Patent Application
20060210975
This invention is related to the area of angiogenesis and anti-angiogenesis. In particular, it relates to genes which are characteristically expressed in tumor endothelial and normal endothelial cells.
It is now widely recognized that tumors require a blood supply for expansive growth. This recognition has stimulated a profusion of research on tumor angiogenesis, based on the idea that the vasculature in tumors represents a potential therapeutic target. However, several basic questions about tumor endothelium remain unanswered. For example, are vessels of tumors qualitatively different from normal vessels of the same tissue? What is the relationship of tumor endothelium to endothelium of healing wounds or other physiological or pathological forms of angiogenesis? The answers to these questions critically impact on the potential for new therapeutic approaches to inhibit angiogenesis in a specific manner.
There is a continuing need in the art to characterize the vasculature of tumors relative to normal vasculature so that any differences can be exploited for therapeutic and diagnostic benefits.
One technique which can be used to characterize gene expression, or more precisely gene transcription, is termed serial analysis of gene expression (SAGE). Briefly, the SAGE approach is a method for the rapid quantitative and qualitative analysis of mRNA transcripts based upon the isolation and analysis of short defined sequence tags (SAGE Tags) corresponding to expressed genes. Each Tag is a short nucleotide sequences (9-17 base pairs in length) from a defined position in the transcript. In the SAGE method, the Tags are dimerized to reduce bias inherent in cloning or amplification reactions. (See, U.S. Pat. No. 5,695,937) SAGE is particularly suited to the characterization of genes associated with vasculature stimulation or inhibition because it is capable of detecting rare sequences, evaluating large numbers of sequences at one time, and to provide a basis for the identification of previously unknown genes.
Yet another aspect of the invention is a method for identification of a ligand involved in endothelial cell regulation. A test compound is contacted with a human protein selected from the group consisting of secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VI, alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midline (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; IK cytokine, down-regulator of HLA II; DnaJ (Hsp40) homolog, subfamily B, member 1; heat shock 70 kD protein 1A; heat shock 70 kD protein 1B; lectin, galactoside-binding, soluble, 1 (galectin 1); heat shock 90 kD protein 1, alpha; DnaJ (Hsp40) homolog, subfamily B, member 1; tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor); heat shock 60 kD protein 1 (chaperonin); heat shock 10 kD protein 1 (chaperonin 10); general transcription factor II, i; heat shock 70 kD protein 6 (HSP70B′); heat shock 105 kD; heat shock 105 kD; eukaryotic translation initiation factor 4A, isoform 2; hypothetical protein similar to mouse Fbw5; DKFZP727M231 protein; dynein, cytoplasmic, light polypeptide; hypothetical protein MGC15875; murine retrovirus integration site 1 homolog; hypothetical protein FLJ22376; smoothelin; vacuolar protein sorting 16 (yeast homolog); peanut (Drosophila)-like 2; hypothetical protein FLJ10350; FK506-binding protein 4 (59 kD); proteasome (prosome, macropain) subunit, beta type, 6; transgelin; sorting nexin 17; ribosomal protein S6 kinase, 90 kD, polypeptide 4; kinesin family member 1C; BTB (POZ) domain containing 2; guanylate cyclase 1, soluble, beta 3; protein-L-isoaspartate (D-aspartate) O-methyltransferase; D-aspartate oxidase; chromosome 9 open reading frame 3; regulator of G-protein signalling 16; voltage-dependent anion channel 3; NS1-binding protein; interferon-induced, hepatitis C-associated microtubular aggregate protein (44 kD); carbonic anhydrase II; protein phosphatase 2, regulatory subunit B (B56), gamma isoform; chromosome 14 open reading frame 3; eukaryotic translation initiation factor 2, subunit 1 (alpha, 35 kD); Rho GTPase activating protein 1; RAP1B, member of RAS oncogene family, profilin 1; DKFZP586L151 protein; hypothetical protein FLJ14987; mitogen-activated protein kinase kinase 1 interacting protein 1; chimerin (chimaerin) 1; hephaestin; KLAA0196 gene product; melanoma-associated antigen recognised by cytotoxic T lymphocytes; HLA class II region expressed gene KE2; histamine N-methyltransferase; hypothetical protein FLJ10842; TIA1 cytotoxic granule-associated RNA-binding protein; N-acylaminoacyl-peptide hydrolase; integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12); DKFZP586J0119 protein; hepatocyte growth factor-regulated tyrosine kinase substrate; regulator of G-protein signalling 1; proteasome (prosome, macropain) subunit, beta type, 7; KIAA1402 protein; crystallin, alpha B; protein kinase C, zeta; protein kinase, cAMP-dependent, regulatory, type II, alpha; homologous to yeast nitrogen permease (candidate tumor suppressor); intestinal cell kinase; GS3955 protein; activated p21cdc42Hs kinase; Rho-associated, oiled-coil-containing protein kinase I; KIAA202 protein; unc-51-like kinase 1; and PDGFA associated protein 1. Binding of a test compound to the human protein is determined. A test compound which binds to the protein is identified as a ligand involved in endothelial cell regulation.
Still another embodiment of the invention provides a method of inhibiting neoangiogenesis in a patient. A molecule comprising an antibody variable region which specifically binds to a TEM protein selected from the group consisting of: secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VI, alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midkine (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; angiopoietin 1; PDGF alpha polypeptide; insulin-like growth factor binding protein; and IK cytokine, down-regulator of HLA II is administered to the patient. Neoangiogenesis in the patient consequently inhibited.
Yet another aspect of the invention is a method of screening for neoangiogenesis in a patient. A body fluid collected from the patient is contacted with a molecule comprising an antibody variable region which specifically binds to a TEM protein selected from the group consisting of: secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VI, alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midkine (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; angiopoietin 1; PDGF alpha polypeptide; insulin-like growth factor binding protein; and IK cytokine, down-regulator of HLA II. Detection of cross-reactive material in the body fluid with the molecule indicates neoangiogenesis in the patient.
Also provided by the present invention is a method of promoting neoangiogenesis in a patient. A TEM protein selected from the group consising of: secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VI, alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midkine (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; angiopoietin 1; PDGF alpha polypeptide; insulin-like growth factor binding protein; and IK cytokine, down-regulator of HLA II and, is administered to a patient in need of neoangiogenesis. Neoangiogenesis in the patient is consequently stimulated.
One embodiment of the invention provides a method of promoting neoangiogenesis in a patient. A nucleic acid molecule encoding a TEM protein selected from the group consising of: secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VI, alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VIII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midkine (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; angiopoietin 1; PDGF alpha polypeptide; insulin-like growth factor binding protein; and IK cytokine, down-regulator of HLA II, is administered to a patient in need of neoangiogenesis. The TEM protein is consequently expressed and neoangiogenesis in the patient is stimulated.
Another embodiment of the invention provides a method of screening for neoangiogenesis in a patient. A TEM protein selected from the group consisting of: secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VI, alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midkine (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; angiopoietin 1; PDGF alpha polypeptide; insulin-like growth factor binding protein; and IK cytokine, down-regulator of HLA II, is detected in a body fluid collected from the patient. Detection of the TEM protein indicates neoangiogenesis in the patient.
Another aspect of the invention is a method of screening for neoangiogenesis in a patient. A nucleic acid encoding a TEM protein selected from the group consisting of: secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VI, alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midkine (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; angiopoietin 1; PDGF alpha polypeptide; insulin-like growth factor binding protein; and IK cytokine, down-regulator of HLA D, is detected in a body fluid collected from the patient. Detection of the TEM protein indicates neoangiogenesis in the patient.
A still further embodiment of the invention is a method to identify candidate drugs for treating tumors. Cells which express one or more TEM genes selected from the group consisting of secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type A, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VI, alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midkine (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; IK cytokine, down-regulator of HLA II; DnaJ (Hsp40) homolog, subfamily B, member 1; heat shock 70 kD protein 1A; heat shock 70 kD protein 1B; lectin, galactoside-binding, soluble, 1 (galectin 1); heat shock 90 kD protein 1, alpha; DnaJ (Hsp40) homolog, subfamily B, member 1; tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor); heat shock 60 kD protein 1 (chaperonin); heat shock 10 kD protein 1 (chaperonin 10); general transcription factor II, i; heat shock 70 kD protein 6 (HSP70B′); heat shock 105 kD; heat shock 105 kD; eukaryotic translation initiation factor 4A, isoform 2; hypothetical protein similar to mouse Fbw5; DKFZP727M231 protein; dynein, cytoplasmic, light polypeptide; hypothetical protein MGC15875; murine retrovirus integration site 1 homolog; hypothetical protein FLJ22376; smoothelin; vacuolar protein sorting 16 (yeast homolog); peanut (Drosophila)-like 2; hypothetical protein FLJ10350; FK506-binding protein 4 (59 kD); proteasome (prosome, macropain) subunit, beta type, 6; transgelin; sorting nexin 17; ribosomal protein S6 kinase, 90 kD, polypeptide 4; kinesin family member 1C; BTB (POZ) domain containing 2; guanylate cyclase 1, soluble, beta 3; protein-L-isoaspartate (D-aspartate) O-methyltransferase; D-aspartate oxidase; chromosome 9 open reading frame 3; regulator of G-protein signalling 16; voltage-dependent anion channel 3; NS1-binding protein; interferon-induced, hepatitis C-associated microtubular aggregate protein (44 kD); carbonic anhydrase II; protein phosphatase 2, regulatory subunit B (B56), gamma isoform; chromosome 14 open reading frame 3; eukaryotic translation initiation factor 2, subunit 1 (alpha, 35 kD); Rho GTPase activating protein 1; RAP1B, member of RAS oncogene family, profilin 1; DKFZP586L151 protein; hypothetical protein FLJ14987; mitogen-activated protein kinase kinase 1 interacting protein 1; chimerin (chimaerin) 1; hephaestin; KIAA0196 gene product; melanoma-associated antigen recognised by cytotoxic T lymphocytes; HLA class II region expressed gene KE2; histamine N-methyltransferase; hypothetical protein FLJ10842; TIA1 cytotoxic granule-associated RNA-binding protein; N-acylaminoacyl-peptide hydrolase; integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12); DKFZP586J0119 protein; hepatocyte growth factor-regulated tyrosine kinase substrate; regulator of G-protein signalling 1; proteasome (prosome, macropain) subunit, beta type, 7; KIAA1402 protein; crystallin, alpha B; protein kinase C, zeta; protein kinase, cAMP-dependent, regulatory, type II, alpha; homologous to yeast nitrogen permease (candidate tumor suppressor); intestinal cell kinase; GS3955 protein; activated p21cdc42Hs kinase; Rho-associated, oiled-coil-containing protein kinase 1; KIAA2002 protein; unc-51-like kinase 1; and PDGFA associated protein 1, are contacted with a test compound. Expression of said one or more TEM genes is determined by hybridization of mRNA of said cells to a nucleic acid probe which is complementary to said mRNA. A test compound is identified as a candidate drug for treating tumors if it decreases expression of said one or more TEM genes. Optionally the cells are endothelial cells. Alternatively or additionally, the cells are recombinant host cells which are transfected with an expression construct which encodes said one or more TEMs. Test compounds which increase expression can be identified as candidates for promoting wound healing.
Yet another embodiment of the invention is a method to identify candidate drugs for treating tumors. Cells which express one or more TEM proteins selected from the group consisting of: secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VL alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midkine (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; IK cytokine, down-regulator of HLA 1; DnaJ (Hsp40) homolog, subfamily B, member 1; heat shock 70 kD protein 1A; heat shock 70 kD protein 1B; lectin, galactoside-binding, soluble, 1 (galectin 1); heat shock 90 kD protein 1, alpha; DnaJ (Hsp40) homolog, subfamily B, member 1; tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor); heat shock 60 kD protein 1 (chaperonin); heat shock 10 kD protein 1 (chaperonin 10); general transcription factor II, i; heat shock 70 kD protein 6 (HSP70B′); heat shock 105 kD; heat shock 105 kD; eukaryotic translation initiation factor 4A, isoform 2; hypothetical protein similar to mouse Fbw5; DKFZP727M231 protein; dynein, cytoplasmic, light polypeptide; hypothetical protein MGC15875; murine retrovirus integration site 1 homolog; hypothetical protein FLJ22376; smoothelin; vacuolar protein sorting 16 (yeast homolog); peanut (Drosophila)-like 2; hypothetical protein FLJ10350; FK506-binding protein 4 (59 kD); proteasome (prosome, macropain) subunit, beta type, 6; transgelin; sorting nexin 17; ribosomal protein S6 kinase, 90 kD, polypeptide 4; kinesin family member 1C; BTB (POZ) domain containing 2; guanylate cyclase 1, soluble, beta 3; protein-L-isoaspartate (D-aspartate) O-methyltransferase; D-aspartate oxidase; chromosome 9 open reading frame 3; regulator of G-protein signalling 16; voltage-dependent anion channel 3; NS1-binding protein; interferon-induced, hepatitis C-associated microtubular aggregate protein (44 kD); carbonic anhydrase II; protein phosphatase 2, regulatory subunit B (B56), gamma isoform; chromosome 14 open reading frame 3; eukaryotic translation initiation factor 2, subunit 1 (alpha, 35 kD); Rho GTPase activating protein 1; RAP1B, member of RAS oncogene family, profilin 1; DKFZP586L151 protein; hypothetical protein FLJ14987; mitogen-activated protein kinase kinase 1 interacting protein 1; chimerin (chimaerin) 1; hephaestin; KIAA0196 gene product; melanoma-associated antigen recognised by cytotoxic T lymphocytes; HLA class II region expressed gene KE2; histamine N-methyltransferase; hypothetical protein FLJ10842; TIA1 cytotoxic granule-associated RNA-binding protein; N-acylaminoacyl-peptide hydrolase; integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12); DKFZP586J0119 protein; hepatocyte growth factor-regulated tyrosine kinase substrate; regulator of G-protein signalling 1; proteasome (prosome, macropain) subunit, beta type, 7; KIAA1402 protein; crystallin, alpha B; protein kinase C, zeta; protein kinase, cAMP-dependent, regulatory, type II, alpha; homologous to yeast nitrogen permease (candidate tumor suppressor); intestinal cell kinase; GS3955 protein; activated p21cdc42Hs kinase; Rho-associated, oiled-coil-containing protein kinase I; KIAA2002 protein; unc-51-like kinase 1; and PDGFA associated protein 1, are contacted with a test compound. The amount of said one or more TEM proteins in said cells is determined. A test compound is identified as a candidate drug for treating tumors if it decreases the amount of one or more TEM proteins in said cells. Optionally the cells are endothelial cells. Alternatively or additionally, the cells are recombinant host cells which are transfected with an expression construct which encodes said one or more TEMs. Alternatively, a test compound which increases the amount of one or more TEM proteins in said cells is identified as a candidate drug for treating wound healing.
According to another aspect of the invention a method is provided to identify candidate drugs for treating tumors. Cells which express one or more TEM proteins selected from the group consisting of: secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VI, alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midkine (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; IK cytokine, down-regulator of LA II; DnaJ (Hsp40) homolog, subfamily B, member 1; heat shock 70 kD protein 1A; heat shock 70 kD protein 1B; lectin, galactoside-binding, soluble, 1 (galectin 1); heat shock 90 kD protein 1, alpha; DnaJ (Hsp40) homolog, subfamily B, member 1; tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor); heat shock 60 kD protein 1 (chaperonin); heat shock 10 kD protein 1 (chaperonin 10); general transcription factor II, i; heat shock 70 kD protein 6 (HSP70B′); heat shock 105 kD; heat shock 105 kD; eukaryotic translation initiation factor 4A, isoform 2; hypothetical protein similar to mouse Fbw5; DKFZP727M231 protein; dynein, cytoplasmic, light polypeptide; hypothetical protein MGC15875; murine retrovirus integration site 1 homolog; hypothetical protein FLJ22376; smoothelin; vacuolar protein sorting 16 (yeast homolog); peanut (Drosophila)-like 2; hypothetical protein FLJ10350; FK506-binding protein 4 (59 kD); proteasome (prosome, macropain) subunit, beta type, 6; transgelin; sorting nexin 17; ribosomal protein S6 kinase, 90 kD, polypeptide 4; kinesin family member 1C; BTB (POZ) domain containing 2; guanylate cyclase 1, soluble, beta 3; protein-L-isoaspartate (D-aspartate) O-methyltransferase; D-aspartate oxidase; chromosome 9 open reading frame 3; regulator of G-protein signalling 16; voltage-dependent anion channel 3; NS I-binding protein; interferon-induced, hepatitis C-associated microtubular aggregate protein (44 kD); carbonic anhydrase II; protein phosphatase 2, regulatory subunit B (B56), gamma isoform; chromosome 14 open reading frame 3; eukaryotic translation initiation factor 2, subunit 1 (alpha, 35 kD); Rho GTPase activating protein 1; RAP 1B, member of RAS oncogene family, profilin 1; DKFZP586L151 protein; hypothetical protein FLJ14987; mitogen-activated protein kinase kinase 1 interacting protein 1; chimerin (chimaerin) 1; hephaestin; KIAA0196 gene product; melanoma-associated antigen recognised by cytotoxic T lymphocytes; HLA class II region expressed gene KE2; histamine N-methyltransferase; hypothetical protein FLJ10842; TIA1 cytotoxic granule-associated RNA-binding protein; N-acylaminoacyl-peptide hydrolase; integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12); DKFZP586J0119 protein; hepatocyte growth factor-regulated tyrosine kinase substrate; regulator of G-protein signalling 1; proteasome (prosome, macropain) subunit, beta type, 7; KIAA1402 protein; crystallin, alpha B; protein kinase C, zeta; protein kinase, cAMP-dependent, regulatory, type II, alpha; homologous to yeast nitrogen permease (candidate tumor suppressor); intestinal cell kinase; GS3955 protein; activated p21cdc42Hs kinase; Rho-associated, oiled-coil-containing protein kinase I; KIAA2002 protein; unc-51-like kinase 1; and PDGFA associated protein 1, are contacted with a test compound. Activity of said one or more TEM proteins in said cells is determined. A test compound is identified as a candidate drug for treating tumors if it decreases the activity of one more TEM proteins in said cells. Optionally the cells are endothelial cells. Alternatively or additionally, the cells are recombinant host cells which are transfected with an expression construct which encodes said one or more TEMs. Optionally the cells are endothelial cells. If a test compound increases the acitivity of one more TEM proteins in said cells it can be identified as a candidate drug for treating wound healing.
An additional aspect of the invention is a method to identify candidate drugs for treating patients bearing tumors. A test compound is contacted with recombinant host cells which are transfected with an expession construct which encodes one or more TEM proteins selected from the group consisting of secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VI, alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midkine (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; IK cytokine, down-regulator of HLA II; DnaJ (Hsp40) homolog, subfamily B, member 1; heat shock 70 kD protein 1A; heat shock 70 kD protein 1B; lectin, galactoside-binding, soluble, 1 (galectin 1); heat shock 90 kD protein 1, alpha; DnaJ (Hsp40) homolog, subfamily B, member 1; tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor); heat shock 60 kD protein 1 (chaperonin); heat shock 10 kD protein 1 (chaperonin 10); general transcription factor II, i; heat shock 70 kD protein 6 (HSP70B′); heat shock 105 kD; heat shock 105 kD; eukaryotic translation initiation factor 4A, isoform 2; hypothetical protein similar to mouse Fbw5; DKFZP727M231 protein; dynein, cytoplasmic, light polypeptide; hypothetical protein MGC15875; murine retrovirus integration site 1 homolog; hypothetical protein FLJ22376; smoothelin; vacuolar protein sorting 16 (yeast homolog); peanut (Drosophila)-like 2; hypothetical protein FLJ10350; FK506-binding protein 4 (59 kD); proteasome (prosome, macropain) subunit, beta type, 6; transgelin; sorting nexin 17; ribosomal protein S6 kinase, 90 kD, polypeptide 4; kinesin family member 1C; BTB (POZ) domain containing 2; guanylate cyclase 1, soluble, beta 3; protein-L-isoaspartate (D-aspartate) O-methyltransferase; D-aspartate oxidase; chromosome 9 open reading frame 3; regulator of G-protein signalling 16; voltage-dependent anion channel 3; NS1-binding protein; interferon-induced, hepatitis C-associated microtubular aggregate protein (44 kD); carbonic anhydrase II; protein phosphatase 2, regulatory subunit B (B56), gamma isoform; chromosome 14 open reading frame 3; eukaryotic translation initiation factor 2, subunit 1 (alpha, 35 kD); Rho GTPase activating protein 1; RAP1B, member of RAS oncogene family; profilin 1; DKFZP586L151 protein; hypothetical protein FLJ14987; mitogen-activated protein kinase kinase 1 interacting protein 1; chimerin (chimaerin) 1; hephaestin; KIAA0196 gene product; melanoma-associated antigen recognised by cytotoxic T lymphocytes; HLA class II region expressed gene KE2; histamine N-methyltransferase; hypothetical protein FLJ10842; TIA1 cytotoxic granule-associated RNA-binding protein; N-acylaminoacyl-peptide hydrolase; integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12); DKFZP586J0119 protein; hepatocyte growth factor-regulated tyrosine kinase substrate; regulator of G-protein signalling 1; proteasome (prosome, macropain) subunit, beta type, 7; KIAA1402 protein; crystallin, alpha B; protein kinase C, zeta; protein kinase, cAMP-dependent, regulatory, type II, alpha; homologous to yeast nitrogen permease (candidate tumor suppressor); intestinal cell kinase; GS3955 protein; activated p21cdc42Hs kinase; Rho-associated, oiled-coil-containing protein kinase I; KIAA2002 protein; unc-51-like kinase 1; and PDGFA associated protein 1. Proliferation of said cells is determined. A test compound which inhibits proliferation of said cells is identified as a candidate drug for treating patients bearing tumors. A test coumpound which stimulates proliferation of said cells is identified as a candidate drug for promoting neoangiogenesis, such as for use in wound healing.
Another aspect of the invention is a method for identifying endothelial cells. One or more antibodies which bind specifically to a TEM protein selected from the group consisting of secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VI, alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midkine (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; IK cytokine, down-regulator of HLA II; DnaJ (Hsp40) homolog, subfamily B, member 1; heat shock 70 kD protein 1A; heat shock 70 kD protein 1B; lectin, galactoside-binding, soluble, 1 (galectin 1); heat shock 90 kD protein 1, alpha; DnaJ (Hsp40) homolog, subfamily B, member 1; tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor); heat shock 60 kD protein 1 (chaperonin); heat shock 105 kD protein 1 (chaperonin 10); general transcription factor II, i; heat shock 70 kD protein 6 (HSP70B′); heat shock 105 kD; heat shock 105 kD; eukaryotic translation initiation factor 4A, isoform 2; hypothetical protein similar to mouse Fbw5; DKFZP727M231 protein; dynein, cytoplasmic, light polypeptide; hypothetical protein MGC15875; murine retrovirus integration site 1 homolog; hypothetical protein FLJ22376; smoothelin; vacuolar protein sorting 16 (yeast homolog); peanut (Drosophila)-like 2; hypothetical protein FLJ10350; FK506-binding protein 4 (59 kD); proteasome (prosome, macropain) subunit, beta type, 6; transgelin; sorting nexin 17; ribosomal protein S6 kinase, 90 kD, polypeptide 4; kinesin family member 1C; BTB (POZ) domain containing 2; guanylate cyclase 1, soluble, beta 3; protein-L-isoaspartate (D-aspartate) O-methyltransferase; D-aspartate oxidase; chromosome 9 open reading frame 3; regulator of G-protein signalling 16; voltage-dependent anion channel 3; NS1-binding protein; interferon-induced, hepatitis C-associated microtubular aggregate protein (44 kD); carbonic anhydrase II; protein phosphatase 2, regulatory subunit B (B56), gamma isoform; chromosome 14 open reading frame 3; eukaryotic translation initiation factor 2, subunit 1 (alpha, 35 kD); Rho GTPase activating protein 1; RAP1B, member of RAS oncogene family, profilin 1; DKFZP586L151 protein; hypothetical protein FLJ14987; mitogen-activated protein kinase kinase 1 interacting protein 1; chimerin (chimaerin) 1; hephaestin; KIAA0196 gene product; melanoma-associated antigen recognised by cytotoxic T lymphocytes; HLA class II region expressed gene KE2; histamine N-methyltransferase; hypothetical protein FLJ10842; TIA1 cytotoxic granule-associated RNA-binding protein; N-acylaminoacyl-peptide hydrolase; integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12); DKFZP586J0119 protein; hepatocyte growth factor-regulated tyrosine kinase substrate; regulator of G-protein signalling 1; proteasome (prosome, macropain) subunit, beta type, 7; KIAA1402 protein; crystallin, alpha B; protein kinase C, zeta; protein kinase, cAMP-dependent, regulatory, type II, alpha; homologous to yeast nitrogen permease (candidate tumor suppressor); intestinal cell kinase; GS3955 protein; activated p21cdc42Hs kinase; Rho-associated, oiled-coil-containing protein kinase I; KIAA2002 protein; unc-51-like kinase 1; and PDGFA associated protein 1, is contacted with a population of cells. Cells in the population which have bound to said antibodies are detected. Cells which are bound to said antibodies are identified as endothelial cells. Optionally cells which have bound to said antibodies are isolated from cells which have not bound.
Still another aspect of the invention is a method for identifying endothelial cells. One or more nucleic acid hybridization probes which are complementary to a TEM gene nucleic acid sequence selected from the group consisting of secreted protein, acidic, cysteine-rich (osteonectin); collagen, type I, alpha 1; collagen, type IV, alpha 1; collagen, type XVIII, alpha 1; fibronectin 1; collagen, type IV, alpha 2; Homo sapiens mRNA; cDNA DKFZp586J021 (from clone DKFZp586J021); collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); collagen, type VI, alpha 2; collagen, type XVIII, alpha 1; collagen, type III, alpha 1 (Ehlers-Danlos syndrome type IV, autosomal dominant); transforming growth factor, beta-induced, 68 Kd; Biglycan; collagen, type VI, alpha 1; small inducible cytokine subfamily B (Cys-X-Cys), member 14 (BRAK); spondin 2, extracellular matrix protein; Fibromodulin; laminin, alpha 4; collagen, type IV, alpha 1; complement component 1, s subcomponent; fibulin 1; frizzled-related protein; lysyl oxidase-like 2; plasminogen activator, urokinase; natural killer cell transcript 4; microfibrillar-associated protein 2; collagen, type VII, alpha 1 (epidermolysis bullosa, dystrophic, dominant and recessive); follistatin-like 1; complement component 1, r subcomponent; Decorin; secreted protein, acidic, cysteine-rich (osteonectin); Thy-1 cell surface antigen; cysteine-rich, angiogenic inducer, 61; immunoglobulin lambda locus; hypothetical protein CAB56184; serine (or cysteine) proteinase inhibitor, clade G (C1 inhibitor), member 1; collagen, type I, alpha 1; collagen, type V, alpha 2; laminin, beta 1; DKFZP586B0621 protein; cysteine knot superfamily 1, BMP antagonist 1; hypothetical protein FLJ23053; hypothetical protein FLJ20397; matrix metalloproteinase 9 (gelatinase B, 92 kD gelatinase, 92 kD type IV collagenase); insulin-like growth factor binding protein 7; collagen, type V, alpha 1; thrombospondin 2; midkine (neurite growth-promoting factor 2); DKFZP564I1922 protein; fibrillin 1 (Marfan syndrome); transforming growth factor, beta 1; serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 1; galactosidase, beta 1; IK cytokine, down-regulator of HLA II; DnaJ (Hsp40) homolog, subfamily B, member 1; heat shock 70 kD protein 1A; heat shock 70 kD protein 1B; lectin, galactoside-binding, soluble, 1 (galectin 1); heat shock 90 kD protein 1, alpha; DnaJ (Hsp40) homolog, subfamily B, member 1; tissue inhibitor of metalloproteinase 1 (erythroid potentiating activity, collagenase inhibitor); heat shock 60 kD protein 1 (chaperonin); heat shock 10 kD protein 1 (chaperonin 10); general transcription factor II, i; heat shock 70 kD protein 6 (HSP70B′); heat shock 105 kD; heat shock 105 kD; eukaryotic translation initiation factor 4A, isoform 2; hypothetical protein similar to mouse Fbw5; DKFZP727M231 protein; dynein, cytoplasmic, light polypeptide; hypothetical protein MGC15875; murine retrovirus integration site 1 homolog; hypothetical protein FLJ22376; smoothelin; vacuolar protein sorting 16 (yeast homolog); peanut (Drosophila)-like 2; hypothetical protein FLJ10350; FK506-binding protein 4 (59 kD); proteasome (prosome, macropain) subunit, beta type, 6; transgelin; sorting nexin 17; ribosomal protein S6 kinase, 90 kD, polypeptide 4; kinesin family member 1C; BTB (POZ) domain containing 2; guanylate cyclase 1, soluble, beta 3; protein-L-isoaspartate (D-aspartate) O-methyltransferase; D-aspartate oxidase; chromosome 9 open reading frame 3; regulator of G-protein signalling 16; voltage-dependent anion channel 3; NS1-binding protein; interferon-induced, hepatitis C-associated microtubular aggregate protein (44 kD); carbonic anhydrase II; protein phosphatase 2, regulatory subunit B (B56), gamma isoform; chromosome 14 open reading frame 3; eukaryotic translation initiation factor 2, subunit 1 (alpha, 35 kD); Rho GTPase activating protein 1; RAP1B, member of RAS oncogene family; profilin 1; DKFZP586L151 protein; hypothetical protein FLJ14987; mitogen-activated protein kinase kinase 1 interacting protein 1; chimerin (chimaerin) 1; hephaestin; KIAA0196 gene product; melanoma-associated antigen recognised by cytotoxic T lymphocytes; HLA class II region expressed gene KE2; histamine N-methyltransferase; hypothetical protein FLJ10842; TIA1 cytotoxic granule-associated RNA-binding protein; N-acylaminoacyl-peptide hydrolase; integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MSK12); DKFZP586J0119 protein; hepatocyte growth factor-regulated tyrosine kinase substrate; regulator of G-protein signalling 1; proteasome (prosome, macropain) subunit, beta type, 7; KIAA1402 protein; crystallin, alpha B; protein kinase C, zeta; protein kinase, cAMP-dependent, regulatory, type II, alpha; homologous to yeast nitrogen permease (candidate tumor suppressor); intestinal cell kinase; GS3955 protein; activated p21cdc42Hs kinase; Rho-associated, oiled-coil-containing protein kinase I; KLAA2002 protein; unc-51-like kinase 1; and PDGFA associated protein 1, is contacted with nucleic acids of a population of cells. Nucleic acids which have specifically hybridized to said nucleic acid hybridization probes are detected. Cells whose nucleic acids specifically hybridized are identified as endothelial cells.
These and other embodiments which will be apparent to those of skill in the art upon reading the specification provide the art with reagents and methods for detection, diagnosis, therapy, and drug screening pertaining to neoangiogenesis and pathological processes involving or requiring neoangiogenesis.
We identified 123 human genes that were expressed at significantly higher levels (>2-fold) in tumor endothelium than in normal endothelium. See Tables 1 and 3, which show extracellular and cytoplasmic tumor endothelial markers (TEMs) respectively. Tables 2 and 4 identify the structure of the genes, proteins, and mRNAs that correspond to the tags identified. See also Tables 5, 6, and 7. Most of these genes were either not expressed or expressed at relatively low levels in Endothelial Cells (ECs) maintained in culture. Interestingly, the tumor endothelium genes were expressed in all tumors tested, regardless of its tissue or organ source. Most tumor endothelium genes were also expressed in corpus luteum and wounds.
It is clear that normal and tumor endothelium are highly related, sharing many endothelial cell specific markers. It is equally clear that the endothelium derived from tumors is qualitatively different from that derived from normal tissues of the same type and is also different from primary endothelial cultures. These genes are characteristically expressed in tumors derived from several different tissue types, documenting that tumor endothelium, in general, is different from normal endothelium. The genes expressed differentially in tumor endothelium are also expressed during other angiogenic processes such as corpus luteum formation and wound healing. It is therefore more appropriate to regard the formation of new vessels in tumors as “neoangiogenesis” rather than “tumor angiogenesis” per se. This distinction is important from a variety of perspectives, and is consistent with the idea that tumors recruit vasculature using much of, or basically the same signals elaborated during other physiologic or pathological processes. That tumors represent “unhealed wounds” is one of the oldest ideas in cancer biology.
Isolated and purified nucleic acids, according to the present invention are those which are not linked to those genes to which they are linked in the human genome. Moreover, they are not present in a mixture such as a library containing a multitude of distinct sequences from distinct genes. They may be, however, linked to other genes such as vector sequences or sequences of other genes to which they are not naturally adjacent. Tags disclosed herein, because of the way that they were made, represent sequences which are 3′ of the 3′ most restriction enzyme recognition site for the tagging enzyme used to generate the SAGE tags. In this case, the tags are 3′ of the most 3′ most NlaIII site in the cDNA molecules corresponding to mRNA. Nucleic acids corresponding to tags may be RNA, cDNA, or genomic DNA, for example. Such corresponding nucleic acids can be determined by comparison to sequence databases to determine sequence identities. Sequence comparisons can be done using any available technique, such as BLAST, available from the National Library of Medicine, National Center for Biotechnology Information. Tags can also be used as hybridization probes to libraries of genomic or cDNA to identify the genes from which they derive. Thus, using sequence comparisons or cloning, or combinations of these methods, one skilled in the art can obtain full-length nucleic acid sequences. Genes corresponding to tags will contain the sequence of the tag at the 3′ end of the coding sequence or of the 3′ untranslated region (UTR), 3′ of the 3′ most recognition site in the cDNA for the restriction endonuclease which was used to make the tags. The nucleic acids may represent either the sense or the anti-sense strand. Nucleic acids and proteins althought disclosed herein with sequence particularity, may be derived from a single individual. Allelic variants which occur in the population of humans are including within the scope of such nucleic acids and proteins. Those of skill in the art are well able to identify allelic variants as being the same gene or protein. Given a nucleic acid, one of ordinary skill in the art can readily determine an open reading frame present, and consequently the sequence of a polypeptide encoded by the open reading frame and, using techniques well known in the art, express such protein in a suitable host. Proteins comprising such polypeptides can be the naturally occurring proteins, fusion proteins comprising exogenous sequences from other genes from humans or other species, epitope tagged polypeptides, etc. Isolated and purified proteins are not in a cell, and are separated from the normal cellular constituents, such as nucleic acids, lipids, etc. Typically the protein is purified to such an extent that it comprises the predominant species of protein in the composition, such as greater than 50, 60 70, 80, 90, or even 95% of the proteins present.
Using the proteins according to the invention, one of ordinary skill in the art can readily generate antibodies which specifically bind to the proteins. Such antibodies can be monoclonal or polyclonal. They can be chimeric, humanized, or totally human. Any functional fragment or derivative of an antibody can be used including Fab, Fab′, Fab2, Fab′2, and single chain variable regions. So long as the fragment or derivative retains specificity of binding for the endothelial marker protein it can be used. Antibodies can be tested for specificity of binding by comparing binding to appropriate antigen to binding to irrelevant antigen or antigen mixture under a given set of conditions. If the antibody binds to the appropriate antigen at least 2, 5, 7, and preferably 10 times more than to irrelevant antigen or antigen mixture then it is considered to be specific.
Techniques for making such partially to fully human antibodies are known in the art and any such techniques can be used. According to one particularly preferred embodiment, fully human antibody sequences are made in a transgenic mouse which has been engineered to express human heavy and light chain antibody genes. Multiple strains of such transgenic mice have been made which can produce different classes of antibodies. B cells from transgenic mice which are producing a desirable antibody can be fused to make hybridoma cell lines for continuous production of the desired antibody. See for example, Nina D. Russel, Jose R. F. Corvalan, Michael L. Gallo, C. Geoffrey Davis, Liise-Anne Pirofski. Production of Protective Human Antipneumococcal Antibodies by Transgenic Mice with Human Immunoglobulin Loci Infection and Immunity April 2000, p. 1820-1826; Michael L. Gallo, Vladimir E. Ivanov, Aya Jakobovits, and C. Geoffrey Davis. The human immunoglobulin loci introduced into mice: V (D) and J gene segment usage similar to that of adult humans European Journal of Immunology 30: 534-540, 2000; Larry L. Green. Antibody engineering via genetic engineering of the mouse: XenoMouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies Journal of Immunological Methods 231 11-23, 1999; Yang X-D, Corvalan J R F, Wang P, Roy CM-N and Davis C G. Fully Human Anti-interleukin-8 Monoclonal Antibodies: Potential Therapeutics for the Treatment of Inflammatory Disease States. Journal of Leukocyte Biology Vol. 66, pp 401-410 (1999); Yang X-D, Jia X-C, Corvalan J R F, Wang P, C G Davis and Jakobovits A. Eradication of Established Tumors by a Fully Human Monoclonal Antibody to the Epidermal Growth Factor Receptor without Concomitant Chemotherapy. Cancer Research Vol. 59, Number 6, pp 1236-1243 (1999); Jakobovits A. Production and selection of antigen-specific fully human monoclonal antibodies from mice engineered with human Ig loci. Advanced Drug Delivery Reviews Vol. 31, pp: 33-42 (1998); Green L and Jakobovits A. Regulation of B cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes. J. Exp. Med. Vol. 188, Number 3, pp: 483-495 (1998); Jakobovits A. The long-awaited magic bullets: therapeutic human monoclonal antibodies from transgenic mice. Exp. Opin. Invest. Drugs Vol. 7(4), pp: 607-614 (1998); Tsuda H, Maynard-Currie K, Reid L, Yoshida T, Edamura K, Maeda N, Smithies O, Jakobovits A. Inactivation of Mouse HPRT locus by a 203-bp retrotransposon insertion and a 55-kb gene-targeted deletion: establishment of new HPRT-Deficient mouse embryonic stem cell lines. Genomics Vol. 42, pp: 413421 (1997); Sherman-Gold, R. Monoclonal Antibodies: The Evolution from '80s Magic Bullets To Mature, Mainstream Applications as Clinical Therapeutics. Genetic Engineering News Vol. 17, Number 14 (August 1997); Mendez M, Green L, Corvalan J, Jia X-C, Maynard-Currie C, Yang X-d, Gallo M, Louie D, Lee D, Erickson K, Luna J, Roy C, Abderrahim H, Kirschenbaum F, Noguchi M, Smith D, Fukushima A, Hales J, Finer M, Davis C, Zsebo K, Jakobovits A. Functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice. Nature Genetics Vol. 15, pp: 146-156 (1997); Jakobovits A. Mice engineered with human immunoglobulin YACs: A new technology for production of fully human antibodies for autoimmunity therapy. Weir's Handbook of Experimental Immunology, The Integrated Immune System Vol. IV, pp: 194.1-194.7 (1996); Jakobovits A. Production of fully human antibodies by transgenic mice. Current Opinion in Biotechnology Vol. 6, No. 5, pp: 561-566 (1995); Mendez M, Abderrahim H, Noguchi M, David N, Hardy M, Green L, Tsuda H, Yoast S, Maynard-Currie C, Garza D, Gemmill R, Jakobovits A, Klapholz S. Analysis of the structural integrity of YACs comprising human immunoglobulin genes in yeast and in embryonic stem cells. Genomics Vol. 26, pp: 294-307 (1995); Jakobovits A. YAC Vectors: Humanizing the mouse genome. Current Biology Vol. 4, No. 8, pp: 761-763 (1994); Arbones M, Ord D, Ley K, Ratech H, Maynard-Curry K, Otten G, Capon D, Tedder T. Lymphocyte homing and leukocyte rolling and migration are impaired in L-selectin-deficient mice. Immunity Vol. 1, No. 4, pp: 247-260 (1994); Green L, Hardy M, Maynard-Curry K, Tsuda H, Louie D, Mendez M, Abderrahim H, Noguchi M, Smith D, Zeng Y, et. al. Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YACs. Nature Genetics Vol. 7, No. 1, pp: 13-21 (1994); Jakobovits A, Moore A, Green L, Vergara G, Maynard-Curry K, Austin H, Klapholz S. Germ-line transmission and expression of a human-derived yeast artificial chromosome. Nature Vol. 362, No. 6417, pp: 255-258 (1993); Jakobovits A, Vergara G, Kennedy J, Hales J, McGuinness R, Casentini-Borocz D, Brenner D, Otten G. Analysis of homozygous mutant chimeric mice: deletion of the immunoglobulin heavy-chain joining region blocks B-cell development and antibody production. Proceedings of the National Academy of Sciences USA Vol. 90, No. 6, pp: 2551-2555 (1993); Kucherlapati et al., U.S. Pat. No. 6,1075,181.
Antibodies can also be made using phage display techniques. Such techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics. Single chain Fv can also be used as is convenient. They can be made from vaccinated transgenic mice, if desired. Antibodies can be produced in cell culture, in phage, or in various animals, including but not limited to cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes.
Antibodies can be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, or the like. Such labeled antibodies can be used for diagnostic techniques, either in vivo, or in an isolated test sample. Antibodies can also be conjugated, for example, to a pharmaceutical agent, such as chemotherapeutic drug or a toxin. They can be linked to a cytokine, to a ligand, to another antibody. Suitable agents for coupling to antibodies to achieve an anti-tumor effect include cytokines, such as interleukin 2 (IL-2) and Tumor Necrosis Factor (TNF); photosensitizers, for use in photodynamic therapy, including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; radionuclides, such as iodine-131 (131I), yttrium-90 (90Y), bismuth-212 (212Bi), bismuth-213 (213Bi), technetium-99m (99mTc), rhenium-186 (186Re), and rhenium-188 (188Re); antibiotics, such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial, plant, and other toxins, such as diphtheria toxin, pseudomonas exotoxin A, staphylococcal enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-alpha toxin, cytotoxin from chinese cobra (naja naja atra), and gelonin (a plant toxin); ribosome inactivating proteins from plants, bacteria and fungi, such as restrictocin (a ribosome inactivating protein produced by Aspergillus restrictus), saporin (a ribosome inactivating protein from Saponaria officinalis), and RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleoside); liposomes containing antitumor agents (e.g., antisense oligonucleotides, plasmids which encode for toxins, methotrexate, etc.); and other antibodies or antibody fragments, such as F(ab).
Those of skill in the art will readily understand and be able to make such antibody derivatives, as they are well known in the art. The antibodies may be cytotoxic on their own, or they may be used to deliver cytotoxic agents to particular locations in the body. The antibodies can be administered to individuals in need thereof as a form of passive immunization.
Characterization of extracellular regions for cell surface and secreted proteins from the protein sequence can be based on the prediction of signal sequence, transmembrane domains and functional domains. Antibodies are preferably specifically immunoreactive with membrane associated proteins, particularly to extracellular domains of such proteins or to secreted proteins. Such targets are readily accessible to antibodies, which typically do not have access to the interior of cells or nuclei. However, in some applications, antibodies directed to intracellular proteins may be useful as well. Moreover, for diagnostic purposes, an intracellular protein may be an equally good target since cell lysates may be used rather than a whole cell assay.
Computer programs can be used to identify extracellular domains of proteins whose sequences are known. Such programs include SMART software (Schultz et al., Proc. Natl. Acad. Sci. USA 95: 5857-5864, 1998) and Pfam software (Bateman et al., Nucleic acids Res. 28: 263-266, 2000) as well as PSORTII. Typically such programs identify transmembrane domains; the extracellular domains are identified as immediately adjacent to the transmembrane domains. Prediction of extracellular regions and the signal cleavage sites are only approximate. It may have a margin of error + or −5 residues. Signal sequence can be predicted using three different methods (Nielsen et al, Protein Engineering 10: 1-6, 1997, Jagla et. al, Bioinformatics 16: 245-250, 2000, Nakai, K and Horton, P. Trends in Biochem. Sci. 24:34-35, 1999) for greater accuracy. Similarly transmembrane (TM) domains can be identified by multiple prediction methods. (Pasquier, et. al, Protein Eng. 12:381-385, 1999, Sonnhammer et al., In Proc. of Sixth Int. Conf. on Intelligent Systems for Molecular Biology, p. 175-182, Ed J. Glasgow, T. Littlejohn, F. Major, R. Lathrop, D. Sankoff, and C. Sensen Menlo Park, Calif.: AAAI Press, 1998, Klein, et. al, Biochim. Biophys. Acta, 815:468, 1985, Nakai and Kanehisa Genomics, 14: 897-911, 1992). In ambiguous cases, locations of functional domains in well characterized proteins are used as a guide to assign a cellular localization.
Putative functions or functional domains of novel proteins can be inferred from homologous regions in the database identified by BLAST searches (Altschul et. al. Nucleic Acid Res. 25: 3389-3402, 1997) and/or from a conserved domain database such as Pfam (Bateman et. al, Nucleic Acids Res. 27:260-262 1999) BLOCKS (Henikoff, et. al, Nucl. Acids Res. 28:228-230, 2000) and SMART (Ponting, et. al, Nucleic Acid Res. 27,229-232, 1999). Extracellular domains include regions adjacent to a transmembrane domain in a single transmembrane domain protein (out-in or type I class). For multiple transmembrane domains proteins, the extracellular domain also includes those regions between two adjacent transmembrane domains (in-out and out-in). For type II transmembrane domain proteins, for which the N-terminal region is cytoplasmic, regions following the transmembrane domain is generally extracellular. Secreted proteins on the other hand do not have a transmembrane domain and hence the whole protein is considered as extracellular.
Membrane associated proteins can be engineered to delete the transmembrane domains, thus leaving the extracellular portions which can bind to ligands. Such soluble forms of transmembrane receptor proteins can be used to compete with natural forms for binding to ligand. Thus such soluble forms act as inhibitors. and can be used therapeutically as anti-angiogenic agents, as diagnostic tools for the quantification of natural ligands, and in assays for the identification of small molecules which modulate or mimic the activity of a TEM:ligand complex.
Alternatively, the endothelial markers themselves can be used as vaccines to raise an immune response in the vaccinated animal or human. For such uses, a protein, or immunogenic fragment of such protein, corresponding to the intracellular, extracellular or secreted TEM of interest is administered to a subject. The immogenic agent may be provided as a purified preparation or in an appropriately expressing cell. The administration may be direct, by the delivery of the immunogenic agent to the subject, or indirect, through the delivery of a nucleic acid encoding the immunogenic agent under conditions resulting in the expression of the immunogenic agent of interest in the subject. The TEM of interest may be delivered in an expressing cell, such as a purified population of tumor endothelial cells or a populations of fused tumor endothelial and dendritic cells. Nucleic acids encoding the TEM of interest may be delivered in a viral or non-viral delivery vector or vehicle. Non-human sequences encoding the human TEM of interest or other mammalian homolog can be used to induce the desired immunologic response in a human subject. For several of the TEMs of the present invention, mouse, rat or other ortholog sequences are described herein or can be obtained from the literature or using techniques well within the skill of the art.
Endothelial cells can be identified using the markers which are disclosed herein as being endothelial cell specific. These include the human markers identified by SEQ ID NOS: 1-123, i.e., the normal, pan-endothelial, and the tumor endothelial markers. Antibodies specific for such markers can be used to identify such cells, by contacting the antibodies with a population of cells containing some endothelial cells. The presence of cross-reactive material with the antibodies identifies particular cells as endothelial. Similarly, lysates of cells can be tested for the presence of cross-reactive material. Any known format or technique for detecting cross-reactive material can be used including, immunoblots, radioimmunoassay, ELISA, immunoprecipitation, and immunohistochemistry. In addition, nucleic acid probes for these markers can also be used to identify endothelial cells. Any hybridization technique known in the art including Northern blotting, RT-PCR, microarray hybridization, and in situ hybridization can be used.
One can identify tumor endothelial cells for diagnostic purposes, testing cells suspected of containing one or more TEMs. One can test both tissues and bodily fluids of a subject. For example, one can test a patient's blood for evidence of intracellular and membrane associated TEMs, as well as for secreted TEMs. Intracellular and/or membrane associated TEMs may be present in bodily fluids as the result of high levels of expression of these factors and/or through lysis of cells expressing the TEMs.
Populations of various types of endothelial cells can also be made using the antibodies to endothelial markers of the invention. The antibodies can be used to purify cell populations according to any technique known in the art, including but not limited to fluorescence activated cell sorting. Such techniques permit the isolation of populations which are at least 50, 60, 70, 80, 90, 92, 94, 95, 96, 97, 98, and even 99% the type of endothelial cell desired, whether normal, tumor, or pan-endothelial. Antibodies can be used to both positively select and negatively select such populations. Preferably at least 1, 5, 10, 15, 20, or 25 of the appropriate markers are expressed by the endothelial cell population.
Populations of endothelial cells made as described herein, can be used for screening drugs to identify those suitable for inhibiting the growth of tumors by virtue of inhibiting the growth of the tumor vasculature.
Populations of endothelial cells made as described herein, can be used for screening candidate drugs to identify those suitable for modulating angiogenesis, such as for inhibiting the growth of tumors by virtue of inhibiting the growth of endothelial cells, such as inhibiting the growth of the tumor or other undesired vasculature, or alternatively, to promote the growth of endothelial cells and thus stimulate the growth of new or additional large vessel or microvasculature.
Inhibiting the growth of endothelial cells means either regression of vasculature which is already present, or the slowing or the absence of the development of new vascularization in a treated system as compared with a control system. By stimulating the growth of endothelial cells, one can influence development of new (neovascularization) or additional vasculature development (revascularization). A variety of model screen systems are available in which to test the angiogenic and/or anti-angiogenic properties of a given candidate drug. Typical tests involve assays measuring the endothelial cell response, such as proliferation, migration, differentiation and/or intracellular interaction of a given candidate drug. By such tests, one can study the signals and effects of the test stimuli. Some common screens involve measurement of the inhibition of heparanase, endothelial tube formation on Matrigel, scratch induced motility of endothelial cells, platelet-derived growth factor driven proliferation of vascular smooth muscle cells, and the rat aortic ring assay (which provides an advantage of capillary formation rather than just one cell type).
Drugs can be screened for the ability to mimic or modulate, inhibit or stimulate, growth of tumor endothelium cells and/or normal endothelial cells. Drugs can be screened for the ability to inhibit tumor endothelium growth but not normal endothelium growth or survival. Similarly, human cell populations, such as normal endothelium populations or tumor endothelial cell populations, can be contacted with test substances and the expression of tumor endothelial markers and/or normal endothelial markers determined. Test substances which decrease the expression of tumor endothelial markers (TEMs) are candidates for inhibiting angiogenesis and the growth of tumors. In cases where the activity of a TEM is known, agents can be screened for their ability to decrease or increase the activity.
For those tumor endothelial markers identified as being secreted proteins, it is desirable to identify drug candidates capable of binding to the secreted TEM protein. For some applications, the identification of drug candidates capable of interfering with the binding of the secreted TEM to its native receptor is desirable. For other applications, the identification of drug candidates capable of mimicking the activity of the native receptor will be desired. Thus, by manipulating the binding of the secreted TEM:receptor complex, one may be able to promote or inhibit futher development of endothelial cells, and hence, vascularization.
Expression can be monitored according to any convenient method. Protein or mRNA can be monitored. Any technique known in the art for monitoring specific genes' expression can be used, including but not limited to ELISAs, SAGE, microarray hybridization, Western blots. Changes in expression of a single marker may be used as a criterion for significant effect as a potential pro-angiogenic, anti-angiogenic or anti-tumor agent. However, it also may be desirable to screen for test substances which are able to modulate the expression of at least 5, 10, 15, or 20 of the relevant markers, such as the tumor or normal endothelial markers. Inhibition of TEM protein activity can also be used as a drug screen. Human and mouse TEMS can be used for this purpose.
Test substances for screening can come from any source. They can be libraries of natural products, combinatorial chemical libraries, biological products made by recombinant libraries, etc. The source of the test substances is not critical to the invention. The present invention provides means for screening compounds and compositions which may previously have been overlooked in other screening schemes. Nucleic acids and the corresponding encoded proteins of the markers of the present invention can be used therapeutically in a variety of modes. TEMs can be used to stimulate the growth of vasculature, such as for wound healing or to circumvent a blocked vessel. The nucleic acids and encoded proteins can be administered by any means known in the art. Such methods include, using liposomes, nanospheres, viral vectors, non-viral vectors comprising polycations, etc. Suitable viral vectors include adenovirus, retroviruses, and sindbis virus. Administration modes can be any known in the art, including parenteral, intravenous, intramuscular, intraperitoneal, topical, intranasal, intrarectal, intrabronchial, etc.
Specific biological antagonists of TEMs can also be used to therapeutic benefit. For example, antibodies, T cells specific for a TEM, antisense to a TEM, and ribozymes specific for a TEM can be used to restrict, inhibit, reduce, and/or diminish tumor or other abnormal or undesirable vasculature growth. Such antagonists can be administered as is known in the art for these classes of antagonists generally. Anti-angiogenic drugs and agents can be used to inhibit tumor growth, as well as to treat diabetic retinopathy, rheumatoid arthritis, psoriasis, polycystic kidney disease (PKD), and other diseases requiring angiogenesis for their pathologies.
The above disclosure generally describes the present invention. All references disclosed herein are expressly incorporated by reference. A more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only, and are not intended to limit the scope of the invention.
Visualization of Vasculature of Colorectal Cancers
The endothelium of human colorectal cancer was chosen to address the issues of tumor angiogenesis, based on the high incidence, relatively slow growth, and resistance to anti-neoplastic agents of these cancers. While certain less common tumor types, such as glioblastomas, are highly vascularized and are regarded as good targets for anti-angiogenic therapy, the importance of angiogenesis for the growth of human colorectal cancers and other common solid tumor types is less well documented.
We began by staining vessels in colorectal cancers using von Willebrand Factor (vWF) as a marker. In each of 6 colorectal tumors, this examination revealed a high density of vessels throughout the tumor parenchyma (Examples in FIGS. 1 A and B). Interestingly, these analyses also substantiated the importance of these vessels for tumor growth, as endothelium was often surrounded by a perivascular cuff of viable cells, with a ring of necrotic cells evident at the periphery (Example in FIG. 1A). Although these preliminary studies suggested that colon tumors are angiogenesis-dependent, reliable markers that could distinguish vessels in colon cancers from the vessels in normal colon are currently lacking. One way to determine if such markers exist is by analyzing gene expression profiles in endothelium derived from normal and neoplastic tissue.
Purification of Endothelial Cells
Global systematic analysis of gene expression in tumor and normal endothelium has been hampered by at least three experimental obstacles. First, endothelium is enmeshed in a complex tissue consisting of vessel wall components, stromal cells, and neoplastic cells, requiring highly selective means of purifying ECs for analysis. Second, techniques for defining global gene expression profiles were not available until recently. And third, only a small fraction of the cells within a tumor are endothelial, mandating the development of methods that are suitable for the analysis of global expression profiles from relatively few cells.
To overcome the first obstacle, we initially attempted to purify ECs from dispersed human colorectal tissue using CD31, an endothelial marker commonly used for this purpose. This resulted in a substantial enrichment of ECs but also resulted in contamination of the preparations by hematopoietic cells, most likely due to expression of CD31 by macrophages. We therefore developed a new method for purifying ECs from human tissues using P1H12, a recently described marker for ECs. Unlike CD31, P1H12 was specifically expressed on the ECs of both colorectal tumors and normal colorectal mucosa. Moreover, immunofluorescence staining of normal and cancerous colon with a panel of known cell surface endothelial markers (e.g. VE-cadherin, CD31 and CD34) revealed that P1H12 was unique in that it stained all vessels including microvessels (see FIG. 2A and data not shown). In addition to selection with P1H12, it was necessary to optimize the detachment of ECs from their neighbors without destroying their cell surface proteins as well as to employ positive and negative affinity purifications using a cocktail of antibodies (FIG. 2B). The ECs purified from normal colorectal mucosa and colorectal cancers were essentially free of epithelial and hematopoietic cells as judged by RT-PCR (FIG. 2C) and subsequent gene expression analysis (see below).
Comparison of Tumor and Normal Endothelial Cell Expression Patterns
To overcome the remaining obstacles, a modification of the Serial Analysis of Gene Expression (SAGE) technique was used. SAGE associates individual mRNA transcripts with 14 base pair tags derived from a specific position near their 3′ termini. The abundance of each tag provides a quantitative measure of the transcript level present within the mRNA population studied. SAGE is not dependent on pre-existing databases of expressed genes, and therefore provides an unbiased view of gene expression profiles. This feature is particularly important in the analysis of cells that constitute only a small fraction of the tissue under study, as transcripts from these cells are unlikely to be well represented in extant EST databases. We adapted the SAGE protocol so that it could be used on small numbers of purified ECs obtained from the procedure outlined in FIG. 2B.
A library of ˜100,000 tags from the purified ECs of a colorectal cancer, and a similar library from the ECs of normal colonic mucosa from the same patient were generated. These ˜193,000 tags corresponded to over 32,500 unique transcripts. Examination of the expression pattern of hematopoietic, epithelial and endothelial markers confirmed the purity of the preparations (FIG. 2D).
Tumor Endothelium Markers are Neo-Angiogenic
Finally, we asked whether these transcripts were expressed in angiogenic states other than that associated with tumorigenesis. We thus performed in situ hybridizations on corpus luteum tissue as well as healing wounds. Although there were exceptions, we found that these transcripts were generally expressed both in the corpus luteum and in the granulation tissue of healing wounds. In all tissues studied, expression of the genes was either absent or exclusively confined to the EC compartment.
The disclosure of each reference cited is expressly incorporated herein.
27. Transcript copies per cell were calculated assuming an average cell contains 300,000 transcripts.
This application claims the benefit of U.S. provisional applications Ser. No. 60/393,023, filed Jul. 2, 2002, and Ser. No. 60/458,964, filed Apr. 1, 2003.
The U.S. government retains certain rights in the invention by virtue of the provisions of National Institutes of Heath grants CA57345 and CA43460, which supported this work.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US03/16250 | 7/2/2003 | WO | 12/3/2005 |
