AU2015201325B2 - Methods for diagnosing and treating cancers - Google Patents

Abstract

The disclosure provides, among other things, molecular markers for diagnosing neoplasias or categorizing the neoplastic state of a patient. The disclosure also provides methods for using the molecular markers in designing, screening and targeting therapeutic agents for the treatment of cancers.

Description

METHODS FOR DIAGNOSING AND TREATING CANCERS

RELATED APPLICATIONS

The present application is a divisional application of Australian Application No. 2008296927, which is incorporated in its entirety herein by reference.

This application claims the benefit of priority of U.S. Provisional Application number 60/967,665 filed September 6, 2007, the entire teachings of which are incorporated herein by reference in their entirety.

FUNDING

Work described herein was funded, in part, by grant numbers U01 CA-88130 and ROl CA120237 from the National Institutes of Health. The United States government has certain rights in the invention.

BACKGROUND

Any discussion of the prior art throughout the specification should in no way be considered as an admission that such prior art is widely known or forms part of common general knowledge in the field.

Modem molecular biology has made it possible to identify proteins and nucleic acids that are specifically associated with certain physiological states. Improved systems for identifying high quality candidate molecular markers in large volumes of gene expression data may help to unlock the power of such tools and increase the likelihood of identifying a molecular marker for cancers. Effective molecular markers for cancers could potentially revolutionize the diagnosis, management and overall health impact of cancers. In addition, molecular markers may be used in screening for, generating and targeting therapeutic agents for cancers. Thus, there remains a need for novel molecular markers for detecting cancers and therapeutic agents in treating cancers.

Unless the context clearly requires otherwise, throughout the description and the claims, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense as opposed to an exclusive or exhaustive sense; that is to say, in the sense of “including, but not limited to”.

SUMMARY OF THE INVENTION

According to a first aspect, the present invention provides a method for detecting whether a subject has a neoplasia of a tissue type selected from endometrium, kidney, stomach, pancreas and uterus, comprising: (a) selecting a subject from a population, wherein the subject is suspected of harbouring a neoplasia of a tissue type selected from endometrium, kidney, stomach, pancreas and uterus; (b) obtaining a biological sample from said subject; and (c) detecting in the biological sample a ColoUp2 polypeptide, wherein the wherein the ColoUp2 polypeptide is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2, wherein the presence of said polypeptide is indicative of said neoplasia; and (d) wherein if the subject is found to have a neoplasia, treating said subject by: i. surgically removing said neoplasia from said subject; or ii. administering to said subject chemotherapy.

According to a second aspect, the present invention provides a method of treating a subject having a neoplasia, wherein the neoplasia is of a tissue type selected from endometrium, kidney, stomach, pancreas, and uterus; wherein the subject has been determined to have increased levels of ColoUp2 polypeptide; wherein the ColoUp2 polypeptide is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2; wherein the method comprises the step of treating the subject by: i. surgically removing said neoplasia from said subject; or ii. administering to said subject chemotherapy.

In a further aspect, this application provides ColoUp2 (also referred to as colon cancer secreted protein-2 (CCSP2)) as a molecular marker that is useful in the detection or diagnosis of neoplasias including cancers or precancers. In certain embodiments, molecular markers described in the application are helpful in distinguishing normal subjects from those who are likely to develop or likely to harbor a neoplasia. In other aspects, the invention provides molecular markers that may be useful in distinguishing subjects who are either normal or precancerous from those who have cancers. In another embodiment, the application provides markers that help in staging cancers in patients. In certain specific embodiments, the - application contemplates the use of the molecular markers described herein for the detection, diagnosis, and Staging of a neoplasia of a tissue type selected from, endometrium, kidney,. Ittag*. stomach, pancreas, breast, prostate, ovary, uteres, and thyroid. I» certain embodiments, the molecular markers disclosed herein may be used for identifying or targeting ami-neoplastic agents directed against neoplasms. hi one .aspect, the application provides a method of screening a subject for a condition associated with increased levels of Cololip2 by detecting in a .'biological sample an amount of €οΙοΙ)»2 and comparing the amount of Coloi ip2 found in the subject to one or more of the following: a predetermined standard, the amount of ColoUp2 detected in. a normal sample from the subled,, the subject's historical baseline level of Co1oUp2, or the ColoUp2 level detected in a different, normal subject (a control subject). Detection of a level of ColoUpS in the subject that is greater Chun that of the predetermined standard or 'that is increased from a subject's past: baseline is indicative of a condition such as neoplasias. In certain aspects, as increase in the amount of Coioljp2 as compared to the subject’s historical baseline would he indicative of a now neoplasia, or progression of an existing neoplasia. Similarly, a t leetease in the amount of C'oloUp2 as compared to the subject’s historical baseline would be indicative of regression on an existing neoplasia. Exemplary neoplasias may be of a tissue typo, selected from endometrium, kidney,: lung, stomach, pancreas, breast, prostate, ovary, uterus, and thyroid, . .

In yet another aspect, the application provides a method of determining whether a subject is likely to develop a neoplasia (canoes· or pr «cancer) or is more likely to harbor a neoplasia by detecting the presence or absence of the molecular markers as set. forth in any ofSEQ ID NOs: I and 3 -4. Detection of these markers is also helpful in staging the neoplasia. In yet another aspect, the application provides markers that are useful m distinguishing normal ami precancerous subjects from those subjects having a cancer. In certain embodiments, the application contemplates determining the levels of a ColoUpZ polypeptide. Optionally, the ColoUp2 polypeptide is a secreted polypeptide.

In certain embodiments, a secreted CokVUp2 polypeptide is selected horn: at) a secreted polypeptide produced by the expression of a nucleic acid that is at least 95% identical to the nuclesc acid sequence of SEQ ID NO: 2; b) a secreted polypeptide produced by the expression of a nucleic acid that is a naturally occurring variant of SEQ ID NO: 2; e) a secreted polypepiideproduced by the expression of a nucleic acid that hybridizes under stringent conditions to a nucleic acid sequence of SEQ ID NO: 2; d) a secreted polypeptide basing a sequence that is at least 95% identical to the anuno acid sequence of SEQ ID NO; 1; and e) a secreted polypeptide having a sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO; 4. Optionally,, the secreted CeIo:Up2 polypeptide is produced by the expression of a , nucleic acid having the sequence of SEQ ID NO; 2, and preferably the secreted Colbtfp polypeptide is produced by the expression of a,nuclei crack! sequeoee that is at least 98%, 99% or 100% .identical to the nucleic acid sequence of SEQ ID NO; 2.

In certain embodiments, the secreted Oo!ot-p2 polypeptide has an amino acid sequence that is at least 9b%, 99% or 100% identical to·' ap amino acid sequence selected frogs SEQ ID NO: I and SEQ ID NO: 4,

In certaih aspect, the invention providevan inremnoussay for determining the presence of any one of the polypeptides haying an amino acid sequence as set forth in SEQ ID NOs: ί and 3-4 in a biological sample. The method includes obtaining· a biological sample and contacting the sample with: an antibody specific for -a polypeptide having an amino acid sequence as set forth In SEQ ID NOs:: 1 and 3-4 and detecting the binding of the antibody;

In some aspects, the application provides methods for the detection of a moiecular marker in a bi ologica! sample such as blood, i.ncludi i ig blood if actions: sueh as serum or plasma. For instance,· the blood sample obtained from a patient may he further processed such as by ffactionation to; obtain blood scroop and the serum may then be enriched for certaih polypeptides.. The serum so enriched Is then contacted with an antibody that Is reactive with an epitope of the desired marker polypeptide

In yet another embodiment, the application provides methods for· determining the appropriate therapeutic protocol for a subject. For example, detection of a neoplasia provides· the treating physician valuable infomiation In determining whether intensive:or invasive protocols: such as surgery or chemotherapy·would: be heeded for effective diagnosis or treatment. Such detection would he helpful not only tor patients not previously diagnosed with a neoplasia but also in those: cases where a patient lias previously received or is currently receiving therapy for eatinefe the presence or absence or a change in the level of the molecular markers (e.g., ColoUp2) set forth herein may be indicative that the subject Is liMy to have a relapse or a progressive, or a persistent a cancer.

In yet other embodiments, the application provides a Ml for detecting neoplasias in a biological sample, Such kits include one or more antibodies that are capable of interacting with an epitope specified by erne of SEQ ID NDs; I and 3-4- In more preferred embodiments, the antibodies may be delectably labeled, such as fer example with an enzyme, a tlutirescent snbsfance, a chemiluminesceht substance, a chromophore, a radioactive isotope or a oomplexing agent.

In certain aspects, the application provides methods for inhibiting the growth or proliferation of a neoplasia in a subject, the method comprising administering to the subject an agent that decreases the amount of a Ct>!oUp2 .polypeptide present m or 'jiie^uced.fey;the-^edplasi'a* Optionally, the polypeptide is a secreted polypeptide (e.p., SEQ ID NO: 1 or 4), Optionally, the agent is an siRNA probe (or an RNAi construct) that hybridizes to an mRNA encoding a CoSoUp2 polypeptide. In preferred embodiments, the siH'NA probe hybridizes to a nucleic acid that ss at least 90%, 95%, 9S%, 99% or 100% identical to a oudeie acid sequence of SEQ ID NO: 3, Optionally, the agent is an. antisense probe that hybridises to a: nucleic acid encoding a ColoUp2 polypeptide. In preferred embodiments, the antisense probe hybridizes to a nucleic acid that is at least 90%, 95%, 9fc%, 99%* or 100% identical to a nucleic acid sequence of SEQ ID NO: 2. In certain embodiments, the agent comprises a nucleic acid vector that causes the production of a sIRNA or an antisense probe that hybridizes to a nucleic acid encoding a ColoUp2 polypeptide. Exemplary neoplasias rnav be of a tissue type selected from endometrium. kidney, lung, stomach, pancreas, breast, prostate, ovary, uterus, and thyroid.

In certain aspects., the application provides a method lor inhibiting the growth or proliferation of a cell of a neoplasm m a subject, the method comprising administering to the subject an agent that binds to and antagonizes a CoIoUp2 polypeptide (e.g., a polypeptide selected from SEQ ID NQs: 1 and 3 % h< some embodiments, the agent comprises an antibody that binds to a CoioUp2 polypeptide. Optionally, the antibody is a monoclonal antibody, a polyclonal antibody or a single chain antibody. Optionally, the antibody is a humanized antibody. In certain embodiments, the agent is a small molecule that binds to a ColoUp2 polypeptide, and preferably a small molecule that inhibits an activity of a CoiolIp2 polypeptide. For example, binding (which may be assayed as ceil surface binding) by a secreted CoioUp2 polypeptide (e.g., SBQ ID NO: 1 or 4).

In certain aspetS* Molecular markers of neoplasias may be used to: target therapeutic agents to cells of a neoplasia. In certain .embodiments, a theTapeutie agent that is targeted to a neoplasia comprises a targeting moiety and an active moiety, Wbdreih the targeting moiety binds to a ColplJp2 polypeptide ami wherein the active moiety facilitates the killing or growth inhibition of a eel! of a neoplasia. Optionally, the targeting moiety comprises tm antibody. lit preferred embodinientSs the antibody binds to a polypeptide selected from SBQ ID NOs; I and 3-4, Optionally, the antibody is selected from a monoclonal antibody, a polyclonal antibody, a single chain antibody. In certain embodiments, the antibody is a humanized antibody. The nuive moiety .maybe, for example, .a toxin, a chemotherapeutic agent, or an agent that sensitizes the cell to a ehemoiherapeutic agent or radiation. Exemplary neoplasias may be of a tissue type selected horn endometrium, kidney, hmg, stomach, pancreas, breast, prostate, ovary, uterus, and thyroid. in certain embodiments, the application provides methods of identifying a candidate agent for treating cancers, the method comprising: identifying a candidate agent that binds to and/or inhibits an activity of a Col.o\.)p2 polypeptide in certain embodiments, the method may further comprise testing the candidate agent for antineoplastic effects on a cell of a neoplasia or a cell line deri ved from a neoplasia. The method may further comprise testing the candidate agent for anti-neoplastic effects on a mouse xenograft comprising cells of a human cancer or cells of a cell line derived from a cancer cell line. The candidate agent may be essentially any molecule or complex .material of interest, in clod big,: tor example, a slENA probe, -m antisense probe, an antibody and a small molecule.

In certain embodiments, the application provides use of an agent which reduces expression of the Co!oUp2 gene or reduce activity of a ColoUpa polypeptide in the manufacture of a medicament tor treating a neoplasia in a subject. Exemplary neoplasias may be of a tissue type selected fiom mtdOmetriurn, kidney* lung* stomach, pancreas, breast, prostate, ovary, uterus, and thyroid.

The embodiments and practices of the present invention, other embodiments, add their features arid eharheferistles, will be apparent from the description:, figures and claims that follow, with all of the claims ’hereby being incorporated by this reference into this Summary.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure. I shows the amino add sequence (SEC) ID: NO: 1} of secreted CdoOp2 protein.

Figure 2 shows the nucleic acid sequence t'SEQ ID NO; 2} of €o!oUp2,

Figure 3 shows the amino add sequence {SEQ ID NO; 3) of Full-length GoIolJp2 protein,

Figure 4 shows detection of VS epitope-tagged CeioUpS protein levels in transfected SW4dO cells and VacodSOeelis..(24 howry and 48 bourn a her transfection). Expression of egliepe4&amp;gged ColoOp.2 protein in transfected cells by Western Mot (right panel), and secretion of epitope-tagged €oloUp2 protein in growth media by serial Imronoopredpitstion and Western blot (left panel)

Figure S shows a western blot of VS tagged GoloDpa protein fey ^fi ve arshbody. I..ane 1: media supernote from SW48Q colon cancer cells transfected with an empty expression vector, Lane 2: media sepernate ton €h;dolIp2-VS expressing cells. Lane 3 ; size markers. Lane 4 .shows assay of senna from a mouse Kenografted with control SW4S9 cells eomespondiog to lane 1, Lanes 5 attd b show detection of bircalating ColoUp2 proteins in blood front two truce bearing human colon cancer xenografts drop ColpDpd- VS expressing SW48C) colon cells shown; ip lane 2.. (felotipl is secreted .as an SSICD apd a companion 3SKD size protein.

Figure 6 shows, in the upper panel, the purification of ColoUp2 protein. Shown is a Coomassie blue staining of 2.i()og (lane 2a) and SOGng (lane 3a) of a purified ColoUp2 protein preparation. Size markers ere In lane la.

Figure f shows the amino acid sequence (SEQ ID NO: 4) of the approximately SS kDa C-fermioaf fragment of €ofefjp2 that is a prosnineni secreted and serum ibrm o! ONoUp2,

Figure 8 «hows ColoUp2 expression in various cancer types m dm blot experiments.

Figure 9. shows; Colo;Up2 expression in various cancer types in Immune» histochemicaJ experiments. For example, top lefl: is endometrial cancer, top center is pancreatic cancer, top right is stomach cancer middle left is colon cancer, middle center is ovarian cancer, middle right is renal cell cancer, bottom left is breast cancer, bottom center is lung cancer, and boiioni right is prostate cancer, DETAILED DESCRIΡΊ10Ν t .IMMimns:

For convenience, certain terms employed m the spedlkatlon, examples, ant! appended; claims are collected here. Unless defined' otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

The tullcles "a” and are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, On element5' means one element or more than one eiesnou.

The term ”tmhbodyft as used herein is intended to include whole antibodies, e.g., of any isotyne (IgG, IgA, IgM, Igb, etc), and includes fragments thereof which are also specifically reactive with a vertebrate, e.g., mammalian, protein. Antibodies can be fragmented using conventional techniques and the fragments screened tor utility and/or interaction with a specific epitope of interest. Thus, the term includes segments of pr o to ο I y t i e a 11 y - cfoa ved or reeornbinaruly-prepared portions of an antibody molecule that are capable of selectively reacting with a certain protein. Morn limiting examples of such proteolytic and/or recombinant fragments mclude Fab, Ffab:)2, Faff , Fv. and single chain antibodies (scFv) containing a VTL] and/or VfHj domain joined by a peptide linker. The seFv's may be covalently or non-covalently linked to form antibodies having, two or more binding sties. The term antibody also includes polyclonal, monoclonal, or oilier purified preparations of antibodies and recombinant, an t i bodies..

The term v<ColoUp2'" is used to refer to a nucleic acid encoding a ColoUp2 protein or a Cok>Up2 protein itself, as well as distinguishable fragments of such nucleic acids and proteins, longer nuciesc acids and polypeptides that comprise distinguishable fragments or full length nucleic adds ior polypeptide and variants thereof! Variants include polypeptides that are at least identical to the human CdlollpS protein sequence referred to in the application, and nucleic acids encoding such variant polypeptides,. In addition, variants include different post-translational modifications. such as glyooxylations, methylaiions, etc. Particularly preferred vplants;inelurle any pa&amp;ra% dqe«^'dg''V-aiipts, such as allelic differences, amiations that: occur in a neoplasia and secreted or processed forms. The terms 'Variants” and “Ikagmentsf are overlapping.

As used herein, the phrase “gene expression” or “protein expression” includes any information pertaining to the amount of gene transcript or protein present in a sample, as well as information about the rate at which genes or proteins are produced or are accumulating or being degraded (e.g., reporter gene data, data from nuclear runoff experiments, pulse-chase data etc.). Certain kinds of data might he viewed as relating to both gene and protein expression. For example, protein levels in a cell are reflective of the level of protein as well as the level of transcription, and such data Is mtended to be included by the phrase “gene or protein expression information”. Such infoihmtipn may be given in the form of amounts per ceil amounts, relative to a control gene ;or protein, in rrnltless measures, eicg the term “information” is not to be limited to any particular means of representation and is intended to mean any representation that provides relevant information. The term “expression levels" refers to a quantity reflected in or derivable from the gene or protein expression data, whether the data is directed to gene transcript accumulation or protein, accumulation or protein synthesis rates, etc.

The term “detection” is used herein to refer to arty process of observing a marker, in a biological sample, whether or not the marker is actually detected. In other words, the act of probing a sample for a marker is a “detection” even if the marker is determined to be not present or below the level of sensitivity. Detection may be a quantitative, scmi-qnantitativc or non-quantitative observation.

The terms “healthy'', “normal” and Dmnmeoplastlc” are used interchangeably herein to refer to a subject or particular cell or tissue that is devoid (at least to the limit of detection) of a disease condition, such as a neoplasia, that is associated with increased expression of a Cololjpd gene.

The ienn '‘including’* is used herein to mean, and is used interchangeably with, the phrase ’‘including hut not limited td>.

As used herein, the term "nucleic acid" refers to polynucleotides such as deoxyribonucleic acid (DMA), and, where appropriate, ribonucleic acid (RNA). The term should also: ho nndmtood to include analogs of either RMA .qr DNA made ffom: nucleotide: analog^, and, as applicable ;to the emhoditoept being described, singlestranded f such as sense or antisense) and. douhie-skanded polynucleotides.

The term ‘‘or” is used herein to mean, and is used ioieretmegeably with, the term "and/or", unless context clearly indicates otherwise.

The. lerp "percent IdmfetF' refers to sequence identity between two amino acid sequences or between two liueledtide sequences. Memos can each be heteopmed by comparing a position ip each sequence which may he aligned for: .purposes· of comparison. When an equivalent position, in the compared-sequencessfis· Occupied by the same base or amino acid, then the molecules are identical at that position;, when the equivalent site occupied by the same .or a similar amino acid residue similar in steric and/or electronic nature), then the molecules can be referred to as homologous (sumlar} at that position. Expression as a percentage of homology/sitmlanty or identity refers to a function of the number of identical nr sinular amino acids at positions shared by the compared sequences. Various elignmorst algorithms and/or prograrns may he used, including FASTA. BLAST or EH’TEBZ. FASTA and BLAST are available as a part of the QCQ sequence::aE»dysis package (University of Wisconsin, Madison, W is .), and cap he used With, e.g„ default settings. Eld'FRET is available: through the National Center lor Biotechnology Tnfemmtton, National Library of Medicine, National institutes ..of Health, Bethesda, Md. in one embodiment* the percent identity of two sequences can be determined by the GCG: program with a gap weight of 1: e.g., each aminp aetd gap is weighted as if it were a single am ino acid or hueleotide mismatch between, the two sequences.

The terms "polypeptide" and "'protein" are used interchangeably herein.

The term ’*purified protein" refers to a preparation of a protein or proteins whsch are preferably isolated from, or otherwise substartdally tree of, other proteins normally associated with the protends) in a ceil or cell lysate. The term "substantially free of other cellular proteins” (also referred to herein as "substantially free of other coota.minai.ing proteins") is defined as encompassing individual preparations of each of the component proteins comprising less than 20% (by dry weight) contaminating protein, and preferably comprises less than S% contaminating protein. Functional forms of each of the component proteins ears be prepared as purified preparations by using a cloned gene as described in the attached examples. By "purified”, it is meant, when referring to component protein preparations: used to generate a reconstituted, protein mixture, that the indicated molecule is preheat in: the substantial absence of other .biological maeremoieeoles, such as ether proteins fparticularly other proteins which may substantially mask, diminish, confuse or alter the characteristics of Ure component proteins either as purified prepar ations or in their function in the subject ruconsfituted mixture;). The term "purified" as used herein preferably means at least. 80% by dry weight more preferably' In the range of 85% by weight, more preferably 95-00%: by weight, and proai. preferably at least 90.8% by weight, of biological maerotnoleoules of the same type present (but water, bufferSj and other small molecules, especially mpiecutes havingm molecular weight pf less; than 5(100, ean be present). The; term "pur®” as msd. teem prefmb'ly.bas· lhe;»pe ««merieaf.· limits as " purified ” form ed I aid y above. A is any nueleie acid that has been pkeed adjacent to another nimleio: acid by recombinant: BMA techniques, A, %ueomMna®t nueleie aeiiT also includes any nueleie acid that has been placed next to a second nucleic acid by a laboratory genetic teehntepe sodh as, for «maniple, transfonuation and Integration, transposon hopping or viral insertion. In general, a recombined nueleie add is not naturally located adjacent to the second nucleic acid.

The .term "reco.rnhi.n.a.rtt protein"' refers to: a protein that is produced by expression front a recombinant nueleie add. A Sisa.mplcT tneludes: any material that is Obtained or prepared tor detection of a molecular marker, or any material that is contacted with a detection: reagent or detection device tor the purpose of detecting a molecular marker. A ‘'subj.eer is any organism of interest, generally a mammalian subject, such as a mouse, and preferably a human subject. 2- Overview

In certain aspects, the hwemion relates to metliods for deterrotoiag whether a subject is likely or unlikely to have a neoplasia (e g., ttoTtor, cancer, anil preeafteer) and markers that may be used to make such determination sail to selected and/or target: ahti-meoplasue therapeutic agents. In other aspects, the invention relates to. methods for determining whether a: patient is likely or unlikely to have a neoplasia, in tissues ineIodiugs bat is not limited to, stomach, pancreas, lung, hreasf, Uterus, ovary, kidney, thyroid, prostate, and endornelrium. la feriber aspects, the itwention relates to methods for monitoring a neoplasia in a subject. In further aspects, the;invention relates to methods lor staging a subject's neoplasia A neoplasia includes any eaneetoas or precaocereas growth located in or derived from any tissue.

Early defection of eaneers, coupled with appropriate mterveuttoe, is important for increatong: patient survival rates, Ih addition, patients who receive surgical Or pharroaetmfical therapy for cancers may .experience a relapse end it would he advantageous tis have aivalteriiadve system for detemiining whether suers patients have a recurrent or relapsed cancer, As an exampte,.a cancer diagnostic system would facilitate. monitoring an increase, decrease or persistence, of a cancer in a patient known to have a eancer. A patient tmdergdiftg chetriOthetopy may he. monitored to assess the effectiveness of the therapy.

Accordingly, in certain embodiments* the invention provides molecular markers (e.g.„ ColoUp2) that distinguish between cells that are not part of a ttooplasia {e,g.5: cancer or preeaneer), referred to herein as “healthy eelifo5, and cells that are part of a neoplasia, referred to herein as “neoplasia ceils-f, For example, certain molecular markers, including ColoUp?, are expressed at sigrufieantly higher levels in cancer cell hues.or tissues (e.g , stomach, pancreas, lung, breast, uterus, ovary, kidney, thyroid, prostate, and endometrium), Optionally, molecular markers of the invention which distinguish between healthy cells and cells of a neoplasia can be used for screening apparently healthy patients to deiemrine whether tins patient: is at Increased risk for (predisposed to) developing a cancer, Furthermore, preferred molecular markers are those that are actually present in the serum of an animal having a neoplasia, and sn general, a secreted protein wall generally occur in the serum if it is secreted from a cell contacting a blood vessel* or a compartment ifc diffeslonai contact with a blood vessel.

In certain specific embodiments, the invention provides methods tor using the GoloUpi molecular marker for determining whether a patient has or does not have a condition characterized by increased expression of Colollps nucleic acids or proteins described herein. In certain embodiments* the invention provides methods lor determining: whether a patient is or is not likely to have a neoplasia. In further emfeodimenis, the invention provides methods ibr determining whether the patient is having a relapse or determining whether a patient's neoplasia is responding to treatment

In certain embodiments, a preferred molecular marker for rise in a diagnostic test that employs a body fluid, sample, such ns a blood or urine sample, or an excreted sample material* such as stool, is a secreted protein* such: as the secreted portion, of a. ColoUp.'! protein. In certain embodiments* a preferred molecular marker for use in a. diagnostic test to distinguish subjects likely to have a neoplasia fe.g., cancer or pmoanccr) from those not likely to have a neoplasia:.½ a gene product of the ColoUp2 gene, Examples of suitable gene products include proteins, both secreted and not secreted and transcripts. In embodiments employing proteins that are secreted,: such as CoioUp2* a .preferred embodiment of the diagnostic, test is a test for the presence of the protein m a body fluid* such as urine or blood or an excreted material, such as stool. It should be noted, however, that intracellular protein may be present in a body fluid if there is significant cell lysis or through some other process. Likewise* secreted proteins are likely to he adherent, even if at a relatively low level, to the- ceils: in which they were produced.

In certain aspects* die disclosure relates to ColoUp? as a diagnostic marker and therapeutic target for cancers.. The ColoUp2 nucleic acid sequence encodes a Iklidength protein of 755 amino acids. Thu application also discloses certain polyahorphisms that have been observed* for exaniple at nucleotide .1.1.2 G€G~*A;CC (Ala-Thr); m 480 GAA—GGA (GImG!y):j and at nt. 2220 CAG-CGG (Gln-Arg). The sequence of ColoUpz protein is similar to that of alpha 3 type VI collagen, isoionn 2 precursor, In addition, a few domains are identified in the Co!oUp2 protein such as a vim· Wlltehrand factor type A domain (vWF) and an EGF-hke domain. The vWF domain is found in various plasma proteins such as some complement factors, the integrim. certain collagen, and other extracellular proteins. Proteins with vWF domains participate in numerous biological events which involve mieraehen with a large array of Uganda for example, cell adhesion, migration, homing, pattern formation, and signal traosdtrotsO». The EGF-Uke domain consisting of about 30-40 amino acid residues has been found many proteins. The functional significance of EGF domains is not yet dear, However, a common feature is that these EGF-like repeats are found in the extracellular domain of membrane-hound proteins m in proteins:known to he secreted.

As demonstrated herein, ColoUp2 is .secreted from both the apical and basdlateral surfaces of intestinal cells, and can be round in the blood in two different ferni%a fuII4eugi.it secreted· form and a Cb terminal fragment {approximately 55 EDf).

In. certain eoibodimeut:S:i the application provides Isolated, purified or recombinant GotoOpS noeleic acids. In certain embodiments, such mieloie acidsmay encode a complete or partial Co!oUp2 polypeptide or such nucleic acids may also be probes or primers usethI for methods involving detection or amplification of Colotipa nucleic acids. Irr certain embodiments, a ColoUp2 nucleic acid is single-stranded or unable-stranded and composed of natural imcleie acids, nucleotide analogs, or mixtures thereof in oestaln embodiments, the application provides isolated, purified or recombinant nucleic acids comprising a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence of SEQ ID NO: 2, or a complement thereof, and optionally at least 95%. 97%, 98%, 99%, 99,3%, 99.5%, 99.7% or 100% identical to a nucleic acid of SEQ ID NO: 2, or a complement thereof. In certain preferred embodiments, the application! provides a isolated, purified or recombinant nucleic acids comprising a nucleic acid sequence that is at least 90%, 95%. 97%, 9S%, 99%, 99 3%,, 99,5%, 99.7% or 100% identical to a nucleic acid of SEQ ID NO, 2. or a complement thereof. In certain embodiments, the application provides isolated, purified or PeeOmbtUam'nuoIei:e..acids comprising a nucleic acid sequence that encodes a polypeptide that is at least 90% identical to an amino acid sequence of any of SEQ ID NOs: I or 3-4, or a complement thereof, and optionally at least 95%, 97%, 98%, 99%, 99.3%, 99,5%, 99.7% or 100% identical to an amino acid sequence of any of SEQ ID NOs: I or· 3-4, or a complement thereof; in further embodiments, the application provides expression constructs, vectors and cells comprising a Ciolotlpi nucleic acid. Expression constructs are bttekfe acid constructs that are designed to permit expression: of an expressible nucleic add (e.g.„ a ColoUp2 nucleic acid) in a suitable cell type or in vitro expression system, A variety or expression construct systems are, in view of ibis specification, well known in the art, and such systems generally include a promoter that Is operabiy linked to the expressible nucleic acid. The promoter may boa eonsfiiuii ve promoter, as in the ease of many vim! promoters, or die promoter may be a conditional promoter, as in the case of the prokaryotic iad-repress ible.. lETO-inducible promoter and as in the case of the eukaryotic tctracydine-'induerblc promoter, Sectors refer to any nucleic acid that is capable of transporting another nucleic acid to winch it has been bnked between different cells of viruses. One type of vector:is an episome, i .e„.a nucleic acid capable of extra-chromosomal replications such as a plasmid, Episome-type vectors typically carry an origin of replication that directs replication of the vector in a host ceil. Another type of vector is an integrative vector that is designed to reebmbine with the generic material of a host cell. Vectors may be both autonomously replicating and integrative, and the properties of a vector may differ depending on the cellular context (i,e,5 a: vector .may he autonomously replicating in one host cell type and purely integrative In another host cell type). Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”. Vectors that carry an expression construct are generally expression vectors. Vectors have been designed for a variety of cell types. For example, in the bacterium E. colt, commonly used vectors include pUC plasmids. pBR.322 plasmids. pBlueScript and ΚΊ13 plasmids, in insect cells (e.g. SF-9, SF-21 and High-Five cells)., commonly used vectors include BacFakd (Clontech) and BaculoOold (PhanrringcuS (both Clonfeeh and Pkarmingen are divisions of Becton. Dickinson and Co.. Franklin Lakes. Mew Jersey), hi mammalian cells (e.g. Chinese hamster ovary (CBD) dells, ¥aeo cells and human embryonic kidney (HEIC) cuffs), commonly used vectors include pCMV vectors (Stratugene, lae., La Jolla, .California), arid pRK. vectors. In certain embodiments, the application provides cefls that comprise a ColoUp2 nucleic acid, particularly a recombinant ColoUp2 nucleic acid, such as m expression construct or vector that comprises a Coloi!p2 nucleic acid.

Cells may be eukaryotic or prokaryotic, depending on the anticipated use. Prokaryotic cells, especially E, coli, areparticularly useful for storing -a«d;t^lie&amp;fbg. nucleic adds* partieularly nucleic acids carried os. plasmid or viral vestors. Bacterial cells are also partrculariy useful for expressing nucleic acids to produce large quantities of recombinant protein, but bacterial cells do not usually mimic eukaryotic post-translational modifications, such as glycosylations or lipld-modi flections, and so will tend to be less suitable tor production of proteins in which the posn-frinislational modification state is significant Eukaryotic cells, and especially cell types such as msec! cells that work with baculovlrus-based protein expression systems, and Chinese hamster ovary cells, are good systems for 'expressing eukaryotic proteins that have significant post-translational modifications. .Eukaryotic cells are also useful for studying various aspects of the hmefien of eukaryotic proteins.

In certain aspects the application further provides, .methods ter preparing: CuloUp2 polypeptides, hi general; such methods comprise obtaining a Cell that comprises a nucleic acid encoding a ColoUpl polypeptide, and culturing the cell under conditions that cause production of the ColoUp2 polypeptide. Polypeptides produced in this manner may be obtained from the appropriate cel! or culture fraction. For example, secreted proteins are most readily obtained from the culture supernatant, soluble miracdlutur proteins arc most readily obtained from the soluble traction of a cell lysate, and membrane proteins are roost readily obtained horn a membrane fraction. However, proteins of each type can generally be found in all three types of cell or culture fraction. Crude cellular or culture fractions may be subjected to funner punficaiion procedures to obtain substantially purified Cok>Up2 polypeptides. Common purification procedures include affinity purification (e.g., with hexahisridioe-iagged polypeptides}, ion exchange chromatography, reverse phase chromatography, gel filtration chromatography, etc.

In certain aspects, the application provides recombinant, isolated,, substanrially pimped dr purified ColoUp2 polypeptides. In certain embodiments, such polypeptides may encode a complete or partial Co!oUp2 polypeptide. In certain embed intents, a CoioUp2 polypeptide is composed of natural amino acids, amino -add analogs, or mixtures thereof ColoUpa polypeptides may also include one or more post-translational modifications, such as glycosylation, phosphorylation, lipid modification, acetylation, etc. In certain embodiments, the application provides isolated, substantially purified, purified or recombinant polypeptides corppriaing m aniino acid ser|aeftoe that is at least 90% identical to art amine add ..'sequence· of any of SEQ ID NOs; 1 or 3-4 and optionally*! least 95%, 97%, 98%, 99%, 99.3%, 99.5% or 99.7% identical to a nucleic acid of any of SEQ ID NOs: 1 or 3-4, In certain preferred embodiments, the application provides an isolated, substantially purified, purified or reconibinant polypeptide coMprising an anitoo acid sequence: 'feat differs. from SEQ ID NGs; I or 3-4 by no more than. or deletions.

Optionally, a polypeptide of the invention comprises an additional moiety, such as an additional pclyT.5ept.ide sequence or other added compound, with a particular function, such as an epitope tap that facilitates detection of the recombinant polypeptide with an antibody, a purification rftplety that facilitates purification (e.g,, by affinity purification), a detection moiety, that facilitates detection of the polypeptide in vivo or in vitro, or an antigenic moiety that Increases the antigenicity of the polypeptide so as to facilitate antibody production. Often, a single moiety will provide multiple functionalities. For example, an epitope tag will generally also assist in purification, because an antibody that recognizes the epitope can be used in an affinity purification procedure as well. Examples of commonly used epitope tags are', an BA tag, a bexahistidme tag, a ¥5 tag, a Olu-Glu fag, a e-mye tag, a VSV-C3 tag, a FLAG tag, an enierokmase cleavage site tag and a T7 tag. Commonly used purification moieties include: a box ab I at id me tag. a glutathione-S-transfenise domain, a cellulose binding domain and a biotin tag. Commonly used detection moieties include fluorescent proteins (e.g., green fluorescent proteins), a biotin lag, arid chromogenic/fluorogenic· enzymes (e.g., beta-galaetosidase and ktcifcrase). Commonly used antigenic moieties include the keyhole limpet hernocyanin sod serum albumins. Note that these moieties need not be polypeptides and need not be connected to the polypeptide by a traditional peptide bond.

Another aspect of the invention pertains to an antibody specifically reactive with a Co!oUp2 polypeptide that is effective for decreasing a biological activity of the polypeptides. For example, by using immunogen*» derived from a Cok>Up2 polypeptide, e.g., based on the cDNA .sequences, anlfq>rotein/anti-peptide antisera or monoclonal antibodies can be made by standard ..protocols (See, for example, Antibodies·: A. Laboratory Manual eel, by Harlow and: Lane (Cold Spring Harbor Press: 1988)), A. mammal* such as a mouse, a hamster or rabbit can be immunized with an immunogenic form of the peptide (e.g., a €otoUp2 polypeptide or an antigenic fragment which is capable of eliciting an antibody response, or' a fusion protein). Techniques for conferring immunogemetfy on a protein or peptide mduclb confngaiieti to carriers or other techniques well known in the art. An imminiogenie portion of a CofoUp2 polypeptide can be administered in the presence of adjuvant. The progress of immunization can be monitored by detection of antibody titers in plasma or scan». Standard EUSA or other immunoassays can be used with the irmuunogen. as antigen to assess tbs levels of antibodies. In a preferred embodiment, the,subject antibodies are imrnnnospeeiiic for antigenic determinates of a Colo!Jp2 polypeptide: of a mammal, e..g,5 antigenic determinants of a protein set; forth in SEQ ID NOs: I and M,

In one embodiment, antibodies are specific for the secreted proteins as encoded by nucleic acid sequences as set forth in SEQ ID NO; 2. hi another embodiment, the antibodies are irnmuooreaetjve with one or more proteins having an amino acid sequence that is at least 80% identical to an amino acid sequence as set forth in SEQ ID NOs: 1 and 3-d. in other embodiments, an antibody is immunoreadive with one or more proteins having an amino acid sequence that is at least 85%, 90%. 93%. 98%, 99%, 99.5%,, 99.5%, 99.7%, identical or 100% identical to ah amino acid sequence as set forth in SEQ ID NOs: 1 or 3-4. In cerium preferred •embod:imen.ts,:'the invention provides an antibody that binds to an epitope including the C-terminal portion of the polypeptide of SEQ ID NOs: 1 or 3-4. In certain preferred embodiments, the invention provides an antibody that binds to an epitope of a ColoUp2 polypeptide that is prevalent in the Mood of an animal having a cancer, such as SEQ ID NO: 1 or 4.

Following immunization of an animal with an antigenic preparation of a CoioUpz polypeptide, ami~ColoUp2 antisera can he obtained and, if desired, polyclonal ami-€oloUp2 antibodies can be isolated front the serum. To produce monoclonal antibodies, antibody-producing cells (lymphocytes) can he harvested from an immunized animal and fused by standard somatic cell fusion procedures with immortalizing cells such as myeloma cells to yield frybndpma cells, Bueb techniques are well known 1» the art, and include, far e&amp;aifrpie, the feyfendoma technique (originally developed by Kohler and Milstein, (1975} Nature, 256; 495-497), the human B cell hybridoma technique (Kozbar et at, (1983) Immunology Today, 4; 77.), and the EBY-hybridoma technique to produce human monoclonal antibodies (Cole et ah, (1985} Monoclonal Antibodies and Ca&amp;cer Therapy, AlaaR, Lists, Inc. pp. 77-96). Hybridoma cells can be screened tmmunochemjeally for production of antibodies specifically reactis'e wnh a mammalian CofoUp2 polypeptide of the present invention and monoclonal antibodies isolated from a culture comprising such hybridoma cells. In otie entbodimem anti-human ColoUp?, antibodies specifically react with the protein ©«coded by a nuclei© acid having SEQ ID NO: 2; snore preferably the antibodies specifically react with a secreted GoloUp2 protein that is produced by the: expression of a nucleic acid having a sequence of SEQ ID NO: 2.

The term ‘'antibody” as used hereto is intended to include fragments thereof which are also specifically reactive wtth one of the subject GoloUp2 polypeptides. Antibodies can be fragmented using eonvcnhonal techmqtws add the fragments screened for utility in the sanie manner as described above fo; whole antibodies. For example. Ffubjy fragments: ©an he generated: by treating antibody with pepsin. The resulting Fiabv? fragment can be treated to reduce disulfide bridges to produce Fab fragments. Tbs antibody of the present invention is further intended tc include bispecific, single-chain, and chiroeric and humanized molecules having affinity for a Cc!oUp2 polypeptide conferred fry at least one CDR region of the antibody. In preferred embodiments, the antibodies, the antibody- further comprises a label attached thereto and able to be detected, (e.g, the label can, be a radioisotope, fluorescent .compound,-enzyme or enzyme co- factor),

In certain preferred embodiments, an antibody of the invention is a monoclonal antibody, and in certain embodiments the invention makes available methods for generating novel antibodies. For example, a method for generating a monoclonal antibody that binds specifically to a ColoUp2 polypeptide may comprise administering to a mouse m amount of an immunogenic composition: comprising the Golollpi liolypeptide effective to stimulate a detectable immune response^ obtaining antibody-producing cells (e.g., veils from the spleen) from the mouse and fusing the antibody^mdueing ceils with myeloma cells to phtaia antifepdy-predhemg hyhridbmaa, and testing the antibody-producing hyhridomas to identify a hyhrldema that produces a monocolonal antibody that binds specifically to the ColuUp2 polypeptide. Once obtained, a hybridoma can be propagated in a cell culture, optionally in culture conditions, where the hybridoma-derived cells produce the iinanocinnal antibody that binds specifically to the €olaUf>2. polypeptide. The monoclonal antibody may be -purified fem the cell eutare,

Anti-ColoUp'2 antibodies can he used, e.g,, to detect ColoUp2 polypeptides in biological samples and/or to monitor ColoUp2 polypeptide levels in an individual, for determining whether or not said patient is likely to develop a cancer or is more likely to harbor a cancer, or allowing deterntisiabon of the efficacy of a given treatment regimen for an individual afflicted with cancer. The level of Co!oUp2 polypeptide may be measured in a variety of Sample types such as, for example, In cells, stools, and/or in bodily fund, suds as in whole blood samples, blood serum, blood plasma and urine. The adjective ''specifically reactive with" as used in reference fo an antibody Is intended to mean, as is generally understood in the an, dun the antibody i$ suflcienily selective between the antigen of interest {e.g,, a ColoUpS polypeptide) and other antigens that are not of Interest that the-antibody is useful lor, at minimum, detecting the presence of the antigen of interest in a particular type of biological sample. In certain methods employing the antibody, a higher degree of specificity in binding may be desirable, for example, an antibody for use in detecting a low abundance protein of interest pt the presence of one or more very high abundance protein that are not of interest may perform better if if has a higher degree of selectivity between the. antigen of interest and other cross-reactants. Monoclonal antibodies generally have a greater tendency (as compared to polyclonal antibodies) to uiscrmhnate effectively between the desired antigens and cross-reacting polypeptides.: In addition, an antibody that is effective at selectively identifying an antigen of interest tn one type of biological sample (e.g., a stool sample) may not be as effective for selectively identifying the same antigen m a different type of biological sample (e.g., a blood sample). Likewise, an antibody that is effective at identifying an antigen of interest in a purified protein preparation that is devoid of other biological contaminants may not be as effective at identifying an antigen of interest in a crude biological sample, such as a blood or urine sample, Accordingly, in preferred embodiments, the application provides antibodies that have demonstrated specificity for an antigen of interest (e.g., a Cok>Up2 polypeptide)-ip a: sample type that is likel y to be the sample type of choice for use of the antibody, in a particularly preferred embodiment, the application provides antibodies that bind specifically to a CofoUfiZ polypeptide in a protein preparation irons biped (optionally serum or plasma)' from a patient that has a cancer or that: hind specifically in a crude blood sample (optiprally aerede serem or plasma sample)

One «haracterislk that influences the specificity of an antibody; anti gen interaction is the affinity of the antibody for the antigen. Although the desired specificity may be reached with a range of different affinities, generally preferred antibodies will have on affinity (a dissociation constant) ofahoiti 1010"',. Iff5, H3r or less.

In addition, the techniques used to screen antibodies in order to identify'' a. desirable antibody may influence the properties of the antibody obtained. For example, an antibody to be used for certain therapeutic purposes will preferably be :able to target a particular cell type, Accordingly, to obtain antibodies of this. type,: it may be desirable to screen for antibodies this? bind tb cells that express the antigen of interest ic.g., by fluorescence acre uteri ceil sorting) likewise, if an antibody is to be used tor bindtng.an antigen in solution, it may be desirable to test Solution binding. A variety of different techniques are available (or testing anhbodv:antigen interactions to identify partieulirly desirable antibodies. Such teehniqnes: include, EbfSAs, surface plasmon resonance binding assays (e.g., the Bracers binding assay, Bk-eora. AB, Uppsala, Sweden), sandwich assays (e.g.. the paramagnetic bead system of 1GEN International, Inc., Gaithersburg, Maryland), western blots, ururtenopi'eoiphation assays and nnniunohismehernistry.

Another application of ,anti-Cp1p.Up2' antibodies of the present invention is in the: iMoiunoloiieal screening of eDMA libraries: constructed in expression vectors such as gtli, gU8~23. ZAP. and C3B:bS, 'Messenger libraries of this type, having coding sequences inserted in the correct reading frame and orientation, can produce fusion proteins. For instances gti I will produce fusion proteins whose amino termini consist of B-gajaciosidase amino acid sequences and whose carhoxy termini consist of a foreign polypeptide. Antigenic epitopes of a €oioU$>2 polypeptide, e,g,, other ortitologs of a particular protein or other para logs from the same species, ears then be detected with aufibodies, such as, for example, reacting nitrocellulose filters lifted from infected plates with the appropriate anti-ColoUp'2 antibodies. Positive phage detected by this assay can then he isolated from the infected plate Thus, the presence .of QoloUp2 homo logs can be detected and cloned irotn other animals, as can alternate Isoferras (including splice variants) ftett .hitmans, 4, Mctlloas...feh..Detectjnv,,Mqlecn|a

In certain embodiments, the invention provides methods for detecting molecular markers, such as proteins or nucleic acid transetipis of the CofoOpT markers described herein. In certain embodiments, a method of die invention comprises providing a biological sample aud: probing die biological sample for the presence of a Col o!Jp:2 marker, Informalign regarding: the presence or absence of the CcloUp2 marker, and optionally the quantitative level of the Co)oUp2 marker, may then be used to drm.v inferences about the nature of she biological sample and, if the biological sample was obtained from a subject,

Samples for use with the methods described beretb may be essentially any biological materia! of interest, for example, a sample may be a tissue sample ik>m a subject, a bald, sample from o subject, a solid or semi-solid sample from a subject, a · primary cell culture Or tissue culture of materials defived from a subject, cells ftoni a coll line, or medium or other extracellular material from a coll or tissue endure, or a xenograft (meaoiog; a sample of a cancer fern a fitM subject, such as a human, that has been cultured in a second subject, e g., on immunocompromised moose). The term 'sample" as used herein is intended to encompass both a biological material obtained directly from a subject (which may he described as the primary Sample} as well as. any manipulated forms or portions of a primary sample. For example, in certaiu embodiments, a preferred fluid sample is a blood, sample, fit this; ease, llte term sample is intended to encompass not only the blood as obtained directly from the patient hat also fractions of the blood, such as plasma, serum, cell fractions fe,g,, platelets, erythrocytes, lysnphoeytes),:proteih .preparations, nucleic acid preparations. etc. A sample may also be obtained by contacting a biological material with an exogenous liquid, resulting in the production of a lavage liquid containing some portion of the contacted biological material. 'Fnrtfeermore, the term “sample” is intended to encompass the primary sample after it has been mixed with one or more additive, such as preservatives, eheknmx, atdt-dotimg factors, etc. In certain embodiments, a fluid sample is a urine sample, In. eertain embodiments, a prefared solid or semi-solid sample is a stool satsple. In oertsin eiPbsdiments, a preferred tissue sampte is a biopsy from a tissue known to harbor or suspectedofharboring a neoplasia. In certain embodiments, a preferred cell editor© sanple Is a; sample comprising cultured cells of a cancer ©ell line. A gubjeet is preferably a human: subject, hot it: is expected that the: molecular markers disclosed: herein, and particularly their horuologs from other auirnalx, are of simlldr utility in other animalm In certain embodiments, it maty be possible to detect a marker directly in an organism without obtaining a separate portion of biological: material. In such instances, the term Sample is intended to encompass that portion of biological material that i.\ contacted with a reagent or device involved m the detection process, in certain embodiments, a method of the mveptlen comprises detecting the presence of a CploilpA protetu in a sample. Optionally, the method Involves obtaining a quantitative measure of the CoioUp2 protein in tee sample. In view* of this specification, one of skill in the art will recognise a wide range of techniques that may be employed to detect and optionally quantitate the presence of a protein, hi preferred embodiments, a Co!oUp2 protein is detected with an antibody. Suitable antibodies arc described in a separate section belpw, In many embodiments, an antibody"based detection assay involves hpaging the sample and the antibody Into contact so that the antibody has an opportunity to bind to proteins having the corresponding, epitope. In many embodiments, an antibody-based detection assay also typically involves a system for detecting the presence of antibddy-epitepe complexes, teernhy aehieymg.a detection of the presence of the prqietes haying the corresponding epitope. Antibodies may be used in a variety of detection techniques, ineluditig enzyme-linked immunosorbent assays (hLlSAs), smmunoprecipaations. Western blots. Antibody-independent techniques for 'identifying a protein may also he employed. For example, mass speotrosePpy, particularly coupled with liquid -.iis^i»a.ta:graph^ permits detection and quantifieoion of large numbers of proteins in a sample, Two-dimensional gel electrophoresis may also be used to identify proteins, and may be coupled with mass spectroscopy or-other detection techniques, such as N~ terminal protein sequencing, RNA aptamers with specific binding for the protein of interest; may also be generated and used as a detection reagent.

In certain preferred embodiments, methods of the invention involve detection pf a secreted form of a CoIoUpd protein.

Samples should generally he prepared in a manner that is consistent with the detection spfem to be employed. For example, a sample to he used in a protein, detection system: should generally be prepared in the absence of proteases, likewise*, a sample; to be used in a. nueieic acid detectmnsystem should generally he prepared in the ahseuce -pi npeleases, In many instances, a sample for use in an antibody-based detection system will not be subjected to substarniai pTOpamtory steps. For example, urine may he used directly, as may saliva and blood, although blood will, in certain preferred embodiments, be separated into It actions such as plasma and serum.

In certain embodiments, a method of the invention comprises detecting the presence, of a ColpUf 2 expressed npelele acid, sueh as an rnRNA, ip a sample. Optionally, the method involves obtaining a qosniitadye measure of the; Coloppl expressed nueieie acid in tire sample. One of skill in the an will recogmze a wide range of techniques that may be employed to detect and optionally quantitate the presence of a nucleic acid. Nucleic acid detection systems generally Involve preparing a purified nucleic acid fraction of a sample, add subjecting,the sample te a direct detection assay or arcarnpllileatfon process followed by a detection assay. Anmlibcation may be achieved, tor example, by polytuerase chant reaction 1PCR), reverse transcriptase (RT) and coupled RT-PCR. Detection of a nucleic add is generally accomplished by probing the purified nucleic acid traction with a probe that hybridizes to the nucleic acid of interest, and In many instances 'detection involves an amplification as well blorshem blots, dot blots, tpioroan'ays, quantitative; PGR: and quantitative RT-PCR are all well known methods for detecting a nucleic acid in a sample, to certain embodiments, the invention provides nucleic acid probes that; bind specifically to a CoioUp2 nucleic aeid. Such probes may be labeled with, for example, a fluorescent moiety, a radionuclide, an enrpne or an affinity tag such as a biotin moiety. For example, the TaqtVian®· system employs nucleic acid probes that are labeled in such a way that the fluorescent signal is quenched when the probe is free in solution and bright when the probe is incorporated into a larger nucleic acid.

In certain embodiments, the application provides methods for imaging tumors or cancerous tissues by targeting aotihodtes. to the €olo!Jj>2 marker:, ThO: €bloUp2 marker may be targeted using monoclonal antibodies which may be labeled with radioisotopes for sliuleal.imaging of tumors or with toxic agents to destroy them.

In other enthodlhients, the application provides methods for adhiinistetihg a imaging agent comprising a targeting moiety and an uefive moiety. The targeting: moiety may he an antibody. Fab. F(Ah)2s a single chain antibody or other binding agent that interaeta with an epitope specified by a polypeptide sequence having an amino acid sequence as set forth ih SHQ ID NOs; l or 3:-4, lire active nkuetv may be a radioactive agent, such as: radioactive heavy metals such as Iron chelates, radioactive chelates of gadolinium or manganese, positron emitters of oxygen, nitrogen, non, carbon, or gallium, 4?K, '"Fe, ‘‘'Co. ;>7Cu, ^Ga, Ga, f~'L ^T, s>ii, ijfi,,or <;ν70. The imaging agent is administered in an amount effective tor diagnostic use in a mammal such, as a human and the localisation and acepmuhtion of the .imaging· agent is then detected, The loealkation and accumulation Of the imaging agent may be detected by radioscintigraphy, nuclear magnetic resonance imaging;, computed tomography or positron emission tomography.

Innnunoscindgmphy using monoclonal antibodies directed at the ColoUp2 marker rnay be used to detect and/or diagnose tumors op cancerous tissues. For exaniple, monodmtai antibodies against the ColoUpl marker labeled with ..^Technetium, ”5Indium, !"'Iodine-may be effectively used foi such .imaging. As will be evident to the .skilled artisan, the amount of radioisotope to be administered is dependent upon the radioisotope, Tfeds&amp; MVing. Ordinary- 'skill in the art can readily IbrJpulatefbe amoiiut of the Imaging: a§#fi to be administered based upon the specific activity and energy of a given radionuclide used as the;: active moiety. Typically 0,1 ~ 100 milbcuries per dose of imaging agent, preferably I-10 milheuries. most often 2-5 rniHieuries are administered. Thus, compositions according to the present invention useful as Imaging agents comprising a targefing molety conjugated: to a radioactive moiety comprise 0.1-100 miHiennex, In some embodiments preferably 1-10 millt curies, in some embodiments, prefemhfy 2-5 mlilieeries, in some embodiments more preferably 1 ~S mitliames, 5.

In certain embodiments, the invention relates to methods for identifying CoioUpa proteins that elicit tm Immune response in subjects, in one aspect these ismmrnogenie ColoUp2 polypeptides have an amino acid sequence that ts at least 00%. 95%, or 98-99% identical to the ammo acid sequences ns set forth in SEQ ID NOa; 1 or 3 -4. In certain embodiments, such proteins may be suitable as components· in a vaccine or for the generation of antibodies that may be used to treat cancers.

In certain embodiments. ColoUp2 proteins that elicit a Immoral response may be Identified as fellows. Sera and/or tissue are obtained from a subject that has been treated for a cancer by immunotherapy. Proteins from the cancer tissue sample will be contacted will» antibodies (either punned or in crude serum) to identify proteins that react with foe antibodies. The sera or tissue may he obtained, for example, from, a center involved in cancer immunotherapy, in one embodiment, CofoUp2 proteins that elicit: a humoral response· may be identified by contacting proteins isolated from a cancer sample with antibodies obtained bom the serum (or simply serum itself or fractions thereof) of a subject having a cancer. Proteins that react with an antibody from the subject having a cancer are likely to be proteins that elicit a humoral response, Optionally, the reactivity of proteins is tested against serum or antibodies from a subject not having a cancer as a comparison, and preferably the antibodies or serum are from the same subject, but at a point m time when the subject did not have a cancer.

For these methods, proteins may be analyzed m any of the various methods, described herein or by other methods that, in view of this specification, are considered to be appropriate by one of skill m the art.

As discussed above, exemplary Co!oIJp2 polypeptides Include SEQ ID HOs; 1 and 3-4 Colotip2 polypeptides are further understood to include variants* such as variants of SEQ ID hi Os: 1 and 3 -4.

in another aspect, the invention provides polypeptides that are agonists or antagonists of a CoioUp2 polypeptide. Variants and. fragments of a CJoloOpS polypeptide may have a hyperactive or constitutive activity, or, altentatively, act to prevent a ColoUp2 polypeptide from performing one or more functions. For example,, a truncated form lacking one or more domain may have a dominant negative cffecL it is. also· posMfelb to of the subject CoioUp2 polypeptides lor such purposes as enhancing feerapsutie or prophylactic efficacy, or stability (e,g,s ex vivo shelf life and; resistance to proteolytte degradation in vivo), Such modified polypeptides, when designed to retain at least one activity of the. natprally-ocajrring form of the protein, ate: considered' functional equivalents of 'the· ColoUp.2 polypeptides described In more detail herein. Sue!) modified polypeptides am he produced, for fostanee, fey amino acid substitution, deletion, or addition.

For instance, it. is reasonable to expect, for example, that an isolated, replacement of a leucine with an tsoleacine or valine, rin aspartate with a gluiamatfe; a foroonine with a serine, or a slroilarroplueeroem of an: amlrro aeid with: a structurally related amino acid (.1,0., conservative: mutations! will not have a major effect on the biological activity of the resulting molecule. Conservative repiuccnrenta are those that take place within a family of amino acids that are related in their side chains.. Genetically encoded amino acids arc can be divided into four families' (1} acidic = aspartate, glutamate; (2) basic :™ lysine, arginine, histidine; (3) nonpolar :::: alanine, valine, leucine, isoleucine, proline, phenylalanine, rnethionme, tryptophan; and (4) uncharged polar = glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. Phenylalanine, tryptophan, and tyrosme are sometimes classified jointly as aromatic ηοΐΐηο acids. In similar fashion, the amino acid repertoire can he grouped as. (1} acinic aspartate, glutamate; (2) basic ::: lysine, arginine histidine, (3} aliphatic = glycine, aianme, valine, leucine, ssoleueme, serine, threonine, with serrne and threonine optionally fee grouped, separately as altpbatiofoydrbxyfe: :(4) aromatic “ phenylalanine, tyrosine, tryptophan; (5} amide.™ asparagine, glutamine· and (fi) sulfur -containing mefofoome. (see, for example. Biochemistry, 2nd ed,, Ed;, by L Stryer, W.H, Freeman and Co., 1981}, Whether a change in the amino acid sequence of a polypeptide results in a functional homolog can be readily determined fey assessing the ability of the variant polypeptide to prod nee a response in.cells in a fashion similar to the wild-type protein. For instance, such variant forms of a Cofol.lpx polypeptide cart he assessed, e.g., for their ability id bind to another polypeptide.. e.g„ another OoloUpS polypeptide. Polypeptides in which more than one replacement has taken place can readily be tested m the same manner.

This invention further contemplates a method of generating sets of combinatorial mutants of the subject ColoUp2 polypeptides, as well as trudcatlon mutants, and. is especially osefol for Idemilying potential variant sequences (shg,* homologsj, The purpose of screening such combinatorial libraries is to generate, for example.. ColoUpc horooiogs which can act as either agonists or antagonist* :b|f alternatively, which possess novel activities all together. Combinatoriaiiy-derived homologs can be generated which have a selective potency relative to a naturally occurring ColoUpd polypeptide. Such proteins* when expressed from recombinant PKA construets, can be: used in gene therapy protocols,

Likewise, mutagenesis can give rise to homologs which have Intracellular half-lives, dramatically different than the corresponding wild-type protein. For example, the altered protein on be rendered either more stable or less stable to proteolytic degradation &amp;t other cdiulas process which result in destruction of, or otherwise inactivation of the ColoijpS polypeptide of interest. Such homology and the genes whsih encode them, can be utilised to alter the levels of a Colotlp2 protein of interest by modulating the half-life of the protein. For instance, a short half-life cars give rise to more transient biological effects and. when part of an inducible expression system, can allow tighter control of recombinant Cololip'.?. polypeptide levels within the cell. As .above, such proteins, and particularly their recombinant nucleic arid constructs, can be used in gene therapy protocols.

In similar fashion, homologs of a ColoUpc polypeptide can. he generated by the present combinatorial approach to act as antagonists, m that they are able to interfere with the ability of the corresponding wild-type protein to function.

Alternatively, -Giber forms of mutagenesis can be utilized to generate a combinatorial library. For example, a. CololipS protein homolog (both agonist and antagonist forms) can be generated and isolated from a library by screening using, for example, alanine scanning mutagenesis and the like (Rof et ah, {1994} Biochemistry' 33:1565-1572; Wang et ah, Π994) i. Biol. Chem. 269:3095-3099; Balmt et ah, (1993)

Gene 137:109-1! 8; Grodherg et al,, (199.3) Ear. .1. Biochein. 218:597-601, Nagashima ei a!., (1993) J, Biol Chem. 268:2888-2892; Logman et al, (1991) Biochemistry 30; 10832-10838; and Cunningham et al, (1989? Science 244:1081-1085), by linker scanning mutagenesis (Gustid el aL, (1993) Virology 193.653-660, Brown et al, (1992) Mol Ceil Biol. 12:2644-2652; McKmght et al, (1982) Science 232:316); by saturation mutagenesis (Meyers et al, (1986) Science 232:613); by PCR mutagenesis (Leung et al, (1989) Method Cell Mol Biol: 1::11-19): or by random mutagenesis, including chemical matagerteais, etc; (Miller ot al, (1992) A Short Course in Bacterial Genetics, CSHL Press, Cold Spring Harbor, NY; and Greener el el... (1994} Strategies m Mol Biol 7:32-34). Linker scanning mutagenesis, particularly in a combinatorial setting, is an attractive method (or identifying truncated thioactive) forms of a Coioupl polypeptide.

The i:nvm6^,aIsO::pipyid^':-feir.red'S.cti0fi: of thc jnbjeci ColoUpd polypeptides to generate ««metres:, e.g,, ppptide Or non-peptide agents, which ar® able tptnirnic the behavior· or hiologjcai activity of the authentic protein. Such umtagenie; techniques as described. above, as well as: the ihioredewin sySMn,· are also paslieulsfly useful for mapping the: detetmipants of a ColoGpS polypeptide which participate in protein-protein interactions involved ί??, for example, cancers, 9- €<>ioUp2 nueiesc acids

In certain aspects, the invention provides, nucleic aeidS: that encode GoloDpS proteins. In one aspect, the nucleic acid sequences: are at least 90%, 95%, of 98-9936 i:deo:tiea| to the nucleic acid sequences. as set lorth in SBQ 10 19Q: 2, In some embodiments, s.ucJi mueieie acids include nucleic acids that are differential!y expressed in cancer samples versus a control sample In further embodiments, Co!oUp2 nucleic acids encode proteins that arc differentially present or absent (or at a different level or in aherud form) in the blood of a subject having a caneer versus a subject not having s caneer. In yet additional embodiments, CMot.JpS nucleic acids include jmdeR acids encoding proteins that are differentially expressed (including altered forms etc.) in cancer samples versus a control sample. Coiolip.?, nucleic acids are further understood to include nucleic adds that encode variants, such as variants of SEQ ID NO: 2 and nucleic acids encoding SEQ ID NOs' 1 or 3-4, Mariaut nucleotide sequences include sequences that differ by one or more nucleotide substitutions, additions or deletions- such as allelic variants: and will, therefore, include coding sequences that -differ from the nucleotide sequence of the coding sequence due to the degeneracy of the genetic code. In other embodiments, variants will also include sequences that will hybndi.ee under highly stringent conditions to a nucleotide sequence of SEQ ID NO: 2 or nucleic acids encoding SEQ ID NOs: 1 or 3~ 4.

One of ordinary skill in the: art will tsnderMand readily that appropriate stringency conditions which promote DMA hybridization can be varied For example, one could perform the hybridization at 6,0 x sodium chioride/sodmm citrate {SSC) at about 45 "C, foHowed by a wash of 2,0 x SSC at SO nC. For example, the salt, concentration in the wash step ears, fee selected from a low stringency of about 2.0 x SSC at Sfe °€ ip: a: high stringency of gbptxf 0.2 x SSC at 50 :*€. In addition, the temperature in the wash stop can fee increased from low stringency conditions at room temperature, about 22 to high stringency conditions at about 65 QCX Both temperature and salt may he varied,, or temperature or salt concentration may fee held constant while the other variable is changed. In doe enfoqdiment, the invention pro'v ides nutdeie acids which hybridize under low stringency conditions of 6 x SSC at room temperature followed by a wash at 2 x SSC at room temperature,

Co!oUp2 nuclem acids include nucleic acids which differ from an identified sequence due to degeneracy in the genetic code. For example, a number of amino acids are designated by more than one triplet. Codons that specify the same amino add, or synonyms (for example, CAU and CAC are synonyms for histidine) may result in “siienr mutations which do not affect the amino acid sequence of the protein. However, it ts expected that DNA sequence polymorphisms that do lead to changes in the amino acid sequences of the subject proteins will exist among mammalian cells. This is particularly likely in the case of nucleic acids derived from cancer samples and proteins that elicit a humoral response in subjects having a cancer. One skilled in the art will appreciate that these variations In one or more nucleoftdes (up to about 5-5% of the nucleotides) of the nucleic, acids encoding a particular protein may exist among individuals of a given species due to natural allelic variation. .Any and all such nucleotide variations and resulting amino acid polymorphisms are within the scope of this invention. M certain embodiments:, oligonucleotides of the invention may he used as .d$iftpsid' i*8gpfttS· I#-#t©6* the presence or absence of the target DMA or IRMA sequences to which they specifically bind, such as for determining; the level of expression of a gene- of the invention or tor determining whether a gene of the invention contains: a genetic lesion, 1, BMdUntetl^f^ in certain aspects, the invention relates to RNAi, rihoryme, antisense and other nucleic acid-related methods and' eoutposibons for manipulating (typically decreasing} CblriUpS expreasipn or activity.

Certain.emhodiments of the invention make use of materials and methods tor aRectiing hriockdxttwn of SbloUpI gene % means of -ENA interference (R NAi). R NAi is a process of sequence-specific post-transcriptional gene repression which can occur in eukaryotic ceils, in general, this process involves degradation of an mRNA of a particular sequence induced by double-stranded RNA {dsRNA) that is honudogposto that sequence. Any selected gene may he repressed by introducing a dsRNA which corresponds to ail or a substantial part of the mRNA for that gene, ft appears that when a long dsRNA is expressed, it is initially processed by a ribunucieasc Hi into shorter dsRNA oligonucleotides of as lew as 21 to 22 base pairs In length.. Accordingly, RNAi may be effected by introduction or expression of relatively shun homologous dstMAs,

The double stranded oligonucleotides used to effect RNAi are preferably less than 30 base pairs rn length and, more preferably, comprise about 25, 24. 23. 22, 21, 20. 19, IS or 1? base pairs of ribonucleic acid. Optionally the dsRNA oligonucleotides of the invention may include 3! overhang ends. Exemplary 2-nucleotide 3' overhangs may be composed of ribonucleotide residues of any type and may even be composed of2s-deoxythymidine resides, which lowers the cost of RNA synthesis arid may enhance nuclease resistance of si RN As in the cell culture medium and wnhin transfected ceils (see Blbashi et al. (2001) Nature 411 - 494-8). Longer dsRNAs of 50, 75, 100 or even 500 base parts or more may also be utilized in certain embodiments of the invention. Exemplar)' concentrations of dsRNAs for effecting RNA1 are #pot 0.05 άΜ, 0..1 hM, 0.5 #C 1,0 nM, 1.5 Ml 25 hM or 100 hM, although other concentrations may be otliiged xiependkg upon the nature of the cells treated, the gene target and other factors readily dsscemabhe die skilletl brtisah. Exemplary dsRNAs may he synthesized chemically or produced in vitro or in vivo using appropriate expression vectors. Exemplars' synthetic RNAs include; 21 nucleotide RNAs chemically synthesized using methods known in the art (e,g. Expedite RNA phophoramidites and thymidine phosphor&amp;midite (Proligo, Germany). Synthetic oligonucleotides are preferably deproiected and gd~purihed using methods known in the art (see e,g. Obashir ct at, (2001) Genes Dev, 15: 188--21)0). Longer RNAs may be transcribed from.promoters, such as T? RNA polymerase promoters, known in the art, A single RNA target, placed in both possible orientations downstream oi an in vitro promoter, will transcribe both strands of the target to creme a dsRNA oligonucleotide of the desired target sequence, Any of the above RNA species wdl he designed to include a portion of nucleic acsd sequence represented In a ColoUp2- mael-cie acid, such as, for example, a nucleic acid that hybridizes, under stringent and/or physiological conditions, to SIR) ID NO: 2 and a complement thereof

The specific sequence utilized in design of the oligonucleotides may he any contiguous sequence of nucleotides contained within the expressed gene message of the target. Programs and algorithms, known m the art, may he used to select appropriate target sequences. In addition, optimal sequences may be selected utilizing programs designed to predict the secondary structure of a specified single stranded nucleic acid sequence and allowing selection of those sequences likely to occur in exposed single stranded regions of a folded mRNA. Methods and compositions Jbf designing appropriate oligonucleotides may he found, for example, in ii.S. Patent Nos. 6,25.1,.588, the contents of which are incorporated herein by reference. Messenger RNA (mRNA) is generally thought of as a linear molecule winch contains the Information fer directing protein; synthesis within the sequence of ribonucleotides, however studies have revealed a number of secondary and tertiary-structures that exist In most mRNAs. Secondary structure elements in RNA are formed largely by Watson-Criek type interactions between different regions of the same RNA molecule.

Important secondary structural elements include intramolecular double stranded regions, hanpm loops, bulges I» duplex RNA and internal loops. Tertiary structural elements are formed when secondary .structural elements come in contact with each other or with single stranded regions to produce a more complex three dimensional structure, A number of researchers have measured the binding energies of a large number of RNA duplex structures and have derived a set of rules which can he used to predict the secondary structure of RNA (see e.g. Jaeger et al. {1989} Free. Natl. Acad. Sea, USA 86:7706 (1989); and Turner et al. (1988} Annu, Rev, Biophys. Biophys, Chem. 17:167) , The rules arc useful in identification of RNA structural elements and, in particular, for identifying single stranded RNA regions which may represent preferred 'segments of the n-RNA to target for silencing RNAi. ribozynieor antisense technologies, Accordingly, preferred segments of the mR.NA target can be identified for design of the RNAi mediating dsR.NA oligonucleotides as well as for design of appropriate riboxyme and hammerheadrtbozynm compositions of: tbs invention. i'hc dsRNA oligonucleotides may be introduced into the cell by transfection with an heterologous target germ usmg carder compositions such as liposomes, vvlucli arc known in the ad- e,g, iipofeciamine 2000 (lift Technologies) as described by the manufacturer for adherent cell lines. Transfection of dsRNA oligonucleotides for targeting endogenous genes may be carried out using Oligofevtamine (Life Technologies). Transfection efficiency may be checked using fluorescence microscopy for mammalian cell lines after co-tmnsiec!ion of hGFP-cncoding pAD3 (Kehlenback et ah (1998) .1 Cell Biol 141: 863--74), The effectiveness of the RNAi •nay be assessed by any of a number of assays following introduction of the dsRNAs. These include Western blot analysis using antibodies which recognize the CokdJpT gene product following sufficient tune for turnover of the endogenous pool after new protein synthesis is repressed, reverse transcriptase polymerase chain reaction and .Northern blot analysis to determine the level of existing ColoUp2 target mRNA.

Further compositions, methods and applications of RNAi technology are provided in il.S. Patent Application Nos. 6,278,039, 5,723,750 and 5,244,805, which are incorporated herein by reference.

Rxbozyme molecules designed 10 catalytic-ally cleave CoIoUp2 mRNA transcripts can also be used to prevent translation of subject OoloUp2 mRNAs and/or expression of ColoUp2 {see, e.g., PCX International Publication WO90/11 364„ published October 4, 1990; Saryer et al, (1990) Science 247:1222-1:225 and ITS. Pateur No, 5,093,246), Ribozvmes are :«nzymatfe RNA. molecules capable of catalyzing the specific cleavage of RNA. (For a review, see Rossi {1994) Current Biology 4; 469-471). The mechanism of ribnzyme: action, involves sequence specific hybridization of the ribozyn» molecule to CunfelemetUary target RNA,· followed by an eBdrOnuclcblytic cleavage event. The composition of ribozyme molecules preferably ismludesoxw or more sequences eemptoeniary- to a C2olnD'p:2 mllNA. axfo the well known catalytic sequence responsible for mRNA cleavage or a functionally equivalent sequence {see, e,g,, iiS> Pat, hip, 5)093.,244, which is .Incorporated, herein by reference in its entirety).

While «hozymes that cleave nifeNA at site speeific redoghitloxi sequences can be used to destroy target mRNAs, the use of hammerhead' is preip!Pr9&amp;:

Ifemmethead dhozymes cleave mRNAs at locations: dictated by flanking regions: tbat form eojnpfententary base pairs with the target jnRNA,. Preferably, the target tuRN A has: the fol lowing sequence of two bases: 5NU QrT. The consfrnctioxr and px'od action of hafo-nfofheaid: - riboxyitj^ is well known in the .aft' and is described more folly in Haseloff and Gerlach {{1988} Nafore 334:585-591; and see FCT Applo, No, WOSR/QSSSli the contents of which are incorporated herein hy reference). Hammerhead ribozyme sequences can be embedded in a stable RNA such as a transfer RNA ttRNA) to increase cleavage efficiency «t al. {1995)

Pros:., Natl, Acad. Set'. USA, 92: 6175-79; be Feyter, and G.androxt, Methods in Molecular Biology. Vo). 74, Chapter 43, "Expressing Riboz.vmes in Plums”, Edited by lumen P. €, Humana Press foe., Totowa, N.i). fo particular, RNA polymerase: tit-mediated expression of xRNA fusion rihozymes ere well known in the art ( see Kawasaki et al, {1998) Mature 393: 234-9: Kuwabara et at, (1998) Nature Biofeehnoi· 16: 961-5: and Kuwahara et al (1998) Mol. Cell 2: 617-27; Koseki et: al, ()999) I Virol 73: 1868-??, Kuwabara et al. (1999) Proc Nad Acad Sa USA 96: 1886-91; Taxuibe ex al <2000) Nature 406: 473-4). There are typically a nxmfeet of potential hatmfterhe&amp;d.' ribozyme cleavage sites within a given target cf>NA sequence;.

Preferably the ribosiyrae is engineered so that the cleavage recognition Site is located near the $* end of the target mflNA- to increase efficiency and minimise the intracellular accumulation of non-fenetfonal tftRNA transcripts, Furthenuore, the use of any cleavage recognition site located hi the targe! sequence encoding different orations of the 0-terminal amino acid domains of* for example* long and short forms iff target would allow the selective targeting of one or the other form of the target, and thus, have a selective effect on one form of the target gene product.

Gene targeting rihozymes necessarily contain a hybridizing region complementary to two regions, each of at least 5 and preferably each 6, 7, 8, 9, It), '1 L 12, 13, Id. if, 16, 17, 18. 19 or 20 contiguous nucleotides in length of a ColoUp2 mRNA, such as art mRNA of a sequence represented in 8 EQ ID NO: 2. in addition, rihozymes possess highly specific cuderihonuclcase activity, which auioeaialyticnUy cleaves the target sense mRNA. The present invention extends to rihozyntes which hybridize id a sense mRNA encoding a ColoUp? gene such as a therapeutic drug target candidate gene, thereby hybridising to the sense mRNA and cleaving it, such that it is no longer capable of being iranshued to synthesize a functional polypeptide product

Riboxymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.) and should be delivered to cells which express the target gene in vivo. A preferred method of delivery involves: using a DNA construct "encoding'' the ribozyme under the control of a strong constitutive pot Π! or poi (1 promoter, so that transfected cells will produce sufficient quantities of the ribozyrne to destroy endogenous target' niessages and inhibit translation. Because rihozymes:, unlike antisense molecules, are catalytic,; a lower: intracellular concentration. Is required for efficiency. A further aspect of the invention relates to the use of the isolated "aid-sense” nucleic acids to inhibit expression, e.g., by inhibiting transcription and/or translation of a. subject C;oloUp2 nucleic acid. The antisense nucleic acids may bind to the potential drug target by conventional base pair complementarity, or, for example, hr the ease of binding to DNA duplexes, through specific Interactions in the major groove of the double helix. In general, these methods refer to the range of techniques generally employed in the art, ami include any methods that rely on specific binding to ol igonuclcotide sequences.

An antisense construct of the present invention cm he delivered, for example, as an expression plasmid which, when transcribed in the cell, produces RNA which is complementary to at least a unique portion of the cellular mRNA which encodes a Cololip2 polypeptide. Alternatively, the antisense construct is an oligonucleotide probe, which is generated ex vivo and which, when introduced into ike cell causes inhibitions erf expression by hybridizing with the mRNA and/or genomic sequences of a ColoUp2 nucleic acid. Such oligonucleotide probes are preferably modified oligonucleotides, which are resistant to endogenous nucleases, e.g., exonucleases and/or endonucleases, and are therefore stable in vivo. Exemplary nucleic acid molecules for use as antisense oligonucleotides are pbosphommidate, phosphothioate and methylphosphonate analogs of DMA (see also US. Patents 5,176,996; 5,264,564; and 5,255,775). Additionally, general approaches to constructing oligomers useful in antisense therapy have been reviewed, for example, by Van der Kroi ei si. (1988} BioTeehniques 6:958-076-. and Stein et a). (1988) Cancer Res 48:2659- 2668.

With respect ίο antisense ON A, ollgodecixyrsbonueleotides derived from the translation initiation site, e,g,5 between tiro -10 and ι 10 regions of the Colo! Ip2: g:ene, are· preferred. Antisense approaches involve the design of oligonucleotides (either DMA or RNA} that are complementary to mRNA encoding the Co!oUp2 polypeptide. The antisense oligonucleotides will bind to the mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required, in the case of double-stranded antisense nucleic acids, a single strand of the duplex DMA may thus be tested, or triplex formation may be assayed. The ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with ;m RNA it may contain and still form s stable duplex (or triplex, as the case may he). One .skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the S! end of the mRNA, e.g., the 5: untranslated sequence up to and including the AUG initiation codon, should work most efficiently at Inhibiting translation. However, sequences complementary io the 3' untranslated .sequences of mRNAs have recently fees® show® to fee effective at inhibiting translation of mRNAs as well, (Wagner, R, 1994. Nature 372:333).

Therefore, oligonucleotides eemplementary to either the S' or 3’ untranslated, noncoding regions of a gene could be used in an antisense approach to inhibit translation of that mRNA. Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon. Antisense oligonucleotides complementary to rnRHA coding regions are less efficient inhibitors of translation -but could also he used in apeordauce with the: invention. Whether designed to hybridize to the 5\ 3' or coding region of mRNA, antisehse nucleic acids should hr: at least six nucleotides in length, and are preferably less that about 100 and more preferably loss than about 50, 25, 17 or 10 nucleotides m length.

The antisense oligonucleotides can he DMA or RINA or chimeric mixtures or derivatives or stodified versions thereof^ single-sir&amp;nded: or douhle-sirauded. The Oligonucleotide can be modified at the: base moiety, sugar moiety, or phosphate: backbone, for example, to improve stability of the moleealc, hyhbdiiatioiu. etc, The· oligonucleotide may include other appended groups, such as peptides (e.g.r tor targeting host cell receptors)., or agents focihtatipg transport across the: cell mcmhmne (see, p;g., Letsinger et ;ah,: 1089» IToc, Natl. Acad, Set, U.S,A, S6:6|53-6S5S'. Lemahre et ah, I9S7, Proe. Natl. Acad. Sci $4:648-652; PCT Publication Mo, WO&amp;8/098IO, published December 15. 1988} or the blood-· oram barner fsee, e;g,,: PCT Publication Ho W039/10134, published April 25, 1988), hybndmationTtiggered cleavage: agents. (See. e.g.:. Krol et a!., 1988, BIoTechniques 6:958- 976) of intercalating agents. (See, e.g.,Τρη, 1:98·$,· PhannCRns, Si539tS49). To this end, the oligonucleotide may be conjugated to another molecule, e.g,:, a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including but not limited to S-tlyorouraed, 5-hrprnouradl, o-ohlorouracii, SAodouraetl, hypoxanifeine, xantine, 4-acetyicytosinc, 5-iearboxyhsxiroxytieihyl) uracil, S-carboxymetfrylaminomefhyl-2-ihioundine, 5-carbo.KynuUlndamlnomethyluracd, dihydrourad 1, beta-D-galactosylqueosine, inosine, N6- jsopeniehyfadenlne, 1 -methylguaninn, I-meihylmoaine, 2,2-dimeihylguaoioe, 2-methyladenine, 2-methyl guanine, 3-methylcytostne, 5-rttethylcytosiiie, Nb-adenme, 7-methylguanine, beta- Ο- matmosy Iqueosine,. <'$'Bnethoxyearhoxynt e th y 1 urafci I,. 5-rneihoxytn'aei 1 , 2~ methyl tine-N6- .isopemeuylarfeniue, ttr^il-S^Xyacetic acid (v), wybutoxosina, pseudouracil, queosine, 2-thtoeytosiae, S-rnethyl-2~rhiouraeil, 2-thiouracil, 4-thtouracil, 5-methyknacth uracil-5- oxyaeefie acid tnethylesfer, uracilS oxyacelic acid (4¾ 5.*m€thyl-2-ihfouraciL 3-0»amkio-3~ M-^arboxypropyl) uracil, (acp3')w, dad 2 jh-diamiot^unpe.

The antisense oligonucleotide may also comprise at least one modified sugar moiety selected fern the group including but as limited to arabinose, 2~ fluoroarabtoose, xylulose, and hexose. The antisense oligonucleotide can also contain a neutral pepiide-Iike backbone. Such molecules are termed peptide nucleic- acid {PN;-\)-oiigotners .and ate described,. e,g., in Perry-θ'Keefe et al. {1996} Proo, Nail, Acad, Sot, D,.S,A. 93:14670 and lit figlpm ot at. (1993) Nature 365:566, One advantage of PNA oligomers is their capability to bind tu complementary DMA essentially independently from the ionic strength of the medium due to the neutral backbone of the ON A. in yet another embodiment, the antisense oligonucleotide comprises at least one modified phosphate backbone selected, from the group consisting of a phoisphorOfMoate, a pbosphorodiihioate, a phosphoramidoihkuite, a phoaphorarmdatc, &amp; phosphordiamidate, a methylphosphonate,. an alkyl phosphotri ester, and a fonoacetal or analog thereof. A further aspect of the invention relates to the use of DNA enzymes' to othihh expression of ColoOp2 gene. ION A enzymes incorporate some of the mechanistic features of both antisense and ..rihozyme technologies. DNA enzymes are designed so that they recognize a particular target nucleic acid sequence, mud’! like an antisense oligonucleotide,, however much tike a rihozyme they are catalytic and specifically cleave the target nucleic acid.

There arc currently two basic types of DNA enzymes, and both of these were identified by Sathojo and Joyce (see, for example, US Patent No. 6110462). The !0~ 23 DNA enzyme comprises a loop structure which connect two arms. The two arms provide specificity by recognizing the particular target nucleic acid sequence while the loop structure provides catalytic function under physiological conditions.

Briefly, to design an ideal DMA enzyme that specifically recognizes and cleaves a target nucleic acid, one of skill in the art must first identify the unique target sequence. This can be clone using the same approach as outlined for antisense oligonucleotides, Preferably, the unique or substantially sequence is a QIC rich of approximately 18 to 22 nneleotides,- High G/C content, helps insure a stronger interaction between 'the DNA enzyme and the target .sequence, When synthesizing the IMA enzyme, the specific antisense recognition sequence that will target the enzyme to the message, is divided so that it comprises fee two iUrms of fee DMA carry me, and the DNA enzyme loop is placed between the two speel it c arms.

Methods of making and administering DNA enzymes can he found, for example, in US 6.110462. Similarly, methods of delivery DNA nhozyrnes in vitro or in vivo include methods of delivery RMA, riboxyme, as outlined in detail above. Additionally, one of skill in the art wdl recognize that, like antisense oligonucleotide, DNA enzymes can be optionally modified to improve stability and improve resistance to degradation.

Antisense RMA and DMA, rihozyme, KMAi eonstfuets: of the invention may he prepared by any method known in the art for the synthesis of DNA and RNA molecules, ’including techniques for chemically synthesizing oiigodedxyrihonnclcotldes and oligoribbnucleofidcs well known in the art sped as for example solid phase phosghoratnidite chemical synthesis, Alternatively, DMA molecules may he generated by in: vitro and in Vivo transcription Of DNA sequences: encoding the antisense RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase.promoters such as the T? or SP6 polymerase ptSfeOters, Alternatively, antisense cDNA constructs that synthesize antisense RNA· eoostlfefiveiy of ipdueihly, depending 0» the promoter used, can be introduced stably into cell lines, Moreover, various well-known modifications to nucleic acid molecules may be introduced as a means of increasing intracellular stability and half-life, Possible modifications include but are. not limited to the addition of flanking sequences of ribonucleotides or deoxyribonuclcptides to the 5· and/or .3-5 ends of the; nrofeeule of fee use of phosphorothioate or 2: 0> methyl rather than phosphor!;esterase linkages'within the oli|odeox>mb0nucieotide backbone. 8· in certain aspects, the pres*» i&amp;v^tion.rpmvideS: metheds of treating cancers and methods of identifying therapeutics for .treatment of cancers. As described herein, the term '"cancer* includes a tumor inside an individual, a tumor xenograft, or a tumor cultured in vitro Exemplary forms of cancers (tumors) include, but are not limited ίο., prostate cancer, hi alder cancer, lung capeer (indudmg either small eel! or nornsmell cell cancer), kidney cancer, liver cancer, breast danced eerviea! caMeer,: endometrial or other uterine cancer, ovarian cancer, testicular cancer, cancer of the pedis, cancer of the vagina, cancer of the urethra, gal! bladder cancer, esophageal cancer, or pancreatic cancer. Additional exemplary Conns of cancer include, but are not limited to. cancer of skeletal or smooth muscle, stomach cancer, cancer of the small intestine, caseer of the salivary gland, anal cancer, rectal cancer, thyroid cancer, parathyroid eancer, pituitary cancer, and nasoplmryugeaf eancer. Farther exemplary forms of eaneer include cancers comprising ColoUp’-Kxpressmg cells. 'Ire certain mofeodinmms, the tumor is a metastatic tumor.

SpecifSh exemplary forms of cancers include, hot arb not limited to, endometrial cancer, renal cancer, adeuoeareinoina of the lung* stomach cancer, pancreas cancer, breast cancer, prostate cancer or ovarian eancer.

In certain embodiments, the subject cancer therapeutics may inhibit the expression of a €elolip2 protein, Such InhiMtory effects Can he at the transoriptionai level:, at the translational level, or at the post-tramdafional level. In certain embodimepts, such therapeutics may affect the function of a C.olollpS polypeptide such as one selected from the: group consisting of SEQ ID NOs; 1 and 3~4. In other Embodiments, such therapeutics may be targeted to a cancer by binding to a ColoUp2 protein with or without affecting the activity of the ColoUp2 protein For example, an aptaiTier that hinds to a Col0lfp2 protein may be eoningated to an anfheancer tberapeptie so ag io target the therapeutic to cancer cells. In certain, embodiments, the noti-GolollpS antibodies as described above may he used in the therapy of cancers. Such anti-ColoUpV antibodies may be conjugated with radio-nucleotides or cytotoxic agents. Anti-Cololip2 antibodies for cancer therapy may also include antibodies against Cell surface exposed epitopes of a ColoUpe protein,.

Optionally, the method further includes additional anil-neoplastic chemotherapeutic compounds that inhibit tumor cells in m% additive or synergistic mariner with the disclosed CoU>Up2-iargeted tfierggeotics. A wide array of conventional compounds have been shown to have anti-neoplastic activities. These-'Compounds have been, used,as pharmaceutical agents in chemotherapy to shrink solid tumors, prevent uielasiases md further growth, or decrease the number of malignant, ceils in leukemic or bone marrow rsaligmmetes, Although chemotherapy has been effective in treating various types of malignancies, many anti-neopiastic compounds induce undesirable side effects. It has been shown that when two or more different treatments are combined, the treatments may work syuergisticaiiy and allow reduction of dosage of each of the treatments, thereby reducing the detrimental side effects' exerted by each compound at higher dosages. In other instances, malignancies that are refractory to a treatment may respond to a combination therapy of two or more di fit-rent treatments.

In cemnn embodiments, candidate therapeutics may he identified on the basis of their ability to modulate the expression oi a CuioUp2 protein. To illustrate, the assay may detect agents which modulate the promoter activity of a Colotdpz gene, hr certain embodiments, -candidate therapeutics pray be identified on the basis of their ability -to modulate the binding of a ColdUp2 polypeptide to an associated protein or hgand. In a further embodiment, the assay detects agents which modulate the intrinsic biological activity of a ColoUp2 polypeptide. A variety of assay formats will suffice and, in light of the present disclosure, those not expressly described herein will nevertheless be comprehended by one of ordinary skill in the art. Assay formats which approximate such conditions as formation of protein complexes, ligand binding, protein activity, or promoter activity can be generated in many different loans, and include assays based on cell*free systems, e.g., purified proiems or cell lysates, as well as cell-based assays which utilize intact cells. Agents to be tested may be.generated in essentially any way, such as. for example, by production in bacteria, yeast or other organisms (e.g,, natural products), produced chemically (e.g.. small molecules, including peptidomimetics), or produced recombiaantly. in a preferred embodiment, the test agent Is a small organic molecule* e.g.„ other than a peptide or oligonucleotide, having a molecular weight, of less than about 2,000 daltons. :

In many drug screening programs which test libraries of compounds and natural extracts, high throughput assays are desirable in order to maximize the number of compounds surveyed in a given period of time. Assays of the presmut invention which are performed in cell-free systems, such as may he developed with purified or semi-purified .proteins or with lysates, are often preferred as "primary" screens is that they can he generated to permit rapid development and relatively easy detection of an alteration in a molecular target which rs mediated by a test compound. Moreover, the effects of cellular toxicity and/or hioavailabdiry of the test compound can be generally ignored in the in vitro system, the assay instead being focused primarily on the effect {.f the drug on the molecular target as may he manifest in an alteration of binding affinity with other proteins or changes in enzymatic properties of the molecular target.

In an exemplary binding assay, die compound of interest is contacted with a mixture comprising a ColoUp2 polypeptide artd at least one. interacting polypeptide or ligand. Detection and quantification of bound ColoUp2 polypeptide complexes provides a nx\ms for determining the compound's efficacy at inhibiting or potentiating mte? action,: The efficacy of the compound can be assessed by generating dose response curves: tom data obtained using various .concentrations of the test compound. Moreover, a control assay car? also be performed to provide a baseline for comparison, In the control assay, the binding is quantitated in the absence of the test compound. Complex formation between a Co!cUp2 polypeptide and an irtteraetor may be detected by a variety of techniques, many of which are.· effectively described above. For instance, modulation m the formation of complexes can he quantitated using, for example, delectably labeled proteins (eg., radiolabeled, fluoreseentiy labeled, or enzymatically labeled), by immunoassay, or by chromatographic detection. Surface plasmon resonance systems, such as those available from BiaCore, Inc., may also bo used to detect protein-protein interaction.

Often, it will be desirable to immobilize one of the polypeptides to facilitate separation of complexes tom uneomplexed forms of one of the proteins, as well as to accommodate automation of the assay. In an illustrative embodiment, a fusion protein can he provided which adds a domain that permits the protein to be bound to m insoluble matrix. For example, GST-fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St Louis, MO) or glutathione derivaiized microtiire plates,-^hi'ch. ape the» CGipfeiped with a potential interacting protein, e,g., an 3SS-labeled polypeptide, and the: test compound: and incubated under conditions conducive io complex formation.

The ColoUp2 marker and/or profile may he Used to screen lor therapeutics lor a disease slate such as cancers. Cell surface proteins associated with a disease state may be diminished or ehmtnated by trsatmeru with certain, test compounds. Such test compounds may: he: useful as therapeutics for the disease state. In addition, certain test compounds may increase the presence of cell surface proteins that are normally present on healthy cells hut diminished or absent in diseased cells, Such test, compounds may also be useful as therapeutics of eancers. Particularly: preferred tberapeulies will cause the eel) surface protein profile of a diseased cell to more closely resemble the cell surface protein profileOfa healthy eelh

In further embodiments, the differences between healthy and cancer tissue samples may he analyzed to identify targets for· therapeutic screening, and a screen may be designed to identify compounds that bind or otherwise affect the activity of the given target. For example, over-expression, of GoIoUpS is delected Ip various caneer types, such ax cancers of endometrium, feidney, lung, stomach, pancreas, breast, prostate, ovary, uterus, and thyroid, 'Therapeutics that rhmimsh this overexpression may fee useful as cancer therapeutics. in certain embodiments, a method for selecting an. appropriate cancer therapeutic for a subject Is a computer-assisted method. Such a method may comprise obtaining a coll surface protein profile or measuring a marker protein m a sample from a subject. The output signal may then fee compared agamst a database comprising output signal i.nfomrtuion iirorn a plurality of subjects and further comprising clinical stains information from a plurality of subjects. It is contemplated that one ofay use a computer interface: to identify in the database any dlnioa! eoudfeionS: correlated mth the. protein profile or murker. Accordingly, one may select a targeted therapeutic to ameliorate or prevent the correlated condition. 9. Tumor Vaccines TMe frosbwnt of cMcer with femor vaccines has been a goal of physici ans and scientists ever since effective: immunization against infectious disease with vaccines was developed, In fee past, major donor antigens had not been moleddisily characterized. Recent advances are, -however, beginning to define potential nioieeular targets and strategies Mil this bad evolved with the principle that T~ce!I mediated responses are a usoM target for approaches to cancer immutiizaiiom In addition, these antigens are not truly -foreign- and tumor antigens St more with a selt/altercd self paradigm, compared to a non-self paradigm for ami gens recognised in infectious diseases. Antigens that have been used in the art include the glycoiipids and glyeoproieins e.g.. gangliosides, the developmental antigens, e.g,, MAGE, tyrosinase, melmoA and gp?5; and mutant oncogene products, e,g„ pS3, ras; arid HEE-2/neu. Yaecme possibilities Include purified proteins and glycoiipids. peptides, cDNA expressed ih various vectors, and a range of immune adjuvants. Any ColoUp2 protein may be selected for use in a tumor vaccinee, although as noted above, ColoUpS proteins that elicit a humoral response in subjects having a cancer are preferred.

Yet Mother aspect of the present invention relates to the modification of tumor cells, and/or the immune response to tumor cells in a patient by adwufostering a vaccine fo enhance the aritbfomor immune response m a host, The present mventioti provides, for examples, tumor vaccines based on administration of expression vectors encoding a Co!oUp2 gene, or portions thereof or immunogenic preparations of polypeptides.

In general, it Is noted that malignant Imnsfomtation of cells is Commonly associated wife phenotypic changes. Such changes can include loss, gam, or aitemtion in the level of expression of certain proteins. It has been observed feat, in some situations the hum ane system may be capable of recognizing a tumor as foreign and, as Midi, mounting an immune response against fee tumor (Kripke. M., Adv. Cancer Res 34, 69-75 (1981)), This hypothesis is based in pain on the existence of phenotypic differences between tumor cells and normal cells., which is supported by the identification of tumor associated antigens (TAAs) t'Schreibe?, H., ct ai. Arm. Rev. Immunol. 6, 465-483 (1988)). TAAs are thought to distinguish a transformed cell from its normal counterpart. For example, three genes encoding TAAs expressed in melanoma cells» MAGE-1, MAGE-2 and MAGE-3, tee 'been cloned (van dor Braggen, P., et a!. Science 254, 16434647 {1991)). That tumor cells under certain circumstances can be re^gpimi-M ibfdg* is also supported by the existence ofT cells which can raecgmze and respond to nppor associated antigens presented by MMC; molecules. Such TA A “Speci fic T lymphocytes have been demonstrated to be present iit JhddtPmuM and arc capable of recognizing and slunul&amp;tmg an immune response against tornor cells when properly stimulated in vitro (Rosenberg,, S-A., ratal. Science 233, 1318-1321 (1986); Rosenberg. SAueaiid. -Lite, Ami. Rev. immuno!.4s 681-709 (1986)), In the case of melanoma cells both the tyrosinase gene (Briehard, ¥„ el at. I, Exp, Med, 178:489 (1993)) and the Mel an-A gene (Coulie et ah Exp. Med. 180:35)) have been identified as genes coding for antigens recognized on melanoma cells by

Indiseiiou of T lymphocytes us often a sgrtificant : early-step in a host's Imtnnne response. Activation of T cells resnlts in cytokine production, T cell proliferation, and generation of 1“ eel I “mediated effector functions T cell activation requires an antigen- specife signal, often called a primary activation signal, which: results fern stimulation of a clonal I y-distributed T cell receptor (TeR) present on the surface of the T eel fi This antigerpspeeihe, signal is usua lly in the form of .an, antigenic peptide bound either to a major histocompatibility complex (MHO) class 1 protein or an MHC class 11 protein present on the surface of an antigen presenting cell (APC). €D4-s-, helper T ceils recognize peptides associated with class 11 molecules which are found bn a limited number of eel! types, primarily B cells, and

dendritic cells. In -most cases class X! moleeules present peptides derived' from proteins:taken In contrast, £1)8A cytotoxicT cells (CTL) recognize peptides associated with class 1 moleeules. Class I molecules are found on almost alt cell types, and, in most eases, present peptides derived from: endogenously symhesized proteins.

The importance of!' cells in tumor Immunity has several implications.·which are important ;:in the development of auti-fiinmr vaccines. Since .antigens are processed and presented before they are recognized by T cells, they may be derived irom any protein of tire tumor cell, whether extracellular or intracellular. In addition, the primary amino acid segnenes of flic antigen is more important than the threedimensional structure of the antigen. Tumor vaccine strategies may use. the tumor eels itself as a source ofaptigea, or may he desigtied to enhance responses against speeifk gene products. {Pardoif, D. I f SB . Annals of the Hew York Academy of Sciences: 690:301).

The present, invention provides for various tumor vaccination methods and reagents which can he used to elicit an anti-tumor response against transformed cells which express/display -a :Cok»lip2 polypeptide, or which have been. engineered Ip present art antigen of a GololipB polypeptide. In general, the tumor vaecinC: Strategies of the present invention fad into two categories: (i) strategies that use the tumor cell itself aa a source of tumor antigen, and {?.) antigen-specific vaccine strategics that are designed to generate hfirndhe responses 'against specific antigens of a ColoUp2 polypeptide.

In genera!, a CoioUpe vaccine polypeptide will include at least a portion of the CcloUpB polypeptide, optionally including a site of mutation which, when occunidg in the full-length protein, results in loss of ns biological activity. Where the cancer vaccine comprises a sufficient, portion of a CoioUpB protein, the protein can he further mutated to render the vaccine polypeptide biologically inactive.

In one embodiment, a tumor ceil winch otherwise does not express a mutant Gol»Up2 polypeptide earth© rendered mirniinogenic as target tor CTL recognition by association of a Co!oUp2 vaccine polypeptide. For example, this can be accomplished by the use of gene transfer vectors. Such gene transfer 'vectors may be administered in any biologically effective carrier, e,g.s any formulation Or composition capable of effectively delivering the GoloOpB vaccine gene to cells in vivo, Alternatively, cells from the patient or other host organism cart be transfected with the tumor vaccine construct ex vivo, allowed to express the Co!oUp2 protein, and, preferably after inactivation by radiation or the like, administered to an uuUs'idual. In particular, viral vectors represent, an attractive method for delivery of tumor vaccine antigens because viral proteins are expressed de novo in infected cells, are degraded within the cytosol, and are transported to the endoplasmic reticulum where the degraded peptide products associate with MHO class 1 molecules before display on the cell surface (Spooner et ah (1995) Gene Therapy 2; 171).

Approaches include insertion of the subject gene into viral vectors including recombinant retroviruses, adenovirus, adeoo-associated virus, vaccinia virus, and herpes simplex virus- I, or plasmids. Viral vectors transfect cells directly; plasmid DNA can he delivered with the help of, for example, eafiomc liposomes (lipofectin) or deriyatlxed (e,g,, antibody conjugated), polylysmc conjugates,, gramacidin S, aittSeial viral envelopes cm tuber such intraoellulsr carriers, as well as direct injection of the gene construe! or CaPCM precipitation carried; opt in vivo, It will be appreciated that because foansdociion; of appropriate target cells represents the: critical first step id gene transfer, ehdide of the partieniar gene delivery systeth; wit! depend cm such factors as: the phenotype of the Intended target and the route of admirustration, s.g. locally or syafOrnieally. M addition fo viral transfer methods, such as those illnstrated.above, non-vira! methods can also he employed to cause: expression of a sufyeet Colodp2 polypeptide in the tissue of an animal in order to click a cellular immune response. Most nonviral methods of gene transfer rely on normal mechanisms used by mammalian cells for the uptake and intracellular transport of macromolecules. In preferred embodiments, nonviral gene delivery systems of the present invention .rely on enddcytle pathways for the uptake of the \ accme gene fey the targeted cell Exemplary gene delivery systems of thus type include liposomal derived systems, poly-lysine conjugates, and artificial viral envelopes

In another embodiment a mutant ColoUp2 peptide of the present invention may he directly delivered to the patient, Although such expression constructs as exemplified above have been shown to be an efficient means hy which to obtain expression of peptides in the context; of class 1 molecules, vaccination with isolated peptides has also been shown to result tn class I express toe of the peptides in some cases, for example, the use of synthetic peptide fragments containing CTL epitopes which are presented by class ! molecules has been shown to he an effective vaccine against infceion with lymphocytic choriomeningitis varus (Schultz et at 1991. free. Math Acad;, Sei USA 88:2283) or sersdai virus (hast et al. 1991. Proe Natl Acad Set. 88:2283), Subcutaneous administration of a CTL epitope has also been found to render .mice resistant to challenge with human papillomavirus 16-transformed tumor cells (Feifkamp et al. (1993) Eur. I, !mtmmol.23:2242-2249}, it is contemplated that such peptides may be presented in the context of tumor cell class 1 -antigens or by other, host-derived class I bearing cells (Huang ei ah 1994. Science 264:961).

The Colollp2 proteins, and portions thereof, may be used u- the preparation·: of vaccines prepared by known techniques (e.f, U.S. Patents 4,365,69?; 4,528,217 arsd 4,573,495) Such polypeptides displaying antigenic, regions capable of eliciting protective immune response are selected and incorporated in m appropriate .carrier. Alternatively, an antitumor antigenic portion of a ColotJp2 protein may be incorporated info a larger protein by expression of fused proteins.

In other embodiments, the carcinoma Cell. itself can be used :as the source: of antitumor CdloUpt antigens, See, lor review, Fatdell, D. 199.3, Annals of the Mew York Academy of Sciences 090:391. For example, cells: which: have been identified Ihfbp^i ptoafypipg'^ expressing a mutant (MoF!p2 protein can be used ter.gencrate a CXL response against a:tunaar. For exam.pi:e, :turuowiiilthraung lymphocytes (TILs) may be derived from tumor biopsies which have such a phenotype.'. Following such protocols as described by Horn et al. (1991) .1 hiummotherap 10:153, TILs can be isolated from tumor specimens and grown in the presence of imerleukm-2 in order to generate oligoelooal populations of activated T-Iyrnphocytea that are cytolytic to the tumor cells expressing tire mutant €olotip2 protein.

In other embodiments, whole cell vaccmes can be used to treat caneer patients. Such vaccines can include, tor example, irradiated autologous or allogenic tumor cells which express (endogenously or recombinamly) a mutant ColoUp? polypeptide (or fragment thereof), or lysates of such cells.

10 9hatm<K)euriM

In certain aspects, any of the Co:oUp2 therapeutic agents of the present invention (e.g., antibodies, peptides, polypeptides, small molecules, RNAs constructs, antisense probes, and tumor vaccines) may be formulated with a pharmaceutically acceptable carrier. Such thertrpcubc .agct5:(5 can be administered alone or as a component of a pharmaceutical fbnmilatlou (Composition), The compounds may be formulated for administration in any convenient way tor use in human or veterinary medicine. Wetting agents., cm nisi iters and lubricants, snob as: sodium lauryl sulfate and iaagnosium stearate, as well ax coloring agents, release agents, coating agents. sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present, in the compositions.

Formulations of the subject therapeutic agents include those suitable for oral/’ nasal, topical, parenteral, rectal, and/or intravagmsl administration. The formulations may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art of pharmacy. The arnoonl of active in^edieot which can he combined with a carrier niatedal to produce a single dosage lorm will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier materia) to produce a single dosage form will generally be that amount of the compound which produces a fkerapeut tc effect.

The therapeutic agents may be administered in any conventional manner, including or&amp;nasaily, .subcutaneously, intrapcritoneally or mtramuscularly. The vaccine may "further comprise, as discussed infra, an adjuvant iu order to increase the irnmunogcmclty of the vaccine preparation. In some eases,, it may be advantageous, to couple one or more of the ColoUp2 therapeutic agents to a earner, in particular a rnacromoiecular carrier. The carrier can be a polymer to which the Coiotip2 therapeutic agent is bound by hydrophobic non-covalent interaction, such as a plastic, c.g,, polystyrene, or a polymer to which the agent is covalently bound, such as a polysaccharide, or a polypeptide, e.g., bovine serum albumin, ovalbumin or keyhole limpet bemocyunin The carrier should preferably be non-toxic and non- allergenic.

In addition, the formulations may also contain one or more stabilizer, exemplary being carbohydrates such as sorbitol., mannitol, starch, sucrose, dextrin, and glucose, proteins such as albumin or casein, and buffers such a® alkaline metal phosphate and the like.

In cluneal settings, the Co;oljp2 therapeutic agent of the proscot invention cars be introduced into a patient by any of a number of methods, each of which ss familiar in the an. for instance, a pharmaceutical preparation of the gene delivery system or peptide cars be introduced systernieally, c.g... by intravenous injection, and specific transduction of the protein in the target ceils occurs predominantly from specukny of transfection provided: by the gene delivery vehicle, cel I-type or tissue-type expression due to the transcriptional regulatory sequences* in other embodiments, initial delivery of the recombinant gene is more limited with introduction into the arson a; being quite localized,

Suhabfe phamiaeeuticaj vehicles lor administration id a patient are known to those skilled in the art. For parenteral admioistiafipti, the ColotipS therapeutic agents will usually be dissolved or suspended in sterile water or saline. For enteral ^ministration* the ColoUpx therapeutic agent can be incorporated into an Inert eamet m tablet, liquid, or capsular form. The preparation may also be emulsified or the active ingredient encapsulated in Hposorne vehicles, hi addition to inhibiting growth of a tumor at its original the CoioUp2 .therapeutic agents? of the current invention may also ire usee! in a method for preventing or treating metastatic spread of a tumor or preventing Or treating recurrence of a tumor,

Toxicity· and therapeutic efficacy of the ColoUpa: therapeutic compounds (agents) can be determined by standard pfmmiacoolieal procedures In eel! cultures or experimental animals, e.g., for determining The Ld50 (The Dose Lethal To 50% Of The Population) Arid Tine EdSO (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index,and it can he expressed as the ratio LD50/ED50, Compounds which exhibit large, therapeutic induces are preferred. While compounds that exhibit toxic side effects may be used, care should be takers to design a deb very system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies eao be used in formulating a range of dosage for use nr humans. The dosage of such compounds lies ureter ably within a range of circulating concentrations that include the EDSO with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the mute of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose cab be estimated initially bom cell culture assays. A dose may be formulated m animal models to achieve a circulating plasma concentration range that includes the IC50 (he,, the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined m cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by high· performance IkpscI chromsiogrspfsy.

Tie invention now being generally described, It will be more readily understood by reference to the following examples, which are included merely- for parpr*ss&amp; of illustration of certain aspects and embodiments of the present invention, and nrit not huended to limit the invention,

EXEMPLIFICATION

The invention now hemg generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of d hast rah on of certain aspects nod embodiments of the present invention, and are not intended to limit the invention.

IfedmpfeJ.; CfeoU^

The ColoUp2 transcript was inserted into a cDNA expression vector with a C-termmal V5 epitope tag. Figure 4 shows a summary of the behavior of fire tagged protein expressed· by transfection of the vector into SW480 and VaeodOO cells. An anti VS western blot shows feed arrows) expression of the transfected tagged protein detected in the lysate of a pellet of transfected cells (lysates western pane!* lanes labeled ColoUp2/V5) which Is absent in cells transfected with a control empty expression vector (lanes labeled peDNAT!) Moreover, serial immunoprecipitation and western blotting of V5 tugged protein from media in which V400 and SW480 cells were growing, (which had been clarified by cesttrifegattotr prior to intmuitopreespitatioo) also clearly demonstrates-sseteiio»-of the Co!oUp2 protein into the growth medium (panel labeled medium IP-western). Antibody bands front the immunoprecipifation are also present on the IP-western blot. Detection of secreted ColoUpx protein was shown in cells assayed both 24 hours and 48 hours after transfection.

Cell lines derived from other cancer tissues (e,g., endometrium, Mdney, lung, stomach, pancreas, breast, prostate, ovary, uterus, and thyroid) can be similarly analyzed to confirm that ColoUp2 is a secreted protein is these cancer types.

Bxampi s .2.g ColoUg,2 nr^ in.the. blood. of m i oe. . To detempne iMt ColoU|>2 proteifrs: are eiteetiue sprOlogie markers of cancers, we derived iraasfeeled eell lines that stably expressed and secreted VS-epitope tagged GolotJpS proteins. These ceils lines were then injected into athymle mice arid .grown as tumor xenografts. Mice were sacrificed and serum was obtained. VS-tagged proteins· were theft precipitated from the scrum using beads conjugated to ante V 5 antibodies. Precipitated scrum proteins 'Were run out on SDS-PAGB, and visna&amp;ed by western blotting rising HRP*conjugated "anti-V5 antibodies, ^thereby eliminating Visualisation of any contaminating moose immunoglobulin). Figure S shows detection of cuculaUng Cok>Up2 protein in mouse serum. The ColoUp2 protein is secreted as 2 bands of 85K.D and S5K.D in size, of winch the 35K.D band predonfinates jn the serum.. The 3?ΚΙ3 band is presumably a processed form of the 85K.D band. This ..observation demonstrates that, in this mouse model. CoioUpl. is indeed a secreted marker Of cancers, and that ColoUpd can gain access to and circulate stably in patient serum. This observation provides the surprising result that a processed fragment of Co1oUp2 is the predominant serum loon of the protein and therefore detection reagents targeted to this portion would he particularly suitable .Tor diagnostic testing. A time course experiment showed that OoloIJpS protein was detectable: in mouse blood at the earliest time assayed, 1 week after injection of Co!oUp2 secreting colon cancer cells, at which time xenograft tumor volume as only 100m nr.

Cell lines derived from oilier cancer tissues fa.g,5 endometrium. kidney, lung, stomach, pancreas, breast, prostate, ovary, uterus, and thyroid) can be similarly analyzed In mice to confirm that Cok>Up2 proteins can .be detected in the blood of mice,

Iwampje3J;G^ in order to develop monoclonal antibodies against native ColoUpI proteins, we devised a protocol for purification on Ni-NTA agarose (QIaGEN) nickel beads of recombinant His -tagged ColoUpz proteins from the media supernote of SW480 cells engineered to express these proteins. Currently we have purified ColoUp2 proteins to sufficient purity to generate, antibodies. As shown in figure 6. a Coomassie blue stained gel of purified ColoUp2 shows only the S5K.D and 55K..D size bands that optrespond to the togged <Μο13ρ2 proteins visualfoed m western blot. Thus, we have purl bed Colol)p2 to sufficient. homogeneity and yield. Sealed up purification of the proteins from a 50 liter media preparation should yield 2,5 mg of protein, more than adequate for immunizing mice and screening fusion supem&amp;tes for development of monoclonal antibodies specific for native €ok>Up2.

The protein sequence of C-temnnal fragment of ColoOp2 that is secreted by human cell lines and detected as predominant fragment in blood (4Si aa) was determined. As described above;, we have found on western blots rmd on purified preparations of Cderrnidal epitope tagged (VS~l-iis opitbpe) CoIolIpS protein secreted by transfoeted human colon cancer cells, both a full sized: haud: of approximately 90 kDa and a Smaller approximately 55 kDa GMfmmal fragment (as demonstrated by the retention of the C-terminal epitope tag). Moreover, when these edb; were injected into athymie mice, the 55 kDa C-torminai tagged protein was the predominant species defected as eireuiahng in the mouse blood, when mouse serum is analysed: by serial innnunopredpitation and western blot analysis directed against the V5 lag. 2'be precise location of the cleavage site accounting: for the Cuerminai fragment was established by excising the acrylamide gel hand containing the purified C-terminal fragment and performing mass spectroscopy analysis of tryptic fragments from the. protein. A peptide of sequence AYLAAHCPFYSWK (SEQ ID NO; 5) was present only in the digest of the 55KD fragment, but was absent Mm the digest of the foil length protein, dem.onsinn.ing that this peptide corresponded to the unique amino termtnus of the 55KD fragment. The complete sequence of the 55KD C-temtinal fragment ;s shown in Figure 7,

The sequences of secreted CoioUp2 protein in cell lines derived from other cancer tissues (e.g., endometrium, kidney, lung, stomach,. pancreas, breast, prostate, ovary, uterus, and thyroid) can be similarly analyzed. S: QiAmm expressfoq.pf

We describe here»'» expression hfCofol!p2 M fcijf expanded list .of human cancers, indicating that Co!ollp2 is useful as a diagnostic bicmarker (e.g,, in Mood or other bodily fluids) for o&amp;jceis with usefulness for early detection Of or for monnoring response to therapy of these cancer types.

Initial analysis of ColoUp.2 expression across a spectrum of human cancers was done by hybridization of a Cok>Up2 cDNA probe to a dot blot mesnhranc eontaioing RMA from cancer tissues: and paired normal. tissues across a variety of human cancers. The findings of this study arc summarised in Table 1 This study detected an increase in ColoUp2 level m 70% of colon .cancers and 50¾ of rectal cancers. The sensilri ity of this experiment was limited by the high background on the blot and by the presumed adorixiere of normal and fomor cells in die cancer Specimen, Thus, due to the high background on the blot, the magnitude: of foe increase in CoioUp2 detected in· colon cancer ajpp^^.'nineh.^glier-'tbM that we. had previously demonstrated by quantitative analysis of CoioUpa in colon cancers using real-time PCR methods. Nonetheless, using this dot blot approach, wo did detect evidence for CoioUp2 induction m a subset clustering around .10% of casus of hang -cancer and around 20% of breast cancers and endometrial, (uterine); cancers, and 13% of ovarian cancers. An example of the dot blot is shown in .Figure fo 1'able 1. A summary of dot blot results showing CoioUp2 expression in various cancels.

To provide better sensitivity for analysis of Golotfp2 expression, we developed an immunohistochemical assay for ColoOp3 protein expression. Tins assay allows direct visualization of the rnalignani cells m a tumor sample, and determination of the presence or absence of ColoUp‘2 protein in the cancer cells. Slices were cut from paraffin blocks of adenocarcinomas .from § different common capper' types: and were stained with an antibody 148 -1 -1 A2T34--AT0 feat we have, shown by western blot analysts .is-sgoerik lor binding only to Coiolip2 and that we. have found has; utility for ifemnoehistoehermcai detection of ColoUpz in formalin fixed· paraffin embedded samples, The results of this study are summarized In Table: 2, In partioular, the study demonstrated ColoUp2 expression in 100% of endometrial and 95% of renal cancep, a level exceeding even the 1S% rate of ColoIJp2«ppsiti ve turners demonstrated by colon cancels. Thus, detection of CoioUp? hi blood or other bodily thuds may also have utility as a diagnostic biomurker of endometrial or renal cancers, with usefulness lor early detection of or tor monitoring response to therapy of endometrial or renal cancers.: IP addition, ColoI)p2 expression: was: detected in 55% of adchoCarcindiPas of the: lung, 4:2% of gastric cancers, 39%. of pancreatic cancep, 22% of prostate cancers, and !S% of ovarian cancers. Thus, in some instances, defection of Golotlp2 in blood or other bodily fluids may also have utility as a diagnostic biomarker of adenocarcinoma of the lung, of gastric cancer, of pancreatic cancer, of prostate cancer, or of ovarian cancer, with usefulness ibr early detection of or for monitoring response to therapy of certain cases of these cancer types. Photographs of representative irurnunostained slides of different tumors {with the. innounost aiuing i'eprcsentipg GoioUp2; proteln) are shown in Figure 9.

Table 2. A summary of in'HTnmohtSiochemicai results showing Cok>Up2 expression m various cancers.

INCORFOR ATiON BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In ease of contlsc·, the present application..including soy definitions herein, will control.

While specific . embodiments of the subject invention have been discussed» the above speclikat ion is illustrative ^and not restrictive. Many variations of the invention wiil become apparent to those skilled in the art upon review of this specification and the claims below. The full scope of the invention should be determined by reference to the claims, along with their hull scope of equivalents, and the specification, along with such variations.

Claims (32)

1. What is claimed is:

   1. A method for detecting whether a subject has a neoplasia of a tissue type selected from endometrium, kidney, stomach, pancreas and uterus, comprising: (a) selecting a subject from a population, wherein the subject is suspected of harbouring a neoplasia of a tissue type selected from endometrium, kidney, stomach, pancreas and uterus; (b) obtaining a biological sample from said subject; and (c) detecting in the biological sample a ColoUp2 polypeptide, wherein the wherein the ColoUp2 polypeptide is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2, wherein the presence of said polypeptide is indicative of said neoplasia; and (d) wherein if the subject is found to have a neoplasia, treating said subject by: i. surgically removing said neoplasia from said subject; or ii. administering to said subject chemotherapy.
2. 2. The method of claim 1, wherein said biological sample is selected from whole blood, blood plasma, blood serum, urine, and stool samples.
3. 3. The method of claim 1 or claim 2, wherein said biological sample is a serum sample.
4. 4. The method of claim 3, wherein said serum sample is enriched for ColoUp2.
5. 5. The method of any one of claims 1 to 4, wherein the ColoUp2 polypeptide is detected by contacting the biological sample with an antibody that interacts with the ColoUp2 polypeptide.
6. 6. The method of claim 5, wherein the antibody interacts with an epitope on SEQ ID NO: 1 or a portion thereof.
7. 7. The method of claim 5 or claim 6, wherein the antibody is detectably labeled.
8. 8. The method of claim 7, wherein the label is selected from an enzyme, a fluorescent substance, a chemiluminescent substance, a chromophore, a radioactive isotope and a complexing agent.
9. 9. The method of any one of claims 1 to 8, further comprising determining the amount of said ColoUp2 polypeptide in the biological sample.
10. 10. The method of any one of claims 1 to 9, wherein the presence of the ColoUp2 polypeptide aids in determining a therapeutic protocol to be administered to the subject having said neoplasia.
11. 11. The method of any one of claims 1 to 10, wherein if ColoUp2 is detected in the biological sample in step (c), said method comprises surgically removing said neoplasia from said subject.
12. 12. The method of any one of claims 1 to 10, wherein if ColoUp2 is detected in the biological sample in step (c), said method comprises treating said subject with chemotherapy.
13. 13. The method of any one of claims 1 to 12, wherein the subject is suspected of harboring a neoplasia of an endometrium tissue type.
14. 14. The method of any one of claims 1 to 12, wherein the subject is suspected of harboring a neoplasia of a kidney tissue type.
15. 15. The method of any one of claims 1 to 12, wherein the subject is suspected of harboring a neoplasia of a stomach tissue type.
16. 16. The method of any one of claims 1 to 12, wherein the subject is suspected of harboring a neoplasia of a pancreas tissue type.
17. 17. The method of any one of claims 1 to 12, wherein the subject is suspected of harboring a neoplasia of a uterus tissue type.
18. 18. A method of treating a subject having a neoplasia, wherein the neoplasia is of a tissue type selected from endometrium, kidney, stomach, pancreas, and uterus; wherein the subject has been determined to have increased levels of ColoUp2 polypeptide; wherein the ColoUp2 polypeptide is encoded by a nucleic acid sequence that is at least 90% identical to the nucleic acid sequence of SEQ ID NO: 2; wherein the method comprises the step of treating the subject by: i. surgically removing said neoplasia from said subject; or ii. administering to said subject chemotherapy.
19. 19. The method of claim 18, wherein the subject has a neoplasia of an endometrium tissue type.
20. 20. The method of claim 18, wherein the subject has a neoplasia of a kidney tissue type.
21. 21. The method of claim 18, wherein the subject has a neoplasia of a stomach tissue type.
22. 22. The method of claim 18, wherein the subject has a neoplasia of a pancreas tissue type.
23. 23. The method of claim 18, wherein the subject has a neoplasia of a uterus tissue type.
24. 24. The method of any one of claims 18 to 23, wherein the method comprises the step of treating the subject by surgically removing said neoplasia from said subject.
25. 25. The method of any one of claims 18 to 23, wherein the method comprises the step of treating the subject by administering to said subject chemotherapy.
26. 26. The method of any one of claims 1 to 25, wherein the ColoUp2 polypeptide is encoded by a nucleic acid sequence that is at least 95% identical to the nucleic acid sequence of SEQ ID NO: 2.
27. 27. The method of any one of claims 1 to 26, wherein the ColoUp2 polypeptide is encoded by SEQ ID NO: 2.
28. 28. The method of any one of claims 1 to 27, wherein the ColoUp2 polypeptide is encoded by a nucleic acid sequence that hybridizes under stringent conditions to a nucleic acid sequence of SEQ ID NO: 2.
29. 29. The method of any one of claims 1 to 28, wherein the ColoUp2 polypeptide has an amino acid sequence that is at least 95% identical to the amino acid sequence as set forth in SEQ ID NO: 1.
30. 30. The method of any one of claims 1 to 29, wherein the ColoUp2 polypeptide has an amino acid sequence that is at least 95% identical to the amino acid sequence as set forth in SEQ ID NO: 3.
31. 31. The method of any one of claims 1 to 30, wherein said neoplasia is a cancer.
32. 32. The method of any one of claims 1 to 31, wherein said neoplasia is a metastatic cancer.