Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
  • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
  • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
  • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
  • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
  • G01N33/57407—Specifically defined cancers
  • G01N33/57449—Specifically defined cancers of ovaries
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
  • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
  • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/156—Polymorphic or mutational markers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q2600/00—Oligonucleotides characterized by their use
  • C12Q2600/158—Expression markers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/04—Screening involving studying the effect of compounds C directly on molecule A (e.g. C are potential ligands for a receptor A, or potential substrates for an enzyme A)

Definitions

• the invention relates to the identification of nucleic acid and protein expression profiles and nucleic acids, products, and antibodies thereto that are involved in angiogenesis; and to the use of such expression profiles and compositions in diagnosis and therapy of angiogenesis.
• the invention further relates to methods for identifying and using agents and/or targets that modulate angiogenesis.
• vasculogenesis the development of an interactive vascular system comprising arteries and veins
• angiogenesis the generation of new blood vessels
• angiogenesis is limited in a normal adult to the placenta, ovary, endometrium and sites of wound healing.
• angiogenesis or its absence, plays an important role in the maintenance of a variety of pathological states. Some of these states are characterized by neovascularization, e.g., cancer, diabetic retinopathy, glaucoma, and age related macular degeneration.
• Angiogenesis has a number of stages (see, e.g., Folkman, J.Natl Cancer Inst.
• the early stages of angiogenesis include endothelial cell protease production, migration of cells, and proliferation. The early stages also appear to require some growth factors, with NEGF, TGF- ⁇ , angiostatin, and selected chemokines all putatively playing a role.
• Later stages of angiogenesis include population of the vessels with mural cells (pericytes or smooth muscle cells), basement membrane production, and the induction of vessel bed specializations.
• the final stages of vessel formation include what is known as “remodeling", wherein a forming vasculature becomes a stable, mature vessel bed.
• the process is highly dynamic, often requiring coordinated spatial and temporal waves of gene expression.
• the complex process may be subject to disruption by interfering with one or more critical steps.
• the lack of understanding of the dynamics of angiogenesis prevents therapeutic intervention in serious diseases such as those indicated.
• the present invention provides solutions to both.
• the present invention provides compositions and methods for detecting or modulating angiogenesis associated sequences.
• the invention provides a method of detecting an angiogenesis- associated transcript in a cell in a patient, the method comprising contacting a biological sample from the patient with a polynucleotide that selectively hybridized to a sequence at least 80% identical to a sequence as shown in Tables 1-8.
• the biological sample is a tissue sample.
• the biological sample comprises isolated nucleic acids, which are often mRNA.
• the method further comprises the step of amplifying nucleic acids before the step of contacting the biological sample with the polynucleotide.
• the polynucleotide comprises a sequence as shown in Tables 1-8.
• the polynucleotide can be labeled, for example, with a fluorescent label and can be immobilized on a solid surface.
• the patient is undergoing a therapeutic regimen to treat a disease associated with angiogenesis or the patient is suspected of having an angiogenesis- associated disorder.
• the invention comprises an isolated nucleic acid molecule consisting of a polynucleotide sequence as shown in Tables 1-8.
• the nucleic acid molecule can be labeled, for example, with a fluorescent label,
• the invention provides an expression vector comprising an isolated nucleic acid molecule consisting of a polynucleotide sequence as shown in Tables 1- 8 or a host cell comprising the expression vector.
• the isolated nucleic acid molecule encodes a polypeptide having an amino acid sequence as shown in Table 8.
• the invention provides an isolated polypeptide which is encoded by a nucleic acid molecule having polynucleotide sequence as shown in Tables 1-8.
• the isolated polypeptide has an amino acid sequence as shown in Table 8.
• the invention provides an antibody that specifically binds a polypeptide that has an amino acid sequence as shown in Table 8 or which is encoded by a nucleotide sequence of Tables 1-8 .
• the antibody can be conjugated or fused to an effector component such as a fluorescent label, a toxin, or a radioisotope.
• the antibody is an antibody fragment or a humanized antibody.
• the invention provides a method of detecting a cell undergoing angiogenesis in a biological sample from a patient, the method comprising contacting the biological sample with an antibody that specifically binds to a polypeptide that has an amino acid sequence as shown in Table 8 or which is encoded by a nucleotide sequence of Tables 1-8 .
• the antibody is further conjugated or fused to an effector component, for example, a fluorescent label.
• the invention provides a method of detecting antibodies specific to angiogenesis in a patient, the method comprising contacting a biological sample from the patient with a polypeptide which is encoded by a nucleotide sequence of Tables 1-8.
• the invention also provides a method of identifying a compound that modulates the activity of an angiogenesis-associated polypeptide, the method comprising the steps of: (i) contacting the compound with a polypeptide that comprises at least 80% identity to an amino acid sequence as shown in Table 8 or which is encoded by a nucleotide sequence of Tables 1-8; and (ii) detecting an increase or a decrease in the activity of the polypeptide.
• the polypeptide has an amino acid sequence as shown in Table 8 or is a polypeptide encoded by a nucleotide sequence of Tables 1-8.
• the polypeptide is expressed in a cell.
• the invention also provides a method of identifying a compound that modulates angiogenesis, the method comprising steps of: (i) contacting the compound with a cell undergoing angiogenesis; and (ii) detecting an increase or a decrease in the expression of a polypeptide sequence as shown in Table 8 or a polypeptide which is encoded by a nucleotide sequence of Tables 1-8.
• the detecting step comprises hybridizing a nucleic acid sample from the cell with a polynucleotide that selectively hybridizes to a sequence at least 80% identical to a sequence as shown in Tables 1-8.
• the method further comprises detecting an increase or decrease in the expression of a second sequence as shown in Table 8 or a polypeptide which is encoded by a nucleotide sequence of Tables 1-8 .
• the invention provides a method of inhibiting angiogenesis in a cell that expresses a polypeptide at least 80% identical to a sequence as shown in Table 8 or which is 80% identical to a polypeptide encoded by a nucleotide sequence of Tables 1-8 , the method comprising the step of contacting the cell with a therapeutically effective amount of an inhibitor of the polypeptide.
• the polypeptide has an amino acid sequence shown in Table 8 or is a polypeptide which is encoded by a nucleotide sequence of Tables 1-8 .
• the inhibitor is an antibody.
• the invention provides a method of activating angiogenesis in a cell that expresses a polypeptide at least 80% identical to a sequence as shown in Table 8 or at least 80%) identical to a polypeptide which is encoded by a nucleotide sequence of Tables 1-8 , the method comprising the step of contacting the cell with a therapeutically effective amount of an activator of the polypeptide.
• the polypeptide has an amino acid sequence shown in Table 8 or is a polypeptide which is encoded by a nucleotide sequence of Tables 1-8.
• Tables 1-8 provide nucleotide sequence of genes that exhibit changes in expression levels as a function of time in tissue undergoing angiogenesis compared to tissue that is not. DESCRIPTION OF THE SPECIFIC EMBODIMENTS
• the present invention provides novel methods for diagnosis and treatment of disorders associated with angiogenesis (sometimes referred to herein as angiogenesis disorders or AD), as well as methods for screening for compositions which modulate angiogenesis.
• disorder associated with angiogenesis or “disease associated with angiogenesis” herein is meant a disease state which is marked by either an excess or a deficit of blood vessel development.
• Angiogenesis disorders associated with increased angiogenesis include, but are not limited to, cancer and proliferative diabetic retinopathy.
• Pathological states for which it may be desirable to increase angiogenesis include stroke, heart disease, infertility, ulcers, wound healing, ischemia, and scleradoma.
• Solid tumors typically require angiogenesis to support or sustain growth, e.g., breast, colon, lung, brain, bladder, and prostate tumors.
• Other AD include, e.g., arthritis, inflammatory bowel disease, diabetis retinopathy, macular degeneration, atherosclerosis, and psoriasis. Also provided are methods for treating AD. Definitions
• angiogenesis protein or “angiogenesis polynucleotide” refers to nucleic acid and polypeptide polymorphic variants, alleles, mutants, and interspecies homologs that: (1) have an amino acid sequence that has greater than about 60% amino acid sequence identity, 65%, 70%, 75%, 80%, 85%, 90%, preferably 91%, 92%, 93%, 94%, 95%, 96%, 97%o, 98% or 99% or greater amino acid sequence identity, preferably over a region of over a region of at least about 25, 50, 100, 200, 500, 1000, or more amino acids, to an angiogenesis protein sequence of Table 8; (2) bind to antibodies, e.g., polyclonal antibodies, raised against an immunogen comprising an amino acid sequence of Table 8, and conservatively modified variants thereof; (3) specifically hybridize under stringent hybridization conditions to an anti-sense strand corresponding to a nucleic acid sequence of Tables 1-8 and conservatively modified variants thereof; (4) have a nucleic acid
• a polynucleotide or polypeptide sequence is typically from a mammal including, but not limited to, primate, e.g., human; rodent, e.g., rat, mouse, hamster; cow, pig, horse, sheep, or any mammal.
• An "angiogenesis polypeptide” and an “angiogenesis polynucleotide,” include both naturally occurring or recombinant.
• a “full length" angiogenesis protein or nucleic acid refers to an agiogenesis polypeptide or polynucleotide sequence, or a variant thereof, that contains all of the elements normally contained in one or more naturally occurring, wild type angiogenesis polynucleotide or polypeptide sequences. The “full length” may be prior to, or after, various stages of post- translation processing.
• Bio sample as used herein is a sample of biological tissue or fluid that contains nucleic acids or polypeptides, e.g., of an angiogenic protein. Such samples include, but are not limited to, tissue isolated from primates, e.g., humans, or rodents, e.g., mice, and rats. Biological samples may also include sections of tissues such as biopsy and autopsy samples, and frozen sections taken for histologic purposes.
• a biological sample is typically obtained from a eukaryotic organism, most preferably a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
• a mammal such as a primate e.g., chimpanzee or human; cow; dog; cat; a rodent, e.g., guinea pig, rat, mouse; rabbit; or a bird; reptile; or fish.
• Providing a biological sample means to obtain a biological sample for use in methods described in this invention. Most often, this will be done by removing a sample of cells from an animal, but can also be accomplished by using previously isolated cells (e.g., isolated by another person, at another time, and/or for another purpose), or by performing the methods of the invention in vivo. Archival tissues, having treatment or outcome histroy, will be particularly useful.
• nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., about 70% identity, preferably 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region (e.g., SEQ ID NOS: 1-229), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection (see, e.g., NCBI web site http://www.ncbi.nlm.nih.gov/BLAST/ or the like).
• sequences are then said to be "substantially identical.”
• This definition also refers to, or may be applied to, the compliment of a test sequence.
• the definition also includes sequences that have deletions and/or additions, as well as those that have substitutions.
• the preferred algorithms can account for gaps and the like.
• identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
• sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
• sequence comparison algorithm test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
• default program parameters can be used, or alternative parameters can be designated.
• the sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
• a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol.
• BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
• This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence.
• T is referred to as the neighborhood word score threshold (Altschul et al, supra).
• a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Nat'l. Acad. Sci. USA 90:5873- 5787 (1993)).
• BLAST algorithm One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• P(N) the smallest sum probability
• a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
• nucleic acid sequences or polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the antibodies raised against the polypeptide encoded by the second nucleic acid, as described below.
• a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
• Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complements hybridize to each other under stringent conditions, as described below.
• Yet another indication that two nucleic acid sequences are substantially identical is that the same primers can be used to amplify the sequences.
• a "host cell” is a naturally occurring cell or a transformed cell that contains an expression vector and supports the replication or expression of the expression vector.
• Host cells may be cultured cells, explants, cells in vivo, and the like.
• Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, insect, amphibian, or mammalian cells such as CHO, HeLa, and the like (see, e.g., the American Type Culture Collection catalog or web site, www.atcc.org).
• polypeptide peptide
• protein protein
• amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer.
• amino acid refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids.
• Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, ⁇ - carboxyglutamate, and O-phosphoserine.
• Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, i.e., an ⁇ carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid.
• Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid.
• Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the UPAC-iUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes. "Conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein.
• the codons GCA, GCC, GCG and GCU all encode the amino acid alanine.
• the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
• Such nucleic acid variations are "silent variations," which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
• each codon in a nucleic acid can be modified to yield a functionally identical molecule. Accordingly, each silent variation of a nucleic acid which encodes a polypeptide is implicit in each described sequence with respect to the expression product, but not with respect to actual probe sequences.
• amino acid sequences one of skill will recognize that individual substitutions, deletions or additions to a nucleic acid, peptide, polypeptide, or protein sequence which alters, adds or deletes a single amino acid or a small percentage of amino acids in the encoded sequence is a "conservatively modified variant" where the alteration results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the invention.
• the following eight groups each contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M) (see, e.g., Creighton, Proteins (1984)).
• Macromolecular structures such as polypeptide structures can be described in terms of various levels of organization. For a general discussion of this organization, see, e.g., Alberts et al, Molecular Biology of the Cell (3 rd ed., 1994) and Cantor and Schimmel, Biophysical Chemistry Part I: Tlie Conformation of Biological Macromolecules (1980).
• Primary structure refers to the amino acid sequence of a particular peptide.
• “Secondary structure” refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains. Domains are portions of a polypeptide that form a compact unit of the polypeptide and are typically 25 to approximately 500 amino acids long.
• Typical domains are made up of sections of lesser organization such as stretches of ⁇ -sheet and ⁇ -helices.
• Tetiary structure refers to the complete three dimensional structure of a polypeptide monomer.
• Quaternary structure refers to the three dimensional structure formed, usually by the noncovalent association of independent tertiary units. Anisotropic terms are also known as energy terms.
• a “label” or a “detectable moiety” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, chemical, or other physical means.
• useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins which can be made detectable, e.g., by incorporating a radiolabel into the peptide or used to detect antibodies specifically reactive with the peptide.
• effector or “effector moiety” or “effector component” is a molecule that is bound (or linked, or conjugated), either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds, to an antibody.
• the "effector” can be a variety of molecules including, for example, detection moieties including radioactive compounds, fluroescent compounds, an enzyme or substrate, tags such as epitope tags, a toxin; a chemotherapeutic agent; a lipase; an antibiotic; or a radioisotope emitting "hard” e.g., beta radiation.
• a "labeled nucleic acid probe or oligonucleotide” is one that is bound, either covalently, through a linker or a chemical bond, or noncovalently, through ionic, van der Waals, electrostatic, or hydrogen bonds to a label such that the presence of the probe may be detected by detecting the presence of the label bound to the probe.
• method using high affinity interactions may achieve the same results where one of a pair of binding partners binds to the other, e.g., biotin, streptavidin.
• nucleic acid probe or oligonucleotide is defined as a nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
• a probe may include natural (i.e., A, G, C, or T) or modified bases (7-deazaguanosine, inosine, etc.).
• the bases in a probe may be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
• probes may be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
• probes may bind target sequences lacking complete complementarity with the probe sequence depending upon the stringency of the hybridization conditions.
• the probes are preferably directly labeled as with isotopes, chromophores, lumiphores, chromogens, or indirectly labeled such as with biotin to which a streptavidin complex may later bind. By assaying for the presence or absence of the probe, one can detect the presence or absence of the select sequence or subsequence.
• recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
• recombinant cells express genes that are not found within the native (non- recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
• heterologous when used with reference to portions of a nucleic acid indicates that the nucleic acid comprises two or more subsequences that are not found in the same relationship to each other in nature.
• the nucleic acid is typically recombinantly produced, having two or more sequences from unrelated genes arranged to make a new functional nucleic acid, e.g., a promoter from one source and a coding region from another source.
• a heterologous protein indicates that the protein comprises two or more subsequences that are not found in the same relationship to each other in nature (e.g., a fusion protein).
• a “promoter” is defined as an array of nucleic acid control sequences that direct transcription of a nucleic acid.
• a promoter includes necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
• a promoter also optionally includes distal enhancer or repressor elements, which can be located as much as several thousand base pairs from the start site of transcription.
• a “constitutive” promoter is a promoter that is active under most environmental and developmental conditions.
• An “inducible” promoter is a promoter that is active under environmental or developmental regulation.
• operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
• An "expression vector” is a nucleic acid construct, generated recombinantly or synthetically, with a series of specified nucleic acid elements that permit transcription of a particular nucleic acid in a host cell.
• the expression vector can be part of a plasmid, virus, or nucleic acid fragment.
• the expression vector includes a nucleic acid to be transcribed operably linked to a promoter.
• sequenceselectively (or specifically) hybridizes to refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA).
• stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acids, but to no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
• T m thermal melting point
• Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30°C for short probes (e.g., 10 to 50 nucleotides) and at least about 60°C for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization.
• Exemplary stringent hybridization conditions can be as following: 50% formamide, 5x SSC, and 1% SDS, incubating at 42°C, or, 5x SSC, 1% SDS, incubating at 65°C, with wash in 0.2x SSC, and 0.1% SDS at 65°C.
• a temperature of about 36°C is typical for low stringency amplification, although annealing temperatures may vary between about 32°C and 48°C depending on primer length.
• a temperature of about 62°C is typical, although high stringency annealing temperatures can range from about 50°C to about 65°C, depending on the primer length and specificity.
• Typical cycle conditions for both high and low stringency amplifications include a denaturation phase of 90°C - 95°C for 30 sec - 2 min., an annealing phase lasting 30 sec. - 2 min., and an extension phase of about 72°C for 1 - 2 min. Protocols and guidelines for low and high stringency amplification reactions are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
• Nucleic acids that do not hybridize to each other under stringent conditions are still substantially identical if the polypeptides which they encode are substantially identical. This occurs, for example, when a copy of a nucleic acid is created using the maximum codon degeneracy permitted by the genetic code. In such cases, the nucleic acids typically hybridize under moderately stringent hybridization conditions.
• Exemplary "moderately stringent hybridization conditions" include a hybridization in a buffer of 40%> formamide, 1 M NaCl, % SDS at 37°C, and a wash in IX SSC at 45°C. A positive hybridization is at least twice background.
• Those of ordinary skill will readily recognize that alternative hybridization and wash conditions can be utilized to provide conditions of similar stringency. Additional guidelines for determining hybridization parameters are provided in numerous reference, e.g., and Current Protocols in Molecular Biology, ed. Ausubel, et al
• the phrase "functional effects" in the context of assays for testing compounds that modulate activity of an angiogenesis protein includes the determination of a parameter that is indirectly or directly under the influence of the angiogenesis protein, e.g., a functional, physical, or chemical effect, such as the ability to increase or decrease angiogenesis. It includes binding activity, the ability of cells to proliferate, expression in cells undergoing angiogenesis, and other characteristics of angiogenic cells. "Functional effects” include in vz ' tr ⁇ , in vivo, and ex vivo activities.
• determining the functional effect is meant assaying for a compound that increases or decreases a parameter that is indirectly or directly under the influence of an angiogenesis protein sequence, e.g., functional, physical and chemical effects.
• Such functional effects can be measured by any means known to those skilled in the art, e.g., changes in spectroscopic characteristics (e.g., fluorescence, absorbance, refractive index), hydrodynamic (e.g., shape), chromatographic, or solubility properties for the protein, measuring inducible markers or transcriptional activation of the angiogenesis protein; measuring binding activity or binding assays, e.g.
• angiogenesis assays known to those of skill in the art such as an in vitro assays, e.g., in vitro endothelial cell tube formation assays, and other assays such as the chick CAM assay, the mouse corneal assay, and assays that assess vascularization of an implanted tumor.
• the functional effects can be evaluated by many means known to those skilled in the art, e.g., microscopy for quantitative or qualitative measures of alterations in morphological features, e.g., tube or blood vessel formation, measurement of changes in RNA or protein levels for angio genesis-associated sequences, measurement of RNA stability, identification of downstream or reporter gene expression (CAT, luciferase, ⁇ -gal, GFP and the like), e.g., via chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and ligand binding assays.
• CAT reporter gene expression
• Inhibitors are used to refer to activating, inhibitory, or modulating molecules identified using in vitro and in vivo assays of angiogenic polynucleotide and polypeptide sequences.
• Inhibitors are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity or expression of angiogenesis proteins, e.g., antagonists.
• Activators are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or up regulate angiogenesis protein activity.
• Inhibitors, activators, or modulators also include genetically modified versions of angiogenesis proteins, e.g., versions with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, antibodies, small chemical molecules and the like.
• Such assays for inhibitors and activators include, e.g., expressing the angiogenic protein in vitro, in cells, or cell membranes, applying putative modulator compounds, and then determining the functional effects on activity, as described above.
• Activators and inhibitors of angiogenesis can also be identified by incubating angiogenic cells with the test compound and determining increases or decreases in the expression of 1 or more angiogenesis proteins, e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50 or more angiogenesis proteins, such as angiogenesis proteins comprising the sequences set out in Table 8.
• Samples or assays comprising angiogenesis proteins that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) are assigned a relative protein activity value of 100%.
• Inhibition of a polypeptide is achieved when the activity value relative to the control is about 80%, preferably 50% > , more preferably 25-0%.
• Activation of an angiogenesis polypeptide is achieved when the activity value relative to the control (untreated with activators) is 110%, more preferably 150%), more preferably 200-500% (i.e., two to five fold higher relative to the control), more preferably 1000-3000%) higher.
• Antibody refers to a polypeptide comprising a framework region from an immunoglobulin gene or fragments thereof that specifically binds and recognizes an antigen.
• the recognized immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes., as well as the myriad immunoglobulin variable region genes.
• Light chains are classified as either kappa or lambda.
• Heavy chains are classified as gamma, mu, alpha, delta, or epsilon, which in turn define the immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively.
• the antigen-binding region of an antibody will be most critical in specificity and affinity of binding.
• An exemplary immunoglobulin (antibody) structural unit comprises a tetramer.
• Each tetramer is composed of two identical pairs of polypeptide chains, each pair having one "light” (about 25 kD) and one "heavy” chain (about 50-70 kD).
• the N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids primarily responsible for antigen recognition.
• the terms variable light chain (V L ) and variable heavy chain (VH) refer to these light and heavy chains respectively.
• Antibodies exist, e.g., as intact immunoglobulins or as a number of well- characterized fragments produced by digestion with various peptidases.
• pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab) 5 2 , a dimer of Fab which itself is a light chain joined to V JJ -C H I by a disulfide bond.
• the F(ab)' 2 may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)' 2 dimer into an Fab' monomer.
• the Fab' monomer is essentially Fab with part of the hinge region (see Fundamental Immunology (Paul ed., 3d ed. 1993).
• antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology.
• antibody also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries (see, e.g., McCafferty et al, Nature 348:552-554 (1990))
• antibodies e.g., recombinant, monoclonal, or polyclonal antibodies
• many technique known in the art can be used (see, e.g., Kohler & Milstein,
• phage display technology can be used to identify antibodies and heteromeric Fab fragments that specifically bind to selected antigens (see, e.g. , McCafferty et al. , Nature
• a “chimeric antibody” is an antibody molecule in which (a) the constant region, or a portion thereof, is altered, replaced or exchanged so that the antigen binding site
• variable region is linked to a constant region of a different or altered class, effector function and/or species, or an entirely different molecule which confers new properties to the chimeric antibody, e.g., an enzyme, toxin, hormone, growth factor, drug, etc.; or (b) the variable region, or a portion thereof, is altered, replaced or exchanged with a variable region having a different or altered antigen specificity.
• Kits for use in diagnostic and/or prognostic applications Expression of angiogenesis-associated sequences
• the expression levels of genes are determined in different patient samples for which diagnosis information is desired, to provide expression profiles.
• An expression profile of a particular sample is essentially a "fingerprint" of the state of the sample; while two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is unique to the state of the cell. That is, normal tissue may be distinguished from AD tissue.
• tissue may be distinguished from AD tissue.
• a particular treatment regime may be evaluated: does a chemotherapeutic drug act to down-regulate angiogenesis, and thus tumor growth or recurrence, in a particular patient.
• diagnosis and treatment outcomes may be done or confirmed by comparing patient samples with the known expression profiles.
• Angiogenic tissue can also be analyzed to determine the stage of angiogenesis in the tissue.
• these gene expression profiles allow screening of drug candidates with an eye to mimicking or altering a particular expression profile; for example, screening can be done for drugs that suppress the angiogenic expression profile. This may be done by making biochips comprising sets of the important angiogenesis genes, which can then be used in these screens.
• angiogenic nucleic acid sequences can be administered for gene therapy purposes, including the administration of antisense nucleic acids, or the angiogenic proteins (including antibodies and other modulators thereof) administered as therapeutic drugs.
• angiogenesis sequences include those that are up-regulated (i.e. expressed at a higher level) in disorders associated with angiogenesis, as well as those that are down- regulated (i.e. expressed at a lower level).
• the angiogenesis sequences are from humans; however, as will be appreciated by those in the art, angiogenesis sequences from other organisms may be useful in animal models of disease and drug evaluation; thus, other angiogenesis sequences are provided, from vertebrates, including mammals, including rodents (rats, mice, hamsters, guinea pigs, etc.), primates, farm animals (including sheep, goats, pigs, cows, horses, etc). Angiogenesis sequences from other organisms may be obtained using the techniques outlined below.
• Angiogenesis sequences can include both nucleic acid and amino acid sequences.
• the angiogenesis sequences are recombinant nucleic acids.
• recombinant nucleic acid herein is meant nucleic acid, originally formed in vitro, in general, by the manipulation of nucleic acid e.g., using polymerases and endonucleases, in a form not normally found in nature.
• an isolated nucleic acid, in a linear form, or an expression vector formed in vitro by ligating DNA molecules that are not normally joined, are both considered recombinant for the purposes of this invention.
• nucleic acid once a recombinant nucleic acid is made and reintroduced into a host cell or organism, it will replicate non-recombinantly, i.e. using the in vivo cellular machinery of the host cell rather than in vitro manipulations; however, such nucleic acids, once produced recombinantly, although subsequently replicated non-recombinantly, are still considered recombinant for the purposes of the invention.
• a "recombinant protein” is a protein made using recombinant techniques, i. e. through the expression of a recombinant nucleic acid as depicted above.
• a recombinant protein is distinguished from naturally occurring protein by at least one or more characteristics.
• the protein may be isolated or purified away from some or all of the proteins and compounds with which it is normally associated in its wild type host, and thus may be substantially pure.
• an isolated protein is unaccompanied by at least some of the material with which it is normally associated in its natural state, preferably constituting at least about 0.5%, more preferably at least about 5% by weight of the total protein in a given sample.
• a substantially pure protein comprises at least about 75% by weight of the total protein, with at least about 80% being preferred, and at least about 90% being particularly preferred.
• the definition includes the production of an angiogenesis protein from one organism in a different organism or host cell.
• the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high expression promoter, such that the protein is made at increased concentration levels.
• the protein may be in a form not normally found in nature, as in the addition of an epitope tag or amino acid substitutions, insertions and deletions, as discussed below.
• the angiogenesis sequences are nucleic acids.
• angiogenesis sequences are useful in a variety of applications, including diagnostic applications, which will detect naturally occurring nucleic acids, as well as screening applications; for example, biochips comprising nucleic acid probes to the angiogenesis sequences can be generated.
• diagnostic applications which will detect naturally occurring nucleic acids, as well as screening applications; for example, biochips comprising nucleic acid probes to the angiogenesis sequences can be generated.
• nucleic acid or oligonucleotide or grammatical equivalents herein means at least two nucleotides covalently linked together.
• a nucleic acid of the present invention will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramidate, phosphorothioate, phosphorodithioate, or O- methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); and peptide nucleic acid backbones and linkages.
• Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non- ribose backbones, including those described in U.S. Patent Nos.
• nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids.
• Modifications of the ribose-phosphate backbone may be done for a variety of reasons, for example to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip.
• nucleic acid analogs may find use in the present invention.
• mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
• PNA peptide nucleic acids
• These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in two advantages.
• the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (Tm) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4°C drop in T m for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9°C.
• Tm melting temperature
• RNA typically exhibit a 2-4°C drop in T m for an internal mismatch.
• the non-ionic PNA backbone the drop is closer to 7-9°C.
• hybridization of the bases attached to these backbones is relatively insensitive to salt concentration.
• PNAs are not degraded by cellular enzymes, and thus can be more stable.
• the nucleic acids may be single stranded or double stranded, as specified, or contain portions of both double stranded or single stranded sequence.
• the depiction of a single strand also defines the sequence of the complementary strand; thus the sequences described herein also provide the complement of the sequence.
• the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid may contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, isoguanine, etc.
• nucleoside includes nucleotides and nucleoside and nucleotide analogs, and modified nucleosides such as amino modified nucleosides.
• nucleoside includes non-naturally occurring analog structures.
• angiogenesis sequence can be initially identified by substantial nucleic acid and/or amino acid sequence homology to the angiogenesis sequences outlined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions.
• the angiogenesis screen typically includes comparing genes identified in a modification of an in vitro model of angiogenesis as described in Hiraoka, Cell 95:365 (1998) with genes identified in controls.
• Samples of normal tissue and tissue undergoing angiogenesis are applied to biochips comprising nucleic acid probes. The samples are first microdissected, if applicable, and treated as is known in the art for the preparation of mRNA. Suitable biochips are commercially available, for example from Affymetrix. Gene expression profiles as described herein are generated and the data analyzed.
• the genes showing changes in expression as between normal and disease states are compared to genes expressed in other normal tissues, including, but not limited to lung, heart, brain, liver, breast, kidney, muscle, prostate, small intestine, large intestine, spleen, bone and placenta.
• those genes identified during the angiogenesis screen that are expressed in any significant amount in other tissues are removed from the profile, although in some embodiments, this is not necessary. That is, when screening for drugs, it is usually preferable that the target be disease specific, to minimize possible side effects.
• angiogenesis sequences are those that are upregulated in angiogenesis disorders; that is, the expression of these genes is higher in the disease tissue as compared to normal tissue.
• Up-regulation means at least about a two-fold change, preferably at least about a three fold change, with at least about five-fold or higher being preferred.
• All accession numbers herein are for the GenBank sequence database and the sequences of the accession numbers are hereby expressly incorporated by reference. GenBank is known in the art, see, e.g., Benson, DA, et al., Nucleic Acids Research 26:1-7 (1998) and http://www.ncbi.nlm.nih.gov/.
• angiogenesis sequences are those that are down-regulated in the angiogenesis disorder; that is, the expression of these genes is lower in angiogenic tissue as compared to normal tissue.
• Down-regulation as used herein means at least about a two-fold change, preferably at least about a three fold change, with at least about five-fold or higher being preferred.
• Angiogenesis sequences according to the invention may be classified into discrete clusters of sequences based on common expression profiles of the sequences.
• Expression levels of angiogenesis sequences may increase or decrease as a function of time in a manner that correlates with the induction of angiogenesis.
• expression levels of angiogenesis sequences may both increase and decrease as a function of time.
• expression levels of some angiogenesis sequences are temporarily induced or diminished during the switch to the angiogenesis phenotype, followed by a return to baseline expression levels.
• Tables 1-8 provides genes, the mRNA expression of which varies as a function of time in angiogenesis tissue when compared to normal tissue.
• angiogenesis sequences are those that are induced for a period of time, typically by positive angiogenic factors, followed by a return to the baseline levels. Sequences that are temporarily induced provide a means to target angiogenesis tissue, for example neovascularized tumors, at a particular stage of angiogenesis, while avoiding rapidly growing tissue that require perpetual vascularization.
• positive angiogenic factors include ⁇ FGF, ⁇ FGF, NEGF, angiogenin and the like.
• Induced angiogenesis sequences also are further categorized with respect to the timing of induction. For example, some angiogenesis genes may be induced at an early time period, such as within 10 minutes of the induction of angiogenesis. Others may be induced later, such as between 5 and 60 minutes, while yet others may be induced for a time period of about two hours or more followed by a return to baseline expression levels.
• angiogenesis sequences that are inhibited or reduced as a function of time followed by a return to "normal" expression levels.
• Inhibitors of angiogenesis are examples of molecules that have this expression profile. These sequences also can be further divided into groups depending on the timing of diminished expression. For example, some molecules may display reduced expression within 10 minutes of the induction of angiogenesis. Others may be diminished later, such as between 5 and 60 minutes, while others may be diminished for a time period of about two hours or more followed by a return to baseline. Examples of such negative angiogenic factors include thrombospondin and endostatin to name a few.
• angiogenesis sequences that are induced for prolonged periods. These sequences are typically associated with induction of angiogenesis and may participate in induction and/or maintenance of the angiogenesis phenotype.
• angiogenesis sequences the expression of which is reduced or diminished for prolonged periods in angiogenic tissue.
• These sequences are typically angiogenesis inhibitors and their diminution is correlated with an increase in angiogenesis.
• the ability to identify genes that undergo changes in expression with time during angiogenesis can additionally provide high-resolution, high-sensitivity datasets which can be used in the areas of diagnostics, therapeutics, drug development, biosensor development, and other related areas.
• the expression profiles can be used in diagnostic or prognostic evaluation of patients with angiogenesis-associated disease.
• subcellular toxicological information can be generated to better direct drug structure and activity correlation (see, Anderson, L., "Pharmaceutical Proteomics: Targets, Mechanism, and Function," paper presented at the IBC Proteomics conference, Coronado, CA (June 11-12, 1998)).
• Subcellular toxicological information can also be utilized in a biological sensor device to predict the likely toxicological effect of chemical exposures and likely tolerable exposure thresholds (see, U.S. Patent No. 5,811,231). Similar advantages accrue from datasets relevant to other biomolecules and bioactive agents (e.g., nucleic acids, saccharides, lipids, drugs, and the like).
• bioactive agents e.g., nucleic acids, saccharides, lipids, drugs, and the like.
• the present invention provides a database that includes at least one set of data assay data.
• the data contained in the database is acquired , e.g., using array analysis either singly or in a library format.
• the database can be in substantially any form in which data can be maintained and transmitted, but is preferably an electronic database.
• the electronic database of the invention can be maintained on any electronic device allowing for the storage of and access to the database, such as a personal computer, but is preferably distributed on a wide area network, such as the World Wide Web.
• the focus of the present section on databases that include peptide sequence data is for clarity of illustration only. It will be apparent to those of skill in the art that similar databases can be assembled for any assay data acquired using an assay of the invention.
• compositions and methods for identifying and/or quantitating the relative and/or absolute abundance of a variety of molecular and macromolecular species from a biological sample undergoing angiogenesis i.e., the identification of angiogenesis-associated sequences described herein, provide an abundance of information, which can be correlated with pathological conditions, predisposition to disease, drug testing, therapeutic monitoring, gene-disease causal linkages, identification of correlates of immunity and physiological status, among others.
• data generated from the assays of the invention is suited for manual review and analysis, in a preferred embodiment, prior data processing using highspeed computers is utilized.
• U.S. Patents 6,023,659 and 5,966,712 disclose a relational database system for storing biomolecular sequence information in a manner that allows sequences to be catalogued and searched according to one or more protein function hierarchies.
• U.S. Patent 5,953,727 discloses a relational database having sequence records containing information in a format that allows a collection of partial-length DNA sequences to be catalogued and searched according to association with one or more sequencing projects for obtaining full-length sequences from the collection of partial length sequences.
• Patent 5,706,498 discloses a gene database retrieval system for making a retrieval of a gene sequence similar to a sequence data item in a gene database based on the degree of similarity between a key sequence and a target sequence.
• U.S. Patent 5,538,897 discloses a method using mass spectroscopy fragmentation patterns of peptides to identify amino acid sequences in computer databases by comparison of predicted mass spectra with experimentally-derived mass spectra using a closeness-of-fit measure.
• U.S. Patent 5,926,818 discloses a multidimensional database comprising a functionality for multi-dimensional data analysis described as on-line analytical processing (OLAP), which entails the consolidation of projected and actual data according to more than one consolidation path or dimension.
• OLAP on-line analytical processing
• Patent 5,295,261 reports a hybrid database structure in which the fields of each database record are divided into two classes, navigational and informational data, with navigational fields stored in a hierarchical topological map which can be viewed as a tree structure or as the merger of two or more such tree structures.
• the present invention provides a computer database comprising a computer and software for storing in computer-retrievable form assay data records cross-tabulated, e.g., with data specifying the source of the target-containing sample from which each sequence specificity record was obtained.
• at least one of the sources of target-containing sample is from a control tissue sample known to be free of pathological disorders.
• At least one of the sources is a known pathological tissue specimen, e.g., a neoplastic lesion or another tissue specimen to be analyzed for angiogenesis.
• the assay records cross-tabulate one or more of the following parameters for each target species in a sample: (1) a unique identification code, which can include, e.g., a target molecular structure and/or characteristic separation coordinate (e.g., electrophoretic coordinates); (2) sample source; and (3) absolute and/or relative quantity of the target species present in the sample.
• the invention also provides for the storage and retrieval of a collection of target data in a computer data storage apparatus, which can include magnetic disks, optical disks, magneto-optical disks, DRAM, SRAM, SGRAM, SDRAM, RDRAM, DDR RAM, magnetic bubble memory devices, and other data storage devices, including CPU registers and on-CPU data storage arrays.
• a computer data storage apparatus can include magnetic disks, optical disks, magneto-optical disks, DRAM, SRAM, SGRAM, SDRAM, RDRAM, DDR RAM, magnetic bubble memory devices, and other data storage devices, including CPU registers and on-CPU data storage arrays.
• the target data records are stored as a bit pattern in an array of magnetic domains on a magnetizable medium or as an array of charge states or transistor gate states, such as an array of cells in a DRAM device (e.g., each cell comprised of a transistor and a charge storage area, which may be on the transistor).
• the invention provides such storage devices, and computer systems built therewith, comprising a bit pattern encoding a protein expression fingerprint record comprising unique identifiers for at least 10 target data records cross-tabulated with target source.
• the invention preferably provides a method for identifying related peptide or nucleic acid sequences, comprising performing a computerized comparison between a peptide or nucleic acid sequence assay record stored in or retrieved from a computer storage device or database and at least one other sequence.
• the comparison can include a sequence analysis or comparison algorithm or computer program embodiment thereof (e.g.
• FASTA, TFASTA, GAP, BESTFIT and/or the comparison may be of the relative amount of a peptide or nucleic acid sequence in a pool of sequences determined from a polypeptide or nucleic acid sample of a specimen.
• the invention also preferably provides a magnetic disk, such as an IBM- compatible (DOS, Windows, Windows95/98/2000, Windows NT, OS/2) or other format (e.g., Linux, SunOS, Solaris, AIX, SCO Unix, VMS, MV, Macintosh, etc.).
• a magnetic disk such as an IBM- compatible (DOS, Windows, Windows95/98/2000, Windows NT, OS/2) or other format (e.g., Linux, SunOS, Solaris, AIX, SCO Unix, VMS, MV, Macintosh, etc.).
• floppy diskette or hard (fixed, Winchester) disk drive comprising a bit pattern encoding data from an assay of the invention in a file format suitable for retrieval and processing in a computerized sequence analysis, comparison, or relative quantitation method.
• the invention also provides a network, comprising a plurality of computing devices linked via a data link, such as an Ethernet cable (coax or lOBaseT), telephone line, ISDN line, wireless network, optical fiber, or other suitable signal tranmission medium, whereby at least one network device (e.g., computer, disk array, etc.) comprises a pattern of magnetic domains (e.g., magnetic disk) and/or charge domains (e.g., an array of DRAM cells) composing a bit pattern encoding data acquired from an assay of the invention.
• a network device e.g., computer, disk array, etc.
• a pattern of magnetic domains e.g., magnetic disk
• charge domains e.g., an array of DRAM cells
• the invention also provides a method for transmitting assay data that includes generating an electronic signal on an electronic communications device, such as a modem, ISDN terminal adapter, DSL, cable modem, ATM switch, or the like, wherein the signal includes (in native or encrypted format) a bit pattern encoding data from an assay or a database comprising a plurality of assay results obtained by the method of the invention.
• the invention provides a computer system for comparing a query target to a database containing an array of data structures, such as an assay result obtained by the method of the invention, and ranking database targets based on the degree of identity and gap weight to the target data.
• a central processor is preferably initialized to load and execute the computer program for alignment and/or comparison of the assay results. Data for a query target is entered into the central processor via an I/O device. Execution of the computer program results in the central processor retrieving the assay data from the data file, which comprises a binary description of an assay result.
• the target data or record and the computer program can be transferred to secondary memory, which is typically random access memory (e.g., DRAM, SRAM, SGRAM, or SDRAM).
• Targets are ranked according to the degree of correspondence between a selected assay characteristic (e.g., binding to a selected affinity moiety) and the same characteristic of the query target and results are output via an I/O device.
• a central processor can be a conventional computer (e.g., Intel Pentium, PowerPC, Alpha, PA-8000, SPARC, MIPS 4400, MIPS 10000, VAX, etc);
• a program can be a commercial or public domain molecular biology software package (e.g., UWGCG Sequence Analysis Software, Darwin);
• a data file can be an optical or magnetic disk, a data server, a memory device (e.g., DRAM, SRAM, SGRAM, SDRAM, EPROM, bubble memory, flash memory, etc.);
• an I/O device can be a terminal comprising a video display and a keyboard, a modem, an ISDN terminal adapter, an Ethernet port, a punched card reader, a magnetic strip reader, or other suitable I/O device.
• the invention also preferably provides the use of a computer system, such as that described above, which comprises: (1) a computer; (2) a stored bit pattern encoding a collection of peptide sequence specificity records obtained by the methods of the invention, which may be stored in the computer; (3) a comparison target, such as a query target; and (4) a program for alignment and comparison, typically with rank-ordering of comparison results on the basis of computed similarity values.
• a computer system such as that described above, which comprises: (1) a computer; (2) a stored bit pattern encoding a collection of peptide sequence specificity records obtained by the methods of the invention, which may be stored in the computer; (3) a comparison target, such as a query target; and (4) a program for alignment and comparison, typically with rank-ordering of comparison results on the basis of computed similarity values.
• Angiogenesis proteins of the present invention may be classified as secreted proteins, transmembrane proteins or intracellular proteins.
• the angiogenesis protein is an intracellular protein.
• Intracellular proteins may be found in the cytoplasm and/or in the nucleus or associated with the intracellular side of the plasma membrane. Intracellular proteins are involved in all aspects of cellular function and replication (including, e.g., signaling pathways); aberrant expression of such proteins often results in unregulated or disregulated cellular processes (see, e.g., Molecular Biology of the Cell, 3rd Edition, Alberts, Ed., Garland Pub., 1994).
• intracellular proteins have enzymatic activity such as protein kinase activity, protein phosphatase activity, protease activity, nucleotide cyclase activity, polymerase activity and the like.
• Intracellular proteins also serve as docking proteins that are involved in organizing complexes of proteins, or targeting proteins to various subcellular localizations, and are involved in maintaining the structural integrity of organelles.
• Src-homology-2 (SH2) domains bind tyrosine-phosphorylated targets in a sequence dependent manner.
• PTB domains which are distinct from SH2 domains, also bind tyrosine phosphorylated targets.
• SH3 domains bind to proline-rich targets.
• PH domains, tetratricopeptide repeats and WD domains have been shown to mediate protein-protein interactions.
• these motifs can be identified on the basis of primary sequence; thus, an analysis of the sequence of proteins may provide insight into both the enzymatic potential of the molecule and/or molecules with which the protein may associate.
• the angiogenesis sequences are transmembrane proteins.
• Transmembrane proteins are molecules that span a phospholipid bilayer of a cell. They may have an intracellular domain, an extracellular domain, or both.
• the intracellular domains of such proteins may have a number of functions including those already described for intracellular proteins.
• the intracellular domain may have enzymatic activity and/or may serve as a binding site for additional proteins.
• the intracellular domain of transmembrane proteins serves both roles.
• certain receptor tyrosine kinases have both protein kinase activity and SH2 domains.
• autophosphorylation of tyrosines on the receptor molecule itself creates binding sites for additional SH2 domain containing proteins.
• Transmembrane proteins may contain from one to many transmembrane domains.
• receptor tyrosine kinases certain cytokine receptors, receptor guanylyl cyclases and receptor serine/threonine protein kinases contain a single transmembrane domain.
• various other proteins including channels and adenylyl cyclases contain numerous transmembrane domains.
• Many important cell surface receptors such as G protein coupled receptors (GPCRs) are classified as "seven transmembrane domain" proteins, as they contain 7 membrane spanning regions. Characteristics of transmembrane domains include approximately 20 consecutive hydrophobic amino acids that may be followed or flanked by charged amino acids.
• the localization and number of transmembrane domains within the protein may be predicted (see, e.g. PSORT web site http://psort.nibb.ac.jp/).
• extracellular domains of transmembrane proteins are diverse; however, conserved motifs are found repeatedly among various extracellular domains. conserveed structure and/or functions have been ascribed to different extracellular motifs. Many extracellular domains are involved in binding to other molecules. In one aspect, extracellular domains are found on receptors. Factors that bind the receptor domain include circulating ligands, which may be peptides, proteins, or small molecules such as adenosine and the like. For example, growth factors such as EGF, FGF and PDGF are circulating growth factors that bind to their cognate receptors to initiate a variety of cellular responses. Other factors include cytokines, mitogenic factors, neurotrophic factors and the like.
• Extracellular domains also bind to cell-associated molecules. In this respect, they mediate cell-cell interactions.
• Cell- associated ligands can be tethered to the cell for example via a glycosylphosphatidylinositol (GPI) anchor, or may themselves be transmembrane proteins.
• Extracellular domains also associate with the extracellular matrix and contribute to the maintenance of the cell structure.
• Angiogenesis proteins that are transmembrane are particularly preferred in the present invention as they are readily accessible targets for immunotherapeutics, as are described herein.
• transmembrane proteins can be also useful in imaging modalities.
• Antibodies may be used to label such readily accessible proteins in situ. Alternatively, antibodies can also label intracellular proteins, in which case samples are typically permeablized to provide acess to intracellular proteins.
• transmembrane protein can be made soluble by removing transmembrane sequences, for example through recombinant methods.
• transmembrane proteins that have been made soluble can be made to be secreted through recombinant means by adding an appropriate signal sequence.
• the angiogenesis proteins are secreted proteins; the secretion of which can be either constitutive or regulated. These proteins have a signal peptide or signal sequence that targets the molecule to the secretory pathway.
• Secreted proteins are involved in numerous physiological events; by virtue of their circulating nature, they serve to transmit signals to various other cell types.
• the secreted protein may function in an autocrine manner (acting on the cell that secreted the factor), a paracrine manner (acting on cells in close proximity to the cell that secreted the factor) or an endocrine manner (acting on cells at a distance).
• Angiogenesis proteins that are secreted proteins are particularly preferred in the present invention as they serve as good targets for diagnostic markers, e.g., for blood or serum tests.
• An angiogenesis sequence is typically initially identified by substantial nucleic acid and/or amino acid sequence homology or linkage to the angiogenesis sequences outlined herein. Such homology can be based upon the overall nucleic acid or amino acid sequence, and is generally determined as outlined below, using either homology programs or hybridization conditions. Typically, linked sequences on a mRNA are found on the same molecule. As detailed in the definitions, percent identity can be determined using an algorithm such as BLAST. A preferred method utilizes the BLASTN module of WU- BLAST-2 set to the default parameters, with overlap span and overlap fraction set to 1 and 0.125, respectively. The alignment may include the introduction of gaps in the sequences to be aligned.
• sequences which contain either more or fewer nucleotides than those of the nucleic acids of the figures it is understood that the percentage of homology will be determined based on the number of homologous nucleosides in relation to the total number of nucleosides. Thus, for example, homology of sequences shorter than those of the sequences identified herein and as discussed below, will be determined using the number of nucleosides in the shorter sequence.
• the nucleic acid homology is determined through hybridization studies.
• nucleic acids which hybridize under high stringency to a nucleic acid of Tables 1-8 , or its complement, or is also found on naturally occurring mRNAs is considered an angiogenesis sequence.
• less stringent hybridization conditions are used; for example, moderate or low stringency conditions may be used, as are known in the art; see Ausubel, supra, and Tijssen, supra.
• angiogenesis nucleic acid sequences of the invention are fragments of larger genes, i.e. they are nucleic acid segments. "Genes" in this context includes coding regions, non-coding regions, and mixtures of coding and non-coding regions. Accordingly, as will be appreciated by those in the art, using the sequences provided herein, extended sequences, in either direction, of the angiogenesis genes can be obtained, using techniques well known in the art for cloning either longer sequences or the full length sequences; see Ausubel, et al, supra. Much can be done by informatics and many sequences can be clustered to include multiple sequences, e.g., systems such as UniGene (see, http://www.ncbi.nlm.nih.gov/UniGene/).
• angiogenesis nucleic acid Once the angiogenesis nucleic acid is identified, it can be cloned and, if necessary, its constituent parts recombined to form the entire angiogenesis nucleic acid coding regions or the entire mRNA sequence.
• the recombinant angiogenesis nucleic acid Once isolated from its natural source, e.g., contained within a plasmid or other vector or excised therefrom as a linear nucleic acid segment, the recombinant angiogenesis nucleic acid can be further-used as a probe to identify and isolate other angiogenesis nucleic acids, for example extended coding regions. It can also be used as a "precursor" nucleic acid to make modified or variant angiogenesis nucleic acids and proteins.
• angiogenesis nucleic acids of the present invention are used in several ways.
• nucleic acid probes to the angiogenesis nucleic acids are made and attached to biochips to be used in screening and diagnostic methods, as outlined below, or for administration, for example for gene therapy, vaccine, and/or antisense applications.
• the angiogenesis nucleic acids that include coding regions of angiogenesis proteins can be put into expression vectors for the expression of angiogenesis proteins, again for screening purposes or for administration to a patient.
• nucleic acid probes to angiogenesis nucleic acids are made.
• the nucleic acid probes attached to the biochip are designed to be substantially complementary to the angiogenesis nucleic acids, i.e. the target sequence (either the target sequence of the sample or to other probe sequences, for example in sandwich assays), such that hybridization of the target sequence and the probes of the present invention occurs.
• this complementarity need not be perfect; there may be any number of base pair mismatches which will interfere with hybridization between the target sequence and the single stranded nucleic acids of the present invention.
• the sequence is not a complementary target sequence.
• substantially complementary herein is meant that the probes are sufficiently complementary to the target sequences to hybridize under normal reaction conditions, particularly high stringency conditions, as outlined herein.
• a nucleic acid probe is generally single stranded but can be partially single and partially double stranded.
• the strandedness of the probe is dictated by the structure, composition, and properties of the target sequence.
• the nucleic acid probes range from about 8 to about 100 bases long, with from about 10 to about 80 bases being preferred, and from about 30 to about 50 bases being particularly preferred. That is, generally whole genes are not used. In some embodiments, much longer nucleic acids can be used, up to hundreds of bases.
• more than one probe per sequence is used, with either overlapping probes or probes to different sections of the target being used. That is, two, three, four or more probes, with three being preferred, are used to build in a redundancy for a particular target.
• the probes can be overlapping (i.e. have some sequence in common), or separate.
• PCR primers may be used to amplify signal for higher sensitivity.
• nucleic acids can be attached or immobilized to a solid support in a wide variety of ways.
• immobilized and grammatical equivalents herein is meant the association or binding between the nucleic acid probe and the solid support is sufficient to be stable under the conditions of binding, washing, analysis, and removal as outlined below.
• the binding can typically be covalent or non-covalent.
• non- covalent binding and grammatical equivalents herein is meant one or more of electrostatic, hydrophilic, and hydrophobic interactions. Included in non-covalent binding is the covalent attachment of a molecule, such as, streptavidin to the support and the non-covalent binding of the biotinylated probe to the streptavidin.
• covalent binding and grammatical equivalents herein is meant that the two moieties, the solid support and the probe, are attached by at least one bond, including sigma bonds, pi bonds and coordination bonds. Covalent bonds can be formed directly between the probe and the solid support or can be formed by a cross linker or by inclusion of a specific reactive group on either the solid support or the probe or both molecules. Immobilization may also involve a combination of covalent and non-covalent interactions.
• the probes are attached to the biochip in a wide variety of ways, as will be appreciated by those in the art. As described herein, the nucleic acids can either be synthesized first, with subsequent attachment to the biochip, or can be directly synthesized on the biochip.
• the biochip comprises a suitable solid substrate.
• substrate or “solid support” or other grammatical equivalents herein is meant a material that can be modified to contain discrete individual sites appropriate for the attachment or association of the nucleic acid probes and is amenable to at least one detection method.
• the number of possible substrates are very large, and include, but are not limited to, glass and modified or functionalized glass, plastics (including acrylics, polystyrene and copolymers of styrene and other materials, polypropylene, polyethylene, polybutylene, polyurethanes, TeflonJ, etc.), polysaccharides, nylon or nitrocellulose, resins, silica or silica- based materials including silicon and modified silicon, carbon, metals, inorganic glasses, plastics, etc.
• the substrates allow optical detection and do not appreciably fluorescese.
• a preferred substrate is described in copending application entitled Reusable Low Fluorescent Plastic Biochip, U.S. Application Serial No. 09/270,214, filed March 15, 1999, herein incorporated by reference in its entirety.
• the substrate is planar, although as will be appreciated by those in the art, other configurations of substrates may be used as well.
• the probes may be placed on the inside surface of a tube, for flow-through sample analysis to minimize sample volume.
• the substrate may be flexible, such as a flexible foam, including closed cell foams made of particular plastics.
• the surface of the biochip and the probe may be derivatized with chemical functional groups for subsequent attachment of the two.
• the biochip is derivatized with a chemical functional group including, but not limited to, amino groups, carboxy groups, oxo groups and thiol groups, with amino groups being particularly preferred.
• the probes can be attached using functional groups on the probes.
• nucleic acids containing amino groups can be attached to surfaces comprising amino groups, for example using linkers as are known in the art; for example, homo-or hetero-bifunctional linkers as are well known (see 1994 Pierce Chemical Company catalog, technical section on cross-linkers, pages 155-200, incorporated herein by reference).
• additional linkers such as alkyl groups (including substituted and heteroalkyl groups) may be used.
• oligonucleotides are synthesized as is known in the art, and then attached to the surface of the solid support. As will be appreciated by those skilled in the art, either the 5 ' or 3' terminus may be attached to the solid support, or attachment may be via an internal nucleoside.
• the immobilization to the solid support may be very strong, yet non-covalent.
• biotinylated oligonucleotides can be made, which bind to surfaces covalently coated with streptavidin, resulting in attachment.
• the oligonucleotides may be synthesized on the surface, as is known in the art.
• photoactivation techniques utilizing photopolymerization compounds and techniques are used.
• the nucleic acids can be synthesized in situ, using well known photolithographic techniques, such as those described in WO 95/25116; WO 95/35505; U.S. Patent Nos. 5,700,637 and 5,445,934; and references cited within, all of which are expressly incorporated by reference; these methods of attachment form the basis of the Affimetrix GeneChipTM technology.
• amplification-based assays are performed to measure the expression level of angiogenesis-associated sequences. These assays are typically performed in conjunction with reverse transcription.
• an angiogenesis-associated nucleic acid sequence acts as a template in an amplification reaction (e.g., Polymerase Chain Reaction, or PCR).
• an amplification reaction e.g., Polymerase Chain Reaction, or PCR.
• the amount of amplification product will be proportional to the amount of template in the original sample. Comparison to appropriate controls provides a measure of the amount of angiogenesis-associated RNA.
• Methods of quantitative amplification are well known to those of skill in the art. Detailed protocols for quantitative PCR are provided, e.g., in Innis et al. (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press, Inc. N.Y.).
• a TaqMan based assay is used to measure expression.
• TaqMan based assays use a fluorogenic oligonucleotide probe that contains a 5' fluorescent dye and a 3 ' quenching agent. The probe hybridizes to a PCR product, but cannot itself be extended due to a blocking agent at the 3' end.
• the 5' nuclease activity of the polymerase e.g., AmpliTaq
• This cleavage separates the 5' fluorescent dye and the 3' quenching agent, thereby resulting in an increase in fluorescence as a function of amplification (see, for example, literature provided by Perkin-Elmer, e.g., www2.perkin- elmer.com).
• LCR ligase chain reaction
• angiogenesis nucleic acids e.g., encoding angiogenesis proteins are used to make a variety of expression vectors to express angiogenesis proteins which can then be used in screening assays, as described below.
• Expression vectors and recombinant DNA technology are well known to those of skill in the art (see, e.g., Ausubel, supra, and Gene Expression Systems, Fernandez & Hoeffler, Eds, Academic Press, 1999) and are used to express proteins.
• the expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome. Generally, these expression vectors include transcriptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the angiogenesis protein.
• control sequences refers to DNA sequences used for the expression of an operably linked coding sequence in a particular host organism.
• Control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a ribosome binding site.
• Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.
• Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence.
• DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide;
• a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or
• a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation.
• "operably linked” means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase.
• Transcriptional and translational regulatory nucleic acid will generally be appropriate to the host cell used to express the angiogenesis protein; for example, transcriptional and translational regulatory nucleic acid sequences from Bacillus are preferably used to express the angiogenesis protein in Bacillus. Numerous types of appropriate expression vectors, and suitable regulatory sequences are known in the art for a variety of host cells.
• transcriptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcriptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
• the regulatory sequences include a promoter and transcriptional start and stop sequences.
• Promoter sequences encode either constitutive or inducible promoters.
• the promoters may be either naturally occurring promoters or hybrid promoters. Hybrid promoters, which combine elements of more than one promoter, are also known in the art, and are useful in the present invention.
• an expression vector may comprise additional elements.
• the expression vector may have two replication systems, thus allowing it to be maintained in two organisms, for example in mammalian or insect cells for expression and in a procaryotic host for cloning and amplification.
• the expression vector contains at least one sequence homologous to the host cell genome, and preferably two homologous sequences which flank the expression construct.
• the integrating vector may be directed to a specific locus in the host cell by selecting the appropriate homologous sequence for inclusion in the vector. Constructs for integrating vectors are well known in the art (e.g., Fernandez & Hoeffler, supra). See also Kitamura, et al. (1995) PNAS 92:9146-9150.
• the expression vector contains a selectable marker gene to allow the selection of transformed host cells.
• Selection genes are well known in the art and will vary with the host cell used.
• the angiogenesis proteins of the present invention are produced by culturing a host cell transformed with an expression vector containing nucleic acid encoding an angiogenesis protein, under the appropriate conditions to induce or cause expression of the angiogenesis protein.
• Conditions appropriate for angiogenesis protein expression will vary with the choice of the expression vector and the host cell, and will be easily ascertained by one skilled in the art through routine experimentation or optimization.
• the use of constitutive promoters in the expression vector will require optimizing the growth and proliferation of the host cell, while the use of an inducible promoter requires the appropriate growth conditions for induction.
• the timing of the harvest is important.
• the baculoviral systems used in insect cell expression are lytic viruses, and thus harvest time selection can be crucial for product yield.
• Appropriate host cells include yeast, bacteria, archaebacteria, fungi, and insect and animal cells, including mammalian cells.
• yeast Of particular interest are Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora, BHK, CHO, COS, HeLa cells, HUV ⁇ C (human umbilical vein endothelial cells), THPl cells (a macrophage cell line) and various other human cells and cell lines.
• the angiogenesis proteins are expressed in mammalian cells.
• Mammalian expression systems are also known in the art, and include retroviral and adenoviral systems.
• mammalian promoters Of particular use as mammalian promoters are the promoters from mammalian viral genes, since the viral genes are often highly expressed and have a broad host range. Examples include the SV40 early promoter, mouse mammary tumor virus LTR promoter, adenovirus major late promoter, herpes simplex virus promoter, and the CMV promoter (see, e.g., Fernandez & Hoeffler, supra). Typically, transcription termination and polyadenylation sequences recognized by mammalian cells are regulatory regions located 3' to the translation stop codon and thus, together with the promoter elements, flank the coding sequence. Examples of transcription terminator and polyadenlytion signals include those derived form SV40.
• angiogenesis proteins are expressed in bacterial systems.
• Bacterial expression systems are well known in the art. Promoters from bacteriophage may also be used and are known in the art.
• synthetic promoters and hybrid promoters are also useful; for example, the tac promoter is a hybrid of the trp and lac promoter sequences.
• a bacterial promoter can include naturally occurring promoters of non-bacterial origin that have the ability to bind bacterial RNA polymerase and initiate transcription. In addition to a functioning promoter sequence, an efficient ribosome binding site is desirable.
• the expression vector may also include a signal peptide sequence that provides for secretion of the angiogenesis protein in bacteria.
• the protein is either secreted into the growth media (gram-positive bacteria) or into the periplasmic space, located between the inner and outer membrane of the cell (gram-negative bacteria).
• the bacterial expression vector may also include a selectable marker gene to allow for the selection of bacterial strains that have been transformed. Suitable selection genes include genes which render the bacteria resistant to drugs such as ampicillin, chloramphenicol, erythromycin, kanamycin, neomycin and tetracycline. Selectable markers also include biosynthetic genes, such as those in the histidine, tryptophan and leucine biosynthetic pathways. These components are assembled into expression vectors. Expression vectors for bacteria are well known in the art, and include vectors for Bacillus subtilis, E.
• the bacterial expression vectors are transformed into bacterial host cells using techniques well known in the art, such as calcium chloride treatment, electroporation, and others.
• angiogenesis proteins are produced in insect cells.
• Expression vectors for the transformation of insect cells, and in particular, baculovirus-based expression vectors, are well known in the art.
• angiogenesis protein is produced in yeast cells.
• yeast expression systems are well known in the art, and include expression vectors for Saccharomyces cerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha, Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P. pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica.
• the angiogenesis protein may also be made as a fusion protein, using techniques well known in the art.
• the angiogenesis protein may be fused to a carrier protein to form an immunogen.
• the angiogenesis protein may be made as a fusion protein to increase expression, or for other reasons.
• the nucleic acid encoding the peptide may be linked to another nucleic acid for expression purposes. Fusion with detection epitope tags can be made, e.g., with FLAG, His 6, myc, HA, etc.
• the angiogenesis nucleic acids, proteins and antibodies of the invention are labeled.
• labeled herein is meant that a compound has at least one element, isotope or chemical compound attached to enable the detection of the compound.
• labels fall into three classes: a) isotopic labels, which may be radioactive or heavy isotopes; b) immune labels, which may be antibodies, antigens, or epitope tags and c) colored or fluorescent dyes.
• the labels may be incorporated into the angiogenesis nucleic acids, proteins and antibodies at any position.
• the label should be capable of producing, either directly or indirectly, a detectable signal.
• the detectable moiety may be a radioisotope, such as H, C, P, S, or I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
• a radioisotope such as H, C, P, S, or I
• a fluorescent or chemiluminescent compound such as fluorescein isothiocyanate, rhodamine, or luciferin
• an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.
• Any method known in the art for conjugating the antibody to the label may be employed, including those methods described by Hunter et al, Nature, 144:945 (1962); David et al, Biochemistry, 13:1014 (1974); Pain et
• angiogenesis protein of the present invention also provides angiogenesis protein sequences.
• An angiogenesis protein of the present invention may be identified in several ways. "Protein” in this sense includes proteins, polypeptides, and peptides.
• the nucleic acid sequences of the invention can be used to generate protein sequences. There are a variety of ways to do this, including cloning the entire gene and verifying its frame and amino acid sequence, or by comparing it to known sequences to search for homology to provide a frame, assuming the angiogenesis protein has an identifiable motif or homology to some protein in the database being used.
• the nucleic acid sequences are input into a program that will search all three frames for homology. This is done in a preferred embodiment using the following NCBI Advanced
• the program is blastx or blastn.
• the database is nr.
• the input data is as "Sequence in FASTA format”.
• the organism list is "none”.
• the “expect” is 10; the filter is default.
• the “descriptions” is 500, the “alignments” is 500, and the “alignment view” is pairwise.
• the "Query Genetic Codes” is standard (1).
• the matrix is BLOSUM62; gap existence cost is 11 , per residue gap cost is 1 ; and the lambda ratio is .85 default. This results in the generation of a putative protein sequence.
• angiogenesis proteins are amino acid variants of the naturally occurring sequences, as determined herein.
• the variants are preferably greater than about 75% homologous to the wild-type sequence, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90%.
• the homology will be as high as about 93 to 95 or 98%).
• nucleic acids homology in this context means sequence similarity or identity, with identity being preferred. This homology will be determined using standard techniques well known in the art as are outlined above for the nucleic acid homologies.
• Angiogenesis proteins of the present invention may be shorter or longer than the wild type amino acid sequences.
• included within the definition of angiogenesis proteins are portions or fragments of the wild type sequences, herein.
• the angiogenesis nucleic acids of the invention may be used to obtain additional coding regions, and thus additional protein sequence, using techniques known in the art.
• the angiogenesis proteins are derivative or variant angiogenesis proteins as compared to the wild-type sequence. That is, as outlined more fully below, the derivative angiogenesis peptide will often contain at least one amino acid substitution, deletion or insertion, with amino acid substitutions being particularly preferred. The amino acid substitution, insertion or deletion may occur at any residue within the angiogenesis peptide.
• angiogenesis proteins of the present invention are amino acid sequence variants. These variants typically fall into one or more of three classes: substitutional, insertional or deletional variants. These variants ordinarily are prepared by site specific mutagenesis of nucleotides in the DNA encoding the angiogenesis protein, using cassette or PCR mutagenesis or other techniques well known in the art, to produce DNA encoding the variant, and thereafter expressing the DNA in recombinant cell culture as outlined above. However, variant angiogenesis protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques.
• Amino acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or interspecies variation of the angiogenesis protein amino acid sequence.
• the variants typically exhibit the same qualitative biological activity as the naturally occurring analogue, although variants can also be selected which have modified characteristics as will be more fully outlined below.
• the mutation per se need not be predetermined.
• random mutagenesis may be conducted at the target codon or region and the expressed angiogenesis variants screened for the optimal combination of desired activity.
• Techniques for making substitution mutations at predetermined sites in DNA having a known sequence are well known, for example, Ml 3 primer mutagenesis and PCR mutagenesis. Screening of the mutants is done using assays of angiogenesis protein activities.
• Amino acid substitutions are typically of single residues; insertions usually will be on the order of from about 1 to 20 amino acids, although considerably larger insertions may be tolerated. Deletions range from about 1 to about 20 residues, although in some cases deletions may be much larger.
• substitutions, deletions, insertions or any combination thereof may be used to arrive at a final derivative. Generally these changes are done on a few amino acids to minimize the alteration of the molecule. However, larger changes may be tolerated in certain circumstances. When small alterations in the characteristics of the angiogenesis protein are desired, substitutions are generally made in accordance with the amino acid substitution chart provided in the definition section. Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those provided in the definition of "conservative substitution".
• substitutions may be made which more significantly affect: the structure of the polypeptide backbone in the area of the alteration, for example the alpha-helical or beta-sheet structure; the charge or hydrophobicity of the molecule at the target site; or the bulk of the side chain.
• the substitutions which in general are expected to produce the greatest changes in the polypeptide's properties are those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
• leucyl isoleucyl, phenylalanyl, valyl or alanyl
• a cysteine or proline is substituted for (or by) any other residue
• a residue having an electropositive side chain e.g. lysyl, arginyl, or histidyl
• an electronegative residue e.g. glutamyl or aspartyl
• a residue having a bulky side chain e.g. phenylalanine, is substituted for (or by) one not having a side chain, e.g. glycine.
• variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analog, although variants also are selected to modify the characteristics of the angiogenesis proteins as needed.
• the variant may be designed such that the biological activity of the angiogenesis protein is altered. For example, glycosylation sites may be altered or removed.
• Covalent modifications of angiogenesis polypeptides are included within the scope of this invention.
• One type of covalent modification includes reacting targeted amino acid residues of an angiogenesis polypeptide with an organic derivatizing agent that is capable of reacting with selected side chains or the N-or C-terminal residues of an angiogenesis. polypeptide.
• Derivatization with bifunctional agents is useful, for instance, for crosslinking angiogenesis polypeptides to a water-insoluble support matrix or surface for use in the method for purifying anti-angiogenesis polypeptide antibodies or screening assays, as is more fully described below.
• crosslinking agents include, e.g., 1,1- bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), bifunctional maleimides such as bis-N- maleimido-l,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
• 1,1- bis(diazoacetyl)-2-phenylethane glutaraldehyde
• N-hydroxysuccinimide esters for example, esters with 4-azidosalicylic acid
• homobifunctional imidoesters including disuccinimidyl esters such as 3,3'-dithiobis(
• Another type of covalent modification of the angiogenesis polypeptide included within the scope of this invention comprises altering the native glycosylation pattern of the polypeptide.
• "Altering the native glycosylation pattern” is intended for purposes herein to mean deleting one or more carbohydrate moieties found in native sequence angiogenesis polypeptide, and/or adding one or more glycosylation sites that are not present in the native sequence angiogenesis polypeptide.
• Glycosylation patterns can be altered in many ways. For example the use of different cell types to express angiogenesis-associated sequences can result in different glycosylation patterns.
• Addition of glycosylation sites to angiogenesis polypeptides may also be accomplished by altering the amino acid sequence thereof.
• the alteration may be made, for example, by the addition of, or substitution by, one or more serine or threonine residues to the native sequence angiogenesis polypeptide (for O-linked glycosylation sites).
• the angiogenesis amino acid sequence may optionally be altered through changes at the DNA level, particularly by mutating the DNA encoding the angiogenesis polypeptide at preselected bases such that codons are generated that will translate into the desired amino acids.
• Another means of increasing the number of carbohydrate moieties on the angiogenesis polypeptide is by chemical or enzymatic coupling of glycosides to the polypeptide.
• Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo-and exo-glycosidases as described by Thotakura et al., Meth. Enzymol., 138:350 (1987).
• Another type of covalent modification of angiogenesis comprises linking the angiogenesis polypeptide to one of a variety of nonproteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, in the manner set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.
• nonproteinaceous polymers e.g., polyethylene glycol, polypropylene glycol, or polyoxyalkylenes
• Angiogenesis polypeptides of the present invention may also be modified in a way to form chimeric molecules comprising an angiogenesis polypeptide fused to another, heterologous polypeptide or amino acid sequence.
• a chimeric molecule comprises a fusion of an angiogenesis polypeptide with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind.
• the epitope tag is generally placed at the amino-or carboxyl-terminus of the angiogenesis polypeptide. The presence of such epitope-tagged forms of an angiogenesis polypeptide can be detected using an antibody against the tag polypeptide.
• the epitope tag enables the angiogenesis polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.
• the chimeric molecule may comprise a fusion of an angiogenesis polypeptide with an immunoglobulin or a particular region of an immunoglobulin.
• a fusion could be to the Fc region of an IgG molecule.
• tag polypeptides and their respective antibodies are well known in the art.
• poly-histidine poly-his
• poly-histidine-glycine poly-his-gly
• HIS6 and metal chelation tags the flu HA tag polypeptide and its antibody 12CA5 [Field et al, Mol Cell Biol, 8:2159-2165 (1988)]
• the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al, Molecular and Cellular Biology, 5:3610-3616 (1985)]
• Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al, Protein Engineering, 3(6):547-553 (1990)].
• tag polypeptides include the Flag-peptide [Hopp et al, BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin et al, Science, 255:192-194 (1992)]; tubulin epitope peptide [Skinner et al, J. Biol. Chem., 266:15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al, Proc Natl. Acad. Sci. USA, 87:6393-6397 (1990)].
• angiogenesis protein Also included with an embodiment of angiogenesis protein are other angiogenesis proteins of the angiogenesis family, and angiogenesis proteins from other organisms, which are cloned and expressed as outlined below.
• probe or degenerate polymerase chain reaction (PCR) primer sequences may be used to find other related angiogenesis proteins from humans or other organisms.
• particularly useful probe and/or PCR primer sequences include the unique areas of the angiogenesis nucleic acid sequence.
• preferred PCR primers are from about 15 to about 35 nucleotides in length, with from about 20 to about 30 being preferred, and may contain inosine as needed.
• the conditions for the PCR reaction are well known in the art (e.g., Innis, PCR Protocols, supra).
• angiogenesis proteins can be made that are longer than those encoded by the nucleic acids of the figures, e.g., by the elucidation of extended sequences, the addition of epitope or purification tags, the addition of other fusion sequences, etc.
• Angiogenesis proteins may also be identified as being encoded by angiogenesis nucleic acids.
• angiogenesis proteins are encoded by nucleic acids that will hybridize to the sequences of the sequence listings, or their complements, as outlined herein.
• the angiogenesis protein when the angiogenesis protein is to be used to generate antibodies, e.g., for immunotherapy or immunodiagnosis, the angiogenesis protein should share at least one epitope or determinant with the full length protein.
• epitope or determinant herein is typically meant a portion of a protein which will generate and/or bind an antibody or T-cell receptor in the context of MHC.
• antibodies made to a smaller angiogenesis protein will be able to bind to the full-length protein, particularly linear epitopes.
• the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.
• the epitope is selected from a protein sequence set out in Table 8. Methods of preparing polyclonal antibodies are known to the skilled artisan (e.g., Coligan, supra; and Harlow & Lane, supra). Polyclonal antibodies can be raised in a mammal, e.g., by one or more injections of an immunizing agent and, if desired, an adjuvant.
• the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
• the immunizing agent may include a protein encoded by a nucleic acid of the figures or fragment thereof or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
• adjuvants examples include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
• the immunization protocol may be selected by one skilled in the art without undue experimentation.
• the antibodies may, alternatively, be monoclonal antibodies.
• Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
• a hybridoma method a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
• the lymphocytes may be immunized in vitro.
• the immunizing agent will typically include a polypeptide encoded by a nucleic acid of Tables 1- 8 , or fragment thereof, or a fusion protein thereof.
• peripheral blood lymphocytes are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired.
• the lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell [Goding, Monoclonal Antibodies: Principles and Practice, Academic Press, (1986) pp. 59-103].
• Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed.
• the hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
• a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells.
• the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.
• the antibodies are bispecific antibodies. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens or that have binding specificities for two epitopes on the same antigen.
• one of the binding specificities is for a protein encoded by a nucleic acid Tables 1-8 or a fragment thereof, the other one is for any other antigen, and preferably for a cell-surface protein or receptor or receptor subunit, preferably one that is tumor specific.
• tetramer-type technology may create multivalent reagents.
• the antibodies to angiogenesis protein are capable of reducing or eliminating a biological function of an angiogenesis protein, as is described below. That is, the addition of anti-angiogenesis protein antibodies (either polyclonal or preferably monoclonal) to angiogenic tissue (or cells containing angiogenesis) may reduce or eliminate the angiogenesis activity. Generally, at least a 25% decrease in activity is preferred, with at least about 50% being particularly preferred and about a 95-100% decrease being especially preferred.
• the antibodies to the angiogenesis proteins are humanized antibodies (e.g., Xenerex Biosciences, Mederex, Inc., Abgenix, Inc., Protein Design Labs,Inc.)
• Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab')2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin.
• Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non- human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• CDR complementary determining region
• donor antibody such as mouse, rat or rabbit having the desired specificity, affinity and capacity.
• Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues.
• Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
• a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the framework (FR) regions are those of a human immunoglobulin consensus sequence.
• the humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].
• Fc immunoglobulin constant region
• a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human. These non-human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method of Winter and co-workers [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
• humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non- human species.
• humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
• Human antibodies can also be produced using various techniques known in the art, including phage display libraries [Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)].
• the techniques of Cole et al. and Boerner et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol., 147(l):86-95 (1991)].
• human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Patent Nos.
• immunotherapy is meant treatment of angiogenesis with an antibody raised against angiogenesis proteins.
• immunotherapy can be passive or active.
• Passive immunotherapy as defined herein is the passive transfer of antibody to a recipient (patient).
• Active immunization is the induction of antibody and or T-cell responses in a recipient (patient).
• Induction of an immune response is the result of providing the recipient with an antigen to which antibodies are raised.
• the antigen may be provided by injecting a polypeptide against which antibodies are desired to be raised into a recipient, or contacting the recipient with a nucleic acid capable of expressing the antigen and under conditions for expression of the antigen, leading to an immune response.
• angiogenesis proteins against which antibodies are raised are secreted proteins as described above.
• antibodies used for treatment bind and prevent the secreted protein from binding to its receptor, thereby inactivating the secreted angiogenesis protein.
• the angiogenesis protein to which antibodies are raised is a transmembrane protein.
• antibodies used for treatment bind the extracellular domain of the angiogenesis protein and prevent it from binding to other proteins, such as circulating ligands or cell-associated molecules.
• the antibody may cause down-regulation of the transmembrane angiogenesis protein.
• the antibody may be a competitive, non- competitive or uncompetitive inhibitor of protein binding to the extracellular domain of the angiogenesis protein.
• the antibody is also an antagonist of the angiogenesis protein. Further, the antibody prevents activation of the transmembrane angiogenesis protein.
• the antibody when the antibody prevents the binding of other molecules to the angiogenesis protein, the antibody prevents growth of the cell.
• the antibody may also be used to target or sensitize the cell to cytotoxic agents, including, but not limited to TNF- ⁇ , TNF- ⁇ , IL-1, INF- ⁇ and IL-2, or chemotherapeutic agents including 5FU, vinblastine, actinomycin D, cisplatin, methotrexate, and the like.
• the antibody belongs to a sub-type that activates serum complement when complexed with the transmembrane protein thereby mediating cytotoxicity or antigen-dependent cytotoxicity (ADCC).
• ADCC antigen-dependent cytotoxicity
• angiogenesis is treated by administering to a patient antibodies directed against the transmembrane angiogenesis protein.
• Antibody-labeling may activate a co-toxin, localize a toxin payload, or otherwise provide means to locally ablate cells.
• the antibody is conjugated or fused to an effector moiety.
• the effector moiety can be any number of molecules, including labelling moieties such as radioactive labels or fluorescent labels, or can be a therapeutic moiety.
• the therapeutic moiety is a small molecule that modulates the activity of the angiogenesis protein.
• the therapeutic moiety modulates the activity of molecules associated with or in close proximity to the angiogenesis protein.
• the therapeutic moiety may inhibit enzymatic activity such as protease or coUagenase activity associated with angiogenesis, or be an attractant of other cells, such as NK cells.
• the therapeutic moiety can also be a cytotoxic agent.
• targeting the cytotoxic agent to angiogenesis tissue or cells results in a reduction in the number of afflicted cells, thereby reducing symptoms associated with angiogenesis.
• Cytotoxic agents are numerous and varied and include, but are not limited to, cytotoxic drugs or toxins or active fragments of such toxins. Suitable toxins and their corresponding fragments include diphtheria A chain, exotoxin A chain, ricin A chain, abrin A chain, curcin, crotin, phenomycin, enomycin and the like.
• Cytotoxic agents also include radiochemicals made by conjugating radioisotopes to antibodies raised against angiogenesis proteins, or binding of a radionuclide to a chelating agent that has been covalently attached to the antibody.
• Targeting the therapeutic moiety to transmembrane angiogenesis proteins not only serves to increase the local concentration of therapeutic moiety in the angiogenesis afflicted area, but also serves to reduce deleterious side effects that may be associated with the therapeutic moiety.
• the angiogenesis protein against which the antibodies are raised is an intracellular protein.
• the antibody may be conjugated or fused to a protein which facilitates entry into the cell.
• the antibody enters the cell by endocytosis.
• a nucleic acid encoding the antibody is administered to the individual or cell.
• an antibody thereto contains a signal for that target localization, i.e., a nuclear localization signal.
• the angiogenesis antibodies of the invention specifically bind to angiogenesis proteins.
• “specifically bind” herein is meant that the antibodies bind to the protein with a K d of at least about 0.1 mM, more usually at least about 1 ⁇ M, preferably at least about 0.1 ⁇ M or better, and most preferably, 0.01 ⁇ M or better. Selectivity of binding is also important.
• the angiogenesis protein is purified or isolated after expression.
• Angiogenesis proteins may be isolated or purified in a variety of ways known to those skilled in the art depending on what other components are present in the sample. Standard purification methods include electrophoretic, molecular, immunological and chromato graphic techniques, including ion exchange, hydrophobic, affinity, and reverse- phase HPLC chromatography, and chromatofocusing.
• the angiogenesis protein may be purified using a standard anti-angiogenesis protein antibody column. Ultrafiltration and diafiltration techniques, in conjunction with protein concentration, are also useful. For general guidance in suitable purification techniques, see Scopes, R., Protein Purification, Springer- Verlag, NY (1982).
• angiogenesis proteins and nucleic acids are useful in a number of applications. They may be used as immunoselection reagents, as vaccine reagents, as screening agents, etc.
• the RNAexpression levels of genes are determined for different cellular states in the angiogenesis phenotype. Expression levels of genes in normal tissue (i.e., not undergoing angiogenesis) and in angiogenesis tissue (and in some cases, for varying severities of angiogenesis that relate to prognosis, as outlined below) are evaluated to provide expression profiles.
• An expression profile of a particular cell state or point of development is essentially a "fingerprint" of the state. While two states may have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is reflective of the state of the cell.
• differential expression refers to qualitative or quantitative differences in the temporal and/or cellular gene expression patterns within and among cells and tissue.
• a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, e.g., normal versus angiogenic tissue.
• Genes may be turned on or turned off in a particular state, relative to another state thus permitting comparison of two or more statese.
• a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques. Some genes will be expressed in one state or cell type, but not in both.
• the difference in expression may be quantitative, e.g., in that expression is increased or decreased; i.e., gene expression is either upregulated, resulting in an increased amount of transcript, or downregulated, resulting in a decreased amount of transcript.
• the degree to which expression differs need only be large enough to quantify via standard characterization techniques as outlined below, such as by use of Affymetrix GeneChipTM expression arrays, Lockhart, Nature Biotechnology, 14: 1675- 1680 (1996), hereby expressly incorporated by reference.
• Other techniques include, but are not limited to, quantitative reverse transcriptase PCR, Northern analysis and RNase protection.
• the change in expression is at least about 50%, more preferably at least about 100%, more preferably at least about 150%, more preferably at least about 200%, with from 300 to at least 1000%) being especially preferred.
• Evaluation may be at the gene transcript, or the protein level.
• the amount of gene expression may be monitored using nucleic acid probes to the DNA or RNA equivalent of the gene transcript, and the quantification of gene expression levels, or, alternatively, the final gene product itself (protein) can be monitored, e.g., with antibodies to the angiogenesis protein and standard immunoassays (ELIS As, etc.) or other techniques, including mass spectroscopy assays, 2D gel electrophoresis assays, etc.
• Proteins corresponding to angiogenesis genes i.e., those identified as being important in an angiogenesis phenotype, can be evaluated in an angiogenesis diagnostic test.
• gene expression monitoring is performed simultaneously on a number of genes. Multiple protein expression monitoring can be performed as well. Similarly, these assays may be performed on an individual basis as well.
• angiogenesis nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of angiogenesis sequences in a particular cell.
• the assays are further described below in the example. PCR techniques can be used to provide greater sensitivity.
• nucleic acids encoding the angiogenesis protein are detected.
• DNA or RNA encoding the angiogenesis protein may be detected, of particular interest are methods wherein an mRNA encoding an angiogenesis protein is detected.
• Probes to detect mRNA can be a nucleotide/deoxynucleotide probe that is complementary to and hybridizes with the mRNA and includes, but is not limited to, oligonucleotides, cDNA or RNA. Probes also should contain a detectable label, as defined herein.
• the mRNA is detected after immobilizing the nucleic acid to be examined on a solid support such as nylon membranes and hybridizing the probe with the sample.
• RNA probe digoxygenin labeled riboprobe
• various proteins from the three classes of proteins as described herein are used in diagnostic assays.
• the angiogenesis proteins, antibodies, nucleic acids, modified proteins and cells containing angiogenesis sequences are used in diagnostic assays. This can be performed on an individual gene or corresponding polypeptide level.
• the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow momtoring for expression profile genes and/or corresponding polypeptides.
• angiogenesis proteins including intracellular, transmembrane or secreted proteins, find use as markers of angiogenesis. Detection of these proteins in putative angiogenesis tissue allows for detection or diagnosis of angiogenesis.
• antibodies are used to detect angiogenesis proteins.
• a preferred method separates proteins from a sample by electrophoresis on a gel (typically a denaturing and reducing protein gel, but may be another type of gel, including isoelectric focusing gels and the like). Following separation of proteins, the angiogenesis protein is detected, e.g., by immunoblotting with antibodies raised against the angiogenesis protein. Methods of immunoblotting are well known to those of ordinary skill in the art.
• antibodies to the angiogenesis protein find use in in situ imaging techniques, e.g., in histology (e.g., Methods in Cell Biology: Antibodies in Cell Biology, volume 37 (Asai, ed. 1993)).
• cells are contacted with from one to many antibodies to the angiogenesis protein(s).
• the presence of the antibody or antibodies is detected.
• the antibody is detected by incubating with a secondary antibody that contains a detectable label.
• the primary antibody to the angiogenesis protein(s) contains a detectable label, for example an enzyme marker that can act on a substrate.
• each one of multiple primary antibodies contains a distinct and detectable label. This method finds particular use in simultaneous screening for a plurality of angiogenesis proteins. As will be appreciated by one of ordinary skill in the art, many other histological imaging techniques are alsoprovided by the invention.
• the label is detected in a fluorometer which has the ability to detect and distinguish emissions of different wavelengths.
• a fluorescence activated cell sorter FACS
• FACS fluorescence activated cell sorter
• antibodies find use in diagnosing angiogenesis from biological samples, such as blood, urine, sputum, or other bodily fluids.
• biological samples such as blood, urine, sputum, or other bodily fluids.
• certain angiogenesis proteins are secreted/circulating molecules. Blood samples, therefore, are useful as samples to be probed or tested for the presence of secreted angiogenesis proteins.
• Antibodies can be used to detect an angiogenesis protein by previously described immunoassay techniques including ELISA, immunoblotting (Western blotting), immunoprecipitation, BIACORE technology and the like. Conversely, the presence of antibodies may indicate an immune response against an endogenous angiogenesis protein.
• in situ hybridization of labeled angiogenesis nucleic acid probes to tissue arrays is done.
• tissue samples including angiogenesis tissue and/or normal tissue
• In situ hybridization (see, e.g., Ausubel, supra) is then performed.
• the skilled artisan can make a diagnosis, a prognosis, or a prediction based on the findings. It is further understood that the genes which indicate the diagnosis may differ from those which indicate the prognosis and molecular profiling of the condition of the cells may lead to distinctions between responsive or refractory conditions or may be predictive of outcomes.
• the angiogenesis proteins, antibodies, nucleic acids, modified proteins and cells containing angiogenesis sequences are used in prognosis assays.
• gene expression profiles can be generated that correlate to angiogenesis severity, in terms of long term prognosis. Again, this may be done on either a protein or gene level, with the use of genes being preferred.
• angiogenesis probes may be attached to biochips for the detection and quantification of angiogenesis sequences in a tissue or patient. The assays proceed as outlined above for diagnosis. PCR method may provide more sensitive and accurate quantification.
• members of the three classes of proteins as described herein are used in drug screening assays.
• the angiogenesis proteins, antibodies, nucleic acids, modified proteins and cells containing angiogenesis sequences are used in drug screening assays or by evaluating the effect of drug candidates on a "gene expression profile" or expression profile of polypeptides.
• the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent (e.g., Zlokarnik, et al., Science 279, 84-8 (1998); Heid, Genome Res 6:986-94, 1996).
• the angiogenesis proteins, antibodies, nucleic acids, modified proteins and cells containing the native or modified angiogenesis proteins are used in screening assays. That is, the present invention provides novel methods for screening for compositions which modulate the angiogenesis phenotype or an identified physiological function of an angiogenesis protein. As above, this can be done on an individual gene level or by evaluating the effect of drug candidates on a "gene expression profile".
• the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring for expression profile genes after treatment with a candidate agent, see Zlokarnik, supra. Having identified the differentially expressed genes herein, a variety of assays may be executed.
• assays may be run on an individual gene or protein level. That is, having identified a particular gene as up regulated in angiogenesis, test compounds can be screened for the ability to modulate gene expression or for binding to the angiogenic protein. "Modulation" thus includes both an increase and a decrease in gene expression. The preferred amount of modulation will depend on the original change of the gene expression in normal versus tissue undergoing angiogenesis, with changes of at least 10%, preferably 50%>, more preferably 100-300%, and in some embodiments 300-1000% or greater.
• a gene exhibits a 4-fold increase in angiogenic tissue compared to normal tissue, a decrease of about four-fold is often desired; similarly, a 10-fold decrease in angiogenic tissue compared to normal tissue often provides a target value of a 10-fold increase in expression to be induced by the test compound.
• the amount of gene expression may be monitored using nucleic acid probes and the quantification of gene expression levels, or, alternatively, the gene product itself can be monitored, e.g., through the use of antibodies to the angiogenesis protein and standard immunoassays. Proteomics and separation techniques may also allow quantification of expression.
• gene expression or protein monitoring of a number of entitites is monitored simultaneously. Such profiles will typically invove a plurality of those entitites described herein..
• the angiogenesis nucleic acid probes are attached to biochips as outlined herein for the detection and quantification of angiogenesis sequences in a particular cell.
• PCR may be used.
• a series e.g., of microtiter plate, may be used with dispensed primers in desired wells. A PCR reaction can then be performed and analyzed for each well.
• Expression momtoring can be performed to identify compounds that modify the expression of one or more angiogenesis-associated sequences, e.g., a polynucleotide sequence set out in Tables 1-8 .
• a test modulator is added to the cells prior to analysis.
• screens are also provided to identify agents that modulate angiogenesis, modulate angiogenesis proteins, bind to an angiogenesis protein, or interfere with the binding of an angiogenesis protein and an antibody or other binding partner.
• test compound or “drug candidate” or “modulator” or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for the capacity to directly or indirectly alter the angiogenesis phenotype or the expression of an angiogenesis sequence, e.g. , a nucleic acid or protein sequence.
• modulators alter expression profiles, or expression profile nucleic acids or proteins provided herein.
• the modulator suppresses an angiogenesis phenotype, for example to a normal tissue fingerprint.
• a modulator induced an angiogenesis phenotype is another embodiment.
• a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations.
• one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.
• a modulator will neutralize the effect of an angiogenesis protein.
• neutralize is meant that activity of a protein is inhibited or blocked and thereby has substantially no effect on a cell.
• combinatorial libraries of potential modulators will be screened for an ability to bind to an angiogenesis polypeptide or to modulate activity.
• new chemical entities with useful properties are generated by identifying a chemical compound (called a “lead compound”) with some desirable property or activity, e.g., inhibiting activity, creating variants of the lead compound, and evaluating the property and activity of those variant compounds.
• HTS high throughput screening
• high throughput screening methods involve providing a library containing a large number of potential therapeutic compounds (candidate compounds). Such "combinatorial chemical libraries" are then screened in one or more assays to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds” or can themselves be used as potential or actual therapeutics.
• a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis by combining a number of chemical "building blocks” such as reagents.
• a linear combinatorial chemical library such as a polypeptide (e.g., mutein) library
• a polypeptide e.g., mutein
• Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks (Gallop et al (1994) J. Med. Chem. 37(9): 1233-1251).
• combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent No. 5,010,175, Furka (1991) Int. J. Pept Prot Res., 37: 487-493, Houghton et al (1991) Nature, 354: 84-88), peptoids (PCT Publication No WO 91/19735, 26 Dec. 1991), encoded peptides (PCT Publication WO 93/20242, 14 Oct. 1993), random bio-oligomers (PCT Publication WO 92/00091, 9 Jan. 1992), benzodiazepines (U.S. Pat. No.
• Patent 5,539,083) antibody libraries (see, e.g., Vaughn et al (1996) Nature Biotechnology, 14(3): 309-314), and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al, (1996) Science, 27 A: 1520-1522, and U.S. Patent No. 5,593,853), and small organic molecule libraries (see, e.g., benzodiazepines, Baum (1993) C&EN, Jan 18, page 33; isoprenoids, U.S. Patent No. 5,569,588; thiazolidinones and metathiazanones, U.S. Patent No. 5,549,974; pyrrolidines, U.S.
• Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA).
• a number of well known robotic systems have also been developed for solution phase chemistries. These systems include automated workstations like the automated synthesis apparatus developed by Takeda Chemical Industries, LTD. (Osaka, Japan) and many robotic systems utilizing robotic arms (Zymate II, Zymark Corporation, Hopkinton, Mass.; Orca, Hewlett-Packard, Palo Alto, Calif), which mimic the manual synthetic operations performed by a chemist. Any of the above devices are suitable for use with the present invention. The nature and implementation of modifications to these devices (if any) so that they can operate as discussed herein will be apparent to persons skilled in the relevant art.
• the assays to identify modulators are amenable to high throughput screening. Preferred assays thus detect enhancement or inhibition of angiogenesis gene transcription, inhibition or enhancement of polypeptide expression, and inhibition or enhancement of polypeptide activity.
• High throughput assays for the presence, absence, quantification, or other properties of particular nucleic acids or protein products are well known to those of skill in the art.
• binding assays and reporter gene assays are similarly well known.
• U.S. Patent No. 5,559,410 discloses high throughput screening methods for proteins
• U.S. Patent No. 5,585,639 discloses high throughput screening methods for nucleic acid binding (i.e., in arrays)
• U.S. Patent Nos. 5,576,220 and 5,541,061 disclose high throughput methods of screening for ligand/antibody binding.
• high throughput screening systems are commercially available (see, e.g., Zymark Corp., Hopkinton, MA; Air Technical Industries, Mentor, OH; Beckman Instruments, Inc. Fullerton, CA; Precision Systems, Inc., Natick, MA, etc.). These systems typically automate entire procedures, including all sample and reagent pipetting, liquid dispensing, timed incubations, and final readings of the microplate in detector(s) appropriate for the assay.
• These configurable systems provide high throughput and rapid start up as well as a high degree of flexibility and customization. The manufacturers of such systems provide detailed protocols for various high throughput systems.
• Zymark Corp. provides technical bulletins describing screening systems for detecting the modulation of gene transcription, ligand binding, and the like.
• modulators are proteins, often naturally occurring proteins or fragments of naturally occurring proteins.
• cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts may be used.
• libraries of proteins may be made for screening in the methods of the invention.
• Particularly preferred in this embodiment are libraries of bacterial, fungal, viral, and mammalian proteins, with the latter being preferred, and human proteins being especially preferred.
• Paticularly useful test compound will be directed to the class of proteins to which the target belongs, e.g., substrates for enzymes or ligands and receptors.
• modulators are peptides of from about 5 to about 30 amino acids, with from about 5 to about 20 amino acids being preferred, and from about 7 to about 15 being particularly preferred.
• the peptides may be digests of naturally occurring proteins as is outlined above, random peptides, or "biased” random peptides.
• randomized or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they may incorporate any nucleotide or amino acid at any position.
• the synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.
• the library is fully randomized, with no sequence preferences or constants at any position.
• the library is biased. That is, some positions within the sequence are either held constant, or are selected from a limited number of possibilities.
• the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, etc., or to purines, etc.
• Modulators of angiogenesis can also be nucleic acids, as defined above.
• nucleic acid modulating agents may be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids.
• digests of procaryotic or eucaryotic genomes may be used as is outlined above for proteins.
• the candidate compounds are organic chemical moieties, a wide variety of which are available in the literature.
• the sample containing a target sequence to be analyzed is added to the biochip.
• the target sequence is prepared using known techniques.
• the sample may be treated to lyse the cells, using known lysis buffers, electroporation, etc., with purification and/or amplification such as PCR performed as appropriate.
• an in vitro transcription with labels covalently attached to the nucleotides is performed.
• the nucleic acids are labeled with biotin-FITC or PE, or with cy3 or cy5.
• the target sequence is labeled with, for example, a fluorescent, a chemiluminescent, a chemical, or a radioactive signal, to provide a means of detecting the target sequence's specific binding to a probe.
• the label also can be an enzyme, such as, alkaline phosphatase or horseradish peroxidase, which when provided with an appropriate substrate produces a product that can be detected.
• the label can be a labeled compound or small molecule, such as an enzyme inhibitor, that binds but is not catalyzed or altered by the enzyme.
• the label also can be a moiety or compound, such as, an epitope tag or biotin which specifically binds to streptavidin.
• the streptavidin is labeled as described above, thereby, providing a detectable signal for the bound target sequence. Unbound labeled streptavidin is typically removed prior to analysis.
• these assays can be direct hybridization assays or can comprise "sandwich assays", which include the use of multiple probes, as is generally outlined in U.S. Patent Nos. 5,681,702, 5,597,909, 5,545,730, 5,594,117, 5,591,584, 5,571,670, 5,580,731, 5,571,670, 5,591,584, 5,624,802, 5,635,352, 5,594,118, 5,359,100, 5,124,246 and 5,681,697, all of which are hereby incorporated by reference.
• the target nucleic acid is prepared as outlined above, and then added to the biochip comprising a plurality of nucleic acid probes, under conditions that allow the formation of a hybridization complex.
• hybridization conditions may be used in the present invention, including high, moderate and low stringency conditions as outlined above.
• the assays are generally run under stringency conditions which allows formation of the label probe hybridization complex only in the presence of target. Stringency can be controlled by altering a step parameter that is a thermodynamic variable, including, but not limited to, temperature, formamide concentration, salt concentration, chaotropic salt concentration pH, organic solvent concentration, etc.
• the reactions outlined herein may be accomplished in a variety of ways. Components of the reaction may be added simultaneously, or sequentially, in different orders, with preferred embodiments outlined below.
• the reaction may include a variety of other reagents. These include salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which may be used to facilitate optimal hybridization and detection, and/or reduce nonspecific or background interactions. Reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may also be used as appropriate, depending on the sample preparation methods and purity of the target.
• the assay data are analyzed to determine the expression levels, and changes in expression levels as between states, of individual genes, forming a gene expression profile.
• Screens are performed to identify modulators of the angiogenesis phenotype.
• screening is performed to identify modulators that can induce or suppress a particular expression profile, thus preferably generating the associated phenotype.
• screens can be performed to identify modulators that alter expression of individual genes.
• screening is performed to identify modulators that alter a biological function of the expression product of a differentially expressed gene. Again, having identified the importance of a gene in a particular state, screens are performed to identify agents that bind and/or modulate the biological activity of the gene product.
• screens can be done for genes that are induced in response to a candidate agent.
• a screen as described above can be performed to identify genes that are specifically modulated in response to the agent. Comparing expression profiles between normal tissue and agent treated angiogenesis tissue reveals genes that are not expressed in normal tissue or angiogenesis tissue, but are expressed in agent treated tissue.
• agent-specific sequences can be identified and used by methods described herein for angiogenesis genes or proteins. In particular these sequences and the proteins they encode find use in marking or identifying agent treated cells.
• a test compound is administered to a population of angiogenic cells, that have an associated angiogenesis expression profile.
• administration or “contacting” herein is meant that the candidate agent is added to the cells in such a manner as to allow the agent to act upon the cell, whether by uptake and intracellular action, or by action at the cell surface.
• nucleic acid encoding a proteinaceous candidate agent i. e.
• a peptide may be put into a viral construct such as an adenoviral or retroviral construct, and added to the cell, such that expression of the peptide agent is accomplished, e.g., PCT US97/01019.
• a viral construct such as an adenoviral or retroviral construct
• Regulatable gene therapy systems can also be used.
• the cells can be washed if desired and are allowed to incubate under preferably physiological conditions for some period of time. The cells are then harvested and a new gene expression profile is generated, as outlined herein.
• angiogenesis tissue may be screened for agents that modulate, e.g., induce or suppress the angiogenesis phenotype.
• a change in at least one gene, preferably many, of the expression profile indicates that the agent has an effect on angiogenesis activity.
• the effects of the test compounds upon the function of the anagiogenesis polypeptides can be measured by examining parameters described above.
• a suitable physiological change that affects activity can be used to assess the influence of a test compound on the polypeptides of this invention.
• the functional consequences are determined using intact cells or animals, one can also measure a variety of effects such as, in the case of angiogenesis associated with tumors, tumor growth, neovascularization, hormone release, transcriptional changes to both known and uncharacterized genetic markers (e.g., northern blots), changes in cell metabolism such as cell growth or pH changes, and changes in intracellular second messengers such as cGMP.
• mammalian angiogenesis polypeptide is typically used, e.g., mouse, preferably human.
• angiogenesis assays are known to those of skill in the art.
• Various models have been employed to evaluate angiogenesis (e.g., Croix et al, Science 289:1197- 1202, 2000 and Kahn et al, Amer. J. Pathol. 156:1887-1900).
• Assessement of angiogenesis in the presence of a potential modulator of angiogenesis can be performed using cell-cultre- based angiogenesis assays, e.g., endothelial cell tube formation assays, as well as other bioassays such as the chick CAM assay, the mouse corneal assay, and assays measuring the effect of administering potential modulators on implanted tumors.
• the chick CAM assay is described by O'Reilly, et al. Cell 79: 315-328, 1994. Briefly, 3 day old chicken embryos with intact yolks are separated from the egg and placed in a petri dish. After 3 days of incubation, a methylcellulose disc containing the protein to be tested is applied to the CAM of individual embryos. After about 48 hours of incubation, the embryos and CAMs are observed to determine whether endothelial growth has been inhibited.
• the mouse corneal assay involves implanting a growth factor-containing pellet, along with another pellet containing the suspected endothelial growth inhibitor, in the cornea of a mouse and observing the pattern of capillaries that are elaborated in the cornea.
• Angiogenesis can also be measured by determining the extent of neovascularization of a tumor.
• carcinoma cells can be subcutaneously inoculated into athymic nude mice and tumor growth then monitored.
• the cancer cells are treated with an angiogenesis inhibitor, such as an antibody, or other compound that is exogenously administered, or can be transfected prior to inoculation with a polynucleotide inhibitor of angiogenesis.
• Immunoassays using endothelial cell-specific antibodies are typically used to stain for vascularization of tumor and the number of vessels in the tumor. Assays to identify compounds with modulating activity can be performed in vitro.
• an angiogenesis polypeptide is first contacted with a potential modulator and incubated for a suitable amount of time, e.g., from 0.5 to 48 hours.
• the angiogenesis polypeptide levels are determined in vitro by measuring the level of protein or mRNA.
• the level of protein is measured using immunoassays such as western blotting, ELISA and the like with an antibody that selectively binds to the angiogenesis polypeptide or a fragment thereof.
• amplification e.g., using PCR, LCR, or hybridization assays, e.g., northern hybridization, RNAse protection, dot blotting, are preferred.
• the level of protein or mRNA is detected using directly or indirectly labeled detection agents, e.g., fluorescently or radioactively labeled nucleic acids, radioactively or enzymatically labeled antibodies, and the like, as described herein.
• a reporter gene system can be devised using the angiogenesis protein promoter operably linked to a reporter gene such as luciferase, green fluorescent protein, CAT, or ⁇ -gal.
• a reporter gene such as luciferase, green fluorescent protein, CAT, or ⁇ -gal.
• the reporter construct is typically transfected into a cell. After treatment with a potential modulator, the amount of reporter gene transcription, translation, or activity is measured according to standard techniques known to those of skill in the art.
• screens may be done on individual genes and gene products (proteins). That is, having identified a particular differentially expressed gene as important in a particular state, screening of modulators of the expression of the gene or the gene product itself can be done.
• the gene products of differentially expressed genes are sometimes referred to herein as "angiogenesis proteins".
• the angiogenesis protein comprises a sequence shown in Table 8.
• the angiogenesis protein may be a fragment, or alternatively, be the full length protein to a fragment shown herein.
• the angiogenesis protein is a fragment of approximately 14 to 24 amino acids long. More preferably the fragment is a soluble fragment.
• an angiogenesis protein is conjugated or fused to an immunogenic agent or BSA.
• screening for modulators of expression of specific genes is performed. Typically, the expression of only one or a few genes are evaluated.
• screens are designed to first find compounds that bind to differentially expressed proteins. These compounds are then evaluated for the ability to modulate differentially expressed activity. Moreover, once initial candidate compounds are identified, variants can be further screened to better evaluate strucutre activity relationships.
• binding assays are done.
• purified or isolated gene product is used; that is, the gene products of one or more differentially expressed nucleic acids are made.
• antibodies are generated to the protein gene products, and standard immunoassays are run to determine the amount of protein present.
• cells comprising the angiogenesis proteins can be used in the assays.
• the methods comprise combining an angiogenesis protein and a candidate compound, and determining the binding of the compound to the angiogenesis protein.
• Preferred embodiments utilize the human angiogenesis protein, although other mammalian proteins may also be used, for example for the development of animal models of human disease.
• variant or derivative angiogenesis proteins may be used.
• the angiogenesis protein or the candidate agent is non-diffusably bound to an insoluble support having isolated sample receiving areas (e.g. a microtiter plate, an array, etc.).
• the insoluble supports may be made of any composition to which the compositions can be bound, is readily separated from soluble material, and is otherwise compatible with the overall method of screening.
• the surface of such supports may be solid or porous and of any convenient shape.
• suitable insoluble supports include microtiter plates, arrays, membranes and beads. These are typically made of glass, plastic (e.g., polystyrene), polysaccharides, nylon or nitrocellulose, teflonTM, etc.
• Microtiter plates and arrays are especially convenient because a large number of assays can be carried out simultaneously, using small amounts of reagents and samples.
• the particular manner of binding of the composition is not crucial so long as it is compatible with the reagents and overall methods of the invention, maintains the activity of the composition and is nondiffusable.
• Preferred methods of binding include the use of antibodies (which do not sterically block either the ligand binding site or activation sequence when the protein is bound to the support), direct binding to "sticky" or ionic supports, chemical crosslinking, the synthesis of the protein or agent on the surface, etc. Following binding of the protein or agent, excess unbound material is removed by washing. The sample receiving areas may then be blocked through incubation with bovine serum albumin (BSA), casein or other innocuous protein or other moiety.
• BSA bovine serum albumin
• the angiogenesis protein is bound to the support, and a test compound is added to the assay.
• the candidate agent is bound to the support and the angiogenesis protein is added.
• Novel binding agents include specific antibodies, non-natural binding agents identified in screens of chemical libraries, peptide analogs, etc. Of particular interest are screening assays for agents that have a low toxicity for human cells. A wide variety of assays may be used for this purpose, including labeled in vitro protein-protein binding assays, electrophoretic mobility shift assays, immunoassays for protein binding, functional assays (phosphorylation assays, etc.) and the like.
• the determination of the binding of the test modulating compound to the angiogenesis protein may be done in a number of ways.
• the compound is labelled, and binding determined directly, e.g., by attaching all or a portion of the angiogenesis protein to a solid support, adding a labelled candidate agent (e.g., a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support.
• a labelled candidate agent e.g., a fluorescent label
• washing off excess reagent e.g., a fluorescent label
• Various blocking and washing steps may be utilized as appropriate.
• labeled herein is meant that the compound is either directly or indirectly labeled with a label which provides a detectable signal, e.g. radioisotope, fluorescers, enzyme, antibodies, particles such as magnetic particles, chemiluminescers, or specific binding molecules, etc.
• Specific binding molecules include pairs, such as biotin and streptavidin, digoxin and antidigoxin, etc.
• the complementary member would normally be labeled with a molecule which provides for detection, in accordance with known procedures, as outlined above.
• the label can directly or indirectly provide a detectable signal.
• only one of the components is labeled, e.g., the proteins (or proteinaceous candidate compounds) can be labeled.
• more than one component can be labeled with different labels, e.g., 125 I for the proteinsand a fluorophor for the compound.
• Proximity reagents e.g., quenching or energy transfer reagents are also useful.
• the binding of the test compound is determined by competitive binding assay.
• the competitor is a binding moiety known to bind to the target molecule (i.e. an angiogenesis protein), such as an antibody, peptide, binding partner, ligand, etc. Under certain circumstances, there may be competitive binding between the compound and the binding moiety, with the binding moiety displacing the compound.
• the test compound is labeled.
• Either the compound, or the competitor, or both, is added first to the protein for a time sufficient to allow binding, if present.
• Incubations may be performed at a temperature which facilitates optimal activity, typically between 4 and 40°C. Incubation periods are typically optimized, e.g., to facilitate rapid high throughput screening. Typically between 0.1 and 1 hour will be sufficient. Excess reagent is generally removed or washed away. The second component is then added, and the presence or absence of the labeled component is followed, to indicate binding.
• the competitor is added first, followed by the test compound.
• Displacement of the competitor is an indication that the test compound is binding to the angiogenesis protein and thus is capable of binding to, and potentially modulating, the activity of the angiogenesis protein.
• either component can be labeled.
• the test compound is added first, with incubation and washing, followed by the competitor. The absence of binding by the competitor may indicate that the test compound is bound to the angiogenesis protein with a higher affinity.
• the presence of the label on the support, coupled with a lack of competitor binding may indicate that the test compound is capable of binding to the angiogenesis protein.
• the methods comprise differential screening to identity agents that are capable of modulating the activitity of the angiogenesis proteins.
• the methods comprise combining an angiogenesis protein and a competitor in a first sample.
• a second sample comprises a test compound, an angiogenesis protein, and a competitor.
• the binding of the competitor is determined for both samples, and a change, or difference in binding between the two samples indicates the presence of an agent capable of binding to the angiogenesis protein and potentially modulating its activity. That is, if the binding of the competitor is different in the second sample relative to the first sample, the agent is capable of binding to the angiogenesis protein.
• differential screening is used to identify drug candidates that bind to the native angiogenesis protein, but cannot bind to modified angiogenesis proteins.
• Positive controls and negative controls may be used in the assays.
• control and test samples are performed in at least triplicate to obtain statistically significant results. Incubation of all samples is for a time sufficient for the binding of the agent to the protein. Following incubation, samples are washed free of non-specifically bound material and the amount of bound, generally labeled agent determined. For example, where a radiolabel is employed, the samples may be counted in a scintillation counter to determine the amount of bound compound.
• the invention provides methods for screening for a compound capable of modulating the activity of an angiogenesis protein.
• the methods comprise adding a test compound, as defined above, to a cell comprising angiogenesis proteins.
• Preferred cell types include almost any cell.
• the cells contain a recombinant nucleic acid that encodes an angiogenesis protein.
• a library of candidate agents are tested on a plurality of cells.
• the assays are evaluated in the presence or absence or previous or subsequent exposure of physiological signals, for example hormones, antibodies, peptides, antigens, cytokines, growth factors, action potentials, pharmacological agents including chemotherapeutics, radiation, carcinogenics, or other cells (i.e. cell-cell contacts).
• the determinations are determined at different stages of the cell cycle process. In this way, compounds that modulate angiogenesis agents are identified. Compounds with pharmacological activity are able to enhance or interfere with the activity of the angiogenesis protein. Once identified, similar structures are evaluated to identify critical structural feature of the compound.
• a method of inhibiting angiogenic cell division is provided. The method comprises administration of an angiogenesis inhibitor.
• a method of inhibiting angiogenesis is provided. The method comprises administration of an angiogenesis inhibitor.
• methods of treating cells or individuals with angiogenesis are provided. The method comprises administration of an angiogenesis inhibitor.
• an angiogenesis inhibitor is an antibody as discussed above. In another embodiment, the angiogenesis inhibitor is an antisense molecule.
• the activity of an angiogenesis-associated protein is downregulated, or entirely inhibited, by the use of antisense polynucleotide, i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., an angiogenesis protein mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and or stability of the mRNA.
• antisense polynucleotide i.e., a nucleic acid complementary to, and which can preferably hybridize specifically to, a coding mRNA nucleic acid sequence, e.g., an angiogenesis protein mRNA, or a subsequence thereof. Binding of the antisense polynucleotide to the mRNA reduces the translation and or stability of the mRNA.
• antisense polynucleotides can comprise naturally-occurring nucleotides, or synthetic species formed from naturally-occurring subunits or their close homologs. Antisense polynucleotides may also have altered sugar moieties or inter-sugar linkages. Exemplary among these are the phosphorothioate and other sulfur containing species which are known for use in the art. Analogs are comprehended by this invention so long as they function effectively to hybridize with the angiogenesis protein mRNA. See, e.g., Isis Pharmaceuticals, Carlsbad, CA; Sequitor, Inc., Natick, MA.
• antisense polynucleotides can readily be synthesized using recombinant means, or can be synthesized in vitro. Equipment for such synthesis is sold by several vendors, including Applied Biosystems. The preparation of other oligonucleotides such as phosphorothioates and alkylated derivatives is also well known to those of skill in the art.
• Antisense molecules as used herein include antisense or sense oligonucleotides. Sense oligonucleotides can, e.g., be employed to block trancription by binding to the anti-sense strand.
• the antisense and sense oligonucleotide comprise a single- stranded nucleic acid sequence (either RNA or DNA) capable of binding to target mRNA (sense) or DNA (antisense) sequences for angiogenesis molecules.
• a preferred antisense molecule is for an angiogenesis sequences in Tables 1-8 , or for a ligand or activator thereof.
• Antisense or sense oligonucleotides, according to the present invention comprise a fragment generally at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.
• ribozymes can be used to target and inhibit transcription of angiogenesis-associated nucleotide sequences.
• a ribozyme is an RNA molecule that catalytically cleaves other RNA molecules.
• Different kinds of ribozymes have been described, including group I ribozymes, hammerhead ribozymes, hairpin ribozymes,
• hairpin ribozymes are described, e.g., in Hampel et al. (1990) Nucl. Acids Res. 18: 299-304; Hampel et al. (1990) European Patent Publication No. 0 360 257; U.S. Patent No. 5,254,678.
• Methods of preparing are well known to those of skill in the art (see, e.g., Wong-Staal et al, WO 94/26877; Ojwang et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6340-6344; Yamada et al. (1994) Human Gene Therapy 1: 39-45; Leavitt et al.
• Polynucleotide modulators of angiogenesis may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753.
• Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.
• conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
• a polynucleotide modulator of angiogenesis may be introduced into a cell containing the target nucleic acid sequence, e.g., by formation of an polynucleotide-lipid complex, as described in WO 90/10448. It is understood that the use of antisense molecules or knock out and knock in models may also be used in screening assays as discussed above, in addition to methods of treatment.
• methods of modulating angiogenesis in cells or organisms comprise administering to a cell an anti-angiogenesis antibody that reduces or eliminates the biological activity of an endogeneous angiogenesis protein.
• the methods comprise administering to a cell or organism a recombinant nucleic acid encoding an angiogenesis protein. This may be accomplished in any number of ways. In a preferred embodiment, for example when the angiogenesis sequence is down-regulated in angiogenesis, such state may be reversed by increasing the amount of angiogenesis gene product in the cell.
• the gene therapy techniques include the inco ⁇ oration of the exogenous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby inco ⁇ orated by reference in its entireity.
• EHR enhanced homologous recombination
• the activity of the endogeneous angiogenesis gene is decreased, for example by the administration of a angiogenesis antisense nucleic acid or other inhibitor, such as RNAi.
• the angiogenesis eproteins of the present invention may be used to generate polyclonal and monoclonal antibodies to angiogenesis proteins.
• the angiogenesis proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify angiogenesis antibodies useful for production, diagnostic, or therapeutic purposes.
• the antibodies are generated to epitopes unique to a angiogenesis protein; that is, the antibodies show little or no cross-reactivity to other proteins.
• the angiogenesis antibodies may be coupled to standard affinity chromatography columns and used to purify angiogenesis proteins.
• the antibodies may also be used as blocking polypeptides, as outlined above, since they will specifically bind to the angiogenesis protein.
• the invention provides methods for identifying cells containing variant angiogenesis genes, e.g., determining all or part of the sequence of at least one endogeneous angiogenesis genes in a cell. This may be accomplished using any number of sequencing techniques.
• the invention provides methods of identifying the angiogenesis genotype of an individual, e.g., determining all or part of the sequence of at least one angiogenesis gene of the individual. This is generally done in at least one tissue of the individual, and may include the evaluation of a number of tissues or different samples of the same tissue.
• the method may include comparing the sequence of the sequenced angiogenesis gene to a known angiogenesis gene, i.e., a wild-type gene.
• the sequence of all or part of the angiogenesis gene can then be compared to the sequence of a known angiogenesis gene to determine if any differences exist. This can be done using any number of known homology programs, such as Bestfit, etc.
• the presence of a a difference in the sequence between the angiogenesis gene of the patient and the known angiogenesis gene correlates with a disease state or a propensity for a disease state, as outlined herein.
• the angiogenesis genes are used as probes to determine the number of copies of the angiogenesis gene in the genome.
• the angiogenesis genes are used as probes to determine the chromosomal localization of the angiogenesis genes, information such as chromosomal localization finds use in providing a diagnosis or prognosis in particular when chromosomal abnormalities such as translocations, and the like are identified in the angiogenesis gene locus.
• a therapeutically effective dose of an angiogenesis protein or modulator thereof is administered to a patient.
• therapeutically effective dose herein is meant a dose that produces effects for which it is administered. The exact dose will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (e.g., Ansel et al, Pharmaceuitcal Dosage Forms and Drug Delivery, Lippincott, Williams & Wilkins Publishers, ISBN:0683305727; Lieberman (1992) Pharmaceutical Dosage Forms (vols. 1-3), Dekker, ISBN 0824770846, 082476918X, 0824712692, 0824716981; Lloyd (1999) The Art, Science and Technology of Pharmaceutical Compounding, Amer.
• a "patient” for the pu ⁇ oses of the present invention includes both humans and other animals, particularly mammals. Thus the methods are applicable to both human therapy and veterinary applications.
• the patient is a mammal, preferably a primate, and in the most preferred embodiment the patient is human.
• angiogenesis proteins and modulators thereof of the present invention can be done in a variety of ways as discussed above, including, but not limited to, orally, subcutaneously, intravenously, intranasally, transdermally, intraperitoneally, intramuscularly, intrapulmonary, vaginally, rectally, or intraocularly.
• the angiogenesis proteins and modulators may be directly applied as a solution or spray.
• the pharmaceutical compositions of the present invention comprise an angiogenesis protein in a form suitable for administration to a patient.
• the pharmaceutical compositions are in a water soluble form, such as being present as pharmaceutically acceptable salts, which is meant to include both acid and base addition salts.
• “Pharmaceutically acceptable acid addition salt” refers to those salts that retain the biological effectiveness of the free bases and that are not biologically or otherwise undesirable, formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like, and organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid and the like.
• inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like
• organic acids such as acetic acid, propionic acid, glycolic acid, pyruvic acid,
• “Pharmaceutically acceptable base addition salts” include those derived from inorganic bases such as sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts and the like. Particularly preferred are the ammonium, potassium, sodium, calcium, and magnesium salts.
• Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
• compositions may also include one or more of the following: carrier proteins such as serum albumin; buffers; fillers such as microcrystalline cellulose, lactose, corn and other starches; binding agents; sweeteners and other flavoring agents; coloring agents; and polyethylene glycol.
• compositions can be administered in a variety of unit dosage forms depending upon the method of administration.
• unit dosage forms suitable for oral administration include, but are not limited to, powder, tablets, pills, capsules and lozenges.
• angiogenesis protein modulators e.g., antibodies, antisense constructs, ribozymes, small organic molecules, etc
• angiogenesis protein modulators when administered orally, should be protected from digestion. This is typically accomplished either by complexing the molecule(s) with a composition to render it resistant to acidic and enzymatic hydrolysis, or by packaging the molecule(s) in an appropriately resistant carrier, such as a liposome or a protection barrier. Means of protecting agents from digestion are well known in the art.
• compositions for administration will commonly comprise an angiogenesis protein modulator dissolved in a pharmaceutically acceptable carrier, preferably an aqueous carrier.
• a pharmaceutically acceptable carrier preferably an aqueous carrier.
• aqueous carriers can be used, e.g., buffered saline and the like. These solutions are sterile and generally free of undesirable matter.
• These compositions may be sterilized by conventional, well known sterilization techniques.
• the compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate and the like.
• the concentration of active agent in these formulations can vary widely, and will be selected primarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the patient's needs (e.g. , Remington 's Pharmaceutical Science, 15th ed., Mack Publishing Company, Easton, Pennsylvania (1980) and Goodman and Gillman, The Pharmacologial Basis of Therapeutics, (Rardman, J.G, Limbird, L.E, Molinoff, P.B., Ruddon, R.W, and Gilman, A.G.,eds) TheMcGraw-Hill Companies, Inc., 1996).
• a typical pharmaceutical composition for intravenous administration would be about 0.1 to 10 mg per patient per day. Dosages from 0.1 up to about 100 mg per patient per day may be used, particularly when the drug is administered to a secluded site and not into the blood stream, such as into a body cavity or into a lumen of an organ. Substantially higher dosages are possible in topical administration.
• Actual methods for preparing parenterally administrable compositions will be known or apparent to those skilled in the art, e.g., Remington 's Pharmaceutical Science and Goodman and Gillman, The Pharmacologial Basis of Therapeutics, supra.
• compositions containing modulators of angiogenesis proteins can be administered for therapeutic or prophylactic treatments.
• compositions are administered to a patient suffering from a disease (e.g., a cancer) in an amount sufficient to cure or at least partially arrest the disease and its complications.
• An amount adequate to accomplish this is defined as a "therapeutically effective dose.” Amounts effective for this use will depend upon the severity of the disease and the general state of the patient's health. Single or multiple administrations of the compositions may be administered depending on the dosage and frequency as required and tolerated by the patient. In any event, the composition should provide a sufficient quantity of the agents of this invention to effectively treat the patient.
• prophylactically effective dose An amount of modulator that is capable of preventing or slowing the development of cancer in a mammal is referred to as a "prophylactically effective dose.”
• the particular dose required for a prophylactic treatment will depend upon the medical condition and history of the mammal, the particular cancer being prevented, as well as other factors such as age, weight, gender, administration route, efficiency, etc.
• prophylactic treatments may be used, e.g., in a mammal who has previously had cancer to prevent a recurrence of the cancer, or in a mammal who is suspected of having a significant likelihood of developing cancer.
• angiogenesis protein-modulating compounds can be administered alone or in combination with additional angiogenesis modulating compounds or with other therapeutic agent, e.g., other anti-cancer agents or treatments.
• additional angiogenesis modulating compounds e.g., other anti-cancer agents or treatments.
• one or more nucleic acids e.g., polynucleotides comprising nucleic acid sequences set forth in Tables 1-8 , such as antisense polynucleotides or ribozymes, will be introduced into cells, in vitro or in vivo.
• the present invention provides methods, reagents, vectors, and cells useful for expression of angiogenesis-associated polypeptides and nucleic acids using in vitro (cell-free), ex vivo or in vivo (cell or organism-based) recombinant expression systems.
• the particular procedure used to introduce the nucleic acids into a host cell for expression of a protein or nucleic acid is application specific. Many procedures for introducing foreign nucleotide sequences into host cells may be used. These include the use of calcium phosphate transfection, spheroplasts, electroporation, liposomes, microinjection, plasma vectors, viral vectors and any of the other well known methods for introducing cloned genomic DNA, cDNA, synthetic DNA or other foreign genetic material into a host cell (see, e.g., Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Inc., San Diego, CA (Berger), F.M.
• angiogenesis proteins and modulators are administered as therapeutic agents, and can be formulated as outlined above.
• angiogenesis genes (including both the full-length sequence, partial sequences, or regulatory sequences of the angiogenesis coding regions) can be administered in a gene therapy application. These angiogenesis genes can include antisense applications, either as gene therapy (i.e. for inco ⁇ oration into the genome) or as antisense compositions, as will be appreciated by those in the art.
• Angiogenesis polypeptides and polynucleotides can also be administered as vaccine compositions to stimulate HTL, CTL and antibody responses..
• Such vaccine compositions can include, for example, lipidated peptides (e.g., Vitiello, A. et al, J.
• PLG poly(DL-lactide-co-glycolide)
• MAPs multiple antigen peptide systems
• peptides formulated as multivalent peptides peptides for use in ballistic delivery systems, typically crystallized peptides, viral delivery vectors
• Toxin-targeted delivery technologies also known as receptor mediated targeting, such as those of Avant Immunotherapeutics, Inc. (Needham, Massachusetts) may also be used.
• Vaccine compositions often include adjuvants.
• Many adjuvants contain a substance designed to protect the antigen from rapid catabolism, such as aluminum hydroxide or mineral oil, and a stimulator of immune responses, such as lipid A, Bortadella pertussis or Mycobacterium tuberculosis derived proteins.
• adjuvants are commercially available as, for example, Freund's Incomplete Adjuvant and Complete Adjuvant (Difco Laboratories, Detroit, MI); Merck Adjuvant 65 (Merck and Company, Inc., Rahway, NJ); AS-2 (SmithKline Beecham, Philadelphia, PA); aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate; salts of calcium, iron or zinc; an insoluble suspension of acylated tyrosine; acylated sugars; cationically or anionically derivatized polysaccharides; polyphosphazenes; biodegradable microspheres; monophosphoryl lipid A and quil A.
• Freund's Incomplete Adjuvant and Complete Adjuvant Difco Laboratories, Detroit, MI
• Merck Adjuvant 65 Merck and Company, Inc., Rahway, NJ
• AS-2 SmithKline Beecham, Philadelphia, PA
• aluminum salts such as aluminum hydroxide gel (alum) or aluminum phosphate
• Cytokines such as GM-CSF, interleukin-2, -7, -12, and other like growth factors, may also be used as adjuvants.
• Vaccines can be administered as nucleic acid compositions wherein DNA or RNA encoding one or more of the polypeptides, or a fragment thereof, is administered to a patient. This approach is described, for instance, in Wolff et. al, Science 247:1465 (1990) as well as U.S. Patent Nos. 5,580,859; 5,589,466; 5,804,566; 5,739,118; 5,736,524; 5,679,647; WO 98/04720; and in more detail below.
• DNA-based delivery technologies include "naked DNA”, facilitated (bupivicaine, polymers, peptide-mediated) delivery, cationic lipid complexes, and particle-mediated (“gene gun”) or pressure-mediated delivery (see, e.g., U.S. Patent No. 5,922,687).
• the peptides of the invention can be expressed by viral or bacterial vectors.
• expression vectors include attenuated viral hosts, such as vaccinia or fowlpox. This approach involves the use of vaccinia virus, for example, as a vector to express nucleotide sequences that encode angiogenic polypeptides or polypeptide fragments. Upon introduction into a host, the recombinant vaccinia virus expresses the immunogenic peptide, and thereby elicits an immune response.
• Vaccinia vectors and methods useful in immunization protocols are described in, e.g., U.S. Patent No. 4,722,848.
• BCG Bacille Calmette Guerin
• BCG vectors are described in Stover et al, Nature 351:456-460 (1991).
• a wide variety of other vectors useful for therapeutic administration or immunization e.g. adeno and adeno-associated virus vectors, retroviral vectors, Salmonella typhi vectors, detoxified anthrax toxin vectors, and the like, will be apparent to those skilled in the art from the description herein (see, e.g., Shata et al. (2000) Mol Med Today, 6: 66-71; Shedlock et al, J Leukoc Biol 68,:793-806, 2000; Hipp et al, In Vivo 14:571-85, 2000).
• Methods for the use of genes as DNA vaccines are well known, and include placing an angiogenesis gene or portion of an angiogenesis gene under the control of a regulatable promoter or a tissue-specific promoter for expression in an angiogenesis patient.
• the angiogenesis gene used for DNA vaccines can encode full-length angiogenesis proteins, but more preferably encodes portions of the angiogenesis proteins including peptides derived from the angiogenesis protein.
• a patient is immunized with a DNA vaccine comprising a plurality of nucleotide sequences derived from an angiogenesis gene.
• angiogenesis-associated genes or sequence encoding subfragments of an angiogenesis protein are introduced into expression vectors and tested for their immunogenicity in the context of Class I MHC and an ability to generate cytotoxic T cell responses.
• This procedure provides for production of cytotoxic T cell responses against cells which present antigen, including intracellular epitopes.
• the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine.
• adjuvant molecules include cytokines that increase the immunogenic response to the angiogenesis polypeptide encoded by the DNA vaccine. Additional or alternative adjuvants are available.
• angiogenesis genes find use in generating animal models of angiogenesis.
• angiogenesis gene identified When the angiogenesis gene identified is repressed or diminished in angiogenesic tissue, gene therapy technology, e.g., wherein antisense RNA directed to the angiogenesis gene will also diminish or repress expression of the gene.
• Animal models of angiogenesis find use in screening for modulators of an angiogenesis- associated sequence or modulators of angiogenesis.
• transgenic animal technology including gene knockout technology for example as a result of homologous recombination with an appropriate gene targeting vector, will result in the absence or increased expression of the angiogenesis protein.
• tissue-specific expression or knockout of the angiogenesis protein may be necessary. It is also possible that the angiogenesis protein is overexpressed in angiogenesis.
• transgenic animals can be generated that overexpress the angiogenesis protein.
• promoters of various strengths can be employed to express the transgene.
• the number of copies of the integrated transgene can be determined and compared for a determination of the expression level of the transgene. Animals generated by such methods find use as animal models of angiogenesis and are additionally useful in screening for modulators to treat angiogenesis or to evaluate a therapeutic entity.
• kits for Use in Diagnostic and/or Prognostic Applications
• kits are also provided by the invention.
• such kits may include any or all of the following: assay reagents, buffers, angiogenesis-specific nucleic acids or antibodies, hybridization probes and/or primers, antisense polynucleotides, ribozymes, dominant negative angiogenesis polypeptides or polynucleotides, small molecules inhibitors of angiogenesis-associated sequences etc.
• a therapeutic product may include sterile saline or another pharmaceutically acceptable emulsion and suspension base.
• kits may include instructional materials containing directions (i.e., protocols) for the practice of the methods of this invention.
• instructional materials typically comprise written or printed materials they are not limited to such. Any medium capable of storing such instructions and communicating them to an end user is contemplated by this invention.
• Such media include, but are not limited to electromc storage media (e.g., magnetic discs, tapes, cartridges, chips), optical media (e.g., CD ROM), and the like.
• Such media may include addresses to internet sites that provide such instructional materials.
• kits for screening for modulators of angiogenesis-associated sequences can be prepared from readily available materials and reagents.
• such kits can comprise one or more of the following materials: an angiogenesis-associated polypeptide or polynucleotide, reaction tubes, and instructions for testing angiogenic-associated activity.
• the kit contains biologically active angiogenesis protein.
• kits and components can be prepared according to the present invention, depending upon the intended user of the kit and the particular needs of the user. Diagnosis would typically involve evaluation of a plurality of genes or products. The genes will be selected based on correlations with important parameters in disease which may be identified in historical or outcome data.
• Example 1 Tissue Preparation. Labeling Chips, and Finee ⁇ rints Purify total RNA from tissue using TRIzol Reagent
• TRIzol is added directly to frozen tissue, which is then homogenize. Following homogenization, insoluble material is removed by centrifugation at 7500 x g for 15 min in a Sorvall superspeed or 12,000 x g for 10 min. in an Eppendorf centrifuge at 4°C. The clear homogenate is transferred to a new tube for use. The samples may be frozen now at -60° to -70°C (and kept for at least one month).
• the homogenate is mixed with 0.2ml of chloroform per 1ml of TRIzol reagent used in the original homogenization and incubated at room temp, for 2-3 minutes.
• the aqueous phase is then separated by centrifugation and transferred to a fresh tube and the RNA precipitated using isopropyl alcohol.
• the pellet is isolated by centrifugation, washed, air-dried, resuspended in an appropriate volume of DEPC H 2 0, and the absorbance measured.
• RNA Purification of poly A+ mRNA from total RNA is performed as follows. Heat an oligotex suspension to 37°C and mixing immediately before adding to RNA. The Elution Buffer is heated at 70°C. Warm up 2 x Binding Buffer at 65°C if there is precipitate in the buffer. Mix total RNA with DEPC-treated water, 2 x Binding Buffer, and Oligotex according to Table 2 on page 16 of the Oligotex Handbook. Incubate for 3 minutes at 65°C. Incubate for 10 minutes at room temperature. Centrifuge for 2 minutes at 14,000 to 18,000 g. Remove supernatant without disturbing Oligotex pellet. A little bit of solution can be left behind to reduce the loss of Oligotex.
• RNA synthesis using Gibco's "Superscript Choice System for cDNA Synthesis" kit First Strand cDNA synthesis is performed as follows. Use 5ug of total RNA or lug of poly A+ mRNA as starting material. For total RNA, use 2ul of Superscript RT. For polyA+ mRNA, use lul of Superscript RT. Final volume of first strand synthesis mix is 20ul. RNA must be in a volume no greater than lOul. Incubate RNA with lul of lOOpmol T7-T24 oligo for 10 min at 70C. On ice, add 7 ul of: 4ul 5X 1 st Strand Buffer, 2ul of 0. IM DTT, and 1 ul of 1 OmM dNTP mix. Incubate at 37C for 2 min then add Superscript RT. Incubate at 37C for 1 hour.
• In vitro Transcription (INT) and labeling with biotin is performed as follows: Pipet 1.5ul of cD ⁇ A into a thin- wall PCR tube. Make ⁇ TP labeling mix by combining 2ul T7 lOxATP (75mM) (Ambion); 2ul T7 lOxGTP (75mM) (Ambion); 1.5ul T7 lOxCTP (75mM) (Ambion); 1.5ul T7 lOxUTP (75mM) (Ambion); 3.75ul lOmM Bio-11-UTP (Boehringer- Mannheim/Roche or Enzo); 3.75ul lOmM Bio-16-CTP (Enzo); 2ul lOx T7 transcription buffer (Ambion); and 2ul lOx T7 enzyme mix (Ambion).
• the final volume is 20ul. Incubate 6 hours at 37°C in a PCR machine.
• the R ⁇ A can be furthered cleaned. Fragmentation is performed as follows. 15 ug of labeled R ⁇ A is usually fragmented. Try to minimize the fragmentation reaction volume; a 10 ul volume is recommended but 20 ul is all right. Do not go higher than 20 ul because the magnesium in the fragmentation buffer contributes to precipitation in the hybridization buffer. Fragment R ⁇ A by incubation at 94 C for 35 minutes in 1 x Fragmentation buffer (5 x Fragmentation buffer is 200 mM Tris-acetate, pH 8.1 ; 500 mM KOAc; 150 mM MgOAc).
• the labeled R ⁇ A transcript can be analyzed before and after fragmentation. Samples can be heated to 65°C for 15 minutes and electrophoresed on 1% agarose/TBE gels to get an approximate idea of the transcript size range
• 200 ul (lOug cRNA) of a hybridization mix is put on the chip. If multiple hybridizations are to be done (such as cycling through a 5 chip set), then it is recommended that an initial hybridization mix of 300 ul or more be made.
• the hybridization mix is: fragment labeled RNA (50ng/ul final cone); 50 pM 948-b control oligo; 1.5 pM BioB; 5 pM BioC; 25 pM BioD; 100 pM CRE; 0. Img/ml herring sperm DNA; 0.5mg/ml acetylated BSA; and 300 ul with lxMES hyb buffer.
• the hybridization reaction includes non- biotinylated IVT (purified by RNeasy columns); INT antisense R ⁇ A 4 ⁇ g: ⁇ l; random Hexamers (1 ⁇ g/ ⁇ l) 4 ⁇ l and water to 14 ul.
• the reaciton is incubated at 70°C, 10 min.
• Reverse transcription is performed in the following reaction: 5X First Strand (BRL) buffer, 6 ⁇ l; 0.1 M DTT, 3 ⁇ l; 50X d ⁇ TP mix, 0.6 ⁇ l; H 2 O, 2.4 ⁇ l; Cy3 or Cy5 dUTP (lmM), 3 ⁇ l; SS RT II (BRL), 1 ⁇ l in a final volume of 16 ⁇ l.
• R ⁇ A degradation is performed as follows. Add 86 ⁇ l H2O, 1.5 ⁇ l IM ⁇ aOH/ 2mM EDTA and incubate at 65°C, 10 min.. For U-Con 30, 500 ⁇ l TE/sample spin at 7000g for 10 min, save flow through for purification. For Qiagen purification, suspend u-con recovered material in 500 ⁇ l buffer PB and proceed using Qiagen protocol. For D ⁇ Ase digestion, add 1 ul of 1/100 dil of D ⁇ Ase/30ul Rx and incubate at 37°C for 15 min. Incubate at 5 min 95 °C to denature the DNAse/
• Cot-1 DNA 10 ⁇ l; 50X dNTPs, 1 ⁇ l; 20X SSC, 2.3 ⁇ l; Na pyro phosphate, 7.5 ⁇ l; lOmg/ml Herring sperm DNA; lul of 1/10 dilution to 21.8 final vol. Dry in speed vac. Resuspend in 15 ⁇ l H20. Add 0.38 ⁇ l 10% SDS. Heat 95°C, 2 min and slow cool at room temp, for 20 min. Put on slide and hybridize overnight at 64°C.
• Example 2 A model of angiogenesis is used to determine expression in angiogenesis
• human umbilical vein endothelial cells were obtained, e.g., as passage 1 (pl) frozen cells from Cascade Biologies (Oregon) and grown in maintenance medium: Medium 199 (Life Technologies) supplemented with 20% pooled human serum, 100 mg/ml heparin and 75 mg/ml endothelial cell growth supplements (Sigma) and gentamicin (Life Technologies).
• RNA was collected, e.g., at 0, 2, 6, 15, 24, 48, and 96 hours of culture.
• the fibrin clots were placed in Trizol (Life Technologies) and disrupted using a Tissuemizer. Thereafter standard procedures were used for extracting the RNA (e.g., Example 1).
• Angiogenesis associated sequences thus identified are shown in Tables 1-8 .
• Accession numbers include expression sequence tags (ESTs).
• genes within an expression profile also termed expression profile genes, include ESTs and are not necessarily full length.
• D55640 gb Human monocyte PABL (pseudoautosomal boundary-like sequence) mRNA, clone Mo2.
• solute carrier family 7 (cationic amino acid transporter, y+ system), member 6
• ubiquitin protein ligase E3A human papilloma virus E6-associated protein, Angelman syndrome
• RNA II DNA directed polypeptide B (140kD)
• amyloid beta (A4) precursor protein protease nexin-ll, Alzheimer disease
• CACNA1F gene, complete eds; HSP27 pseudogene, complete sequence; and JM1 protein, JM2 protein, and Hb2E genes, complete eds
• RNA II DNA directed polypeptide J (13.3kD)

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