US20100028256A1 - Differential gene expression in physiological and pathological angiogenesis - Google Patents

Differential gene expression in physiological and pathological angiogenesis Download PDF

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US20100028256A1
US20100028256A1 US12/514,297 US51429707A US2010028256A1 US 20100028256 A1 US20100028256 A1 US 20100028256A1 US 51429707 A US51429707 A US 51429707A US 2010028256 A1 US2010028256 A1 US 2010028256A1
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Brad St. Croix
Steven Seaman
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Definitions

  • the present disclosure relates to the field of angiogenesis and endothelial cell markers and in particular, to pathological angiogenesis endothelial markers and organ-specific endothelial markers and methods of their uses.
  • tumor angiogenesis Inhibition of tumor angiogenesis is an anticancer strategy that has gained widespread support from biologists and clinicians.
  • Dr. Judah Folkman introduced the concept of an “angiogenic switch” driving tumor growth and malignant progression.
  • Angiogenesis can occur under “normal” physiological conditions, such as during growth and development or wound healing, as well as under “pathological” conditions, such as in the transition of tumors from a dormant state to a malignant state.
  • pathological such as in the transition of tumors from a dormant state to a malignant state.
  • the dependency of solid tumors on new vessel growth has made tumor vessels an appealing target for cancer therapy.
  • Angiogenesis-based tumor therapy has several theoretical advantages over traditional cancer therapies (such as radiation and chemotherapy).
  • Anti-angiogenesis therapy targets endothelial cells that line tumor vessels instead of the tumor cells themselves.
  • Tumor cells evolve resistance to cancer therapies due to genomic instability (high variation) and rapid generation time (days).
  • endothelial cells have a higher genomic stability (low variation) and a longer generation time (months) compared to tumor cells.
  • Endothelial cells are less likely to “escape” therapy because they will not undergo mitosis at such a rapid rate and carry any drug resistance variation through to the next generation within the lifespan of the therapy.
  • the genomic stability of endothelial cells coupled with their longevity make them an attractive target for therapies directed against them.
  • TEMs Tumor endothelial markers
  • St. Croix et al. employed serial analysis of gene expression (SAGETM) technology to compare small populations of normal and tumor-derived endothelial cells. The comparison revealed 79 genes that are potentially involved in angiogenesis. Of these, 46 genes were specifically expressed at least ten times higher in tumor-associated endothelium as compared to normal endothelium from the same patient.
  • SAGETM serial analysis of gene expression
  • bevacizumab an antibody that neutralizes vascular endothelial growth factor (VEGF; one of the many proteins involved in the development of a new network of blood vessels)
  • VEGF vascular endothelial growth factor
  • a remaining challenge is to identify markers that can differentiate pathological and physiological angiogenesis in order to selectively deliver therapeutic agents to diseased tissues while minimizing the potential side effects of the targeted therapy.
  • angiogenesis-specific endothelial markers including some specific for pathological angiogenesis. Endothelial cells were isolated from normal, regenerating, and tumor-bearing livers. Gene expression profiles amongst the multiple samples were compared by performing serial analysis of gene expression (SAGE) on the isolated endothelial cells. The identification of markers highly specific for physiological or pathological angiogenesis has significant implications for the development of selective vascular targeted therapies. Thus, methods of reducing or inhibiting pathological angiogenesis in a subject are disclosed.
  • the method includes administering a therapeutically effective amount of a composition that includes one or more binding agents (such as an antibody) that specifically binds to one or more of the following pathological angiogenesis marker proteins: Vscp, CD276, ETSvg4 (Pea3), CD137(4-1BB), MiRP2, Ubiquitin D (Fat10), Doppel (prion-PLP), Apelin, Plgf, Ptprn (IA-2), CD109, Ankylosis, and collagen VIII ⁇ 1, thereby inhibiting pathological angiogenesis in the subject.
  • the binding agent is conjugated to one or more therapeutic molecules, such as chemotherapy agents, cytoxins, radionucleotides or a combination thereof.
  • the method includes detecting at least one expression product including one or more of: Vscp, CD276, ETSvg4 (Pea3), CD137(4-1BB), MiRP2, Ubiquitin D (Fat10), Doppel (prion-PLP), Apelin, Plgf, Ptprn (IA-2), CD109, Ankylosis, and collagen VIII, 1 in a sample obtained from the subject.
  • Detection of the at least one expression product can indicate the presence of pathological angiogenesis in the subject.
  • liver-specific endothelial markers are 27 brain-specific endothelial markers and 15 liver-specific endothelial markers. These organ-specific endothelial markers can serve as therapeutic targets to allow molecular agents to be selectively delivered to specific anatomical sites. Similarly, these organ-specific endothelial markers can serve as diagnostic targets to allow diagnostic agents (such as imaging agents) to be selectively delivered to specific anatomical sites. Thus, methods of delivering a therapeutic agent to organ-specific endothelial cells are provided.
  • Methods for delivering a therapeutic or diagnostic agent to brain endothelial cells.
  • the method includes administering a therapeutically effective amount of a composition that includes a therapeutic binding agent that preferentially binds to one or more brain endothelial marker proteins.
  • a therapeutic binding agent that preferentially binds to one or more brain endothelial marker proteins.
  • Such a method can evoke a therapeutic response in the brain endothelial cells or permit detection of the cells.
  • brain endothelial markers may also facilitate the selectively delivery of therapeutic agents across the blood-brain barrier to underlying neuronal cells via transcytosis.
  • the one or more brain endothelial markers can include Glucose transporter GLUT-1, Organic anion transporter 2, Pleiotrophin, ATPase class V, type 10A, Peptidoglycan recognition protein 1, Organic anion transporter 14, Forkhead box Q1, Organic anion transporter 3, SN2 (Solute carrier family 38, member 5), Inter-alpha (globulin) inhibitor H5, Solute carrier 38 member 3, Zinc finger protein of the cerebellum 2, Testican-2,3-HMG-CoA synthase 2, Progestin and adipoQ receptor family member V, APC down-regulated 1 Drapc1, GDPD phosphodiesterase family Accession No.
  • NM — 001042671 putative transmembrane protein Accession No. NM — 029001, DES2 lipid desaturase/C4-hyroxylase, Kelch repeat and BTB (POZ) domain, Lipolysis stimulated receptor, Glutathione S-transferase alpha 4, TNF receptor superfamily member 19, T-box 1, putative secreted protein Accession No. XM — 620023 or combinations thereof.
  • the method includes administering a therapeutically effective amount of a composition that includes a binding agent that specifically binds to one or more liver endothelial marker proteins (e.g., deoxyribonuclease 1-like 3, LZP oncoprotein induced transcript 3, putative transmembrane protein Accession No. NM — 023438, CD32 15, putative G-protein coupled receptor NM — 033616, C-type lectin-like receptor 2, C-type lectin domain family 4 member g 16, Plexin C1, Wnt9B, Accession No.
  • liver endothelial marker proteins e.g., deoxyribonuclease 1-like 3, LZP oncoprotein induced transcript 3, putative transmembrane protein Accession No. NM — 023438, CD32 15, putative G-protein coupled receptor NM — 033616, C-type lectin-like receptor 2, C-type lectin domain family 4 member g 16, Plexin C1, Wnt9B, Accession
  • AK144596 GATA-binding protein 4, MBL-associated serine protease-3, Renin binding protein, putative transmembrane protein Accession No. NM — 144830, or Retinoic acid receptor, beta
  • a therapeutic agent such as a method can evoke a therapeutic response in the liver endothelial cells or permit detection of the cells.
  • FIG. 1A includes digital images of heart tissue stained with immunofluorescently-labeled CD105 (left panel), VE-cadherin (middle panel) or both CD105 and VE-cadherin (right panel). Scale bar, 20 ⁇ m.
  • FIG. 1B is a digital image of liver tissue stained with immunofluorescently-labeled CD105. Scale bar, 20 ⁇ m.
  • FIG. 1C is a bar graph showing the relative amount of VE-cadherin detected by quantitative polymerase chain reaction (QPCR) in cDNA isolated from unfractionated normal whole tissues (WT), purified endothelial cells (ECs) isolated from normal tissues (N-ECs) or purified ECs isolated from tumors (T-ECs).
  • QPCR quantitative polymerase chain reaction
  • FIG. 1D is a schematic of a model used to identify genes expressed during pathological, but not physiological angiogenesis. ECs were isolated from normal resting livers, regenerating livers, or tumor bearing livers.
  • FIGS. 2A , 2 B and 2 C are bar graphs illustrating the expression of various genes in resting normal ECs, regenerating liver ECs and tumor ECs, respectively.
  • the expression of the various genes was evaluated by real-time Q-PCR and compared with that of Srnp70, a gene expressed at nearly identical levels in all ECs as detected by SAGE.
  • FIGS. 3A-31 are digital images of various mRNA expressed by ECs in vivo detected by staining samples with Oatp2 (a), CD276 (b), ETSvg4 (c), Apelin (d), CD109 (e), MiRP2 (f), CD137 (g), Doppel (h) and Vscp (i).
  • (a) is representative of a brain endothelial marker in brain tissue
  • (d-f) depict SW620 tumors grown subcutaneously
  • (g-h) depict KM12 tumors grown in the liver.
  • a dilute counterstain was applied to the sections to highlight the lack of detectable expression in the non-ECs of the tumors.
  • Scale bars 50 ⁇ M.
  • FIG. 4A includes digital images of human colon samples stained with immunoflurescently labeled CD276 and von Willebrand factor (vWF).
  • CD276 was expressed predominantly by the tumor vessels of the colorectal cancer, but was also expressed at a lower level by the tumor cells themselves. Expression of CD276 in normal colonic mucosa was undetectable (top middle panel). As a control, vessels were stained for vWF, which co-localized with CD276 only in the tumor sample. Scale bar, 100 ⁇ m.
  • FIG. 4B includes digital images of angiogenic vessels of the developing corpus luteum stained with immunoflurescently-labeled CD276.
  • CD276 expression was undetectable in the angiogenic vessels of the developing corpus luteum.
  • Sections were counterstained with DAPI which is shown in the left panels to highlight the epithelial cells. Scale bar, 200 ⁇ m.
  • FIG. 5 includes digital images of vessels of human colorectal cancer.
  • In situ hybridization revealed that CD276 mRNA is expressed predominantly in the vessels of human colorectal cancer (middle panel) with a pattern of staining similar to that of the control endothelial marker VEGFR2 (left panel).
  • the tumor cells also display positive staining, albeit less intense.
  • T tumor
  • the extracellular staining around the normal crypts represents non-specific binding of the in situ hybridization reagents to the mucous (right panel) and is also present in control sections. Scale bars, 50 ⁇ M.
  • FIG. 6A is a digital image of an immunoblot including colorectal tumor (T) and normal (N) colonic mucosa samples. Immunoblotting with a CD276 monoclonal antibody revealed an upregulation of CD276 protein in colorectal tumors (T) compared to normal (N) colonic mucosa.
  • FIG. 6B is a digital image of an immunoblot including lung tumor (T) and normal (N) adjacent lung tissue samples.
  • Immunoblotting with a CD276 monoclonal antibody revealed an upregulation of CD276 protein in lung tumors (T) compared to normal (N) adjacent lung tissue.
  • the normal tissues in A and B were classified as normal based on gross morphology, but microscopic disease or inflammatory host cells may have contributed to the low level CD276 expression observed in these tissues.
  • FIGS. 6C-6L are digital images of various samples stained with a polyclonal CD276 antibody.
  • Immunohistochemical staining with a polyclonal CD276 antibody revealed a vessel-like pattern in colorectal cancer (C-E), non-small cell lung cancer (F-H), esophageal cancer (I-J), bladder cancer (K) and breast cancer (L).
  • C-E colorectal cancer
  • F-H non-small cell lung cancer
  • I-J esophageal cancer
  • K bladder cancer
  • breast cancer L
  • FIG. 7 is a digital image of amplification products generated in tumor cell lines or tumor endothelial cells in the presence of VE-cadherin, Ubiquitin D or ⁇ -actin primers. RT-PCR was used to verify that Ubiquitin D is expressed by the tumor endothelial cells (TECs) and not the tumor cells themselves.
  • TECs tumor endothelial cells
  • FIG. 8 is a digital image of an immunoblot including protein extracts from three subjects with either normal colonic mucosa (N) or colorectal tumors (T). CD137 expression was elevated in protein extracts of human colorectal cancer.
  • FIG. 9A includes digital images of LEM and BEM genes identified by SAGE are expressed by ECs in vivo. Localization of mRNA in ECs was demonstrated for the brain endothelial markers GLUT-1 (BEM1) and organic anion transporter 2 (BEM2), and the liver endothelial markers deoxyribonuclease 1-like 3 (LEM1) and oncogenes induced transcript 3 (LEM2).
  • BEM1 brain endothelial markers GLUT-1
  • BEM2 organic anion transporter 2
  • LEM1 deoxyribonuclease 1-like 3
  • LEM2 oncogenes induced transcript 3
  • the BEMs are selectively expressed in brain endothelium whereas the LEMs are selectively expressed in liver endothelium.
  • the endothelial control probe, VEGFR2 stains both brain and liver endothelium.
  • FIG. 9B includes digital images of localization of Apelin and Doppel mRNA in subcutaneous implanted LLC tumors.
  • Angiogenesis is critical for the progression of many diseases, including age-related macular degeneration and cancer. Markers that can distinguish physiological and pathological angiogenesis are needed in order to selectively deliver anti-angiogenic or vascular targeting agents to diseased tissues and minimize the potential side effects of the targeted therapy. Physiological and pathological angiogenesis are morphologically distinct. However, the extent of differential gene expression between these cellular states has remained elusive. Most of the well-studied molecules thought to regulate tumor angiogenesis, such as vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), the angiopoietins, and their receptors, also regulate normal physiological angiogenesis.
  • VEGF vascular endothelial growth factor
  • bFGF basic fibroblast growth factor
  • angiopoietins and their receptors
  • angiogenesis-specific endothelial markers including thirteen that are specific for pathological angiogenesis.
  • the genes specific for pathological angiogenesis were primarily cell surface molecules. Therefore, this disclosure provides several molecules that can be used for the therapeutic targeting of tumor vessels.
  • a binding agent specific to one or more of the disclosed pathological angiogenesis endothelial marker proteins can be used for targeted drug delivery to the tumor site.
  • linking or conjugating the binding agent to a chemical or radioactive toxin can provide a targeted cytotoxic therapy.
  • a binding agent specific to one or more of the disclosed pathological angiogenesis endothelial marker proteins is labeled with an imaging tag, such as a fluorophore, thereby providing diagnostic imaging agents.
  • a therapeutically effective amount of a binding agent that specifically binds to at least one of the disclosed pathological angiogenesis endothelial marker proteins is administered to a subject.
  • pathological angiogenesis in the subject is thereby reduced or inhibited. Additional methods of diagnosing or treating a tumor are also provided.
  • the present disclosure also provides twenty-seven brain-specific endothelial markers and fifteen liver-specific endothelial markers. These organ-specific endothelial markers can aid in the selective delivery of therapeutic and diagnostic agents to specific anatomical sites.
  • methods are disclosed for delivering a therapeutic or diagnostic agent to brain endothelial cells.
  • the method includes administering a therapeutically effective amount of a binding agent, such as an antibody, that specifically binds to at least one of the disclosed brain endothelial markers, thereby evoking a therapeutic response in the brain endothelial cells or permitting imaging of the brain endothelial cells.
  • a binding agent such as an antibody
  • the binding agent upon binding at least one of the disclosed brain endothelial markers, would enable the delivery of the agent, via mechanisms such as transcytosis, across the blood-brain barrier to the particular cells underlying the brain endothelium, such as neuronal cells.
  • the method includes delivering a therapeutic agent to liver endothelial cells by administering a therapeutically effective amount of a binding agent that specifically binds to at least one of the disclosed liver endothelial marker proteins, thereby evoking a therapeutic response in the liver endothelial cells or permitting imaging of the liver endothelial cells.
  • an agent such as a composition that includes a binding agent that specifically binds to one or more of the disclosed pathological angiogenesis endothelial marker proteins (such as those listed in Tables 8 and 9) by any effective route.
  • routes of administration include, but are not limited to, oral, injection (such as subcutaneous, intramuscular, intradermal, intraperitoneal, intravenous, and intratumoral), sublingual, rectal, transdermal, intranasal, vaginal and inhalation routes.
  • Agent Any protein, nucleic acid molecule, compound, small molecule, organic compound, inorganic compound, or other molecule of interest.
  • Agent can include a therapeutic agent, a diagnostic agent or a pharmaceutical agent.
  • a therapeutic or pharmaceutical agent is one that alone or together with an additional compound induces the desired response (such as inducing a therapeutic or prophylactic effect when administered to a subject).
  • a pharmaceutical agent such as an antibody to any of the proteins listed in Tables 8 and 9 conjugated to a therapeutic agent significantly reduces angiogenesis.
  • Angiogenesis A physiological process involving the growth of new blood vessels from pre-existing vessels.
  • Angiogenesis can occur under normal physiological conditions such as during growth and development or wound healing (known as physiological angiogenesis) as well as pathological conditions such as in the transition of tumors from a dormant state to a malignant state (known as pathological angiogenesis).
  • Ankylosis The ANK protein, the product of the progressive ankylosis (ank) gene, is a multipass transmembrane protein that is highly conserved in vertebrates.
  • the ANK protein has been shown to control pyrophosphate levels in cells and may act as a pyrophosphate transporter that stimulates the elaboration of extracellular pyrophosphate from intracellular stores.
  • the term ankylosis includes any ankylosis gene, cDNA, mRNA, or protein from any organism and that is ankylosis and is increased during pathological angiogenesis relative to either normal or physiological angiogenesis conditions. In one example, ANK protein is expressed during pathological angiogenesis.
  • GenBank Accession Nos.: DQ832285, NM — 020332, AK083135, BC054379, AY358503, and NM — 054027 disclose ankylosis nucleic acid sequences
  • GenBank Accession Nos.: AAF88038, Q9JHZ2, XP — 001132013, NP — 473368, and Q9HCJ1 disclose ankylosis protein sequences.
  • ankylosis includes a full-length wild-type (or native) sequence, as well as ankylosis allelic variants, fragments, homologs or fusion sequences that retain the ability to be preferentially expressed during pathological angiogenesis and/or modulate pathological angiogenesis.
  • ankylosis has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to ankylosis.
  • ankylosis has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No.
  • DQ832285, NM — 020332, AK083135, BC054379, AY358503, or NM — 054027 and retains ankylosis activity (e.g., the capability to be expressed during pathological angiogenesis and/or modulate pathological angiogenesis).
  • ankylosis activity e.g., the capability to be expressed during pathological angiogenesis and/or modulate pathological angiogenesis.
  • Antibody A polypeptide ligand including at least a light chain or heavy chain immunoglobulin variable region which specifically recognizes and binds an epitope of an antigen, such as an endothelial marker or a fragment thereof.
  • Antibodies are composed of a heavy and a light chain, each of which has a variable region, termed the variable heavy (V H ) region and the variable light (V L ) region. Together, the V H region and the V L region are responsible for binding the antigen recognized by the antibody.
  • an antibody specifically binds to one of the proteins listed in Tables 8 and 9.
  • scFv protein is a fusion protein in which a light chain variable region of an immunoglobulin and a heavy chain variable region of an immunoglobulin are bound by a linker, while in dsFvs, the chains have been mutated to introduce a disulfide bond to stabilize the association of the chains.
  • the term also includes genetically engineered forms such as chimeric antibodies (for example, humanized murine antibodies), heteroconjugate antibodies (such as, bispecific antibodies). See also, Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, Ill.); Kuby, J., Immunology, 3 rd Ed., W.H. Freeman & Co., New York, 1997.
  • a naturally occurring immunoglobulin has heavy (H) chains and light (L) chains interconnected by disulfide bonds.
  • H heavy chain
  • L light chain
  • ⁇ lambda
  • k kappa
  • IgM immunoglobulin heavy chain classes
  • Each heavy and light chain contains a constant region and a variable region, (the regions are also known as “domains”).
  • the heavy and the light chain variable regions specifically bind the antigen.
  • Light and heavy chain variable regions contain a “framework” region interrupted by three hypervariable regions, also called “complementarity-determining regions” or “CDRs”.
  • CDRs complementarity-determining regions
  • the extent of the framework region and CDRs have been defined (see, Kabat et al., Sequences of Proteins of Immunological Interest , U.S. Department of Health and Human Services, 1991, which is hereby incorporated by reference).
  • the Kabat database is now maintained online.
  • the sequences of the framework regions of different light or heavy chains are relatively conserved within a species.
  • the framework region of an antibody that is the combined framework regions of the constituent light and heavy chains, serves to position and align the CDRs in three-dimensional space.
  • the CDRs are primarily responsible for binding to an epitope of an antigen.
  • the CDRs of each chain are typically referred to as CDR1, CDR2, and CDR3, numbered sequentially starting from the N-terminus, and are also typically identified by the chain in which the particular CDR is located.
  • a V H CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found
  • a V L CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.
  • An antibody that binds RET will have a specific V H region and the V L region sequence, and thus specific CDR sequences.
  • Antibodies with different specificities i.e. different combining sites for different antigens
  • SDRs specificity determining residues
  • V H refers to the variable region of an immunoglobulin heavy chain, including that of an Fv, scFv, dsFv or Fab.
  • V L refers to the variable region of an immunoglobulin light chain, including that of an Fv, scFv, dsFv or Fab.
  • a “monoclonal antibody” is an antibody produced by a single clone of B-lymphocytes or by a cell into which the light and heavy chain genes of a single antibody have been transfected.
  • Monoclonal antibodies are produced by methods known to those of skill in the art, for instance by making hybrid antibody-forming cells from a fusion of myeloma cells with immune spleen cells.
  • Monoclonal antibodies include humanized monoclonal antibodies.
  • polyclonal antibody is an antibody that is derived from different B-cell lines.
  • Polyclonal antibodies are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognising a different epitope. These antibodies are produced by methods known to those of skill in the art, for instance, by injection of an antigen into a suitable mammal (such as a mouse, rabbit or goat) that induces the B-lymphocytes to produce IgG immunoglobulins specific for the antigen which are then purified from the mammal's serum.
  • a suitable mammal such as a mouse, rabbit or goat
  • a “chimeric antibody” has framework residues from one species, such as human, and CDRs (which generally confer antigen binding) from another species, such as a murine antibody that specifically binds an endothelial marker.
  • a “humanized” immunoglobulin is an immunoglobulin including a human framework region and one or more CDRs from a non-human (for example a mouse, rat, or synthetic) immunoglobulin.
  • the non-human immunoglobulin providing the CDRs is termed a “donor,” and the human immunoglobulin providing the framework is termed an “acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin.
  • Constant regions need not be present, but if they are, they are substantially identical to human immunoglobulin constant regions, e.g., at least about 85-90%, such as about 95% or more identical.
  • Humanized immunoglobulins can be constructed by means of genetic engineering (see for example, U.S. Pat. No. 5,585,089).
  • Binding affinity Affinity of one molecule for another, such as an antibody for an antigen (for example, the antigens shown in Tables 8 and 9).
  • affinity is calculated by a modification of the Scatchard method described by Frankel et al., Mol. Immunol., 16:101-106, 1979.
  • binding affinity is measured by an antigen/antibody dissociation rate.
  • a high binding affinity is measured by a competition radioimmunoassay.
  • a high binding affinity is at least about 1 ⁇ 10 ⁇ 8 M.
  • a high binding affinity is at least about 1.5 ⁇ 10 ⁇ 8 , at least about 2.0 ⁇ 10 ⁇ 8 , at least about 2.5 ⁇ 10 ⁇ 8 , at least about 3.0 ⁇ 10 ⁇ 8 , at least about 3.5 ⁇ 10 ⁇ 8 , at least about 4.0 ⁇ 10 ⁇ 8 , at least about 4.5 ⁇ 10 ⁇ 8 , or at least about 5.0 ⁇ 10 ⁇ 8 M.
  • Biological activity An expression describing the beneficial or adverse effects of an agent on living matter. When the agent is a complex chemical mixture, this activity is exerted by the substance's active ingredient or pharmacophore, but can be modified by the other constituents. Activity is generally dosage-dependent and it is not uncommon to have effects ranging from beneficial to adverse for one substance when going from low to high doses.
  • a specific binding agent significantly reduces the biological activity of the one or more pathological angiogenesis marker proteins (such as those listed in Table 9) which in turn inhibits pathological angiogenesis.
  • Cancer Malignant neoplasm that has undergone characteristic anaplasia with loss of differentiation, increase rate of growth, invasion of surrounding tissue, and is capable of metastasis.
  • CD276 A member of the B7 family of immunoregulatory molecules that can be induced on T-cells, macrophages and dendritic cells by a variety of inflammatory cytokines. Its homology to other co-stimulatory molecules indicates it may have an immunoregulatory role. In particular examples, expression of CD276 is increased during pathological angiogenesis.
  • the term CD276 includes any CD276 gene, cDNA, mRNA, or protein from any organism and that is CD276 and is expressed during pathological angiogenesis.
  • Nucleic acid and protein sequences for CD276 are publicly available.
  • GenBank Accession Nos.: DQ832276, NM — 001024736, AK031354, AK155114, NM — 133983, and NM — 025240 disclose CD276 nucleic acid sequences
  • GenBank Accession Nos.: NP — 598744, NP — 079516, and AAK15438 disclose CD276 protein sequences.
  • CD276 includes a full-length wild-type (or native) sequence, as well as CD276 allelic variants, fragments, homologs or fusion sequences that retain the ability to be expressed during pathological angiogenesis and/or modulate pathological angiogenesis.
  • CD276 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to CD276.
  • CD276 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No.
  • DQ832276 NM — 001024736, NP — 598744, NP — 079516 and AAK15438, or NM — 025240 and retains CD276 activity (such as the capability to be expressed during pathological angiogenesis and/or modulate pathological angiogenesis).
  • Chemotherapy In cancer treatment, chemotherapy refers to the administration of one or a combination of compounds to kill or slow the reproduction of rapidly multiplying cells.
  • Chemotherapuetic agents include but are not limited to: 5-fluorouracil (5-FU), azathioprine, cyclophosphamide, antimetabolites (such as Fludarabine), antineoplastics (such as Etoposide, Doxorubicin, methotrexate, and Vincristine), carboplatin, cis-platinum and the taxanes, such as taxol and taxotere.
  • Such agents can be co-administered with the disclosed endothelial marker molecules to a subject.
  • chemotherapeutic agents can also be administered prior to or subsequent to administration of the disclosed modified endothelial marker molecules to a subject or can be conjugated to the disclosed endothelial markers (e.g., Tables 8 and 9).
  • chemotherapeutic agents are co-administered with radiation therapy, along with the disclosed endothelial molecules for treatment of a tumor.
  • Chimeric antibody An antibody which includes sequences derived from two different antibodies, which typically are of different species. Most typically, chimeric antibodies include human and murine antibody domains, generally human constant regions and murine variable regions, murine CDRs and/or murine SDRs. For example, the variable segments of the genes from a mouse monoclonal antibody can be joined to human constant segments, such as kappa and gamma 1 or gamma 3.
  • a chimeric antibody is a hybrid protein composed of the variable or antigen-binding domain from a mouse antibody and the constant or effector domain from a human antibody (such as an antibody that recognizes one of the disclosed pathological angiogenesis endothelial markers listed in Table 9), although other mammalian species can be used, or the variable region can be produced by molecular techniques.
  • Methods of making chimeric antibodies are well known in the art, for example, see U.S. Pat. No. 5,807,715.
  • a therapy decreases a tumor (such as the size of a tumor, the number of tumors, the metastasis of a tumor, or combinations thereof), or one or more symptoms associated with a tumor, for example as compared to the response in the absence of the therapy (such as a therapy administered to affect tumor size by inhibiting pathological angiogenesis via administration of a binding agent capable of binding to one or more of the pathological angiogenesis markers listed in Table 9).
  • a therapy decreases the size of a tumor, the number of tumors, the metastasis of a tumor, or combinations thereof, subsequent to the therapy, such as a decrease of at least 10%, at least 20%, at least 50%, or even at least 90%.
  • Such decreases can be measured using the methods disclosed herein as well as those known in the art.
  • Endothelial cell Cells that line the interior surface of blood vessels, forming an interface between circulating blood in the lumen and the rest of the vessel wall. For example, endothelial cells line the entire circulatory system. Further, both blood and lymphatic capillaries are composed of a single layer of endothelial cells.
  • AK144596 is a protein that is expressed in liver endothelial cells.
  • the term expression product with Accession No. AK144596 includes any expression product with Accession No. AK144596 gene, cDNA, mRNA, or protein from any organism and that is an expression product with Accession No. AK144596 capable of delivering a therapeutic agent specifically to liver endothelial cells.
  • Nucleic acid and protein sequences for expression product with Accession No. AK144596 are publicly available.
  • GenBank Accession No: AK144596 discloses an expression product with Accession No. AK144596 nucleic acid sequence.
  • an expression product with Accession No. AK144596 includes a full-length wild-type (or native) sequence, as well as an expression product with Accession No. AK144596 allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to liver endothelial cells.
  • an expression product with Accession No. AK144596 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to an expression product with Accession No. AK144596.
  • an expression product with Accession No. AK144596 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No. AK144596 and retains expression product with Accession No. AK144596 activity (e.g., the capability to serve as a liver endothelial cell marker).
  • FOXQ1 Forkhead box Q1: A member of the evolutionarily conserved winged helix (WH)/forkhead transcription factor gene family. The protein regulates the expression of other genes.
  • FOXQ1 protein is expressed in brain endothelial cells.
  • the term FOXQ1 includes any FOXQ1 gene, cDNA, mRNA, or protein from any organism and that is FOXQ1 capable of delivering a therapeutic agent specifically to brain endothelial cells.
  • Nucleic acid and protein sequences for FOXQ1 are publicly available.
  • GenBank Accession Nos.: NM — 008239, AK147202, AF010405, AF225950, and NM — 033260 disclose FOXQ1 nucleic acid sequences
  • GenBank Accession Nos.: NP — 032265, AAH53850, and NP — 150285 disclose FOXQ1 protein sequences.
  • FOXQ1 includes a full-length wild-type (or native) sequence, as well as FOXQ1 allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to brain endothelial cells.
  • FOXQ1 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to FOXQ1.
  • FOXQ1 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession Nos. NM — 008239, AK147202, AF010405, AF225950, or NM — 033260 and retains FOXQ1 (e.g., the capability to serve as a brain endothelial cell marker).
  • Humanized antibodies An immunoglobulin including a human framework region and one or more CDRs from a non-human (such as a mouse, rat, or synthetic) immunoglobulin.
  • a humanized antibody binds to the same antigen as the donor antibody that provides the CDRs.
  • a humanized antibody specifically binds to one of the proteins listed in Tables 8 and 9.
  • the non-human immunoglobulin providing the CDRs is termed a “donor” and the human immunoglobulin providing the framework is termed an “acceptor.”
  • all the CDRs are from the donor immunoglobulin in a humanized immunoglobulin. Constant regions need not be present, but if they are, they are substantially identical to human immunoglobulin constant regions, for instance, at least about 85-90%, such as about 95% or more identical.
  • the donor CDRs of a humanized antibody can have a limited number of substitutions using amino acids from the acceptor CDR.
  • all parts of a humanized immunoglobulin, except possibly the CDRs are substantially identical to corresponding parts of natural human immunoglobulin sequences.
  • the acceptor framework of a humanized immunoglobulin or antibody can have a limited number of substitutions by amino acids taken from the donor framework.
  • Humanized or other monoclonal antibodies can have additional amino acid substitutions which have substantially no effect on antigen binding or other immunoglobulin functions. Exemplary conservative substitutions are described above (see also U.S. Pat. No. 5,585,089).
  • Humanized immunoglobulins can be constructed by means of genetic engineering, for example, see U.S. Pat. Nos. 5,225,539 and 5,585,089, herein incorporated by reference.
  • Hybridization To form base pairs between complementary regions of two strands of DNA, RNA, or between DNA and RNA, thereby forming a duplex molecule.
  • Hybridization conditions resulting in particular degrees of stringency will vary depending upon the nature of the hybridization method and the composition and length of the hybridizing nucleic acid sequences. Generally, the temperature of hybridization and the ionic strength (such as the Na+ concentration) of the hybridization buffer will determine the stringency of hybridization. Calculations regarding hybridization conditions for attaining particular degrees of stringency are discussed in Sambrook et al., (1989) Molecular Cloning, second edition, Cold Spring Harbor Laboratory, Plainview, N.Y. (chapters 9 and 11). The following is an exemplary set of hybridization conditions and is not limiting:
  • Immunoassay A biochemical test that measures the level of a substance in a biological sample (such as serum or urine), using the reaction of an antibody or antibodies to its antigen.
  • the assay takes advantage of the specific binding of an antibody to its antigen.
  • the antibodies selected ideally have a high affinity for the antigen (if there is antigen available, a very high proportion of it will bind to the antibody). Both the presence of antigen or antibodies can be measured. For instance, when detecting pathological angiogenesis the presence of a pathological angiogenesis marker can be measured.
  • Detecting the quantity of antibody or antigen can be achieved by a variety of methods.
  • One of the most common is to label the antigen or antibody.
  • the label can include an enzyme (e.g., luciferase or ⁇ -gal), radioisotopes (such as 125 I) or a fluorophore.
  • Other techniques include Western Blot.
  • Kelch repeat and BTB (POZ) domain In one example, the Kelch repeat and BTB (POZ) domain is expressed in brain endothelial cells.
  • the term Kelch repeat and BTB (POZ) domain includes any Kelch repeat and BTB (POZ) domain gene, cDNA, mRNA, or protein from any organism and that is Kelch repeat and BTB (POZ) domain capable of delivering a therapeutic agent specifically to brain endothelial cells.
  • Kelch repeat and BTB (POZ) domain Nucleic acid and protein sequences for Kelch repeat and BTB (POZ) domain are publicly available.
  • GenBank Accession Nos.: XM — 486083, XM — 979486, XM — 921147, NM — 014867, and AB018254 disclose Kelch repeat and BTB (POZ) domain nucleic acid sequences
  • GenBank Accession Nos.: XP — 926240, XP — 486083, and NP — 055682 disclose ankylosis protein sequences.
  • Kelch repeat and BTB (POZ) domain includes a full-length wild-type (or native) sequence, as well as Kelch repeat and BTB (POZ) domain allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to brain endothelial cells.
  • Kelch repeat and BTB (POZ) domain has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to Kelch repeat and BTB (POZ) domain.
  • Kelch repeat and BTB (POZ) domain has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession Nos.
  • Kelch repeat and BTB (POZ) domain activity e.g., the capability to deliver therapeutic agents to brain endothelial cells.
  • Label A detectable compound.
  • a label is conjugated directly or indirectly to another molecule, such as an antibody or a protein, to facilitate detection of that molecule.
  • the label can be capable of detection by ELISA, spectrophotometry, flow cytometry, or microscopy.
  • Specific, non-limiting examples of labels include fluorophores, chemiluminescent agents, enzymatic linkages, and radioactive isotopes. Methods for labeling and guidance in the choice of labels appropriate for various purposes are discussed for example in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, N.Y., 1989) and Ausubel et al.
  • a label is conjugated to a binding agent that specifically binds to one or more of the pathological angiogenesis endothelial markers disclosed in Table 9 to allow for the detection/screening for pathological angiogenesis and/or the presence of a tumor in a subject.
  • Malignant Cells that have the properties of anaplasia invasion and metastasis.
  • Mammal This term includes both human and non-human mammals. Examples of mammals include, but are not limited to: humans, pigs, cows, goats, cats, dogs, rabbits and mice.
  • MBL-associated serine protease-3 MASP-3 transcripts encode serine proteases that display distinct substrate specificity and associate with Mannan-binding lectin complexes.
  • MBL-associated serine protease-3 is preferentially expressed in liver endothelial cells.
  • MBL-associated serine protease-3 includes any MBL-associated serine protease-3 gene, cDNA, mRNA, or protein from any organism and that is a MBL-associated serine protease-3 capable of delivering a therapeutic agent specifically to liver endothelial cells.
  • Exemplary nucleic acid and protein sequences for MBL-associated serine protease-3 are publicly available.
  • GenBank Accession Nos.: AB049755, AK031598, NM — 139125, NM — 001879, and NM — 001031849 disclose MBL-associated serine protease-3 nucleic acid sequences
  • GenBank Accession Nos.: NP 624302, NP — 001870, and NP — 001027019 disclose MBL-associated serine protease-3 protein sequences.
  • a MBL-associated serine protease-3 sequence includes a full-length wild-type (or native) sequence, as well as MBL-associated serine protease-3 allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to liver endothelial cells.
  • MBL-associated serine protease-3 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a MBL-associated serine protease-3.
  • a MBL-associated serine protease-3 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No.
  • AB049755, AB049755, AK031598, NM — 139125, NM — 001879, or NM — 001031849 and retains MBL-associated serine protease-3 activity (e.g., the capability to deliver therapeutic agents to liver endothelial cells).
  • MiRP2 The MiRP2 gene encodes a small integral membrane subunit that assembles with HERG, a pore-forming protein, to form a potassium voltage-gated channel. MiRP2 alters the function of the channel. Channels formed with mutant MiRP1 subunits display slower activation, faster deactivation, and increased drug sensitivity.
  • MiRP2 is expressed during pathological angiogenesis.
  • the term MiRP2 includes any MiRP2 gene, cDNA, mRNA, or protein from any organism and that is MiRP2 and is expressed during pathological angiogenesis.
  • Exemplary nucleic acid and protein sequences for MiRP2 are publicly available.
  • GenBank Accession Nos.: DQ832280, NM — 020574, AK008744, and NM — 005472 disclose MiRP2 nucleic acid sequences
  • GenBank Accession Nos.: NP — 065599, BAB25871, and NP — 005463 disclose MiRP2 protein sequences.
  • MiRP2 includes a full-length wild-type (or native) sequence, as well as MiRP2 allelic variants, fragments, homologs or fusion sequences that retain the ability to be expressed during pathological angiogenesis and/or modulate pathological angiogenesis.
  • MiRP2 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to MiRP2.
  • MiRP2 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession Nos.
  • Neoplasm Abnormal growth of cells.
  • Normal Cell Non-tumor cell, non-malignant, uninfected cell.
  • Oncoprotein induced transcript 3 (Oit3): Encodes a secreted ZP domain-containing protein.
  • oncoprotein induced transcript 3 is expressed in liver endothelial cells.
  • the term oncoprotein induced transcript 3 includes any oncoprotein induced transcript 3 gene, cDNA, mRNA, or protein from any organism and that is a oncoprotein induced transcript 3 capable of delivering a therapeutic agent specifically to liver endothelial cells.
  • Oncoprotein induced transcript 3 is also referred to in the literature as LZP.
  • Oncoprotein induced transcript 3 nucleic acid and protein sequences are publicly available.
  • GenBank Accession Nos.: NM — 010959, AF356506, AY180915, NM — 152635, and AY013707 disclose oncoprotein induced transcript 3 nucleic acid sequences
  • GenBank Accession Nos.: AAO22058, NP — 035089, NP — 689848, and AAG40096 disclose oncoprotein induced transcript 3 protein sequences.
  • a oncoprotein induced transcript 3 sequence includes a full-length wild-type (or native) sequence, as well as oncoprotein induced transcript 3 allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to liver endothelial cells.
  • oncoprotein induced transcript 3 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a native oncoprotein induced transcript 3.
  • oncoprotein induced transcript 3 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession Nos. NM — 010959, AF356506, AY180915, NM — 152635, or AY013707 and retains oncoprotein induced transcript 3 activity.
  • compositions and formulations suitable for pharmaceutical delivery of one or more therapeutic agents such as one or more compositions that include a binding agent that specifically binds to at least one of the disclosed pathological angiogenesis marker proteins.
  • parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutical compositions to be administered can contain minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate, sodium lactate, potassium chloride, calcium chloride, and triethanolamine oleate.
  • Plexin C1 A large transmembrane receptor.
  • plexin-C1 has been shown to bind the GPI-anchored semaphorin Sema7A and the soluble viral semaphorins SemaVA (A39R) and SemaVB (AHV).
  • Plexin C1 engagement by SemaVA inhibits integrin-mediated dendritic cell adhesion and chemotaxis in vitro, suggesting a role for plexin C1 in dendritic cell migration.
  • plexin C1 is expressed in liver endothelial cells.
  • the term plexin C1 includes any plexin C1 gene, cDNA, mRNA, or protein from any organism and that is a plexin C1 capable of delivering a therapeutic agent specifically to liver endothelial cells.
  • Exemplary nucleic acid and protein sequences for plexin C1 are publicly available.
  • GenBank Accession Nos.: NM — 018797, XM — 622776, AB208934, and NM — 005761 disclose plexin C1 nucleic acid sequences
  • GenBank Accession Nos.: NP — 061267, XP — 622776, BAD92171, and NP — 005752 disclose plexin C1 protein sequences.
  • a plexin C1 sequence includes a full-length wild-type (or native) sequence, as well as plexin C1 allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to liver endothelial cells.
  • plexin C1 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a plexin C1.
  • a plexin C1 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No. NM — 018797, XM — 622776, AB208934, or NM — 005761 and retains plexin C1 activity (e.g., the capability to deliver therapeutic agents to liver endothelial cells).
  • PCR Polymerase Chain Reaction
  • a nucleic acid molecule for example, a nucleic acid molecule in a sample or specimen.
  • a biological sample collected from a subject is contacted with a pair of oligonucleotide primers, under conditions that allow for the hybridization of the primers to nucleic acid template in the sample.
  • the primers are extended under suitable conditions, dissociated from the template, and then re-annealed, extended, and dissociated to amplify the number of copies of the nucleic acid.
  • the product of a PCR can be characterized by electrophoresis, restriction endonuclease cleavage patterns, oligonucleotide hybridization or ligation, and/or nucleic acid sequencing, using standard techniques.
  • Preimplantation protein 4 The Prei4 gene is expressed during mouse preimplantation embryogenesis. It is a putative glycerophosphodiester phosphodiesterase. In one example, Prei4 is expressed in brain endothelial cells.
  • the term Prei4 includes any Prei4 gene, cDNA, mRNA, or protein from any organism and that is Prei4 capable of delivering a therapeutic agent specifically to brain endothelial cells.
  • Nucleic acid and protein sequences for Prei4 are publicly available.
  • GenBank Accession Nos.: NM — 001042671, NM — 028802, BC006887, and NM — 019593 disclose Prei4 nucleic acid sequences
  • GenBank Accession Nos.: NP — 001036136, NP — 062539, and Q9NPB8 disclose Prei4 protein sequences.
  • Prei4 includes a full-length wild-type (or native) sequence, as well as Prei4 allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to brain endothelial cells.
  • Prei4 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to Prei4.
  • Prei4 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession Nos. NM — 001042671, NM — 028802, BC006887, or NM — 019593 and retains Prei4 activity (e.g., the capability to deliver therapeutic agents to brain endothelial cells).
  • Nucleic acid probes and primers can be readily prepared based on the nucleic acid molecules provided in this disclosure.
  • a probe includes an isolated nucleic acid attached to a detectable label or reporter molecule.
  • Exemplary labels include radioactive isotopes, enzyme substrates, co-factors, ligands, chemiluminescent or fluorescent agents, haptens, and enzymes.
  • Primers are short nucleic acid molecules such as DNA oligonucleotides, 10 nucleotides or more in length. Longer DNA oligonucleotides can be about 15, 17, 20, or 23 nucleotides or more in length. Primers can be annealed to a complementary target DNA strand by nucleic acid hybridization to form a hybrid between the primer and the target DNA strand, and then the primer extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other nucleic-acid amplification methods known in the art.
  • PCR polymerase chain reaction
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, ( ⁇ 1991, Whitehead Institute for Biomedical Research, Cambridge, Mass.).
  • Progestin and adipoQ receptor family member V (Paqr5): An integral membrane protein that binds progesterone. Paqr5 is a putative G-protein coupled receptor involved in signal transduction in response to steroids such as progesterone.
  • Paqr5 is expressed in brain endothelial cells.
  • Progestin and adipoQ receptor family member V includes any Progestin and adipoQ receptor family member V gene, cDNA, mRNA, or protein from any organism and that is Progestin and adipoQ receptor family member V capable of delivering a therapeutic agent specifically to brain endothelial cells.
  • Exemplary nucleic acid and protein sequences for Progestin and adipoQ receptor family member V are publicly available.
  • GenBank Accession Nos: NM — 028748, AK035475, AY424283, and NM — 017705 disclose Progestin and adipoQ receptor family member V nucleic acid sequences
  • GenBank Accession Nos.: NP — 083024, BAC29072, AAR08371, and NP — 060175 disclose Progestin and adipoQ protein sequences.
  • Progestin and adipoQ receptor family member V includes a full-length wild-type (or native) sequence, as well as Progestin and adipoQ receptor family member V allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to brain endothelial cells.
  • Progestin and adipoQ receptor family member V has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to Progestin and adipoQ receptor family member V.
  • Progestin and adipoQ receptor family member V has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession Nos.
  • NM — 028748, NM — 028748, AK035475, AY424283, or NM — 017705 retains Progestin and adipoQ receptor family member V activity (e.g., the capability to deliver therapeutic agents to brain endothelial cells).
  • PTPRN (IA-2) is a major autoantigen in type 1 diabetes. Autoantibodies against PTPRN appear years before the development of clinical disease. PTPRN is an enzymatically inactive member of the transmembrane protein tyrosine phosphatase family and is an integral component of secretory granules in neuroendocrine cells. PTPRN is an important regulator of dense core vesicle number and glucose-induced and basal insulin secretion.
  • Ptprn is expressed during pathological angiogenesis.
  • the term Ptprn includes any Ptprn gene, cDNA, mRNA, or protein from any organism and that is Ptprn and is preferentially expressed during pathological angiogenesis. Ptprn is also known in the literature as IA-2.
  • Exemplary nucleic acid and protein sequences for Ptprn are publicly available.
  • GenBank Accession Nos.: DQ832283, NM — 008985, AK041296, NM — 002846, and L18983 disclose Ptprn nucleic acid sequences
  • GenBank Accession Nos.: NP — 033011, NP — 002837, and AAA90974 disclose Ptprn (IA-2) protein sequences.
  • Ptprn includes a full-length wild-type (or native) sequence, as well as Ptprn allelic variants, fragments, homologs or fusion sequences that retain the ability to be preferentially expressed during pathological angiogenesis and/or modulate pathological angiogenesis.
  • Ptprn has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to Ptprn.
  • Ptprn has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession Nos.
  • DQ832283, NM — 008985, AK041296, NM — 002846, or L18983 retains Ptprn activity (e.g., the capability to be expressed during pathological angiogenesis and/or modulate pathological angiogenesis).
  • Putative G-protein coupled receptor NM — 033616 or Component of Sp100-rs (Csprs): A putative G-protein coupled receptor.
  • putative G-protein coupled receptor NM — 033616 is expressed in liver endothelial cells.
  • the term putative G-protein coupled receptor NM — 033616 includes any putative G-protein coupled receptor NM — 033616 gene, cDNA, mRNA, or protein from any organism and that is a putative G-protein coupled receptor NM — 033616 capable of delivering a therapeutic agent specifically to liver endothelial cells.
  • Exemplary nucleic acid and protein sequences for putative G-protein coupled receptor NM — 033616 are publicly available.
  • GenBank Accession Nos.: NM — 033616, AK037063, and XM — 979370 disclose putative G-protein coupled receptor NM — 033616 nucleic acid sequences
  • GenBank Accession Nos.: NP — 291094 and XP — 984464 disclose putative G-protein coupled receptor NM — 033616 protein sequences.
  • a putative G-protein coupled receptor NM — 033616 sequence includes a full-length wild-type (or native) sequence, as well as putative G-protein coupled receptor NM — 033616 allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to liver endothelial cells.
  • putative G-protein coupled receptor NM — 033616 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a putative G-protein coupled receptor NM — 033616.
  • a putative G-protein coupled receptor NM — 033616 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession Nos. NM — 033616, AK037063, or XM — 979370 and retains putative G-protein coupled receptor NM — 033616 activity (e.g., the capability to target agents to liver endothelial cells).
  • Putative transmembrane protein Accession No. NM — 023438 A putative transmembrane protein.
  • putative transmembrane protein Accession No. NM — 023438 is expressed in liver endothelial cells.
  • the term putative transmembrane protein Accession No. NM — 023438 includes any putative transmembrane protein Accession No. NM — 023438 gene, cDNA, mRNA, or protein from any organism and that is a putative transmembrane protein Accession No. NM — 023438 capable of delivering a therapeutic agent specifically to liver endothelial cells.
  • Exemplary nucleic acid and protein sequences for putative transmembrane protein Accession No. NM — 023438 are publicly available.
  • GenBank Accession Nos.: NM — 023438, NM — 207313, and BN000149 disclose putative transmembrane protein Accession No. NM — 023438 nucleic acid sequences
  • GenBank Accession Nos.: NP — 075927, NP — 997196, and CAD80169 disclose putative transmembrane protein Accession No. NM — 023438 protein sequences.
  • a putative transmembrane protein Accession No. NM — 023438 sequence includes a full-length wild-type (or native) sequence, as well as putative transmembrane protein Accession No. NM — 023438 allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to liver endothelial cells.
  • putative transmembrane protein Accession No. NM — 023438 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a putative transmembrane protein Accession No. NM — 023438.
  • NM — 023438 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession Nos. NM — 023438, NM — 207313, and BN000149 and retains putative transmembrane protein Accession No. NM — 023438 activity (e.g., the capability to target agents to liver endothelial cells).
  • Putative transmembrane protein Accession No. NM — 029001 A putative transmembrane protein.
  • putative transmembrane protein Accession No. NM — 029001 is expressed in brain endothelial cells.
  • the term putative transmembrane protein Accession No. NM — 029001 includes any putative transmembrane protein Accession No. NM — 029001 gene, cDNA, mRNA, or protein from any organism and that is a putative transmembrane protein Accession No. NM — 029001 capable of delivering a therapeutic agent specifically to brain endothelial cells.
  • Exemplary nucleic acid and protein sequences for putative transmembrane protein Accession No. NM — 029001 are publicly available.
  • GenBank Accession Nos.: NM — 029001, NM — 024930, and AB181393 disclose putative transmembrane protein Accession No. NM — 029001 nucleic acid sequences
  • GenBank Accession Nos.: NP — 083277, NP — 079206, and BAD93238 disclose putative transmembrane protein Accession No. NM — 029001 protein sequences.
  • a putative transmembrane protein Accession No. NM — 029001 sequence includes a full-length wild-type (or native) sequence, as well as putative transmembrane protein Accession No. NM — 029001 allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to brain endothelial cells.
  • putative transmembrane protein Accession No. NM — 029001 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a putative transmembrane protein Accession No. NM — 029001.
  • NM — 029001 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No. NM — 029001, NM — 024930, or AB181393 and retains putative transmembrane protein Accession No. NM — 029001 activity (e.g., the capability to target agents to brain endothelial cells).
  • NM — 144830 encodes a putative transmembrane protein.
  • putative transmembrane protein Accession No. NM — 144830 is expressed in liver endothelial cells.
  • the term putative transmembrane protein Accession No. NM — 144830 includes any putative transmembrane protein Accession No. NM — 144830 gene, cDNA, mRNA, or protein from any organism and that is putative transmembrane protein Accession No. NM — 144830 capable of delivering a therapeutic agent specifically to liver endothelial cells.
  • Exemplary nucleic acid and protein sequences for putative transmembrane protein Accession No. NM — 144830 are publicly available.
  • GenBank Accession Nos.: NM — 144830, AK154217, NM — 145041, and XM — 001133074 disclose putative transmembrane protein Accession No. NM — 144830 nucleic acid sequence and GenBank Accession Nos.: NP — 659079, BAE32441, NP — 659478, and XP — 001133074 disclose putative transmembrane protein Accession No. NM — 144830 protein sequences.
  • putative transmembrane protein Accession No. NM — 144830 includes a full-length wild-type (or native) sequence, as well as putative transmembrane protein Accession No. NM — 144830 allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to liver endothelial cells.
  • putative transmembrane protein Accession No. NM — 144830 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to putative transmembrane protein Accession No. NM — 144830.
  • NM — 144830 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No. NM — 144830, AK154217, NM — 145041, or XM — 001133074 and retains putative transmembrane protein Accession No. NM — 144830 activity (e.g., the capability to target agents to liver endothelial cells).
  • Putative secreted protein Accession No. XM — 620023 encodes a putative secreted protein.
  • putative secreted protein Accession No. XM — 620023 is expressed in brain endothelial cells.
  • the term putative secreted protein Accession No. NM — 620023 includes any putative secreted protein Accession No. XM — 620023 gene, cDNA, mRNA, or protein from any organism and that is a putative secreted protein Accession No. NM — 620023 capable of delivering a therapeutic agent specifically to brain endothelial cells.
  • Exemplary nucleic acid and protein sequences for putative secreted protein Accession No. XM — 620023 are publicly available.
  • GenBank Accession Nos.: XM — 620023, AK128180, and BX648118 disclose secreted protein Accession No. XM — 620023 nucleic acid sequences
  • GenBank Accession Nos.: XP — 620023, BAC87313, and CAH56187 disclose putative secreted protein Accession No. XM — 620023 protein sequences.
  • a putative secreted protein Accession No. XM — 620023 sequence includes a full-length wild-type (or native) sequence, as well as putative secreted protein Accession No. XM — 620023 allelic variants, fragments, homologs or fusion sequences that retain the ability to deliver therapeutic agents specifically to brain endothelial cells.
  • putative secreted protein Accession No. XM — 620023 has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to a putative secreted protein Accession No. XM — 620023.
  • XM — 620023 has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession Nos. XM — 620023, BAC87313, and CAH56187 and retains putative secreted protein Accession No. NM — 620023 activity (e.g., the capability to target agents to brain endothelial cells).
  • Sample Biological specimens containing genomic DNA, cDNA, RNA, or protein obtained from the cells of a subject, such as those present in peripheral blood, urine, saliva, semen, tissue biopsy, surgical specimen, fine needle aspriates, amniocentesis samples and autopsy material.
  • a sample includes lung, colon, breast or liver cancer cells obtained from a subject.
  • Sequence identity The identity/similarity between two or more nucleic acid sequences, or two or more amino acid sequences, is expressed in terms of the identity or similarity between the sequences. Sequence identity can be measured in terms of percentage identity; the higher the percentage, the more identical the sequences are. Sequence similarity can be measured in terms of percentage similarity (which takes into account conservative amino acid substitutions); the higher the percentage, the more similar the sequences are. Homologs or orthologs of nucleic acid or amino acid sequences possess a relatively high degree of sequence identity/similarity when aligned using standard methods. This homology is more significant when the orthologous proteins or cDNAs are derived from species that are more closely related (such as human and mouse sequences), compared to species more distantly related (such as human and C. elegans sequences).
  • NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215:403-10, 1990) is available from several sources, including the National Center for Biological Information (NCBI, National Library of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) and on the Internet, for use in connection with the sequence analysis programs blastp, blastn, blastx, tblastn and tblastx. Additional information can be found at the NCBI web site.
  • NCBI National Center for Biological Information
  • BLASTN is used to compare nucleic acid sequences
  • BLASTP is used to compare amino acid sequences.
  • the options can be set as follows: -i is set to a file containing the first nucleic acid sequence to be compared (such as C: ⁇ seq1.txt); -j is set to a file containing the second nucleic acid sequence to be compared (such as C: ⁇ seq2.txt); -p is set to blastn; -o is set to any desired file name (such as C: ⁇ output.txt); -q is set to ⁇ 1; -r is set to 2; and all other options are left at their default setting.
  • the following command can be used to generate an output file containing a comparison between two sequences: C: ⁇ B12seq-i c: ⁇ seq1.txt-j c: ⁇ seq2.txt-p blastn-o c: ⁇ output.txt-q ⁇ 1-r 2.
  • the options of B12seq can be set as follows: -i is set to a file containing the first amino acid sequence to be compared (such as C: ⁇ seq1.txt); -j is set to a file containing the second amino acid sequence to be compared (such as C: ⁇ seq2.txt); -p is set to blastp; -o is set to any desired file name (such as C: ⁇ output.txt); and all other options are left at their default setting.
  • the following command can be used to generate an output file containing a comparison between two amino acid sequences: C: ⁇ B12seq-i c: ⁇ seq1.txt-j c: ⁇ seq2.txt-p blastp-o c: ⁇ output.txt. If the two compared sequences share homology, then the designated output file will present those regions of homology as aligned sequences. If the two compared sequences do not share homology, then the designated output file will not present aligned sequences.
  • the number of matches is determined by counting the number of positions where an identical nucleotide or amino acid residue is presented in both sequences.
  • 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while 75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2.
  • the length value will always be an integer.
  • the Blast 2 sequences function is employed using the default BLOSUM62 matrix set to default parameters, (gap existence cost of 11, and a per residue gap cost of 1). Homologs are typically characterized by possession of at least 70% sequence identity counted over the full-length alignment with an amino acid sequence using the NCBI Basic Blast 2.0, gapped blastp with databases such as the nr or swissprot database. Queries searched with the blastn program are filtered with DUST (Hancock and Armstrong Comput. Appl. Biosci. 10: 67-70, 1994). Other programs use SEG. In addition, a manual alignment can be performed. Proteins with even greater similarity will show increasing percentage identities when assessed by this method, such as at least 75%, 80%, 85%, 90%, 95%, or 99% sequence identity to any protein listed in Tables 8 and 9.
  • the alignment should be performed using the Blast 2 sequences function, employing the PAM30 matrix set to default parameters (open gap 9, extension gap 1 penalties). Proteins with even greater similarity to the reference sequence will show increasing percentage identities when assessed by this method, such as at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. When less than the entire sequence is being compared for sequence identity, homologs will typically possess at least 75% sequence identity over short windows of 10-20 amino acids, and can possess sequence identities of at least 85%, 90%, 95% or 98% depending on their identity to the reference sequence. Methods for determining sequence identity over such short windows are described at the NCBI web site.
  • nucleic acid molecules hybridize to each other under stringent conditions. Stringent conditions are sequence-dependent and are different under different environmental parameters. Nucleic acid sequences that do not show a high degree of identity may nevertheless encode identical or similar (conserved) amino acid sequences, due to the degeneracy of the genetic code. Changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid molecules that all encode substantially the same protein. Such homologous nucleic acid sequences can, for example, possess at least 60%, 70%, 80%, 90%, 95%, 98%, or 99% sequence identity determined by this method.
  • homologous nucleic acid sequences can possess at least 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity to the nucleic acid sequences that encode endothelial cell proteins listed in Tables 8 and 9.
  • homologous proteins can possess at least 60%, 70%, 80%, 90%, 95%, 98% or 99% sequence identity to the endothelial cell proteins listed in Tables 8 and 9.
  • An alternative (and not necessarily cumulative) indication that two nucleic acid sequences are substantially identical is that the polypeptide which the first nucleic acid encodes is immunologically cross reactive with the polypeptide encoded by the second nucleic acid.
  • Serial analysis of gene expression A technique that can be used to characterize gene expression, or more precisely gene transcription.
  • SAGE Serial analysis of gene expression
  • SAGE Tags short defined sequence tags
  • Each Tag is a short nucleotide sequence (such as 9-33 base pairs in length) from a defined position in the transcript.
  • 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 sequence, evaluating large numbers of sequences at one time, and to provide a basis for the identification of previously unknown genes.
  • Specific Binding Agent An agent that binds substantially only to a defined target such as a protein, enzyme, polysaccharide, oligonucleotide, DNA, RNA, recombinant vector or a small molecule.
  • a protein-specific binding agent binds substantially only the defined protein, or to a specific region within the protein.
  • a “specific binding agent” includes antibodies and other agents that bind substantially to a specified polypeptide. Exemplary antibodies include monoclonal or polyclonal antibodies that are specific for the polypeptide, as well as immunologically effective portions (“fragments”) thereof.
  • a “specific binding agent” is capable of binding to at least one of the disclosed physiological or pathological angiogenesis endothelial marker proteins.
  • the “specific binding agent” is an antibody specific for at least one of the disclosed physiological or pathological angiogenesis endothelial marker proteins.
  • the “specific binding agent” is capable of interacting with at least one of the organ-specific endothelial marker proteins.
  • a particular agent binds substantially only to a specific polypeptide may readily be made by using or adapting routine procedures.
  • One suitable in vitro assay makes use of the Western blotting procedure (described in many standard texts, including Harlow and Lane, Using Antibodies: A Laboratory Manual, CSHL, New York, 1999).
  • the specific binding agent is capable of binding to a mRNA or small molecule that results in pathological angiogenesis being inhibited.
  • Subject Living multicellular vertebrate organisms, a category which includes both human and veterinary subjects that are in need of the desired biological effect, such as treatment of a tumor. Examples include, but are not limited to: humans, apes, dogs, cats, mice, rats, rabbits, horses, pigs, and cows.
  • Therapeutically Effective Amount An amount of a composition that alone, or together with an additional therapeutic agent(s) (for example a chemotherapeutic agent), induces the desired response (e.g., treatment of a tumor).
  • an additional therapeutic agent(s) for example a chemotherapeutic agent
  • the preparations disclosed herein are administered in therapeutically effective amounts.
  • a desired response is to decrease tumor size or metastasis in a subject to whom the therapy is administered. Tumor metastasis does not need to be completely eliminated for the composition to be effective.
  • a composition can decrease metastasis by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of the tumor), as compared to metastasis in the absence of the composition.
  • a composition can decrease the number of cancer cells by a desired amount, for example by at least 20%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 98%, or even at least 100% (elimination of detectable cancer cells), as compared to the number of cancer cells in the absence of the composition.
  • a therapeutically effective amount of a specific binding agent for at least one of the disclosed pathological angiogenesis protein markers, or cancer cells lysed by a therapeutic molecule conjugated to the agent can be administered in a single dose, or in several doses, for example daily, during a course of treatment.
  • the therapeutically effective amount can depend on the subject being treated, the severity and type of the condition being treated, and the manner of administration.
  • a therapeutically effective amount of such agent can vary from about 1 ⁇ g-10 mg per 70 kg body weight if administered intravenously and about 10 ⁇ g -100 mg per 70 kg body weight if administered intratumorally.
  • Treating a disease refers to a therapeutic intervention that ameliorates a sign or symptom of a disease or pathological condition, such a sign or symptom of a tumor. Treatment can also induce remission or cure of a condition, such as a tumor. In particular examples, treatment includes preventing a disease, for example by inhibiting the full development of a disease, such as preventing development of a tumor (such as a metastasis). Prevention of a disease does not require a total absence of a tumor. For example, a decrease of at least 50% can be sufficient.
  • Tumor A neoplasm. Includes solid and hematological (or liquid) tumors.
  • solid tumors such as sarcomas and carcinomas, include, but are not limited to: fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, and other sarcomas, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, lymphoid malignancy, pancreatic cancer, breast cancer, lung cancers, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, chor
  • Unit dose A physically discrete unit containing a predetermined quantity of an active material calculated to individually or collectively produce a desired effect, such as a therapeutic effect.
  • a single unit dose or a plurality of unit doses can be used to provide the desired effect, such as treatment of a tumor, for example a metastatic tumor.
  • a unit dose includes a desired amount of an agent that decreases or inhibits pathological angiogenesis.
  • Vscp Encodes an SH2-containing protein.
  • Vscp is expressed during pathological angiogenesis.
  • the term Vscp includes any Vscp gene, cDNA, mRNA, or protein from any organism and that is Vscp and is expressed during pathological angiogenesis.
  • Vscp Exemplary nucleic acid and protein sequences for Vscp are publicly available.
  • GenBank Accession Nos.: DQ832275, XM — 357399, AK032598, XM — 375698, and XM — 939275 disclose Vscp nucleic acid sequences
  • GenBank Accession Nos.: XP — 357399, XP — 375698, and XP — 944368 disclose Vscp protein sequences.
  • Vscp includes a full-length wild-type (or native) sequence, as well as Vscp allelic variants, fragments, homologs or fusion sequences that retain the ability to be expressed during pathological angiogenesis and/or modulate pathological angiogenesis.
  • Vscp has at least 80% sequence identity, for example at least 85%, 90%, 95%, or 98% sequence identity to Vscp.
  • Vscp has a sequence that hybridizes under very high stringency conditions to a sequence set forth in GenBank Accession No.
  • Vscp activity e.g., the capability to be expressed during pathological angiogenesis and/or modulate pathological angiogenesis.
  • Western blot A method in molecular biology/biochemistry/immunogenetics to detect protein in a biological sample, such as a tissue homogenate or extract.
  • Gel electrophoresis can be employed to separate denatured proteins by mass. Following separation, the proteins are transferred out of the gel and onto a membrane (typically nitrocellulose), where they are “probed” using antibodies specific to the protein. As a result, the amount of protein in the sample can be examined and compared to other protein levels.
  • Other techniques also using antibodies allow detection of proteins in tissues (immunohistochemistry) and cells (immunocytochemistry).
  • pathological angiogenesis is associated with the increased expression of at least thirteen endothelial cell proteins (such as the pathological angiogenesis marker proteins listed in Table 9). It is also demonstrated that these proteins are not increased during physiological angiogenesis. In addition, expression levels of various endothelial cell proteins have been found to be dependent upon the organ in which the proteins are expressed. Based on these observations, methods of treating pathological angiogenesis, such as pathological angiogenesis associated with a tumor, are disclosed. Further, methods of delivering a therapeutic agent to a specific organ to treat a disease are disclosed.
  • the method includes administering a therapeutically effective amount of a composition to a subject in which the composition includes a specific binding agent that preferentially binds to one or more pathological angiogenesis marker proteins listed in Table 9 or a subset thereof, such as at least 1, at least 2, at least 3, at least 5, at least 10, or at least 12 (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 of those listed).
  • the one or more pathological angiogenesis marker proteins are Vscp, CD276, MiRP2, Ptprn (IA-2), ankylosis or combinations thereof.
  • the specific binding agent can be an antibody to one or more of the pathological angiogenesis marker proteins conjugated to a therapeutic molecule, such as a cytotoxin, chemotherapeutic reagent, radionucleotide or a combination thereof.
  • Pathological angiogenesis is a physiological process involving the growth of new blood vessels under pathological conditions.
  • pathological angiogenesis is involved in the transition of tumors from a dormant state to a malignant state. Inhibition of pathological angiogenesis does not require 100% inhibition, but can include at least a reduction (such as a reduction of at least 10% or at least 25%) if not a complete inhibition of new blood vessels associated with a specific pathological condition.
  • inhibiting pathological angiogenesis can be used to treat a tumor.
  • Treatment of a tumor by reducing new blood vessel growth can include preventing or delaying the development of the tumor in a subject (such as preventing metastasis of a tumor), and also includes reducing signs or symptoms associated with the presence of such a tumor (for example by reducing the size or volume of the tumor or a metastasis thereof).
  • Such reduced growth can in some examples decrease or slow metastasis of the tumor, or reduce the size or volume of the tumor by at least 10%, at least 20%, at least 50%, or at least 75%.
  • pathological angiogenesis can be inhibited to treat cancer such as a liver, breast, colon and lung cancer.
  • inhibition of pathological angiogenesis includes reducing the invasive activity of the tumor in the subject, for example by reducing the ability of the tumor to metastasize by reducing or inhibiting new blood vessel growth.
  • treatment using the methods disclosed herein prolongs the time of survival of the subject.
  • Specific binding agents are agents that bind with higher affinity to a molecule of interest, than to other molecules.
  • a specific binding agent can be one that binds with high affinity to one of the proteins listed in Tables 8 and 9, but does not substantially bind to another protein.
  • a specific binding agent binds to one of the proteins listed in Tables 8 and 9 with a binding affinity in the range of 0.1 to 20 nM.
  • specific binding agents include antibodies, ligands, recombinant proteins, peptide mimetics, and soluble receptor fragments.
  • a specific binding agent is an antibody, such as a monoclonal or polyclonal antibody. Methods of making antibodies that can be used clinically are known in the art. Particular antibodies and methods that can be used to produce them are described in detail below.
  • a specific binding agent is a cell surface receptor ligand.
  • Many cell surface receptors have natural ligands that often bind the receptors with high affinity.
  • the ligands that can be either soluble or cell surface bound, can be used to direct cytotoxic agents to tumors.
  • VEGF has been fused to the toxin gelonin and used in preclinical models to prevent the growth of several tumor types.
  • the ligand is cell surface receptor itself and a recombinant protein including the extracellular portion of the ligand can be used as a specific binding agent.
  • the extracellular domain can be fused to a toxin or labeled with an agent that allows detection of the tumor endothelium.
  • the cell surface ligand 4-1BBL can be used as a specific binding agent for CD137.
  • the ligand for CD276 or CD109 can be used.
  • small molecular weight inhibitors or antagonists of the receptor protein can be used to regulate pathological angiogenesis.
  • small molecular weight inhibitors or antagonists of the MiRP2 protein are used to inhibit pathological angiogenesis.
  • the function of secreted proteins that participate in angiogenesis may be altered by using antibodies that recognize the secreted proteins, or soluble recombinant receptor fragments.
  • An example of this is bevacizumab (Avastin), a monoclonal antibody that recognizes VEGF which has been approved by the FDA for the treatment of human metastatic colorectal cancer and non-small cell lung cancer.
  • the VEGF-trap is a receptor fusion protein that also binds to and blocks VEGF and is also currently in clinical development.
  • Specific binding agents can be therapeutic, for example by reducing or inhibiting the biological activity of a protein.
  • a specific binding agent that binds with high affinity to one of the proteins listed in Tables 8 and 9, may substantially reduce the biological function of the protein (for example, the ability of the protein to promote pathological angiogenesis).
  • a specific binding agent is conjugated to a therapeutic molecule, for example an anti-tumor agent. In this way, the specific binding agent permits targeting of the therapeutic molecule to the cells of interest, such as vascular endothelium.
  • Such agents can be administered in therapeutically effective amounts to individuals in need thereof, such as a subject having a tumor.
  • Therapeutic molecules include agents that can be used to treat a disease, such as a tumor.
  • a therapeutic molecule is one that alone or together with an additional compound induces the desired therapeutic response.
  • One or more therapeutic molecules can be conjugated directly or indirectly to a specific binding agent, such as an antibody that binds to one of the proteins listed in Tables 8 and 9.
  • a specific binding agent such as an antibody that binds to CD276, or Vscp can be conjugated to an anti-tumor agent.
  • a therapeutic agent is an anti-tumor agent such as a cytotoxin, chemotherapeutic reagent, radionucleotide or a combination thereof.
  • anti-tumor agent such as a cytotoxin, chemotherapeutic reagent, radionucleotide or a combination thereof.
  • suitable chemotherapeutic agents for coupling to antibodies to achieve an anti-tumor effect include fluorouracil, doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin.
  • the anti-tumor agent 5-fluorouracil can be conjugated to a specific binding agent to treat a tumor such as breast cancer.
  • Non-limiting examples of suitable toxins include 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).
  • the anti-tumor agent diphtheria toxin can be conjugated to a specific binding agent such as CD276 to treat a tumor such as cancer.
  • a therapeutic agent is a ribosome inactivating protein from plants, bacteria and fungi.
  • suitable ribosome inactivating proteins for coupling to specific binding agents include restrictocin (a ribosome inactivating protein produced by Aspergillus restrictus ), saporin (a ribosome inactivating protein from Saponaria officinalis ), and RNase.
  • a therapeutic composition that includes a therapeutically effective amount of a binding agent specific for one or more of the disclosed organ-specific or pathological angiogenesis marker proteins (as listed in Tables 8 and 9) further includes therapeutically effective amounts of one or more other biologically active compounds.
  • biologically active compounds include, but are not limited to: anti-neoplastic agents (such as chemotherapeutics), antibiotics, alkylating agents, antioxidants, adjuvants, and so forth (such as those listed below under “additional treatments”).
  • compositions including a therapeutically effective amount of a binding agent specific for one or more of the disclosed pathological angiogenesis or organ-specific marker proteins and the other biologically active compounds can also be administered separately (instead of in a single composition).
  • ligands or antibodies that target them may be directly shuttled across the endothelial layer into the underlying tissue by a process known as transcytosis.
  • transcytosis a process known as transcytosis.
  • site-directed pharmacodelivery may be accomplished by use of cell surface endothelial markers specific for certain organs, such as liver or brain endothelium. Drugs can be conjugated to antibodies for selective delivery. A higher local concentration of drug may result in higher efficacy with fewer side effects.
  • antibodies directed to a particular endothelial marker do not naturally enter a transcytotic pathway, they can be forced to do so, for example through the generation of a bispecific antibody that targets both the endothelial marker and a protein present in caveolae, such as Caveolin-1.
  • subjects are initially screened to determine if they have increased expression levels of the disclosed pathological angiogenesis markers in their serum, whether they have a tumor that has increased expression levels of the disclosed pathological angiogenesis markers or a combination thereof.
  • the pathological angiogenesis markers provided herein can be used to screen subjects to determine if they are candidates for the disclosed therapies (see Section III.B).
  • a tumor is an abnormal growth of tissue that results from excessive cell division.
  • a particular example of a tumor is cancer.
  • the current application provides methods for the treatment (such as the prevention or reduction of metastasis) of tumors (such as cancers).
  • the tumor is treated in vivo, for example in a mammalian subject, such as a human subject.
  • Exemplary tumors that can be treated using the disclosed methods include, but are not limited to: cancers of the liver, breast, colon, and lung, including metastases of such tumors to other organs.
  • methods of delivering a therapeutic or diagnostic agent to a specific organ to treat a disease include administering a therapeutically effective amount of a composition that includes a binding agent that preferentially binds to one or more organ-specific endothelial marker proteins provided in Table 8 and a therapeutic agent to evoke a therapeutic response in the specific organ.
  • a therapeutic agent is delivered to the brain via a composition including a specific binding agent (such as an antibody) to one or more of the disclosed brain endothelial marker proteins in Table 8 and a therapeutic agent to evoke a desired therapeutic response.
  • the one or more brain endothelial marker proteins is Glucose transporter GLUT-1, Organic anion transporter 2, Pleiotrophin, ATPase class V, type 10A, Peptidoglycan recognition protein 1, Organic anion transporter 14, Forkhead box Q1, Organic anion transporter 3, SN2 (Solute carrier family 38, member 5), Inter-alpha (globulin) inhibitor H5, Solute carrier 38 member 3, Zinc finger protein of the cerebellum 2, Testican-2,3-HMG-CoA synthase 2, Progestin and adipoQ receptor family member V, APC down-regulated 1 Drapc1, GDPD phosphodiesterase family Accession No.
  • NM — 001042671 putative transmembrane protein Accession No. NM — 029001, DES2 lipid desaturase/C4-hyroxylase, Kelch repeat and BTB (POZ) domain, Lipolysis stimulated receptor, Glutathione S-transferase alpha 4, TNF receptor superfamily member 19, T-box 1 or putative secreted protein Accession No. XM — 620023).
  • the one or more brain endothelial marker proteins include GDPD phosphodiesterase family Accession No. NM — 001042671, Forkhead box Q1 (FOXQ1), putative transmembrane protein Accession No.
  • the desired therapeutic response is to reduce the growth of brain tumor cells or even kill the brain tumor cells (for example the therapeutic agent inducing cells to undergo apoptosis). Such reduced growth can in some examples decrease or slow metastasis of the brain tumor, or reduce the size or volume of the brain tumor.
  • the desired therapeutic response is to treat a disease of the brain such as depression or a stroke.
  • a therapeutic agent is delivered to the liver via a composition including a specific binding agent to the one or more liver endothelial marker proteins and a therapeutic agent to evoke a desired therapeutic response.
  • the specific binding agent is an antibody that specifically binds to one or more of the liver endothelial marker proteins disclosed in Table 8.
  • the one or more liver endothelial marker proteins is deoxyribonuclease 1-like 3, LZP oncoprotein induced transcript 3, putative transmembrane protein Accession No.
  • the one or more liver endothelial marker proteins includes oncoprotein induced transcript 3, putative transmembrane protein Accession No.
  • NM — 023438 putative G-protein coupled receptor NM — 033616, Plexin C1, MBL-associated serine protease-3, Accession No. AK144596, putative transmembrane protein Accession No. NM — 144830 or combinations thereof such as at least 1, at least 2, at least 3, or at least 5 (for example, 1, 2, 3, 4, 5, 6, or 7).
  • the desired therapeutic response is to reduce the growth of liver tumor cells or even kill the liver tumor cells (for example the therapeutic agent inducing cells to undergo apoptosis). Such reduced growth can in some examples decrease or slow metastasis of the liver tumor, or reduce the size or volume of a liver tumor.
  • the desired therapeutic response is to treat a liver disease.
  • a diagnostic agent is delivered to a specific organ such as the brain or liver via a composition including a specific binding agent such as an antibody to one or more of the disclosed organ-specific endothelial marker proteins in Table 8.
  • a diagnostic agent can be delivered to the brain via a specific binding agent that is capable of binding to one or more of the disclosed brain endothelial marker proteins to identify brain endothelial cells or to identify a tumor.
  • the vessels in tumors are often tortuous and dilated compared to normal vessels.
  • organ-specific vessel markers can be used to detect tumors in a particular organ such as the liver or brain.
  • compositions are routine, and can be determined by a skilled clinician.
  • the disclosed therapies (such as those that include a binding agent specific for one or more of the disclosed pathological angiogenesis marker proteins listed in Table 9 or the organ-specific markers listed in Table 8) can be administered via injection, intratumorally, orally, topically, transdermally, parenterally, or via inhalation or spray.
  • a composition is administered intravenously to a mammalian subject, such as a human.
  • the method includes daily administration of at least 1 ⁇ g of the composition to the subject (such as a human subject).
  • a human can be administered at least 1 ⁇ g or at least 1 mg of the composition daily, such as 10 ⁇ g to 100 ⁇ g daily, 100 ⁇ g to 1000 ⁇ g daily, for example 10 ⁇ g daily, 100 ⁇ g daily, or 1000 ⁇ g daily.
  • the subject is administered at least 1 ⁇ g (such as 1-100 ⁇ g) intravenously of the composition including a binding agent that specifically binds to one or more of the disclosed organ-specific or pathological angiogenesis marker proteins.
  • the subject is administered at least 1 mg intramuscularly (for example in an extremity) of such composition.
  • the dosage can be administered in divided doses (such as 2, 3, or 4 divided doses per day), or in a single dosage daily.
  • the subject is administered the therapeutic composition that includes a binding agent specific for one or more of the disclosed organ-specific or pathological angiogenesis marker proteins on a multiple daily dosing schedule, such as at least two consecutive days, 10 consecutive days, and so forth, for example for a period of weeks, months, or years.
  • the subject is administered the therapeutic composition that a binding agent specific for one or more of the disclosed organ-specific or pathological angiogenesis marker proteins daily for a period of at least 30 days, such as at least 2 months, at least 4 months, at least 6 months, at least 12 months, at least 24 months, or at least 36 months.
  • compositions such as those that include a binding agent specific for one or more of the disclosed pathological angiogenesis or organ-specific marker proteins, can further include one or more biologically active or inactive compounds (or both), such as anti-neoplastic agents and conventional non-toxic pharmaceutically acceptable carriers, respectively.
  • a therapeutic composition that includes a therapeutically effective amount of a binding agent specific for one or more of the disclosed pathological angiogenesis or organ-specific marker proteins further includes one or more biologically inactive compounds.
  • biologically inactive compounds include, but are not limited to: carriers, thickeners, diluents, buffers, preservatives, and carriers.
  • the pharmaceutically acceptable carriers useful for these formulations are conventional (see Remington's Pharmaceutical Sciences, by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition (1995)). In general, the nature of the carrier will depend on the particular mode of administration being employed.
  • parenteral formulations can include injectable fluids that include pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • pharmaceutically and physiologically acceptable fluids such as water, physiological saline, balanced salt solutions, aqueous dextrose, glycerol or the like as a vehicle.
  • conventional non-toxic solid carriers can include, for example, pharmaceutical grades of mannitol, lactose, starch, or magnesium stearate.
  • pharmaceutical compositions to be administered can include minor amounts of non-toxic auxiliary substances, such as wetting or emulsifying agents, preservatives, and pH buffering agents and the like, for example sodium acetate or sorbitan monolaurate.
  • the subject prior to, during, or following administration of a therapeutic amount of an agent that reduces or inhibits pathological angiogenesis due to the interaction of a binding agent with one or more of the disclosed pathological angiogenesis marker proteins, the subject can receive one or more other therapies.
  • the subject receives one or more treatments to remove or reduce the tumor prior to administration of a therapeutic amount of a composition including a binding agent specific for one or more of the disclosed pathological angiogenesis marker proteins.
  • Such therapies include, but are not limited to, surgical treatment for removal or reduction of the tumor (such as surgical resection, cryotherapy, or chemoembolization), as well as anti-tumor pharmaceutical treatments which can include radiotherapeutic agents, anti-neoplastic chemotherapeutic agents, antibiotics, alkylating agents and antioxidants, kinase inhibitors, and other agents.
  • additional therapeutic agents can that can be used include microtubule binding agents, DNA intercalators or cross-linkers, DNA synthesis inhibitors, DNA and/or RNA transcription inhibitors, antibodies, enzymes, enzyme inhibitors, and gene regulators. These agents (which are administered at a therapeutically effective amount) and treatments can be used alone or in combination. Methods and therapeutic dosages of such agents are known to those skilled in the art, and can be determined by a skilled clinician.
  • Microtubule binding agent refers to an agent that interacts with tubulin to stabilize or destabilize microtubule formation thereby inhibiting cell division.
  • microtubule binding agents that can be used in conjunction with the disclosed therapy include, without limitation, paclitaxel, docetaxel, vinblastine, vindesine, vinorelbine (navelbine), the epothilones, colchicine, dolastatin 15, nocodazole, podophyllotoxin and rhizoxin. Analogs and derivatives of such compounds also can be used and are known to those of ordinary skill in the art. For example, suitable epothilones and epothilone analogs are described in International Publication No.
  • Taxoids such as paclitaxel and docetaxel, as well as the analogs of paclitaxel taught by U.S. Pat. Nos. 6,610,860; 5,530,020; and 5,912,264 can be used.
  • Suitable DNA and/or RNA transcription regulators including, without limitation, actinomycin D, daunorubicin, doxorubicin and derivatives and analogs thereof also are suitable for use in combination with the disclosed therapies.
  • DNA intercalators and cross-linking agents that can be administered to a subject include, without limitation, cisplatin, carboplatin, oxaliplatin, mitomycins, such as mitomycin C, bleomycin, chlorambucil, cyclophosphamide and derivatives and analogs thereof.
  • DNA synthesis inhibitors suitable for use as therapeutic agents include, without limitation, methotrexate, 5-fluoro-5′-deoxyuridine, 5-fluorouracil and analogs thereof.
  • Suitable enzyme inhibitors include, without limitation, camptothecin, etoposide, formestane, trichostatin and derivatives and analogs thereof.
  • Suitable compounds that affect gene regulation include agents that result in increased or decreased expression of one or more genes, such as raloxifene, 5-azacytidine, 5-aza-2′-deoxycytidine, tamoxifen, 4-hydroxytamoxifen, mifepristone and derivatives and analogs thereof.
  • Kinase inhibitors include Gleevac, Iressa, and Tarceva that prevent phosphorylation and activation of growth factors.
  • anti-tumor agents for example anti-tumor agents, that may or may not fall under one or more of the classifications above, also are suitable for administration in combination with the disclosed therapies.
  • agents include adriamycin, apigenin, rapamycin, zebularine, cimetidine, and derivatives and analogs thereof.
  • the therapeutic composition (such as one including a binding agent specific for one or more of the disclosed pathological angiogenesis marker proteins) is injected into the subject in the presence of an adjuvant.
  • An adjuvant is an agent that when used in combination with an immunogenic agent augments or otherwise alters or modifies a resultant immune response.
  • an adjuvant increases the titer of antibodies induced in a subject by the immunogenic agent.
  • the one or more peptides are administered to the subject as an emulsion with IFA and sterile water for injection (for example an intravenous or intramuscular injection).
  • Incomplete Freund's Adjuvant (Seppic, Inc.) can be used as the Freund's Incomplete Adjuvant (IFA) (Fairfield, N.J.).
  • IFA is provided in 3 ml of a mineral oil solution based on mannide oleate (Montanide ISA-51).
  • the peptide(s) is mixed with the Montanide ISA.51 and then administered to the subject.
  • Other adjuvants can be used, for example, Freund's complete adjuvant, B30-MDP, LA-15-PH, montanide, saponin, aluminum hydroxide, alum, lipids, keyhole lympet protein, hemocyanin, a mycobacterial antigen, and combinations thereof.
  • the subject receiving the therapeutic peptide composition is also administered interleukin-2 (IL-2), for example via intravenous administration.
  • IL-2 interleukin-2
  • IL-2 Chiron Corp., Emeryville, Calif.
  • IL-2 is administered at a dose of at least 500,000 IU/kg as an intravenous bolus over a 15 minute period every eight hours beginning on the day after administration of the peptides and continuing for up to 5 days. Doses can be skipped depending on subject tolerance.
  • compositions can be co-administered with a fully human antibody to cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4).
  • anti-CTLA-4 cytotoxic T-lymphocyte antigen-4
  • subjects receive at least 1 mg/kg anti-CTLA-4 (such as 3 mg/kg every 3 weeks or 3 mg/kg as the initial dose with subsequent doses reduced to 1 mg/kg every 3 weeks).
  • At least a portion of the tumor (such as a metastatic tumor) is surgically removed (for example via cryotherapy), irradiated, chemically treated (for example via chemoembolization) or combinations thereof, prior to administration of the disclosed therapies (such as administration of a binding agent specific for one or more of the disclosed pathological angiogenesis marker proteins).
  • a subject having a metastatic tumor can have all or part of the tumor surgically excised prior to administration of the disclosed therapies (such as one including a binding agent specific for one or more of the disclosed pathological angiogenesis marker proteins).
  • the subject has a metastatic tumor and is administered radiation therapy, chemoembolization therapy, or both, prior to administration of the disclosed therapies (such as one including a binding agent specific for one or more of the disclosed pathological angiogenesis marker proteins).
  • the disclosed pathological angiogenesis marker proteins can be used as “surrogate” markers of angiogenesis that can also be used to detect the efficacy of other previously disclosed anti-angiogenic agents in clinical trials.
  • Subjects can be screened prior to initiating the disclosed therapies, for example to determine whether the subject has pathological angiogenesis, a tumor, or a combination thereof.
  • the presence of one or more of the disclosed pathological angiogenesis marker proteins listed in Table 9 can indicate that the subject has pathological angiogenesis and the tumor associated with the angiogenesis can be treated using the methods provided herein.
  • the pathological angiogenesis marker proteins are detected in a serum sample, such as pathological angiogenesis markers known to be secreted (e.g., Apelin, sCD137 and plgf), or cell surface molecules that are susceptible to enzymatic cleavage at the cell surface (e.g., CD276, MiRP2, Doppel, PTPRN, CD109 or ankylosis).
  • the proteins are detected in a tumor biopsy.
  • the presence of the respective pathological angiogenesis marker proteins can be used to diagnose, or determine the prognosis of, a tumor in a subject.
  • pathological angiogenesis can be screened for by detecting at least one expression product including one or more of: Vscp, CD276, ETSvg4 (Pea3), CD137(4-1BB), MiRP2, Ubiquitin D (Fat10), Doppel (prion-PLP), Apelin, Plgf, Ptprn (IA-2), CD109, Ankylosis, and collagen VIII, 1, in a sample obtained from the subject.
  • detection of the at least one expression product indicates pathological angiogenesis in the subject.
  • detection of the at least one expression product indicates the presence of a tumor, such as cancer.
  • the biological sample can be incubated with an antibody that specifically binds to one or more of the disclosed pathological angiogenesis marker proteins.
  • the primary antibody can include a detectable label.
  • the primary antibody can be directly labeled, or the sample can be subsequently incubated with a secondary antibody that is labeled (for example with a fluorescent label).
  • the label can then be detected, for example by microscopy, ELISA, flow cytometery, or spectrophotometry.
  • the biological sample is analyzed by Western blotting for the presence of at least one of the disclosed pathological angiogenesis marker proteins (see Table 9).
  • the level of expression of at least one of the disclosed angiogenesis marker proteins can be compared to the level of expression of such proteins in a control (e.g., non-cancer sample) or reference value.
  • the antibody that specifically binds an endothelial marker (such as those listed in Table 9) is directly labeled with a detectable label.
  • each antibody that specifically binds an endothelial marker (the first antibody) is unlabeled and a second antibody or other molecule that can bind the human antibody that specifically binds the respective endothelial marker is labeled.
  • a second antibody is chosen that is able to specifically bind the specific species and class of the first antibody.
  • the first antibody is a human IgG
  • the secondary antibody can be an anti-human-IgG.
  • Other molecules that can bind to antibodies include, without limitation, Protein A and Protein G, both of which are available commercially.
  • Suitable labels for the antibody or secondary antibody include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, magnetic agents and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase.
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin.
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin.
  • a non-limiting exemplary luminescent material is luminol; a non-limiting exemplary magnetic agent is gadolinium, and non-limiting exemplary radioactive labels include 125 I, 131 I, 35 S or 3 H.
  • endothelial markers can be assayed in a biological sample by a competition immunoassay utilizing endothelial marker standards labeled with a detectable substance and an unlabeled antibody that specifically binds the desired endothelial marker.
  • the biological sample such as serum
  • the labeled endothelial marker standards and the antibody that specifically binds the desired endothelial marker are combined and the amount of labeled endothelial marker standard bound to the unlabeled antibody is determined.
  • the amount of endothelial marker in the biological sample is inversely proportional to the amount of labeled endothelial marker standard bound to the antibody that specifically binds the endothelial marker.
  • a subject is screened by determining whether that have increased levels of one or more of the disclosed pathological angiogenesis marker proteins in their serum (for example relative to a level present in a serum sample from a subject not having a tumor), for example using an antibody that specifically binds one or more of the disclosed pathological angiogenesis markers (such as those described below).
  • the presence of nucleic acids can be determined.
  • the biological sample can be incubated with primers that permit the amplification of one or more of the pathological angiogenesis marker mRNAs, under conditions sufficient to permit amplification of such products (see, for example, primer sequences provided in Example 1).
  • Exemplary methods include SAGE and PCR.
  • the biological sample is incubated with probes that can bind to one or more of the disclosed pathological angiogenesis marker nucleic acid sequences (such as cDNA, genomic DNA, or RNA (such as mRNA)) under high stringency conditions.
  • the resulting hybridization products can then be detected using methods known in the art.
  • a subject is screened by determining whether that have increased levels of one or more the disclosed pathological angiogenesis marker nucleic acids in their serum (for example relative to a level present in adjacent non-tumor cells from the same subject), for example detecting mRNA expression of one or more the disclosed pathological angiogenesis markers.
  • antibodies which specifically bind to the disclosed endothelial marker proteins. These antibodies can be monoclonal or polyclonal. They can be chimeric or humanized. 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 in the methods provided herein. 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 appropriate antigen at least 2, at least 5, at least 7 or 10 times more than to irrelevant antigen or antigen mixture, then it is considered to be specific.
  • monoclonal antibodies are generated to the endothelial cell markers disclosed in Tables 8 and 9. These monoclonal antibodies each include a variable heavy (V H ) and a variable light (V L ) chain and specifically bind to the specific endothelial cell markers.
  • V H variable heavy
  • V L variable light
  • the antibody can bind the specific endothelial cell markers with an affinity constant of at least 10 6 M ⁇ 1 , such as at least 10 7 M ⁇ 1 , at least 10 8 M ⁇ 1 , at least 5 ⁇ 10 8 M ⁇ 1 , or at least 10 9 M ⁇ 1 .
  • the specific antibodies can include a V L polypeptide having amino acid sequences of the complementarity determining regions (CDRs) that are at least about 90% identical, such as at least about 95%, at least about 98%, or at least about 99% identical to the amino acid sequences of the specific endothelial marker proteins and a V H polypeptide having amino acid sequences of the CDRs that are at least about 90% identical, such as at least about 95%, at least about 98%, or at least about 99% identical to the amino acid sequences of the specific endothelial marker proteins.
  • CDRs complementarity determining regions
  • the sequence of the specificity determining regions of each CDR is determined. Residues that are outside the SDR (non-ligand contacting sites) are substituted. For example, in any of the CDR sequences, at most one, two or three amino acids can be substituted.
  • the production of chimeric antibodies, which include a framework region from one antibody and the CDRs from a different antibody, is well known in the art.
  • humanized antibodies can be routinely produced.
  • the antibody or antibody fragment can be a humanized immunoglobulin having CDRs from a donor monoclonal antibody that binds one of the disclosed endothelial marker proteins and immunoglobulin and heavy and light chain variable region frameworks from human acceptor immunoglobulin heavy and light chain frameworks.
  • the humanized immunoglobulin specifically binds to one of the disclosed endothelial marker proteins with an affinity constant of at least 10 7 M ⁇ 1 , such as at least 10 8 M ⁇ 1 at least 5 ⁇ 10 8 M ⁇ 1 or at least 10 9 M ⁇ 1 .
  • human monoclonal antibodies to the disclosed specific endothelial marker proteins in Tables 8 and 9 are produced.
  • Human monoclonal antibodies can be produced by transferring donor complementarity determining regions (CDRs) from heavy and light variable chains of the donor mouse immunoglobulin into a human variable domain, and then substituting human residues in the framework regions when required to retain affinity.
  • CDRs donor complementarity determining regions
  • the use of antibody components derived from humanized monoclonal antibodies obviates potential problems associated with the immunogenicity of the constant regions of the donor antibody. For example, when mouse monoclonal antibodies are used therapeutically, the development of human anti-mouse antibodies (HAMA) leads to clearance of the murine monoclonal antibodies and other possible adverse events.
  • HAMA human anti-mouse antibodies
  • Chimeric monoclonal antibodies, with human constant regions, humanized monoclonal antibodies, retaining only murine CDRs, and “fully human” monoclonal antibodies made from phage libraries or transgenic mice have all been used to reduce or eliminate the murine content of therapeutic monoclonal antibodies.
  • the antibody may be of any isotype, but in several embodiments the antibody is an IgG, including but not limited to, IgG 1 , IgG 2 , IgG 3 and IgG 4 .
  • the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 65% identical to the sequence of the donor immunoglobulin heavy chain variable region framework.
  • the sequence of the humanized immunoglobulin heavy chain variable region framework can be at least about 75%, at least about 85%, at least about 99% or at least about 95%, identical to the sequence of the donor immunoglobulin heavy chain variable region framework.
  • Human framework regions, and mutations that can be made in a humanized antibody framework regions, are known in the art (see, for example, in U.S. Pat. No. 5,585,089).
  • Antibodies such as murine monoclonal antibodies, chimeric antibodies, and humanized antibodies, include full length molecules as well as fragments thereof, such as Fab, F(ab′) 2 , and Fv, which include a heavy chain and light chain variable region and are capable of binding the epitopic determinant. These antibody fragments retain some ability to selectively bind with their antigen or receptor.
  • fragments include: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab′, the fragment of an antibody molecule can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab′ fragments are obtained per antibody molecule; (3) (Fab′) 2 , the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab′) 2 is a dimer of two Fab′ fragments held together by two disulfide bonds; (4) Fv, a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody (such as scFv), defined as a genetically engineered molecule containing the variable region of the light chain, the variable region of the heavy chain, linked by
  • Fv antibodies are typically about 25 kDa and contain a complete antigen-binding site with three CDRs per each heavy chain and each light chain.
  • the V H and the V L can be expressed from two individual nucleic acid constructs in a host cell. If the V H and the V L are expressed non-contiguously, the chains of the Fv antibody are typically held together by noncovalent interactions.
  • the Fv can be a disulfide stabilized Fv (dsFv), wherein the heavy chain variable region and the light chain variable region are chemically linked by disulfide bonds.
  • dsFv disulfide stabilized Fv
  • the Fv fragments include V H and V L chains connected by a peptide linker.
  • These single-chain antigen binding proteins are prepared by constructing a structural gene comprising DNA sequences encoding the V H and V L domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli . The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains.
  • Methods for producing scFvs are known in the art (see Whitlow et al., Methods: a Companion to Methods in Enzymology , Vol. 2, page 97, 1991; Bird et al., Science 242:423, 1988; U.S. Pat. No. 4,946,778; Pack et al., Bio/Technology 11:1271, 1993; and Sandhu, supra).
  • Antibody fragments can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment.
  • Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods.
  • antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′) 2 .
  • This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab′ monovalent fragments.
  • an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly (see U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647, and references contained therein; Nisonhoff et al., Arch. Biochem. Biophys. 89:230, 1960; Porter, Biochem. J. 73:119, 1959; Edelman et al., Methods in Enzymology , Vol. 1, page 422, Academic Press, 1967; and Coligan et al. at sections 2.8.1-2.8.10 and 2.10.1-2.10.4).
  • cleaving antibodies such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.
  • conservative variants of the antibodies can be produced. Such conservative variants employed in antibody fragments, such as dsFv fragments or in scFv fragments, will retain critical amino acid residues necessary for correct folding and stabilizing between the V H and the V L regions, and will retain the charge characteristics of the residues in order to preserve the low pI and low toxicity of the molecules. Amino acid substitutions (such as at most one, at most two, at most three, at most four, or at most five amino acid substitutions) can be made in the V H and the V L regions to increase yield. Conservative amino acid substitution tables providing functionally similar amino acids are well known to one of ordinary skill in the art.
  • the following six groups are examples of amino acids that are considered to be conservative substitutions for one another: 1) Alanine (A), Serine (S), Threonine (T); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).
  • Antibodies are commercially available for many of the endothelial markers disclosed herein (see Tables 1-4).
  • Binding agents such as antibodies of this disclosure, can be conjugated or linked to an effector molecule, such as a therapeutic agent (such as an anti-tumor agent) or a diagnostic agent (such as a fluorescent moiety), using any number of methods known to those of skill in the art (for example, see Harlow and Lane, Using Antibodies: A Laboratory Manual , CSHL, New York, 1999; Yang et al., Nature, 382:319-24, 1996). Both covalent and noncovalent attachment means can be used.
  • the procedure for attaching an effector molecule to an antibody varies according to the chemical structure of the effector.
  • Polypeptides typically contain a variety of functional groups; such as carboxylic acid (COOH), free amine (—NH 2 ) or sulfhydryl (—SH) groups, which are available for reaction with a suitable functional group on an antibody to result in the binding of the effector molecule.
  • the antibody is derivatized to expose or attach additional reactive functional groups.
  • the derivatization can involve attachment of any of a number of linker molecules such as those available from Pierce Chemical Company, Rockford, Ill.
  • the linker can be any molecule used to join the antibody to the effector molecule (e.g., therapeutic agent or diagnostic agent).
  • the linker is capable of forming covalent bonds to both the antibody and to the effector molecule.
  • Suitable linkers are well known to those of skill in the art and include, but are not limited to, straight or branched-chain carbon linkers, heterocyclic carbon linkers, or peptide linkers.
  • the linkers can be joined to the constituent amino acids through their side groups (such as through a disulfide linkage to cysteine) or to the alpha carbon amino and carboxyl groups of the terminal amino acids.
  • immunoconjugates can include linkages that are cleavable in the vicinity of the target site. Cleavage of the linker to release the effector molecule from the antibody may be prompted by enzymatic activity or conditions to which the immunoconjugate is subjected either inside the target cell or in the vicinity of the target site.
  • a linker which is cleavable under conditions present at the tumor site for example, when exposed to tumor-associated enzymes or acidic pH
  • EMT6 cells were a kind gift of Dr. Robert S. Kerbel
  • KM12SM cells were a kind gift of Judith J. Fidler
  • HCT116 cells were from the DCT tumor repository (NCI, Frederick)
  • LS174T, SW620, CT26 and LLC were from the American Type Culture Collection (Manassas, Va.).
  • Tumor cell lines were maintained in DMEM containing 10% fetal bovine serum. Tumors were made by inoculating 5 ⁇ 10 5 ⁇ 1 ⁇ 10 6 cells subcutaneously or intrasplenically. To produce liver metastasis by intrasplenic injection, the spleen was exteriorized through a left lateral incision prior to tumor cell injection.
  • the tumor cell suspension was allowed to enter the portal circulation over a period of five minutes, after which the spleen was removed and the skin sutured.
  • the liver was exposed through a midline abdominal incision and the two anterior lobes were exteriorized and the suspensory ligaments severed.
  • the left lateral and caudal lobes were gently tied off using 6-0 sterile silk prior to excision leaving a 3 mm stump above the silk.
  • the procedure results in the removal of ⁇ 70% of liver volume.
  • the remaining liver was placed back into the peritoneal cavity and the peritoneal cavity and skin are sutured.
  • biotinylated rat anti-mouse CD105 (eBioscience, San Diego, Calif.) was added; to label ECs from spleen, CT26 tumors or LLC tumors biotinylated goat anti-mouse VE-cadherin (R&D Systems, Minneapolis, Minn.) was added, and to label ECs from brain, muscle, EMT6 tumors and SW620 a mixture of both antibodies was added.
  • SAGE libraries were constructed using the I-SAGE Long Kit (Invitrogen) and a previously established MicroSAGE protocol by St. Croix et al. (available from John Hopkins Oncology Center, Baltimore, Md. 21231) which is herein incorporated by reference in its entirety. Ditags were PCR amplified using biotinylated primers to facilitate efficient linker removal and Mme-I enzyme was purchased from New England Biolabs (Ipswich, Mass.). SAGE tags used to identify various endothelial cell markers are included in Tables 5A-5D. Some genes have multiple tags due to alternative polyadenylation sites, internal polyA stretches, and antisense transcripts.
  • SAGE tags used to identify Brain Endothelial Markers.
  • SAGE tags used to identify Liver Endothelial Markers.
  • SEQ ID NO: Acc# SAGE Tags 57 NM_007870 TGTAACCTGAAGAAATA (122) 58 NM_007870 CAGATAGCTTAGACCTA* (38) 59 NM_007870 GGTGATTTCAACGCCGG (16) 60 NM_007870 GTGCTTGCTTGTGTGCA* (15) 61 NM_007870 CCAAATCTGTCCTGTTG* (6) 62 NM_010959 CAGGCAAACCACTCATA (28) 63 NM_010959 ATCTCCTAGATACCTAA (26) 64 NM_010959 AAAGGACTGGCTGGCTG (5) 65 NM_023438 GGGTGGGTGAAGGCAGA (16) 66 AK150613 TTACTTTAATAGTAAAA (66) 67 AK150613 GTACAGTGTAGATAATT (32) 68 AK150613 TATAGGCTTTCTAAAAA* (6)
  • Quantitative PCR Quantitative PCR. mRNA was purified using the Quick Prep Micro mRNA purification kit (Amersham, Piscataway, N.J.). Single-stranded cDNA was generated using Superscript III first strand synthesis system (Invitrogen) following the manufacturer's directions. Quantitative PCR was performed with an MX4000 using Brilliant SYBR Green QPCR Master Mix and threshold cycle numbers were obtained using MX4000 software v4.20 (Stratagene, La Jolla, Calif.). Primer sets for each sequence analyzed are included in Table 5E below. Endothelial cells used in QPCR are provided in Table 5F.
  • VE-cadherin VE-cad
  • Antibodies against the endothelial selection markers CD105, VE-cadherin (VE-cad) or both were used in the positive selection to immunopurify the endothelial cells.
  • Endothelial cells were derived from the host strain indicated and then used to generate cDNA for QPCR. Nude: NCr nu/nu.
  • Gene expression was normalized to that of the 70 Kd U1 small nuclear ribonucleoprotein polypeptide A (Srnp70), a gene that is uniformly expressed in all ECs as assessed by SAGE. Relative expression was calculated using the formula 2 (Rt-Et) /2 (Rn-En) where Rt is the threshold cycle number observed in the experimental sample for Srnp70, Et is the threshold cycle number observed in the experimental sample for the gene of interest (GOI), R n is the average threshold cycle number observed for Srnp70 in all the N-EC samples and E n is the average threshold cycle number observed for the GOI in all the N-EC samples.
  • Digoxigenin (DIG)-labeled antisense RNA probes were generated by PCR amplification of 500-600 basepair products incorporating T7 promoters into the antisense primers.
  • In vitro transcription was performed with DIG RNA labeling reagents and T7 RNA polymerase according to the manufacturer's instructions (Roche, Indianapolis, Ind.). Tumors and normal tissues were dissected, embedded in OCT, frozen in a dry ice-methanol bath, and cryosectioned at 10 ⁇ m.
  • RNA probes 100 ng/ml diluted in ISH solution (Dako, Carpinteria, Calif.) overnight at 55° C. After washing three times with 2 ⁇ SSC, sections were incubated at 37° C. with RNase Cocktail (Ambion, Austin, Tex.) diluted 1:200 in 2 ⁇ SSC. Slides were stringently washed twice in 2 ⁇ SSC/50% deionized formamide (American Bioanalytical, Natick, Mass.) and then once with 0.1 ⁇ SSC at 55° C.
  • DAKO peroxidase blocking reagent
  • DAKO horseradish peroxidase-rabbit anti-DIG antibody
  • DAKO horseradish peroxidase-rabbit anti-FITC
  • DAKO horseradish peroxidase-rabbit anti-FITC
  • GenPoint Kit Biotin-tyramide
  • DAKO alkaline phosphatase rabbit anti-biotin
  • VE-cadherin was detected using goat anti-mouse VE-cadherin followed by rhodamine-streptavidin (Vector Laboratories, Burlingame, Calif.).
  • vWF was detected using a biotin-linked donkey anti-rabbit antibody (Jackson Immunoresearch Laboratories) followed by rhodamine-streptavidin (Vector Laboratories, Burlingame, Calif.). Images were captured using a Nikon Eclipse E600 microscope.
  • CD276 expression vector was made by excising a human CD276 cDNA from an EST (accession number BC7472032) using the restriction enzymes EcoR1 and Not1 and cloning the fragment into the same sites of the expression vector pcDNA3.1 (+) (Invitrogen). Sequencing of the CD276/pcDNA3 vector revealed that it contained a full length CD276 cDNA corresponding to transcript variant 1 (accession number NM — 001024736). CD276/pcDNA3 was transfected into 293 cells using lipofectamine, and stable transfectants selected with Geneticin. To generate extracts for immunoblotting, colorectal tissues stored at ⁇ 80° C.
  • Immunoblots were probed with a monoclonal anti-CD276 antibody (eBioscience) or an anti-actin antibody (Chemicon) followed by an HRP-conjugated anti-mouse secondary antibody (Jackson), and visualized using the ECL plus system (Amersham) according to the supplier's instructions.
  • This example describes methods used to immunopurify endothelial cells (ECs) from various tissue types.
  • CD105 Endoglin
  • VE-cadherin VE-cadherin
  • FIG. 1A immunofluorescence staining of heart tissue demonstrated co-localization of CD105 (green) with VE-cadherin (red) in the heart vessels.
  • FIG. 1B demonstrates immunofluorscence staining of liver tissue with CD105 (green).
  • CD105 was determined to be a preferred marker in liver because CD105 stained all the endothelium including sinusoidal ECs whereas VE-cadherin did not.
  • the cell isolation involved tissue dissociation, the removal of non-ECs, and the positive selection of ECs using magnetic beads coupled to either anti-VE-cadherin or anti-CD105 antibodies, the choice depending on the tissue being dissociated (see Example 1, Material and Methods).
  • QPCR analysis was performed on cDNA generated directly from unfractionated normal whole tissues (WT), purified ECs isolated from normal tissues (N-ECs) or ECs isolated from tumors.
  • WT unfractionated normal whole tissues
  • N-ECs purified ECs isolated from normal tissues
  • ECs isolated from tumors As illustrated in FIG. 1C , a marked enrichment of endothelial-specific genes such as VE-cadherin was found in each of the purified fractions compared to unfractionated whole tissues, but little contamination by hematopoietic cells, as judged by CD45 expression.
  • VE-cadherin was enriched 110 to 530-fold in the endothelial fractions.
  • the modest level of VE-cadherin found in the unfractionated heart and lung sample is presumably due to a higher proportion of ECs in these tissues.
  • Gene expression was normalized to that of the Eif4h, a gene found to be uniformly expressed in all cells as assessed by SAGE (Velculescu et al. Nat. Genet. 23: 387-8, 1999). Unfractionated brain was used to calibrate relative expression because this tissue had the lowest VE-cadherin expression levels.
  • FIG. 1D provides a model used to identify genes expressed during pathological but not physiological angiogenesis.
  • ECs were isolated from normal resting livers, regenerating livers, or tumor bearing livers.
  • This example illustrates methods used to identify 27 brain and 15 liver specific endothelial cell markers.
  • VE-cadherin VE-cad
  • Antibodies against the endothelial selection markers CD105, VE-cadherin (VE-cad) or both were used in the positive selection to immunopurify the endothelial cells.
  • Endothelial cells were derived from the host strain indicated, and the number of SAGE tags obtained for each library is indicated. These SAGE libraries utilized a 21 nucleotide “long tag” which facilitates the mapping of genes directly to genomic DNA even when EST or cDNA sequence was unavailable (Saha et al., Nat. Biotechnol. 20: 508-12, 2002).
  • all endothelial cell libraries were normalized to 100,000 tags except for kidney which was normalized to 30,000 tags due to the lower number of tags obtained for the kidney endothelial cell library. As illustrated in Table 6, 700,189 tags were obtained from these 7 normal EC libraries.
  • This SAGE library was constructed from hematopoietic cells that had been purified from collagenase dispersed KM12SM tumors using a mixture of magnetic beads coupled to anti-F480, anti-CD45, anti-CD68 and anti-CD19 antibodies.
  • the unfractionated (Unfrac.) liver control was derived from 37,162 SAGE tags originating from C57BL/6 whole liver and is publicly available at SAGEmap (World Wide Web address of ncbi.nlm.nih.gov/projects/SAGE/).
  • the unfractionated intestine control was derived from 115,942 SAGE tags originating from microscope-dissected small intestine of a late gestation embryo also available at SAGEmap.
  • the endothelial libraries are the same as those found in Table 6.
  • Tables 7A and 7B Multiple endothelial-specific transcripts in the 7 normal EC libraries.
  • BEMs Brain Endothelial Markers
  • LAMs Liver Endothelial Markers
  • CD32 is a low affinity Fc ⁇ -receptor that is a known marker of liver sinusoidal ECs (Muro et al. Am. J. Pathol. 143:105-20, 1993).
  • This example illustrates the expression of various markers in resting normal ECs, regenerating liver ECs and tumor ECs.
  • ECs were isolated from liver at 24-, 48- or 72-hours following partial hepatectomy, the period during which endothelial growth is thought to occur (Michalopoulos & DeFrances. Science 276:60-66, 1997). In total, 395,234 SAGE tags were isolated from regenerating liver (See Table 6). Gene expression patterns of regenerating liver ECs were compared with a combined set of EC libraries derived from all non-proliferating normal organs including resting liver (see FIG. 1D ). This comparison revealed 12 genes that were overexpressed in regenerating liver ECs compared to non-angiogenic ECs (Table 9), which were referred to as physiological angiogenesis endothelial markers.
  • At least seven of these genes may be involved in regulating progression through the cell cycle, consistent with the fact that these ECs are dividing.
  • the most abundant physiological angiogenesis marker is an ubiquitin-conjugating enzyme, Ube2c. Its human counterpart, UbcH10, is involved in progression through the G1 phase of the cell cycle (Townsley et al. Proc. Natl. Acad. Sci. U.S.A. 94:2362-7, 1997; and Rape & Kirschner. Nature 432:588-95, 2004).
  • Protein regulator of cytokinesis 1 PRC1 is a mitotic spindle-associated CDK substrate that is involved in cytokinesis (Jiang et al. Mol. Cell.
  • DNA topoisomerase II-alpha Top2a
  • Thymidine Kinase 1 TK1
  • Ki67 antigen markers of proliferating cells
  • Oatp2 Organic-anion-transporter 2
  • Ube2c Ube2c
  • TRAFaf1 DNA topoisomerase II ⁇
  • Top2a physiological angiogenesis markers
  • Vscp, CD276, Ptprn and CD137 pathological angiogenesis markers.
  • Oatp2 samples were normalized to the average expression in intestinal, heart and kidney ECs.
  • Each of the disclosed pathological angiogenesis genes detected by QPCR had a similar pattern of expression to that predicted by the SAGE analysis, with levels of expression barely detectable in regenerating liver endothelium ( FIG. 2A and FIG. 2C ). Most of the genes were overexpressed in the ECs of all of the tumors examined, although 6 of the genes (Ankylosis, Apelin, MiRP2, CD109, Doppel and Ubiquitin D) were overexpressed in the vessels of only a subset of the tumor types. Ubiquitin D was only expressed in the vessels of mouse tumors (CT26, EMT6 and LLC), but was essentially undetectable by QPCR in tissue culture-derived tumor cells.
  • RT-PCR was used to verify that Ubiquitin D is expressed by the tumor endothelial cells (TECs) and not the tumor cells themselves.
  • TECs tumor endothelial cells
  • mRNA was extracted from CT26, EMT6 and LLC tumor cell lines grown in tissue culture, the corresponding tumor cells isolated from tumors grown in vivo, or the corresponding TECs isolated from the same tumors.
  • tumors were dispersed with collagenase and endothelial cells and hematopoietic cells were removed using magnetic dynabeads coupled to CD105 and CD45. Tumor endothelial cells were isolated as described in the Examples (such as Example 1).
  • PCR amplification of VE-cadherin was used as a control to verify the endothelial origin of the purified tumor endothelial cells, and ⁇ -actin was used as housekeeping control to ensure the presence of similar amounts of template in each of the samples.
  • Ubiquitin D mRNA was essentially undetectable when RT-PCR was performed on in vivo tumor cell-enriched fractions or the tumor cell lines grown in tissue culture indicating that such expression is not due to the presence of contaminating tumor cells.
  • Table 10 illustrates in situ hybridization results of BEMs and LEMs in normal adult brain and liver tissues.
  • Expression of BEM or LEM mRNA was analyzed in resting adult brain and liver tissues and scored as negative ( ⁇ ), moderately positive (+), moderate to strongly positive (++) or strongly positive (+++) based on the staining intensity of endothelial cells.
  • brain and liver tissues were placed next to each other in frozen tissue blocks so that the two tissues could be sectioned together and processed simultaneously.
  • Four brain endothelial markers were localized to ECs throughout the brain whereas expression in liver was undetectable (Table 10).
  • an analysis of five liver endothelial markers revealed that each was readily detectable in liver endothelium but not brain endothelium.
  • Liver endothelial markers were expressed predominantly in the sinusoidal ECs with a pattern of staining similar to that of the endothelial control VEGFR2 (Table 10). However, LEM5, a previously uncharacterized putative G-protein coupled receptor, was also found in the larger vessels of central veins, portal veins and hepatic arteries
  • Liver large Liver capillaries vessel ECs ⁇ (Sinusoidal (CV, Brain ECs) PV & HA) ECs Controls CD31 + +++ + VEGFR2 +++ ⁇ ++ Brain BEM1 (GLUT-1) ⁇ ⁇ +++ Endothelial BEM2 (Oat2) ⁇ ⁇ ++ Markers BEM3 (Ptn) ⁇ ⁇ ++ ⁇ BEM4 (Atp10a) ⁇ ⁇ + Liver LEM1 Endothelial (Dnase113) +++ ⁇ ⁇ Markers LEM2 (Oit3) +++ ⁇ ⁇ LEM5 (Csprs) ++ ++ ⁇ LEM6 (Clec1b) + ⁇ ⁇ LEM8 (Plxnc1) +++ ⁇ ⁇ * ⁇ CV: central vein; PV: portal vein;
  • FIG. 3 panel a
  • various tumor endothelial markers in tumor tissues including CD276, ETSvg4, Apelin, CD109, MiRP2, CD137, Doppel and Vscp, as illustrated in FIG. 3 panel b through i, respectively.
  • a dilute counterstain was applied to the sections to highlight the lack of detectable expression in the non-ECs of the tumors.
  • CD276 a tumor endothelial marker
  • CD276 was expressed predominantly by the tumor vessels of the colorectal cancer, but was also expressed at a lower level by the tumor cells themselves. Expression of CD276 in normal colonic mucosa was undetectable (top middle panel). As a control, vessels were stained for vWF, which co-localized with CD276 only in the tumor sample.
  • the human corpus luteum was stained to determine if the normal angiogenic vessels of this tissue express CD276. Unlike the vWF control, CD276 expression was undetectable in the angiogenic vessels of the developing corpus luteum (see FIG. 4B ). Sections were counterstained with DAPI (left panels of FIG. 4B ) to highlight the epithelial cells.
  • This example illustrates that CD276 mRNA is expressed in human colorectal cancer and indicates that CD276 can be used for tumor-specific vascular targeting.
  • Riboprobes against human CD276 were generated and mRNA in situ hybridization on normal and malignant colorectal tissues was performed.
  • CD276 mRNA was most prominent in the tumor vessels, with a pattern of expression similar to that of the endothelial control VEGFR2 (left panel). CD276 expression was also detected in the tumor cells themselves, albeit at a lower level. In contrast, CD276 expression was undetectable in normal colonic mucosa, and an analysis of the tumor margin showed a striking on/off pattern of staining at the tumor/normal border ( FIG. 5 , right panel). For instance, the margin between tumor (T) tissue and normal (N) colonic mucosa CD276 staining abruptly ends (right panel). Further, extracellular staining around the normal crypts was observed and represents non-specific binding of the in situ hybridization reagents to the mucous (right panel); similar staining was also detected in control sections.
  • CD276 mRNA is expressed in human colorectal cancer and indicate that CD276 is a target for tumor-specific vascular targeting.
  • CD276 Protein is Overexpressed in Human Tumors
  • This example illustrates that CD276 is overexpressed in human tumors and indicates that CD276 is a target for tumor-specific vascular targeting.
  • CD276 protein expression patterns were evaluated using anti-CD276 antibodies.
  • the overall level of CD276 was assessed in extracts taken from 12 normal and 12 malignant colorectal tissues, 10 of which were derived from the same patient (P1-P10).
  • CD276 was clearly elevated in 11 of the 12 tumors, while the remaining matched normal/tumor pair (case P7) displayed unaltered expression.
  • CD276 protein migrated at a size similar to that observed in 293 cells transfected with the 4IgG-containing form of CD276 (293/CD276).
  • the faint product present in 293 parent cells may represent low-level endogenous CD276 expression which was also detected at the mRNA level in these cells by RT-PCR.
  • CD276 protein expression levels were assessed in 6 lung tumor samples. As illustrated in FIG. 6B , CD276 protein expression levels were increased in each of the lung tumor samples as compared with protein levels detected in patient-matched control samples. All tumor samples appeared to overexpress the predominant 4-IgG form of CD276, as exogenous overexpression of this form in transfected 293 cells resulted in a product of similar size ( FIG. 6A ).
  • FIGS. 6I-6L Similar expression patterns were observed using an independent monoclonal antibody.
  • CD276 overexpression was frequently detected in the tumor cells while normal epithelium was uniformly negative.
  • the highest tumor-cell expression levels of CD276 were found in lung and breast cancer where they matched that found in tumor endothelium ( FIGS. 6F , 6 G and 6 L).
  • This example describes methods that can be used to significantly reduce pathological angiogenesis, for example as a means to treat a tumor, such as cancer.
  • a tumor such as cancer.
  • Similar methods can be used with any of the pathological angiogenesis inhibitors shown in Table 9 to treat any tumor that expresses the target angiogenesis protein.
  • pathological angiogenesis can be reduced or inhibited by administering a therapeutically effective amount of a composition, wherein the composition includes a specific binding agent that preferentially binds to one or more pathological angiogenesis marker proteins comprising Vscp, CD276, ETSvg4 (Pea3), CD137(4-1BB), MiRP2, Ubiquitin D (Fat10), Doppel (prion-PLP), Apelin, Plgf, Ptprn (IA-2), CD109, Ankylosis, and collagen VIII ⁇ 1, thereby inhibiting pathological angiogenesis in the subject.
  • a specific binding agent that preferentially binds to one or more pathological angiogenesis marker proteins comprising Vscp, CD276, ETSvg4 (Pea3), CD137(4-1BB), MiRP2, Ubiquitin D (Fat10), Doppel (prion-PLP), Apelin, Plgf, Ptprn (IA-2), CD109, Ankylosis, and collagen VIII ⁇ 1,
  • a subject who has been diagnosed with a disease associated with or caused by pathological angiogenesis such as a tumor is identified.
  • a therapeutic effective dose of the composition including the specific binding agent is administered to the subject.
  • a therapeutic effective dose of a specific binding agent to one or more of the disclosed pathological angiogenesis markers is administered to the subject to inhibit pathological angiogenesis.
  • the specific binding agent is an antibody conjugated to a therapeutic molecule (such as therapeutic molecule is a cytotoxin, chemotherapeutic reagent, radionucleotide or a combination thereof).
  • a therapeutically effective amount of an agent is the amount sufficient to prevent, reduce, and/or inhibit, and/or treat the disorder (e.g., cancer) in a subject without causing a substantial cytotoxic effect in the subject.
  • naked antibodies are administered at 5 mg per kg every two weeks or 10 mg per kg every two weeks depending upon the cancer.
  • the antibodies are administered continuously.
  • antibodies or antibody fragments conjugated to cytotoxic agents are administered at 50 ⁇ g per kg given twice a week for 2 to 3 weeks.
  • pathological angiogenesis can be screened for by detecting at least one expression product comprising one or more of: Vscp, CD276, ETSvg4 (Pea3), CD137(4-1BB), MiRP2, Ubiquitin D (Fat10), Doppel (prion-PLP), Apelin, Plgf, Ptprn (IA-2), CD109, Ankylosis, and collagen VIII, 1, in a sample obtained from the subject and compared to a control (sample obtained from a subject without pathological angiogenesis) or reference value.
  • detection of the at least one expression product indicates pathological angiogenesis in the subject.
  • detection of the at least one expression product indicates the presence of a tumor such as cancer.
  • the expression product can be RNA or protein.
  • An RNA expression product can be detected by SAGE or PCR by methods described above (see, for example, Example 1).
  • a protein expression product can be detected by Western blot or immunoassay (see, for example, Example 1).
  • the disclosure is not limited to particular methods of detection.
  • a therapeutic agent can be delivered to organ-specific cells by administering a therapeutically effective amount of a composition, wherein the composition includes a binding agent that preferentially binds to one or more of the disclosed brain endothelial marker proteins or liver endothelial markers and the therapeutic agent, thereby evoking a therapeutic response in the organ-specific endothelial cells.
  • the one or more brain endothelial markers can include Glucose transporter GLUT-1, Organic anion transporter 2, Pleiotrophin, ATPase class V, type 10A, Peptidoglycan recognition protein 1, Organic anion transporter 14, Forkhead box Q1, Organic anion transporter 3, SN2 (Solute carrier family 38, member 5), Inter-alpha (globulin) inhibitor H5, Solute carrier 38 member 3, Zinc finger protein of the cerebellum 2, Testican-2,3-HMG-CoA synthase 2, Progestin and adipoQ receptor family member V, APC down-regulated 1 Drapc1, GDPD phosphodiesterase family Accession No.
  • liver endothelial markers can include liver endothelial marker proteins such as deoxyribonuclease 1-like 3, LZP oncoprotein induced transcript 3, putative transmembrane protein Accession No.
  • NM — 023438 CD32 15, putative G-protein coupled receptor NM — 033616, C-type lectin-like receptor 2, C-type lectin domain family 4 member g 16, Plexin C1, Wnt9B, Accession No. AK144596, GATA-binding protein 4, MBL-associated serine protease-3, Renin binding protein, putative transmembrane protein Accession No. NM — 144830, or Retinoic acid receptor, beta.
  • a subject who is in need of delivery of a therapeutic agent to either a brain endothelial cell or a liver endothelial cell is identified.
  • a therapeutic effective dose of the composition including the specific binding agent is administered to the subject.
  • a therapeutic effective dose of a specific binding agent to one or more of the disclosed pathological angiogenesis markers is administered to the subject to inhibit tumor growth in the brain or liver.
  • the specific binding agent can be an antibody to one or more of the organ-specific endothelial markers in which the antibody is conjugated to the therapeutic agent such as a cytotoxin, chemotherapeutic reagent, radionucleotide or a combination thereof.

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WO2008057632A1 (fr) 2008-05-15
CA3025354A1 (fr) 2008-05-15
CA2669260A1 (fr) 2008-05-15
US8440411B2 (en) 2013-05-14
US20110207141A1 (en) 2011-08-25
CA2669260C (fr) 2019-01-15

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