Patent Application: US-201314065587-A

Abstract:
to gain a better understanding of breast tumor angiogenesis , breast endothelial cells were isolated and evaluated for gene expression patterns . when transcripts from breast ecs derived from normal and malignant breast tissues were compared , genes that were specifically elevated in tumor - associated breast endothelium were revealed . these results confirm that neoplastic and normal endothelium in human breast are distinct at the molecular level , and have significant implications for the development of anti - angiogenic therapies in the future .

Description:
using sage ( serial analysis of gene expression ) profiling , the present inventors were able to identify previously unrecognized , angiogenesis - specific markers that discriminate between non - proliferative and pathologic endothelial cells . in addition , a set of previously identified angiogenesis - specific markers from other tumor types ( colon and / or brain ) were found to be expressed in breast tumor endothelium as well . we identified 111 human genes that were expressed at significantly higher levels in breast tumor endothelium than in normal breast endothelium . see table 1 . additional such genes which can be used similarly to the 11 human genes are shown in table 2 . we have named these markers bems ( breast tumor endothelial markers ). bems that are expressed in both colon and breast tumor epithelium are identified in table 3 . bems that are expressed in both brain and breast tumor epithelium are identified in table 4 . bems that are expressed in each of brain , colon , and breast tumor epithelium are identified in table 5 . endothelial cells ( ecs ) represent only a minor fraction of the total cells within normal or tumor tissues , and only those ec transcripts expressed at the highest levels would be expected to be represented in libraries constructed from unfractionated tissues . the genes described in the current study should therefore provide a valuable resource for basic and clinical studies of human breast angiogenesis in the future . isolated and purified nucleic acids , according to the present invention are those which are not linked to those genes to which they are linked in the human genome . moreover , they are not present in a mixture such as a library containing a multitude of distinct sequences from distinct genes . they may be , however , linked to other genes such as vector sequences or sequences of other genes to which they are not naturally adjacent . the nucleic acids may represent either the sense or the anti - sense strand . nucleic acids and proteins although disclosed herein with sequence particularity , may be derived from a single individual . allelic variants which occur in the population of humans are included within the scope of such nucleic acids and proteins . those of skill in the art are well able to identify allelic variants as being the same gene or protein . given a nucleic acid , one of ordinary skill in the art can readily determine an open reading frame present , and consequently the sequence of a polypeptide encoded by the open reading frame and , using techniques well known in the art , express such protein in a suitable host . proteins comprising such polypeptides can be the naturally occurring proteins , fusion proteins comprising exogenous sequences from other genes from humans or other species , epitope tagged polypeptides , etc . isolated and purified proteins are not in a cell , and are separated from the normal cellular constituents , such as nucleic acids , lipids , etc . typically the protein is purified to such an extent that it comprises the predominant species of protein in the composition , such as greater than 50 , 60 70 , 80 , 90 , or even 95 % of the proteins present . using the proteins according to the invention , one of ordinary skill in the art can readily generate antibodies which specifically bind to the proteins . such antibodies can be monoclonal or polyclonal . they can be chimeric , humanized , or totally human . any functional fragment or derivative of an antibody can be used including fab , fab ′, fab2 , fab ′ 2 , and single chain variable regions . so long as the fragment or derivative retains specificity of binding for the endothelial marker protein it can be used . antibodies can be tested for specificity of binding by comparing binding to appropriate antigen to binding to irrelevant antigen or antigen mixture under a given set of conditions . if the antibody binds to the appropriate antigen at least 2 , 5 , 7 , and preferably 10 times more than to irrelevant antigen or antigen mixture then it is considered to be specific . techniques for making such partially to fully human antibodies are known in the art and any such techniques can be used . according to one particularly preferred embodiment , fully human antibody sequences are made in a transgenic mouse which has been engineered to express human heavy and light chain antibody genes . multiple strains of such transgenic mice have been made which can produce different classes of antibodies . b cells from transgenic mice which are producing a desirable antibody can be fused to make hybridoma cell lines for continuous production of the desired antibody . see for example , nina d . russel , jose r . f . corvalan , michael l . gallo , c . geoffrey davis , liise - anne pirofski . production of protective human antipneumococcal antibodies by transgenic mice with human immunoglobulin loci infection and immunity april 2000 , p . 1820 - 1826 ; michael l . gallo , vladimir e . ivanov , aya jakobovits , and c . geoffrey davis . the human immunoglobulin loci introduced into mice : v ( d ) and j gene segment usage similar to that of adult humans european journal of immunology 30 : 534 - 540 , 2000 ; larry l . green . antibody engineering via genetic engineering of the mouse : xenomouse strains are a vehicle for the facile generation of therapeutic human monoclonal antibodies journal of immunological methods 231 11 - 23 , 1999 ; yang x - d , corvalan j r f , wang p , roy c m - n and davis c g . fully human anti - interleukin - 8 monoclonal antibodies : potential therapeutics for the treatment of inflammatory disease states . journal of leukocyte biology vol . 66 , pp 401 - 410 ( 1999 ); yang x - d , jia x - c , corvalan j r f , wang p , c g davis and jakobovits a . eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy . cancer research vol . 59 , number 6 , pp 1236 - 1243 ( 1999 ); jakobovits a . production and selection of antigen - specific fully human monoclonal antibodies from mice engineered with human ig loci . advanced drug delivery reviews vol . 31 , pp : 33 - 42 ( 1998 ); green l and jakobovits a . regulation of b cell development by variable gene complexity in mice reconstituted with human immunoglobulin yeast artificial chromosomes . j . exp . med . vol . 188 , number 3 , pp : 483 - 495 ( 1998 ); jakobovits a . the long - awaited magic bullets : therapeutic human monoclonal antibodies from transgenic mice . exp . opin . invest . drugs vol . 7 ( 4 ), pp : 607 - 614 ( 1998 ); tsuda h , maynard - currie k , reid l , yoshida t , edamura k , maeda n , smithies o , jakobovits a . inactivation of mouse hprt locus by a 203 - bp retrotransposon insertion and a 55 - kb gene - targeted deletion : establishment of new hprt - deficient mouse embryonic sbem cell lines . genomics vol . 42 , pp : 413 - 421 ( 1997 ); sherman - gold , r . monoclonal antibodies : the evolution from &# 39 ; 80s magic bullets to mature , mainstream applications as clinical therapeutics . genetic engineering news vol . 17 , number 14 ( august 1997 ); mendez m , green l , corvalan j , jia x - c , maynard - currie c , yang x - d , gallo m , louie d , lee d , erickson k , luna j , roy c , abderrahim h , kirschenbaum f , noguchi m , smith d , fukushima a , hales j , finer m , davis c , zsebo k , jakobovits a . functional transplant of megabase human immunoglobulin loci recapitulates human antibody response in mice . nature genetics vol . 15 , pp : 146 - 156 ( 1997 ); jakobovits a . mice engineered with human immunoglobulin yacs : a new technology for production of fully human antibodies for autoimmunity therapy . weir &# 39 ; s handbook of experimental immunology , the integrated immune system vol . iv , pp : 194 . 1 - 194 . 7 ( 1996 ); jakobovits a . production of fully human antibodies by transgenic mice . current opinion in biotechnology vol . 6 , no . 5 , pp : 561 - 566 ( 1995 ); mendez m , abderrahim h , noguchi m , david n , hardy m , green l , tsuda h , yoast s , maynard - currie c , garza d , bemmill r , jakobovits a , klapholz s . analysis of the structural integrity of yacs comprising human immunoglobulin genes in yeast and in embryonic sbem cells . genomics vol . 26 , pp : 294 - 307 ( 1995 ); jakobovits a . yac vectors : humanizing the mouse genome . current biology vol . 4 , no . 8 , pp : 761 - 763 ( 1994 ); arbones m , ord d , ley k , ratech h , maynard - curry k , otten g , capon d , tedder t . lymphocyte homing and leukocyte rolling and migration are impaired in l - selectin - deficient mice . immunity vol . 1 , no . 4 , pp : 247 - 260 ( 1994 ); green l , hardy m , maynard - curry k , tsuda h , louie d , mendez m , abderrahim h , noguchi m , smith d , zeng y , et . al . antigen - specific human monoclonal antibodies from mice engineered with human ig heavy and light chain yacs . nature genetics vol . 7 , no . 1 , pp : 13 - 21 ( 1994 ); jakobovits a , moore a , green l , vergara g , maynard - curry k , austin h , klapholz s . germ - line transmission and expression of a human - derived yeast artificial chromosome . nature vol . 362 , no . 6417 , pp : 255 - 258 ( 1993 ); jakobovits a , vergara g , kennedy j , hales j , mcguinness r , casentini - borocz d , brenner d , otten g . analysis of homozygous mutant chimeric mice : deletion of the immunoglobulin heavy - chain joining region blocks b - cell development and antibody production . proceedings of the national academy of sciences usa vol . 90 , no . 6 , pp : 2551 - 2555 ( 1993 ); kucherlapati et al ., u . s . pat . no . 6 , 1075 , 181 . antibodies can also be made using phage display techniques . such techniques can be used to isolate an initial antibody or to generate variants with altered specificity or avidity characteristics . single chain fv can also be used as is convenient . they can be made from vaccinated transgenic mice , if desired . antibodies can be produced in cell culture , in phage , or in various animals , including but not limited to cows , rabbits , goats , mice , rats , hamsters , guinea pigs , sheep , dogs , cats , monkeys , chimpanzees , apes . antibodies can be labeled with a detectable moiety such as a radioactive atom , a chromophore , a fluorophore , or the like . such labeled antibodies can be used for diagnostic techniques , either in vivo , or in an isolated test sample . antibodies can also be conjugated , for example , to a pharmaceutical agent , such as chemotherapeutic drug or a toxin . they can be linked to a cytokine , to a ligand , to another antibody . suitable agents for coupling to antibodies to achieve an anti - tumor effect include cytokines , such as interleukin 2 ( il - 2 ) and tumor necrosis factor ( tnf ); photosensitizers , for use in photodynamic therapy , including aluminum ( iii ) phthalocyanine tetrasulfonate , hematoporphyrin , and phthalocyanine ; radionuclides , such as iodine - 131 ( 131 i ), yttrium - 90 ( 90 y ), bismuth - 212 ( 212 bi ), bismuth - 213 ( 213 bi ), technetium - 99m ( 99m tc ), rhenium - 186 ( 186 re ), and rhenium - 188 ( 188 re ); antibiotics , such as doxorubicin , adriamycin , daunorubicin , methotrexate , daunomycin , neocarzinostatin , and carboplatin ; bacterial , plant , and other toxins , such as diphtheria toxin , pseudomonas exotoxin a , staphylococcal enterotoxin a , abrin - a toxin , ricin a ( deglycosylated ricin a and native ricin a ), tgf - alpha toxin , cytotoxin from chinese cobra ( naja naja atra ), and gelonin ( a plant toxin ); ribosome inactivating proteins from plants , bacteria and fungi , such as restrictocin ( a ribosome inactivating protein produced by aspergillus restrictus ), saporin ( a ribosome inactivating protein from saponaria officinalis ), and rnase ; tyrosine kinase inhibitors ; ly207702 ( a difluorinated purine nucleoside ); liposomes containing antitumor agents ( e . g ., antisense oligonucleotides , plasmids which encode for toxins , methotrexate , etc . ); and other antibodies or antibody fragments , such as f ( ab ). those of skill in the art will readily understand and be able to make such antibody derivatives , as they are well known in the art . the antibodies may be cytotoxic on their own , or they may be used to deliver cytotoxic agents to particular locations in the body . the antibodies can be administered to individuals in need thereof as a form of passive immunization . characterization of extracellular regions for the cell surface and secreted proteins from the protein sequence is based on the prediction of signal sequence , transmembrane domains and functional domains . antibodies are preferably specifically immunoreactive with membrane associated proteins , particularly to extracellular domains of such proteins or to secreted proteins . such targets are readily accessible to antibodies , which typically do not have access to the interior of cells or nuclei . however , in some applications , antibodies directed to intracellular proteins may be useful as well . moreover , for diagnostic purposes , an intracellular protein may be an equally good target since cell lysates may be used rather than a whole cell assay . computer programs can be used to identify extracellular domains of proteins whose sequences are known . such programs include smart software ( schultz et al ., proc . natl . acad . sci . usa 95 : 5857 - 5864 , 1998 ) and pfam software ( babeman et al ., nucleic acids res . 28 : 263 - 266 , 2000 ) as well as psortii . typically such programs identify transmembrane domains ; the extracellular domains are identified as immediately adjacent to the transmembrane domains . prediction of extracellular regions and the signal cleavage sites are only approximate . it may have a margin of error + or − 5 residues . signal sequence can be predicted using three different methods ( nielsen et al , protein engineering 10 : 1 - 6 , 1997 , jagla et . al , bioinformatics 16 : 245 - 250 , 2000 , nakai , k and horton , p . trends in biochem . sci . 24 : 34 - 35 , 1999 ) for greater accuracy . similarly transmembrane ( tm ) domains can be identified by multiple prediction methods . ( pasquier , et . al , protein eng . 12 : 381 - 385 , 1999 , sonnhammer et al ., in proc . of sixth int . conf . on intelligent systems for molecular biology , p . 175 - 182 , ed j . glasgow , t . littlejohn , f . major , r . lathrop , d . sankoff , and c . sensen menlo park , calif . : aaai press , 1998 , klein , et . al , biochim . biophys . acta , 815 : 468 , 1985 , nakai and kanehisa genomics , 14 : 897 - 911 , 1992 ). in ambiguous cases , locations of functional domains in well characterized proteins are used as a guide to assign a cellular localization . putative functions or functional domains of novel proteins can be inferred from homologous regions in the database identified by blast searches ( altschul et . al . nucleic acid res . 25 : 3389 - 3402 , 1997 ) and / or from a conserved domain database such as pfam ( babeman et . al , nucleic acids res . 27 : 260 - 262 1999 ) blocks ( henikoff , et . al , nucl . acids res . 28 : 228 - 230 , 2000 ) and smart ( ponting , et . al , nucleic acid res . 27 , 229 - 232 , 1999 ). extracellular domains include regions adjacent to a transmembrane domain in a single transmembrane domain protein ( out - in or type i class ). for multiple transmembrane domains proteins , the extracellular domain also includes those regions between two adjacent transmembrane domains ( in - out and out - in ). for type ii transmembrane domain proteins , for which the n - terminal region is cytoplasmic , regions following the transmembrane domain is generally extracellular . secreted proteins on the other hand do not have a transmembrane domain and hence the whole protein is considered as extracellular . membrane associated proteins can be engineered to delete the transmembrane domains , thus leaving the extracellular portions which can bind to ligands . such soluble forms of transmembrane receptor proteins can be used to compete with natural forms for binding to ligand . thus such soluble forms act as inhibitors and can be used therapeutically as anti - angiogenic agents , as diagnostic tools for the quantification of natural ligands , and in assays for the identification of small molecules which modulate or mimic the activity of a bem : ligand complex . alternatively , the endothelial markers themselves can be used as vaccines to raise an immune response in the vaccinated animal or human . for such uses , a protein , or immunogenic fragment of such protein , corresponding to the intracellular , extracellular or secreted bem of interest is administered to a subject . the immogenic agent may be provided as a purified preparation or in an appropriately expressing cell . the administration may be direct , by the delivery of the immunogenic agent to the subject , or indirect , through the delivery of a nucleic acid encoding the immunogenic agent under conditions resulting in the expression of the immunogenic agent of interest in the subject . the bem of interest may be delivered in an expressing cell , such as a purified population of breast tumor endothelial cells or a population of fused breast tumor endothelial and dendritic cells . nucleic acids encoding the bem of interest may be delivered in a viral or non - viral delivery vector or vehicle . non - human sequences encoding the human bem of interest or other mammalian homolog can be used to induce the desired immunologic response in a human subject . for several of the bems of the present invention , mouse , rat or other ortholog sequences can be obtained from the literature or using techniques well within the skill of the art . endothelial cells can be identified using the markers which are disclosed herein as being endothelial cell specific . antibodies specific for such markers can be used to identify such cells , by contacting the antibodies with a population of cells containing some endothelial cells . the presence of cross - reactive material with the antibodies identifies particular cells as endothelial . similarly , lysates of cells can be tested for the presence of cross - reactive material . any known format or technique for detecting cross - reactive material can be used including , immunoblots , radioimmunoassay , elisa , immunoprecipitation , and immunohistochemistry . in addition , nucleic acid probes for these markers can also be used to identify endothelial cells . any hybridization technique known in the art including northern blotting , rt - pcr , microarray hybridization , and in situ hybridization can be used . one can identify breast tumor endothelial cells for diagnostic purposes , testing cells suspected of containing one or more bems . one can test both tissues and bodily fluids of a subject . for example , one can test a patient &# 39 ; s blood for evidence of intracellular and membrane associated bems , as well as for secreted bems . of particular interest in this context is the testing of breast duct fluid . intracellular and / or membrane associated bems may be present in bodily fluids as the result of high levels of expression of these factors and / or through lysis of cells expressing the bems . populations of various types of endothelial cells can also be made using the antibodies to endothelial markers of the invention . the antibodies can be used to purify cell populations according to any technique known in the art , including but not limited to fluorescence activated cell sorting . such techniques permit the isolation of populations which are at least 50 , 60 , 70 , 80 , 90 , 92 , 94 , 95 , 96 , 97 , 98 , and even 99 % the type of endothelial cell desired , whether normal , tumor , or pan - endothelial . antibodies can be used to both positively select and negatively select such populations . preferably at least 1 , 5 , 10 , 15 , 20 , or 25 of the appropriate markers are expressed by the endothelial cell population . populations of endothelial cells made as described herein , can be used for screening drugs to identify those suitable for inhibiting the growth of tumors by virtue of inhibiting the growth of the tumor vasculature . populations of endothelial cells made as described herein , can be used for screening candidate drugs to identify those suitable for modulating angiogenesis , such as for inhibiting the growth of tumors by virtue of inhibiting the growth of endothelial cells , such as inhibiting the growth of the tumor or other undesired vasculature , or alternatively , to promote the growth of endothelial cells and thus stimulate the growth of new or additional large vessel or microvasculature . inhibiting the growth of endothelial cells means either regression of vasculature which is already present , or the slowing or the absence of the development of new vascularization in a treated system as compared with a control system . by stimulating the growth of endothelial cells , one can influence development of new ( neovascularization ) or additional vasculature development ( revascularization ). a variety of model screening systems are available in which to test the angiogenic and / or anti - angiogenic properties of a given candidate drug . typical tests involve assays measuring the endothelial cell response , such as proliferation , migration , differentiation and / or intracellular interaction with a given candidate drug . by such tests , one can study the signals and effects of the test stimuli . some common screens involve measurement of the inhibition of heparanase , endothelial tube formation on matrigel , scratch induced motility of endothelial cells , platelet - derived growth factor driven proliferation of vascular smooth muscle cells , and the rat aortic ring assay ( which provides an advantage of capillary formation rather than just one cell type ). drugs can be screened for the ability to mimic or modulate , inhibit or stimulate , growth of tumor endothelium cells and / or normal endothelial cells . drugs can be screened for the ability to inhibit tumor endothelium growth but not normal endothelium growth or survival . similarly , human cell populations , such as normal endothelium populations or breast tumor endothelial cell populations , can be contacted with test substances and the expression of breast tumor endothelial markers and / or normal endothelial markers determined . test substances that decrease the expression of breast tumor endothelial markers ( bems ) are candidates for inhibiting angiogenesis and the growth of tumors . in cases where the activity of a bem is known , agents can be screened for their ability to decrease or increase the activity . for those breast tumor endothelial markers identified as containing transmembrane regions , it is desirable to identify drug candidates capable of binding to the bem receptors found at the cell surface . for some applications , the identification of drug candidates capable of blocking the bem receptor from its native ligand will be desired . for some applications , the identification of a drug candidate capable of binding to the bem receptor may be used as a means to deliver a therapeutic or diagnostic agent . for other applications , the identification of drug candidates capable of mimicking the activity of the native ligand will be desired . thus , by manipulating the binding of a transmembrane bem receptor : ligand complex , one may be able to promote or inhibit further development of endothelial cells and hence , vascularization . for those breast tumor endothelial markers identified as being secreted proteins , i . e ., extracellular , it is desirable to identify drug candidates capable of binding to the secreted bem protein . for some applications , the identification of drug candidates capable of interfering with the binding of the secreted bem it is native receptor . for other applications , the identification of drug candidates capable of mimicking the activity of the native receptor will be desired . thus , by manipulating the binding of the secreted bem : receptor complex , one may be able to promote or inhibit further development of endothelial cells , and hence , vascularization . expression can be monitored according to any convenient method . protein or mrna can be monitored . any technique known in the art for monitoring specific genes &# 39 ; expression can be used , including but not limited to elisas , sage , microarray hybridization , western blots . changes in expression of a single marker may be used as a criterion for significant effect as a potential pro - angiogenic , anti - angiogenic or anti - tumor agent . however , it also may be desirable to screen for test substances that are able to modulate the expression of at least 5 , 10 , 15 , or 20 of the relevant markers , such as the tumor or normal endothelial markers inhibition of bem protein activity can also be used as a drug screen . test substances for screening can come from any source . they can be libraries of natural products , combinatorial chemical libraries , biological products made by recombinant libraries , etc . the source of the test substances is not critical to the invention . the present invention provides means for screening compounds and compositions that may previously have been overlooked in other screening schemes . nucleic acids and the corresponding encoded proteins of the markers of the present invention can be used therapeutically in a variety of modes . bems can be used to stimulate the growth of vasculature , such as for wound healing or to circumvent a blocked vessel . the nucleic acids and encoded proteins can be administered by any means known in the art . such methods include , using liposomes , nanospheres , viral vectors , non - viral vectors comprising polycations , etc . suitable viral vectors include adenovirus , retroviruses , and sindbis virus . administration modes can be any known in the art , including parenteral , intravenous , intramuscular , intraperitoneal , topical , intranasal , intrarectal , intrabronchial , etc . specific biological antagonists of bems can also be used to therapeutic benefit . for example , antibodies , t cells specific for a bem , antisense to a bem , interferance rna to a bem , and ribozymes specific for a bem can be used to restrict , inhibit , reduce , and / or diminish tumor or other abnormal or undesirable vasculature growth . such antagonists can be administered as is known in the art for these classes of antagonists generally . anti - angiogenic drugs and agents can be used to inhibit tumor growth , as well as to treat diabetic retinopathy , rheumatoid arthritis , psoriasis , polycystic kidney disease ( pkd ), and other diseases requiring angiogenesis for their pathologies . mouse counterparts to human bems can be used in mouse cancer models or in cell lines or in vitro to evaluate potential anti - angiogenic or anti - tumor compounds or therapies . their expression can be monitored as an indication of effect . mouse bems can be used as antigens for raising antibodies which can be tested in mouse tumor models . mouse bems with transmembrane domains are particularly preferred for this purpose . mouse bems can also be used as vaccines to raise an immunological response in a human to the human ortholog . the above disclosure generally describes the present invention . all references disclosed herein are expressly incorporated by reference in their entireties . a more complete understanding can be obtained by reference to the following specific examples which are provided herein for purposes of illustration only , and are not intended to limit the scope of the invention . function of bem proteins was determined using bioinformatics tools . bems that are putative functional receptors with short cytoplasmic tails make particularly interesting targets . protein kinases were identified among the bems . these are particularly good druggable targets , especially for small molecules . kinases with non - 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