WO2006108095A9 - Serum biomarker for disease and methods of using same - Google Patents

Abstract

The present invention relates to methods of diagnosing abnormal conditions subjects, with the methods comprising determining the levels of a biomarker in a sample the subject, where the biomarker is selected from the group consisting of a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:1 and a peptide having a mass:charge ratio of about 771.88 daltons. The levels of biomarker in the test subject are then compared to normal levels of the biomarker. Any differences between the levels of the biomarker in the test subject and normal levels are indicative of the presence of an abnormal condition in said subject.

Description

SERUM BIOMARKER FOR DISEASE AND METHODS OF

USING SAME

Inventors:

Steven J. Meltzer Takatsugu Kan and Emanuel Petricoin Related Applications

[0001] This application claims priority to United States Provisional Application No. 60/668,732, filed April 6, 2005, the contents of which are incorporated by reference.

Statement Regarding Federally Sponsored Research or Development

[0002] Part of the work performed during development of this invention utilized U.S. Government funds from the National Institutes of Health Grant No. CA77057. The U.S. Government has certain rights in this invention.

Background of the Invention

Field of the Invention

[0003] The present invention relates to methods of diagnosing abnormal conditions subjects, with the methods comprising deteπnining the levels of a biomarker in a sample the subject, where the biomarker is selected from the group consisting of a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:1 and a peptide having a massxharge ratio of about 771.88 daltons. The levels of biomarker in the test subject are then compared to normal levels of the biomarker. Any differences between the levels of the biomarker in the test subject and normal levels are indicative of the presence of an abnormal condition in said subject.

Background of the Invention

[0004] Currently, colorectal cancer is the second leading cause of cancer-related deaths in the Western world. Although data clearly indicate that early detection is critical to enhanced survival rates, current early detection procedures are invasive or incomplete. For example, current methods of screening or diagnosing subjects for colorectal cancer include the fecal occult blood test (FOBT), x-ray using double contrast between barium enema and air (DCBE), sigmoidoscopy, virtual colonoscopy, and colonoscopy. While FOBT is rapid and non-invasive, the results are plagued by low specificity as well as low sensitivity. Sigmoidoscopy, on the other hand, is an invasive procedure that visually examines only the lower third of the colon using a lighted, flexible endoscope, while a related method, colonoscopy, is a somewhat dangerous, expensive, painful procedure that examines the entire colon. In both cases, biopsy samples can be taken during the procedure for further testing, but only during a colonoscopy can precancerous polyps be removed. Both of these procedures confer risks of perforation, bleeding, infection, and in the case of colonoscopy in particular, cardiac or respiratory arrest. In addition, emerging imaging techniques such as magnetic resonance imaging and computerized tomography are available but can be costly and time- consuming for the patient and healthcare provider.

[0005] Accordingly, there is a need in the art for approaches that have value in early detection and treatment of colorectal cancer that are cost effective, rapid, safe, and minimally invasive or noninvasive. Additional utility would be conferred by an approach that would also serve as the basis for establishing prognosis, monitoring patient treatment, and detecting relapse, as well as for the discovery of therapeutic interventions in colorectal cancer.

Summary of the Invention

[0006] The present invention relates to methods of diagnosing abnormal conditions in subjects, with the methods comprising determining the levels of a biomarker in a sample in the subject, where the biomarker is selected from the group consisting of a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:1 and a peptide having a mass: charge ratio of approximately 771.88 daltons. The levels of biomarker in the test subject are then compared to normal levels of the biomarker. Any differences between the levels of the biomarker in the test subject and normal levels are indicative of the presence of an abnormal condition in said subject.

[0007] The present invention also relates to methods of monitoring the progression of abnormal conditions in subjects, with the methods comprising determining the levels of a biomarker in a sample in the subject at a first and second time point, where the biomarker is selected from the group consisting of a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:1 and a peptide having a massxharge ratio of about 771.88 daltons. The levels of biomarker at each time point are then compared to determine differences of the biomarker over time. Any differences between the levels of the biomarker over time are indicative of the progression, regression or stasis of the abnormal condition in the subject.

[0008] The invention also relates to antibodies or fragments thereof that bind to an antigen, with the antigen being selected from the group consisting of a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:1 and a peptide having a massxharge ratio of about 771.88 daltons.

[0009] The invention also relates to methods of treating abnormal conditions in subjects in need of treatment thereof, with the methods comprising administering to these subjects a therapeutically effective amount of a substance that reduces the activity levels of molecules selected from the group consisting of a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:1 and a peptide having a mass:charge ratio of approximately 771.88 daltons.

Brief Description of the Drawings

[0010] FIGURE 1 depicts the ion trap mass spectrum of serum samples after albumin extraction and subsequent purification.

[0011] FIGURE 2 depicts the ion trap mass spectrum of the sample at an elution time t = 22.79 minutes.

[0012] FIGURE 3 depicts the ion trap mass spectra at t = 22.79 min with mass to charge ratio (m/z) of 771.88.

Detailed Description of the Invention

[0013] The present invention relates to methods of diagnosing abnormal conditions in subjects comprising determining the levels of a biomarker in a sample from the subject and comparing the levels of biomarker in the test subject to normal levels of the biomarker. Any differences between the levels of the biomarker in the test subject and normal levels are indicative of the presence of an abnormal condition in the subject.

[0014] As used herein the term "diagnose" means to confirm the results of other tests or to simply confirm suspicions that the subject may have a particular abnormal condition. In other words, the diagnostic tests of the present invention are used in conjunction with other tests, regardless of timing of the other tests. A "test," on the other hand, is used to indicate a screening method where the subject or the healthcare provider has no indication that the subject may, in fact, have a particular disease or particular abnormal condition. Thus a test may be a screening method where a patient exhibits some general symptom. For example, a patient may exhibit a symptom such as weight loss, bloody stool, abdominal pain, anemia, change in appetite, etc. that does not clearly indicate a specific abnormal condition. The testing methods could then be used to determine if the subject needs additional diagnostic procedures to properly diagnose the condition that may be causing the general symptom(s). The methods of testing herein may be used for a definitive diagnosis, or the tests may be used to assess a subject's likelihood or probability of developing a disease or abnormal condition.

[0015] The methods of the present invention, therefore, may be used for diagnostic or screening purposes. Both diagnostic and testing can be used to "stage" a condition or disease in a subject. As used herein, the term "stage" is used to indicate that the abnormal condition or disease can be categorized, either arbitrarily or rationally, into degrees of severity. The categorization may be based upon any quantitative characteristic that can be separated, such as, but not limited to, a numerical value of a biomarker, or it may be based upon qualitative characteristics that can be separated. The term "stage" may or may not involve disease progression. In addition, the assay or measurement may be used to stratify a population into relevant cohorts of similarly classified individuals, such as for a clinical trial or other study.

[0016] As used herein, an "abnormal condition" is used to mean a disease, or aberrant cellular or metabolic condition. Examples of abnormal conditions in which the methods can be used include but are not limited to, dysplasia, neoplastic growth and abnormal cell proliferation. In one embodiment, the abnormal condition comprises neoplastic growth. In a more specific embodiment, the abnormal condition comprises a carcinoma. In an even more specific embodiment, the abnormal condition comprises either squamous cell carcinoma or adenocarcinoma. The invention is not limited to the type of neoplasm, such as an adenoma, polyp, or carcinoma. For example, the adenoma or carcinoma may be a carcinoma of the digestive tract or any associated glands or organs, including, but not limited to, the throat, the salivary glands, the esophagus, the stomach, the small intestine, the large intestine, or the pancreas. Additional forms of cancer include, but are not limited to, lung cancer, prostate cancer and breast cancer. In one embodiment, the abnormal condition that is being diagnosed, monitored or tested is colon cancer or colorectal cancer. [0017] The levels of biomarker of the subject may be assessed in vivo or in vitro, from a sample from the subject. As used herein, a sample can be any environment that may be suspected of containing the antigen of interest. Thus, a sample includes, but is not limited to, a solution, a cell or a portion thereof, tissue culture medium, a body fluid, a tissue or portion thereof, and an organ or portion thereof. Examples of cells include, but are not limited to, bacteria, yeast, plant, insect, avian, fish, reptilian, amphibian, and mammalian such as, for example, bovine, ovine, equine, porcine, canine, feline, human and nonhuman primates. Other examples include non-animal organisms that may harbor similar antigens of interest, include but are not limited to molds, viruses, and other model systems for the study of biological processes. The scope of the invention should not be limited by the cell type assayed or the media in which these cells are cultured or processed (e.g., for the production of cellular or tissue lysates). Examples of biological samples to be assayed include, but are not limited to, blood, plasma, serum, urine, saliva, milk, seminal plasma, synovial fluid, interstitial fluid, cerebrospinal fluid, lymphatic fluids, bile, and amniotic fluid, tissue culture medium, tissue homogenates, cell lysates, chemical solutions. In one embodiment, the sample is a serum sample taken from the subject. The scope of the methods of the present invention should not be limited by the type of sample assayed. The terms "subject" "patient" and "organism" are used interchangeably herein and are used to mean any animal. In one embodiment the animal is a mammal. In a more particular embodiment, the animal is a human or nonhuman primate.

[0018] The samples may or may not have been removed from their native environment. Thus, the portion of sample assayed need not be separated or removed from the rest of the sample or from a subject that may contain the sample. Of course, the sample may also be removed from its native environment. For example, the sample may be a tissue section or serum sample. Furthermore, the sample may be processed prior to being assayed. For example, the sample may be diluted or concentrated; the sample may be purified and/or at least one compound, such as an internal standard, may be added to the sample. The sample may also be physically altered (e.g., centrifugation, affinity separation) or chemically altered (e.g., adding an acid, base or buffer, heating) prior to or in conjunction with the methods of the current invention. Processing also includes freezing and/or preserving the sample prior to assaying. [0019] The present invention relates to detecting and measuring levels of biomarker, where the biomarker is selected from the group consisting of a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:1 and a peptide having a massxharge ratio of approximately 771.88 daltons. In one embodiment, the biomarker is a peptide having a massxharge ratio of approximately 771.88 daltons. In a more specific embodiment, the biomarker is a peptide have a massxharge ratio of exactly 771.88.

[0020] In another embodiment, the biomarker is a peptide selected from the group consisting of a polypeptide comprising an amino acid at least 80% identical to the amino acid of SEQ ID NO: 1 and a polypeptide comprising an amino acid at least 80% identical to the amino acid ofSEQ ID NO:2.

[0021] The amino acid sequence of SEQ ID NO:1 and SEQ ID NO:2 are below.

SEQ ID NO:1 v ledrhlwnds hplkl

SEQ ID NO:2 mlsidlqlss icvprmqlkt cyveeirgw ledrhlwnds hplklggwrp aslrswg

[0022] Examples of an assay used in the methods of the present invention to assess the levels of biomarker include, but are not limited to, immunoassays, spectrophotometric assays and electrophoresis assays. Examples of immunoassays include, but are not limited to, immunosorbence assays and competitive binding assays. Specific embodiments of some of the assays listed include, but are not limited to, direct and indirect assays, as well as binary and tertiary sandwich assays. In one embodiment, the assay is an immunosorbence assay. In more specific embodiments, the immunosorbence assay is a colorimetric assay, an enzyme- linked immunosorbence assay (ELISA), a planar array or a radioimmunoassay. Other examples of assays that may be used in the methods of the present invention include, but are not limited to, bead or particle-based immunoassays, chemiluminescence assays, surface plasmon resonance (SPR) based assays, fluorescence assays, rolling-circle amplification assays, assays using dendrimers, and other enzyme or non-enzymatic amplification schemes.

[0023] The methods of the present invention may utilize antibodies or functional fragments thereof that are specific for the biomarker of the present invention. Accordingly, the invention also relates to antibodies or functional fragments thereof that bind to an epitope on the biomarker of the present invention, with the epitope residing on a molecule that is selected from the group consisting of a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:1 and a peptide having a massxharge ratio of between 750 and 790 daltons. In specific embodiments, the epitope resides on a peptide having a massxharge ratio of about 771.88 daltons. In another specific embodiment, the epitope resides on a peptide with a massxharge ratio of exactly 771.88 daltons. In yet another embodiment, the epitope resides on a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1. In still more specific embodiments, the epitope resides on a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:2.

[0024] As used herein, the term "antibody" is used to mean immunoglobulin molecules and functional fragments thereof, regardless of the source or method of producing the fragment. As used herein, a "functional fragment" of an immunoglobulin is a portion of the immunoglobulin molecule that specifically binds to a binding target. Thus, the term "antibody" as used herein encompasses whole antibodies, such as antibodies with isotypes that include but are not limited to IgG, IgM, IgA, IgD, IgE and IgY , and even single-chain antibodies found in some animals e.g., camels. Whole antibodies may be monoclonal or polyclonal, and they may be humanized or chimeric. The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. Rather the term "monoclonal antibody" refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. The term "antibody" also encompasses functional fragments of immunoglobulins, including but not limited to Fab fragments, Fab' fragments, F(ab')₂ fragments and Fd fragments. "Antibody" also encompasses fragments of immunoglobulins that comprise at least a portion of a V_L and/or V_H domain, such as single chain antibodies, a single-chain Fv (scFv), disulfide-linked Fvs and the like.

[0025] The antibodies used in the present invention may be monospecific, bispecific, trispecific or of even greater multispecifϊcity. In addition, the antibodies may be monovalent, bivalent, trivalent or of even greater multivalency. Furthermore, the antibodies of the invention may be from any animal origin including, but not limited to, birds and mammals. In specific embodiments, the antibodies are human, murine, rat, sheep, rabbit, goat, guinea pig, horse, or chicken. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described in United States Patent No. 5,939,598, which is herein incorporated by reference.

[0026] The antibodies used in the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide to which they recognize or specifically bind. Or the antibodies may be described based upon their ability to bind to specific conformations of the antigen, or specific modification (e.g., cleavage or chemical, natural or otherwise, modification of sequence).

[0027] The specificity of the antibodies used in present invention may also be described or specified in terms their binding affinity towards the antigen (epitope) or of by their cross- reactivity. Specific examples of binding affinities encompassed in the present invention include but are not limited to those with a dissociation constant (Kd) less than 5^χlO^~2 M, 10^"2 M, 5^χl(T³ M, 1(T³ M, 5xlO^"4 M, 1(T⁴ M, 5xlO^"5 M, 10^'5 M, 5xlO^"6 M, 10^"6 M, 5^χlO^"7 M, 10^"7 M, 5^χlO^'8 M, 10-⁸ M, 5xlO^"9 M, 10^"9 M, 5xl0^"10 M, 10^"10 M, 5XlO^"11 M, 10^"11 M, 5xlO^"12 M, 10^"12 M, 5^χlO^"13 M, 10^"13 M, 5^χlO^"14 M, 10^"14 M, 5X10⁴⁵ M, or 10^"15 M.

[0028] The antibodies used in the invention also include derivatives that are modified, for example, by covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response. Examples of modifications to antibodies include but are not limited to, glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other composition, such as a signaling moiety, a label, etc. In addition, the antibodies may be linked or attached to solid substrates, such as, but not limited to, beads, particles, glass surfaces, plastic surfaces, ceramic surfaces or metal surfaces. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, biotinylation, farnesylation, formylation, inhibition of glycosylation by tunicaniycin and the like. Additionally, the derivative may contain one or more non-classical or synthetic amino acids.

[0029] The antibodies used in the present invention may be generated by any suitable method known in the art. Polyclonal antibodies can be produced by various procedures well known in the art. For example, the biomarker or fragment thereof can be administered to various host animals including, but not limited to, rabbits, goats, chickens, mice, rats, to induce the production of sera containing polyclonal antibodies specific for the antigen. Various adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.

[0030] Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et at, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al, in: Monoclonal Antibodies and T-CeIl Hybridomas 563-681 (Elsevier, N. Y., 1981) (both of which are incorporated by reference).

[0031] Methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art such as, but not limited to, immunizing a mouse, hamster, or rat. Additionally, newer methods to produce rabbit and other mammalian monoclonal antibodies may be available to produce and screen for antibodies. In short, methods of producing and screening antibodies, and the animals used therein, should not limit the scope of the invention. Once an immune response is detected, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP2/0 available from the ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones can then be assayed by methods known in the art for cells that secrete antibodies capable of binding a biomarker of the present invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones. In addition, antibodies can be produced using a variety of alternate methods, including but not limited to bioreactors and standard tissue culture methods, to name a few.

[0032] The antibodies used the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In a particular embodiment, such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library. Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with the antigen of interest, such as using a labeled antigen or antigen bound or captured to a solid surface or bead. The phage used in these methods are typically filamentous phage including, but not limited to, fd and M 13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein. Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al, J. Immunol. Methods 182:41-50 (1995); Ames et al, J. Immunol. Methods 184:177-186 (1995); Kettleborough et al, Eur. J. Immunol. 24:952-958 (1994); Persic et al, Gene 187 9-18 (1997); Burton et al, Advances in Immunology 57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO 95/15982; WO 95/20401; and United States Patent Nos. 5,698,426; 5,223,409; 5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698; 5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108, all of which are incorporated by reference.

[0033] Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab')₂ fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')₂ fragments). F(ab')₂ fragments contain the variable region, the light chain constant region and the CHl domain of the heavy chain.

[0034] Other methods, such as recombinant techniques, may be used to produce Fab, Fab' and F(ab')₂ fragments and are disclosed in PCT publication WO 92/22324; Mullinax et al, BioTechniques 12(6):864-869 (1992); and Sawai et al, AJRI 34:26-34 (1995); and Better et al, Science 240:1041-1043 (1988), which are herein incorporated by reference. After phage selection, for example, the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria. [0035] Examples of techniques which can be used to produce other types of fragments, such as scFvs and include those described in United States Patent Nos. 4,946,778 and 5,258,498; Huston et al, Methods in Enzymology 203:46-88 (1991); Shu et al, Proc. Nat'lAcad. Sd. (USA) 90:7995-7999 (1993); and Skena et al, Science 240:1038-1040 (1988), all of which are incorporated by reference. For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al, BioTechniques 4:214 (1986); Gillies et al, J. Immunol. Methods 125:191- 202(1989); United States Patent Nos. 5,807,715; 4,816,567; and 4,816,397, all of which are herein incorporated by reference. Humanized antibodies are antibody molecules from non- human species antibody that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, and possibly improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. {See United States Patent No. 5,585,089; Riechmann et al, Nature 332:323 (1988), both of which are herein incorporated by reference. Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; United States Patent Nos. 5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al, Protein Engineering 7(6):805-814 (1994); Roguska. et al, Proc. Nat'l. Acad. Set 91:969-913 (1994)), and chain shuffling (United States Patent No. 5,565,332), all of which are hereby incorporated by reference.

[0036] Completely human antibodies may be particularly desirable for therapeutic treatment or diagnosis of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also. U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated by reference.

[0037] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995), which is hereby incorporated by reference. For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; United States Patent Nos. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771; and 5,939,598, which are incorporated by reference.

[0038] Still another approach for generating human antibodies utilizes a technique referred to as guided selection. In guided selection, a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et ah, Biotechnology 12:899-903 (1988), herein incorporated by reference).

[0039] The invention may also be used to screen antibodies that have been developed as potential therapeutics, such as, but not limited to, humanized antibodies. Vaccination studies may be undertaken that have the intent of generating antibodies in the subject that bind and antagonize the effects of the biomarker of the present invention. The compositions of the present invention may be used to compare the affinity or other characteristics of generated antibodies.

[0040] In one embodiment of the present invention, an antibody or functional fragment thereof would be bound to a solid support (such as glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylases, natural and modified celluloses, polyacrylamides, agaroses, and magnetite). The nature of the carrier can be either soluble to some extent or insoluble for the purposes of the present invention. The support material may have virtually any possible structural configuration so long as the coupled molecule is capable of binding to the biomarker. Thus, the support configuration may be spherical, as in a bead, or cylindrical, as in the inside surface of a test tube, or the external surface of a rod. Alternatively, the surface may be flat such as a sheet, test strip, etc. Those skilled in the art will note many other suitable carriers for binding antibody, or will be able to ascertain the same by use of routine experimentation.

[0041] For example, ELISA assays utilize a capture molecule that initially binds to the biomarker is or can be bound to the wells of culture plate. In this embodiment, the culture plate is acting as the carrier for the binding agent, i.e., the antibody. Subsequently, a labeled detection molecule (which may recognize the capture molecule or the biomarker) may be added to the test environment. One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the signal detected as well as other variations of ELISAs known in the art. As used herein the term "capture molecule" is used mean a binding agent that immobilizes the biomarker by its binding to the biomarker. Further, a biomarker is "immobilized" if the biomarker or biomarker-capture molecule complex is separated or is capable of being separated from the remainder of the sample. When the capture molecule is coated to a well or other surface, a detection molecule may be added following the addition of the biomarker of interest to the wells. As used herein, a detection molecule is used to mean a molecule, such as an antibody or receptor, comprising a label. In a specific embodiment, the methods of the present invention comprise the use of a capturing antibody and a detection antibody to detect the biomarker. In a more specific embodiment, the capture antibody and the detection antibody are the same antibodies with the same binding specificities. In another specific embodiment, the capture antibody and the detection antibody are different antibodies.

[0042] A label, as used herein, is intended to mean a chemical compound or ion that possesses or comes to possess or is capable of generating a detectable signal. The labels of the present invention may be conjugated to the primary binding agent, e.g., primary antibody, or secondary binding agent, e.g., secondary antibody, the biomarker or a surface onto which the label and/or binding agent is attached. Examples of labels includes, but are not limited to, radiolabels, such as, for example, ³H and ³²P, that can be measured with radiation-counting devices; pigments, biotin, dyes or other chromogens that can be visually observed or measured with a spectrophotometer; spin labels that can be measured with a spin label analyzer; and fluorescent labels (fluorophores), where the output signal is generated by the excitation of a suitable molecular adduct and that can be visualized by excitation with light that is absorbed by the dye or can be measured with standard fluorometers or imaging systems. Examples of suitable radioisotopic labels include ¹¹¹In, ¹²⁵1, ¹³¹I, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷Co, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd etc. Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd, ⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, ⁵⁶Fe, etc.

[0043] Additional examples of labels include, but are not limited to, a phosphorescent dye, a tandem dye and a particle. The label can be a chemiluminescent substance, where the output signal is generated by chemical modification of the signal compound; a metal-containing substance; or an enzyme, where there occurs an enzyme-dependent secondary generation of signal, such as the formation of a colored product from a colorless substrate. The term label also includes a "tag" or hapten that can bind selectively to a conjugated molecule such that the conjugated molecule, when added subsequently along with a substrate, is used to generate a detectable signal. For example, one can use biotin as a label and subsequently use an avidin or streptavidin conjugate of horseradish peroxidate (FIRP) to bind to the biotin label, and then use a colorimetric substrate (e.g., tetramethylbenzidine (TMB)) or a fluorogenic substrate such as Amplex Red reagent (Molecular Probes, Inc.) to detect the presence of HRP. Numerous labels are know by those of skill in the art and include, but are not limited to, particles, fluorophores, haptens, enzymes and their colorimetric, fluorogenic and chemiluminescent substrates and other labels that are described in RICHARD P. HAUGLAND, MOLECULAR PROBES HANDBOOK OF FLUORESCENT PROBES AND RESEARCH PRODUCTS (9^th edition, CD-ROM, (September 2002), which is herein incorporated by reference.

[0044] A fluorophore of the present invention is any chemical moiety that exhibits an absorption maximum beyond 280 nm, and when covalently attached to a labeling reagent retains its spectral properties. Fluorophores of the present invention include, without limitation; a pyrene (including any of the corresponding derivative compounds disclosed in US Patent 5,132,432, incorporated by reference), an anthracene, a naphthalene, an acridine, a stilbene, an indole or benzindole, an oxazole or benzoxazole, a thiazole or benzothiazole, a A- amino-7-nitrobenz-2-oxa-l, 3-diazole (NBD), a cyanine (including any corresponding compounds in US Serial Nos. 09/968,401 and 09/969,853, incorporated by reference), a carbocyanine (including any corresponding compounds in US Serial Nos. 09/557,275; 09/969,853 and 09/968,401; U.S.; Patents Nos. 4,981,977; 5,268,486; 5,569,587; 5,569,766; 5,486,616; 5,627,027; 5,808,044; 5,877,310; 6,002,003; 6,004,536; 6,008,373; 6,043,025; 6,127,134; 6,130,094; 6,133,445; and publications WO 02/26891, WO 97/40104, WO 99/51702, WO 01/21624; EP 1 065 250 Al, incorporated by reference), a carbostyryl, a porphyrin, a salicylate, an anthranilate, an azulene, a perylene, a pyridine, a quinoline, a borapolyazaindacene (including any corresponding compounds disclosed in US Patent Nos. 4,774,339; 5,187,288; 5,248,782; 5,274,113; and 5,433,896 , incorporated by reference), a xanthene (including any corresponding compounds disclosed in U.S. Patent No. 6,162,931; 6,130,101; 6,229,055; 6,339,392; 5,451,343 and US serial No. 09/922,333, incorporated by reference), an oxazine (including any corresponding compounds disclosed in US Patent No. 4,714,763, incorporated by reference) or a benzoxazine, a carbazine (including any corresponding compounds disclosed in US Patent No. 4,810,636, incorporated by reference), a phenalenone, a coumarin (including an corresponding compounds disclosed in US Patent Nos. 5,696,157; 5,459,276; 5,501,980 and 5,830,912, incorporated by reference), a benzofuran (including an corresponding compounds disclosed in US Patent Nos. 4,603,209 and 4,849,362, incorporated by reference) and benzphenalenone (including any corresponding compounds disclosed in US Patent No. 4,812,409, incorporated by reference) and derivatives thereof. As used herein, oxazines include resorufins (including any corresponding compounds disclosed in 5,242,805, incorporated by reference), aminooxazinones, diaminooxazines, and their benzo-substituted analogs. [0045] When the fluorophore is a xanthene, the fluorophore is optionally a fluorescein, a rhodol (including any corresponding compounds disclosed in US Patent Nos. 5,227,487 and 5,442,045, incorporated by reference), or a rhodamine (including any corresponding compounds in US Patent Nos. 5,798,276; 5,846,737; US serial no. 09/129,015, incorporated by reference). As used herein, fluorescein includes benzo- or dibenzofluoresceins, seminaphthofluoresceins, or naphthofluoresceins. Similarly, as used herein rhodol includes seminaphthorhodafluors (including any corresponding compounds disclosed in U.S. Patent No. 4,945,171, incorporated by reference). Alternatively, the fluorophore is a xanthene that is bound via a linkage that is a single covalent bond at the 9-position of the xanthene. Preferred xanthenes include derivatives of 3H-xanthen-6-ol-3-one attached at the 9-position, derivatives of 6-amino-3H-xanthen-3-one attached at the 9-position, or derivatives of 6- amino-3H-xanthen-3 -imine attached at the 9-position.

[0046] Fluorophores for use in the invention include, but are not limited to, xanthene (rhodol, rhodamine, fluorescein and derivatives thereof) coumarin, cyanine, pyrene, oxazine and borapolyazaindacene. Most preferred are sulfonated xanthenes, fluorinated xanthenes, sulfonated coumarins, fluorinated coumarins and sulfonated cyanines. The choice of the fluorophore attached to the labeling reagent will determine the absorption and fluorescence emission properties of the labeling reagent and immuno-labeled complex. Physical properties of a fluorophore label include spectral characteristics (absorption, emission and stokes shift), fluorescence intensity, lifetime, polarization and photo-bleaching rate all of which can be used to distinguish one fluorophore from another.

[0047] Typically the fluorophore contains one or more aromatic or heteroaromatic rings, that are optionally substituted one or more times by a variety of substituents, including without limitation, halogen, nitro, cyano, alkyl, perfluoroalkyl, alkoxy, alkenyl, alkynyl, cycloalkyl, arylalkyl, acyl, aryl or heteroaryl ring system, benzo, or other substituents typically present on fluorophores known in the art.

[0048] In one aspect of the invention, the fluorophore has an absorption maximum beyond 480 nm. In a particularly useful embodiment, the fluorophore absorbs at or near 488 nm to 514 nm (particularly suitable for excitation by the output of the argon-ion laser excitation source) or near 546 nm (particularly suitable for excitation by a mercury arc lamp). [0049] Many of fluorophores can also function as chromophores and thus the described fluorophores are also preferred chromophores of the present invention.

[0050] In addition to fluorophores, enzymes also find use as labels. Enzymes are desirable labels because amplification of the detectable signal can be obtained resulting in increased assay sensitivity. The enzyme itself may not produce a detectable signal but is capable of generating a signal by, for example, converting a substrate to produce a detectable signal, such as a fluorescent, colorimetric or luminescent signal. Enzymes amplify the detectable signal because one enzyme on a labeling reagent can result in multiple substrates being converted to a detectable signal. This is advantageous where there is a low quantity of target present in the sample or a fluorophore does not exist that will give comparable or stronger signal than the enzyme. The enzyme substrate is selected to yield the preferred measurable product, e.g. colorimetric, fluorescent or chemiluminescence. Such substrates are extensively used in the art, many of which are described in the MOLECULAR PROBES HANDBOOK, supra.

[0051] In a specific embodiment, a colorimetric or fluorogenic substrate and enzyme combination uses oxidoreductases such as horseradish peroxidase and a substrate such as 3,3'-diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC), which yield a distinguishing color (brown and red, respectively). Other colorimetric oxidoreductase substrates that yield detectable products include, but are not limited to: 2,2-azino-bis(3- ethylbenzothiazoline-6-sulfonic acid) (ABTS), ø-phenylenediamine (OPD), 3,3',5,5'- tetramethylbenzidine (TMB), ø-dianisidine, 5 -aminosalicylic acid, 4-chloro-l-naphthol. Fluorogenic substrates include, but are not limited to, homovanillic acid or 4-hydroxy-3- methoxyphenylacetic acid, reduced phenoxazines and reduced benzothiazines, including Amplex^® Red reagent and its variants (U.S. Pat. No. 4,384,042) and reduced dihydroxanthenes, including dihydrofluoresceins (U.S. Pat. No. 6,162,931, incorporated by reference) and dihydrorhodamines including dihydrorhodamine 123. Peroxidase substrates that are tyramides (U.S. Pat. Nos. 5,196,306; 5,583,001 and 5,731,158, incorporated by reference) represent a unique class of peroxidase substrates in that they can be intrinsically detectable before action of the enzyme but are "fixed in place" by the action of a peroxidase in the process described as tyramide signal amplification (TSA). These substrates are extensively utilized to label targets in samples that are cells, tissues or arrays for their subsequent detection by microscopy, flow cytometry, optical scanning and fluorometry. [0052] Another preferred colorimetric (and in some cases fluorogenic) substrate and enzyme combination uses a phosphatase enzyme such as an acid phosphatase, an alkaline phosphatase or a recombinant version of such a phosphatase in combination with a colorimetric substrate such as 5-bromo-6-chloro-3-indolyl phosphate (BCIP), 6-chloro-3-indolyl phosphate, 5- bromo-6-chloro-3-indolyl phosphate, /?-nitrophenyl phosphate, or o-nitrophenyl phosphate or with a fluorogenic substrate such as 4-methylumbelliferyl phosphate, 6,8-difluoro-7-hydroxy- 4-methylcoumarinyl phosphate (DiFMUP, U.S. Pat. No. 5,830,912, incorporated by reference) fluorescein diphosphate, 3-O-methylfluorescein phosphate, resorufin phosphate, PH-(1, 3-dichloro-9,9-dimethylacridin-2-one-7-yl) phosphate (DDAO phosphate), or ELF 97, ELF 39 or related phosphates (U.S. Pat. Nos. 5,316,906 and 5,443,986, incorporated by reference).

[0053] Glycosidases, in particular beta-galactosidase, beta-glucuronidase and beta- glucosidase, are additional suitable enzymes. Appropriate colorimetric substrates include, but are not limited to, 5-bromo-4-chloro-3-indolyl beta-D-galactopyranoside (X-gal) and similar indolyl galactosides, glucosides, and glucuronides, o-nitrophenyl beta-D- galactopyranoside (ONPG) and p-nitrophenyl beta-D-galactopyranoside. Preferred fluorogenic substrates include resorufin beta-D-galactopyranoside, fluorescein digalactoside (FDG), fluorescein diglucuronide and their structural variants (U.S. Pat. Nos. 5,208,148; 5,242,805; 5,362,628; 5,576,424 and 5,773,236, incorporated by reference), 4- methylumbelliferyl beta-D-galactopyranoside, carboxyumbelliferyl beta-D-galactopyranoside and fluorinated coumarin beta-D-galactopyranosides (U.S. Pat. No. 5,830,912, incorporated by reference).

[0054] Additional enzymes include, but are not limited to, hydrolases such as cholinesterases and peptidases, oxidases such as glucose oxidase and cytochrome oxidases, and reductases for which suitable substrates are known.

[0055] Specific embodiments of the present invention comprise enzymes and their appropriate substrates to produce a chemiluminescent signal, such as, but not limited to, natural and recombinant forms of luciferases and aequorins. Chemiluminescence-producing substrates for phosphatases, glycosidases and oxidases such as those containing stable dioxetanes, luminol, isoluminol and acridinium esters are additionally useful. [0056] Additional embodiments comprise haptens such as biotin. Biotin is useful because it can function in an enzyme system or fluorogenic system to further amplify the detectable signal, and it can function as a tag to be used in affinity chromatography for isolation purposes. For detection purposes, an enzyme conjugate that has affinity for biotin is used, such as avidin-HRP or streptavidin-HRP. Subsequently, a peroxidase substrate is added to produce a detectable signal. Alternatively, a colorimetric or fluorimetric reporter dye or protein that has affinity for biotin is used, such as streptavidin-R-Phycoerythrin.

[0057] Haptens also include hormones, naturally occurring and synthetic drugs, pollutants, allergens, affector molecules, growth factors, chemokines, cytokines, lymphokines, amino acids, peptides, chemical intermediates, nucleotides and the like.

[0058] Fluorescent proteins also find use as labels for the labeling reagents of the present invention. Examples of fluorescent proteins include green fluorescent protein (GFP) and the phycobiliproteins and the derivatives thereof. The fluorescent proteins, especially phycobiliprotein, are particularly useful for creating tandem dye labeled labeling reagents. These tandem dyes comprise a fluorescent protein and a fluorophore for the purposes of obtaining a larger stokes shift wherein the emission spectra is farther shifted from the wavelength of the fluorescent protein's absorption spectra. This is particularly advantageous for detecting a low quantity of a target in a sample wherein the emitted fluorescent light is maximally optimized, in other words little to none of the emitted light is reabsorbed by the fluorescent protein. For this to work, the fluorescent protein and fluorophore function as an energy transfer pair wherein the fluorescent protein emits at the wavelength that the fluorophore absorbs at and the fluorophore then emits at a wavelength farther from the fluorescent proteins than could have been obtained with only the fluorescent protein. A particularly useful combination is the phycobiliproteins disclosed in US Patents 4,520,110; 4,859,582; 5,055,556, incorporated by reference, and the sulforhodamine fluorophores disclosed in 5,798,276, or the sulfonated cyanine fluorophores disclosed in US serial Nos. 09/968/401 and 09/969/853, incorporated by reference; or the sulfonated xanthene derivatives disclosed in 6,130,101, incorporated by reference and those combinations disclosed in US Patent 4,542,104, incorporated by reference. Alternatively, the fluorophore functions as the energy donor and the fluorescent protein is the energy acceptor.

[0059] In one embodiment, the label is a fluorophore selected from the group consisting of fluorescein, coumarins, rhodamines, 5-TMRIA (tetramethylrhodamine-5-iodoacetamide), (9- (2(or4)-(N-(2-maleimdylethyl)-sulfonamidyl)-4(or 2)-sulfophenyl)-2,3,6,7, 12,13,16,17- octahydro-(lH,5H,l lH,15H-xantheno(2,3,4-ij:5,6,7-i'j')diquinolizin-18-mm salt) (Texas Red®), 2-(5-(l -(6-(N-(2-maleimdylethyl)-amino)-6-oxohexyl)- 1 ,3-dihydro-3,3-dimethyl-5- sulfo-2H-indol-2-ylidene)-l,3-propyldienyl)-l-ethyl-3,3-dimethyl-5-sulfo-3H-indolium salt (Cy™3), N,N'-dimethyl-N-(iodoacetyl)-N'-(7-nitrobenz-2-oxa- 1 ,3-diazol-4- yl)ethylenediamine (IANBD amide), ό-acryloyl^-dimethylaminonaphthalene (acrylodan), pyrene, 6-amino-2,3-dihydro-2-(2-((iodoacetyl)amino)ethyl)- 1 ,3 -dioxo- 1 H- benz(de)isoquinoline-5,8-disulfonic acid salt (lucifer yellow), 2-(5-(l-(6-(N-(2- maleimdylethyl)-amino)-6-oxohexyl)-l,3-dihydro-3,3-dimethyl-5-sulfo-2H-indol-2-ylidene)- l,3-pentadienyl)-l-ethyl-3,3-dimethyl-5-sulfo-3H-indolium salt (Cy™5), 4-(5-(4- dimethylaminophenyl)oxazol-2-yl)phenyl-N- (2-bromoacetamidoethyl)sulfonamide (Dapoxyl® (2-bromoacetamidoethyl)sulfonamide)), (N- (4,4-difluoro-l,3,5,7-tetramethyl- 4- bora-3a,4a-diaza-s-indacene- 2-yl)iodoacetamide (BODIPY® 507/545 IA), N-(4,4-difiuoro- 5,7-diphenyl- 4-bora-3a,4a-diaza-s-indacene- 3-propionyl)- N'-iodoacetylethylenediamine (BODIPY 530/550 IA), 5-((((2-iodoacetyl)amino)ethyl) amino)naphthalene-l -sulfonic acid (1,5-LAEDANS), and carboxy-X-rhodamine, 5/6- iodoacetamide (XRIA 5,6). Another example of a label is BODIPY-FL-hydrazide. Other luminescent labels include lanthanides such as europium (Eu3+) and terbium (Tb3+), as well as metal-ligand complexes of ruthenium [Ru(II)], rhenium [Re(I)], or osmium [Os(II)], typically in complexes with diimine ligands such as phenanthroline.

[0060] In one embodiment, the sample would be placed in contact with the bound antibody or functional fragment thereof, under conditions sufficient to permit the biomarker which may be present in the sample to bind to the support-bound antibody or functional fragment thereof. In one specific embodiment, the support would then be incubated in the presence of a labeled antibody, or functional fragment thereof, specific for biomarker under conditions sufficient to permit the labeled antibody or functional fragment thereof to bind to an open binding site on the biomarker. After washing away unbound molecules of the labeled antibody or functional fragment thereof, the amount of label bound to the support is determined. The presence of molecules of labeled antibody or functional fragment thereof bound to the support indicates the presence of biomarker in the sample.

[0061] As will be readily perceived, any of a large number of equivalent assays may be alternatively employed without departing from the spirit of the above-described assay. For example, the assay can be conducted in liquid phase rather than through the use of a solid support. Other variations of immunoassays can be also employed.

[0062] The methods described herein can be used to measure of the levels for the purposes of the present invention. The measurement of the levels of biomarker may be qualitative or quantitative. For example, the levels of biomarker may be quantified is some numerical expression, such as a ratio or a percentage.

[0063] Once the levels of biomarker have been determined, this determination is then compared to normal levels of the biomarker of the present invention. "Normal levels" of the biomarker may be assessed by measuring levels of the biomarker in a known healthy subject, including the same subject that is later screened or being diagnosed. Normal levels may also be assessed over a population sample, where a population sample is intended to mean either multiple samples from a single subject or at least one sample from a multitude of subjects. Normal levels of a biomarker, in terms of a population of samples, may or may not be categorized according to characteristics of the population including, but not limited to, sex, age, weight, ethnicity, geographic location, fasting state, state of pregnancy or post- pregnancy, menstrual cycle, general health of the subject, alcohol or drug consumption, caffeine or nicotine intake and circadian rhythms.

[0064] A difference between normal levels and the measured levels of the biomarker may indicate that the subject has a disease or abnormal condition or has a higher (or lower) probability of developing a disease or abnormal condition than do normal subjects. In addition, the magnitude of difference between measured levels and normal levels of the biomarker may also indicate the severity of disease or abnormal condition or the level of probability of developing a disease or abnormal condition, compared to normal subjects.

[0065] The difference between measured levels of the biomarker and normal levels may be a relative or absolute quantity. In addition, "levels of biomarkers" is used to mean any measure of the quantity of the biomarker such as, but not limited to, mass, concentration and biological activity. Example of biological activities that may be used to quantify biomarkers include, but are not limited to, chemotactic, cytotoxic, enzymatic or other biological activities, such as quantifiable activities that are used, for example, by the National Institute for Biological Standards and Control (NIBSC) in the United Kingdom for the quantification of interferon, cytokine and growth-factor activity. The difference in levels of biomarker may be equal to zero, indicating that the subject is or may be normal, or that there has been no change in levels of biomarker since the previous assay.

[0066] The difference may simply be, for example, a measured fluorescent value, radiometric value, densitometric value, DNA quantification, mass value etc., without any additional measurements or manipulations. Alternatively, the difference may be expressed as a percentage or ratio of the measured value of the biomarker to a measured value of another compound including, but not limited to, a standard or internal standard. The difference may be negative, indicating a decrease in the amount of measured biomarker over normal value or from a previous measurement, or the difference may be positive, indicating an increase in the amount of measured antigen or other metric over normal values or from a previous measurement. The difference may also be expressed as a difference or ratio of the biomarker to itself, measured at a different point in time. The difference may also be determined using in an algorithm, wherein the raw data is manipulated.

[0067] In general, levels of biomarker that are higher than normal levels of biomarker may confirm that the subject has the abnormal condition or that the subject may have a lower probably than normal of not developing the abnormal condition. Conversely, levels of biomarker that are equal to or lower than normal levels of biomarker may confirm that the subject either does not have the abnormal condition or that the subject may have a higher probability than normal of not developing the abnormal condition.

[0068] The present invention also relates to methods of monitoring the progression of abnormal conditions in subjects, with the methods comprising determining the levels of a biomarker in a sample from the subject at a first and second, third, fourth, etc. time points. The levels of biomarker at each time point are then compared to determine differences of the biomarker over time. Any differences between the levels of the biomarker over time are indicative of the progression, regression or stasis of the abnormal condition in the subject.

[0069] As used herein, the phrase "monitor the progression" is used to indicate that the abnormal condition in the subject is being periodically checked to deteπnine if the abnormal condition is progressing (worsening), regressing (improving) or remaining static (no detectable change) in the individual by assaying the levels of biomarker in the subject using the methods of the present invention. The methods of monitoring may be used in conjunction with other monitoring methods or treatment regimens for the abnormal condition and to monitor the efficacy of these treatments. Thus, "monitor the progression" is also intended to indicate assessing the efficacy of a treatment regimen by periodically assessing the levels of biomarker and correlating any differences in the levels of biomarker in the subject over time with the progression, regression or stasis of the abnormal condition. Thus, for example, the methods of the present invention may be used to monitor a subject after polypectomy. In particular, the methods may be used to monitor patients that have had a successful polypectomy, such that the methods can be used to monitor the patient for followup colonoscopy. In another particular embodiment, the methods can be used to monitor patients that have received treatment, such as a tumor removal, but may need followup or concurrent treatment for that cancer. The methods can thus be used to determine a suitable follow up therapeutic regimen, after an initial treatment. Thus, in one embodiment, the present invention provides methods of individualizing a therapeutic regimen, comprising assessing levels of the biomarker and correlating these levels with a likely response to a variety of therapies. For example, patient populations may be stratified according to their response to therapy as their responsiveness correlates to levels of biomarker. Monitoring may include assessing the levels of biomarker at two time points from which a sample is taken, or it may include more time points, where any of the levels of biomarker at one particular time point from a given subject may be compared with the levels of biomarker in the same subject, respectively, at one or more other time points.

[0070] The biomarkers of the present invention include, but are not limited to, a polypeptide of comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1. The terms "polypeptide" and "protein" are used interchangeably herein. In specific embodiments, the biomarker is a polypeptide of comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:1. In another specific embodiment, the polypeptide comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:2. In even more specific embodiments, the biomarker is a polypeptide of comprising an amino acid sequence at least 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% identical to the amino acid sequence of SEQ ID NO:2. As used herein, "isolated polypeptide" is used to mean a polypeptide, which has been removed from its native environment. For example, polypeptides that have been removed or purified from cells are considered isolated. In addition, recombinantly produced polypeptides molecules contained in host cells are considered isolated for the purposes of the present invention. [0071] Protein purification techniques are well known to those of skill in the art. These techniques involve, but are not limited to, the crude fractionation of the cellular milieu to polypeptide and non-polypeptide fractions. Having separated the polypeptide from other proteins, the polypeptide of interest may be further purified using chromatographic and electrophoretic techniques to achieve partial or complete purification (or purification to homogeneity). Analytical methods particularly suited to the preparation of a pure peptide are ion-exchange chromatography, exclusion chromatography; polyacrylamide gel electrophoresis; isoelectric focusing. A particularly efficient method of purifying peptides is fast protein liquid chromatography or HPLC.

[0072] Isolated polypeptides also include "purified" peptides. The term "purified" as it relates to polypeptides or polynucleotides will refer to a polypeptide or polynucleotide molecule that has been subjected to fractionation to remove various other components, and which composition substantially retains its expressed biological activity. Where the term "substantially purified" is used, this designation will refer to a composition in which the polypeptide or polynucleotide forms the major non-solvent component of the composition. For example, a "substantially purified polypeptide" indicates that more than about 50%, about 60%, about 70%, about 80%, about 90%, about 95% of the protein component of a solution or composition is the polypeptide of the present invention.

[0073] Various methods for quantifying the degree of purification of the protein or peptide will be known to those of skill in the art in light of the present disclosure. These include, for example, determining the specific activity of an active fraction, or assessing the amount of polypeptides within a fraction by SDS/PAGE analysis. A preferred method for assessing the purity of a fraction is to calculate the specific activity of the fraction, to compare it to the specific activity of the initial extract, and to thus calculate the degree of purity, herein assessed by a "x-fold purification number" (i.e., 2-fόld, 5-fold, 10-fold, 50-fold, 100-fold, 1000-fold, etc.). The actual units used to represent the amount of activity will, of course, be dependent upon the particular assay technique chosen to follow the purification and whether or not the expressed protein or peptide exhibits a detectable activity.

[0074] Various techniques suitable for use in protein purification will be well known to those of skill in the art. These include, for example, precipitation with ammonium sulphate, PEG, antibodies and the like or by heat or acid pH denaturation of contaminating proteins, followed by centrifugation; chromatography steps such as ion exchange and ion trapping, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focusing; gel electrophoresis; and combinations of such and other techniques. As is generally known in the art, it is believed that the order of conducting the various purification steps may be changed, or that certain steps may be omitted, and still result in a suitable method for the preparation of a substantially purified protein or peptide.

[0075] There is no general requirement that the polypeptide always be provided in their most purified state. Indeed, it is contemplated that less substantially purified products will have utility in certain embodiments. Partial purification may be accomplished by using fewer purification steps in combination, or by utilizing different foπns of the same general purification scheme. For example, it is appreciated that a cation-exchange column chromatography performed utilizing an HPLC apparatus will generally result in a greater "x- fold" purification than the same technique utilizing a low pressure chromatography system. Methods exhibiting a lower degree of relative purification may have advantages in total recovery of protein product, or in maintaining the activity of an expressed protein.

[0076] It is known that the migration of a polypeptide can vary, sometimes significantly, with different conditions of SDS/PAGE and according to how extensively it is glycosylated (Capaldi et al., 1977). It will therefore be appreciated that under differing electrophoresis conditions, the apparent molecular weights of purified or partially purified expression products may vary.

[0077] High Performance Liquid Chromatography (HPLC) is characterized by a very rapid separation with extraordinary resolution of peaks. This is achieved by the use of very fine particles and high pressure to maintain an adequate flow rate. Separation can be accomplished in a matter of minutes. Moreover, only a very small volume of the sample is needed, because the particles are so small and closely packed that the void volume is a very small fraction of the bed volume. Also, the concentration of the sample need not be great because the bands are so narrow that there is very little dilution of the sample.

[0078] Gel chromatography, or molecular sieve chromatography, is a special type of partition chromatography that is based on molecular size. The theory behind gel chromatography is that the column, which is prepared with small particles of an inert substance that contain small pores, separates larger molecules from smaller molecules as they pass through or around the pores, depending on their size. As long as the material of which the particles are made does not adsorb the molecules, the primary factor determining rate of flow is particle size. Hence, molecules are eluted from the column in decreasing size. Gel chromatography is unsurpassed for separating molecules of different size because separation is independent of all other factors such as, but not limited to pH, ionic strength, temperature, etc.

[0079] Affinity Chromatography is a chromatographic procedure that relies on the specific affinity between a substance to be isolated and a molecule to which it can specifically bind in a receptor-ligand type interaction. The column material is synthesized by covalently coupling one of the binding partners to an insoluble matrix. The column material can then specifically adsorb the substance from the solution. Elution occurs by changing the conditions (pH, ionic strength, temperature, etc.) such that binding between the column material and the biomarker will not occur.

[0080] A particular type of affinity chromatography useful in the purification of carbohydrate containing compounds is lectin affinity chromatography. Lectins are a class of substances that bind to a variety of polysaccharides and glycoproteins. Lectins are usually coupled to agarose by cyanogen bromide. Conconavalin A coupled to Sepharose was the first material of this sort to be used and has been widely used in the isolation of polysaccharides and glycoproteins other lectins that have been include lentil lectin, wheat germ agglutinin which has been useful in the purification of N-acetyl glucosaminyl residues and Helix pomatia lectin. Lectins themselves are purified using affinity chromatography with carbohydrate ligands. Lactose has been used to purify lectins from castor bean and peanuts; maltose has been useful in extracting lectins from lentils and jack bean; N-acetyl-D galactosamine is used for purifying lectins from soybean; N-acetyl glucosaminyl binds to lectins from wheat germ; D-galactosamine has been used in obtaining lectins from clams and L-fucose will bind to lectins from lotus.

[0081] The matrix used in affinity chromatography should be a substance that itself does not adsorb molecules to any significant extent and that has a broad range of chemical, physical and thermal stability. The binding agent should be coupled to the matrix in such a way as to not affect its binding properties. The binding agent should also provide relatively tight binding to the biomarker. As a practical matter, it should be possible to elute the biomarker without significant destruction of the polypeptide. One of the most common forms of affinity chromatography is immunoaffmity chromatography. The generation of antibodies that would be suitable for use in accord with the present invention is discussed above. [0082] As used herein, "identity" as it relates to amino acid sequence or polynucleotide sequences is a measure of the identity of nucleotide sequences or amino acid sequences compared to a reference nucleotide or amino acid sequence, usually a wild-type sequence. In general, the sequences are aligned so that the highest order match is obtained. "Identity" per se has an art-recognized meaning and can be calculated using published techniques. (See, e.g., Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York (1988); Biocomputing: Informatics And Genome Projects, Smith, D. W., ed., Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey (1994); von Heinje, G., Sequence Analysis In Molecular Biology, Academic Press (1987); and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York (1991)). While several methods exist to measure identity between two polynucleotide or polypeptide sequences, the term "identity" is well known in the art (Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988)). Methods commonly employed to determine identity or similarity between two sequences include, but are not limited to, those disclosed in Guide to Huge Computers, Martin J. Bishop, ed., Academic Press, San Diego (1994) and Carillo, H. & Lipton, D., Siam J Applied Math 48:1073 (1988). Computer programs may also contain methods and algorithms that calculate identity and similarity. Examples of computer program methods to determine identity and similarity between two sequences include, but are not limited to, GCS program package (Devereux, J., et al., Nucleic Acids Research 12(i):387 (1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol 215:403 (1990)).

[0083] A polypeptide having an amino acid sequence at least, for example, about 95% "identical" to a reference nucleotide sequence encoding a peptide of interest, for example BBl, is understood to mean that the amino acid sequence of the peptide is identical to the reference sequence except that the amino acid sequence may include up to about five mutations per each 100 amino acids of the reference peptide sequence encoding the BBl peptide being used as the reference sequence. In other words, to obtain a polypeptide having an amino acid sequence at least about 95% identical to a reference amino acid sequence, up to about 5% of the amino acids in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to about 5% of the total amino acids in the reference sequence may be inserted into the reference sequence. These mutations of the reference sequence may occur at the N- or C- terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among amino acids in the reference sequence or in one or more contiguous groups within the reference sequence.

[0084] For example, certain amino acids may be substituted for other amino acids in a protein structure without appreciable loss of interactive binding capacity with structures such as, for example, antigen-binding regions of antibodies or binding sites on substrate molecules. Since it is the interactive capacity and nature of a protein that defines that protein's biological functional activity, certain amino acid sequence substitutions can be made in a protein sequence, and the DNA sequence encoding the polypeptide, to obtain a polypeptide with similar, if not identical, properties. It is thus contemplated by the inventors that various changes may be made in the peptide sequences of the disclosed compositions.

[0085] In making such changes, the hydropathic index of amino acids may or may not be considered. The importance of the hydropathic amino acid index in conferring interactive biologic function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporate herein by reference). It is accepted that the relative hydropathic character of the amino acid contributes to the secondary structure of the resultant protein, which in turn may define the interaction of the protein with other molecules, for example, enzymes, substrates, receptors, DNA, antibodies, antigens, and the like.

[0086] Each naturally occurring amino acid has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics (Kyte and Doolittle, 1982), these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (- 0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (4.5). Of course, non-naturually occurring amino acids, which have their own hydrophobicity and charge characteristics may be used in the present invention.

[0087] It is known in the art that certain amino acids may be substituted by other amino acids having a similar hydropathic index or score and still result in a protein with similar biological activity, i.e., still obtain a biological functionally equivalent protein. In making such changes, the substitution of amino acids whose hydropathic indices may be within ±0.2 units in the hydropathic index. In one particular embodiment, amino acids are substituted with alternate amino acids that are within ±0.1 units in the hyphopathic index. In a more particular embodiment, amino acids are substituted with alternate amino acids that are within ±0.05. units in the hyphopathic index.

[0088] It is also understood in the art that the substitution of like amino acids can be made effectively on the basis of hydrophilicity. As detailed in U.S. Pat. No. 4,554,101, incorporated by reference, the following hydrophilicity values have been assigned to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ±0.1); glutamate (+3.0 ±0.1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ±0.1); alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4).

[0089] It is understood that an amino acid can be substituted for another having a similar hydrophilicity value and still obtain a biologically equivalent protein. In making such changes, the substitution of amino acids whose hydrophilicity indices may be within ±0.2 units in the hydrophilicty index. In one particular embodiment, amino acids are substituted with alternate amino acids that are within ±0.1 units in the hydrophilicity index. In a more particular embodiment, amino acids are substituted with alternate amino acids that, are within ±0.05 units in the hydrophilicity index.

[0090] As outlined above, amino acid substitutions are generally therefore based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. Exemplary substitutions which take variations of the foregoing characteristics into consideration are well known to those of skill in the art and include: arginine and lysine; glutamate and aspartate; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine.

[0091] The proteins of the current invention may be expressed in a modified form and may include not only additional fusions, but also secretion signals and other heterologous functional regions. Thus, for instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the protein to improve stability and persistence in the host cell, during purification or during subsequent handling and storage. Also, a region also may be added to the protein to facilitate purification. Such regions may be removed prior to final preparation of the protein. The addition of peptide moieties to proteins to engender secretion or excretion, to improve stability and to facilitate purification, among others, is familiar and routine techniques in the art. A preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins. For example, EP A0464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof. In many cases, the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thereby results, for example, in improved pharmacokinetic properties (EP A0232 262). On the other hand, for some uses, it would be desirable to be able to delete the Fc part after the fusion protein has been expressed, detected and purified in the advantageous manner described.

[0092] The fusion proteins of the current invention can be recovered and purified from recombinant cell cultures by well-known methods including, but not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography ("EDPLC") may also be employed for purification. Well- known techniques for refolding protein may be employed to regenerate active conformation when the fusion protein is denatured during isolation and/or purification.

[0093] Fusion proteins of the present invention include, but are not limited to, products of chemical synthetic procedures and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the fusion proteins of the present invention may be glycosylated or may be non- glycosylated. In addition, fusion proteins of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.

[0094] The fusion proteins may be used in accordance with the present invention for a variety of applications, particularly those useful in detecting or monitoring an analyte. Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms.

[0095] The present invention also relates to methods of immortalizing cells, with the methods comprising administering a polypeptide of the present invention to a cell line. The term "administer" is used to indicate that the compound is introduced into a cell or is contacted with the cell. Thus, "administer" includes introducing a polypeptide via recombinant methods, as well as "traditional" methods for administering a compound to cells. [0096] The invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention. In one embodiment, the therapeutic is an antibody of the invention.

[0097] Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.

[0098] The agonist or antagonists described herein can be administered in vitro, ex vivo, or in vivo to cells which express the receptor of the present invention. Thus, the present invention also relates to arresting cells in the cell cycle comprising administering an antagonist of the biomarker of the present invention to cells. Likewise, the present invention relates to increasing cell proliferation comprising administering the biomarker of the present invention, or agonists thereof, to cells. By administration of an "effective amount" of an agonist or antagonist is intended an amount of the compound that is sufficient to enhance or inhibit a cellular response to the biomarker of the present invention. In particular, by administration of an "effective amount" of an agonist or antagonists is intended an amount effective to enhance or inhibit biomarker-mediated cell proliferation. Of course, where it is desired for cell proliferation to be enhanced, an agonist according to the present invention can be coadministered with another compound. One of ordinary skill will appreciate that effective amounts of an agonist or antagonist can be determined empirically and may be employed in pure form or in pharmaceutically acceptable salt, ester or prodrug form. The agonist or antagonist may be administered in compositions in combination with one or more pharmaceutically acceptable excipients (i.e., carriers).

[0099] It will be understood that, when administered to a human patient, the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgement. The specific therapeutically effective dose level for any particular patient will depend upon factors well known in the medical arts.

[00100] As a general proposition, the total pharmaceutically effective amount of BBl antagonist administered parenterally per dose will be in the range of about 1 μg/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. In particular, this dose is at least 0.01 mg/kg/day. If given continuously, the agonists or antagonists of antagonists of the present invention are typically administered at a dose rate of about 1 μg/kg/hour to about 50 μg/kg/hour, either by 14 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed.

[00101] Dosaging may also be arranged in a patient specific manner to provide a predetermined concentration of an agonist or antagonist in the blood, as determined by the RIA technique. Thus patient dosaging may be adjusted to achieve regular on-going trough blood levels, as measured by RIA, on the order of from 50 to 1000 ng/ml, in particulart about 150 to 500 ng/ml.

[00102] Pharmaceutical compositions are provided comprising an agonist or antagonist and a pharmaceutically acceptable carrier or excipient, which may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray. Importantly, by co-administering an agonist and an addition effective compound, clinical side effects can be reduced by using lower doses of both the ligand and the agonist. It will be understood that the agonist can be "co-administered" either before, after, or simultaneously with the antagonists of the present invention, depending on the exigencies of a particular therapeutic application. By "pharmaceutically acceptable carrier" is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In a specific embodiment, "pharmaceutically acceptable" means approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers include, but are not limited to, sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include, but are not limited to, starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained- release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[00103] In one embodiment, the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.

[00104] The compounds of the invention can be formulated as neutral or salt forms.

Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc. [00105] The term "parenteral" as used herein refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.

[00106] Various delivery systems are known and can be used to administer a compound of the invention, e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, 1987, J Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradeπnal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compounds or compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[00107] In a specific embodiment, it may be desirable to administer the pharmaceutical compounds or compositions of the invention locally to the area in need of treatment; this may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the invention, care must be taken to use materials to which the protein does not absorb.

[00108] In another embodiment, the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, (1989). [00109] In yet another embodiment, the compound or composition can be delivered in a controlled release system. In one embodiment, a pump may be used (see Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, FIa. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Neurol. 25:351; Howard et al., 1989, J. Neurosurg. 71:105). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 1 15-138 (1984)).

[00110] Other controlled release systems are discussed in the review by Langer (1990,

Science 249:1527-1533). The compositions of the invention are also suitably administered by sustained-release systems. Suitable examples of sustained-release compositions include suitable polymeric materials (such as, for example, semi-permeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules), suitable hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, and sparingly soluble derivatives (such as, for example, a sparingly soluble salt).

[00111] Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP

58,481), copolymers of L-glutamic acid and gamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556 (1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J. Biomed Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech. 12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) or poly-D- (-)-3-hydroxybutyric acid (EP 133,988).

[00112] Sustained-release compositions also include liposomally entrapped compositions of the invention (see generally, Langer, Science 249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 317-327 and 353-365 (1989)). Liposomes containing DR5 polypeptide my be prepared by methods known per se: DE 3,218,121; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP 36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl. 83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324. Ordinarily, the liposomes are of the small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal DR5 polypeptide therapy.

[00113] In a specific embodiment where the compound of the invention is a nucleic acid encoding a protein, the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Pat. No.4,980,286), or by direct injection, or by use of microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox-like peptide which is known to enter the nucleus (see, e.g., Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864- 1868), etc. Alternatively, a nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.

[00114] In yet an additional embodiment, the compositions of the invention are delivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989)).

[00115] Pharmaceutical compositions of the present invention for parenteral injection can comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.

[00116] The compounds or pharmaceutical compositions of the invention may be tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans. For example, in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample. The effect of the compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to, rosette formation assays and cell lysis assays. In accordance with the invention, in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.

[00117] The compositions of the invention may be administered alone or in combination with other adjuvants. Adjuvants that may be administered with the compositions of the invention include, but are not limited to, alum, alum plus deoxycholate (ImmunoAg), MTP-PE (Biocine Corp.), QS21 (Genentech, Inc.), BCG, and MPL. In a specific embodiment, compositions of the invention are administered in combination with alum. In another specific embodiment, compositions of the invention are administered in combination with QS-21. Further adjuvants that may be administered with the compositions of the invention include, but are not limited to, Monophosphoryl lipid immunomodulator, Adju Vax 100a, QS-18, CRL1005, Aluminum salts, ME-59, and Virosomal adjuvant technology.

[00118] The compositions of the invention may be administered alone or in combination with other therapeutic agents. Therapeutic agents that may be administered in combination with the compositions of the invention, include but are not limited to, chemotherapeutic agents, antibiotics, antivirals, steroidal and non-steroidal antiinflammatories, conventional immunotherapeutic agents, cytokines, chemokines and/or growth factors. Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g., as through separate intravenous lines into the same individual. Administration "in combination" further includes the separate administration of one of the compounds or agents given first, followed by the second.

[00119] The invention also relates to nucleic acid molecule encoding the polypeptides of the present invention. Nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically. The DNA may be double-stranded or single-stranded. Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand. [00120] The present invention is further directed to fragments of the isolated nucleic acid molecules described herein. A "fragment" of an isolated nucleic acid molecule having the nucleotide sequence coding for the fusion proteins of the current invention is used to indicate fragments at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length, which are useful as diagnostic probes and primers as discussed herein. Of course larger DNA fragments that are 50, 100, 150, 200, 250, 300, 350, 400, or 425 nt in length are also useful according to the present invention, as are fragments corresponding to most, if not all, of the nucleotide sequence that codes for a fusion protein of the current invention. A fragment at least 20 nt in length, for example, is understood to mean a fragment that includes 20 or more contiguous bases from the nucleotide sequence coding for the fusion proteins of the current invention. Generating such DNA fragments would be routine to the skilled artisan. For example, restriction endonuclease cleavage or shearing by sonication could easily be used to generate fragments of various sizes. Alternatively, such fragments could be generated synthetically.

[00121] In another aspect, the invention provides an isolated nucleic acid molecule comprising a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above. "Stringent hybridization conditions" is understood in the art and is used to mean overnight incubation at 42°C in a solution comprising: 50% formamide, 5X SSC (150 mMNaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5X Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1X SSC at about 65⁰C.

[00122] A polynucleotide which hybridizes to a "portion" of a polynucleotide is understood to mean a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 nt of the reference polynucleotide. Such fragments that hybridize to a portion of the reference polynucleotide are useful as fragments.

[00123] Of course, polynucleotides hybridizing to a larger portion of the reference polynucleotide, e.g., a portion 50-300 nt in length, or even to the entire length of the reference polynucleotide, are also useful as probes according to the present invention, as are polynucleotides corresponding to most, if not all, of the reference nucleotide sequences. A portion of a polynucleotide of "at least 20 nt in length," for example, is understood to mean 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide. As indicated, such portions are useful diagnostically either as a probe according to conventional DNA hybridization techniques or as primers for amplification of a target sequence by the polymerase chain reaction (PCR), as described, for instance, in Molecular Cloning, A Laboratory Manual, 3rd. edition, Sambrook, J., Fritsch, E. F. and Maniatis, T., eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N. Y. (2001), the entire disclosure of which is hereby incorporated herein by reference.

[00124] The present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the fusion proteins. Variants may occur naturally, such as a natural allelic variant. An "allelic variant" is understood to mean one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. See, e.g., Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985). Non-naturally occurring variants may be produced using art-known mutagenesis techniques.

[00125] Such variants include those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides. The variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions.

[00126] Thus, the invention contemplates isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least about 80%, 90% or 95% identical to polynucleotides encoding the polypeptides of the current invention. More particularly, the invention contemplates isolated nucleic acid molecules comprising a polynucleotide having a nucleotide sequence at least about 96%, about 97%, about 98% or about 99% identical to polynucleotides encoding the polypeptides of the current invention.

[00127] The present invention also relates to vectors that include DNA molecules of the present invention, host cells that are genetically engineered with vectors of the invention and the production of proteins of the invention by recombinant techniques.

[00128] Host cells can be genetically engineered to incorporate nucleic acid molecules that are free within the nucleus of the cell (transiently transfected) or incorporated within the chromosome of the cell (stably transfected) and express proteins of the present invention. The polynucleotides may be introduced alone or with other polynucleotides. Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention.

[00129] In accordance with this aspect of the invention, the vector may be, for example, a plasmid vector, a single-or double-stranded phage vector, or a single-or double- stranded RNA or DNA viral vector. Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well-known techniques for introducing DNA and RNA into cells. Viral vectors may be replication competent or replication defective. In the latter, case viral propagation generally will occur only in complementing host cells.

[00130] Preferred among vectors, in certain respects, are those for expression of polynucleotides and proteins of the present invention. Generally, such vectors comprise cis- acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed. Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.

[00131] A great variety of expression vectors can be used to express the proteins of the invention. Such vectors include chromosomal, episomal and virus-derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as adeno-associated virus, lentivirus, baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids. All may be used for expression in accordance with this aspect of the present invention. Generally, any vector suitable to maintain, propagate or express polynucleotides or proteins in a host may be used for expression in this regard.

[00132] The DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s) including, for instance, a promoter to direct mRNA transcription. Representatives of such promoters include, but are not limited to, the phage lambda PL promoter, the E. coli lac, trp and tac promoters, HIV promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well- known promoters. In general, expression constructs will contain sites for transcription, initiation and termination and, in the transcribed region, a ribosome binding site for translation. The coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.

[00133] In addition, the constructs may contain control regions that regulate, as well as engender expression. Generally, such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.

[00134] Vectors for propagation and expression generally will include selectable markers. Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose. In this regard, the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing E. coli and other bacteria.

[00135] The vector containing the appropriate DNA sequence, as well as an appropriate promoter, and other appropriate control sequences, may be introduced into an appropriate host using a variety of well-known techniques suitable to expression therein of a desired polypeptide. Representative examples of appropriate hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells. Hosts for of a great variety of expression constructs are well known, and those of skill in the art will be enabled by the present disclosure to select an appropriate host for expressing one of the proteins of the present invention.

[00136] Examples of vectors for use in bacteria include, but are not limited to, pQE70, pQE60 and pQE-9, available from Qiagen (Valencia, CA); pBS vectors, Phagescript vectors, Bluescript vectors, pNHSA, pNHlόa, pNH18A, pNH46A, available from Stratagene (La Jolla, CA); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Amersham- Pharmacia Biotech (Piscataway, NJ); and pEGFP-Cl, pEYFP-Cl, pDsRed2-Cl, pDsRed2- Express-Cl, and pAcGFPl, pAcGFP-Cl, pZsYellow-Cl, available from Clontech (Palo Alto, CA). Examples of eukaryotic vectors include, but are limited to, p W-LNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; pSVK3, pBPV, pMSG and pSVL available from Pharmacia; and pCMVDsRed2-express, pIRES2-DsRed2, pDsRed2- Mito, pCMV-EGFP available from Clontech. Many other commercially available and well- known vectors are available to those of skill in the art. Selection of appropriate vectors and promoters for expression in a host cell is a well-known procedure and the requisite techniques for expression vector construction, introduction of the vector into the host and expression in the host are routine skills in the art.

[00137] The present invention also relates to host cells containing the above-described constructs. The host cell can be a higher eukaryotic cell, such as a mammalian cell, or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell. The host cell can be stably or transiently transfected with the construct.

[00138] Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods in Molecular Biology (1986).

[00139] The current invention also relates to methods of producing a protein, such as biomarkers or antibodies, comprising culturing the host cells of the invention under conditions such that said protein is expressed, and recovering said protein. The culture conditions required to express the proteins of the current invention are dependent upon the host cells that are harboring the polynucleotides of the current invention. The culture conditions for each cell type are well-known in the art and can be easily optimized, if necessary.

[00140] The present invention also relates to kits useful for monitoring an analyte in a sample. The kits of the current invention comprise at least one composition (e.g. biomarker, antibody and/or a fusion polypeptide) of the current invention. The kit may also comprise instructions or written material to aid the user.

[00141] The present invention also relates to methods of determining cell proliferation or differentiation. The methods comprise determining levels of the biomarker of the present invention in a cell sample and comparing these determined levels to levels of biomarker in a control cell population. The control cell population may be non-proliferating cells or proliferating cells. The comparison between determined levels and control levels of the biomarker can then be used to determine a proliferation rate of test cells. Correlating levels of biomarker with proliferation rates in cells may be assessed using additional, well-known proliferation assays.

[00142] Similarly, the present invention also relates to methods of identifying compounds that alter cell proliferation, with the methods comprising treating cells with a candidate compound and assessing the levels of the biomarker of the present invention, in response to the test compound. A difference in levels of the biomarker of the present invention in treated cells versus untreated control cells may indicate that the candidate compound is effective in altering the proliferation rates of cells. For example, a candidate compound that causes an increase in the levels of biomarker over control untreated cells may increase the proliferation rates of the cells. Alternatively, a candidate compound that causes a decrease in the levels of biomarker over control untreated cells may decrease the proliferation rates of the cells.

Examples

[00143] Example 1 — Isolation of the Biomarker of the Present Invention

[00144] The present invention provides a novel serum biomarker for detecting the presence of cancerous cells. The biomarker was identified as a protein/polypeptide differentially expressed in the sera of colorectal cancer patients using mass spectrometry. The methods of extracting the polypeptide marker from a serum sample are described in Mehta, A.I., et al, Disease Markers, 19:1-10 (2003), which is incorporated by reference.

[00145] Briefly, serum samples were collected under full patient consent and

Institutional Review Board approval. Serum was collected before physical evaluation, diagnosis, and treatment and stored at-20°C. The colorectal study set consisted of 20 samples from normal, unaffected patients and 20 patients with diagnosed colorectal cancer. The same personnel were involved in the blood collection, handling, and storage of all biospecimens. In addition, all blood specimens were processed in an identical manner under the same methodology. These subjects were recruited from the general population and had no prior evidence of any cancer for 5 years. [00146] Each sample was accompanied by a verified pathology diagnosis. Briefly, specimens were collected in red-top Vacutainer Tubes and allowed to clot for 1 h on ice, followed by centrifugation at 4 ⁰C for 10 min at 1210g. The serum supernatant was divided in aliquots and stored at-20 ⁰C until needed. Samples were not assayed on one day and were specific samples were selected for analysis on a given day by random selection where the samples were categorized by pathology diagnosis as either cancer or benign.

[00147] Native, diluted serum was introduced into an affinity column so that the carrier protein (albumin) was captured along with any bound molecules. The bound subproteome comprising the carrier protein and their peptide "cargo" was eluted, dissociated, and separated by 1 -dimensional gel electrophoresis.

[00148] Each entire gel lane was cut out, finely subdivided into molecular mass regions, subjected to in-gel trypsin digestion, and prepared for electrospray mass spectrometric analysis.

[00149] About 25 μL of human stage-specific (pooled) cancer serum (~3.1 mg of protein) was diluted to 200 μL with Equilibration Buffer (Millipore) and run through a (Montage) albumin-specific affinity column twice. The bound protein was washed thoroughly with two 200-μL volumes of proprietary wash buffer (provided by the manufacturer). These fractions were combined and labeled as a "flow-through" fraction. The bound proteins were eluted from the column by equilibrating with acetonitrile-H2O- trifiuoroacetic acid (70:30:0.2 by volume) for 30 min, followed by a slow spin-through of the elution mixture, repeated once. The eluate (retentate fraction) was lyophilized to about <10 μL in a HetoVac roto (CT 110) and reconstituted in an H2O-acetonitrile-formic acid (95:5:0.1 by volume) buffer. Samples were desalted with a ZipTip cleanup and reconstituted in a 1:1 mixture of water and sodium dodecyl sulfate sample buffer (20 μL total volume).

[00150] The flow-through and retentate fractions were kept on ice in 20 μL of sample buffer from 25 μL of original serum, and then were heated for 5 min at 95 ⁰C and loaded onto a 1 -dimensional precast gel to separate albumin from the proteins/peptides/fragments of interest. The proteins and fragments were visualized with a Gel Code Blue Stain Reagent (Pierce) according to the manufacturer's protocols. The entire lane was excised from the gel and finely sliced into very small molecular-weight regions (~35 slices/lane). Gel bands were reduced, alkylated, and digested with porcine modified trypsin according to standard protocol and peptides were concentrated and prepped for mass spectrometric analysis.

[00151] Samples were lyophilized to near dryness and reconstituted in 6.3 μL of

Buffer A (H2O-acetonitrile-formic acid; 95:5:0.5 by volume) for MS analysis. Microcapillary reversed-phase tandem MS (μLC-MS/MS) analysis was performed with a Dionex LC Packings liquid chromatography system coupled on-line to a ThermoFinnigan LCQ Classic ion trap mass spectrometer with a modified nanospray source. Reversed-phase separations were performed on an in-house, slurry-packed capillary column. The Cl 8 silica- bonded column was a 10-cm long (75-μm i.d.) fused-silica column packed with 5-μm beads (pore size, 300 A; Vydac). A PepMap C18 cartridge (5-mm; Dionex) acted as a desalting column. Sample was injected in microliter pick-up mode and washed with Buffer A for 5 min before elution with a linear gradient with buffer B (acetonitrile-H2O-formic acid; 95:5:0.1 by volume) up to 85% over 95 min at a flow rate of 200 nL/min. Full MS scans were followed by 4 MS/MS scans of the most abundant peptide ions (in a data-dependent mode), and collision-induced dissociation was performed at a collision energy of 38% with the ion spray voltage set to 1.80 kV, capillary voltage set to 22.80 V and temperature set to 180 ⁰C.

[00152] Figure 1 represents the ion trap mass spectrum of the serum sample. Figure 2 represents the ion trap mass spectrum of the sample at an elution time t = 22.79 minutes. Finally, Figure 3 represents the ion trap mass spectra at t = 22.79 min with mass to charge ratio (m/z) of 771.88.

[00153] Example 2 — Data Analysis of Isolated Chromatographic Peaks

[00154] Data analysis was performed by searching MS/MS spectra against the

European Bioinformatics Institute of the nonredundant proteome set of Swiss-Prot, TrEMBL, and Ensembl entries through the Sequest Bioworks Browser (ThermoFinnigan), with a static modification of +57 Da on cysteine residues and a dynamic modification for oxidation of methionine of +15.9994 Da. Peptides were considered legitimate hits after the correlation scores were filtered and the MS/MS data were manually inspected. The table of correlation scores is below.

Table 1 Charge X_Covτ ΔCn Rap

+2 >2.2 XU -1

+3 >3.5 MU =-1

[00155] Using the above-identified analysis, ceruloplasmin precursor, prothrombin precursor, growth-regulating protein BBl, and complement component C6 precursor were significantly expressed only in colorectal cancer patients. In contrast, F-box protein 3, gelsolin precursor, lamin B receptor and several immune-related proteins, including immunoglobulins, were detected only in unaffected controls. The cell cycle regulator BBl is disclosed in Moats-Staats, B. M., et al, Molecular and Cellular Biology, 14(5): 2936-3945 (1994), which is hereby incoporated by reference.

[00156] Example 3 — Validation ofBiomarker via Western Blotting

[00157] A primary monoclonal or polyclonal antibody that recognizes BB 1 is synthesized according to well-known procedures. For example, rabbits are immunized with a peptide corresponding to an epitope of BBl and the resulting anti-BBl antibody is affinity- purified. The specificity of the antibody is verified against the full-length BBl protein (57 amino acids) extracted from a cellular nuclear extract. Preincubation of the primary antibody with an immunizing synthetic peptide overlapping the antigenic region of interest should successfully compete away the representative band of native BBl. After verification of the specificities of the antibody and competition peptide, this experimental procedure is applied to pooled colorectal cancer and control serum samples.

[00158] Example 4 - Immunoassay to Detect BBl Biomarker in Serum Samples

[00159] Prepared serum samples are heated for 5 min at 95 ⁰C in sample buffer containing 20 mL/L β-mercaptoethanol, followed by centrifugation at 10 00Og for 1 min to remove insoluble material. Samples are then subjected to 1-dimensional electrophoresis and electroblotting at 30 V for 2 h on ice. Membranes are incubated overnight at 4 ⁰C in 50 g/L nonfat dry milk, 75 g/L glycine, and 1 mL/L Tween 20 in water to block unoccupied protein binding sites.

[00160] The blocked membranes are rinsed twice with wash buffer [10 mmol/L Tris

(pH 7.5), 150 mmol/L NaCl, 1 g/L bovine serum albumin, 1 mL/L Tween 20] and then incubated with 1 mg/L primary antibody in wash buffer containing 50 g/L nonfat dry milk, with rocking, for 2 h at room temperature. For peptide blocking/competition assays, 10 μg of primary antibody is incubated with 100 μg of the corresponding immunization peptide in 400 μL of wash buffer for 1 h at room temperature with end-over-end mixing. The peptide- treated antibody solution is diluted to 10 mL (1 mg/L final antibody concentration) in wash buffer containing 50 g/L nonfat dry milk before incubation with polyvinylidene difluoride (PVDF) membrane.

[00161] The membranes are washed 5 times (3 min each) in 50 mL of wash buffer and subsequently incubated in 10 mL of horseradish peroxidase-conjugated goat anti-rabbit IgG (1 :50 000 in wash buffer) for 1 h at room temperature. After the PVDF membranes are washed thoroughly, signals are developed by enhanced chemiluminescence.

Claims

What is Claimed is:

1. A method of diagnosing an abnormal condition in a subject, said method comprising

a) determining the levels of a biomarker in a sample from said subject, said biomarker being selected from the group consisting of

i) a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1 ; and

ii) a peptide having a massxharge ratio of about 771.88 daltons; and

b) comparing the levels of said biomarker in said sample to normal levels of said biomarker,

wherein a difference between the levels of said biomarker from said sample and the normal levels of said biomarker indicates the presence of an abnormal condition in said subject.

2. The method of claim 1, wherein said biomarker is (i).

3. The method of claim 1, wherein said biomarker is (ii).

4. The method of claim 3, wherein said biomarker has a massxharge ratio of 771.88.

5. The method of claim 4, wherein said abnormal condition is colorectal carcinoma.

6. The method of claim 5, wherein said sample is a serum sample.

7. The method of claim 6, wherein said determination of levels of biomarker comprises an assay selected from the group consisting of an immunoassay, a spectrophotometric assay and an electrophoresis assay.

8. The method of claim 7, wherein said assay is an immunoassay, and wherein said immunoassay comprises an antibody or functional fragment thereof that is specific towards said biomarker.

9. The method of claim 8, wherein said antibody or functional fragment thereof is a monoclonal antibody.

10. The method of claim 2, wherein said biomarker comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:1.

11. The method of claim 10, wherein said abnormal condition is colorectal carcinoma.

12. The method of claim 11, wherein said sample is a serum sample.

13. The method claim 12, wherein said determination of levels of biomarker comprises an assay selected from the group consisting of an immunoassay, a spectrophotometric assay and an electrophoresis assay.

14. The method of claim 13, wherein said assay is an immunoassay, and wherein said immunoassay comprises an antibody or functional fragment thereof that is specific towards said biomarker.

15. The method of claim 14, wherein said antibody or functional fragment thereof is a monoclonal antibody.

16. The method of claim 10, wherein said biomarker comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:2.

17. The method of claim 16, wherein said biomarker comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2.

18. A method of monitoring the progression of an abnormal condition in a subject, said method comprising

a) determining the levels of a biomarker in a sample from said subject at a first time point, said biomarker being selected from the group consisting of

i) a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1 ; and

ii) a peptide having a mass:charge ratio of about 771.88daltons; and

b) determining levels of said biomarker in a sample from said subject at a second time point; c) comparing the levels of said biomarker at said first and second time points to determine a difference in the levels of said biomarker over time,

wherein a difference in said levels of said biomarker over time is indicative of the progression of said abnormal condition is said subject.

19. The method of claim 18, wherein said biomarker is (i).

20. The method of claim 18, wherein said biomarker is (ii).

21. The method of claim 20, wherein said biomarker has a massxharge ratio of 771.88.

22. The method of claim 21, wherein said abnormal condition is colorectal carcinoma.

23. The method of claim 22, wherein said sample is a serum sample.

24. The method of claim 23, wherein said determination of levels of biomarker comprises an assay selected from the group consisting of an immunoassay, a spectrophotometric assay and an electrophoresis assay.

25. The method of claim 24, wherein said assay is an immunoassay, and wherein said immunoassay comprises an antibody or functional fragment thereof that is specific towards said biomarker.

26. The method of claim 25, wherein said antibody or functional fragment thereof is a monoclonal antibody.

27. The method of claim 19, wherein said biomarker comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO: 1.

28. The method of claim 27, wherein said abnormal condition is colorectal carcinoma.

29. The method of claim 28, wherein said sample is a serum sample.

30. The method claim 29, wherein said determination of levels of biomarker comprises an assay selected from the group consisting of an immunoassay, a spectrophotometric assay and an electrophoresis assay.

31. The method of claim 30, wherein said assay is an immunoassay, and wherein said immunoassay comprises an antibody or functional fragment thereof that is specific towards said biomarker.

32. The method of claim 31, wherein said antibody or functional fragment thereof is a monoclonal antibody.

33. The method of claim 27, wherein said biomarker comprises an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:2.

34. The method of claim 33, wherein said biomarker comprises an amino acid sequence at least 90% identical to the amino acid sequence of SEQ ID NO:2.

35. An antibody or fragment thereof that binds to an antigen, said antigen being selected from the group consisting of a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO:1 and a peptide having a massxharge ratio of about 771.88 daltons.

36. A method of treating an abnormal condition in a subject in need of treatment thereof, said method comprising administering to said subject a therapeutically effective amount of a substance that reduces the activity levels of molecule selected from the group consisting of a peptide comprising an amino acid sequence at least 80% identical to the amino acid sequence of SEQ ID NO: 1 and a peptide having a massrcharge ratio of about 771.88 daltons.