepristone (RU486) is used as a progesterone antagonist.
  • the binding of a modified progesterone receptor ligand-binding domain to the progesterone antagonist activates transcription by forming a dimer of two transcription factors that then pass into the nucleus to bind DNA.
  • the ligand-binding domain is modified to eliminate the ability of the receptor to bind to the natural ligand.
  • the modified steroid hormone receptor system is further described in U.S. Patent No. 5,364,791 and International Pub. Nos. WO 96/40911 and WO 97/10337.
  • Yet another control system uses ecdysone (a fruit fly steroid hormone) that binds to and activates an ecdysone receptor (cytoplasmic receptor). The receptor then translocates to the nucleus to bind a specific DNA response element (promoter from ecdysone-responsive gene).
  • the ecdysone receptor includes a transactivation domain, DNA-binding domain, and ligand-binding domain to initiate transcription.
  • the ecdysone system is further described in U.S. Patent No. 5,514,578 and International Pub. Nos. WO 97/38117, WO 96/37609, and WO 93/03162.
  • Another control means uses a positive tetracycline-controllable transactivator.
  • This system involves a mutated tet repressor protein DNA-binding domain (mutated tet R-4 amino acid changes which resulted in a reverse tetracycline-regulated transactivator protein, i.e., it binds to a tet operator in the presence of tetracycline) linked to a polypeptide which activates transcription.
  • mutated tet repressor protein DNA-binding domain mutated tet R-4 amino acid changes which resulted in a reverse tetracycline-regulated transactivator protein, i.e., it binds to a tet operator in the presence of tetracycline linked to a polypeptide which activates transcription.
  • In vivo gene therapy may be accomplished by introducing the gene encoding TEM5 polypeptide into cells via local injection of a TEM5 nucleic acid molecule or by other appropriate viral or non- viral delivery vectors. Hefti 1994, Neurobiology 25:1418-35.
  • a nucleic acid molecule encoding a TEM5 polypeptide may be contained in an adeno-associated virus (AAV) vector for delivery to the targeted cells (see, e.g., International Pub. Nos. WO 95/34670 and WO 95/34670).
  • AAV adeno-associated virus
  • the recombinant AAV genome typically contains AAV inverted terminal repeats flanking a DNA sequence encoding a TEM5 polypeptide operably linked to functional promoter and polyadenylation sequences.
  • Alternative suitable viral vectors include, but are not limited to, retrovirus, adenovirus, herpes simplex virus, lentivirus, hepatitis virus, parvovirus, papovavirus, poxvirus, alphavirus, coronavirus, rhabdovirus, paramyxovirus, and papilloma virus vectors.
  • U.S. Patent No. 5,672,344 describes an in vivo viral-mediated gene transfer system involving a recombinant neurotrophic HSV-1 vector.
  • U.S. Patent No. 5,399,346 provides examples of a process for providing a patient with a therapeutic protein by the delivery of human cells that have been treated in vitro to insert a DNA segment encoding a therapeutic protein.
  • Nonviral delivery methods include, but are not limited to, liposome-mediated transfer, naked DNA delivery (direct injection), receptor-mediated transfer (ligand- DNA complex), electroporation, calcium phosphate precipitation, and microparticle bombardment (e.g., gene gun).
  • Gene therapy materials and methods may also include inducible promoters, tissue-specific enhancer-promoters, DNA sequences designed for site-specific integration, DNA sequences capable of providing a selective advantage over the parent cell, labels to identify transformed cells, negative selection systems and expression control systems (safety measures), cell-specific binding agents (for cell targeting), cell-specific internalization factors, and transcription factors to enhance expression by a vector as well as methods of vector manufacture.
  • inducible promoters tissue-specific enhancer-promoters
  • DNA sequences designed for site-specific integration DNA sequences capable of providing a selective advantage over the parent cell, labels to identify transformed cells, negative selection systems and expression control systems (safety measures), cell-specific binding agents (for cell targeting), cell-specific internalization factors, and transcription factors to
  • TEM5 gene therapy or cell therapy can further include the delivery of one or more additional polypeptide(s) in the same or a different cell(s).
  • additional polypeptide(s) in the same or a different cell(s).
  • Such cells may be separately introduced into the patient, or the cells may be contained in a single implantable device, such as the encapsulating membrane described above, or the cells may be separately modified by means of viral vectors.
  • a means to increase endogenous TEM5 polypeptide expression in a cell via gene therapy is to insert one or more enhancer elements into the TEM5 polypeptide promoter, where the enhancer elements can serve to increase transcriptional activity of the TEM5 gene.
  • the enhancer elements used will be selected based on the tissue in which one desires to activate the gene - enhancer elements known to confer promoter activation in that tissue will be selected. For example, if a gene encoding a TEM5 polypeptide is to be "turned on" in T-cells, the lck promoter enhancer element may be used.
  • the functional portion of the transcriptional element to be added may be inserted into a fragment of DNA containing the TEM5 polypeptide promoter (and optionally, inserted into a vector and/or 5' and/or 3' flanking sequences) using standard cloning techniques.
  • This construct known as a "homologous recombination construct," can then be introduced into the desired cells either ex vivo or in vivo.
  • Gene therapy also can be used to decrease TEM5 polypeptide expression by modifying the nucleotide sequence of the endogenous promoter. Such modification is typically accomplished via homologous recombination methods.
  • a DNA molecule containing all or a portion of the promoter of the TEM5 gene selected for inactivation can be engineered to remove and/or replace pieces of the promoter that regulate transcription.
  • the TATA box and/or the binding site of a transcriptional activator of the promoter may be deleted using standard molecular biology techniques; such deletion can inhibit promoter activity thereby repressing the transcription of the corresponding TEM5 gene.
  • the deletion of the TATA box or the transcription activator binding site in the promoter may be accomplished by generating a DNA construct comprising all or the relevant portion of the TEM5 polypeptide promoter (from the same or a related species as the TEM5 gene to be regulated) in which one or more of the TATA box and/or transcriptional activator binding site nucleotides are mutated via substitution, deletion and/or insertion of one or more nucleotides.
  • the TATA box and/or activator binding site has decreased activity or is rendered completely inactive.
  • This construct which also will typically contain at least about 500 bases of DNA that correspond to the native (endogenous) 5' and 3' DNA sequences adjacent to the promoter segment that has been modified, may be introduced into the appropriate cells (either ex vivo or in vivo) either directly or via a viral vector as described herein.
  • the integration of the construct into the genomic DNA of the cells will be via homologous recombination, where the 5' and 3' DNA sequences in the promoter construct can serve to help integrate the modified promoter region via hybridization to the endogenous chromosomal DNA.
  • TEM5 nucleic acid molecules, polypeptides, and agonists and antagonists thereof can be used to treat, diagnose, ameliorate, or prevent a number of diseases, disorders, or conditions, including those recited herein.
  • TEM5 polypeptide agonists and antagonists include those molecules which regulate TEM5 polypeptide activity and either increase or decrease at least one activity of the mature form of the TEM5 polypeptide.
  • Agonists or antagonists may be co-factors, such as a protein, peptide, carbohydrate, lipid, or small molecular weight molecule, which interact with TEM5 polypeptide and thereby regulate its activity.
  • Potential polypeptide agonists or antagonists include antibodies that react with either soluble or membrane-bound forms of TEM5 polypeptides that comprise part or all of the extracellular domains of the said proteins.
  • Molecules that regulate TEM5 polypeptide expression typically include nucleic acids encoding TEM5 polypeptide that can act as anti-sense regulators of expression. Since TEM5 polypeptide expression has been detected in the lung, TEM5 nucleic acid molecules, polypeptides, agonists and antagonists thereof (including, but not limited to, anti-TEM5 selective binding agents) may be useful for the treatment or diagnosis of diseases involving the lung. Examples of such diseases include, but are not limited to, asthma, bronchospasm, and acute respiratory distress syndrome. Other diseases associated with the lung are encompassed within the scope of the invention.
  • TEM5 nucleic acid molecules, polypeptides, agonists and antagonists thereof may be useful for the treatment or diagnosis of diseases involving the heart.
  • diseases include, but are not limited to, arrhythmias, angina, hypertension, myocardial infarction and congestive heart failure.
  • Other diseases associated with the heart are encompassed within the scope of the invention.
  • TEM5 polypeptide expression has been detected in the kidney
  • TEM5 nucleic acid molecules, polypeptides, agonists and antagonists thereof may be useful for the treatment or diagnosis of diseases involving the kidney. Examples of such diseases include, but are not limited to, polycystic kidney disease, and acute renal failure. Other diseases associated with the kidney are encompassed within the scope of the invention.
  • TEM5 polypeptide expression has been detected in the pancreas
  • TEM5 nucleic acid molecules, polypeptides, agonists and antagonists thereof may be useful for the treatment or diagnosis of diseases involving the pancreas. Examples of such diseases include, but are not limited to, diabetes, obesity, and gastro intestinal failure. Other diseases associated with the pancreas are encompassed within the scope of the invention.
  • TEM5 polypeptides may play a role in the regulation of angiogenesis in primary and metastatic tumors. Accordingly, TEM5 nucleic acid molecules, polypeptides, agonists and antagonists thereof (including, but not limited to, anti-
  • TEM5 selective binding agents may be useful as surrogate markers for the treatment or diagnosis of cancer diseases.
  • diseases include, but are not limited to, colorectal cancer, breast cancer, lung cancer, stomach cancer, pancreatic cancer and liver cancer.
  • Other primary and metastatic cancer diseases are encompassed within the scope of the invention.
  • TEM5 polypeptides may also play a role in the control of angiogenesis in inflammatory diseases. Accordingly, TEM5 nucleic acid molecules, polypeptides, agonists and antagonists thereof (including, but not limited to, anti-TEM5 selective binding agents) may be useful for the treatment or diagnosis of inflammatory diseases. Examples of such diseases include, but are not limited to, rheumatoid arthritis and inflammatory bowel disease. Other inflammatory diseases are encompassed within the scope of the invention. Agonists or antagonists of TEM5 polypeptide function may be used
  • cytokines in combination with one or more cytokines, growth factors, antibiotics, anti-inflammatories, and/or chemotherapeutic agents as is appropriate for the condition being treated.
  • Undesirable levels include excessive levels of TEM5 polypeptides and sub-normal levels of TEM5 polypeptides.
  • Nucleic acid molecules of the invention may be used to map the locations of the TEM5 gene and related genes on chromosomes. Mapping may be done by techniques known in the art, such as PCR amplification and in situ hybridization.
  • TEM5 nucleic acid molecules may be useful as hybridization probes in diagnostic assays to test, either qualitatively or quantitatively, for the presence of a TEM5 nucleic acid molecule in mammalian tissue or bodily fluid samples.
  • TEM5 polypeptides may also be employed where it is desirable to inhibit the activity of one or more TEM5 polypeptides. Such inhibition may be effected by nucleic acid molecules that are complementary to and hybridize to expression control sequences (triple helix formation) or to TEM5 mRNA.
  • antisense DNA or RNA molecules which have a sequence that is complementary to at least a portion of a TEM5 gene can be introduced into the cell.
  • Anti-sense probes may be designed by available techniques using the sequence of the TEM5 gene disclosed herein. Typically, each such antisense molecule will be complementary to the start site (5' end) of each selected TEM5 gene.
  • Anti-sense inhibitors provide information relating to the decrease or absence of a TEM5 polypeptide in a cell or organism.
  • gene therapy may be employed to create a dominant-negative inhibitor of one or more TEM5 polypeptides.
  • the DNA encoding a mutant polypeptide of each selected TEM5 polypeptide can be prepared and introduced into the cells of a patient using either viral or non-viral methods as described herein. Each such mutant is typically designed to compete with endogenous polypeptide in its biological role.
  • a TEM5 polypeptide may be used as an immunogen, that is, the polypeptide contains at least one epitope to which antibodies may be raised.
  • Selective binding agents that bind to a TEM5 polypeptide may be used for in vivo and in vitro diagnostic purposes, including, but not limited to, use in labeled form to detect the presence of TEM5 polypeptide in a body fluid or cell sample.
  • the antibodies may also be used to prevent, treat, or diagnose a number of diseases and disorders, including those recited herein.
  • the antibodies may bind to a TEM5 polypeptide so as to diminish or block at least one activity characteristic of a TEM5 polypeptide, or may bind to a polypeptide to increase at least one activity characteristic of a TEM5 polypeptide (including by increasing the pharmacokinetics of the TEM5 polypeptide).
  • TEM5 polypeptides can be used to clone TEM5 ligands using an "expression cloning" strategy.
  • Radiolabeled ( 125 Iodine) TEM5 polypeptide or "affinity/activity- tagged" TEM5 polypeptide can be used in binding assays to identify a cell type, cell line, or tissue that expresses a TEM5 ligand.
  • RNA isolated from such cells or tissues can then be converted to cDNA, cloned into a mammalian expression vector, and transfected into mammalian cells (e.g., COS or 293) to create an expression library.
  • Radiolabeled or tagged TEM5 polypeptide can then be used as an affinity reagent to identify and isolate the subset of cells in this library expressing a TEM5 ligand.
  • DNA is then isolated from these cells and transfected into mammalian cells to create a secondary expression library in which the fraction of cells expressing the TEM5 ligand would be many-fold higher than in the original library. This enrichment process can be repeated iteratively until a single recombinant clone containing the TEM5 ligand is isolated.
  • Isolation of TEM5 ligands is useful for identifying or developing novel agonists and antagonists of the TEM5 signaling pathway.
  • Such agonists and antagonists include TEM5 ligands, anti-TEM5 ligand antibodies, small molecules or antisense oligonucleotides.
  • the TEM5 nucleic acids of the present invention are also useful tools for isolating the corresponding chromosomal TEM5 polypeptide genes.
  • mouse chromosomal DNA containing TEM5 sequences can be used to construct knockout mice, thereby permitting an examination of the in vivo role for TEM5 polypeptide.
  • the human TEM5 genomic DNA can be used to identify heritable tissue-degenerating diseases.
  • TEM5 ⁇ novel, human seven- transmembrane protein, called TEM5 ⁇ , having a nucleotide sequence that is similar to the nucleotide sequence encoding human KIAA1531 (GenBank Accession No. AB040964).
  • a comparison of the amino acid sequences for TEM5 ⁇ and KIAA1531 revealed that the disclosed KIAA1531 nucleotide sequence was only a partial sequence - the polypeptide encoded by the KIAA1531 cDNA sequence lacks approximately 300 amino acids at the amino-terminus of the human TEM5 polypeptide describe herein.
  • this comparison indicated that the KIAA1531 cDNA fragment lacked sequences corresponding to the fourth transmembrane domain of KIAA1531.
  • TEM5 ⁇ cDNA sequence corresponding to the 5' end of the coding sequence, was used as a probe to identify sequences corresponding to the human TEM5 (KIAA1531) gene in a proprietary human genomic DNA database.
  • TEM5 cDNA sequences corresponding to the 5' end of the transcript were isolated from human pancreas Quick-Clone cDNA (Clontech, Palo Alto, CA) by PCR using a plimers derived from the predicted exon sequences of the TEM5 gene (5'-G-G-G- C-G-A-T-G-G-G-T-T-G-A-T-G-G-G-C-3'; SEQ ID NO: 6 and 5'-G-G-T-G-C-A-G-T-G-G-A-T-G-A-G-G-G-3'; SEQ ID NO: 7).
  • a PCR product of 1234 bp was generated in the amplification reaction. This PCR product was subcloned into the pCR 2.1 vector (Invitrogen) and positive clones were sequenced. A consensus sequence for the 5' portion of the human TEM5 gene was derived using sequences obtained from at least three clones. Sequence analysis of this 5' TEM5 fragment indicated that it contained an open reading frame encoding a polypeptide having a methionine residue at its carboxyl terminus followed by a putative signal peptide. In addition, the 5' TEM5 fragment was found to share sequence homology with the corresponding region of the TEM5 ⁇ gene.
  • Figure 1A-1H shows the nucleotide sequence of the human TEM5 gene and the deduced amino acid sequence of the human TEM5 polypeptide.
  • Figures 2A-2C illustrate an amino acid sequence alignment of human TEM5 polypeptide (huTEM5; SEQ ID NO: 2) and human TEM5 ⁇ .
  • the human TEM5 gene shares a 63.5% similarity with the human TEM5 ⁇ gene and human TEM5 polypeptide shares a 60% similarity with human TEM5 ⁇ polypeptide.
  • the structure of human TEM5 polypeptide parallels that of TEM5 ⁇ in that both polypeptides contain a predicted signal peptide sequence in the N-terminus and seven transmembrane domains near the C-terminus, suggesting that TEM5 is a membrane- bound protein.
  • Hodge et al. (International Pub. No. WO 01/09328) describe a nucleotide sequence encoding a G protein coupled receptor they designate as hl5571. The polypeptide encoded by this nucleotide sequence shares sequence identity with human TEM5 polypeptide. While Hodge et al. describe a G protein coupld receptor that shares sequence identity with the TEM5 polypeptide described herein, they do not identify their polypeptide as having TEM activity.
  • Human TEM5 mRNA expression was analyzed by PCR using amplimers derived from the exon sequence of the human TEM5 gene (5'-G-G-G-C-G-A-T-G-G- G-T-T-G-A-T-G-G-G-C-3'; SEQ ID NO: 6 and 5'-G-C-T-G-C-A-G-G-G-A-G-C-G- A-T-T-C-T-G-G-3'; SEQ ID NO: 8).
  • the expected PCR product (647 bp) was detected in heart, lung, liver, kidney, pancreas, placenta, brain, liver, prostate, and testis, colon, and small intestine.
  • TEM5 has been shown to be elevated in the endothelial compartment of blood vessels in colorectal tumors.
  • TEM5 (as well as other members of the TEM family) expression has also been shown in sarcomas and in primary cancers of the lung, breast, brain, and pancreas.
  • TEM expression has been shown in metastatic endothelial tissues.
  • TEM5 mRNA is examined by Northern blot analysis. Multiple human tissue northern blots (Clontech) are probed with a suitable restriction fragment isolated from a human TEM5 polypeptide cDNA clone. The probe is labeled with 32 P-dCTP using standard techniques.
  • Northern blots are prehybridized for 2 hours at 42°C in hybridization solution (5X SSC, 50% deionized formamide, 5X Denhardt's solution, 0.5% SDS, and 100 mg/ml denatured salmon sperm DNA) and then hybridized at 42°C overnight in fresh hybridization solution containing 5 ng/ml of the labeled probe. Following hybridization, the filters are washed twice for 10 minutes at room temperature in 2X SSC and 0.1% SDS, and then twice for 30 minutes at 65°C in 0.1X SSC and 0.1% SDS. The blots are then exposed to autoradiography. The expression of TEM5 mRNA is localized by in situ hybridization.
  • a panel of normal embryonic and adult mouse tissues is fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 5 ⁇ m. Sectioned tissues are permeabilized in 0.2 M HC1, digested with Proteinase K, and acetylated with triethanolamine and acetic anhydride.
  • Sections are prehybridized for 1 hour at 60°C in hybridization solution (300 mM NaCl, 20 mM Tris-HCl, pH 8.0, 5 mM EDTA, IX Denhardt's solution, 0.2% SDS, 10 mM DTT, 0.25 mg/ml tRNA, 25 ⁇ g/ml polyA, 25 ⁇ g/ml polyC and 50% formamide) and then hybridized overnight at 60°C in the same solution containing 10% dextran and 2 x IO 4 cpm/ ⁇ l of a 33 P-labeled antisense riboprobe complementary to the human TEM5 gene.
  • the riboprobe is obtained by in vitro transcription of a clone containing human TEM5 cDNA sequences using standard techniques.
  • sections are rinsed in hybridization solution, treated with RNaseA to digest unhybridized probe, and then washed in 0.1X SSC at 55°C for 30 minutes. Sections are then immersed in NTB-2 emulsion (Kodak, Rochester, NY), exposed for 3 weeks at 4°C, developed, and counterstained with hematoxylin and eosin.
  • Tissue morphology and hybridization signal are simultaneously analyzed by darkfield and standard illumination for brain (one sagittal and two coronal sections), gastrointestinal tract (esophagus, stomach, duodenum, jejunum, ileum, proximal colon, and distal colon), pituitary, liver, lung, heart, spleen, thymus, lymph nodes, kidney, adrenal, bladder, pancreas, salivary gland, male and female reproductive organs (ovary, oviduct, and uterus in the female; and testis, epididymus, prostate, seminal vesicle, and vas deferens in the male), BAT and WAT (subcutaneous, peri- renal), bone (femur), skin, breast, and skeletal muscle.
  • brain one sagittal and two coronal sections
  • gastrointestinal tract esophagus, stomach, duodenum, jejunum, ileum, proximal colon, and distal colon
  • pituitary liver, lung, heart,
  • Example 3 Production of TEM5 Polypeptides A. Expression of TEM5 Polypeptides in Bacteria
  • PCR is used to amplify template DNA sequences encoding a TEM5 polypeptide using primers corresponding to the 5' and 3' ends of the sequence.
  • the amplified DNA products may be modified to contain restriction enzyme sites to allow for insertion into expression vectors.
  • PCR products are gel purified and inserted into expression vectors using standard recombinant DNA methodology.
  • An exemplary vector, such as pAMG21 (ATCC no. 98113) containing the lux promoter and a gene encoding kanamycin resistance is digested with Bam HI and Nde I for directional cloning of inserted DNA.
  • the ligated mixture is transformed into an E. coli host strain by electroporation and transformants are selected for kanamycin resistance. Plasmid DNA from selected colonies is isolated and subjected to DNA sequencing to confirm the presence of the insert.
  • Transformed host cells are incubated in 2xYT medium containing 30 ⁇ g/mL kanamycin at 30°C prior to induction.
  • Gene expression is induced by the addition of N-(3-oxohexanoyl)-dl-homoserine lactone to a final concentration of 30 ng/mL followed by incubation at either 30°C or 37°C for six hours.
  • the expression of T ⁇ M5 polypeptide is evaluated by centrifugation of the culture, resuspension and lysis of the bacterial pellets, and analysis of host cell proteins by SDS-polyacrylamide gel electrophoresis.
  • Inclusion bodies containing TEM5 polypeptide are purified as follows. Bacterial cells are pelleted by centrifugation and resuspended in water. The cell suspension is lysed by sonication and pelleted by centrifugation at 195,000 xg for 5 to 10 minutes. The supernatant is discarded, and the pellet is washed and transferred to a homogenizer. The pellet is homogenized in 5 mL of a Percoll solution (75% liquid Percoll and 0.15 M NaCl) until uniformly suspended and then diluted and centrifuged at 21,600 xg for 30 minutes. Gradient fractions containing the inclusion bodies are recovered and pooled. The isolated inclusion bodies are analyzed by SDS-PAGE.
  • PCR is used to amplify template DNA sequences encoding a TEM5 polypeptide using primers corresponding to the 5' and 3' ends of the sequence.
  • the amplified DNA products may be modified to contain restriction enzyme sites to allow for insertion into expression vectors.
  • PCR products are gel purified and inserted into expression vectors using standard recombinant DNA methodology.
  • An exemplary expression vector, pCEP4 that contains an Epstein-Barr virus origin of replication, may be used for the expression of TEM5 polypeptides in 293-EBNA-l cells. Amplified and gel purified PCR products are ligated into pCEP4 vector and introduced into 293-EBNA cells by lipofection.
  • the transfected cells are selected in lOO ⁇ g/mL hygromycin and the resulting drug-resistant cultures are grown to confluence. The cells are then cultured in serum-free media for 72 hours. The conditioned media is removed and TEM5 polypeptide expression is analyzed by SDS- PAGE.
  • TEM5 polypeptide expression may be detected by silver staining.
  • TEM5 polypeptide is produced as a fusion protein with an epitope tag, such as an IgG constant domain or a FLAG epitope, which may be detected by Western blot analysis using antibodies to the peptide tag.
  • TEM5 polypeptides may be excised from an SDS-polyacrylamide gel, or
  • TEM5 fusion proteins are purified by affinity chromatography to the epitope tag, and subjected to N-terminal amino acid sequence analysis as described herein.
  • TEM5 polypeptide expression constructs are introduced into 293 EBNA or
  • conditioned media are generated from a pool of hygromycin selected 293 EBNA clones.
  • the cells are cultured in 500 cm Nunc Triple Flasks to 80% confluence before switching to serum free media a week prior to harvesting the media.
  • Conditioned media is harvested and frozen at
  • Conditioned media is purified by affinity chromatography as described below.
  • TEM5 polypeptide is eluted from the column with 0.1 M Glycine-HCl at pH 2.7 and immediately neutralized with 1 M Tris-HCl at pH 8.5. Fractions containing TEM5 polypeptide are pooled, dialyzed in PBS, and stored at -70°C.
  • Antibodies to TEM5 polypeptides may be obtained by immunization with purified protein or with TEM5 peptides produced by biological or chemical synthesis. Suitable procedures for generating antibodies include those described in Hudson and Bay, Practical Immunology (2nd ed., Blackwell Scientific Publications).
  • animals typically mice or rabbits
  • a TEM5 antigen such as a TEM5 polypeptide
  • those with sufficient serum titer levels as determined by ELISA are selected for hybridoma production.
  • Spleens of immunized animals are collected and prepared as single cell suspensions from which splenocytes are recovered.
  • the splenocytes are fused to mouse myeloma cells (such as Sp2/0-Agl4 cells), are first incubated in DMEM with 200 U/mL penicillin, 200 ⁇ g/mL streptomycin sulfate, and 4 mM glutamine, and are then incubated in HAT selection medium (hypoxanthine, aminopterin, and thymidine). After selection, the tissue culture supematants are taken from each fusion well and tested for anti-TEM5 antibody production by ELISA.
  • HAT selection medium hyperxanthine, aminopterin, and thymidine
  • anti-TEM5 antibodies may also be employed, such as the immunization of transgenic mice harboring human Ig loci for production of human antibodies, and the screening of synthetic antibody libraries, such as those generated by mutagenesis of an antibody variable domain.
  • a construct encoding a TEM5 polypeptide Fc fusion protein under the control of a liver specific ApoE promoter is prepared.
  • the delivery of this construct is expected to cause pathological changes that are informative as to the function of TEM5 polypeptide.
  • a construct containing the full-length TEM5 polypeptide under the control of the beta actin promoter is prepared.
  • the delivery of this construct is expected to result in ubiquitous expression.
  • PCR is used to amplify template DNA sequences encoding a TEM5 polypeptide using primers that correspond to the 5' and 3' ends of the desired sequence and which incorporate restriction enzyme sites to permit insertion of the amplified product into an expression vector.
  • PCR products are gel purified, digested with the appropriate restriction enzymes, and ligated into an expression vector using standard recombinant DNA techniques.
  • amplified TEM5 polypeptide sequences can be cloned into an expression vector under the control of the human ⁇ -actin promoter as described by Graham et al, 1997, Nature Genetics, 17:272-74 and Ray et al, 1991, Genes Dev. 5:2265-73.
  • reaction mixtures are used to transform an E. coli host strain by electroporation and transformants are selected for drug resistance. Plasmid DNA from selected colonies is isolated and subjected to DNA sequencing to confirm the presence of an appropriate insert and absence of mutation.
  • the TEM5 polypeptide expression vector is purified through two rounds of CsCl density gradient centrifugation, cleaved with a suitable restriction enzyme, and the linearized fragment containing the TEM5 polypeptide transgene is purified by gel electrophoresis.
  • the purified fragment is resuspended in 5 mM Tris, pH 7.4, and 0.2 mM EDTA at a concentration of 2 mg/mL.
  • RNA recovered from spleens is converted to cDNA using the SuperscriptTM Preamplification System (Gibco-BRL) as follows.
  • a suitable primer, located in the expression vector sequence and 3' to the TEM5 polypeptide transgene, is used to prime cDNA synthesis from the transgene transcripts.
  • RNA from transgenic founders and controls is incubated with 1 mM of primer for 10 minutes at 70°C and placed on ice.
  • the reaction is then supplemented with 10 mM Tris-HCl, pH 8.3, 50 mM KCl, 2.5 mM MgCl 2 , 10 mM of each dNTP, 0.1 mM DTT, and 200 U of Superscript R reverse transcriptase.
  • the reaction is stopped by heating for 15 minutes at 72°C and digested with 2U of RNase H for 20 minutes at 37°C. Samples are then amplified by PCR using primers specific for TEM5 polypeptide.
  • transgenic animals Prior to euthanasia, transgenic animals are weighed, anesthetized by isofluorane and blood drawn by cardiac puncture. The samples are subjected to hematology and serum chemistry analysis. Radiography is performed after terminal exsanguination. Upon gross dissection, major visceral organs are subject to weight analysis.
  • tissues i.e., liver, spleen, pancreas, stomach, the entire gastrointestinal tract, kidney, reproductive organs, skin and mammary glands, bone, brain, heart, lung, thymus, trachea, esophagus, thyroid, adrenals, urinary bladder, lymph nodes and skeletal muscle
  • Zn-Formalin 10% buffered Zn-Formalin for histological examination.
  • the tissues are processed into paraffin blocks, and 3 mm sections are obtained. All sections are stained with hematoxylin and exosin, and are then subjected to histological analysis.
  • the spleen, lymph node, and Peyer's patches of both the transgenic and the control mice are subjected to immunohistology analysis with B-cell and T-cell specific antibodies as follows.
  • the formalin fixed paraffin embedded sections are deparaffinized and hydrated in deionized water.
  • the sections are quenched with 3% hydrogen peroxide, blocked with Protein Block (Lipshaw, Pittsburgh, PA), and incubated in rat monoclonal anti-mouse B220 and CD3 (Harlan, Indianapolis, IN).
  • Antibody binding is detected by biotinylated rabbit anti-rat immunoglobulins and peroxidase conjugated streptavidin (BioGenex, San Ramon, CA) with DAB as a chromagen (BioTek, Santa Barbara, CA). Sections are counterstained with hematoxylin.
  • MLN and sections of spleen and thymus from transgenic animals and control littermates are removed.
  • Single cell suspensions are prepared by gently grinding the tissues with the flat end of a syringe against the bottom of a 100 mm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). Cells are washed twice, counted, and approximately 1 x IO 6 cells from each tissue are then incubated for 10 minutes with 0.5 ⁇ g CD16/32(Fc ⁇ IH/II) Fc block in a 20 ⁇ L volume.
  • Samples are then stained for 30 minutes at 2-8°C in a 100 ⁇ L volume of PBS (lacking Ca + and Mg + ), 0.1% bovine serum albumin, and 0.01% sodium azide with 0.5 ⁇ g antibody of FITC or PE-conjugated monoclonal antibodies against CD90.2 (Thy-1.2), CD45R (B220), CDI lb (Mac-1), Gr-1, CD4, or CD8 (PharMingen, San Diego, CA). Following antibody binding, the cells are washed and then analyzed by flow cytometry on a FACScan (Becton Dickinson).

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Abstract

La présente invention concerne des polypeptides du marqueur endothélial tumoral 5 (TEM5) et des molécules d'acide nucléique codant pour ces polypeptides. L'invention concerne également des agents de liaison sélectifs, des vecteurs, des cellules hôtes, ainsi que des procédés permettant de produire ces polypeptides TEM5. En outre, l'invention concerne des compositions pharmaceutiques et des méthodes permettant de diagnostiquer, de traiter, d'améliorer ou de prévenir des maladies, des troubles ou des affections associés aux polypeptides TEM5.
PCT/US2002/035662 2001-11-07 2002-11-07 Molecules du marqueur endothelial tumoral 5 et utilisation associee WO2003046127A2 (fr)

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