WO2007030571A2 - Identification of targets and development of reagents for testing and molecular imaging of human disease - Google Patents

Abstract

The present invention describes both the identification of disease target molecules and the development of imaging reagents and diagnostic assays specific to those molecules. Described herein are methods and reagents for the identification of molecular targets specific to a disease or disease state, methods of imaging technology which can be used, the development of specific molecular imaging reagents, clinical validation of the imaging reagents, and clinical indications for molecular imaging.

Description

IDENTIFICATION OF TARGETS AND DEVELOPMENT OF REAGENTS FOR TESTING AND MOLECULAR IMAGING OF HUMAN DISEASE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional App. Ser. No. 60/714,790, filed' September 6, 2005. This application is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] The methods and reagents described herein generally relate to the identification of molecular targets which are specific to disease and the development of affinity reagents to the targets. The affinity reagent may be utilized as a diagnostic in a diagnostic assay. A contrast or signal emitting source may be incorporated into the reagent, to generate a target-specific imaging reagent.

BACKGROUND OF THE INVENTION

[0003] Medical diagnosis and monitoring utilize a variety of imaging techniques. Standard radiography, fluoroscopy, Magnetic Resonance Imaging (MRI), Computed Tomography (CT), Positron Emission Tomography (PET), Single Photon Emission Computed Tomography (SPECT) and Ultrasound are all useful methods in clinical practice. These techniques all have their unique advantages and drawbacks. The clinical utility of all of these techniques could be greatly enhanced by using contrast or signal emitting reagents which are targeted to disease specific molecules. This approach (molecular imaging) could provide added sensitivity for detection of disease or anatomical extent of disease and specificity to help differentiate anatomical findings due to disease from normal or non-diseased tissue. The potential applications of this approach are clear in every medical discipline and disease category.

[0004] Discovery or selection of molecules to target with such an approach can leverage the published literature, gene expression and protein databases, genomics studies, in vitro models, animal models and genomic level screening of candidates using high throughput scanning of thousands of candidate reagent combinations. Development of specific affinity reagents for such molecules can leverage recent advances in antibody development peptide and small molecule design along with methods for incorporation of contrast or signal emitting materials into reagents. When an affinity reagent is developed for a disease specific target, it may subsequently be linked to or labeled with a variety of different contrast/signal reagents for use with a variety of imaging technologies. In some cases the disease tissue may be obtained from a patient and assessed for the expression of appropriate markers prior to imaging of the patient. This may be useful in cancer staging or monitoring for recurrence where tumor tissue is obtained by biopsy or in surgery. Imaging may also be targeted to a biological process as opposed to a specific antigen. Examples of this would be targeting angiogenesis or macrophage transit into the blood vessel wall.

[0005] The field of molecular imaging represents a crossroads of multiple scientific and clinical disciplines. Clinical expertise is needed to assess needs, identify best indications for application of the technology and design clinical trials. Molecular biology, genomics and bioinformatics expertise is needed for identification of disease specific target molecules. The development of labeled affinity reagents requires expertise in chemistry and an understanding of physics. Radiological expertise is needed to develop the specific imaging protocols and technology use. As with early efforts in applications of genomics to diagnostics, it is critical that development and commercial efforts in this area not be driven only by technology concerns. Instead, clinical value should be the primary consideration for focusing these efforts. These efforts should not be wed to a single technology only, but should utilize the appropriate imaging technology for the specific disease indication. The imaging technology used must meet certain specifications to be useful for a given clinical indication and molecular marker, but a number of technologies may be useful alone or in combination and efforts to develop reagents should not be restricted to a single technology without consideration of all issues.

[0006] There is currently an opportunity to identify the most relevant target molecules for the clinical indications with highest need and take them downstream in focused development efforts to bring them to clinical use. The significant value associated with this effort is in development of clinically useful reagents and protocols and creation of a pipeline for target identification and development which brings a genomic level approach to this field and brings together the diverse disciplines needed to bring new tools to bear on clinical management for the benefit of patients.

[0007] In general the addition of a specific molecular targeting reagent to imaging protocols will be most useful for those diseases where: the anatomical distribution of disease is important (cancer, atherosclerosis, Crohn's), the diagnosis is difficult to make by biopsy or other modalities (early Alzheimer's, Hepatocellular carcinoma, active plaque, Crohn's flare), detection of preclinical disease is important (cancer metastasis, recurrence), detection of disease prior to being detectable by imaging without molecular specific reagent contrast (cancer, Alzheimer's, prostate cancer) and there is difficulty distinguishing diseased tissue from other causes of abnormal anatomical findings (postsurgical changes vs. cancer, quiescent, fixed atherosclerosis vs. inflamed, active plaque).

[0008] Molecular imaging approaches may be utilized in numerous clinical situations in oncology. Diagnosis of disease may be improved in this way. Cancer marker specific imaging affinity reagents may provide added sensitivity for small tumors vs. standard imaging or clinical monitoring. Also, this approach may be more specific and allow distinction of cancer tissue from normal tissue or benign processes. Clearly, it may also provide a less invasive means of diagnosis compared to invasive procedures such as endoscopy or biopsy. Upon diagnosis of cancer, staging and risk stratification is necessary to plan surgery and guide radiation and chemotherapy usage. One possibility is for molecular imaging to be performed before completion of cancer surgery to ensure that there has been complete tumor removal ("molecular imaging margins"). Also, if the expression of the molecular target for imaging has prognostic value, the tumor may be treated differently on this basis alone. Imaging may also be done after cancer surgery to check for complete excision if the imaging target is cancer specific. This can be difficult with non-molecular imaging because of anatomical changes from surgery and sensitivity limitations. When a treatment has been successful in inducing remission, molecular imaging may provide a sensitive way to monitor for recurrence. In one scenario for clinical implementation, the primary tumor tissue may be obtained at the time of biopsy or surgery and assessed for the expression of the molecular target. Subsequently the tumor spread or recurrence can be imaged with the appropriate agent that the primary tumor is found to express. Molecular imaging approaches may be extremely valuable in chemotherapeutic drug trials as patient selection tools or as a surrogate endpoint.

[0009] Hepatocellular carcinoma often occurs in patients with cirrhotic liver disease from hepatis B or C, alcohol or other processes. This presents a difficult diagnostic dilemma as cirrhosis is associated with nodule formation and scarring in the liver tissue which makes detection of a cancer nodule nearly impossible at an early stage using standard anatomical imaging techniques (ultrasound, MRI, CT). Biopsy is not a very helpful modality in this setting either as it is very difficult to know where to sample this very large organ for monitoring for onset of cancer and patients with liver disease often have underlying clotting disorders or platelet abnormalities which create a risk of excessive bleeding. In addition, hepatocellular cancer can occur any time over many years in cirrhotic patients which makes monitoring modality selection critical. Hepatocellular carcinoma can be cured by liver lobar resection if the cancer is clearly restricted to one area. In addition, a patient with this cancer could have a transplant if the risk of spread beyond the liver is extremely low. Molecular imaging may help make this determination.

BRIEF SUMMARY OF THE INVENTION

[0010] Described herein is a target-specific imaging reagent composed of an affinity agent coupled to an imaging agent, where the affinity agent specifically binds to a biological molecule, and where the expression of the biological molecule is predictive of a disease or a disease state. A "disease state" refers to the current status of a disease which may have been previously diagnosed, such prognosis, risk-stratification, assessment of ongoing drug therapy, prediction of outcomes, determining response to therapy, diagnosis of a disease or disease complication, following progression of a disease or providing any information relating to a patient's health status over time.

[0011] Also described are imaging agents detectable by at least one of computed tomography, ultrasound, magnetic resonance, nuclear imaging (PET or SPECT), or optical imaging, where the optical imaging is by Diffuses Optical Tomography, Optical Coherence Tomography, Confocal Laser Scanning Microscopy, Fluorescence Correlation Microscopy, Fluorescence Resonance Energy Transfer, or Fluorescence Lifetime Imaging.

[0012] Also described are affinity agents which can be used, including antibodies, small molecules, or peptides. Such affinity agents can be used with or without the imaging agent to identify the biological molecule in body fluids. Such body fluids include blood or serum.

[0013] Also described are biological molecules which can be used, including cell surface proteins, secreted proteins, cell surface polysaccharides, RNA, or DNA.

[0014] Also described are diseases which can be diagnosed or monitored, including cancer (such as lung cancer, melanoma, breast cancer, prostate cancer, neuroendocrine, stomach cancer, lymphoma, head and neck cancer, pancreatic cancer, ovarian cancer, liver cancer, cancer of the central nervous system, or testicular cancer), cardiovascular disease, a condition caused by hematopoietic stem cell transplantation, neurologic disease, autoimmune disease, chronic inflammatory disease, gynecologic disease, or infectious disease. [0015] In certain embodiments of the invention, the disease is hepatocellular carcinoma or liver cancer and the biological molecule used is a hepatocellular carcinoma antigen, such as Glypican-3 or MAGE-I.

[0016] Also described is a method of diagnosing or monitoring a disease or disease state including administering to a mammal the target-specific imaging reagent as described, imaging said mammal, and diagnosing a disease or disease state.

[0017] Also described is a method of diagnosing and monitoring disease by molecular imaging of at least one protein chosen from proteins 1-7626 depicted in Table 2.

[0018] In this method, the imaging method may be chosen from one of computed tomography, ultrasound, magnetic resonance, nuclear imaging, or optical imaging. The optical imaging may be by Diffuses Optical Tomography, Optical Coherence Tomography, Confocal Laser Scanning Microscopy, Fluorescence Correlation Microscopy, Fluorescence Resonance Energy Transfer, or Fluorescence Lifetime Imaging.

[0019] In the methods of the invention, the disease may be chosen from cancer, cardiovascular disease, a condition caused by hematopoietic stem cell transplantation, neurologic disease, autoimmune disease, chronic inflammatory disease, gynecologic disease, or infectious disease. The cancer may be chosen from lung cancer, melanoma, breast cancer, prostate cancer, neuroendocrine, stomach cancer, lymphoma, head and neck cancer, pancreatic cancer, ovarian cancer, liver cancer, cancer of the central nervous system, or testicular cancer.

[0020] In certain embodiments of the methods, the disease is hepatocellular carcinoma or liver cancer and the protein chosen from Table 2 is a hepatocellular carcinoma antigen, such as Glypican- 3 or MAGE-I.

[0021] In another format of the invention, at least two proteins chosen from proteins 1-7626 depicted in Table 2 are imaged. In another format, at least three or more proteins chosen from proteins 1-7626 depicted in Table 2 are imaged. [0022] In another format of the invention, the target-specific affinity agents of the invention may be used in diagnostic assays to identify the biological agents in blood or serum. The diagnostic assay may be in the form of a kit.

DETAILED DESCRIPTION OF THE INVENTION

Identification of Molecular Targets

[0023] Given the sequencing of the human genome and the extent of annotation of the encoded genes, as well as the emergence of tools which can predict protein localization, there is an opportunity to identify targets for molecular imaging from the human genome sequence. Of particular interest are those genes known or predicted to be expressed as proteins on the cell surface. Such genes can be identified via annotation or protein prediction algorithms. Care should be taken to identify proteins expressed on the cell surface and not simply associated with membranes. The specific amino acid sequence which is extra-cellular can be predicted and specific affinity reagents for these can be developed. Secreted proteins may also be of interest as targets for imaging. Although these proteins may have the potential to diffuse away from the diseased tissue they are likely to have a high concentration in diseased tissue vs. other surrounding tissues and they may accumulate in the disease tissue by binding to receptors of other molecules. These proteins may also accumulate in blood or serum where they can be detected with the affinity agents. Cell surface polysaccharides are also of interest as targets, but are often not possible to predict from genome sequence information. RNA and DNA may also be targets, particularly when the process in question results in apoptosis or necrosis which lead to exposure of these molecular to the extracellular environment.

[0024] Data may be available or may be generated to help identify and prioritize the most appropriate targets for a given clinical indication. Such data may take the form of gene expression, proteomics, glycomics, cell sorting (fluorescence activated cell sorting), sequence databases or other such information. Information may come from public literature or databases or in vitro studies involving diseased tissue, animal models or studies utilizing human subjects.

[0025] The following steps can be taken to identify and prioritize molecular targets for imaging from literature, gene expression or proteomic data sets. [0026] Identification of disease molecular imaging targets for specific indications:

[0027] • Knowledge-based approaches

[0028] o Search published literature to identify relevant potential target information (gene expression, cellular, protein)

[0029] o Search annotation databases of the human genome to identify genes and gene products associated with the clinical indication. For example, Online Mendelian Inheritance in Man (OMIM, Johns Hopkins University, NCBI),

[0030] • Analysis of genomic data sets relevant to target diseases.

[0031] o Identification of molecular targets specific to disease state of interest (expressed in disease and not in relevant control (non-diseased) tissues

[0032] o Assessment of level of expression in diseased tissue vs. control specimens (signal to noise)

[0033] o Assessment of % of diseased specimens with expression of each gene above control sample level

[0034] o Data used may be derived from gene expression (e.g. microarray, SAGE) or proteomic (e.g. ELISA, protein array, mass spec or other)

[0035] o Data can be generated using tissue samples (disease and control) derived from human specimens or animal models or from cell culture / in vitro experiments.

[0036] o Hierarchical clustering of genes to identify pathways or cell type association, choose candidate markers that cover multiple pathways and cell types

[0037] o Identification of simple combinations of gene markers which may provide added value above the use of a single marker [0038] • Using candidate markers derived from and prioritized by the above approaches, further characterize

[0039] o Identification of cellular expression of genes of interest from annotation (Pub

Med, NCBI) or from protein structure and localization prediction algorithms

[0040] o Identification of sub-cellular localization of expressed proteins for identified markers

[0041] o Determine known or predicted amino acid sequence of encoded product

[0042] o Identification of predicted extracellular domain of encoded protein (sequence to target for affinity reagent development)

[0043] In vitro experiments may be done to identify and prioritize candidate targets for molecular imaging of specific diseases. Cell lines, human or animal model diseased and control tissues can be obtained and used for experimentation. Gene expression data can be generated from RNA extracted from these tissues using RNA prepared from whole tissue or from laser capture micro-dissection. Genes can then be identified with appropriate expression patterns as described above. Proteins from such tissues can also be directly analyzed using a variety of approaches such as mass spectrometry or protein arrays. Immunohistochemistry or immunofluorescence can be performed on fixed of frozen sections of these tissues to evaluate these proteins and their distribution. In addition, real-time PCR can be performed on paraffin sections to evaluate gene expression using newly developed methodologies (Paik et al., 2004), Tissue arrays can also be utilized to help define the expression patterns of markers and the specificity for diseased tissue (see Jubb et al. 2003, Tsiambas et al. 2006, Divito et al. 2004).

[0044] For studies involving animal models, animals with and without a disease can have tissues compared or can compare disease to control tissues within individual animals.

[0045] Human tissues can be obtained from biopsies or specimens removed at surgery. [0046] A wide variety of gene expression and genomic data set analytical tools are available and well known which can be applied to data sets to help identify those candidate targets with the most desirable expression features. These include tools which identify individual genes or proteins which are significantly correlated with a disease state as well as clustering tools which can group sets of genes into pathways which have some expression or functional relationship. Examples of such tools can be found in Alizadeh et al. 2000, Golub et al. 1999, Hastie et al. 2000, Perou et al. 2000, Tibshirani et al. 2002, Eisen et al. 1998, Tusher et al. 2001.

[0047] For nucleic acid or peptide sequences which are identified by genomic analysis as having desirable expression features for an imaging target, sequence analysis tools can be applied to predict which of these are secreted or have extracellular domains which may be accessible for targeting. A wide variety of prediction tools exist, some of which can be found in Nielsen et al. 1997, Emanuelsson et al. 2000, Bannai et al. 2002, Horton et al. 1997, Geourjon et al. 2001, Rost et al. 1995, Argos et al. 1982, Kyte et al. 1982, Rost et al. 1993, Kneller et al. 1990, Jonassen et al. 1995, 1997, Altschul et al. 1991, 1994, 1996.

Imaging Technology

[0048] Development of molecular imaging targets for specific disease states can be done without regards to downstream label or contrast agent for imaging technology. Modalities differ with respect to spatial resolution and sensitivity, which are usually mutually exclusive. Suitability of an imaging modality for molecular imaging is judged on the criteria of spatial resolution, anatomical coverage, reproducibility, potential for quantification, support of image-guided drug delivery and, finally, the ability to image molecular targets.

[0049] Computed Tomography (CT) has good spatial resolution. However, there are limitations in imaging with the use of contrast agents and in assessing molecular information. CT scanning exposes the subject to ionizing radiation. The high spatial resolution makes x-ray-based imaging important for hybrid systems such as PET-CT (see below).

[0050] Ultrasound has a wide range of applications but is restricted to anatomical regions that are closer to a surface which is accessible to the ultrasound probe. Ultrasound does allow the use of microbubbles as a contrast agent and other ultrasound contrast agents have recently been developed that allow imaging of smaller molecular targets.

[0051] Magnetic Resonance (MR) can visualize anatomy with good spatial resolution, is applicable to all body regions and will allow reproducible and quantitative imaging. It can also be used for intravascular and needle image-guided drug delivery, but not for a broad range of drugs due to safety aspects. MR can partly assess molecular information, for example through spectroscopy, but is limited by sensitivity. However, highly sensitive contrast agents have recently been used to allow imaging of molecular targets and gene expression. Since MR allows reproducible quantitative imaging without radiation it has significant potential for molecular imaging. Magnetic Resonance Spectroscopy (MRS) is also available as a method of evaluation of molecular content of tissues. This approach has significant advantages in that no target specific reagent needs to be administered in that molecular discrimination is achieved using specific pulse sequences. The potential drawback of this approach is the technical challenge associated with development and implementation of molecular specific pulses and sequences. For further information about the use of MRI for molecular imaging see Artemov et al., 2003a and 2003b.

[0052] Nuclear Imaging, comprising PET (Huang et al., 1980) and SPECT is a molecular imaging technique with excellent sensitivity and whole-body applications with good reproducibility and quantitation. However, its poor spatial resolution makes it unsuitable for image-guided drug delivery, and it requires relatively long scan times. Using nuclear imaging with radiopharmaceutical agents enables drug tracing, including the study of pharmacokinetics in vivo. Recently, PET has produced images of gene expression that show promise for future applications in monitoring of gene therapy.

[0053] PET requires isotopes which are generally short lived and thus a cyclotron needs to be within 2 hours of the scanning site. The advantage of this is a short period of exposure to ionizing radiation. PET is more sensitive than SPECT. SPECT tracers are 99Tc or 201 Tl. SPECT is several-fold inferior to PET with respect to sensitivity and special resolution. Mobile PET units are being developed. There are about 200 PET centers in the US. PET/CT combinations are replacing PET alone. A PET/CT scanner is $2M vs. $1.3M for PET alone.

[0054] PET/CT combined scanning allows anatomical and molecular information to be collected on the same piece of equipment which helps combine sensitivity to specific cells or molecules with anatomical information.

[0055] Optical Imaging is a relatively new imaging technique that, because of its lower penetration depth, is currently limited to endoscopic and microscopic applications in humans and animals. Optical imaging may eventually be used to retrieve information from deeper areas. Potential is seen in screening applications where only a yes/no answer is required rather than spatially resolved information. There are a number of specific optical approaches:

[0056] DOT - Diffuses Optical Tomography, which can penetrate several centimetres.

[0057] OCT - Optical Coherence Tomography

[0058] CSLM - Confocal Laser Scanning Microscopy

[0059] FCM - Fluorescence Correlation Microscopy

[0060] FRET - Fluorescence Resonance Energy Transfer

[0061] FLIM — Fluorescence Lifetime Imaging

[0062] In general, these imaging approaches can be assessed with respect to anatomical resolution, sensitivity, amount of probe needed, difficulty of designing, producing and labelling probe, costs, imaging depth and availability. Some of these features are examined in Table 1. The particular technology selected may differ for specific clinical indications. Detailed descriptions of these technologies and their implementation for clinical use are provided in these references: Nuclear imaging technology:

[0063] Blasberg et al., 2002, Chatziioannou et a!., 2001, Chatziioannou et al., 2002, Chen et al., 2000, Chen et al., 2004, Cherry et al., 2001, Gumming et al., 1998, Del Vecchio et al., 2000, Fischman et al., 1993, Gambhir et al., 2002, Ghambhir et al., in press, Springer- Verlag, Huang et al., 1980. Iyer et al., 2002, Jacobs et al., 2002, Kim et al., 2001, Lamberts et al., 1990, Lardinois et al., 2003, Liang et al., 2002, Lovqvist et al., 2001, Lucignani et al., 2000, McCarthy et al., 2002, Polyakov et al., 2000, Reubi et al., 2000, Rosenthal et al., 1995, Shields et al., 1998, Stolz et al., 1998, Strijckmans et al., 2001, Townsend et al., 2001, Townsend et al., 2001, van Tinteren et al., 2002, Virgolini et al., 2000, and Ziegler et al., 2000.

Optical imaging:

[0064] Bogdanov et al., 2002, Bremer et al., 2001, Bremer et al., 2002, Bremer et al., 2001, Chen et al., 2002, Chen et al., 2004, Contag et al., 1998, Hadjantonakis et al., 2001, Iyer et al., 2002, Kelly et al., 2004, Li et al., 2002, Remington et al., 2002, and Tung et al., 1999.

MRI technology:

[0065] Artemov et al., 2003a, b, and c, Bhorade et al., 2000, Bogdanov et al., 2005, Castillo et al., 1996, Chatham et al., 2001, Dodd et al., 2001, Flacke et al., 2001 , Harisinghani et al., 2003, Jackson et al., 2001, Jacobs et al., 2002, Josephson et al., 1999, Kobayashi et al., 2003, Lewin et al., 2000, Luypaert et al., 2001, Remsen et al., 1996, Winter et al., 2003, and Wunderbaldinger et al., 2002.

[0066]

Table 1

Development of Specific Molecular Imaging Reagents

[0067] Once appropriate antigens are identified that may be expressed locally in the diseased tissue, an affinity reagent/agent which binds in a specific manner to the target is developed. Humanized monoclonal antibodies may be used and avoid problems with sensitization seen with murine antibodies. Small molecules may also be developed that bind to targets using computational tools and databases. Small molecule design technologies may be employed in this way. Some antibodies may have high molecular weight and limited uptake in target sites along with slow blood clearance resulting in high signal to noise (Fischman et al., 1993). Peptides are readily synthesized via solid phase synthesis and other parallel approaches as well as phage display. They are cheaper and more stable for labeling, less immunogenic, and have better tissue penetration and blood clearance than antibodies. A peptide is identified which binds a specific receptor and then is taken downstream for imaging reagent development.

[0068] The term "antibody," as used herein, refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that specifically binds an antigen. As such, the term antibody encompasses not only whole antibody molecules, but also antibody multimers and antibody fragments as well as variants (including derivatives) of antibodies, antibody multimers and antibody fragments. Examples of molecules which are described by the term "antibody" herein include, but are not limited to: single chain Fvs (scFvs), Fab fragments, Fab' fragments, F(ab')2, disulfide linked Fvs (sdFvs), Fvs, and fragments comprising or alternatively consisting of, either a VL or a VH domain. The term "single chain Fv" or "scFv" as used herein refers to a polypeptide comprising a VL domain of antibody linked to a VH domain of an antibody.

[0069] Antibodies of the invention include, but are not limited to, monoclonal, multispecifϊc, human or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, anti- idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), intracellularly-made antibodies (i.e., intrabodies), and epitope-binding fragments of any of the above. The immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG₂, IgG₃, IgG₄, IgAi and IgA₂) or subclass of immunoglobulin molecule. Preferably, an antibody of the invention comprises, or alternatively consists of, a VH domain, VH CDR, VL domain, or VL CDR having an amino acid sequence of any one of those referred to in Table 2, or a fragment or variant thereof. In a preferred embodiment, the immunoglobulin is an IgGl isotype. In another preferred embodiment, the immunoglobulin is an IgG4 isotype. Immunoglobulins may have both a heavy and light chain. An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms.

[0070] Antibodies of the invention may also include multimeric forms of antibodies. For example, antibodies of the invention may take the form of antibody dimers, trimers, or higher-order multimers of monomeric immunoglobulin molecules. Dimers of whole immunoglobulin molecules or of F(ab')₂ fragments are tetravalent, whereas dimers of Fab fragments or scFv molecules are bivalent. Individual monomers within an antibody multimer may be identical or different, i.e., they may be heteromeric or homomeric antibody multimers. For example, individual antibodies within a multimer may have the same or different binding specificities. [0071] Multimerization of antibodies may be accomplished through natural aggregation of antibodies or through chemical or recombinant linking techniques known in the art. For example, some percentage of purified antibody preparations (e.g., purified IgGl molecules) spontaneously form protein aggregates containing antibody homodimers, and other higher-order antibody multimers. Alternatively, antibody homodimers may be formed through chemical linkage techniques known in the art. For example, heterobifunctional crosslinking agents including, but not limited to, SMCC (succinimidyl 4-(maleimidomethyl)cyclohexane-l-carboxylate) and SATA (N- succinimidyl S-acethylthio-acetate) (available, for example, from Pierce Biotechnology, Inc. (Rockford, 111.)) can be used to form antibody multimers. An exemplary protocol for the formation of antibody homodimers is given in Ghetie et al., Proceedings of the National Academy of Sciences USA (1997) 94:7509-7514, which is hereby incorporated by reference in its entirety. Antibody homodimers can be converted to Fab'2 homodimers through digestion with pepsin. Another way to form antibody homodimers is through the use of the autophilic T15 peptide described in Zhao and Kohler, The Journal of Immunology (2002) 25:396-404, which is hereby incorporated by reference in its entirety.

[0072] Alternatively, antibodies can be made to multimerize through recombinant DNA techniques. IgM and IgA naturally form antibody multimers through the interaction with the J chain polypeptide. Non-IgA or non-IgM molecules, such as IgG molecules, can be engineered to contain the J chain interaction domain of IgA or IgM, thereby conferring the ability to form higher order multimers on the non-IgA or non-IgM molecules, (see, for example, Chintalacharuvu et al., (2001) Clinical Immunology 101:21-31. and Frigerio et al., (2000) Plant Physiology 123:1483-94., both of which are hereby incorporated by reference in their entireties.) ScFv dimers can also be formed through recombinant techniques known in the art; an example of the construction of scFv dimers is given in Goel et al., (2000) Cancer Research 60:6964-6971 which is hereby incorporated by reference in its entirety. Antibody multimers may be purified using any suitable method known in the art, including, but not limited to, size exclusion chromatography.

[0073] In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them. In particular, DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues) or synthetic cDNA libraries. The DNA encoding the VH and VL domains are joined together by an scFv linker by PCR and cloned into a phagemid vector (e.g., pCANTAB 6 or pComb 3 HSS). The vector is electroporated in E. coli and the E. coli is infected with helper phage. Phage used in these methods are typically filamentous phage including fd and Ml 3 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII. Phage expressing an antigen binding domain that binds to an antigen of interest (i.e., a GMAD polypeptide or a fragment thereof) can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead. Examples of phage display methods that can be used to make the antibodies of the present invention include, but are not limited to, 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/O1 134; PCT publications WO 90/02809; WO 91/10737; WO 92/01047; WO 92/18719; WO 93/1 1236; WO 95/15982; WO 95/20401; WO97/13844; and U.S. Pat. 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; 5,516,717; 5,780,225; 5,658,727; 5,735,743 and 5,969,108; each of which is incorporated herein by reference in its entirety.

[0074] For some uses, such as for in vitro affinity maturation of an antibody of the invention, it may be useful to express the VH and VL domains of one or more scFvs referred to in Table 2 as single chain antibodies or Fab fragments in a phage display library. For example, the cDNAs encoding the VH and VL domains of the scFvs referred to in Table 2 may be expressed in all possible combinations using a phage display library, allowing for the selection of VH/VL combinations that bind a GMAD polypeptide with preferred binding characteristics such as improved affinity or improved off rates. Additionally, VH and VL segments, the CDR regions of the VH and VL domains of the scFvs, may be mutated in vitro. Expression of VH and VL domains with "mutant" CDRs in a phage display library allows for the selection of VH/VL combinations that bind a GMAD polypeptides with preferred binding characteristics such as improved affinity or improved off rates. [0075] Methodologies for preparing and screening such libraries are known in the art. There are commercially available kits for generating phage display libraries (e.g., the Pharmacia Recombinant Phage Antibody System, catalog no. 27-9400-01 ; and the Stratagene SurfZAP.TM. phage display kit, catalog no. 240612), There are also other methods and reagents that can be used in generating and screening antibody display libraries (see, e.g., Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO 92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO 92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCafferty et al. PCT Publication No. WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370- 1372; Hay et al. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science 246:1275- 1281; McCafferty et al., Nature (1990) 348:552-554; Griffiths et al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. MoI. Biol. 226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991) Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) Proc. Natl. Acad. Sci. USA 88:7978-7982.

[0076] Methods of preparing polyclonal antibodies are known to the skilled artisan (e.g., Coligan, supra; and Harlow & Lane, supra). Polyclonal antibodies can be raised in a mammal, e.g., by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include a protein encoded by a nucleic acid of Table 2 or fragment thereof or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation. [0077] The antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods, such as those described by Kohler & Milstein, Nature 256:495 (1975). In a hybridoma method, a mouse, hamster, or other appropriate host animal, is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The immunizing agent will typically include a polypeptide encoded by a nucleic acid of Table 2, a fragment thereof, or a fusion protein thereof. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (1986)). Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are employed. The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, immortalized cells. For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically will include hypoxanthine, aminopterin, and thymidine ("HAT medium"), which substances prevent the growth of HGPRT-deficient cells.

[0078] Humanized forms of antibodies are chimeric immunoglobulins in which residues from a complementary determining region (CDR) of human antibody are replaced by residues from a CDR of a non-human species such as mouse, rat or rabbit having the desired specificity, affinity and capacity.

[0079] An example of a chimeric humanized antibody is a molecule having a human variable region and a non-human (e.g., murine) 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); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety. Chimeric antibodies comprising one or more CDRs from human species and framework regions from a non-human immunoglobulin molecule (e.g., framework regions from a murine, canine or feline immunoglobulin molecule) (or vice versa) can be produced using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. 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., PNAS 91 :969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,352). In a preferred embodiment, chimeric antibodies comprise a human CDR3 having an amino acid sequence of any one of the VH CDR3s or VL CDR3s of a VH or VL domain of one or more of the scFvs and non-human framework regions or human framework regions different from those of the frameworks in the corresponding scFvs. Often, framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably 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, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 352:323 (1988), which are incorporated herein by reference in their entireties.)

[0080] Human antibodies can be produced using various techniques known in the art, including phage display libraries (Hoogenboom & Winter, J. MoI. Biol. 227:381 (1991); Marks et al., J. MoI. Biol. 222:581 (1991)). The techniques of Cole et al. and Boemer et al. are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, p. 77 (1985) and Boemer et al., J. Immunol. 147(l):86-95 (1991)). Similarly, human antibodies can be made by introducing of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, e.g., in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al., Bio/Technology 10:779-783 (1992); Lonberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994); Fishwild et al., Nature Biotechnology 14:845-51 (1996); Neuberger, Nature Biotechnology 14:826 (1996); Lonberg & Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0081] A number of methods have been devised to generate peptide libraries and methods of using the libraries to identify a peptide sequence that is complementary to a receptor or antibody.

[0082] An early method is the "Merrifield" method, described in Atherton et al., "Solid Phase Peptide Synthesis," IRL Press, (1989), incorporated herein by reference for all purposes. This method has been used to synthesize peptides on a solid support such as pins or rods. The peptides are then screened to determine if they are complementary to a receptor.

[0083] To screen a larger number of polymer sequences, more advanced techniques have been disclosed. For example, Pirrung et al., WO 90/15070, incorporated herein by reference for all purposes, describes a method of synthesizing a large number of polymer sequences on a solid substrate using light directed methods. Dower et al., U.S. application Ser. No. 07/762,522, also incorporated by reference herein for all purposes, describes a method of synthesizing a library of polymers and a method of use thereof. The polymers are synthesized on beads, for example. A first monomer is attached to a pool of beads. Thereafter, the pool of beads is divided, and a second monomer is attached. The process is repeated until a desired, diverse set of polymers is synthesized.

[0084] Houghten et al., "Generation and Use of Synthetic Peptide Combinatorial Libraries for Basic Research and Drug Discovery," Nature (1991) 354:84-86, disclose a method of generating peptide libraries that are used for screening peptides for biological activity (see also, Houghton et al., "The Use of Synthetic Peptide Combinatorial Libraries for the Identification of Bioactive Peptides," Peptide Research (1992) 5:351-358). Houghten synthesized a peptide combinatorial library (SPCL) composed of some 34x10₆ hexapeptides and screened it to identify antigenic determinants that are recognized by a monoclonal antibody. Furka et al., "General Method for Rapid Synthesis of Multicomponent Peptide Mixtures," Int. J. Peptide Protein Res. (1991) 37:487- 493, discusses a method of synthesizing multicomponent peptide mixtures. Furka proposed pooling as a general method for the rapid synthesis of milticomponent peptide mixtures and illustrated its application by synthesizing a mixture of 27 tetrapeptides and 180 pentapeptides. Lam et al., "A new type of synthetic peptide library for identifying ligand-binding activity," Nature (1991) 354:82-84 used pooling to generate a pentapeptide bead library that was screened for binding to a monoclonal antibody. Blake et al. "Evaluation of Peptide Libraries: An Interative Strategy To Analyze the Reactivity of Peptide Mixtures With Antibodies," Bioconjugate Chem. (1992) 3:510-513 discusses, the screening of presumed mixtures of 50,625 tetrapeptides and 16,777,216 hexpeptides to select epitopes recognized by specific antibodies.

[0085] Lam's synthetic peptide library consists of a large number of beads, each bead containing peptide molecules of one kind. Beads that bind a target (e.g., an antibody or streptavidin) are rendered colored or fluorescent. Lam reports that several million beads distributed in 10-15 petri dishes can be screened with a low-power dissecting microscope in an afternoon. Positive beads are washed with 8M guanidine hydrochloride to remove the target protein and then sequenced. The 100-200 μm diameter beads contain 50-200 pmol of peptide, putatively well above their 5 pmol sensitivity limit. Three pentapeptide beads were sequenced daily. The essence of Lam's method is that the identity of positive beads is established by direct sequencing.

[0086] Houghten et al. use a different approach to identify peptide sequences that are recognized by an antibody. Using the nomenclature described herein, Houghten et al. screened an X₆X₅X_4pX_3pX_2pXi_p library and found that the mixture DVX_4pX_3pX_2pXi_p had greatest potency in their inhibition assay. Houghten then synthesized a DVX₄X_3pX_2pXi_p library and identified the most potent amino acid in the third position. After three more iterations, they found that DVPDYA binds to the antibody with a Kd of 30 nM. The essence of Houghten' s method is recursive retrosynthesis, in which the number of pooled positions decreases by one for each iteration.

[0087] In order to detect binding of a receptor or antibody to a peptide, the antibody must be labeled. Antibodies may also be modified with a detectable label, such as an enzymatic, fluorescent, radioisotopic or affinity label to allow for detection and isolation of the antibody.

[0088] Examples of suitable enzymatic labels include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, glucose oxidase or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent materials include biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; examples of bioluminescent materials include luciferase, luciferin, and aequorin; and examples of suitable radioactive material include a radioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi, or other radioisotopes such as, for example, iodine (¹³¹I, ¹²⁵1, ¹²³I, ¹²¹I, carbon (¹⁴C), sulfur (³⁵S), tritium (³H), indium (^115mIn, ^{1 13111}In, ¹¹²In, ^{1 11}In), and technetium (⁹⁹Tc, 99mTc), thallium (²⁰¹Ti), gallium (⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe), fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y, ⁴⁷Sc, ¹⁸⁶Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru, ⁶⁸Ge, ⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se, ¹¹³Sn, and ¹¹⁷Tm.

[0089] Antibodies may also be labeled with Europium. For example, antibodies may be labelled with Europium using the DELFIA Eu-labeling kit (catalog# 1244-302, Perkin Elmer Life Sciences, Boston, Mass.) following manufacturer's instructions.

[0090] Antibodies may be attached to macrocyclic chelators useful for conjugating radiometal ions, including but not limited to, ¹¹¹In, ¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, ¹⁵³Sm, ²¹⁵Bi and ²²⁵Ac to polypeptides. In a preferred embodiment, the radiometal ion associated with the macrocyclic chelators attached to antibodies of the invention is ¹¹¹In. In another preferred embodiment, the radiometal ion associated with the macrocyclic chelator attached to antibodies polypeptides of the invention is Y. In specific embodiments, the macrocyclic chelator is 1,4,7, 10-tetraazacyclododecane-N,N',N",N'"- tetraacetic acid (DOTA). In specific embodiments, the macrocyclic chelator is a-(5-isothiocyanato- 2-m- ethoxyphenyl)-l,4,7,10-tetraazacyclododecane-l,4,7,10-tetraacetic acid. In other specific embodiments, the DOTA is attached to the antibody of the invention via a linker molecule. Examples of linker molecules useful for conjugating a macrocyclic chelator such as DOTA to a polypeptide are commonly known in the art—see, for example, DeNardo et al., Clin Cancer Res. 4(10):2483-90, 1998; Peterson et al., Bioconjug. Chem. 10(4):553-7, 1999; and Zimmerman et al, Nucl. Med. Biol. 26(8):943-50, 1999 which are hereby incorporated by reference in their entirety. In addition, U.S. Pat. Nos. 5,652,361 and 5,756,065, which disclose chelating agents that may be conjugated to antibodies, and methods for making and using them, are hereby incorporated by reference in their entireties. Antibodies can also be labeled with biotin.

Diagnostic and Monitoring Assays

[0091] The affinity reagents/agents of the invention have various utilities, including use as diagnostic and monitoring assays for proteins of the invention, e.g., detecting their expression in specific cells, tissues, or serum.

[0092] For example, antibodies may be used in diagnostic and monitoring assays for the proteins of the invention, e.g., detecting their expression in specific cells, tissues, or serum. Various diagnostic assay techniques known in the art may be used, such as competitive binding assays, direct or indirect sandwich assays and immunoprecipitation assays conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques, CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in the diagnostic and monitoring assays can be labeled with a detectable moiety. The detectable moiety should be capable of producing, either directly or indirectly, a detectable signal. For example, the detectable moiety may be a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase. Any method known in the art for conjugating the antibody to the detectable moiety may be employed, including those methods described by Hunter et al., Nature, 144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al., J Immunol. Metk, 40:219 (1981); and Nygren, J. Histochem. and Cytochem., 30:407 (1982).

[0093] The diagnostic and monitoring assays can be utilized to diagnose and/or monitor cancer, heart disease, hematopoietic stem cell transplantation, neurologic disease, autoimmune and chronic inflammatory disease, gynecologic disease and infectious disease. "Diagnosing" can include detection of any type of activity or progression of a disease, for example, determination of whether the disease is present, identifying the stage of the disease, monitoring the response of the disease to therapy, etc. The term "monitoring" is used herein to describe the use of an affinity agent to provide useful information about an individual or an individual's health or disease status. "Monitoring" can include, determination of prognosis, risk-stratification, selection of drug therapy, assessment of ongoing drug therapy, prediction of outcomes, determining response to therapy, diagnosis of a disease or disease complication, following progression of a disease or providing any information relating to a patient's health status over time, selecting patients most likely to benefit from experimental therapies with known molecular mechanisms of action, selecting patients most likely to benefit from approved drugs with known molecular mechanisms where that mechanism may be important in a small subset of a disease for which the medication may not have a label, screening a patient population to help decide on a more invasive/expensive test, for example, a cascade of tests from a non-invasive blood test to a more invasive option such as biopsy, or testing to assess side effects of drugs used to treat another indication..

[0094] Such diagnostic and monitoring assays can be in the form of a kit. The kit may include reagents for performing the diagnostic assay.

[0095] Development of Imaging Reagent from Target Specific Reagents

[0096] Once an affinity reagent (e.g., antibody or peptide) is available for a molecular imaging target, imaging contrast or signal emitting source is incorporated into the reagent. This can be done for a variety of technologies as discussed above.

[0097] For development of imaging reagents for nuclear techniques (PET and SPECT) an isotope which emits particles is incorporated into or attached to the reagent. For PET, isotopes that emit positrons which collide with electrons to produce two gamma rays. These gamma rays are then detected to generate a signal. Positrons are emitted by 150, 11C, 13N and 18F which are the most commonly used isotopes. Also of potential use are 140, 64Cu, 62Cu, 1241, 76Br, 82Rb and 68Ga. These isotopes are produced in a cyclotron and available in radiopharmacies (Strijckmans 2001, Gambhir 2002). These isotopes can often be substitute for naturally occurring atoms in affinity reagents. Isotopes that emit gamma rays directly (e.g., 99mTc, 111In, 1231, 1311) are used for SPECT imaging (Rosenthal et al. 1995). [0098] Methods for development of signal emitting reagents for nuclear imaging (PET and SPECT) are well established (Bogdanov et al., 2005). Radiolabeling can be achieved by direct labeling (incorporation of label materials into molecules) or chelation of a label (Bogdanov et al. 2005). Radiopeptides have been used for tumor imaging with some success (Lamberts et al., 1990). Octreotide has also been labeled with technicium and is used for tumor imaging (Octreoscan, Mid- South Imaging, Memphis), Introduction of label into a molecule may affect biological or binding properties markedly. These properties must be reassessed after labeling. This may be done in a high throughput manner using cell or tissue preps in 96 or 384 well plates (Reubi et al., 2000). Radiolabeled molecules must also be tested in animals before human testing (Stolz et al., 1998). This will help assess toxicity and tissue distribution and clearance. Clearance of radiopeptides by the kidneys is a key issue which needs improvement. Examples of common methods used to incorporate labels into affinity reagents for PET and SPECT imaging are found in Gumming and Gjedde 1998, Lucignani and Frost 2000, Wagner et al. 1983, Coenen et al. 1987, Kung et al. 1990, Crouzel et al. 1988, Virgolini 2000, Oriuchi and Yang 2001, Lovqvist et al. 2001, Kim 2001.

[0099] For MRI scanning, the most common approaches currently in use include the incorporation or labeling of the affinity reagent with Gadolinium or Supermagnetic Iron Oxide containing compounds. These molecules alter local magnetic resonance characteristics which creates a local signal contrast in the area of reagent accumulation. One strategy involves the use of a biotinylated antibody followed by the administration of Gadolinium complexes (e.g., with liopsomes) which are linked to Avidin (Artemov et al. 2003). Another strategy involves the use of enzyme mediated polymerization of paramagnetic substrates into oligomers of higher relaxivity (Weissleder et al. 2002).

[00100] One can develop reagents which activate with binding (Bogdanov et al., 2002). Also one can develop high relaxivity contrast agents (Artemov et al., 2003, Weissleder et al., 2001, Bogdanov et al., 2002). Other magnetic resonance signal amplification probe technologies are available which utilize receptor mediated internalization or enzyme mediated signal amplification. Additional methods for conjugation of iron oxide or Gadolinium compounds to affinity probes are found in Remson et al. 1996, Kang et al. 2002 and Zhao et al. 2001, Flacke et al. 2001, Li et al. 2002, Sipkins et al. 1998, Sipkins et al. 2000, Weissleder 1991, Artemov et al. 2003, Storrs et al. 1995.

[0100] Scanning using the selected imaging technology using 384 well plates of potential reagent combinations to determine binding characteristics and labeling efficiency may be utilized as a direct approach to evaluation of potential reagents. Tissue or cell arrays can be used to screen reagent combinations. Clonal cell line expressing target on surface in high throughput format could also be used. Animal testing with scanning is used when appropriate models exist. This can be done in vivo or explanted tumor tissue or other explanted tissues may be used to determine binding characteristics. Standard immunohistochemistry or immunofluorescence can be used to test the binding, sensitivity and cell type specificity of the reagent and label combination (Brown et al., 1990; Abbondanzo et al., 1990; Allred et al., 1990, and Sambrook et al. 1989). This can be done using human tissue specimens or animal model tissue specimens in the case of animal reagents.

[0101] In any case, the imaging reagent needs to be produced with materials which provide contrast or can be linked with contrast materials after production. Probes should be biocompatible, lack interference with biological function, generate high signal output and signal-to-noise. PET and SPECT tracers are easier to develop because of need for low concentrations and ease of labeling.

[0102] "Multiplex imaging" may prove to be clinically useful. Such an approach could be used to image more than one molecule in a single scan. It could be done in such a way that the >1 target both contribute signal, but are not resolvable ("one color") or may be done using contrast or signal emitting reagents that allow the >1 targets to be distinguished in the image ("two color"). Simultaneous imaging of multiple markers may be useful to overcome variability in the population (e.g., some tumors express one marker and others express another marker). This approach could also provide a means to provide additional contrast by specifically imaging both the diseased and control tissues. Perhaps also this approach would add to the information value of an imaging protocol. For example, one could image 2 or 3 markers of tumor progression and the worst prognosis would be associated with the presence of more markers or more advanced markers in the process. PET can only measure two targets by injecting and imaging one and then performing a second injection and scan for the second target after the signal from the first has faded. SPECT can image more than one target in a single scan. Other combinations of technologies may be used to measure more than one target simultaneously (combine MRI, CT, Nuclear, Optical).

Clinical Validation of Imaging Reagents

[0103] For any given molecular imaging reagent, clinical utility and efficacy in humans is demonstrated for specific clinical indications to provide support for the position that the test will have an impact on clinical practice. After appropriate safety studies have been performed in primates and then human subjects, patients with the target disease process (or suspected to have it) are recruited along with appropriate control subjects. The performance of the molecular imaging test with respect to disease diagnosis or prognosis is then assessed. The assessment of performance may be related to a gold standard based on the pathological diagnosis of the disease status or other standards such as endoscopic evaluation, other imaging approaches or diagnostic testing. Alternatively or in addition, the test is assessed with respect to its ability to identify patients at risk for events, complications, outcomes or mortality related to the disease process (prognosis). The test may also be shown to identify patients with an increased or decreased responsiveness to therapy (therapy broadly or specific medications). Alternatively, testing can be shown to monitor response to therapy. Various clinical utilities of molecular imaging approaches are discussed and exemplified below in the section on clinical indications for molecular testing.

Clinical Indications for Molecular Imaging

[0104] In general the addition of a specific molecular targeting reagent to imaging protocols will be most useful for those diseases where:

[0105] 1. The anatomical distribution of disease is important (cancer, atherosclerosis, Crohn's)

[0106] 2. The diagnosis is difficult to make by biopsy or other modalities (early Alzheimer's, Hepatocellular carcinoma, active plaque, Crohn's flare)

[0107] 3. Detection of preclinical disease is important (cancer metastasis, recurrence) [0108] 4. Detection of disease prior to being detectable by imaging without molecular specific reagent contrast (cancer, Alzheimer's, prostate cancer)

[0109] 5. There is difficulty distinguishing diseased tissue from other causes of abnormal anatomical findings (post-surgical changes vs. cancer, quiescent, fixed atherosclerosis vs. inflamed, active plaque.

Cancer

[0110] Molecular imaging approaches may be utilized in numerous clinical situations in oncology. Diagnosis of disease may be improved in this way. Cancer marker specific imaging affinity reagents may provide added sensitivity for small tumors vs. standard imaging or clinical monitoring. Also, this approach may be more specific and allow distinction of cancer tissue from normal tissue or benign processes. Clearly, it may also provide a less invasive means of diagnosis compared to invasive procedures such as endoscopy or biopsy. Upon diagnosis of cancer, staging and risk stratification is necessary to plan surgery and guide radiation and chemotherapy usage. Molecular imaging may provide information about the local regional and distant spread of cancer. One possibility is for molecular imaging to be performed before completion of cancer surgery to ensure that there has been complete tumor removal ("molecular imaging margins"). Also, if the expression of the molecular target for imaging has prognostic value, the tumor may be treated differently on this basis alone. Multiple markers could be imaged using imaging reagent with differential labeling which can be resolved with a single scan or could have multiple scans done with different individual reagents. Once treatment has begun, response to therapy may be monitored with molecular imaging. This may provide a means for an earlier assessment of response so that the regimen can be altered earlier if not effective. Imaging may also be done after cancer surgery to check for complete excision if the imaging target is cancer specific. This can be difficult with non-molecular imaging because of anatomical changes from surgery and sensitivity limitations. When a treatment has been successful in inducing remission, molecular imaging may provide a sensitive way to monitor for recurrence. In one scenario for clinical implementation, the primary tumor tissue may be obtained at the time of biopsy or surgery and assessed for the expression of the molecular target. Subsequently the tumor spread or recurrence can be imaged with the appropriate agent that the primary tumor is found to express. Molecular imaging approaches may be extremely valuable in chemotherapeutic drug trials as patient selection tools or as a surrogate endpoint.

Clinical and economic value of molecular imaging for cancer

[0111] Molecular imaging could provide tremendous clinical value for oncologists and patients in those situations described above, These benefits are associated with increased sensitivity and specificity of imaging techniques which could lead to earlier treatment, more appropriate treatment, improved surgical or radiation therapy planning and appropriate cessation or withholding or treatment. These applications may also be associated with significant economic value for the health care system. Early detection of cancer may lead to an increased rate of curative treatment, avoiding the costs of prolonged curative or non-curative therapy. Improvements in staging may help plan more appropriate surgical or radiation therapy which may improve survival or decrease costs of complications. Identification of distant metastasis may obviate the need for unnecessary or fruitless medical or surgical therapy. Imaging may also reduce the need for invasive or expensive diagnostic procedures such as biopsy, endoscopy or bronchoscopy. Earlier assessment of response to chemotherapy could reduce prolonged and costly unnecessary treatment and allow more rapid changes in treatment. A beneficial impact on drug trials could decrease, cost and duration of these expensive trials and increase likelihood of success.

Lung cancer

[0112] Molecular imaging may be useful to diagnose disease in patients at risk, stage disease, monitor response to therapy and remission, Flurodeoxyglucose (FDG) Positron Emission Tomograpy (PET) scanning can reduce need for thoracotomy (van Tinteren et al. 2002). Diatide's Neospect (labeled depreotide) helps assess the need for biopsy with solitary pulmonary nodules. Staging is critical prior to surgery or medical therapy. FDR PET scanning can be useful, but lacks specificity and may be positive with any inflammatory lesion even if not malignant. Scanning may be used to assess local tumor extent (T staging), Nodal spread (N) and Metastasis (M). This can be done for new cancer, cancer under treatment or cancer in remission. Lardinois et al used combined PET-CT imaging for non-small cell lung cancer to assess metastatic disease. Patients received FDG- 18F. This approach provided additional information in 40% of patients beyond that of separate PET and CT scans, along with a significant 35% to 40% improvement in diagnostic accuracy with respect to lung cancer tumor staging.

Melanoma

[0113] As this cancer is present on the skin, optical techniques may be used. The scan may help distinguish benign from malignant skin lesions. Scanning could also be used to stage and monitor the cancer.

Breast

[0114] Breast cancer occurs close to the surface and is amenable to imaging with most modalities including those with more limited penetration of tissue (ultrasound, optical imaging). Molecular probes could be used to aid in screening and diagnosis. Breast tumors metastasize to axillary lymph nodes and a lymph node dissection is often done for staging. Molecular imaging could aid in this staging process by identifying tumor spread to these regional lymph nodes as well as to distant sites though out the body. Molecular imaging may also be appropriate for monitoring of recurrence in treated patients.

Prostate

[0115] Prostate cancer can be assessed with a variety of imaging techniques. Low penetrations techniques such as ultrasound can be used in conjunction with endoscopy. Both CT and MRI have very high false negative results for early prostate cancer which could be improved upon with the addition of a molecular probe using the same or other imaging technologies. Assessment of regional or distant spread could aid in the staging or prostate cancer. This information could guide medical, surgical and radiation therapy. MRI enhanced with lymphotropic monocrystalline iron oxide nanoparticles can be used to detect prostate cancer metastases as small as 2 mm in diameter, in contrast with conventional MRI detection (>8-10 mm). Neuroendocrine Tumors

[0116] These cancers often have a distinct molecular phenotype compared to surrounding tissue. They can occur in almost any anatomical location. Molecular techniques could help diagnose, stage and monitor these tumors. One existing reagent targets the somatostatin receptor (Mallinckrodt OctreoScan).

[0117] Colon cancer is often diagnosed only after is has grown to a size associated with clinical symptoms (bleeding or obstruction) or is large enough to be easily visible on a standard examination (endoscopy, non-molecular imaging study). Unfortunately, at this stage the cancer is too often associated with aggressive local behavior and distant spread. Earlier diagnosis with the help of specific affinity reagents would help guide earlier resection which is more likely to be curative. Colon cancer cannot be distinguished from benign adenoma using anatomical imaging or visualization using endoscopy. Molecular imaging techniques may provide a non-invasive means to make this distinction. Colon cancer is known to be associated with multiple molecular changes ("hits") on the way from benign adenoma to cancer. This information may be used in the design of molecular imaging protocols. One may image more than one molecule or only one molecule associated with malignant transformation. Colon cancer staging involves evaluation of the region around the primary tumor and the liver. Molecular imaging may improve the accuracy of anatomical imaging in staging. This information can help guide medical therapy and surgical planning. In some cases, limited liver resection can be performed if a solitary metastasis exists. After surgery for colon cancer, anatomical changes (inflammation or scarring) may be seen with conventional anatomical imaging techniques. It is difficult to distinguish these findings from cancer persistence or recurrence and specific molecular probes may help identify malignant cells in this situation. CT scanning does not permit distinction between post-surgical changes and tumor recurrence and cannot determine tumor involvement in normal sized lymph nodes (Goldberg et al., 1998).

Stomach

[0118] Cancer of the stomach may be difficult to distinguish from benign ulceration. Gastric ulceration is very common. Biopsy of ulcer edges is performed, however this technique may be subject to sampling error and a negative result can occur in cases of cancer associated with ulceration. Clinical practice when biopsy is negative for cancer is to treat for gastric ulcer and then to reassess for at a later time for refractory cases. Unfortunately, this may result in a delay in diagnosis and treatment which could be costly to the patient. Molecular imaging could help identify malignancy as an underlying cause of ulceration at the time of first diagnosis for at risk patients.

Lymphoma

[0119] Lymphoma can be associated with bulky disease masses which are largely cancerous cells, but also are composed of inflammatory cells. Upon treatment, malignant cells become necrotic and significant inflammation and clean up of residual disease occurs. Standard imaging techniques cannot distinguish non-malignant from malignant causes of masses in these cases. There is a need to evaluate treatment response after completion of therapy especially when there are residual masses.

Head and Neck

[0120] Head and neck cancers can spread to numerous regional lymph nodes and the surgical approach to these patients involves extensive and disfiguring procedures. Molecular imaging may help identify primary lesions, lymph node involvement and distant metastasis. This information may also help guide radiation therapy.

Pancreas

[0121] Pancreatic cancer is very difficult to diagnosis at a curable stage. Symptoms may occur due to mass effects in the abdomen or if the tumor happens to obstruct the pancreatic duct, however most cancers are incurable at the time of diagnosis. Molecular imaging could add significantly to the diagnosis of this cancer at an early stage. This approach could also be used to evaluate spread of tumors at the time of diagnosis to help plan surgical therapy. In the absence of evidence of distant spread, curative surgical therapy may be attempted. Otherwise, palliative treatment may be the only option. Ovary

[0122] Ovary cancer is difficult to diagnose at asymptomatic stage with standard CT or MRI. Addition of specific molecular reagents to imaging protocols may add sensitivity for early stage lesions which are more amenable to curative therapy. Also, benign masses (adenomas and cysts) are very common in and around the ovaries. Molecular imaging may provide a means to distinguish benign and malignant lesions. Ovarian cancer may also be assessed after diagnosis and monitored for recurrence in a sensitive and specific way using molecular imaging approaches. CIS bio International has developed an indium labeled monoclonal antibody to CA 125 which can be used to image ovarian cancers.

Liver

[0123] Hepatocellular carcinoma often occurs in patients with cirrhotic liver disease from hepatis B or C, alcohol or other processes. This presents a difficult diagnostic dilemma as cirrhosis is associated with nodule formation and scarring in the liver tissue which makes detection of a cancer nodule nearly impossible at an early stage using standard anatomical imaging techniques (ultrasound, MRI, CT). Biopsy is not a very helpful modality in this setting either as it is very difficult to know where to sample this very large organ for monitoring for onset of cancer and patients with liver disease often have underlying clotting disorders or platelet abnormalities which create a risk of excessive bleeding. In addition, hepatocellular cancer can occur any time over many years in cirrhotic patients which makes monitoring modality selection critical. Hepatocellular carcinoma can be cured by liver lobar resection if the cancer is clearly restricted to one area. In addition, a patient with this cancer could have a transplant if the risk of spread beyond the liver is extremely low. Molecular imaging may help make this determination.

Central Nervous System — Glioblastoma

[0124] Cancers of the central nervous system present significant diagnostic and treatment challenges to physicians and surgeons. Because of their anatomical location, biopsy based diagnosis of brain masses is technically challenging and associated with complications. Molecular imaging may provide a means to make a firm diagnosis of brain cancer (vs. benign mass) and guide planning of surgery, radiation therapy and medical therapy. In addition, surgical therapy can be curative. However it is critically important to know the precise anatomical distribution of the tumor to plan optimal surgery. One reason for this is that removal of brain tissue results in neurological deficits so precision is key to minimize unnecessary brain loss. In addition, it is critical to ensure that the entire extent of the tumor is removed at the time of surgery. Given these needs, molecular imaging may provide a boost to sensitivity that improves this evaluation. Clearly, molecular imaging information must be combined with precise anatomical imaging. One possibility is to image the patient during surgery using the molecular probe to assess "margins" and determine if the tumor has been completely removed. Glioblastomas are known to be resistant to many chemotherapeutic agents and often require the use of chemotherapy in a trial and error fashion. Molecular imaging may provide a means for earlier evaluation of treatment response which would decrease costly prolonged futile drug trials.

Testicular cancer

[0125] This cancer is treatable with radiation and chemotherapy. Cure is dependent on identification of an appropriate field for radiation and appropriate staging of disease. Molecular imaging could provide a means to stage the disease and also monitor response to therapy and recurrence.

Cardiovascular disease

[0126] Coronary artery disease is an inflammatory disease of the arterial wall. Infiltration of the vessel wall with inflammatory cells, accumulation of lipids and remodeling leads to arterial plaque formation. Atherosclerotic plaques can cause ischemia in tissue served by the diseased artery. This can manifest as angina pectoris, chronic ischemia and cardiac failure, claudication and other signs of peripheral arterial insufficiency. Plaques may suddenly rupture leading to thrombosis and partial or total occlusion of the arterial lumen. In the case of coronary arteries these events lead to unstable angina or myocardial infarction which is a major cause of morbidity and mortality relating to coronary artery disease. Coronary angiography is commonly used to assess the extent and severity of coronary artery disease. This technique provides information on the degree of lumen occlusion by atherosclerotic plaque. However, one cannot determine which lesions in the coronary tree are at high risk for rupture and acute events from this information. The risk of rupture is not related to the degree of stenosis as lesions causing 40-50% vessel occlusion are more likely to cause acute events than those with higher degrees of occlusion. It is known that the risk of plaque rupture is related to the degree of inflammation in the lesion as well as the cellular and extracellular composition (plaques with less collagen and more cholesterol are at higher risk).

[0127] When a patient is found to have coronary artery disease on angiography, it is often unclear how to intervene. When the patient has a recent history of acute coronary events (unstable angina or myocardial infarction) it can sometimes be difficult to identify the culprit vessel. Even more challenging is to identify vessel segments with coronary atherosclerosis which pose a high risk for subsequent plaque rupture. If one could identify these vessel segments, they could be addressed with percutaneous interventions such as angioplasty or stenting. More generally, if one knew that a patient had atherosclerotic lesions at risk for rupture, the patient would be a candidate for the most aggressive medical therapy. This information would be of particular interest in the future when drugs are available for coronary disease that target the underlying inflammatory process.

[0128] Non-invasive imaging techniques can now provide information on the anatomical extent of coronary disease as well as some information on composition. MRI scans can assess degree of lumen occlusion as well as providing some characterization of the cellular and extracellular composition. CT scanning can provide some anatomical information as well as information on calcification of coronary plaques which is know to relate to risk of adverse events.

[0129] Molecular imaging could be applied to coronary atherosclerosis by identification and targeting of molecules associated with active plaques which are at risk from rupture. An ideal target molecule would be highly expressed specifically in vessels with the highest risk of rupture. Such molecules may represent proteases involved in the remodeling process or genes involved in the inflammatory process. They could be cell surface or secreted. The targeting may also be to a cell type. For example, the relative number of macrophage or lymphocytes may have the highest predictive value. Given their exposure to the circulation, endothelial cells may express molecular which are accessible to circulating affinity reagents. [0130] Molecular imaging approaches could provide a number of advantages over available techniques. Sensitivity gains could be made by specific contrast accumulation at the site of vulnerable plaques. Information may also be more specific or predictive of future events by imaging molecules which are specific to highest risk lesions. If combined with anatomical imaging protocols, this approach would provide information on the extent, severity and specific location of atherosclerotic plaques as well as the risk of acute events and progression for each lesion. This information could guide intervention with medical therapy, percutaneous interventions and bypass surgery.

[0131] Such techniques could be useful in the coronary circulation, carotids or peripheral vessels. It is clear that patients with atherosclerosis in any one arterial bed are at risk for disease in other distributions. Therefore, this approach also may allow evaluation of atherosclerosis throughout the body in a single scan in patients at risk or for pre-surgical planning.

[0132] Molecular imaging of vascular disease has wide applicability.

[0133] Gadolinium texaphyrin chelates may accumulate in vulnerable plaques for MR imaging.

[0134] Reagents to MMP inhibitors have been developed for vascular lesion imaging (University of Muenster). Gadolinium labeled affinity reagents to components of thrombus have also been explored (Epix, gadolinium labeled fibrin binding peptide). Activated macrophages are the main cellular effectors of inflammation in atherosclerosis and their presence identifies high-risk lesions. Magnetic nanoparticles (eg, iron oxide) accumulate within human atherosclerotic macrophages and are therefore preferentially found in macrophage-rich carotid plaques. Future areas of exploration in this field may include the development of agents that specifically target oxidized low-density lipoprotein, activated macrophages (as opposed to all macrophages or multiple cell types), or endothelial cell markers such as vascular cell adhesion molecule. Detection of apoptotic cells in atherosclerotic lesions may represent another molecular imaging strategy to identify high-risk lesions. Apoptotic cells are able to bind a number of proteins, such as annexin V (Belochine et al. 2004, Toretsky et al. 2004, Blankenberg et al. 2003, Murakami et al. 2004). Using radiolabeled annexin A5 investigators have imaged apoptosis in patients with acute myocardial infarction.

Hematopoietic Stem Cell Transplantation

[0135] Hematopoietic stem cell transplantation (bone marrow transplant) is performed most commonly in the treatment of leukemia. The management of patients after transplant is largely focused on monitoring for the occurrence of graft versus host disease (GVHD) and for recurrence of the cancer for which the procedure was performed. GVHD results from recognition of host tissues as foreign by the donor immune system. This can result in severe complications and is a major cause of death. Molecular imaging reagents could detect T cells as they respond to the graft. This could provide valuable information to guide therapy. Detection of cancer recurrence is a very difficult clinical problem. By the time a recurrent leukemia is detectable in the circulation by standard methods, it is often incurable. Molecular probes specific to cancer cells could provide a means to detect recurrence of leukemia at an earlier stage.

Neurologic Disease

[0136] The brain and spinal cord are difficult to biopsy to make a tissue diagnosis of disease. This is due to accessibility and risk of complications from damage to the organ. At the same time, there are a number of neurologic diseases involving specific regions of the brain. In each case, it is extremely difficult to diagnose disease prior to significant neurologic dysfunction. Detection of disease at an earlier stage may allow intervention with medical or other therapies which slows progression of disease prior to significant damage.

[0137] Alzheimer's disease is characterized by the formation of amyloid plaques in the brain. There is a characteristic distribution in the frontal lobes. Definitive diagnosis at an early stage is not possible as findings of dementia are not specific and numerous alternative causes must be considered. Even when these have been ruled out, Alzheimer's disease is a diagnosis of exclusion and can only be definitively diagnosed at autopsy. A specific molecular probe would be valuable to make a definitive early diagnosis which would allow early medical therapy. Molecular imaging would also facilitate monitoring of response to therapy. Dojindo Laboratories has developed an MRI reagent for amyloid plaques.

[0138] Multiple Sclerosis is an autoimmune disease of the central nervous system characterized by inflammation and demyelination leading to chronic neurologic disability. The disease is often characterized by flares and periods of remission. MRI scanning is used to detect plaques (characteristic lesions). However, once apparent on a standard MRI scan, the disease has already caused irreversible damage to the brain. It would be very useful if one could detect plaque formation at an earlier stage. Molecular imaging contrast or signal emitting reagents specific to features of the plaque could provide additional sensitivity for early disease.

[0139] Parkinson's Disease and Amyotrophic Lateral Sclerosis are additional diseases of the nervous system that could benefit from the emergence of specific and sensitive molecular imaging reagents. Earlier diagnosis could facilitate earlier therapy or the validation of new treatments which could slow progression of disease.

Autoimmune and chronic inflammatory disease

Inflammatory bowel disease: Crohn's disease and Ulcerative colitis

[0140] These disorders are characterized by inflammation of the gastrointestinal tract. They are currently diagnosed and monitored using clinical evaluation, endoscopy and CT scanning. However, these methods have significant limitations, particularly in the care of patients with Crohn's. Crohn's disease can flare and remit and can occur in any location in the gastrointestinal tract. Endoscopy is limited to accessible regions of the GI tract. Biopsy is needed to make a definitive diagnosis and to distinguish from Ulcerative colitis in many cases. CT scanning can evaluate any portion of the GI tract but is very non-specific. Patients with a history of Crohn's can have findings on CT scanning that represent previous bouts of inflammation or reactions to surgery and these may not be readily distinguished from active disease by a CT image. Imaging reagents specific to markers of active Crohn's disease could provide a means to overcome current limitations. Active Crohn's lesions could be identified in a specific manner anywhere in the GI tract. These lesions would be differentiated from ulcerative colitis and post-surgical findings. The need to CT scanning or endoscopy +/- biopsy could be reduced. Early diagnosis of disease flare (prior to symptoms) could allow proactive use of therapies which could decrease complications and hospitalizations. This approach would also be of value for surgical planning so that the location of active lesions could be assessed prior to surgery.

Autoimmune diabetes

[0141] The status of pancreatic islet cells could be assessed with molecular imaging reagents in patients with type I diabetes or pre-diabetics. In this disease, destruction of these cells leads to loss of insulin production and diabetes. Diabetes becomes clinically apparent only after 90% of islet cells are destroyed by the autoimmune process. Monitoring of islet cell mass with specific imaging reagents would provide a means to follow islet cell destruction and intervene with immunosuppression prior to significant destruction.

Transplantation

[0142] Allograft rejection is a major complication of transplantation leading to graft loss. Current methods to diagnose and monitor rejection rely on invasive biopsy and blood testing. Molecular imaging could provide a means to assess rejection. It could be used in combination with imaging of graft function so that a single procedure may allow assessment of both rejection and graft function.

Gynecologic disease

[0143] Endometriosis causes very significant morbidity in young women and can lead to surgery and infertility. A specific diagnosis of this disease is not possible prior to open biopsy. Specific molecular imaging reagents could allow for diagnosis and surgical planning.

Infectious disease

[0144] Infections can occur in any part of the human body. Diagnosis of infection can be difficult to distinguish from other inflammatory processes or from colonization. Molecular imaging could provide a means to make a diagnosis of infection, identify anatomical extent and monitor therapy. Currently available are Leukoscan Fab against granulocytes; Palatin technologies NeutroSpec anti-CD25 for diagnosis of appendicitis; Draximage's labeled Ciprofloxicin.

[0145] The following examples illustrate various aspects and embodiments of the disclosed invention. These examples in no way limit the scope of the claimed invention.

EXAMPLES

Example 1: Identification of Molecular Imaging Candidate Target Peptide Sequences from the Human Genome

[0146] Analyses were performed to identify candidate molecular imaging target peptides from the human genome. Methods were employed to identify a set of proteins with like expression on cell surfaces or secretion from cells.

[0147] 113,708 records for human proteins were extracted from the Uniprot database (Version number 46). Each record was associated with a Uniprot ID number, an amino acid sequence of the associated protein and Gene Ontology terms (GO terms) associated with the protein. The GO terms represent compiLed information regarding each protein with respect to protein structure and subcellular location. Of these 113,708 peptides, a subset of 7626 were identified which were associated with the GO terms: cell surface, cell septum surface, external side of plasma membrane, extracellular region, extracellular matrix, extracellular space, membrane, external encapsulating structure and cell surface. These proteins are listed in Table 2, which can be found at the end of the specification.

[0148] For each of the 7626 proteins selected (identified as 1-7626), those with annotation terms Signal, Secreted, Extracellular Domain, Integral Membrane, Type I, Type II, Type III, Type IV, Mitochondrial, Golgi, Endoplasmic reticulum, Nuclear, Extracellular, Cytoskeletal, Peroxisomal, GPI-anchor, Microsomal were identified (Tables 2 and 3). These annotation terms confer in some cases increased and in some cases decreased likelihood that the protein is expressed on the cell surface or secreted. In Table 3, the numbers of proteins among the 7626 associated with each term are given. A subgroup of 3163 proteins was identified associated with the terms "signal, secreted, extracellular domain, extracellular" and "GPI-anchor" (Group 1 in Tables 2 and 3). These proteins may have an increased likelihood of being accessible outside the cell. Another subgroup of 3117 proteins was identified which was Group 1, less those proteins associated with the terms "mitochondrial" and "nuclear", which may confer a decreased likelihood of accessibility from outside the cell (Group T). A subgroup of 1973 proteins was identified which was associated with the annotation term "extracellular domain" (Group 3).

Table 3

Example 2: Identification of Molecular Imaging Targets for Disease - Brain Cancer

[0149] Given a set of secreted and cell surface candidate proteins for molecular imaging, those specific proteins which may serve as targets for molecular imaging of brain cancer were sought. Specifically, protein targets for Glioblastoma were identified.

[0150] The database Online Mendelian Inheritance in Man (OMIM) was searched with the work "glioblastoma" and the resulting set of genes identified were evaluated and those that were identified as cell surface or secreted were identified. The resulting set of 35 proteins is shown in Table 4. Table 4

[0151] The PubMed database was searched with combinations of key words "glioblastoma", "gene expression" and "protein". Resulting publications were evaluated and those which identified genes or proteins which are cell surface or secreted and were expressed in a specific manner on glioblastomas or expressed in association with glioblastoma prognosis or invasiveness were identified. The resulting set of 52 proteins is shown in Table 4.

[0152] The SAGE map library (Serial Analysis of Gene Expression, Lash et al. 2000) at the NCBI was analyzed to identify genes specific to Glioblastoma as compared to normal brain tissue. This was done by comparing the frequency of SAGE tags in Glioblastoma tissues (11) to those in normal brain (6). The libraries compared were: Normal brain libraries:

1. SAGE_Duke_BB542_normal_cerebellum(58826 tags)

Brain, normal, greater than 95% white matter. Post-mortem delay of 3:15. normal cerebellum bulk CGAP non-normalized SAGE library method bulk

2. SAGE_Duke_thalamus(48548 tags)

Brain, normal thalamus. Post-mortem delay of 3: 15 normal bulk thalamus CGAP non-normalized SAGE library method bulk

3. SAGE_NHA(5th)(52261 tags)

Brain, normal human astrocyte cells harvested at passage 5 brain normal SAGE astrocyte CGAP non-normalized SAGE library method cell line

4. SAGE_normal_cerebellum(51280 tags) normal cerebellum normal cerebellum bulk CGAP non-normalized SAGE library method bulk

5. SAGE_normal_pool(6th)(63208 tags)

Brain, normal, pooled, brain normal SAGE CGAP non-normalized SAGE library method bulk

6. SAGE_normal_pediatric_cortex_H 1571(77968 tags) frontal cortex normal cortex CGAP non-normalized SAGE library method bulk

Glioblastoma libraries: 1. SAGE_glioma_l 150(62675 tags) glioma brain glioblastoma multiforme CGAP non-normalized SAGE library method bulk

2. SAGE_Brain_glioblastoma_B_H833(100600 tags) Glioblastoma brain glioblastoma bulk short SAGE adult male

3. SAGE_Brain_glioblastoma_B_R336(102322 tags) Glioblastoma brain glioblastoma bulk short SAGE adult male

4. SAGE_Brain_glioblastoma_B_R70(99099 tags) Glioblastoma brain glioblastoma bulk short SAGE adult

5. SAGE_pooled_GBM(61886 tags)

Brain, 5 pooled Duke glioblastoma multiforme primary tumors, brain glioblastoma multiforme SAGE CGAP non-normalized SAGE library method bulk

6. SAGE_Duke_GBM_Hl 110(70087 tags)

Brain, Duke glioblastoma multiforme primary tumor derived from a 51 yo male brain glioblastoma multiforme SAGE CGAP non-normalized SAGE library method bulk

7. SAGE_Duke_H247_Hypoxia(72031 tags) glioblastoma multiforme brain glioblastoma multiforme SAGE cell line CGAP non-normalized SAGE library method cell line

8. SAGE_Duke_H247_normal(60663 tags) glioblastoma multiforme brain glioblastoma multiforme SAGE cell line CGAP non-normalized SAGE library method cell line

9. SAGE_Duke_H392(57582 tags)

Brain, Duke glioblastoma multiforme cell line brain glioblastoma multiforme SAGE CGAP non- normalized SAGE library method cell line

10. SAGE_Duke_H54_EGFRvIII(57400 tags) brain brain glioblastoma multiforme cell line CGAP non-normalized SAGE library method cell line

11. SAGE_Duke_H54_lacZ(67236 tags) brain brain glioblastoma multiforme cell line CGAP non-normalized SAGE library method cell line.

[0153] The genes that were most significantly different between the tissues were identified using the SAGE algorithm. Those genes with increased frequency in glioblastoma libraries at a p- value < 0.011 were selected and then a subset which were cell surface or secreted (N = 54) were identified and are listed in Table 4.

[0154] The identified set of 141 proteins associated with Glioblastoma (Table 4) are candidate targets for molecular imaging reagent development as described.

Example 3: Identification of Molecular Imaging Targets for Hepatic Cancer

[0155] Patients with hepatic failure and cirrhosis are at increased risk for the occurrence of hepatocellular carcinoma. Given the nodular nature of the scarring process in cirrhosis, it is difficult to monitor for the occurrence of cancer in the setting of the cirrhotic liver. Molecular imaging could provide valuable clinical tools for monitoring, diagnosis and planning of treatment of hepatic cancer.

[0156] Candidate cell surface or secreted proteins which are highly expressed and specific to hepatic cancer can be identified by the method described in the invention. Proteins must be expressed specifically in the setting of cancer or pre-malignant transformation of hepatic cells versus normal or cirrhotic hepatic cells.

[0157] Methods for identification of these targets include mining of existing data as described above. These data can take the form of published literature, sequence databases, gene expression databases, or proteomic database. Methods similar to those described in Example 2 can also be used. In addition, data can be generated using animal models of liver cancer, tissue culture or human tissue specimens. Gene expression or proteomic data sets can be generated from these tissues and data from cancerous cells or tissues can be compared to normal liver tissues or control cells or tissues.

[0158] Identified markers of hepatic cancer which are expressed on the cell surface or secreted such as those present in Table 2 and have expression features as described in the specification are candidates for development of molecular imaging reagents. [0159] Further experiments to identify and prioritize protein targets for molecular imaging of hepatic cancer can utilize tissue microarrays which contain hundreds of human tissue samples representing hepatic cancer and control tissues. In this example MAGE-I and Glypican 3 antibodies reactive to human antigen are tested on a tissue microarray consisting of more than 140 cases of hepatocellular carcinoma of various grades (US Biomax, Rockville, MD). To determine the specificity of protein expression in hepatocelluar carcinoma, the controls on the tissue array are normal liver sections and other metastatic adenocarcinoma as well as intrahepatic cholangiocarcinoma.

[0160] Tissue samples are fixed in 4% in neutral phosphate buffered formalin for 24 hours. Then the tissue is dehydrated with ethanol and embedded in paraffin. The paraffin embedded tissue is section and mounted onto a positive charge lass slide. Each tissue section is 5 υm thick and ~1.0 mm in diameter and ~200 cores are mounted onto each slide. Among the 200 cases, there are 140 cases hepatocellular carcinoma grade 1-3, 12 cases of intrahepatic cholangiocarcinoma, and 31 metastic adenocarcinoma. The tissue cores are layered with a thin layer of paraffin to prevent oxidation or moisture condensation. Before the slide is used for immunohistochemistry studies, the tissue microarray is de-paraffinized. The array is rinsed with phosphate buffer saline. In some cases, the endogenous peroxidase activity is blocked. The array goes through an antigen retrieval process to expose the antigenic sites. The array is incubated with normal serum to reduce nonspecific binding. The array is incubated with the primary antibody. In this case, the primary antibodies are against human antigens for MAGE-I and Glypican 3. The MAGE-I is a monoclonal antibody raised in mouse. The Glypican 3 antibody is polyclonal antibody raised in sheep. The array slide is rinsed with phosphate buffer saline. Then the array is incubated with a biotin- conjugated secondary antibody. The secondary antibody is anti-host of the primary antibody. In this case the secondary antibody is against rabbit, mouse and sheep. The array is incubated with an Avidin/Biotinylated Enzyme Complex (ABC). In this case the enzyme is horseradish peroxidase. The array is incubated with diaminobenzidine which is the substrate for the enzyme to produce a insoluble brown product. The array is stained with hematoxylin to differentiate cell structure in the tissue sections. The array is dehydrated and mounted. An image of the immunostained tissue array is acquired through image acquisition software. Image analysis software is used to generate quantitative data. One form of the raw data is percentage of positive cells and intensity of positive signal. Another form of the raw data is scoring the signal from a range of + to +++.

Example 4: Identification of Molecular Imaging Targets for Atherosclerosis

[0161] Atherosclerosis is an inflammatory process of arterial walls which can lead to occlusion and limitation in blood flow. Atherosclerotic plaques can suddenly rupture leading to complete occlusion of a blood vessel which leads to myocardial infarction (heart attack) or stroke. It is not possible to predict which atherosclerotic plaques in which vessels are likely to rupture and lead to acute events. Molecular imaging could enable assessment of both blood vessel anatomy and risk of plaque rupture.

[0162] Candidate cell surface or secreted proteins which are highly expressed and specific to active (at risk) atherosclerotic plaque can be identified by the method described in the invention. Proteins must be specifically expressed in active athrerosclerotic plaque (e.g., with subsequent acute events) versus lower risk atherosclerosis.

[0163] Methods for identification of these targets include mining of existing data as described above. These data can take the form of published literature, sequence databases, gene expression databases, or proteomic database. Methods similar to those described in Example 2 can also be used. In addition, data can be generated using animal models of atherosclerosis, tissue culture or human tissue specimens. Gene expression or proteomic data sets can be generated from these tissues and comparisons or active vs. low-risk plaque can be used to identify candidate markers.

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Claims

1. A target-specific imaging reagent, comprising an affinity agent coupled to an imaging agent, wherein said affinity agent specifically binds to a biological molecule, wherein expression of said biological molecule is predictive of a disease or a disease state.

2. The target-specific imaging reagent of claim 1, wherein said imaging agent is detectable by at least one of the technologies selected from the group consisting of: computed tomography, ultrasound, magnetic resonance, nuclear imaging, and optical imaging.

3. The target-specific imaging reagent of claim 2, wherein said optical imaging is by Diffuses Optical Tomography, Optical Coherence Tomography, Confocal Laser Scanning Microscopy, Fluorescence Correlation Microscopy, Fluorescence Resonance Energy Transfer, or Fluorescence Lifetime Imaging.

4. The target-specific imaging reagent of claim 2, wherein said nuclear imaging is by PET or SPECT.

5. The target-specific imaging reagent of claim 1, wherein said affinity agent is selected from the group consisting of: antibodies, small molecules, and peptides.

6. The target-specific imaging reagent of claim 1, wherein said biological molecule is selected from the group consisting of: cell surface proteins, secreted proteins, cell surface polysaccharides, RNA, and DNA.

7. The target-specific imaging reagent of claim 1, wherein said biological molecule is chosen from the proteins listed in Table 2.

8. The target-specific imaging reagent of claim 1, wherein said disease is selected from the group consisting of: cancer, cardiovascular disease, a condition caused by hematopoietic stem cell transplantation, neurologic disease, autoimmune disease, chronic inflammatory disease, gynecologic disease, orthopedic disease and infectious disease.

9. The target-specific imaging reagent of claim 8, wherein said cancer is selected from the group consisting of: lung cancer, melanoma, breast cancer, prostate cancer, neuroendocrine, stomach cancer, lymphoma, head and neck cancer, pancreatic cancer, ovarian cancer, liver cancer, cancer of the central nervous system, and testicular cancer.

10. A method of diagnosing or monitoring a disease or disease state comprising: administering to a mammal the target-specific imaging reagent of any of claims 1-9, imaging said mammal, and diagnosing or monitoring said disease or disease state.

11. The method of claim 10, wherein said mammal is an animal or a human.

12. A method of diagnosing or monitoring disease by molecular imaging of at least one protein chosen from proteins 1-7626 depicted in Table 2.

13. The method of claim 12 wherein said imaging is conducted using at least one of the technologies selected from the group consisting of: computed tomography, ultrasound, magnetic resonance, nuclear imaging, and optical imaging.

14. The method of claim 13, wherein said optical imaging is by Diffuses Optical Tomography, Optical Coherence Tomography, Confocal Laser Scanning Microscopy, Fluorescence Correlation Microscopy, Fluorescence Resonance Energy Transfer, or Fluorescence Lifetime Imaging.

15. The method of claim 10, wherein said disease is selected from the group consisting of: cancer, cardiovascular disease, a condition caused by hematopoietic stem cell transplantation, neurologic disease, autoimmune disease, chronic inflammatory disease, gynecologic disease, and infectious disease.

16. The method of claim 15, wherein said cancer is selected from the group consisting of: lung cancer, melanoma, breast cancer, prostate cancer, neuroendocrine, stomach cancer, lymphoma, head and neck cancer, pancreatic cancer, ovarian cancer, liver cancer, cancer of the central nervous system, and testicular cancer.

17. The method of claim 12 wherein at least two proteins chosen from proteins 1- 7626 depicted in Table 2 are imaged.

18. The method of claim 12 wherein at least three proteins chosen from proteins 1- 7626 depicted in Table 2 are imaged.

19. A target-specific agent comprising an affinity agent wherein said affinity agent specifically binds to a biological molecule wherein expression of said biological molecule is predictive of a disease or a disease state.

20. A diagnostic assay comprising the agent of claim 19.

21. A kit comprising the diagnostic assay of claim 20.