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
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5091—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing the pathological state of an organism
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • 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
  • C12Q1/48—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving transferase
• • 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
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
• • 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
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
• • 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
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
  • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
• • 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
  • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
  • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
  • G01N33/5044—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics involving specific cell types
  • G01N33/5064—Endothelial cells
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2550/00—Electrophoretic profiling, e.g. for proteome analysis

Definitions

• the invention relates to the identification of expression profiles and the nucleic acids involved in angiogenesis, and to the use of such expression profiles and nucleic acids in diagnosis of angiogenesis
• the invention further relates to methods for identifying candidate agents and/or targets which modulate angiogenesis
• New blood vessel development comprises the formation of veins (vasculogenesis) and arteries (angiogenesis)
• Angiogenesis plays a normal role in embryonic development, as well as menstration, wound healing Angiogenesis also plays a crucial pathogenic role in a variety of disease states, including cancer, proliferative diabetic retinopathy, and maintaining blood flow to chronic inflammatory sites
• Angiogenesis has a number of stages
• 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 VEGF, TGF- ⁇ , angiostatin, and selected chemokmes all putatively playing a role
• Later stages of angiogenesis include the population of the vessels with mural cells (pe cytes 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 present invention provides novel methods for diagnosis and prognosis evaluation for angiogenesis, as well as methods for screening for compositions which modulate angiogenesis. Methods of treatment of disorders associated with angiogenesis, as well as compositions are also provided herein.
• a method of screening drug candidates comprises providing a cell that expresses an expression profile gene or fragments thereof, or fragments thereof.
• Preferred embodiments of the expression profile gene are genes which are differentially expressed in angiogenesis cells, compared to other cells.
• Preferred embodiments of expression profile genes used in the methods herein include but are not limited to the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10; fragments of the proteins of this group are also preferred. It is understood that molecules for use in the present invention may be from any figure or any subset of listed molecules. Therefore, for example, any one or more of the genes listed above can be used in the methods herein.
• a nucleic acid is selected from Tables 1 , 2, 3, 4 or 5.
• Preferred nucleic acids are in Table 4, and most preferably Table 5.
• the method further includes adding a drug candidate to the cell and determining the effect of the drug candidate on the expression of the expression profile gene.
• the method of screening drug candidates includes comparing the level of expression in the absence of the drug candidate to the level of expression in the presence of the drug candidate, wherein the concentration of the drug candidate can vary when present, and wherein the comparison can occur after addition or removal of the drug candidate.
• the cell expresses at least two expression profile genes. The profile genes may show an increase or decrease.
• AMP angiogenesis modulator protein
• the method comprising combining the AMP and a candidate bioactive agent, and determining the binding of the candidate agent to the AMP.
• the AMP is a protein or fragment thereof selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10.
• the proteins is encoded by a nucleic acid selected from Tables 1 , 2, 3, 4 or 5. Preferred nucleic acids are in Table 4, and most preferably Table 5.
• a method for screening for a bioactive agent capable of modulating the activity of an AMP is a method for screening for a bioactive agent capable of modulating the activity of an AMP.
• the method comprises combining the AMP and a candidate bioactive agent, and determining the effect of the candidate agent on the bioactivity of the AMP.
• the AMP is a protein or fragment thereof selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10.
• the proteins is encoded by a nucleic acid selected from Tables 1 , 2, 3, 4 or 5. Preferred nucleic acids are in Table 4, and most preferably Table 5.
• Also provided is a method of evaluating the effect of a candidate angiogenesis drug comprising administering the drug to a transgenic animal expressing or over-expressing the AMP, or an animal lacking the AMP, for example as a result of a gene knockout.
• a method of evaluating the effect of a candidate angiogenesis drug comprising administering the drug to a patient and removing a cell sample from the patient.
• the expression profile of the cell is then determined.
• This method may further comprise comparing the expression profile to an expression profile of a healthy individual.
• the expression profile includes a gene of Table 1 , Table 2, Table 3, Table 4 or Table 5.
• a biochip comprising one or more nucleic acid segments which encode an angiogenesis protein, preferable selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase , or fragment thereof, wherein the biochip comprises fewer than 1000 nucleic acid probes.
• the biochip comprises fewer than 1000 nucleic acid probes.
• at least two nucleic acid segments are included.
• the nucleic acid selected from Tables 1 , 2, 3, 4 or 5.
• Preferred nucleic acids are in Table 4, and most preferably Table 5.
• a method of diagnosing a disorder associated with angiogenesis comprises determining the expression of a gene which encodes an angiogenesis protein preferable selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10, or fragment thereof in a first tissue type of a first individual, and comparing the distribution to the expression of the gene from a second normal tissue type from the first individual or a second unaffected individual.
• the proteins is encoded by a nucleic acid selected from Tables 1 , 2, 3, 4 or 5. Preferred nucleic acids are in Table 4, and most preferably Table 5. A difference in the expression indicates that the first individual has a disorder associated with angiogenesis.
• the present invention provides an antibody which specifically binds to an angiogenesis preferably selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10 or fragment thereof
• an angiogenesis preferably selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10 or fragment thereof
• the proteins is encoded by a nucleic acid selected from Tables 1 , 2, 3, 4 or 5 Preferred nucleic acids are in Table 4, and most preferably Table 5
• the fragment of AAA1 is selected from AAA1 p1 or AAA1 p2
• Other preferred fragments for the angiogenesis proteins are shown in the figures
• a method for screening for a bioactive agent capable of interfering with the binding of a angiogenesis modulating protein (AMP) or a fragment thereof and an antibody which binds to said AMP or fragment thereof comprises combining an AMP or fragment thereof, a candidate bioactive agent and an antibody which binds to said AMP or fragment thereof
• the method further includes determining the binding of said AMP or fragment thereof and said antibody Wherein there is a change in binding, an agent is identified as an interfering agent
• the interfering agent can be an agonist or an antagonist
• the agent inhibits angiogenesis
• a method for inhibiting angiogenesis comprises administering to a cell a composition comprising an antibody to an angiogenesis modulating protein, preferably selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10, or fragment thereof
• the proteins is encoded by a nucleic acid selected from Tables 1 , 2, 3, 4 or 5 Preferred nucleic acids are in Table 4, and most preferably Table 5
• the method can be performed in vitro or in vivo, preferably in vivo to an individual
• the method of inhibiting angiogenesis is provided to an individual with a disorder associated with angiogenesis such as cancer
• methods of inhibiting angiogenesis can be performed by administering an inhibitor of the activity of an angiogenesis protein, including an antisense molecule to the gene or its gene products, and preferable small molecules
• a method provided herein comprises administering to an individual a composition comprising an angiogenesis modulating protein, preferably selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10, or fragment thereof
• angiogenesis modulating protein preferably selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10, or fragment thereof
• the proteins is encoded by a nucleic acid selected from Tables 1 , 2,
• nucleic acids are in Table 4, and most preferably Table 5
• said composition comprises a nucleic acid comprising a sequence encoding an angiogenesis modulating protein, preferably selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10, or fragment thereof.
• the proteins is encoded by a nucleic acid selected from Tables 1, 2, 3, 4 or 5.
• Preferred nucleic acids are in Table 4, and most preferably Table 5.
• compositions capable of eliciting an immune response in an individual.
• a composition provided herein comprises an angiogenesis modulating protein, preferably selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10, or fragment thereof.
• the proteins is encoded by a nucleic acid selected from Tables 1 , 2, 3, 4 or 5. Preferred nucleic acids are in Table 4, and most preferably Table 5.
• said composition comprises a nucleic acid comprising a sequence encoding an angiogenesis modulating protein, preferably selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10, or fragment thereof, and a pharmaceutically acceptable carrier.
• angiogenesis modulating protein preferably selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10, or fragment thereof, and a pharmaceutically acceptable carrier.
• nucleic acid selected from Tables 1 , 2, 3, 4 or 5.
• Preferred nucleic acids are in Table 4, and most preferably Table 5.
• a method of neutralizing the effect of an angiogenesis protein preferably selected from the group consisting of AAA4, AAA1 , Edg-1 , alpha 5 betal integrin, endomucin and matrix metalloproteinase 10, or fragment thereof, comprising contacting an agent specific for said protein with said protein in an amount sufficient to effect neutralization.
• the proteins is encoded by a nucleic acid selected from Tables 1 , 2, 3, 4 or 5. Preferred nucleic acids are in Table 4, and most preferably Table 5.
• a method of treating an individual for a disorder associated with angiogenesis comprises administering to said individual an inhibitor of Edg-1.
• the method comprises administering to a patient having a disorder with angiogenesis an antibody to Edg-1 conjugated to a therapeutic moiety.
• a therapeutic moiety can be a cytotoxic agent or a radioisotope.
• Table 1 provides the Accession numbers for 1774 genes, including expression sequence tags, (incorporated in their entirety here and throughout the application where Accession numbers are provided), whose expression levels change as a function of time in tissue undergoing angiogenesis compared to tissue that is not.
• Table 2 provides the Accession numbers for a preferred subset of 559 genes, including expression sequence tags (incorporated in their entirety here and throughout the application where Accession numbers are provided), whose expression levels change as a function of time in tissue undergoing angiogenesis compared to tissue that is not.
• the sequences are characterized as predicted to encode secreted proteins (SS), or transmembrane proteins (TM) proteins.
• Table 3 provides the Accession numbers for 1916 genes including expression sequence tags
• Table 4 provides a preferred subset of 558 Accession numbers identified in Figure 4 whose expression levels change as a function of time in tissue undergoing angiogenesis compared to tissue that is not.
• Table 5 provides a preferred subset of 20 Accession numbers identified in Figure 4 whose expression levels change as a function of time in tissue undergoing angiogenesis compared to tissue that is not.
• Figure 1 is a graph of expression levels of sequences identified in Figure 1. Expression profiles are clustered into 4 groups. C1 (blue), C2 (red), C3 (green) and C4 (mustard).
• Figure 2 shows an embodiment of a nucleic acid (mRNA) which includes a sequence encoding an angiogenesis protein, AAA4. The start and stop codons are underlined.
• mRNA nucleic acid
• Figure 3 shows the open reading frame of a nucleic acid sequence encoding AAA4. The start and stop codons are underlined.
• FIG 4 shows an embodiment of the amino acid sequence of AAA4.
• the signal peptide is double underlined, and the transmembrane sequence is underlined.
• AAA4 is soluble.
• the signal peptide can be omitted, and the transmembrane domain deleted, inactivated, or truncated.
• Figure 5 shows peptides AAA4p1 and AAA4p2.
• Figure 6 shows the expression of AAA4 in angiogenesis models over time and in other, non- angiogenic tissues.
• Figure 7 shows an embodiment of a nucleic acid sequence encoding an angiogenesis protein, AAA1.
• a putative stop codon is underlined.
• FIG. 8 shows an embodiment of an amino acid sequence for AAA1.
• a transmembrane domain is underlined.
• AAA1 is soluble.
• the transmembrane domain is deleted or inactivated, or AAA1 is truncated to delete the transmembrane domain.
• FIG. 9 shows AAA1 p1 and AAA1 p2.
• FIG 10 shows a graph showing the relative expression of AAA1 in various tissues at different time points.
• “Exp 3” is an angiogenesis model showing tube formation over time using endothelial cells.
• Figure 1 1 shows an embodiment of a nucleic acid, mRNA, which comprises a sequence encoding an angiogenesis protein, Edg-1. The start and stop codons are underlined.
• Figure 12 shows the open reading frame encoding Edg-1 , wherein the start and stop codons are underlined.
• Figure 13 shows an embodiment of an amino acid sequence for an angiogenesis protein, Edg-1 , wherein the transmembrane domains are underlined.
• Edg-1 an angiogenesis protein
• a soluble form of Edg-1 is provided.
• the transmembrane domains are deleted, inactivated, and/or the protein is truncated so as to exclude the domains (with or without re-ligation of remaining soluble regions).
• Figure 14 depicts four peptide sequences provided herein and their respective solubilities.
• Figure 15 shows the expression of Edg-1 over a variety of tissues.
• Figure 16 shows the time course of induction of Edg-1 in a model for angiogenesis (Expt 1 , Expt 2,
• Figure 17 shows an embodiment of a nucleic acid sequence which includes the coding sequence for a tissue remodeling protein, alpha 5 beta 1 integrin (sometimes referred to as VLA-5), wherein the start and stop codon are underlined.
• Figure 18 shows an embodiment of an amino acid sequence of a tissue remodeling protein, alpha 5 beta 1 integrin, wherein a transmembrane domain is underlined.
• Figure 19 shows a bar graph depicting the results of 5 expression profiles of alpha 5 beta 1 integrin throughout the time course of tube formation.
• tube models 1 , 2 and 3 show models which form tube structures from single isolated human endothelial cells; the "EC/PMA” model shows endothelial cells stimulated with pokeweed mitogen antigen, and the body atlas profile shows expression in various normal cell types and tissues.
• Figures 20A and 20B show the results of antagonism of tube formation wherein Figure 20A is an isotype control and Figure 20B shows specific antibody antagonism after 48 hours.
• Figure 21 shows an embodiment of a nucleic acid sequence which includes the coding sequence for an angiogenesis protein, endomucin, wherein the start and stop codon are boxed.
• Figure 22 shows an embodiment of an amino acid sequence of an angiogenesis protein, endomucin, wherein a signal sequence is bolded and a transmembrane domain is underlined.
• Figure 23 shows an embodiment of a nucleic acid sequence which includes the coding sequence for an angiogenesis protein, matrix metalloproteinase 10 (also called stromolysin 2), wherein the start and stop codon are boxed.
• matrix metalloproteinase 10 also called stromolysin 2
• Figure 24 shows expression of matrix metalloproteinase 10 over a variety of tissues.
• Figure 25 shows expression of matrix metalloproteinase 10 over a variety of tissues.
• the present invention provides novel methods for diagnosis 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 vessel development.
• Angiogenesis disorders include, but are not limited to, cancer and proliferative diabetic retinopathy. Also provided are method for treating AD.
• 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.
• the evaluation of 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 may be done or confirmed by comparing patient samples with the known expression profiles.
• these gene expression profiles (or individual genes) 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.
• These methods can also be done on the protein basis; that is, protein expression levels of the angiogenic proteins can be evaluated for diagnostic purposes or to screen candidate agents.
• the 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).
• angiogenesis sequences are from humans; however, as WO 01 /l 1086 PCT/USOO/22061
• 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 ammo 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 by 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 gating DNA molecules that are not normally joined are both considered recombinant for the purposes of this 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 Alternatively, the protein may be made at a significantly higher concentration than is normally seen, through the use of an inducible promoter or high
• 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, as outlined below, nucleic acid analogs are included that may have alternate backbones, comprising, for example, phosphoramidate (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); SRocl et al., Eur. J. Biochem. 81 :579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta
• nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Patent Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991 ); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994);
• nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp169- 176).
• nucleic acid analogs are described in Rawls, C & E News June 2, 1997 page 35. All of these references are hereby expressly incorporated by reference. These 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.
• Particularly preferred are peptide nucleic acids (PNA) which includes peptide nucleic acid analogs.
• 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. First, 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 Tm for an internal mismatch.
• 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 other strand (“Crick"); thus the sequences described herein also includes the complement of the sequence.
• the nucleic acid may be DNA, both genomic and cDNA, RNA or a hybrid, where the nucleic acid contains any combination of deoxyribo- and ribo-nucleotides, and any combination 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. Thus for example the individual units of a peptide nucleic acid, each containing a base, are referred to herein as a nucleoside.
• An 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 included comparing genes identified in an in vitro model of angiogenesis as described in Hiraoka, Cell 95:365 (1998), which is expressly incorporated by reference, 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 preferable that the target be disease specific, to minimize possible side effects
• angiogenesis sequences are those that are up-regulated in angiogenesis disorders, that is, the expression of these genes is higher in the disease tissue as compared to normal tissue
• Up-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
• GenBank GenBank sequence database
• 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/
• these genes were found to be expressed in a limited amount or not at all in heart, brain, lung, liver, breast, kidney, prostate, small intestine and spleen
• 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 Alternatively, 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
• Table 1 depicts 1774 genes, the expression of which varies as a function of time in angiogenesis tissue when compared to normal tissue
• Figure 1 depicts 4 discrete expression profiles of angiogenesis genes identified in Table 1
• a particularly preferred embodiment includes the sequences as described in Table 2 which depicts a preferred subset of 559 angiogenesis sequences, the expression of which is altered in angiogenesis when compared to normal tissue
• An additional embodiment includes the sequences as described in Table 3, which depicts 1916 genes including expression sequence tags (incorporated in their entirety here and throughout the application where Accession numbers are provided), whose expression levels change as a function of time in tissue undergoing angiogenesis compared to tissue that is not
• a preferred embodiment includes the sequences as described in Table 4 which depicts a preferred subset of 558 genes identified in Table 3 whose expression levels change as a function of time in tissue undergoing angiogenesis compared to tissue that is not
• a particularly preferred embodiment includes the sequences as described in Table 5 which provides a preferred subset of 20 Accession numbers identified in Table 3 whose expression levels change as a function of time in tissue undergoing angiogenesis compared to tissue that is not
• angiogenesis sequences are those that are induced for a period of time followed by a return to the baseline levels Sequences that are temporarily induced provide a means to target angiogenesis tissue, for example neovasculanzed tumors, while avoiding rapidly growing tissue that require perpetual vasculanzation
• positive angiogenic factors include aFGF, bFGF, VEGF, 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 with 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 with 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 endostatm 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.
• 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 nucleos. Intracellular proteins are involved in all aspects of cellular function and replication (including, for example, signaling pathways); aberrant expression of such proteins results in unregulated or disregulated cellular processes. For example, many 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 the 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. For example, the intracellular domain may have enzymatic activity and/or may serve as a binding site for additional proteins. Frequently the intracellular domain of transmembrane proteins serves both roles. For example certain receptor tyrosine kinases have both protein kinase activity and SH2 domains. In addition, 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 are classified as "seven transmembrane domain" proteins, as they contain 7 membrane spanning regions.
• transmembrane protein receptors include, but are not limited to insulin receptor, insulin-like growth factor receptor, human growth hormone receptor, glucose transporters, transferrin receptor, epidermal growth factor receptor, low density lipoprotein receptor, epidermal growth factor receptor, leptin receptor, interleukin receptors, e.g. IL-1 receptor, IL-2 receptor, etc.
• Characteristics of transmembrane domains include approximately 20 consecutive hydrophobic amino acids that may be followed by charged amino acids. Therefore, upon analysis of the amino acid sequence of a particular protein, the localization and number of transmembrane domains within the protein may be predicted.
• extracellular domains are involved in binding to other molecules.
• extracellular domains are receptors.
• Factors that bind the receptor domain include circulating ligands, which may be peptides, proteins, or small molecules such as adenosine and the like.
• 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.
• GPI glycosylphosphatidylinositol
• Putative transmembrane angiogenesis proteins include those encoded by the sequences labeled with "Y" in the TM column depicted in Table 2.
• transmembrane proteins that are transmembrane are particularly preferred in the present invention as they are good targets for immunotherapeutics, as are described herein.
• transmembrane proteins can be also useful in imaging modalities.
• 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).
• secreted molecules find use in modulating or altering numerous aspects of physiology.
• Angiogenesis proteins that are secreted proteins are particularly preferred in the present invention as they serve as good targets for diagnostic markers, for example for blood tests.
• Putative secreted angiogenesis proteins include those encoded by the sequences depicted in Table 2 that are labeled with "Y” in the SS column, but a "N” in the TM column.
• angiogenesis sequence is 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.
• a nucleic acid is an "angiogenesis nucleic acid” if the overall homology of the nucleic acid sequence to one of the nucleic acids of Table 1 , Table 2, Table 3, Table 4 or Table 5 is preferably greater than about 75%, more preferably greater than about 80%, even more preferably greater than about 85% and most preferably greater than 90%. In some embodiments the homology will be as high as about 93 to 95 or 98%.
• Homology in this context means sequence similarity or identity, with identity being preferred.
• a preferred comparison for homology purposes is to compare the sequence containing sequencing errors to the correct sequence. This homology will be determined using standard techniques known in the art, including, but not limited to, the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981 ), by the homology alignment algorith of Needleman & Wunsch, J. Mol. Biool. 48:443 (1970), by the search for similarity method of Pearson & Lipman,
• sequences which are used to determine sequence identity or similarity are selected from the sequences set forth in the tables and figures, preferable those represented in Table 4, more preferably those represented in table 5, still more preferably those of Figures 2, 3, 7, 11 , 12, 17, 21 , 23 and fragments thereof.
• sequences utilized herein are those set forth in the tables and figures.
• sequences are naturally occurring allelic variants of the sequences set forth in the tables and figures.
• sequences are sequence variants as further described herein.
• PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments. It can also plot a tree showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. Mol. Evol. 35:351-360 (1987); the method is similar to that described by Higgins & Sharp CABIOS 5:151-153 (1989).
• Useful PILEUP parameters including a default gap weight of 3.00, a default gap length weight of 0.10, and weighted end gaps.
• BLAST algorithm described in Altschul et al offset J. Mol. Biol. 215, 403-410, (1990) and Karlin et al., PNAS USA 90:5873-5787 (1993).
• WU-BLAST-2 program was obtained from Altschul et al., Methods in
• the HSP S and HSP S2 parameters are dynamic values and are established by the program itself depending upon the composition of the particular sequence and composition of the particular database against which the sequence of interest is being searched; however, the values may be adjusted to increase sensitivity.
• a % amino acid sequence identity value is determined by the number of matching identical residues divided by the total number of residues of the "longer" sequence in the aligned region. The "longer" sequence is the one having the most actual residues in the aligned region (gaps introduced by WU- Blast-2 to maximize the alignment score are ignored).
• percent (%) nucleic acid sequence identity is defined as the percentage of nucleotide residues in a candidate sequence that are identical with the nucleotide residues of the nucleic acids of the figures.
• 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 the nucleic acids identified in the figures, or their complements are considered an angiogenesis sequence.
• High stringency conditions are known in the art; see for example Maniatis et al., Molecular Cloning: A Laboratory Manual, 2d Edition, 1989, and Short Protocols in Molecular Biology, ed. Ausubel, et al., both of which are hereby incorporated by reference.
• Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures.
• Tm 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.
• less stringent hybridization conditions are used; for example, moderate or low stringency conditions may be used, as are known in the art; see Maniatis and Ausubel, supra, and
• 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, additional sequences 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 Maniatis et al., and Ausubel, et al., supra, hereby expressly incorporated by reference.
• 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. 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 additional 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 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 either 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, in general, 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.
• 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 be covalent or non-covalent.
• non-covalent binding and grammatical equivalents herein is meant one or more of either 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 any 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).
• 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 covendedly coated with streptavidin, resulting in attachment
• the oligonucleotides may be synthesized on the surface, as is known in the art
• photoactivation techniques utilizing photopolymenzation 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
• angiogenesis nucleic acids 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
• the expression vectors may be either self-replicating extrachromosomal vectors or vectors which integrate into a host genome Generally, these expression vectors include transcnptional and translational regulatory nucleic acid operably linked to the nucleic acid encoding the angiogenesis protein
• control sequences refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism
• the control sequences that are suitable for prokaryotes include a promoter, optionally an operator sequence, and a nbosome 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 preprotem 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
• a nbosome 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
• enhancers do not have to be contiguous Linking is accomplished by ligation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice The transcnptional and translational regulatory nucleic acid will generally be appropriate
• the transcnptional and translational regulatory sequences may include, but are not limited to, promoter sequences, ribosomal binding sites, transcnptional start and stop sequences, translational start and stop sequences, and enhancer or activator sequences.
• the regulatory sequences include a promoter and transcnptional 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.
• the 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.
• 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.
• the 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.
• 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, archaebacte a, fungi, and insect and animal cells, including mammalian cells. Of particular interest are Drosophila melangaster cells, Saccharomyces cerevisiae and other yeasts, E. coli, Bacillus subtilis, Sf9 cells, C129 cells, 293 cells, Neurospora,
• BHK BHK, CHO, COS, HeLa cells, HUVEC (human umbilical vein endothelial cells),THP1 cells (a macrophage cell line) and human cells and lines.
• the angiogenesis proteins are expressed in mammalian cells.
• Mammalian expression systems are also known in the art, and include retroviral systems.
• a preferred expression vector system is a retroviral vector system such as is generally described in PCT/US97/01019 and
• 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.
• 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 nbosome 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 other nucleic acid for expression purposes.
• 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 or antigens; 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 3 H, 14 C, 32 P, 35 S, or 125 l, 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 3 H, 14 C, 32 P, 35 S, or 125 l
• 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
• 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 homology to some protein in the database being used. Generally, the nucleic acid sequences are input into a program that will search all three frames for homology.
• NCBI Advanced BLAST parameters 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
• homology in this context means sequence similarity or identity, with identity being preferred. This homology will be determined using standard techniques 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, in addition, as outlined above, 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 contain at least one ammo acid substitution, deletion or insertion, with ammo acid substitutions being particularly preferred The ammo acid substitution, insertion or deletion may occur at any residue within the angiogenesis peptide
• ammo acid sequence variants are also included within one embodiment of angiogenesis proteins of the present invention.
• ammo acid sequence variants 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
• variant angiogenesis protein fragments having up to about 100-150 residues may be prepared by in vitro synthesis using established techniques
• Ammo acid sequence variants are characterized by the predetermined nature of the variation, a feature that sets them apart from naturally occurring allelic or mterspecies variation of the angiogenesis protein ammo 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, M13 primer mutagenesis and PCR mutagenesis Screening of the mutants is done using assays of angiogenesis protein activities
• Ammo acid substitutions are typically of single residues, insertions usually will be on the order of from about 1 to 20 ammo 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 that are less conservative than those shown in Chart I.
• 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.
• the variants typically exhibit the same qualitative biological activity and will elicit the same immune response as the naturally-occurring analogue, 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.
• Dehvatization 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-1 ,8-octane and agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate.
• 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.
• Addition of glycosylation sites to angiogenesis polypeptides may 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. Such methods are described in the art, e.g., in WO 87/05330 published 11 September 1987, and in Aplin and Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981 ). Removal of carbohydrate moieties present on the angiogenesis polypeptide may be accomplished chemically or enzymatically or by mutational substitution of codons encoding for amino acid residues that serve as targets for glycosylation. Chemical deglycosylation techniques are known in the art and described, for instance, by Hakimuddin, et al., Arch. Biochem.
• 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;
• 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. For a bivalent form of the chimeric molecule, such a fusion could be to the Fc region of an IgG molecule.
• tag polypeptides and their respective antibodies are well known in the art. Examples include poly-histidine (poly-his) or poly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al., Mol. Cell.
• 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
• 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.
• angiogenesis proteins can be made that are longer than those encoded by the nucleic acids of the figures, for example, by the elucidation of additional 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, for example for immunotherapy, the angiogenesis protein should share at least one epitope or determinant with the full length protein.
• epitope or “determinant” herein is meant a portion of a protein which will generate and/or bind an antibody or T-cell receptor in the context of MHC. Thus, in most instances, antibodies made to a smaller angiogenesis protein will be able to bind to the full length protein.
• the epitope is unique; that is, antibodies generated to a unique epitope show little or no cross-reactivity.
• the epitope is selected from AAA4p1 and AAA4p2. In another preferred embodiment the epitope is selected from AAA1p1 and AAA1 p2. In another preferred embodiment the epitope is selected from AAA7p1 , AAA7p2, AAA7p3 and AAA7p1m.
• antibody includes antibody fragments, as are known in the art, including Fab, Fab 2 , single chain antibodies (Fv for example), chimeric antibodies, etc., either produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies.
• Polyclonal antibodies can be raised in a mammal, for example, 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.
• immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
• adjuvants which may be employed 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 Table 1 ,
• PBLs peripheral blood lymphocytes
• 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.
• 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 parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT)
• HGPRT or HPRT hypoxanthine guanine phosphoribosyl transferase
• 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.
• one of the binding specificities is for a protein encoded by a nucleic acid of figure 1 or 3-6 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.
• the antibodies to angiogenesis protein are capable of reducing or eliminating the biological function of angiogenesis protein, as is described below.
• anti-angiogenesis protein antibodies either polyclonal or preferably monoclonal
• angiogenic tissue or cells containing angiogenesis
• anti-angiogenesis protein antibodies may reduce or eliminate the angiogenesis activity.
• 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.
• 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
• 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.
• the 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 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.
• 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.
• such 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(1 ):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.
• transgenic animals e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated.
• 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. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661 ,016, and in the following scientific publications: Marks et al., Bio/Technology 10, 779-783 (1992); Lonberg et al.. Nature 368 856-859 (1994); Morris
• 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.
• 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
• the antibody prevents activation of the transmembrane angiogenesis protein
• the antibody prevents growth of the cell
• the antibody also sensitizes the cell to cytotoxic agents, including, but not limited to TNF- ⁇ , TNF- ⁇ , IL-1 , INF- ⁇ and IL-2, or chemotherapeutic agents including 5FU, vinblastme, actinomycm 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
• angiogenesis is treated by administering to cytotoxic agents, including, but not limited to TNF- ⁇ , TNF- ⁇ , IL-1 , I
• the antibody is conjugated to 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 collagenase activity associated with angiogenesis
• the therapeutic moiety may 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 dipthena A chain, exotox A chain, ncin A chain, abrin A chain, curcm, crotm, phenomycm, enomycm 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 chelatmg 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
• the angiogenesis protein against which the antibodies are raised is an intracellular protein
• the antibody may be conjugated 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.
• angiogenesis antibodies of the invention specifically bind to angiogenesis proteins.
• specifically bind herein is meant that the antibodies bind to the protein with a binding constant in the range of at least 10 "4 - 10 " ⁇ M "1 , with a preferred range being 10 "7 - 10 "9 M “1 .
• 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 chromatographic 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- Veriag, NY (1982). The degree of purification necessary will vary depending on the use of the angiogenesis protein. In some instances no purification will be necessary.
• angiogenesis proteins and nucleic acids are useful in a number of applications.
• the expression levels of genes are determined for different cellular states in the angiogenesis phenotype; that is, the 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 unique to the state of the cell.
• tissue from a particular patient have the gene expression profile of normal or angiogenesis tissue.
• differential expression refers to both qualitative as well as quantitative differences in the genes' temporal and/or cellular expression patterns within and among the cells.
• a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation, in, for example, normal versus angiogenic tissue That is, genes may be turned on or turned off in a particular state, relative to another state As is apparent to the skilled artisan, any comparison of two or more states can be made Such a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which is detectable by standard techniques in one such state or cell type, but is not detectable in both Alternatively, the determination is quantitative in that expression is increased or decreased, that is, the expression of the gene 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 Affymet ⁇ x GeneChip
• this may be done by evaluation at either the gene transcript, or the protein level, that is, 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, for example through the use of antibodies to the angiogenesis protein and standard immunoassays (ELISAs, etc ) or other techniques, including mass spectroscopy assays, 2D gel electrophoresis assays, etc
• ELISAs angiogenesis protein
• ELISAs angiogenesis protein and standard immunoassays
• mass spectroscopy assays i e those identified as being important in an angiogenesis phenotype
• gene expression monitoring is done and a number of genes, i e an expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well Similarly, these assays may be done 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
• DNA or RNA encoding the angiogenesis protein may be detected, of particular interest are methods wherein the mRNA encoding an angiogenesis protein is detected
• the presence of mRNA in a sample is an indication that the angiogenesis gene has been transcribed to form the mRNA, and suggests that the protein is expressed.
• Probes to detect the mRNA can be any nucleotide/deoxynucleotide probe that is complementary to and base pairs 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. Following washing to remove the non-specifically bound probe, the label is detected.
• detection of the mRNA is performed in situ. In this method permeabilized cells or tissue samples are contacted with a detectably labeled nucleic acid probe for sufficient time to allow the probe to hybridize with the target mRNA. Following washing to remove the non-specifically bound probe, the label is detected.
• RNA probe for example a digoxygenin labeled riboprobe (RNA probe) that is complementary to the mRNA encoding an angiogenesis protein is detected by binding the digoxygenin with an anti-digoxygenin secondary antibody and developed with nitro blue tetrazolium and 5-bromo-4-chloro-3-indoyl phosphate.
• any of 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 done on an individual gene or corresponding polypeptide level.
• the expression profiles are used, preferably in conjunction with high throughput screening techniques to allow monitoring 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 or patients allows for a determination or diagnosis of angiogenesis. Numerous methods known to those of ordinary skill in the art find use in detecting angiogenesis.
• antibodies are used to detect angiogenesis proteins.
• a preferred method separates proteins from a sample or patient by electrophoresis on a gel (typically a denaturing and reducing protein gel, but may be any other type of gel including isoelectric focusing gels and the like). Following separation of proteins, the angiogenesis protein is detected 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.
• cells are contacted with from one to many antibodies to the angiogenesis protein(s). Following washing to remove non-specific antibody binding, 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.
• 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, numerous other histological imaging techniques are useful in 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 blood samples.
• 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 the angiogenesis by any of the previously described immunoassay techniques including ELISA, immunoblotting (Western blotting), immunoprecipitation, BIACORE technology and the like, as will be appreciated by one of ordinary skill in the art.
• in situ hybridization of labeled angiogenesis nucleic acid probes to tissue arrays is done.
• arrays of tissue samples, including angiogenesis tissue and/or normal tissue are made.
• In situ hybridization as is known in the art can then be done.
• 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.
• the angiogenesis probes are attached to biochips for the detection and quantification of angiogenesis sequences in a tissue or patient. The assays proceed as outlined above for diagnosis. In a preferred embodiment any 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, Zlokarnik, et al., Science 279, 84-8 (1998), Heid, 1996 #69.
• 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. 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.
• 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, candidate bioactive agents may be screened to modulate this gene's response; preferably to down regulate the gene, although in some circumstances to up regulate the gene.
• 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 desired; a 10 fold decrease in angiogenic tissue compared to normal tissue gives a 10 fold increase in expression for a candidate agent being desired.
• this may be done by evaluation at either the gene or the protein level; that is, 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, for example through the use of antibodies to the angiogenesis protein and standard immunoassays.
• gene expression monitoring is done and a number of genes, i.e. an expression profile, is monitored simultaneously, although multiple protein expression monitoring can be done as well.
• the 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.
• a candidate bioactive agent is added to the cells prior to analysis.
• screens are provided to identify a candidate bioactive agent which modulates angiogenesis, modulates angiogenesis proteins, binds to an angiogenesis protein, or interferes between the binding of an angiogenesis protein and an antibody.
• bioactive agent or “drug candidate” or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, etc., to be tested for bioactive agents that are capable of directly or indirectly altering either the angiogenesis phenotype or the expression of an angiogenesis sequence, including both nucleic acid sequences and protein sequences.
• the bioactive agents modulate the expression profiles, or expression profile nucleic acids or proteins provided herein.
• the candidate agent suppresses an angiogenesis phenotype, for example to a normal tissue fingerprint.
• the candidate agent preferably suppresses a severe angiogenesis phenotype.
• 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 candidate agent will neutralize the effect of an angiogenesis protein.
• neutralize is meant that activity of a protein is either inhibited or counter acted against so as to have substantially no effect on a cell.
• Candidate agents encompass numerous chemical classes, though typically they are organic molecules, preferably small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Preferred small molecules are less than 2000, or less than 1500 or less than 1000 or less than 500 D.
• Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups.
• the candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
• Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. Particularly preferred are peptides.
• Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.
• the candidate bioactive agents are proteins.
• protein herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides.
• the protein may be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
• amino acid or “peptide residue”, as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the invention.
• Amino acid also includes imino acid residues such as proline and hydroxyproline.
• the side chains may be in either the (R) or the (S) configuration. In the preferred embodiment, the amino acids are in the (S) or L-configuration. If non-naturally occurring side chains are used, non-amino acid substituents may be used, for example to prevent or retard in vivo degradations.
• the candidate bioactive agents are 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 procaryotic and eucaryotic 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.
• the candidate bioactive agents 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.
• the candidate bioactive agents are nucleic acids, as defined above.
• nucleic acid candidate bioactive 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 bioactive agents are organic chemical moieties, a wide variety of which are available in the literature.
• the sample containing the target sequences 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 occurring as needed, as will be appreciated by those in the art.
• an in vitro transcription with labels covalently attached to the nucleosides is done.
• 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 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.
• 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.
• reaction may be accomplished in a variety of ways, as will be appreciated by those in the art. Components of the reaction may be added simultaneously, or sequentially, in any order, with preferred embodiments outlined below.
• the reaction may include a variety of other reagents may be included in the assays. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal hybridization and detection, and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used, depending on the sample preparation methods and purity of the target.
• the data is analyzed to determine the expression levels, and changes in expression levels as between states, of individual genes, forming a gene expression profile.
• the screens are done to identify drugs or bioactive agents that modulate the angiogenesis phenotype.
• a preferred embodiment is in the screening of candidate agents that can induce or suppress a particular expression profile, thus preferably generating the associated phenotype. That is, candidate agents that can mimic or produce an expression profile in angiogenesis similar to the expression profile of normal tissue is expected to result in a suppression of the angiogenesis phenotype.
• mimicking an expression profile, or changing one profile to another is the goal.
• screens can be run to alter the expression of the genes individually. That is, screening for modulation of regulation of expression of a single gene can be done; that is, rather than try to mimic all or part of an expression profile, screening for regulation of individual genes can be done. Thus, for example, particularly in the case of target genes whose presence or absence is unique between two states, screening is done for modulators of the target gene expression.
• screening is done to alter the biological function of the expression product of the differentially expressed gene. Again, having identified the importance of a gene in a particular state, screening for agents that bind and/or modulate the biological activity of the gene product can he run as is more fully outlined below.
• screening of candidate agents that modulate the angiogenesis phenotype either at the gene expression level or the protein level can be done.
• screens can be done for novel 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 any of the 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 candidate agent is administered to a population of angiogenic cells, that thus has 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
• 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 reduce or suppress the angiogenesis phenotype.
• a change in at least one gene of the expression profile indicates that the agent has an effect on angiogenesis activity.
• 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 either 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 is as depicted in Figures 4, 8, 13, 18, and 22 or encoded by the sequences shown in figures 2, 3, 7, 12, 17, 21 and 23.
• 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.
• the fragment is from AAA1.
• the fragment includes a non- transmembrane region.
• the AAA1 fragment has an N-terminal Cys to aid in solubility.
• the fragment is selected from AAA1 p1 and AAA1p2.
• the fragment is charged and from the c-terminus of AAA4.
• the c-terminus of the fragment is kept as a free acid and the n-terminus is a free amine to aid in coupling, i.e., to cysteine.
• the fragment is an internal peptide overlapping hydrophilic stretch of AAA4.
• the termini is blocked.
• the fragment of AAA4 is selected from AAA4p1 or AAA4p2.
• the fragment is a novel fragment from the N-terminal.
• the fragment excludes sequence outside of the N-terminal, in another embodiment, the fragment includes at least a portion of the N-terminal.
• N-terminal is used interchangeably herein with “N-terminus” which is further described above.
• angiogenesis proteins are conjugated to an immunogenic agent as discussed herein. In one embodiment the angiogenesis protein is conjugated to BSA.
• screening for modulators of expression of specific genes can be done. This will be done as outlined above, but in general the expression of only one or a few genes are evaluated.
• screens are designed to first find candidate agents that can bind to differentially expressed proteins, and then these agents may be used in assays that evaluate the ability of the candidate agent to modulate differentially expressed activity.
• assays there are a number of different assays which may be run; binding assays and activity assays.
• 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. In general, this is done as is known in the art.
• 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 bioactive agent, and determining the binding of the candidate agent 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 candidate bioactive agent 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 candidate bioactive agent to the angiogenesis protein may be done in a number of ways.
• the candidate bioactive agent is labelled, and binding determined directly. For example, this may be done by attaching all or a portion of the angiogenesis protein to a solid support, adding a labelled candidate agent (for example a fluorescent label), washing off excess reagent, and determining whether the label is present on the solid support.
• a labelled candidate agent for example a fluorescent label
• label 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.
• the proteins may be labeled at tyrosine positions using 25 l, or with fluorophores.
• more than one component may be labeled with different labels; using 125 l for the proteins, for example, and a fluorophor for the candidate agents.
• the binding of the candidate bioactive agent is determined through the use of competitive binding assays.
• the competitor is a binding moiety known to bind to the target molecule (i.e. angiogenesis), such as an antibody, peptide, binding partner, ligand, etc.
• the target molecule i.e. angiogenesis
• the candidate bioactive agent is labeled.
• Either the candidate bioactive agent, 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 any temperature which facilitates optimal activity, typically between 4 and 40°C. Incubation periods are selected for optimum activity, but may also be optimized to facilitate rapid high through put 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 candidate bioactive agent.
• Displacement of the competitor is an indication that the candidate bioactive agent 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 presence of label in the wash solution indicates displacement by the agent.
• the candidate bioactive agent is labeled, the presence of the label on the support indicates displacement.
• the candidate bioactive agent is added first, with incubation and washing, followed by the competitor.
• the absence of binding by the competitor may indicate that the bioactive agent is bound to the angiogenesis protein with a higher affinity.
• the candidate bioactive agent is labeled, the presence of the label on the support, coupled with a lack of competitor binding, may indicate that the candidate agent is capable of binding to the angiogenesis protein.
• the methods comprise differential screening to identity bioactive 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 candidate bioactive agent, 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.
• a preferred embodiment utilizes differential screening to identify drug candidates that bind to the native angiogenesis protein, but cannot bind to modified angiogenesis proteins.
• the structure of the angiogenesis protein may be modeled, and used in rational drug design to synthesize agents that interact with that site.
• Drug candidates that affect angiogenesis bioactivity are also identified by screening drugs for the ability to either enhance or reduce the activity of the protein.
• Positive controls and negative controls may be used in the assays.
• Preferably all 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, all 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.
• reagents may be included in the screening assays. These include reagents like salts, neutral proteins, e.g. albumin, detergents, etc which may be used to facilitate optimal protein-protein binding and/or reduce non-specific or background interactions. Also reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, anti-microbial agents, etc., may be used. The mixture of components may be added in any order that provides for the requisite binding.
• methods for screening for a bioactive agent capable of modulating the activity of angiogenesis proteins comprise the steps of adding a candidate bioactive agent to a sample of angiogenesis proteins, as above, and determining an alteration in the biological activity of angiogenesis proteins.
• “Modulating the activity of angiogenesis proteins” includes an increase in activity, a decrease in activity, or a change in the type or kind of activity present.
• the candidate agent should both bind to angiogenesis proteins(although this may not be necessary), and alter its biological or biochemical activity as defined herein.
• the methods include both in vitro screening methods, as are generally outlined above, and in vivo screening of cells for alterations in the presence, distribution, activity or amount of angiogenesis proteins.
• the methods comprise combining an angiogenesis sample and a candidate bioactive agent, and evaluating the effect on angiogenesis.
• angiogenesis activity or grammatical equivalents herein is meant one of angiogenesis's biological activities, including, but not limited to, its role in angiogenesis.
• angiogenesis activity includes activation of AAA4, AAA1 ,
• An inhibitor of angiogenesis activity is the inhibition of any one or more angiogenesis activities.
• the activity of the angiogenesis protein is increased; in another preferred embodiment, the activity of the angiogenesis protein is decreased.
• bioactive agents that are antagonists are preferred in some embodiments, and bioactive agents that are agonists may be preferred in other embodiments.
• the invention provides methods for screening for bioactive agents capable of modulating the activity of an angiogenesis protein.
• the methods comprise adding a candidate bioactive agent, 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).
• 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.
• angiogenesis protein activity includes at least one of the following: angiogenesis protein activity as defined herein, binding to Edg-1 , activation of Edg-1 , or activation of substrates of Edg-1.
• angiogenesis activity is defined as the unregulated proliferation of angiogenic tissue, or the growth of arteries in tissue.
• angiogenesis activity as defined herein is related to the activity of Edg-1 in the upregulation of Edg-1 in angiogenic tissue.
• angiogenesis protein activity includes at least one of the following: angiogenesis activity, binding to one of AAA4, AAA1 , Edg-1 , alpha 5 beta 1 integrin, endomucin, matrix metalloproteinase 10, or activation of substrates of AAA4, AAA1 , Edg-1 , alpha 5 beta 1 integrin, endomucin, matrix metalloproteinase 10, respectively.
• AAA1 comprises its N-terminal end.
• angiogenesis activity as defined herein is related to the activity of AAA4, AAA1 , Edg-1 , alpha 5 beta 1 integrin, endomucin, matrix metalloproteinase 10, in the upregulation of AAA4, AAA1 , Edg-1 , alpha 5 beta 1 integrin, endomucin, matrix metalloproteinase 10, respectively in angiogenesis tissue.
• a method of inhibiting angiogenic cell division comprises administration of a angiogenesis inhibitor.
• a method of inhibiting angiogenesis comprises administration of an angiogenesis inhibitor.
• methods of treating cells or individuals with angiogenesis comprise administration of an angiogenesis inhibitor.
• an angiogenesis inhibitor is an antibody as discussed above.
• the angiogenesis inhibitor is an antisense molecule.
• Antisense molecules as used herein include antisense or sense oligonucleotides comprising a singe-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 AAA4, AAA1 , Edg-1 , alpha 5 beta 1 integrin, endomucin, or matrix metalloproteinase 10, more preferable the angiogenesis sequences in Table 5, or for a ligand or activator thereof.
• a most preferred antisense molecule is for Edg-1 or for a ligand or activator thereof.
• Antisense or sense oligonucleotides comprise a fragment generally at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.
• the ability to derive an antisense or a sense oligonucleotide, based upon a cDNA sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Krol et al. (BioTechni ⁇ ues 6:958, 1988).
• Antisense molecules 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 sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide- 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.
• the compounds having the desired pharmacological activity may be administered in a physiologically acceptable carrier to a host, as previously described.
• the agents may be administered in a variety of ways, orally, parenterally e.g., subcutaneously, intraperitoneally, intravascularly, etc. Depending upon the manner of introduction, the compounds may be formulated in a variety of ways.
• the concentration of therapeutically active compound in the formulation may vary from about 0.1-100 wt.%.
• the agents may be administered alone or in combination with other treatments, i.e., radiation.
• compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like.
• Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral and topical use can be used to make up compositions containing the therapeutically-active compounds.
• Diluents known to the art include aqueous media, vegetable and animal oils and fats. Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, and skin penetration enhancers can be used as auxiliary agents.
• the invention provides methods for identifying cells containing variant angiogenesis genes comprising determining all or part of the sequence of at least one endogeneous angiogenesis genes in a cell. As will be appreciated by those in the art, this may be done using any number of sequencing techniques. In a preferred embodiment, the invention provides methods of identifying the angiogenesis genotype of an individual comprising 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 is indicative of 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.
• 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. As will be appreciated by those in the art, this may be accomplished in any number of ways.
• the activity of the angiogenesis gene is increased by increasing the amount of angiogenesis in the cell, for example by overexpressing the endogeneous angiogenesis or by administering a gene encoding the angiogenesis sequence, using known gene-therapy techniques, for example.
• the gene therapy techniques include the incorporation of the exogenous gene using enhanced homologous recombination (EHR), for example as described in PCT/US93/03868, hereby incorporated 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.
• the angiogenesis proteins of the present invention may be used to generate polyclonal and monoclonal antibodies to angiogenesis proteins, which are useful as described herein.
• the angiogenesis proteins can be coupled, using standard technology, to affinity chromatography columns. These columns may then be used to purify angiogenesis antibodies.
• the antibodies are generated to epitopes unique to a angiogenesis protein; that is, the antibodies show little or no cross-reactivity to other proteins. These antibodies find use in a number of applications.
• 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.
• a therapeutically effective dose of an angiogenesis proteins and modulator thereof is administered to a patient.
• therapeutically effective dose herein is meant a dose that produces the 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. As is known in the art, adjustments for angiogenesis degradation, systemic versus localized delivery, and rate of new protease synthesis, as well as the age, body weight, general health, sex, diet, time of administration, drug interaction and the severity of the condition may be necessary, and will be ascertainable with routine experimentation by those skilled in the art.
• a "patient” for the purposes of the present invention includes both humans and other animals, particularly mammals, and organisms. Thus the methods are applicable to both human therapy and veterinary applications.
• the patient is a mammal, 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.
• 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, tnmethylamine, diethylamine, t ⁇ ethylamine, t ⁇ propylamine, 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 Additives are well known in the art, and are used in a variety of formulations
• 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 gene therapy applications, as is known in the art
• These angiogenesis genes can include antisense applications, either as gene therapy (i e for incorporation into the genome) or as antisense compositions, as will be appreciated by those in the art
• angiogenesis genes are administered as DNA vaccines, either single genes or combinations of angiogenesis genes Naked DNA vaccines are generally known in the art Brower, Nature Biotechnology, 16 1304-1305 (1998)
• angiogenesis genes of the present invention are used as DNA vaccines
• 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
• expression of the polypeptide encoded by the DNA vaccine, cytotoxic T-cells, helper T-cells and antibodies are induced which recognize and destroy or eliminate cells expressing angiogenesis proteins
• the DNA vaccines include a gene encoding an adjuvant molecule with the DNA vaccine.
• adjuvant molecules such adjuvant molecules
• angiogenesis genes find use in generating animal models of angiogenesis.
• gene therapy technology wherein antisense RNA directed to the angiogenesis gene will also diminish or repress expression of the gene.
• An animal generated as such serves as an animal model of angiogenesis that finds use in screening bioactive drug candidates.
• gene knockout technology for example as a result of homologous recombination with an appropriate gene targeting vector, will result in the absence of the angiogenesis protein.
• tissue-specific expression or knockout of the angiogenesis protein may be necessary.
• 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 bioactive molecules to treat angiogenesis.
• tissue weight Homogenize tissue samples in 1ml of TRIzol per 50mg of tissue using a
• Polytron 3100 homogenizer The generator/probe used depends upon the tissue size. A generator that is too large for the amount of tissue to be homogenized will cause a loss of sample and lower RNA yield. Use the 20mm generator for tissue weighing more than 0.6g. If the working volume is greater than 2ml, then homogenize tissue in a 15ml polypropylene tube (Falcon 2059). Fill tube no greater than 10ml.
• centrifuge for 2 minutes at 14,000 to 18,000 g. If centrifuge has a "soft setting,” then use it. Remove supernatant without disturbing Oligotex pellet. A little bit of solution can be left behind to reduce the loss of Oligotex. Save sup until certain that satisfactory binding and elution of poly A + mRNA has occurred.
• the mRNA Before proceeding with cDNA synthesis, the mRNA must be precipitated. Some component leftover or in the Elution Buffer from the Oligotex purification procedure will inhibit downstream enzymatic reactions of the mRNA.
• RNA Clean up total RNA using Qiaqen's RNeasy kit
• RNA Use 5ug of total RNA or 1 ug of polyA+ mRNA as starting material.
• For total RNA use 2ul of Superscript RT.
• For polyA+ mRNA use 1 ul of Superscript RT.
• Final volume of first strand synthesis mix is 20ul.
• RNA must be in a volume no greater than 10ul.
• Second Strand Synthesis Place 1 sl strand reactions on ice.
• RNeasy clean-up of IVT product follow previous instructions for RNeasy columns or refer to Qiagen's RNeasy protocol handbook.
• cRNA will most likely need to be ethanol precipitated. Resuspend in a volume compatible with the fragmentation step. ⁇ ragmentation
• RNA 15 ug of labeled RNA 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 RNA by incubation at 94 C for 35 minutes in 1 x Fragmentation buffer.
• RNA transcript can be analyzed before and after fragmentation. Samples can be heated to 65C for 15 minutes and electrophoresed on 1 % agarose/TBE gels to get an approximate idea of the transcript size range
• Hybrization Mix fragment labeled RNA (50ng/ul final cone.) 50 pM 948-b control oligo 1.5 pM BioB 5 pM BioC 25 pM BioD
• IVT antisense RNA 4 ⁇ g: ⁇ l Random Hexamers (1 ⁇ g/ ⁇ l): 4 ⁇ l H 2 0: ⁇ l
• Cot-1 DNA 10 ⁇ l 50X dNTPs: 1 ⁇ l 20X SSC: 2.3 ⁇ l
• genes within an expression profile also termed expression profile genes, include ESTs and are not necessarily full length.
• EOS28844 A_AA232837 ESTs Weakly similar to Human pre-mRNA cleavag Y Y Type il (Ncyt YType II (Ncyt Cexo)
• EOS06820 A_RC_AA489245 ESTs, Weakly similar to sperm specific protein [H sapiens]
• EOS01487 1_M31994 Homo sapiens aldehyde dehydrogenase (ALDH1) gene, exon 13 and complete cds
• EOS00044 1_D00596 Homo sapiens gene for thymidylate synthase, exons N N N
• EOS33755 1_U44975 Homo sapiens Kruppel-like zinc finger protein Zf9 m N N N N
• EOS30706 A_R79356 Homo sapiens mRNA for KIAA0544 protein, partial c N N N
• EOS00335 1_D86425 Homo sapiens mRNA for n ⁇ dogen-2 Y N N cr
• EOS02390 1_U48959 Homo sapiens myosin light chain kinase (MLCK) m N N N N
• EOS34005 1_U28811 Human cysteine-nch fibroblast growth factor recepto N N N
• EOS01122 1_L20859 Human leukemia virus receptor 1 (GLVR1) mRNA, c N Y Type ilia (Nc YType Ilia (Ncyt Cexo)
• EOS02575 1_U67963 Human lysophospholipase homolog (HU-K5) mRNA, N N N N
• EOS02421 1JJ51010 Human nicotinamide N-methyltransferase gene, exon 1 and 5' flanking region
• EOS02453 1_U53445 Human ovarian cancer downregulated myosin heavy N N N
• EOS00682 1JHG3543-HT3739 Insulin-Like Growth Factor 2
• E0S33225 1_L00352 low density lipoprotein receptor (familial hypercholes Y Y Type la YType la
• EOS01040 1J.08246 myeloid cell leukemia sequence 1 (BCL2-related) Y Y Type lb (Nex YType lb (Nexo Ccyt)
• EOS01473 1_M31166 pentaxi ⁇ -related gene rapidly induced by IL-1 beta Y N N ft
• EOS04824 A_RC_AA054087 phospholipase A2 group IVC (cytosolic, calcium-ind N Y Type lb (Nex YType lb (Nexo Ccyt)
• EOS32094 1_U84573 procollagen-lysine, 2-oxoglutarate 5-d ⁇ oxygenase (ly N N N
• EOS01040 1J.08246 myeloid cell leukemia sequence 1 (BCL2-related) Y Y Type lb (Nex YType lb (Nexo Ccyt)
• EOS01473 1_M31166 pentaxin-related gene rapidly induced by IL-1 beta Y N N
• EOS04824 A_RC_AA054087 phospholipase A2 group IVC (cytosolic, calcium-ind N Y ⁇ Type lb (Nex YType lb (Nexo Ccyt) r
• EOS32094 1_U84573 procollagen-lysine, 2-oxoglutarate 5-d ⁇ oxygenase (ly N N N
• EOS01861 1_S76965 Protein kinase inhibitor [human neuroblastoma cell I N N N EOS03401 1_Y00815 protein tyrosine phosphatase, receptor type, F Y N N EOS34011 1J.77886 protein tyrosine phosphatase receptor type, K N Y Type lb (Nex YType lb (Nexo Ccyt) EOS00138 1_D26129 nbonuclease, RNase A family, 1 (pancreatic) Y Y Type lb (Nex YType lb (Nexo Ccyt) EOS30425 D_RC_AA243278_ ⁇ ⁇ bosomal protein mitochondnal, L12 N N N N EOS29398 1_J03040 secreted protein acidic, cysteine- ⁇ ch (osteonectm) Y N N EOS01415 1_M24736 selectin E (endothelial
• EOS35279 D83174 collagen-binding protein 2 (colligen 2) Y Y Type la YType la
• EOS25520 R23858 ESTs, Moderately similar to envelope protein [H sap Y N
• EOS30902 AA370302 Homo sapiens mRNA; cDNA DKFZp586l1518 (from Y N N);
• EOS04522 R81003 Homo sapiens serine protease mRNA; complete cds Y N N
• EOS02828 X06256 integrin alpha 5 (fibronectin receptor; alpha polypep N N N
• EOS01473 M31166 pentaxin-related gene rapidly induced by IL-1 beta Y N N
• EOS01124 L20971 phosphodiesterase 4B EOS01124 L20971 phosphodiesterase 4B; cAMP-specific (dunce (Dros N Y Type lb (Nex YType lb (Nexo Ccyt)
• EOS04824 AA054087 phospholipase A2 group IVC (cytosolic; calcium-ind N Y Type lb (Nex YType lb (Nexo Ccyt)
• EOS01480 M31551 plasmi ⁇ ogen activator inhibitor type II (arginine-serp N N N
• EOS33915 L34657 platelet endothelial cell adhesion molecule (CD31 an Y Y Type la YType la
• EOS01415 M24736 selectin E (endothelial adhesion molecule 1) Y Y Type la YType la
• EOS33480 W80846 vesicle-associated membrane protein 5 (myobrevin) N Y Type II (Ncyt YType II (Ncyt Cexo)

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  • 2000-08-11 AU AU69022/00A patent/AU6902200A/en not_active Abandoned
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  • 2000-08-11 JP JP2001515333A patent/JP2003517816A/ja not_active Withdrawn
  • 2000-08-11 EP EP00957393A patent/EP1204764A2/en not_active Withdrawn
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