WO2013036755A1 - Etv1 antibodies and uses thereof - Google Patents

Abstract

The present invention provides monoclonal antibodies that bind to the ETV1 protein. The present invention also provides various methods and compositions relating to provided antibodies. For example, the present invention provides compositions, including pharmaceutical compositions, comprising provided antibodies, or fragments or mimics thereof. The present invention provides various therapeutic and/or diagnostic methods of using provided antibodies and/or compositions.

Description

ETVl ANTIBODIES AND USES THEREOF

Priority Claim

[0001] The present application claims priority to U.S. Provisional Patent Application No.

61/532,940 that was filed on September 9, 2011. The entire contents of this priority application are incorporated herein by reference.

Background

[0002] ETVl is a transcription factor belonging to the large ETS (E-twenty six) family and PEA3 subfamily. ETS family members are defined by a highly conserved DNA binding domain, the ETS domain, which is a winged helix-turn-helix structure that binds to DNA sites with a central GGA DNA sequence. Multiple ETS factors have been found to be associated with cancer. In a condition called Ewing's sarcoma, the ERG ETS transcription factor is fused to the EWS gene (Ida, K., et al, 1995, Int. J. Cancer, 63(4):500-4; incorporated herein by reference). The fusion of TEL to the JAK2 protein results in early pre-B acute lymphoid leukemia (Peeters, P., et al, 1997, Blood, 90(7):2535-40; incorporated herein by reference) and ERG and ETVl are ETS family members which are over-expressed, and whose gene fusions are found, in prostate cancer (Tomlins, S.A., et al, 2005, Science, 310(5748):644-8; incorporated herein by reference).

[0003] There is a need for reagents, in particular monoclonal antibodies, and methods that permit detection of ETVl and/or treatment of diseases, disorders, and conditions associated with ETVl expression.

Summary

[0004] In one aspect the present invention provides antibodies (e.g., human antibodies, humanized antibodies, etc.) which bind to ETVl . As discussed herein, ETVl is associated with certain diseases, disorders, and conditions. Provided antibodies are useful in medicine, for example, in the prophylaxis, treatment, diagnosis, and/or study of ETVl -associated diseases, disorders, and conditions. [0005] In some embodiments, the present invention provides compositions that comprise antibodies that bind to ETV1. In some embodiments, provided antibodies have one or more CDRs found in a reference antibody. In some embodiments, provided antibodies compete with one or more reference antibodies for binding to ETV1.

[0006] In some embodiments, a provided antibody is an antibody produced by a hybridoma deposited with the American Type Culture Collection (ATCC) and selected from the group set forth in Table 1.

[0007] In some embodiments, a reference antibody is an antibody produced by a hybridoma deposited with the American Type Culture Collection (ATCC) and selected from the group set forth in Table 1.

[0008] In some embodiments, provided compositions comprise a provided antibody associated with a payload. In some embodiments, provided compositions comprise a provided antibody associated with a therapeutic and/or a diagnostic payload. In some embodiments, association of a provided antibody and payload is covalent.

[0009] In some embodiments, provided antibodies bind to ETV1 in tissue samples (e.g., a sample of gastrointestinal stromal tissue). In some embodiments, binding by provided antibodies to tissue samples correlates with ETV1 level of expression and/or activity in the samples.

[0010] In some embodiments, the present invention provides therapeutic and/or diagnostic technologies that utilize one or more provided antibodies.

[0011] In some embodiments, the present invention provides methods of identifying useful anti-ETVl antibodies. For example, in some embodiments, such methods involve determining whether a test antibody competes for antigen binding with one or more provided antibodies. In some embodiments, a test antibody is identified as a useful antibody if it cross- competes with one or more provided antibodies (including, for example, with one or more provided reference antibodies).

[0012] In some embodiments, provided ETV1 antibodies are combined with

pharmaceutically acceptable excipients to provide pharmaceutical compositions. The present invention provides pharmaceutical compositions for treatment, prevention, and/or diagnosis of ETV1 -associated diseases, disorders, and conditions.

[0013] In some embodiments, pharmaceutical compositions in accordance with the invention comprise human antibodies capable of binding to the ETV1 protein and capable of inhibiting ETV1 activity in vitro. In some embodiments, pharmaceutical compositions in accordance with the invention comprise human antibodies capable of binding to the ETV1 protein and capable of inhibiting ETV1 activity in vivo. In some embodiments, ETV1 inhibition by provided antibodies in in vitro systems is correlative and/or predictive of ETV1 inhibition by provided antibodies in vivo (e.g., in humans and/or other mammals).

[0014] In some embodiments, pharmaceutical compositions in accordance with the invention may comprise fragments of provided ETV1 antibodies that substantially retain the antigen-binding characteristics of the whole antibody. In some embodiments, pharmaceutical compositions may comprise provided ETV1 antibodies produced by recombinant methods that are well known in the art.

[0015] The present invention also provides methods of using provided ETV1 antibodies to diagnose, prognose and/or treat various disorders, diseases and conditions (e.g.,

gastrointestinal stromal tumor or GIST).

[0016] Thus, in another aspect there is provided a method for determining the likelihood of gastrointestinal stromal tissue being cancerous or being gastrointestinal stromal tumor (GIST), comprising (a) determining a level of expression or activity of ETV1 in a gastrointestinal stromal tissue sample obtained from a subject; (b) comparing the determined ETV1 level with that of a reference correlated with a predetermined probability of being cancerous or being GIST; and (c) based on the comparing, determining that the tissue sample has an increased or decreased probability, relative to the reference, of being cancerous or being GIST. Determining the level of expression or activity involves determining the expression of a ETV1 using a provided antibody. In certain embodiments, an increase in the level of ETV1 correlates with the onset of or progression of a GIST phenotype. In other embodiments, a decrease in the level of ETV1 correlates with the regression of a GIST phenotype. In certain embodiments, determining the level of expression comprises performing Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, ELISA, and/or immunohistochemistry. In a related embodiment, the step (b) of comparing comprises establishing that the determined level in the subject tissue sample is higher than the reference level, and the reference level correlates with that observed in normal tissue, so that step (c) of determining comprises detemining that the tissue sample has an elevated likelihood of being cancerous or GIST. In another related embodiment, the step (a) of determining comprises performing an analysis selected from the group consisting of Northern blotting, Western blotting, protein gel electrophoresis,

immunoprecipitation, ELISA, PCR, RT-PCR, differential display, serial analysis of gene expression, array analysis, immunohistochemistry, or a combination thereof, so that the ETVl level of expression or activity is determined. In additional embodiments, the step (a) of determining comprises performing immunohistochemistry on the tissue sample, using an ETVl antibody or antigen binding fragment thereof. In a particular embodiment, the antibody or antigen binding fragment binds to an amino acid sequence as set forth in SEQ ID NO: 1.

[0017] In yet another aspect, there is provided a method for determining the likelihood of cancerous gastrointestinal stromal tissue responding to therapy, comprising: (a) determining a level of expression or activity of ETVl in a cancerous gastrointestinal stromal tissue sample obtained from a subject; (b) comparing the determined ETVl level with that of a reference correlated with a predetermined probability of responding to a therapeutic regimen; and (c) based on the comparing, determining that the subject from which the tissue sample was obtained has an increased or decreased probability, relative to the reference, of responding to the therapeutic regimen. In a particular embodiment, the reference is an average of responses derived from a population of individuals.

[0018] In yet another aspect, there is provided a methhod for identifying or

characterizing an agent that affects GIST phenotype comprising: (a) contacting cultured GIST tissue or cells with an agent to be characterized; and (b) determining that ETVl level of expression or activity is altered when the agent is present as compared with when it is absent.

[0019] In yet another aspect, there is provided a method of treating GIST by

administering to a subject suffering from or susceptible to GIST a therapy that alters ETVl level of expression or activity. In a particular embodiments, the step of administering comprises administering a therapy that modulates expression or activity of an ETVl regulator so that ETVl level of expression or activity is altered.

[0020] In yet another aspect, there is provided a method of treating GIST in a subject in need thereof comprising administering a therapeutically effective amount of pharmaceutical composition comprising an agent identified by the methods disclosed herein. In certain embodiments, the composition inhibits EVT1 expression. In other embodiments, the

composition promotes the degradation of ETVl protein.

[0021] In yet another aspect, there is provided a kit for use in detecting ETVl expression.

The kit comprises a provided antibody that is capable of detecting the level of ETVl expression or activity, a container comprising the provided antibody for detecting the level of ETVl in a sample, a control, and instructions to provide guidance for carrying out an assay embodied by the kit and for making a determination of ETVl based upon that assay. In certain embodiments, the provided antibody is attached to a substrate, the substrate is applied to a sample from a patient or to a surface that may contain ETVl, and the surface of the substrate is then processed to assess whether specific binding occurs between the antibody and ETVl or other component of the sample. In still another embodiment, the control is a control slide comprising tumor samples for testing reagents in the kit.

[0022] In yet another aspect, there is provided a method comprising the steps of: (a) monitoring ETVl level of expression or activity of ETVl in a subject suffering from GIST; (b) detecting a change in the monitored ETVl level over time; and (c) initiating or altering therapy administered to the subject after detecting the change. In a particular embodiment, the subject is undergoing a first therapy and the step of initiating or altering comprises ceasing the first therapy, modifying the first therapy, adding a second therapy, or combinations thereof.

[0023] In yet another aspect, there is provided a method of inhibiting the growth of ICC cancerous cell with aberrant ETVl expression comprising contacting the cell with a provided ETVl antibody so as to decrease the level or activity of ETVl, thereby inhibiting the growth of the ICC cancerous cell. Definitions

[0024] Amino acid: As used herein, term "amino acid," in its broadest sense, refers to any compound and/or substance that can be incorporated into a polypeptide chain. In some embodiments, an amino acid has the general structure H2N-C(H)(R)-COOH. In some embodiments, an amino acid is a naturally occurring amino acid. In some embodiments, an amino acid is a synthetic amino acid; in some embodiments, an amino acid is a d-amino acid; in some embodiments, an amino acid is an 1-amino acid. "Standard amino acid" refers to any of the twenty standard 1-amino acids commonly found in naturally occurring peptides. "Nonstandard amino acid" refers to any amino acid, other than the standard amino acids, regardless of whether it is prepared synthetically or obtained from a natural source. As used herein, "synthetic amino acid" encompasses chemically modified amino acids, including but not limited to salts, amino acid derivatives (such as amides), and/or substitutions. Amino acids, including carboxy- and/or amino-terminal amino acids in peptides, can be modified by methylation, amidation, acetylation, protecting groups, and/or substitution with other chemical groups that can change the peptide's circulating half-life without adversely affecting their activity. Amino acids may participate in a disulfide bond. Amino acids may comprise one or posttranslational modifications, such as association with one or more chemical entities (e.g., methyl groups, acetate groups, acetyl groups, phosphate groups, formyl moieties, isoprenoid groups, sulfate groups, polyethylene glycol moieties, lipid moieties, carbohydrate moieties, biotin moieties, etc.). The term "amino acid" is used interchangeably with "amino acid residue," and may refer to a free amino acid and/or to an amino acid residue of a peptide. It will be apparent from the context in which the term is used whether it refers to a free amino acid or a residue of a peptide.

[0025] Animal: As used herein, the term "animal" refers to any member of the animal kingdom. In some embodiments, "animal" refers to humans, of either sex and at any stage of development. In some embodiments, "animal" refers to non-human animals, at any stage of development. In certain embodiments, the non-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). In some embodiments, animals include, but are not limited to, mammals, birds, reptiles, amphibians, fish, insects, and/or worms. In certain embodiments, the animal is susceptible to infection by HCV. In some embodiments, an animal may be a transgenic animal, genetically engineered animal, and/or a clone.

[0026] Agonist: As used herein, the term "agonist" refers to an agent that increases or prolongs the duration of the effect of a polypeptide or a nucleic acid. Agonists may include proteins, nucleic acides, carbohydrates, lipids, small molecules, ions, or any other molecules that modulate the effect of the polypeptide or nucleic acide. An agonist may be a direct agonist, in which case it is a molecule that exerts its effect by binding to the polypeptide or nucleic acid, or an indirect agonist, in which case it exerts its effect via a mechanism other than binding to the polypeptide or nucleic acid (e.g., by altering expression or stability of the polypeptide or nucleic acid, by altering the expression or activity of a target of the polypeptide or nucleic acid, by interacting with an intermediate in a pathway involving the polypeptide or nucleic acid, etc.).

[0027] Antagonist: As used herein, the term "antagonist" refers to an agent that decreases or reduces the duration of the effect of a polypeptide or a nucleic acid. Antagonists may include proteins, nucleic acids, carbohydrates, or any other molecules that modulate the effect of the polypeptide or nucleic acid. An antagonist may be a direct antagonist, in which case it is a molecule that exerts its effect by binding to the polypeptide or nucleic acid, or an indirect antagonist, in which case it exerts its effect via a mechanism other than binding to the polypeptide or nucleic acid (e.g. by altering expression or stability of the polypeptide or nucleic acid, by altering the expression or activity of a target of the polypeptide or nucleic acide, by interacting with an intermediate in a pathway involving the polypeptide or nucleic acid, etc.).

[0028] Antibody: As used herein, the term "antibody" refers to any immunoglobulin, whether natural or wholly or partially synthetically produced. All derivatives thereof which maintain specific binding ability are also included in the term. The term also covers any protein having a binding domain which is homologous or largely homologous to an immunoglobulin- binding domain. Such proteins may be derived from natural sources, or partly or wholly synthetically produced. An antibody may be monoclonal or polyclonal. An antibody may be a member of any immunoglobulin class, including any of the human classes: IgG, IgM, IgA, IgD, and IgE. In certain embodiments, an antibody may be a member of the IgG immunoglobulin class. As used herein, the terms "antibody fragment" or "characteristic portion of an antibody" are used interchangeably and refer to any derivative of an antibody which is less than full-length. In general, an antibody fragment retains at least a significant portion of the full-length antibody's specific binding ability. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, scFv, Fv, dsFv diabody, and Fd fragments. An antibody fragment may be produced by any means. For example, an antibody fragment may be enzymatically or chemically produced by fragmentation of an intact antibody and/or it may be recombinantly produced from a gene encoding the partial antibody sequence. Alternatively or additionally, an antibody fragment may be wholly or partially synthetically produced. An antibody fragment may optionally comprise a single chain antibody fragment. Alternatively or additionally, an antibody fragment may comprise multiple chains which are linked together, for example, by disulfide linkages. An antibody fragment may optionally comprise a multimolecular complex. A functional antibody fragment typically comprises at least about 50 amino acids and more typically comprises at least about 200 amino acids. In some embodiments, an antibody may be a human antibody. In some embodiments, an antibody may be a humanized antibody.

[0029] Approximately: As used herein, the term "approximately" or "about," as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In certain embodiments, the term "approximately" or "about" refers to a range of values that fall within 25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%>, 1%), or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0030] Biologically active: As used herein, the phrase "biologically active" refers to a characteristic of any substance that has activity in a biological system (e.g., cell culture, organism, etc.). For instance, a substance that, when administered to an organism, has a biological effect on that organism, is considered to be biologically active. In particular embodiments, where a protein or polypeptide is biologically active, a portion of that protein or polypeptide that shares at least one biological activity of the protein or polypeptide is typically referred to as a "biologically active" portion. [0031] Characteristic portion: As used herein, the term a "characteristic portion" of a substance, in the broadest sense, is one that shares some degree of sequence or structural identity with respect to the whole substance. In certain embodiments, a characteristic portion shares at least one functional characteristic with the intact substance. For example, a "characteristic portion" of a protein or polypeptide is one that contains a continuous stretch of amino acids, or a collection of continuous stretches of amino acids, that together are characteristic of a protein or polypeptide. In some embodiments, each such continuous stretch generally contains at least 2, 5, 10, 15, 20, 50, or more amino acids. In general, a characteristic portion of a substance (e.g., of a protein, antibody, etc.) is one that, in addition to the sequence and/or structural identity specified above, shares at least one functional characteristic with the relevant intact substance. In some embodiments, a characteristic portion may be biologically active.

[0032] Combination therapy: The term "combination therapy", as used herein, refers to those situations in which two or more different pharmaceutical agents are administered in overlapping regimens so that the subject is simultaneously exposed to both agents.

[0033] Diagnostic information: As used herein, diagnostic information or information for use in diagnosis is any information that is useful in determing whether a patient has a disease or condition and/or in classifying the disease or condition into a phenotypic category or any category having significance with regards to the prognosis of, or likely response to, treatment (either treatment in general or any particular treatment) of the disease or condition. Similarly, diagnosis refers to providing any type of diagnostic information, including, but not limited to, whether a subject is likely to have a condition (such as a tumor), information related to the nature or classification of a tumor, information related to prognosis and/or information useful in selecting an appropriate treatment. Selection of treatment may include the choice of a particular chemotherapeutic agent or other treatment modalitiy such as surgery, radiation, etc. a choice about whether to withhold or deliver therapy, etc.

[0034] Dosage form: As used herein, the terms "dosage form" and "unit dosage form" refer to a physically discrete unit of a therapeutic protein (e.g., antibody) for the patient to be treated. Each unit contains a predetermined quantity of active material calculated to produce the desired therapeutic effect. It will be understood, however, that the total dosage of the composition will be decided by the attending physician within the scope of sound medical judgment.

[0035] Dosing regimen: A "dosing regimen" (or "therapeutic regimen"), as that term is used herein, is a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time. In some embodiments, a given therapeutic agent has a recommended dosing regiment, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regime comprises a plurality of doses and at least two different time periods separating individual doses.

[0036] Expression: As used herein, "expression" of a nucleic acid sequence refers to one or more of the following events: (1) production of an R A template from a DNA sequence (e.g., by transcription); (2) processing of an RNA transcript (e.g., by splicing, editing, 5' cap formation, and/or 3' end formation); (3) translation of an RNA into a polypeptide or protein; and/or (4) post-translational modification of a polypeptide or protein.

[0037] Functional: As used herein, a "functional" biological molecule is a biological molecule in a form in which it exhibits a property and/or activity by which it is characterized.

[0038] Gastrointestinal stromal tumor: The term "gastrointestinal stromal tumor" or

"GIST" as used herein refers to tumors located in the gastrointestinal tract (such as the stomach, the small intestine, and the large intestine) or in surrounding organs or tissues (such as the appendix, ampulla vater, rectum, omentum, anus, or the esophagus). In specific embodiments, the tumors comprise at least one cell expressing KIT or comprising a gain of function mutation in KIT, and in further specific embodiments the cell comprises a drug resistance-conferring mutation that arises before or during therapy. In specific embodiments, the tumor arises from at least one intersitial cell of Cajal (ICC) or one or more precursors or pluripotential stem cells thereof. In additional embodiments, the cell also expresses other markers, such as CD34, SMA, desmin, S-100, or any combination thereof.

[0039] Gene: As used herein, the term "gene" has its meaning as understood in the art.

It will be appreciated by those of ordinary skill in the art that the term "gene" may include gene regulatory sequences (e.g., promoters, enhancers, etc.) and/or intron sequences. It will further be appreciated that definitions of gene include references to nucleic acids that do not encode proteins but rather encode functional RNA molecules such as tRNAs, RNAi-inducing agents, etc. For the purpose of clarity we note that, as used in the present application, the term "gene" generally refers to a portion of a nucleic acid that encodes a protein; the term may optionally encompass regulatory sequences, as will be clear from context to those of ordinary skill in the art. This definition is not intended to exclude application of the term "gene" to non-protein- coding expression units but rather to clarify that, in most cases, the term as used in this document refers to a protein-coding nucleic acid.

[0040] Gene product or expression product: As used herein, the term "gene product" or

"expression product" generally refers to an RNA transcribed from the gene (pre-and/or postprocessing) or a polypeptide (pre- and/or post-modification) encoded by an RNA transcribed from the gene.

[0041] Homology: As used herein, the term "homology" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. In some embodiments, polymeric molecules are considered to be "homologous" to one another if their sequences are at least 80%, 85%, 90%), 95%o, or 99% identical. In some embodiments, polymeric molecules are considered to be "homologous" to one another if their sequences are at least 80%>, 85%, 90%>, 95%, or 99% similar.

[0042] Identity: As used herein, the term "identity" refers to the overall relatedness between polymeric molecules, e.g., between nucleic acid molecules (e.g., DNA molecules and/or RNA molecules) and/or between polypeptide molecules. Calculation of the percent identity of two nucleic acid sequences, for example, can be performed by aligning the two sequences for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second nucleic acid sequences for optimal alignment and non-identical sequences can be disregarded for comparison purposes). In certain embodiments, the length of a sequence aligned for comparison purposes is at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%), at least 95%, or substantially 100% of the length of the reference sequence. The nucleotides at corresponding nucleotide positions are then compared. When a position in the first sequence is occupied by the same nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences. The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two nucleotide sequences can be determined using the algorithm of Meyers and Miller (CABIOS, 1989, 4: 11-17), which has been incorporated into the ALIGN program (version 2.0) using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4. The percent identity between two nucleotide sequences can,

alternatively, be determined using the GAP program in the GCG software package using an NWSgapdna.CMP matrix.

[0043] Isolated: As used herein, the term "isolated" refers to a substance and/or entity that has been (1) separated from at least some of the components with which it was associated when initially produced (whether in nature and/or in an experimental setting), and/or (2) produced, prepared, and/or manufactured by the hand of man. Isolated substances and/or entities may be separated from about 50%, about 60%, about 70%>, about 80%>, about 90%>, about 91 >, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% of the other components with which they were initially associated. In some embodiments, isolated agents are about 80%>, about 85%, about 90%>, about 91%>, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, or more than about 99% pure. As used herein, a substance is "pure" if it is substantially free of other components. As used herein, calculation of percent purity of isolated substances and/or entities should not include excipients (e.g., buffer, solvent, water, etc.).

[0044] Marker: A marker, as used herein, refers to a gene whose expression is a characteristic of a particular tumor subclass. The term may also refer to a product of gene expression (e.g., an RNA transcribed from the gene or a translation product of such an RNA), the production of which is characteristic of a particular tumor subclass. In some cases expression or levels of a marker may be the sole criterion used to define the tumor subclass. In other cases expression or levels of a marker may be combined with other criteria to define the tumor subclass. The statistical significance of the presence or absence of a marker may vary depending upon the particular marker. In some cases the detection of a marker is highly specific in that it reflects a high probability that the tumor is of a particular subclass. This specificity may come at the cost of sensitivity (i.e., a negative result may occur even if the tumor is a tumor that would be expected to express the marker). Conversely, markers with a high degree of sensitivity may be less specific that those with lower sensitivity. Thus it will be appreciated that a useful marker need not distinguish tumors of a particular subclass with 100% accuaracy. Furthermore, it will be appreciated that the use of multiple markers may improve the specificity and/or sensitivity with which a tumor can be identified as being of a particular tumor subclass. It is to be understood that a marker for a particular tumor subclass is a gene (or gene product) whose expression is characteristic of a particular tumor subclass (i.e., a gene or gene product) whose expression is characteristic of some or all of the cells in the tumor.

[0045] Mimotope: As used herein, the term "mimotope" refers to a macromolecule which mimics the structure of an epitope. In some embodiments, a mimotope elicits an antibody response identical or similar to that elicited by its corresponding epitope. In some embodiments, an antibody that recognizes an epitope also recognizes a mimotope which mimics that epitope. In some embodiments, a mimotope is a peptide. In some embodiments, a mimotope is a small molecule, carbohydrate, lipid, or nucleic acid. In some embodiments, mimotopes are peptide or non-peptide mimotopes of conserved HCV epitopes. In some embodiments, by mimicking the structure of a defined viral epitope, a mimotope interferes with the ability of HCV virus particles to bind to its natural binding partners (e.g., HCV target receptor, El protein, etc.), e.g., by binding to the natural binding partner itself.

[0046] Nucleic acid: As used herein, the term "nucleic acid," in its broadest sense, refers to any compound and/or substance that is or can be incorporated into an oligonucleotide chain. In some embodiments, a nucleic acid is a compound and/or substance that is or can be incorporated into an oligonucleotide chain via a phosphodiester linkage. In some embodiments, "nucleic acid" refers to individual nucleic acid residues (e.g., nucleotides and/or nucleosides). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising individual nucleic acid residues. As used herein, the terms "oligonucleotide" and "polynucleotide" can be used interchangeably. In some embodiments, "nucleic acid" encompasses RNA as well as single and/or double-stranded DNA and/or cDNA. Furthermore, the terms "nucleic acid," "DNA," "RNA," and/or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. For example, the so-called "peptide nucleic acids," which are known in the art and have peptide bonds instead of phosphodiester bonds in the backbone, are considered within the scope of the present invention. The term "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and/or encode the same amino acid sequence. Nucleotide sequences that encode proteins and/or RNA may include introns. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, backbone modifications, etc. A nucleic acid sequence is presented in the 5' to 3' direction unless otherwise indicated. The term "nucleic acid segment" is used herein to refer to a nucleic acid sequence that is a portion of a longer nucleic acid sequence. In many embodiments, a nucleic acid segment comprises at least 3, 4, 5, 6, 7, 8, 9, 10, or more residues. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxy cytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolo-pyrimidine, 3-methyl adenosine, 5-methylcytidine, C-5 propynyl-cytidine, C-5 propynyl-uridine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl- cytidine, C5-methylcytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8- oxoadenosine, 8-oxoguanosine, 0(6)-methylguanine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); intercalated bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and/or modified phosphate groups (e.g., phosphorothioates and 5'-N-phosphoramidite linkages). In some embodiments, the present invention is specifically directed to "unmodified nucleic acids," meaning nucleic acids (e.g., polynucleotides and residues, including nucleotides and/or nucleosides) that have not been chemically modified in order to facilitate or achieve delivery.

[0047] Patient: As used herein, the term "patient" or "subject" refers to any organism to which a provided composition may be administered, e.g., for experimental, diagnostic, prophylactic, cosmetic, and/or therapeutic purposes. Typical patients include animals (e.g., mammals such as mice, rats, rabbits, non-human primates, and/or humans). In some

embodiments, a patient is a human.

[0048] Pharmaceutically acceptable: The term "pharmaceutically acceptable" as used herein, refers to substances that, within the scope of sound medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0049] Polypeptide: As used herein, a "polypeptide", generally speaking, is a string of at least two amino acids attached to one another by a peptide bond. In some embodiments, a polypeptide may include at least 3-5 amino acids, each of which is attached to others by way of at least one peptide bond. Those of ordinary skill in the art will appreciate that polypeptides sometimes include "non-natural" amino acids or other entities that nonetheless are capable of integrating into a polypeptide chain, optionally.

[0050] Positive or negative subclass: As used herein, a tumor belonging to a positive subclass includes cells with a mutated version of a particular marker gene. A tumor belonging to a mutant negative subclass includes cells with a wild-type version of a particular marker gene. Mutant versions of the KIT marker have been identified. Mutant versions of the PDGFRA marker have been identified. Mutations may result in overexpression or inappropriate expression of the marker gene. Additionally or alternatively mutations may result in an overly activated gene product (e.g., polypeptide). The terms "KIT mutant positive/negative" and KIT

positive/negative" are used interchangeably herein as are the terms "PDGFRA mutant positive/negative" and "PDGFRA positive/negative".

[0051] Prognostic and predictive information: As used herein, the terms prognostic and predictive information are used interchangeably to refer to any information that may be used to foretell any aspect of the course of a disease or condition either in the absence or presence of treatment. Such information may include, but is not limited to, the average life expectancy of a patient, the likelihood that a patient will survive for a given amount of time (e.g., 6 months, 1 year, 5 years, etc.), the likelihood that a patient will be cured of a disease, the likelihood that a patient's disease will respond to a particular therapy (wherein response may be defined in any o f avariety of ways). Prognostic and predictive information are included within the broad category of diagnostic information.

[0052] Protein: As used herein, the term "protein" refers to a polypeptide (i.e., a string of at least two amino acids linked to one another by peptide bonds). Proteins may include moieties other than amino acids (e.g., may be glycoproteins, proteoglycans, etc.) and/or may be otherwise processed or modified. Those of ordinary skill in the art will appreciate that a "protein" can be a complete polypeptide chain as produced by a cell (with or without a signal sequence), or can be a characteristic portion thereof. Those of ordinary skill will appreciate that a protein can sometimes include more than one polypeptide chain, for example linked by one or more disulfide bonds or associated by other means. Polypeptides may contain 1-amino acids, d-amino acids, or both and may contain any of a variety of amino acid modifications or analogs known in the art. Useful modifications include, e.g., terminal acetylation, amidation, methylation, etc. In some embodiments, proteins may comprise natural amino acids, non-natural amino acids, synthetic amino acids, and combinations thereof. The term "peptide" is generally used to refer to a polypeptide having a length of less than about 100 amino acids, less than about 50 amino acids, less than 20 amino acids, or less than 10 amino acids. In some embodiments, proteins are antibodies, antibody fragments, biologically active portions thereof, and/or characteristic portions thereof.

[0053] Reference antibody: As used herein, the term "reference antibody" refers to an antibody 10D12C5B4D4, 2F11G11D10D11, 14D1F8C4E6, 8E6B7C9D10, 13A7E12G5A5, 2F11G11D10B4, and/or 13A7E12G5B8 produced by a hybridoma cell line that secretes human monoclonal antibody ETV1, deposited with the American Type Culture Collection, 10801 University Boulevard, Manassas, VA 20110-2209 under the Budapest Treaty in accordance with Table 1. These cell lines will be maintained at an authorized depository and replaced in the event of mutation, nonviability or destruction for a period of at least five years after the most recent request for release of a sample was received by the depository, for a period of at least thirty years after the date of the deposit, or during the enforceable life of the related patent, whichever period is longest. All restrictions on the availability to the public of these cell lines will be irrevocably removed upon the issuance of a patent from the application. Table 1: ETV1 monoclonal antibody ATCC deposits

[0054] Refractory: The term "refractory" as used herein, refers to any subject that does not respond with an expected clinical efficacy following the administration of provided compositions as normally observed by practicing medical personnel.

[0055] Response: As used herein, a response to treatment may refer to any beneficial alteration in a subject's condition that occurs as a result of treatment. Such alteration may include stabilization of the condition (e.g., prevention of deterioration that would have taken place in the absence of the treatment), amelioration of symptoms of the condition, and/or improvement in the prospects for cure of the condition, etc. One may refer to a subject's response or to a tumor's response. In general these concepts are used interchangeably herein. Tumor or subject response may be measured according to a wide variety of criteria, including clinical criteria and objective criteria. Techniques for assessing response include, but are not limited to, clinical examination, chest X-ray CT scan, MRI, ultrasound, endoscopy, laparoscopy, presence or level of tumor markers in a sample obtained from a subject, cytology, and/or histology. Many of these techniques attempt to determine the size of a tumor or otherwise dertermine the total tumor burden. Methods and guidelines for assessing response to treatment are discussed in Therasse et. al, "New guidelines to evaluate the response to treatment in solid tumors", European Organization for Research and Treatment of Cancer, National Cancer Institute of the United States, National Cancer Institute of Canada, J. Natl. Cancer Inst.,

92(3):205-16, 2000. The exact response criteria can be selected in any appropriate manner, provided that when comparing groups of tumors and/or patients, the groups to be compared are assessed based on the same or comparable criteria for determining response rate. One of ordinary skill in the art will be able to select appropriate criteria.

[0056] Sample: As used herein, a sample obtained from a subject may include, but is not limited to, any or all of the following: a cell or cells, a portion of tissue, blood, serum, ascites, urine, saliva, and other body fluids, secretions, or excretions. The term "sample" also includes any material derived by processing such a sample. Derived samples may include nucleotide molecules or polypeptides extracted from the sample or obtained by subjecting the sample to techniques such as amplification or reverse transcription of mRNA, etc.

[0057] Small Molecule: In general, a "small molecule" is a molecule that is less than about 5 kilodaltons (kD) in size. In some embodiments, the small molecule is less than about 4 kD, 3 kD, about 2 kD, or about 1 kD. In some embodiments, the small molecule is less than about 800 daltons (D), about 600 D, about 500 D, about 400 D, about 300 D, about 200 D, or about 100 D. In some embodiments, a small molecule is less than about 2000 g/mol, less than about 1500 g/mol, less than about 1000 g/mol, less than about 800 g/mol, or less than about 500 g/mol. In some embodiments, small molecules are non-polymeric. In some embodiments, in accordance with the present invention, small molecules are not proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, polysaccharides, glycoproteins, proteoglycans, etc.

[0058] Specific binding: As used herein, the term refers to an interaction between a target polypeptide (or, more generally, a target molecule) and a binding agent such as provided antibodies. The interaction is typically dependent upon the presence of a particular structural feature of the target molecule such as an antigenic determinant or epitope recognized by the binding molecule. For example, if an antibody is specific for epitope A, the presence of a polypeptide containing epitope A or the presence of free unlabeled A in a reaction containing both free labeled A and the antibody thereto, will reduce the amount of labeled A that binds to the antibody. It is to be understood that specificity need not be absolute. For example, it is well known in the art that numerous antibodies cross-react with other epitopes in addition to those present in the target molecule. Such cross-reactivity may be acceptable depending upon the application for which the antibody is to be used. One of ordinary skill in the art will be able to select antibodies having a sufficient degree of specificity to perform appropriately in any given application (e.g., for detection of a target molecule, for therapeutic purposes, etc.). It is also to be understood that specificity may be evaluated in the context of additional factors such as the affinity of the binding molecule for the target molecule versus the affinity of the binding molecule for other targets (e.g., competitors). If a binding molecule exhibits a high affinity for a target molecule that it is desired to detect and low affinity for non-target molecules, the antibody will likely be an acceptable reagent for immunodiagnostic purposes. Once the specificity of a binding molecule is established in one or more contexts, it may be employed in other, preferably similar, contexts without necessarily re-evaluating its specificity.

[0059] Stage of cancer: As used herein, the term "stage of cancer" refers to a qualitative or quantitative assessment of the level of advancement of a cancer. Criteria used to determine the stage of a cancer include, but are not limited to, the size of the tumor and the extent of metastases (e.g., localized or distant).

[0060] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, go to completion and/or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0061] Substantial homology: The phrase "substantial homology" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be "substantially homologous" if they contain homologous residues in corresponding positions. Homologous residues may be identical residues. Alternatively, homologous residues may be non-identical residues will appropriately similar structural and/or functional characteristics. For example, as is well known by those of ordinary skill in the art, certain amino acids are typically classified as "hydrophobic" or "hydrophilic" amino acids., and/or as having "polar" or "non-polar" side chains Substitution of one amino acid for another of the same type may often be considered a "homologous" substitution. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al, Basic local alignment search tool, J. Mol. Biol, 215(3): 403-410, 1990; Altschul, et al, Methods in Enzymology; Altschul, et al., "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis, et al., Bioinformatics : A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al, (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying homologous sequences, the programs mentioned above typically provide an indication of the degree of homology. In some

embodiments, two sequences are considered to be substantially homologous if at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are homologous over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0062] Substantial identity: The phrase "substantial identity" is used herein to refer to a comparison between amino acid or nucleic acid sequences. As will be appreciated by those of ordinary skill in the art, two sequences are generally considered to be "substantially identical" if they contain identical residues in corresponding positions. As is well known in this art, amino acid or nucleic acid sequences may be compared using any of a variety of algorithms, including those available in commercial computer programs such as BLASTN for nucleotide sequences and BLASTP, gapped BLAST, and PSI-BLAST for amino acid sequences. Exemplary such programs are described in Altschul, et al., Basic local alignment search tool, J. Mol. Biol., 215(3): 403-410, 1990; Altschul, et al, Methods in Enzymology; Altschul et al, Nucleic Acids Res. 25:3389-3402, 1997; Baxevanis et al., Bioinformatics : A Practical Guide to the Analysis of Genes and Proteins, Wiley, 1998; and Misener, et al, (eds.), Bioinformatics Methods and Protocols (Methods in Molecular Biology, Vol. 132), Humana Press, 1999. In addition to identifying identical sequences, the programs mentioned above typically provide an indication of the degree of identity. In some embodiments, two sequences are considered to be substantially identical if at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more of their corresponding residues are identical over a relevant stretch of residues. In some embodiments, the relevant stretch is a complete sequence. In some embodiments, the relevant stretch is at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500 or more residues.

[0063] Suffering from: An individual who is "suffering from" a disease, disorder, or condition (e.g., GIST or prostate cancer) has been diagnosed with and/or exhibits one or more symptoms of the disease, disorder, or condition. Early-stage GIST and prostate tumors are frequently asymptomatic. In some embodiments, an individual who is suffering from GIST or prostate cancer has GIST or prostrate tumors, but does not display any symptoms of GIST or prostate cancer and/or has not been diagnosed with GIST or prostate cancer. In some embodiments, an individual who is suffering from GIST or prostate cancer is an individual who has upregulated ETV1 expression.

[0064] Susceptible to: An individual who is "susceptible to" a disease, disorder, or condition (e.g., GIST or prostate cancer) is at risk for developing the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition does not display any symptoms of the disease, disorder, or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition has not been diagnosed with the disease, disorder, and/or condition. In some embodiments, an individual who is susceptible to a disease, disorder, or condition is an individual who displays conditions associated with development of the disease, disorder, or condition (e.g., the individual has elevated levels of ETV1 expression). In some embodiments, a risk of developing a disease, disorder, and/or condition is a population-based risk (e.g., carrier of KIT exon 9 mutation, carrier of KIT exon 11 mutation, carrier of KIT exon 13 mutation; etc.). [0065] Symptoms are reduced: According to the present invention, "symptoms are reduced" when one or more symptoms of a particular disease, disorder or condition is reduced in magnitude (e.g., intensity, severity, etc.) or frequency. For purposes of clarity, a delay in the onset of a particular symptom is considered one form of reducing the frequency of that symptom. Many GIST patients with smaller tumors have no symptoms. Larger tumors can cause symptoms that are related to the increased mass being accommodated in the abdominal cavity. To give but a few examples, exemplary symptoms of GIST may include, but are not limited to, digestive discomfort, sensations of abdominal fullness, abdominal pain, visible enlargement of the abdomen, vomiting or diarrhea, bowel obstruction, perforation of the stomach or gut lining, black or tarry stools, vomiting of blood, anemia fatigue, and weight loss. It is not intended that the present invention be limited only to cases where the symptoms are eliminated. The present invention specifically contemplates treatment such that one or more symptoms is/are reduced (and the condition of the subject is thereby "improved"), albeit not completely eliminated.

[0066] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and/or elicits a desired biological and/or pharmacological effect, when administered to a subject.

[0067] Therapeutically effective amount: As used herein, the term "therapeutically effective amount" refers to an amount of a therapeutic protein (e.g., ETV1 antibody) which confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). In particular, the "therapeutically effective amount" refers to an amount of a therapeutic protein or composition effective to treat, ameliorate, or prevent a desired disease or condition, or to exhibit a detectable therapeutic or preventative effect, such as by ameliorating symptoms associated with the disease, preventing or delaying the onset of the disease, and/or also lessening the severity or frequency of symptoms of the disease. A therapeutically effective amount is commonly administered in a dosing regimen that may comprise multiple unit doses. For any particular therapeutic protein, a therapeutically effective amount (and/or an appropriate unit dose within an effective dosing regimen) may vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and/or unit dose) for any particular patient may depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and/or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts.

[0068] Treatment: As used herein, the term "treatment" (also "treat" or "treating") refers to any administration of a substance (e.g., provided compositions) that partially or completely alleviates, ameliorates, relives, inhibits, delays onset of, reduces severity of, and/or reduces incidence of one or more symptoms, features, and/or causes of a particular disease, disorder, and/or condition (e.g., GIST or prostate cancer). Such treatment may be of a subject who does not exhibit signs of the relevant disease, disorder and/or condition and/or of a subject who exhibits only early signs of the disease, disorder, and/or condition. Alternatively or additionally, such treatment may be of a subject who exhibits one or more established signs of the relevant disease, disorder and/or condition. In some embodiments, treatment may be of a subject who has been diagnosed as suffering from the relevant disease, disorder, and/or condition. In some embodiments, treatment may be of a subject known to have one or more susceptibility factors that are statistically correlated with increased risk of development of the relevant disease, disorder, and/or condition.

[0069] Sample: The term "sample" as used herein is taken broadly to include cell or tissue samples removed from a patient (e.g., a sample of gastrointestinal stromal tissue). The term may include cells (or their progeny) derived from a tumor that may be located elsewhere in the body (e.g., cells in the bloodstream or at a site of metastasis), or any material derived by processing such a sample. Derived samples may include nucleic acids or proteins extracted from the sample or obtained by subjecting the sample to techniques such as amplification or reverse transcription of m NA, etc.

[0070] Tumor subclass: A tumor subclass is the group of tumors that display one or more phenotypic or genotypic characteristics that distinguish members of the group from other tumors. [0071] Vector: As used herein, "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is associated. In some embodiment, vectors are capable of extra-chromosomal replication and/or expression of nucleic acids to which they are linked in a host cell such as a eukaryotic and/or prokaryotic cell. Vectors capable of directing the expression of operatively linked genes are referred to herein as "expression vectors."

Brief Description of the Drawing

[0072] The Figures described below, that together make up the Drawing, are for illustration purposes only, not for limitation.

[0073] FIG. 1 depicts an exemplary Western Blot result illustrating binding of provided monoclonal antibodies to ETVl in a G882 cell line with and without imatinib (IMAT) added. 400ug of protein were loaded per lane in an 11% SDS-polyacrylamide gel. The secondary antibody used was Jackson Catalog #: 115-035-003 in a 1 :5000 dilution. Primary ETVl monoclonal antibodies were loaded as follows - 10D12C5B4D4 (lane 1), 2F11G11D10D11 (lane 2), 14D1F8C4E6 (lane 3), 8E6B7C9D10 (lane 4), 13A7E12G5A5 (lane 5),

2F11G11D10B4 (lane 6), 13A7E12G5B8 (lane 7). Lane 8 was loaded with serum in a 1 :500 dilution.

[0074] FIG. 2 depicts an exemplary result illustrating ETVl is universally highly expressed and required for tumor growth and survival in GIST. FIG. 2A depicts an exemplary result illustrating a Venn diagram of GIST-signature genes from three datasets. FIG. 2B depicts an exemplary result illustrating expression of ETVl in multiple tumor types from the ExpO dataset. Box, 25-75 percentile; error bar, 10-90 percentile; dots, outliers. FIG. 2C depicts an exemplary result illustrating ETVl and KIT mRNA levels by qRT-PCR of GIST and non-GIST samples, whose details are described in Full Methods. Mean±SD, n=3. FIG. 2D depicts an exemplary result illustrating immunoblotting of selected tumor tissues and cell lines from FIG. 2C. FIG. 2E depicts an exemplary result illustrating growth curves of GIST and U20S cells after shRNA-mediated ETVl suppression compared to control. Mean±SEM, n=3. FIG. 2F depicts an exemplary result illustrating tumor volume over time in SCID mice implanted with GIST882 cells after shRNA-mediated ETVl suppression compared to scrambled shRNA controls. Mean±SEM, * p<0.05; n=7, 10, 8 for scrambled, ETVlshl, and ETVlsh2 respectively. FIG. 2G depicts an exemplary result illustrating ETV1 mR A levels of preimplanted GIST882 cells and explanted xenografts at week 10. Mean±SD.

[0075] FIG. 3 depicts an exemplary result illustrating ETV1 is expressed in the subtypes of ICCs susceptible to oncogenesis and is required for their development. FIG. 3 A depicts an exemplary result illustrating a schematic showing localization of ICC-MY (yellow arrowheads), ICC-IM (yellow arrows) and ICC-SMP (white arrowheads) in the large intestine. M: mucosa, CM: circular muscle, LM: longitudinal muscle. All three ICC subtypes express Kit (red). FIG. 3B depicts an exemplary result illustrating co-immunofluorescence (divided into two microscopy fields) of Kit (red), Etvl (green) and DAPI (blue) of the large intestine of wild-type mice. FIG. 3C depicts an exemplary result illustrating co-immunofluorescence of Kit (red), Pgp9.5 (green), and DAPI (blue) of the large intestine of Etvl+/+ and Etvl-/- mice. FIG. 3D depicts an exemplary result illustrating representative deconvoluted whole-mount Kit-immunofluorescence images of the large intestine of Etvl+/+ and Etvl-/- mice. A single microscopy field focused to the ICC-MY and ICC-SMP planes are shown. Scale bar, 20 μιη.

[0076] FIG. 4 depicts an exemplary result illustrating ETV1 regulates GIST-signature genes predominantly through enhancer binding. FIG. 4A depicts an exemplary result illustrating a ranked list of ETV1 regulated genes generated based on the average fold-change by the two ETV1 hairpins in two cell lines. FIG. 4B depicts an exemplary result illustrating a heatmap of expression of the 48 genes with average downregulation > 1.7-fold. For each gene, table shows p- value of GIST vs. other tumor types from the ExpO dataset, calculated by Oncomine™ (NS: p>0.05), and the presence of ETV1 binding sites from ChlP-Seq analysis. FIG. 4C depicts an exemplary result illustrating GSEA plots of the shETVl ranked list using three gene sets: GIST signature genes from ExpO dataset, ICC-MY and ICC-DMP signature genes in mouse small intestine. ES, enrichment score; FDR, false discovery rate. FIG. 4D depicts an exemplary result illustrating pie charts of genomic structure and distribution of ETV1 ChlP-Seq peaks. TSS, transcription start site; TES, transcription end site. FIG. 4E depicts an exemplary result illustrating representative ChlP-Seq reads in top ETV1 target genes. FIG. 4F depicts an exemplary result illustrating the consensus sequence motif identified in the ETV1 binding sites by the MEME program. FIG. 4G depicts an exemplary result illustrating pie chart of genes with ETVl binding sites divided into promoter only, enhancer only and both. FIG. 4H depicts an exemplary result illustrating plot of percent of all genes, genes averagely downregulated 1.4-fold by shETVl (n=410), and genes averagely upregulated 1.4-fold by shETVl (n=380) with promoter only, enhancer only and both promoter and enhancer ETVl binding. Fold enrichment over all genes is shown above the plots.

[0077] FIG. 5 depicts an exemplary result illustrating KIT signalling synergizes with

ETVl in GIST tumorigenesis by stabilization of ETVl protein. FIG. 5 A depicts an exemplary result illustrating immunoblots of GIST882 cells treated with the imatinib (1 μΜ) and PD325901 (100 nM) for the indicated time points. FIG. 5B depicts an exemplary result illustrating immunoblots of GIST882 cells treated for 2 hours with imatinib or PD325901 in combination with cyclohexamide (10 μg/ml) or MG132 (10 μΜ). FIG. 5C depicts an exemplary result illustrating a Venn diagram of genes downregulated by 1.4-fold by shETVl and by imatinib in GIST882 cells. P-value: Fisher's exact test based on number of expressed genes. FIG. 5D depicts an exemplary result illustrating percent of all genes, imatinib-downregulated genes, shETVl -downregulated genes, and overlapping genes with ETVl enhancer peaks. FIG. 5E depicts an exemplary result illustrating an immunoblot of NIH3T3 cells expressing ETVl and either KITwt or ΚΙΤΔ560 two hours after treatment with PD325901, imatinib, or MG132. FIG. 5F depicts an exemplary result illustrating growth of xenografts of engineered NIH3T3 cells stabilizing the indicated genes (n=12, Mean ±SEM). FIG. 5G depicts an exemplary result illustrating a photograph of 4 representative explanted xenografts at 4 weeks after implanting. Scale bar 1 cm. FIG. 5H depicts an exemplary result illustrating a model of the role of ETVl in ICC maintenance and GIST oncogenesis. Normal level of KIT activation by KIT ligand (red triangle) stabilizes ETVl transcription factor through the MAPK pathway, and results in physiological ETVl transcriptional output critical for ICC development (middle). In the absence of ETVl, there is decreased ICC development, which phenocopies genetic loss of KIT signalling (left). Activating mutation of KIT (e.g. ΚΙΤΔ560) leads to constitutive activation of the KIT- MAPK signalling pathway, increased stabilization and augmented ETVl transcriptional output that promotes tumorigenesis (right).

[0078] FIG. 6 depicts an exemplary result illustrating ETVl expression levels from GIST containing microarray expression datasets. FIG. 6A depicts an exemplary result illustrating expression of ETV1 (37156_at, Affymetrix U95A-Av2) of individual tumor samples of 9 sarcoma types from Segal dataset. FIG. 6B depicts an exemplary result illustrating expression of ETV1 (IMAGE:81320, Stanford spotted platform) of individual tumor samples of 6 sarcoma types from Nielsen dataset. FIG. 6C depicts an exemplary result illustrating expression of ETV1 (22191 l at, Affymetrix HG_U133_Plus_2) of 32 GIST samples from Yamaguchi dataset annotated by location. FIG. 6D depicts an exemplary result illustrating expression of ETV1 (22191 l at, Affymetrix HG_U133_Plus_2) of 29 GIST samples from Ostrowski dataset annotated by TKI mutation status.

[0079] FIG. 7 depicts an exemplary result illustrating ETV1 immunofluorescence in four

GIST (GIST 1-4) samples and a leiomyosarcoma (LMS) control sample. Paraffin embedded human GIST samples were stained with ETVl-specific primary antibody and Alexa-594 anti- rabbit secondary antibody. Alexa 594: left column; DAPI: middle column; Alexa/DAPI merge: right column. Scale bar = 20 um.

[0080] FIG. 8 depicts an exemplary result illustrating effect of shETVl on ETV1 protein and mRNA levels and cell cycle profile. FIG. 8 A depicts an exemplary result illustrating ETV1 mRNA and protein levels in GIST48, GIST882, and U20S cells 4-days after shRNA-mediated ETV1 knockdown. FIG. 8B depicts an exemplary result illustrating cell-cycle analysis of GIST48 and GIST882 cells 5 days after infection with shETVl viruses containing scrambled or the ETVl-specific shRNAs. Cells were infected at MOI=5, fixed 5 days after infection and stained with propidium iodide.

[0081] FIG. 9 depicts an exemplary result illustrating an assessment for ETV1 translocation and amplification in GIST. FIG. 9A-B depicts an exemplary result illustrating representative FISH of the ETV1 locus in two GIST clinicial specimens. Green are two probes in the gene body and downstream of ETV1 and red are two probes upstream of ETV1. There's no evidence of "breakaway" or amplification of ETV1. FIG. 9C depicts an exemplary result illustrating qRT-PCR of same cDNA as in FIG. 2C, which used primers targeting ETV1 exons 6 and 7. THis qRT-PCR used primers targeting exons 2 and 3. UP arrows indicate regions of translocation in prostate cancers. Comparing to FIG. 2C, there is no evidence that early exons before translocation hotspots are significantly under-expressed compared to exons 6 and 7.

Mean i SD, n=3.

[0082] FIG. 10 depicts an exemplary result illustrating expression of ETV1 in the murine small intestine. FIG. 10A depicts an exemplary result illustrating the myenteric ICC network (ICC-MY, yellow arrowheads) is localized adjacent to the myenteric plexus (MP) between the circular (CM) and longitudinal (LM) muscle layers. The deep muscle plexus ICC network (ICC- DMP, white arrowheads) is localized just below the mucosa (M) in the circular muscle layer. FIG. 10B depicts an exemplary result illustrating both ICC subsets express Kit though ICC-DMP stains much more dimly. Co-immunofluorescence of Kit (red), Etvl (green), and DAPI (blue) shows that the ICC-MY, but not ICC-DMP express Etvl . FIG. IOC depicts an exemplary result illustrating Kit and Etvl mR A expression level from expression profiles ICC-MY (n=3), ICC- DMP (n=3), and muscle (n=2) isolated from mouse small intestine (GSE7809, Mean ± SD).

[0083] FIG. 11 depicts an exemplary result illustrating Etvl is required for ICC-MY but not ICC-DMP development of the murine small intestine. FIG. 11 A depicts an exemplary result illustrating co-immunofluorescence of Kit (red), Pgp9.5 (green), and DAPI (blue) of the small intestine of Etvl+/+ and Etvl-/- mice showing significant loss of ICC-MY (yellow arrowheads) and preservation of ICC-DMP (white arrowheads). FIG. 1 IB depicts an exemplary result illustrating whole mount Kit immunofluorescence taken at the ICC-MY plane and ICC-DMP plane, also showing loss of ICC-MYs and preservation of ICC-DMPs. Scale bar 20uM.

[0084] FIG. 12 depicts an exemplary result illustrating Etvl is required for ICC-IM development of the murine gastric fundus. FIG. 12A depicts an exemplary schematic showing ICC-IMs present in both the circular and longitudinal muscle layers. FIG. 12B depicts an exemplary result illustrating co-immunofluorescence of Kit (red), Pgp9.5 (green), and DAPI (blue) of the gastric fundus of Etvl+/+ and Etvl-/- mice showing significant loss of ICC-IM. FIG. 12C depicts an exemplary result illustrating whole mount Kit immunofluorescence taken at the circular muscle ICC-IM and longitudinal muscle ICC-IM planes, also showing loss of ICC- IMs. Scale bar 20 uM.

[0085] FIG. 13 depicts an exemplary result illustrating Etvl is required for ICC-MY development of the murine cecum. FIG. 13A depicts an exemplary schematic of ICC types of the cecum. Compared to the rest of the large intestine, ICC-IM and ICC-SMP is very sparse (see FIG. 3). FIG. 13B depicts an exemplary result illustrating co-immunofluorescence of Kit (red), Pgp9.5 (green), and DAPI (blue) of the cecum of Etvl+/+ and Etvl-/- mice showing significant loss of ICC-MY. FIG. 13C depicts an exemplary result illustrating whle mount Kit

immunofluorescence taken at the ICC-MY plane also showing loss of ICC-MY. Scale bar 20 uM.

[0086] FIG. 14 depicts an exemplary result illustrating quantification of ICC subclasses remaining in Etvl-/- mice expressed as precent of Etvl-/- mice. To quantify ICC subsets, immunofluorescence images from at least four fields each from two Etvl+/+ and Etvl-/- mice (at least eight fields total) of the stomach, small intestine, cecum, and large intestine were examined. A percentage of ICCs were obtained by dividing the number of KIT-positive immunostaining cells with the number of DAPI-positive nuclei. Plotted is the ratio of percent of ICC 's in Etvl-/- mice compared to Etvl+/+ mice (Mean ± SD).

[0087] FIG. 15 depicts an exemplary result illustrating Kit and Anol

immunofluorescence in GI tract of Etvl+/+ and Etvl-/- mice. Kit and Anol label all ICC subsets with similar intensity except in ICC-DMP of the small intestine where Kit staining is faint and Anol is stronger (FIG. 15B, white arrows). FIG. 15A depicts an exemplary result illustrating global loss of ICC-MY and ICC-IM in stomach of Etvl-/- mice. FIG. 15B depicts an exemplary result illustrating global loss of ICC-MY and ICC-IM in small intestine of Etvl-/- mice. FIG. 15C depicts an exemplary result illustrating global loss of ICC-MY and ICC-IM in large intestine of Etvl-/- mice. ICC-DMP (FIG. 15B) are preserved in the small intestine, and ICC-SMP (FIG. 15C) are preserved in the colon (white arrows). Scale bar, 20 um.

[0088] FIG. 16 depicts an exemplary result illustrating whole mont Pgp9.5

immunofluorescence of small and large intestine to examine the neuronal myenteric plexus in Etvl+/+ and Etvl-/- mice. Whole mount Z-stack of Pgp9.6 staining of the small and large intestine was performed and an exemplary Z-section of the myenteric plexus is shown. Scale bar 20 uM.

[0089] FIG. 17 depicts an exemplary result illustrating overlap of expressed probes (total

38985) perturbed 1.7-fold by ETV1 shR A in GIST48 and GIST 882 cells. FIG. 17A and FIG. 17B depict an exemplary result illustrating a Venn diagram of probes downregulated by ETVshl (blue), downregulated by ETVlsh2 (orange), upregulated by ETVlshl (red) and upregulated by ETVlsh2 (green), all 1.7-fold compared to scrambled shR A. FIG. 17C depicts an exemplary result illustrating a Venn diagram of probes downregulated by either ETVlshl or ETVlsh2 in GIST48 (blue) and GIST882 (orange). FIG. 17D depicts an exemplary result illustrating a Venn diagram of probes upregulated by either ETVlshl or ETVlsh2 in GIST28 (green) and GIST882 (red). P-values for significance of overlap were calculated using Fisher Exact Test.

[0090] FIG. 18 depicts an exemplary result illustrating qRT-PCR validation of top downregulated genes by ETV1 knockdown. GIST48 and GIST882 cells were infected with shScr, ETVlshl, and ETVlsh2 lentiviruses in an independent experiment from the expression profiling experiment. RNA was harvested 4 days after lentiviral infection without drug selection and qRT-PCR performed. Data represents 2-AACt using RPL27 as housekeeping gene control and shScr as normalization sample. Mean ± SD, n=3.

[0091] FIG. 19 depicts an exemplary result illustrating GSEA enrichment of GIST- signature datasets. FIG. 19A depicts an exemplary result illustrating enrichment plots of Segal GIST-signature gene sets on the "shETVl" ranked list. FIG. 19B depicts an exemplary result illustrating enrichment plots of Nielsen GIST-signature gene sets on the "shETVl" ranked list.

[0092] FIG. 20 depicts an exemplary result illustrating qPCR ChIP validation of ETV1 binding to DUSP6 and GPR20 locus in GIST48 and GIST882 cells (Mean ± SD, n=3).

[0093] FIG. 21 depicts an exemplary result illustrating mRNA level of ETV1 by qRT-

PCT in GIST882 cells after imatinib treatment for the indicated time points. Samples normalized to endogenous transcript (Mean ± SD, n=3).

[0094] FIG. 22 depicts an exemplary result illustrating soft agar colony formation assays of NIH3T3 cells transduced with different permutations of ETV1 and KIT. NIH3T3 cells transduced with EGFP or ETV1 expression virus were subsequently transduced with different combinations of empty vector, wild-type KIT, or activating mutant KIT (Δ560). 5,000 cells/well of 6-well plate were seeded in triplicate of each transduced line. FIG. 22A depicts an exemplary result illustrating photographs of representative wells of soft agar colony formation 3 weeks after plating 5,000 cells/well. Scale bar 2mm. FIG. 22B depicts an exemplary result illustrating colongy quantification (Mean ± SD, n=3).

Detailed Description of Certain Embodiments

[0095] As described herein, the present invention provides useful anti-ETVl antibodies with particular structural and/or functional characteristics, as well as compositions and methods relating to such antibodies.

ETVl

[0096] ETVl is a transcription factor belonging to the large ETS (E-twenty six) family and PEA3 subfamily. ETS family members are defined by a highly conserved DNA binding domain, the ETS domain, which is a winged helix-turn-helix structure that binds to DNA sites with a central GGA DNA sequence. Multiple ETS factors have been found to be associated with cancer. In a condition called Ewing's sarcoma, the ERG ETS transcription factor is fused to the EWS gene (Ida, K., et al, 1995, Int. J. Cancer, 63(4):500-4; incorporated herein by reference). The fusion of TEL to the JAK2 protein results in early pre-B acute lymphoid leukemia (Peeters, P., et al, 1997, Blood, 90(7):2535-40; incorporated herein by reference) and ERG and ETVl are ETS family members which are over-expressed, and whose gene fusions are found, in prostate cancer (Tomlins, S.A., et al, 2005, Science, 310(5748):644-8; incorporated herein by reference).

[0097] As described herein, ETVl (SEQ ID NO: 1) is universially highly expressed in gastroinstestinal stromal tumors (GISTs) and is required for growth of imatinib-sensitive and resistant GIST cell lines. Activated KIT, through MEK, prolongs ETVl protein stability and cooperates with ETVl to promote tumorigenesis GIST arises from interstitial cells of Cajal (ICCs) exhibiting high levels of endogenous ETVl expression that, when coupled with an activating KIT mutation, drives an oncogenic ETS transcriptional program. This oncogenic role for ETVl in GIST differs from classical models of other ETS-dependent tumors such as prostate cancer, melanoma and Ewing sarcoma where genomic translocation or amplification drives aberrant ETS expression and promote tumorigenesis (Tomlins, S.A., et al, 2005, Science, 310(5748):644-8; Mertens, F. et al, 2009, Semin. Oncol, 26, 312-23; Jane-Valbuena, J. et al, 2010, Cancer Res., (70), 2075-84; each of which is incorporated herein by reference). The fact that ETVl is universally highly expressed in all GISTs makes it immediately useful as a candidate diagnostic biomarker, because the current standard of KIT immunreactivity is negative in about 5% of all GISTs (Miettinen, M. et al, 2006, Arch. Pathol. Lab. Med., 130: 1466-78; incorporated herein by reference).

Table 2: Human ETVl

MDGFYDQQVPYMVTNSQRGR CNEKPTNVRKRKFINRD

LAHDSEELFQDLSQLQETWLAEAQVPDNDEQFVPDYQA

ESLAFHGLPLKIKKEPHSPCSEISSACSQEQPFKFSYGEKC

LYNVSAYDQKPQVGMRPSNPPTPSSTPVSPLHHASPNST

HTPKPDRAFPAHLPPSQSIPDSSYPMDHRFRRQLSEPCNSF

PPLPTMPREGRPMYQRQMSEPNIPFPPQGFKQEYHDPVY

EHNTMVGSAASQSFPPPLMIKQEPRDFAYDSEVPSCHSIY

MRQEGFLAHPSRTEGCMFEKGPRQFYDDTCVVPEKFDG

DIKQEPGMYREGPTYQRRGSLQLWQFLVALLDDPSNSHF

IAWTGRGMEFKLIEPEEVARRWGIQKNRPAMNYDKLSRS

LRYYYEKGIMQKVAGERYVYKFVCDPEALFSMAFPDNQ

RPLLKTDMERHINEEDT VPL SHFDE SM AYMPEGGC CNPH

PYNEGYVY (SEQ ID NO: 1)

Polyclonal ETVl Antibodies

[0098] There currently exists a need for effective human monoclonal ETVl antibodies for use in diagnostic and therapeutic applications. Polyclonal ETVl antibodies (e.g., rabbit polyclonal antibody commercially available as ab81806 from Abeam of Cambridge, MA and PRB-362C from Covance of Princeton, NJ) show reasonable ETVl specificity on Western Blots (with multiple nonspecific bands) and limited utility (dirty) for immunohistochemistry (IHC). In addition to performing poorly in IHC tests, polyclonal antibodies have limited usefulness in human therapeutics and diagnostics. The usefulness of monoclonal antibodies stems from their specificity of binding, their homogeneity, and their ability to be produced in unlimited quantities from cultured hybridoma. Provided ETV1 Monoclonal Antibodies

[0099] As described herein, the present invention provides monoclonal ETV1 antibodies that show certain structural (i.e., sequence) relationship with described reference antibodies and/or have particular functional attributes, including for example certain improved functional attributes as compared with described reference antibodies.

[0100] In some embodiments, the present invention provides antibodies whose amino acid sequence, show specified levels of homology and/or identity with provided reference antibodies. In some embodiments, the present invention provides antibodies whose amino acid sequence, show specified levels of homology and/or identity with Mab 10D12C5B4D4, Mab 2F11G11D10D11, Mab 14D1F8C4E6, Mab 8E6B7C9D10, Mab 13A7E12G5A5, Mab

2F11G11D10B4, and/or Mab 13A7E12G5B8. In some embodiments provided antibodies show at least 80%, at least 90%>, at least 95%, at least 99% identity with reference antibodies deposited in the American Type Culture Collection (ATCC) as set forth in Table 1.

[0101] In some embodiments, the present invention includes a cell line expressing one or more provided antibodies described herein, whose amino acid sequence show specified levels of homology and/or identity with provided reference antibodies. In some embodiments a cell line will express provided antibodies that show at least 80%>, at least 90%>, at least 95%, at least 99% identity with reference antibodies deposited in the American Type Culture Collection (ATCC) as set forth in Table 1.

[0102] In some embodiments, the present invention provides antibodies that specificially bind to an epitope of ETV1 recognized by a provided reference antibody.

[0103] In some embodiments, the present invention provides antibodies or fragments therof that bind the peptide sequence CNPHPYNEQYVY (SEQ ID NO: 31). In some embodiments, the present invention provides antibodies or fragments thereof that bind a peptide sequence that shares at least 90%, at least 95%, at least 99% identity with the peptide sequence CNPHPYNEQYVY (SEQ ID NO: 31).

[0104] In some embodiments, the present invention provides antibodies or fragments thereof that bind the peptide sequence of SEQ ID NO: 1. In some embodiments, the present invention provides antibodies or fragments thereof that bind a peptide sequence that shares at least 80%, at least 90%, at least 95%, at least 99% identity with SEQ ID NO: 1.

[0105] In some embodiments, the present invention includes a pharmaceutical composition comprising one or more of provided antibodies or fragments thereof as described herein.

[0106] In some embodiments, provided antibodies have one or more CDRs and/or one or more FRs that are identical in sequence to a corresponding CDR or FR of reference antibodies deposited in the American Type Culture Collection (ATCC) as set forth in Table 1. In some embodiments, provided antibodies have one or more CDRs and/or FRs showing a specified degree of homology and/or identity with the corresponding CDRs ad/or FRs of provided reference antibodies as discussed below. In some embodiments, all CDRs and FRs of a provided antibody show at least the specified level of homology and/or identity. In some embodiments, a provided antibody has CDR and FR sequences that together contain no more than 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 substitutions as compared with the provided reference antibodies.

[0107] In some embodiments, a provided VH complementarity determining region 1

(CDR1) of the present invention shows at least 80%>, at least 85%, at least 90%, at least 95%, or at least 99% identity with the VH CDR1 region of a reference antibody. In some embodiments, a provided CDR1 has an amino acid sequence that is identical to that of a reference antibody and/or does not contain any amino acid substitutions as compared with the CDR1 sequence of a reference antibody deposited in the American Type Culture Collection (ATCC) as set forth in Table 1. In some embodiments, a provided CDR1 has one or more amino acid substitutions as compared to the CDR1 sequence of a provided reference antibody. In some embodiments, a provided CDR1 will have two or more amino acid substitutions as compared to the CDR1 sequence of a provided reference antibody. In some embodiments, a provided CDR1 has 1, 2, 3, 4 or 5 substitutions and in some embodiments 1, 2, or 3 substitutions as compared with the CDR1 sequence of a provided reference antibody.

[0108] In some embodiments, a provided VH complementarity determining region 2

(CDR2) of the present invention shows at least 80%, at least 85%, at least 90%, at least 95%, or at least 99% identity with the VH CDR2 region of a reference antibody. In some embodiments, a provided CDR2 has an amino acid sequence that is identical to that of a reference antibody and/or does not contain any amino acid substitutions as compared with the CDR2 sequence of a reference antibody deposited in the American Type Culture Collection (ATCC) as set forth in Table 1. In some embodiments, a provided CDR2 has one or more amino acid substitutions as compared to the CDR2 sequence of a provided reference antibody. In some embodiments, a provided CDR2 will have two or more amino acid substitutions as compared to the CDR2 sequence of a provided reference antibody. In some embodiments, a provided CDR2 has 1, 2, 3, 4 or 5 substitutions and in some embodiments 1, 2, or 3 substitutions as compared with the CDR2 sequence of a provided reference antibody.

[0109] In some embodiments, a provided VH complementarity determining region 3

(CDR3) of the present invention shows at least 80%>, at least 85%, at least 90%>, at least 95%, or at least 99% identity with the VH CDR3 region of a reference antibody. In some embodiments, a provided CDR3 has an amino acid sequence that is identical to that of a reference antibody and/or does not contain any amino acid substitutions as compared with the CDR3 sequence of a reference antibody deposited in the American Type Culture Collection (ATCC) as set forth in Table 1. In some embodiments, a provided CDR3 has one or more amino acid substitutions as compared to the CDR3 sequence of a provided reference antibody. In some embodiments, a provided CDR3 will have two or more amino acid substitutions as compared to the CDR3 sequence of a provided reference antibody. In some embodiments, a provided CDR3 has 1, 2, 3, 4 or 5 substitutions and in some embodiments 1, 2, or 3 substitutions as compared with the CDR3 sequence of a provided reference antibody.

[0110] In some embodiments, a provided VH framework region 1 (FR1) shows at least

80%, at least 85%, at least 90%, at least 95%, or at least 99% percent identity with the VH FR1 region of a reference antibody deposited in the American Type Culture Collection (ATCC) as set forth in Table 1. In some embodiments, the FR1 has a sequence that does not have any amino acid substitutions as compared to a provided reference antibody; in some particular

embodiments, the FR1 has an amino acid sequence identical to that of a provided reference antibody. In some embodiments, the FR1 has a sequence including one or more amino acid substitutions as compared to the FR1 sequence of a provided reference antibody. In some embodiments, the FR1 has a sequence including two or more amino acid substitutions as compared to the FR1 sequence of a provided reference antibody.

[0111] In some embodiments, a provided VH framework region 2 (FR2) shows at least

80%, at least 85%, at least 90%, at least 95%, or at least 99% percent identity with the VH FR2 region of a reference antibody deposited in the American Type Culture Collection (ATCC) as set forth in Table 1. In some embodiments, the FR2 has a sequence that does not have any amino acid substitutions as compared to a provided reference antibody; in some particular

embodiments, the FR2 has an amino acid sequence identical to that of a provided reference antibody. In some embodiments, the FR2 has a sequence including one or more amino acid substitutions as compared to the FR2 sequence of a provided reference antibody. In some embodiments, the FR2 has a sequence including two or more amino acid substitutions as compared to the FR2 sequence of a provided reference antibody.

[0112] In some embodiments, a provided VH framework region 3 (FR3) shows at least

80%, at least 85%, at least 90%, at least 95%, or at least 99% percent identity with the VH FR3 region of a reference antibody deposited in the American Type Culture Collection (ATCC) as set forth in Table 1. In some embodiments, the FR3 has a sequence that does not have any amino acid substitutions as compared to a provided reference antibody; in some particular

embodiments, the FR3 has an amino acid sequence identical to that of a provided reference antibody. In some embodiments, the FR3 has a sequence including one or more amino acid substitutions as compared to the FR3 sequence of a provided reference antibody. In some embodiments, the FR3 has a sequence including two or more amino acid substitutions as compared to the FR3 sequence of a provided reference antibody.

[0113] In some embodiments, a provided VH framework region 4 (FR4) shows at least

80%, at least 85%, at least 90%, at least 95%, or at least 99% percent identity with the VH FR4 region of a reference antibody deposited in the American Type Culture Collection (ATCC) as set forth in Table 1. In some embodiments, the FR4 has a sequence that does not have any amino acid substitutions as compared to a provided reference antibody; in some particular

embodiments, the FR4 has an amino acid sequence identical to that of a provided reference antibody. In some embodiments, the FR4 has a sequence including one or more amino acid substitutions as compared to the FR4 sequence of a provided reference antibody. In some embodiments, the FR4 has a sequence including two or more amino acid substitutions as compared to the FR4 sequence of a provided reference antibody.

[0114] In some embodiments, a provided antibody comprises at least two of a VH CDR1,

CDR2 and/or CDR3 as defined above. In some embodiments, a provided antibody comprises all three of a VH CDR1, CDR2 and CDR3 as defined above.

[0115] In some embodiments, a provided antibody comprises at least two of a VH FR1,

FR2, FR3 and/or FR4 as defined above. In some embodiments, a provided antibody comprises at least three of a VH FR1, FR2, FR3 and/or FR4 as defined above. In some embodiments, a provided antibody comprises all four of a VH FR1, FR2, FR3 and FR4 as defined above.

[0116] In some embodiments, a provided antibody comprises at least one of a VH CDR1,

CDR2 and/or CDR3 as defined above and at least one of a VH FR1, FR2, FR3 and/or FR4 as defined above. In some embodiments, a provided antibody comprises at least two of a VH CDR1, CDR2 and/or CDR3 as defined above and at least two of a VH FR1, FR2, FR3 and/or FR4 as defined above. In some embodiments, a provided antibody comprises all three of a VH CDR1, CDR2 and CDR3 as defined above and all four of a VH FR1, FR2, FR3 and FR4 as defined above.

[0117] In some embodiments, a provided VL CDR1, CDR2 and/or CDR3 is defined in the same way as the provided VH CDRs except that the definition is made with reference to the VL CDRs of the reference antibody.

[0118] In some embodiments, a provided VL FR1, FR2, FR3 and/or FR4 is defined in the same way as the provided VH FRs except that the definition is made with reference to the VL FRs of the reference antibody.

[0119] In some embodiments, a provided antibody comprises at least two of a VL CDR1,

CDR2 and/or CDR3 as defined above. In some embodiments, a provided antibody comprises all three of a VL CDR1, CDR2 and CDR3 as defined above. [0120] In some embodiments, a provided antibody comprises at least two of a VL FRl,

FR2, FR3 and/or FR4 as defined above. In some embodiments, a provided antibody comprises at least three of a VL FRl, FR2, FR3 and/or FR4 as defined above. In some embodiments, a provided antibody comprises all four of a VL FRl, FR2, FR3 and FR4 as defined above.

[0121] In some embodiments, a provided antibody comprises at least one of a VL CDRl,

CDR2 and/or CDR3 as defined above and at least one of a VL FRl, FR2, FR3 and/or FR4 as defined above. In some embodiments, a provided antibody comprises at least two of a VL CDRl, CDR2 and/or CDR3 as defined above and at least two of a VL FRl, FR2, FR3 and/or FR4 as defined above. In some embodiments, a provided antibody comprises all three of a VL CDRl, CDR2 and CDR3 as defined above and all four of a VL FRl, FR2, FR3 and FR4 as defined above.

[0122] In some embodiments, a provided antibody comprises at least 1, 2 or 3 of a VH

CDRl, CDR2 and/or CDR3 as defined above and at least 1, 2 or 3 of a VL CDRl, CDR2 and/or CDR3 as defined above. In some embodiments, a provided antibody comprises all three of a VH CDRl, CDR2 and CDR3 as defined above and all three of a VL CDRl, CDR2 and CDR3 as defined above.

[0123] In some embodiments, a provided antibody comprises at least 1, 2 or 3 of a VH

CDRl, CDR2 and/or CDR3 as defined above; at least 1, 2, 3 or 4 of a VH FRl, FR2, FR3 and/or FR4 as defined above; at least 1, 2 or 3 of a VL CDRl, CDR2 and/or CDR3 as defined above; and at least 1, 2, 3 or 4 of a VL FRl, FR2, FR3 and/or FR4 as defined above. In some embodiments, a provided antibody comprises all three of a VH CDRl, CDR2 and CDR3 as defined above; all four of a VH FRl, FR2, FR3 and FR4 as defined above; all three of a VL CDRl, CDR2 and CDR3 as defined above; and all four of a VL FRl, FR2, FR3 and FR4 as defined above.

[0124] In some embodiments, the present invention as described herein provides at least

1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90- fold or greater affinity for binding purified ETV1 than Abeam rabbit polyclonal (ab81806) as measured by Kd. [0125] In some embodiments, the present invention as described herein provides at least

1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90- fold or greater affinity for binding purified ETV1 than Abeam rabbit polyclonal (ab81806) as measured by quantitative Western Blotting.

[0126] In some embodiments, the present invention as described herein provides at least

1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90- fold or greater affinity for binding purified ETV1 than Covance rabbit polyclonal (PRB-362C) as measured by Kd.

[0127] In some embodiments, the present invention as described herein provides at least

1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90- fold or greater affinity for binding purified ETV1 than Covance rabbit polyclonal (PRB-362C) as measured by quantitative Western Blotting.

Humanized and Veneered Antibodies

[0128] As noted above, in certain embodiements when using a provided antibody for therapeutic purposes it may prove advantageous to use a humanized or veneered antibody to reduce any potential immunogenic reaction. In general, humanized or veneered antibodies minimize unwanted immunological responses that limit the duration and effectiveness of therapeutic applications of non-human antibodies in human recipients.

[0129] A number of methods for preparing humanized antibodies comprising an antigen binding portion derived from a non-human antibody have been described in the art. In particular, antibodies with rodent variable regions and their associated complementarity-determining regions (CDRs) fused to human constant domains have been described (e.g., see Winter et al, Nature 349:293, 1991; Lobuglio et al, Proc. Nat. Acad. Sci. USA 86:4220, 1989; Shaw et al, J. Immunol. 138:4534, 1987; and Brown et al, Cancer Res. 47:3577, 1987). Rodent CDRs grafted into a human supporting framework region (FR) prior to fusion with an appropriate human antibody constant domain (e.g., see Riechmann et al., Nature 332:323, 1988; Verhoeyen et al., Science 239: 1534, 1988; and Jones et al. Nature 321 :522, 1986) and rodent CDRs supported by recombinantly veneered rodent FRs have also been described (e.g., see EPO Patent Pub. No. 519,596).

[0130] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Such antibodies can be produced using transgenic mice that are incapable of expressing endogenous immunoglobulin heavy and light chains genes, but which can express human heavy and light chain genes (e.g., see Lonberg and Huszar Int. Rev. Immunol. 13:65-93, 1995 and U.S. Patent Nos. 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016).

[0131] Veneered versions of the inventive antibodies may also be used in the methods of the present invention. The process of veneering involves selectively replacing FR residues from, e.g., a murine heavy or light chain variable region, with human FR residues in order to provide an antibody that comprises an antigen binding portion which retains substantially all of the native FR protein folding structure. Veneering techniques are based on the understanding that the antigen binding characteristics of an antigen binding portion are determined primarily by the structure and relative disposition of the heavy and light chain CDR sets within the antigen- association surface (e.g., see Davies et al, Ann. Rev. Biochem. 59:439, 1990). Thus, antigen association specificity can be preserved in a humanized antibody only wherein the CDR structures, their interaction with each other and their interaction with the rest of the variable region domains are carefully maintained. By using veneering techniques, exterior (e.g., solvent- accessible) FR residues which are readily encountered by the immune system are selectively replaced with human residues to provide a hybrid molecule that comprises either a weakly immunogenic, or substantially non-immunogenic veneered surface.

Pharmaceutical Compositions

[0132] The present invention also provides compositions comprising one or more provided antibodies. In some embodiments the present invention provides at least one antibody and at least one pharmaceutically acceptable excipient. Such pharmaceutical compositions may optionally comprise and/or be administered in combination with one or more additional therapeutically active substances. In some embodiments, provided pharmaceutical compositions are useful in medicine. In some embodiments, provided pharmaceutical compositions are useful as prophylactic agents (i.e., vaccines) in the treatment or prevention of GIST formation or of negative ramifications associated or correlated with GIST. In some embodiments, provided pharmaceutical compositions are useful in therapeutic applications, for example in individuals suffering from or susceptible to GIST. In some embodiments, pharmaceutical compositions are formulated for administration to humans.

[0133] For example, pharmaceutical compositions provided here may be provided in a sterile injectable form (e.g., a form that is suitable for subcutaneous injection or intravenous infusion). For example, in some embodiments, pharmaceutical compositions are provided in a liquid dosage form that is suitable for injection. In some embodiments, pharmaceutical compositions are provided as powders (e.g., lyophilized and/or sterilized), optionally under vacuum, which are reconstituted with an aqueous diluent (e.g., water, buffer, salt solution, etc.) prior to injection. In some embodiments, pharmaceutical compositions are diluted and/or reconstituted in water, sodium chloride solution, sodium acetate solution, benzyl alcohol solution, phosphate buffered saline, etc. In some embodiments, powder should be mixed gently with the aqueous diluent (e.g., not shaken).

[0134] In some embodiments, provided pharmaceutical compositions comprise one or more pharmaceutically acceptable excipients (e.g., preservative, inert diluent, dispersing agent, surface active agent and/or emulsifier, buffering agent, etc.). In some embodiments,

pharmaceutical compositions comprise one or more preservatives. In some embodiments, pharmaceutical compositions comprise no preservative.

[0135] In some embodiments, pharmaceutical compositions are provided in a form that can be refrigerated and/or frozen. In some embodiments, pharmaceutical compositions are provided in a form that cannot be refrigerated and/or frozen. In some embodiments,

reconstituted solutions and/or liquid dosage forms may be stored for a certain period of time after reconstitution (e.g., 2 hours, 12 hours, 24 hours, 2 days, 5 days, 7 days, 10 days, 2 weeks, a month, two months, or longer). In some embodiments, storage of antibody compositions for longer than the specified time results in antibody degradation. [0136] Liquid dosage forms and/or reconstituted solutions may comprise particulate matter and/or discoloration prior to administration. In some embodiments, a solution should not be used if discolored or cloudy and/or if particulate matter remains after filtration.

[0137] Compositions of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In some embodiments, such preparatory methods include the step of bringing active ingredient into association with one or more excipients and/or one or more other accessory ingredients, and then, if necessary and/or desirable, shaping and/or packaging the product into a desired single- or multi-dose unit.

[0138] A pharmaceutical composition in accordance with the invention may be prepared, packaged, and/or sold in bulk, as a single unit dose, and/or as a plurality of single unit doses. As used herein, a "unit dose" is discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to a dose which would be administered to a subject and/or a convenient fraction of such a dose such as, for example, one-half or one -third of such a dose.

[0139] Relative amounts of active ingredient, pharmaceutically acceptable excipient, and/or any additional ingredients in a pharmaceutical composition in accordance with the invention may vary, depending upon the identity, size, and/or condition of the subject treated and/or depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient.

[0140] Pharmaceutical compositions of the present invention may additionally comprise a pharmaceutically acceptable excipient, which, as used herein, may be or comprise solvents, dispersion media, diluents, or other liquid vehicles, dispersion or suspension aids, surface active agents, isotonic agents, thickening or emulsifying agents, preservatives, solid binders, lubricants and the like, as suited to the particular dosage form desired. Remington's The Science and Practice of Pharmacy, 21st Edition, A. R. Gennaro, (Lippincott, Williams & Wilkins, Baltimore, MD, 2006) discloses various excipients used in formulating pharmaceutical compositions and known techniques for the preparation thereof. Except insofar as any conventional excipient medium is incompatible with a substance or its derivatives, such as by producing any undesirable biological effect or otherwise interacting in a deleterious manner with any other component(s) of the pharmaceutical composition, its use is contemplated to be within the scope of this invention.

Conjugates Generally

[0141] Multifunctional agents described herein comprise multiple entities, each having at least one function. Certain embodiments of contemplated multifunctional agents comprise a targeting entity and at least one of the following entities: a detection entity and a therapeutic entity. In some embodiments, a multifunctional agent of the provided antibodies contains a targeting entity and a therapeutic entity but not a detection entity. In some embodiments, a multifunctional agent of the provided antibodies contains a targeting entity and a detection entity but not a therapeutic entity. In some embodiments, a multifunctional agent of the invention contains a targeting entity, a therapeutic entity and a detection entity. In any of contemplated embodiments, the entities of an agent are conjugated to one another. Conjugation of various entities to form a multifunctional agent is not limited to particular modes of conjugation. For example, two entities may be covalently conjugated directly to each other. Alternatively, two entities may be indirectly conjugated to each other, such as via a linker entity. In some embodiments, a multifunctional agent may include different types of conjugation within the agent, such that some entities of the agent are conjugated via direct conjugation while other entities of the agent are indirectly conjugated via one or more linkers. In some embodiments, a multifunctional agent of the invention comprises a single type of a linker entity. In other embodiments, a multifunctional agent of the invention comprises more than one type of linker entities. In some embodiments, a multifunctional agent includes a single type of linker entities but of varying length.

[0142] In many of the embodiments described herein, association between or amongst entities contained in a multifunctional agent is covalent. As will be appreciated by one skilled in the art, the moieties may be attached to each other either directly or indirectly (e.g., through a linker, as described above). [0143] In certain embodiments, where one entity (such as a targeting entity) and a second entity of a multifunctional agent are directly covalently linked to each other, such direct covalent conjugation can be through a linkage (e.g., a linker or linking entity) such as an amide, ester, carbon-carbon, disulfide, carbamate, ether, thioether, urea, amine, or carbonate linkage.

Covalent conjugation can be achieved by taking advantage of functional groups present on the first entity and/or the second entity of the multifunctional agent. Alternatively, a non-critical amino acid may be replaced by another amino acid that will introduce a useful group (such as amino, carboxy or sulfhydryl) for coupling purposes. Alternatively, an additional amino acid may be added to at least one of the entities of the multifunctional agent to introduce a useful group (such as amino, carboxy or sulfhydryl) for coupling purposes. Suitable functional groups that can be used to attach moieties together include, but are not limited to, amines, anhydrides, hydroxyl groups, carboxy groups, thiols, and the like. An activating agent, such as a

carbodiimide, can be used to form a direct linkage. A wide variety of activating agents are known in the art and are suitable for conjugating one entity to a second entity.

[0144] In other embodiments, entities of a multifunctional agent embraced by the present invention are indirectly covalently linked to each other via a linker group. Such a linker group may also be referred to as a linker or a linking entity. This can be accomplished by using any number of stable bifunctional agents well known in the art, including homofunctional and heterofunctional agents (for examples of such agents, see, e.g., Pierce Catalog and Handbook). The use of a bifunctional linker differs from the use of an activating agent in that the former results in a linking moiety being present in the resulting conjugate (agent), whereas the latter results in a direct coupling between the two moieties involved in the reaction. The role of a bifunctional linker may be to allow reaction between two otherwise inert moieties. Alternatively or additionally, the bifunctional linker that becomes part of the reaction product may be selected such that it confers some degree of conformational flexibility to the provided antibody (e.g., the bifunctional linker comprises a straight alkyl chain containing several atoms, for example, the straight alkyl chain contains between 2 and 10 carbon atoms). Alternatively or additionally, the bifunctional linker may be selected such that the linkage formed between a provided antibody and therapeutic agent is cleavable, e.g., hydrolysable (for examples of such linkers, see e.g. U.S. Pat. Nos. 5,773,001; 5,739,116 and 5,877,296, each of which is incorporated herein by reference in its entirety). Such linkers, for example, may be used when higher activity of certain entities, such as a targeting agent (e.g., the provided ETV1 antibodies) and/or of a therapeutic entity is observed after hydrolysis of the conjugate. Exemplary mechanisms by which an entity may be cleaved from a multifunctional agent include hydrolysis in the acidic pH of the lysosomes (hydrazones, acetals, and cis-aconitate-like amides), peptide cleavage by lysosomal enzymes (the capthepsins and other lysosomal enzymes), and reduction of disulfides). Another mechanism by which such an entity is cleaved from the multifunctional agent includes hydrolysis at

physiological pH extra- or intra-cellularly. This mechanism applies when the crosslinker used to couple one entity to another entity is a biodegradable/bioerodible component, such as polydextran and the like.

[0145] For example, hydrazone-containing multifunctional agents can be made with introduced carbonyl groups that provide the desired release properties. Multifunctional agents can also be made with a linker that comprises an alkyl chain with a disulfide group at one end and a hydrazine derivative at the other end. Linkers containing functional groups other than hydrazones also have the potential to be cleaved in the acidic milieu of lysosomes. For example, multifunctional agents can be made from thiol-reactive linkers that contain a group other than a hydrazone that is cleavable intracellularly, such as esters, amides, and acetals/ketals.

[0146] Another example of class of pH sensitive linkers are the cis-aconitates, which have a carboxylic acid group juxtaposed to an amide group. The carboxylic acid accelerates amide hydrolysis in the acidic lysosomes. Linkers that achieve a similar type of hydrolysis rate acceleration with several other types of structures can also be used.

[0147] Another potential release method for conjugates of the provided antibodies is the enzymatic hydrolysis of peptides by the lysosomal enzymes. In one example, a provided antibody is attached via an amide bond to para-aminobenzyl alcohol and then a carbamate or carbonate is made between the benzyl alcohol and the therapeutic agent. Cleavage of the peptide leads to collapse of the amino benzyl carbamate or carbonate, and release of the therapeutic agent. In another example, a phenol can be cleaved by collapse of the linker instead of the carbamate. In another variation, disulfide reduction is used to initiate the collapse of a para- mercaptobenzyl carbamate or carbonate. [0148] Useful linkers which may be used as a linking entity of a multifunctional agent provided herein include, without limitation: polyethylene glycol, a copolymer of ethylene glycol, a polypropylene glycol, a copolymer of propylene glycol, a carboxymethylcellulose, a polyvinyl pyrrolidone, a poly-l,3-dioxolane, a poly-l,3,6-trioxane, an ethylene/maleic anhydride copolymer, a polyaminoacid, a dextran n- vinyl pyrrolidone, a poly n- vinyl pyrrolidone, a propylene glycol homopolymer, a propylene oxide polymer, an ethylene oxide polymer, a polyoxyethylated polyol, a polyvinyl alcohol, a linear or branched glycosylated chain, a polyacetal, a long chain fatty acid, a long chain hydrophobic aliphatic group.

[0149] Embraced also herein are multifunctional agents that include at least one entity which involves non-covalent association. Examples of non-covalent interactions include, but are not limited to, hydrophobic interactions, electrostatic interactions, dipole interactions, van der Waals interactions, and hydrogen bonding. Irrespective of the nature of the binding, interaction, or coupling, the association between a first entity and a second entity is, in some embodiments, selective, specific and strong enough so that the second entity contained in the agent does not dissociate from the first entity before or during transport/delivery to and into the target. Thus, Association amongst multiple entities of a multifunctional agent may be achieved using any chemical, biochemical, enzymatic, or genetic coupling known to one skilled in the art.

Therapeutic Conjugates

[0150] As described herein, a multifunctional agent of the provided antibodies comprises multiple entities that are conjugated together to form an agent, which offers a broad range of utility or application. The term "function" as applied to "multifunctional agents" of the instant invention broadly refers to a functionally discernable molecular structure associated with certain utility. Examples of functionalities in the context of the present disclosure include, without limitation, utility associated with targeting, utility associated with therapeutic effects (e.g., cytotoxic and/or cytostatic effects, anti-proliferative effects, anti-angiogenic effects, etc.), utility associated with detection or labeling, etc., each of which is discussed in more detail below. Functionality associated with diagnostic purposes is generally referred to as modality.

[0151] In a broad sense, an "entity" is a molecular structure or module having at least one function. An entity when attached to an agent may be generally referred to as a moiety. For example, a targeting entity is a molecular structure which can be contained in an agent which affects or controls the site of action by specifically interacting with, or has affinity for, a target of interest. As an example, a target may be a molecule or molecular complex present on a cell surface, e.g., certain cell types, tissues, etc. Thus, a corresponding targeting entity can, by virtue of affinity, specifically or preferentially interact with such a target. Use of targeting moieties for agents such as therapeutic agents is known in the art. The nature of a target-targeting entity interaction varies depending on the binding pair, and is within the knowledge of the art. In the context of the present application, many of the embodiments involve a target which is a tumor or tumor cells. That is, at the molecular level, a target is a molecule or cellular constituent that is present (e.g., preferentially expressed) on a tumor cell, such that it can specifically or

preferentially bind to a targeting entity/moiety upon contact. Various components (e.g., entities) of the multifunctional agent of the present invention are discussed in more detail below.

[0152] Useful targeting entities can be any molecules that have specificity for at least one target of interest. In some embodiments, a target is a tumor cell or tumor cells. More specifically, at the molecular level, a target is a molecule or molecular complex present on tumor cells. Thus, contemplated targeting entities exert specificity for such target (e.g., tumor cells) and are able to localize to and bind to the target. In some embodiments of the provided antibodies, a target is a tumor specific antigen. In some embodiments, a target may be ETV1 or a related family member thereof. In some embodiments, contemplated targeting entities localize to tumor cells and retain the association with tumor cells over a period of time. In some embodiments, contemplated targeting entities bind to at least one receptor present on the surface of tumor cells and are subsequently internalized.

[0153] In a number of embodiments, the provided antibodies provide for multifunctional agents comprising a ETV1 target entity which essentially consists of a provided antibody (e.g., provided ETV1 antibodies). In such embodiments, therefore, the multifunctional agents according to the provided antibodies are antibody conjugates. Non- limiting embodiments of useful conjugates of the provided antibodies are provided below.

[0154] In some embodiments, conjugates of the provided antibodies comprise a provided antibody and a nucleic acid molecule that is useful as a therapeutic (e.g., anti-cancer) agent. A variety of chemical types and structural forms of nucleic acid can be suitable for such strategies. These include, by way of non-limiting example, DNA, including single-stranded (ssDNA) and double-stranded (dsDNA); RNA, including, but not limited to ssR A, dsR A, tR A, mR A, rR A, enzymatic RNA; RNA:DNA hybrids, triplexed DNA (e.g., dsDNA in association with a short oligonucleotide), and the like.

[0155] In some embodiments, the nucleic acid agent is between about 5 and 2000 nucleotides long. In some embodiments, the nucleic acid agent is at least about 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more nucleotides long. In some embodiments, the nucleic acid agent is less than about 2000, 1900, 1800, 1700, 1600, 1500, 1400, 1300, 1200, 1100, 1000, 900, 800, 700, 600, 500, 450, 400, 350, 300, 250, 200, 150, 100, 50, 45, 40, 35, 30, 25, 20 or fewer nucleotides long.

[0156] In some embodiments, the nucleic acid agent comprises a promoter and/or other sequences that regulate transcription. In some embodiments, the nucleic acid agent comprises an origin of replication and/or other sequences that regulate replication. In some embodiments, the nucleic acid agent does not include a promoter and/or an origin of replication. [0157] Nucleic acid anti-cancer agents suitable for use in the practice of the present invention include those agents that target genes associated with tumorigenesis and cell growth or cell transformation (e.g., proto-oncogenes, which code for proteins that stimulate cell division), angiogenic/anti-angiogenic genes, tumor suppressor genes (which code for proteins that suppress cell division), genes encoding proteins associated with tumor growth and/or tumor migration, and suicide genes (which induce apoptosis or other forms of cell death), especially suicide genes that are most active in rapidly dividing cells.

[0158] Examples of genes associated with tumorigenesis and/or cell transformation include MLL fusion genes, BCR-ABL, TEL-AML1, EWS-FLI1, TLS-FUS, PAX3- FKHR, Bcl- 2, AMLl-ETO, AML1-MTG8, Ras, Fos PDGF, RET, APC, NF-1, Rb, p53, MDM2 and the like; overexpressed genes such as multidrug resistance genes; cyclins; beta-Catenin; telomerase genes; c-myc, n-myc, Be 1-2, Erb-Bl and Erb-B2; and mutated genes such as Ras, Mos, Raf, and Met. Examples of tumor suppressor genes include, but are not limited to, p53, p21, RBI, WT1, NF1, VHL, APC, DAP kinase, pi 6, ARF, Neurofibromin, and PTEN. Examples of genes that can be targeted by nucleic acid agents useful in anti-cancer therapy include genes encoding proteins associated with tumor migration such as integrins, selectins, and metalloproteinases; anti- angiogenic genes encoding proteins that promote formation of new vessels such as Vascular Endothelial Growth Factor (VEGF) or VEGFr; anti-angiogenic genes encoding proteins that inhibit neovascularization such as endostatin, angiostatin, and VEGF-R2; and genes encoding proteins such as interleukins, interferon, fibroblast growth factor (a-FGF and(P-FGF), insulinlike growth factor (e.g., IGF-1 and IGF-2), Platelet-derived growth factor (PDGF), tumor necrosis factor (TNF), Transforming Growth Factor (e.g., TGF-a and TGF-β, Epidermal growth factor (EGF), Keratinocyte Growth Factor (KGF), stem cell factor and its receptor c-Kit (SCF/c- Kit) ligand, CD40L/CD40, VLA-4 VCAM-1, ICAM-l/LFA-1, hyalurin/CD44, and the like. As will be recognized by one skilled in the art, the foregoing examples are not exclusive.

[0159] Nucleic acid agents suitable for conjugation with the provided antibodies may have any of a variety of uses including, for example, use as anti-cancer or other therapeutic agents, probes, primers, etc. Nucleic acid agents may have enzymatic activity (e.g., ribozyme activity), gene expression inhibitory activity (e.g., as antisense or siRNA agents, etc), and/or other activities. Nucleic acids agents may be active themselves or may be vectors that deliver active nucleic acid agents (e.g., through replication and/or transcription of a delivered nucleic acid). For purposes of the present specification, such vector nucleic acids are considered "therapeutic agents" if they encode or otherwise deliver a therapeutically active agent, even if they do not themselves have therapeutic activity.

[0160] In certain embodiments, conjugates of the provided antibodies comprise a nucleic acid therapeutic agent that comprises or encodes an antisense compound. The terms "antisense compound or agent," "antisense oligomer," "antisense oligonucleotide," and "antisense oligonucleotide analog" are used herein interchangeably, and refer to a sequence of nucleotide bases and a subunit-to-subunit backbone that allows the antisense compound to hybridize to a target sequence in an R A by Watson-Crick base pairing to form an RNA oligomer

heteroduplex within the target sequence. The oligomer may have exact sequence

complementarity within the target sequence or near complementarity. Such antisense oligomers may block or inhibit translation of the mRNA containing the target sequence, or inhibit gene transcription. Antisense oligomers may bind to double-stranded or single-stranded sequences.

[0161] Examples of antisense oligonucleotides suitable for use in the practice of the present invention include, for example, those mentioned in the following reviews: R.A Stahel et al, Lung Cancer, 2003, 41 : S81-S88; K.F. PiroUo et al, Pharmacol. Ther., 2003, 99: 55-77; A.C. Stephens and R.P. Rivers, Curr. Opin. Mol. Ther., 2003, 5: 118- 122; N.M. Dean and C.F.

Bennett, Oncogene, 2003, 22: 9087-9096; N. Schiavone et al, Curr. Pharm. Des., 2004, 10: 769- 784; L. Vidal et al, Eur. J. Cancer, 2005, 41 : 2812- 2818; T. Aboul-Fadl, Curr. Med. Chem., 2005, 12: 2193-2214; M.E. Gleave and B.P. Monia, Nat. Rev. Cancer, 2005, 5: 468-479; Y.S. Cho-Chung, Curr. Pharm. Des., 2005, 11 : 2811-2823; E. Rayburn et al, Lett. Drug Design & Discov., 2005, 2: 1-18; E.R. Rayburn et al, Expert Opin. Emerg. Drugs, 2006, 11 : 337-352; I. Tamm and M. Wagner, Mol. Biotechnol., 2006, 33: 221-238 (each of which is incorporated herein by reference in its entirety).

[0162] Examples of suitable antisense oligonucleotides include, for example oblimersen sodium (also known as Genasense™ or G31239, developed by Genta, Inc., Berkeley Heights, NJ), a phosphorothioate oligomer targeted towards the initiation codon region of the be 1-2 mRNA. Be 1-2 is a potent inhibitor of apoptosis and is overexpressed in many cancer including follicular lymphomas, breast cancer, colon cancer, prostate cancer, and intermediate/high-grade lymphomas (C.A. Stein et al, Semin. Oncol, 2005, 32: 563-573; S.R. Frankel, Semin. Oncol, 2003, 30: 300-304). Other suitable antisense oligonucleotides include GEM-231 (HYB0165, Hybridon, Inc., Cambridge, MA), which is a mixed backbone oligonucleotide directed against cAMP-dependent protein kinase A (PKA) (S. Goel et al, Clin. Cancer Res., 203, 9: 4069-4076); Affinitak (ISIS 3521 or aprinocarsen, ISIS pharmaceuticals, Inc., Carlsbad, CA), an antisense inhibitor of PKCalpha; OGX-011 (Isis 112989, Isis Pharmaceuticals, Inc.), a 2'-methoxyethyl modified antisense oligonucleotide against clusterin, a glycoprotein implicated in the regulation of the cell cycle, tissue remodeling, lipid transport, and cell death and which is overexpressed in cancers of breast, prostate and colon; ISIS 5132 (Isis 112989, Isis Pharmaceuticals, Inc.), a phosphorothioate oligonucleotide complementary to a sequence of the 3 '-mistranslated region of the c-raf-1 niRNA (S.P. Henry et al, Anticancer Drug Des., 1997, 12: 409-420; B.P. Monia et al, Proc. Natl. Acad. Sci. USA, 1996, 93: 15481- 15484; CM. Rudin et al, Clin. Cancer Res., 2001, 7: 1214-1220); ISIS 2503 (Isis Pharmaceuticals, Inc.), a phosphorothioate oligonucleotide antisense inhibitor of human H-ras mRNA expression (J. Kurreck, Eur. J. Biochem., 2003, 270: 1628-1644); oligonucleotides targeting the X-linked inhibitor of apoptosis protein (XIAP), which blocks a substantial portion of the apoptosis pathway, such as GEM 640 (AEG 35156, Aegera Therapeutics Inc. and Hybridon, Inc.) or targeting survivin, an inhibitor of apoptosis protein (IAP), such as ISIS 23722 (Isis Pharmaceuticals, Inc.), a 2'-0-methoxyethyl chimeric oligonucleotide; MG98, which targets DNA methyl transferase; and GTI-2040 (Lorus

Therapeutics, Inc. Toronto, Canada), a 20-mer oligonucleotide that is complementary to a coding region in the mRNA of the R2 small subunit component of human ribonucleotide reductase.

[0163] Other suitable antisense oligonucleotides include antisense oligonucleotides that are being developed against Her-2/neu, c-Myb, c-Myc, and c-Raf (see, for example, A. Biroccio et al, Oncogene, 2003, 22: 6579-6588; Y. Lee et al, Cancer Res., 2003, 63: 2802-2811; B. Lu et al, Cancer Res., 2004, 64: 2840-2845; K.F. PiroUo et al, Pharmacol. Ther., 2003, 99: 55-77; and A. Rait et al, Ann. N. Y. Acad. Sci., 2003, 1002: 78-89).

[0164] In certain embodiments, conjugates of the provided antibodies comprise a nucleic acid anti-cancer agent that comprises or encodes an interfering RNA molecule. The terms "interfering RNA" and "interfering RNA molecule" are used herein interchangeably, and refer to an RNA molecule that can inhibit or downregulate gene expression or silence a gene in a sequence-specific manner, for example by mediating RNA interference (RNAi). RNA

interference (RNAi) is an evolutionarily conserved, sequence-specific mechanism triggered by double-stranded RNA (dsRNA) that induces degradation of complementary target single- stranded mRNA and "silencing" of the corresponding translated sequences (McManus and Sharp, 2002, Nature Rev. Genet., 2002, 3: 737). RNAi functions by enzymatic cleavage of longer dsRNA strands into biologically active "short-interfering RNA" (siRNA) sequences of about 21-23 nucleotides in length (Elbashir et al, Genes Dev., 2001, 15: 188). RNA

interference has emerged as a promising approach for therapy of cancer.

[0165] An interfering RNA suitable for use in the practice of the present invention can be provided in any of several forms. For example, an interfering RNA can be provided as one or more of an isolated short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), or short hairpin RNA (shRNA).

[0166] Examples of interfering RNA molecules suitable for use in the present invention include, for example, the iRNAs cited in the following reviews: O. Milhavet et al., Pharmacol. Rev., 2003, 55: 629-648; F. Bi et al, Curr. Gene. Ther., 2003, 3: 411- 417; P.Y. Lu et al, Curr. Opin. Mol. Ther., 2003, 5: 225-234; I. Friedrich et al, Semin. Cancer Biol, 2004, 14: 223-230; M. Izquierdo, Cancer Gene Ther., 2005, 12: 217-227; P.Y. Lu et al, Adv. Genet., 2005, 54: 117- 142; G.R. Devi, Cancer Gene Ther., 2006, 13: 819-829; M.A. Behlke, Mol. Ther., 2006, 13: 644- 670; and L.N. Putral et al, Drug News Perspect., 2006, 19: 317-324 (the contents of each of which are incorporated herein by reference in their entirety).

[0167] Other examples of suitable interfering RNA molecules include, but are not limited to, p53 interfering RNAs (e.g., T.R. Brummelkamp et al, Science, 2002, 296: 550-553; M.T. Hemman et al, Nat. Genet., 2003, 33: 396-400); interfering RNAs that target the bcr-abl fusion, which is associated with development of chronic myeloid leukemia and acute lymphoblastic leukemia (e.g., M. Scherr et al, Blood, 2003, 101 : 1566-1569; M.J. Li et al, Oligonucleotides, 2003, 13: 401-409), interfering RNAs that inhibit expression of NPM-ALK, a protein that is found in 75% of anaplastic large cell lymphomas and leads to expression of a constitutively active kinase associated with tumor formation (U. Ritter et al., Oligonucleotides, 2003, 13: 365- 373); interfering R As that target oncogenes, such as Raf-1 (T.F. Lou et al, Oligonucleotides, 2003, 13: 313- 324), K-Ras (T.R. Brummelkamp et al, Cancer Cell, 2002, 2: 243-247), erbB-2 (G. Yang et al, J. Biol. Chem., 2004, 279: 4339-4345); interfering RNAs that target b-catenin protein, whose over-expression leads to transactivation of the T-cell factor target genes, which is thought to be the main transforming event in colorectal cancer (M. van de Wetering et al., EMBO Rep., 2003, 4: 609-615).

[0168] In certain embodiments, conjugates of the provided antibodies comprise a nucleic acid therapeutic agent that is a ribozyme. As used herein, the term "ribozyme" refers to a catalytic RNA molecule that can cleave other RNA molecules in a target-specific marmer Ribozymes can be used to downregulate the expression of any undesirable products of genes of interest. Examples of ribozymes that can be used in the practice of the present invention include, but are not limited to, ANGIOZYMETM (RPI.4610, Sima Therapeutics, Boulder, CO), a ribozyme targeting the conserved region of human, mouse, and rat vascular endothelial growth factor receptor (VEGFR)-1 mRNA, and Herzyme (Sima Therapeutics).

[0169] In certain embodiments, entities or moieties within conjugates of the provided antibodies comprise a photosensitizer used in photodynamic therapy (PDT). In PDT, local or systemic administration of a photosensitizer to a patient is followed by irradiation with light that is absorbed by the photosensitizer in the tissue or organ to be treated. Light absorption by the photosensitizer generates reactive species (e.g., radicals) that are detrimental to cells. For maximal efficacy, a photosensitizer typically is in a form suitable for administration, and also in a form that can readily undergo cellular internalization at the target site, often with some degree of selectivity over normal tissues.

[0170] While some photosensitizers (e.g., Photofrin®, QLT, Inc., Vancouver, BC,

Canada) have been delivered successfully as part of a simple aqueous solution, such aqueous solutions may not be suitable for hydrophobic photosensitizer drugs, such as those that have a tetra- or poly-pyrrole-based structure. These drugs have an inherent tendency to aggregate by molecular stacking, which results in a significant reduction in the efficacy of the

photosensitization processes (Siggel et al, J. Phys. Chem., 1996, 100: 2070-2075). Approaches to minimize aggregation include liposomal compositions (e.g., for benzoporphyrin derivative monoacid A, BPDMA, Verteporfin®, QLT, Inc., Vancouver, Canada; and zinc phthalocyanine, CIBA-Geigy, Ltd., Basel, Switzerland), and conjugation of photosensitizers to biocompatible block copolymers (Peterson et al, Cancer Res., 1996, 56: 3980-3985) and/or antibodies

(Omelyanenko et al, Int. J. Cancer, 1998, 75: 600-608).

[0171] Conjugates of the provided antibodies associated with a photosensitizer can be used as new delivery systems in PDT. In addition to reducing photosensitizer aggregation, delivery of photosensitizers according to the present invention exhibits other advantages such as increased specificity for target tissues/organ and cellular internalization of the photosensitizer.

[0172] Photosensitizers suitable for use in the present invention include any of a variety of synthetic and naturally occurring molecules that have photosensitizing properties useful in PDT. In certain embodiments, the absorption spectrum of the photosensitizer is in the visible range, typically between 350 nm and 1200 nm, preferably between 400 nm and 900 nm, e.g., between 600 nm and 900 nm. Suitable photosensitizers that can be coupled to toxins according to the present invention include, but are not limited to, porphyrins and porphyrin derivatives (e.g., chlorins, bacteriochlorins, isobacteriochlorins, phthalocyanines, and naphthalocyanines);

metalloporphyrins, metallophthalocyanines, angelicins, chalcogenapyrrillium dyes, chlorophylls, coumarins, flavins and related compounds such as alloxazine and riboflavin, fullerenes, pheophorbides, pyropheophorbides, cyanines (e.g., merocyanine 540), pheophytins, sapphyrins, texaphyrins, purpurins, porphycenes, phenothiaziniums, methylene blue derivatives,

naphthalimides, nile blue derivatives, quinones, perylenequinones (e.g., hypericins, hypocrellins, and cercosporins), psoralens, quinones, retinoids, rhodamines, thiophenes, verdins, xanthene dyes (e.g., eosins, erythrosins, rose bengals), dimeric and oligomeric forms of porphyrins, and prodrugs such as 5 -aminolevulinic acid (R.W. Redmond and J.N. Gamlin, Photochem.

PhotobioL, 1999, 70: 391-475).

[0173] Exemplary photosensitizers suitable for use in the present invention include those described in U.S. Pat. Nos. 5,171,741; 5,171,749; 5,173,504; 5,308,608; 5,405,957; 5,512,675; 5,726,304; 5,831,088; 5,929,105; and 5,880,145 (the contents of each of which are incorporated herein by reference in their entirety). [0174] In certain embodiments, conjugates of the provided antibodies comprise a radiosensitizer. As used herein, the term "radiosensitizer" refers to a molecule, compound or agent that makes tumor cells more sensitive to radiation therapy. Administration of a

radiosensitizer to a patient receiving radiation therapy generally results in enhancement of the effects of radiation therapy. Ideally, a radiosensitizer exerts its function only on target cells. For ease of use, a radiosensitizer should also be able to find target cells even if it is administered systemically. However, currently available radiosensitizers are typically not selective for tumors, and they are distributed by diffusion in a mammalian body. ETV1 antibody conjugates of the present invention can be used as a new delivery system for radiosensitizers.

[0175] A variety of radiosensitizers are known in the art. Examples of radiosensitizers suitable for use in the present invention include, but are not limited to, paclitaxel (TAXOL®), carboplatin, cisplatin, and oxaliplatin (Amorino et al, Radiat. Oncol. Investig. 1999; 7: 343-352; Choy, Oncology, 1999, 13: 22-38; Safran et al, Cancer Invest, 2001, 19: 1-7; Dionet et al, Anticancer Res., 2002, 22: 721-725; Cividalli et al, Radiat. Oncol. Biol. Phys., 2002, 52: 1092- 1098); gemcitabine (Gemzar®) (Choy, Oncology, 2000, 14: 7-14; Mornex and Girard, Annals of Oncology, 2006, 17: 1743- 1747); etanidazole (Nitrolmidazole®) (Inanami et al., Int. J. Radiat. Biol, 2002, 78: 267- 274); misonidazole (Tamulevicius et al, Br. J. Radiology, 1981, 54: 318- 324; Palcic et al, Radiat. Res., 1984, 100: 340-347), tirapazamine (Masunaga et al, Br. J.

Radiol, 2006, 79: 991-998; Rischin et al, J. Clin. Oncol, 2001, 19: 535-542; Shulman et al, Int. J. Radiat. Oncol. Biol. Phys., 1999, 44: 349-353); and nucleic acid base derivatives, e.g., halogenated purines or pyrimidines, such as 5-fluorodeoxyuridine (Buchholz et al, Int. J. Radiat. Oncol. Biol. Phys., 1995, 32: 1053-1058).

[0176] In certain embodiments, conjugates of the provided antibodies comprise a radioisotope. Examples of suitable radioisotopes include any α-, β- or γ-emitter, which, when localized at a tumor site, results in cell destruction (S.E. Order, "Analysis, Results, and Future Prospective of the Therapeutic Use of Radiolabeled Antibody in Cancer Therapy", Monoclonal Antibodies for Cancer Detection and Therapy, R.W. Baldwin et al. (Eds.), Academic Press, 1985). Examples of such radioisotopes include, but are not limited to, iodine-131 (1311), iodine- 125 (1251), bismuth-212 (212Bi), bismuth-213 (213Bi), astatine-211 (211At), rhenium-186 (186Re), rhenium-188 (188Re), phosphorus-32 (32P), yttrium-90 (90yY), samarium- 153

(153Sm), and lutetium-177 (177Lu).

[0177] In certain embodiments, conjugates of the provided antibodies comprise a superantigen or biologically active portion thereof. Superantigens constitute a group of bacterial and viral proteins that are extremely efficient in activating a large fraction of the T-cell population. Superantigens bind directly to the major histocompatibility complex (MHC) without being processed. In fact, superantigens bind unprocessed outside the antigenbinding groove on the MHC class II molecules, thereby avoiding most of the polymorphism in the conventional peptide-binding site.

[0178] A superantigen-based tumor therapeutic approach has been developed for the treatment of solid tumors. In this approach, a targeting moiety (e.g., a provided antibody or fragments thereof) is conjugated to a superantigen, providing a targeted superantigen. If the antibody, or antibody fragment, recognizes a tumor-associated antigen, the targeted superantigen, bound to tumors cells, can trigger superantigen-activated cytotoxic T-cells to kill the tumor cells directly by superantigen-dependent cell mediated cytotoxicity. (See, e.g., Sogaard et al, (1996) "Antibody-targeted superantigens in cancer immunotherapy," Immunotechnology, 2(3): 151- 162, the entire contents of which are herein incorporated by reference.)

[0179] Superantigen-based tumor therapeutics have had some success. For example, fusion proteins with wild-type staphylococcal enterotoxin A (SEA) have been investigated in clinical trials of colorectal and pancreatic cancer (Giantonio et al., J. Clin. Oncol., 1997, 15: 1994-2007; Alpaugh et a., Clin. Cancer Res., 1998, 4: 1903-1914; Cheng et al, J. Clin. Oncol, 2004, 22: 602-609; the entire contents of each of which are herein incorporated by reference); staphylococcal superantigens of the enterotoxin gene cluster (egc) have been studied for the treatment of non-small cell lung cancer (Terman et al., Clin. Chest Med., 2006, 27: 321-324, the entire contents of which are herein incorporated by reference), and staphylococcal enterotoxin B has been evaluated for the intravesical immunotherapy of superficial bladder cancer (Perabo et al., Int. J. Cancer, 2005, 115: 591-598, the entire contents of which are herein incorporated by reference). [0180] A superantigen, or a biologically active portion thereof, can be associated to provided antibodies to form a conjugate of the provided antibodies comprising a provided antibody and a superantigen, and used in a therapy, e.g., an anti-cancer therapy, as described herein.

[0181] Examples of superantigens suitable for use in the present invention include, but are not limited to, staphylococcal enterotoxin (SE) (e.g., staphylococcal enterotoxin A (SEA) or staphylococcal enterotoxin E (SEE)), Streptococcus pyogenes exotoxin (SPE), Staphylococcus aureus toxic shock-syndrome toxin (TSST-1), streptococcal mitogenic exotoxin (SME), streptococcal superantigen (SSA), and staphylococcal superantigens of the enterotoxin gene cluster. As known to one skilled in the art, the three-dimensional structures of the above listed superantigens can be obtained from the Protein Data Bank. Similarly, the nucleic acid sequences and the amino acid sequences of the above listed superantigens and other superantigens can be obtained from GenBank.

[0182] In certain embodiments, conjugates of the provided antibodies may be used in directed enzyme prodrug therapy. In a directed enzyme prodrug therapy approach, a

directed/targeted enzyme and a prodrug are administered to a subject, wherein the targeted enzyme is specifically localized to a portion of the subject's body where it converts the prodrug into an active drug. The prodrug can be converted to an active drug in one step (by the targeted enzyme) or in more than one step. For example, the prodrug can be converted to a precursor of an active drug by the targeted enzyme. The precursor can then be converted into the active drug by, for example, the catalytic activity of one or more additional targeted enzymes, one or more non-targeted enzymes administered to the subject, one or more enzymes naturally present in the subject or at the target site in the subject (e.g., a protease, phosphatase, kinase or polymerase), by an agent that is administered to the subject, and/or by a chemical process that is not

enzymatically catalyzed (e.g., oxidation, hydrolysis, isomerization, epimerization, etc.).

[0183] Different approaches have been used to direct/target the enzyme to the site of interest. For example, in ADEPT (antibody-directed enzyme prodrug therapy), an antibody designed/developed against a tumor antigen is linked to an enzyme and injected in a subject, resulting in selective binding of the enzyme to the tumor. When the discrimination between tumor and normal tissue enzyme levels is sufficient, a prodrug is administered to the subject. The prodrug is converted to its active form by the enzyme only within the tumor. Selectivity is achieved by the tumor specificity of the antibody and by delaying prodrug administration until there is a large differential between tumor and normal tissue enzyme levels. Early clinical trials are promising and indicate that ADEPT may become an effective treatment for all solid cancers for which tumor-associated or tumor-specific antibodies are known. Tumors have also been targeted with the genes encoding for prodrug activating enzymes. This approach has been called virus-directed enzyme prodrug therapy (VDEPT) or more generally GDEPT (gene-directed enzyme prodrug therapy, and has shown good results in laboratory systems. Other versions of directed enzyme prodrug therapy include PDEPT (polymer-directed enzyme prodrug therapy), LEAPT (lectin-directed enzyme-activated prodrug therapy), and CDEPT (clostridial-directed enzyme prodrug therapy). A conjugate according to the present invention, which comprises a prodrug activating enzyme associated with a reduced lysine chlorotoxin polypeptide, can be used in a similar way.

[0184] Nonlimiting examples of enzyme/prodrug/active drug combinations suitable for use in the present invention are described, for example, in Bagshawe et al., Current Opinions in Immunology, 1999, 11 : 579-583; Wilman, "Prodrugs in Cancer Therapy", Biochemical Society Transactions, 14: 375-382, 615th Meeting, Belfast, 1986; Stella et al, "Prodrugs: A Chemical Approach To Targeted Drug Delivery", in "Directed Drug Delivery", Borchardt et al, (Eds), pp. 247-267 (Humana Press, 1985). Nonlimiting examples of enzyme/prodrug/active anti-cancer drug combinations are described, for example, in Rooseboom et al, Pharmacol. Reviews, 2004, 56: 53-102.

[0185] Examples of prodrug activating enzymes include, but are not limited to, nitroreductase, cytochrome P450, purine-nucleoside phosphorylase, thymidine kinase, alkaline phosphatase, β-glucuronidase, carboxypeptidase, penicillin amidase, β-lactamase, cytosine deaminase, and methionine γ-lyase.

[0186] Examples of anti-cancer drugs that can be formed in vivo by activation of a prodrug by a prodrug activating enzyme include, but are not limited to, 5-(aziridin-l-yl)- 4- hydroxyl-amino-2-nitro-benzamide, isophosphoramide mustard, phosphoramide mustard, 2- fluoroadenine, 6-methylpurine, ganciclovir-triphosphate nucleotide, etoposide, mitomycin C, p- [N,N-bis(2-chloroethyl)amino]phenol (POM), doxorubicin, oxazolidinone, 9- aminocamptothecin, mustard, methotrexate, benzoic acid mustard, doxorubicin, adriamycin, daunomycin, carminomycin, bleomycins, esperamicins, melphalan, palytoxin, 4- desacetylvinblastine-3-carboxylic acid hydrazide, phenylenediamine mustard, 4'- carboxyphthalato(l,2-cyclohexane-diamine) platinum, taxol, 5-fluorouracil, methylselenol, and carbonothionic difluoride.

[0187] In certain embodiments, a therapeutic (e.g., anti-cancer) agent within a conjugate of the provided antibodies comprises an anti-angiogenic agent. Antiangiogenic agents suitable for use in the present invention include any molecule, compound, or factor that blocks, inhibits, slows down, or reduces the process of angiogenesis, or the process by which new blood vessels form by developing from preexisting vessels. Such a molecule, compound, or factor can block angiogenesis by blocking, inhibiting, slowing down, or reducing any of the steps involved in angiogenesis, including (but not limited to) steps of (1) dissolution of the membrane of the originating vessel, (2) migration and proliferation of endothelial cells, and (3) formation of new vasculature by migrating cells.

[0188] Examples of anti-angiogenic agents include, but are not limited to, bevacizumab

(AVASTIN®), celecoxib (CELEBREX®), endostatin, thalidomide, EMD121974 (Cilengitide), TNP-470, squalamine, combretastatin A4, interferon-a, anti-VEGF antibody, SU5416, SU6668, PTK787/2K 22584, Marimistal, AG3340, COL-3, Neovastat, and BMS-275291.

[0189] Anti-angiogenic agents may be used in a variety of therapeutic contexts, including, but not limited to, anti-cancer therapies and therapies for macular degeneration.

[0190] In certain embodiments, a therapeutic (e.g., anti-cancer) agent within a conjugate of the provided antibodies comprises a specific inhibitor of a number of receptor tyrosine kinase (RTK) enzymes including, but not limited to, c-KIT tyrosine kinase and PDGFRa. Examples of RTK inhibitors include, but are not limited to, imatinib, GLEEVEC™, sunitinib, SUTENT®, and SU 11248. [0191] According to another embodiment, the provided antibodies relate to a method of treating the growth or metastasis of tumor/cancer cells; and carcinomas (e.g, squamous cell carcinomas, multiple myeloma, melanoma, glioma, glioblastomas, leukemia, sarcomas, leiomyomas, mesothelioma, GIST, and carcinomas of the lung, breast, ovary, cervix, liver, biliary tract, gastrointestinal tract, pancreas, prostate, and head and neck, wherein said method comprises administering to a patient in need thereof a provided antibody, or pharmaceutically acceptable composition thereof.

Administration

[0192] Provides ETVl antibodies in accordance with the invention and pharmaceutical compositions thereof in accordance with the present invention may be administered according to any appropriate route and regimen. In some embodiments, a route or regimen is one that has been correlated with a positive therapeutic benefit. In some embodiments, a route or regimen is one that has been approved by the FDA and/or EMEA.

[0193] In some embodiments, the exact amount administered may vary from subject to subject, depending on one or more factors as is well known in the medical arts. Such factors may include, for example, one or more of species, age, general condition of the subject, severity of the infection, particular composition, its mode of administration, its mode of activity, the disorder being treated and the severity of the disorder; the activity of the specific ETVl antibody employed; the specific pharmaceutical composition administered; the half-life of the composition after administration; the age, body weight, general health, sex, and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed and the like. Pharmaceutical compositions may be formulated in dosage unit form for ease of administration and uniformity of dosage. It will be understood, however, that the total daily usage of the compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. [0194] Pharmaceutical compositions of the present invention may be administered by any route, as will be appreciated by those skilled in the art. In some embodiments, pharmaceutical compositions of the present invention are administered by oral (PO), intravenous (IV), intramuscular (IM), intra-arterial, intramedullary, intrathecal, subcutaneous (SQ),

intraventricular, transdermal, interdermal, intradermal, rectal (PR), vaginal, intraperitoneal (IP), intragastric (IG), topical (e.g., by powders, ointments, creams, gels, lotions, and/or drops), mucosal, intranasal, buccal, enteral, vitreal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; as an oral spray, nasal spray, and/or aerosol, and/or through a portal vein catheter.

[0195] In specific embodiments, ETV1 antibodies in accordance with the present invention and/or pharmaceutical compositions thereof may be administered intravenously, for example, by intravenous infusion. In specific embodiments, ETV1 antibodies in accordance with the present invention and/or pharmaceutical compositions thereof may be administered by intramuscular injection. In specific embodiments, ETV1 antibodies in accordance with the present invention and/or pharmaceutical compositions thereof may be administered by subcutaneous injection. In specific embodiments, ETV1 antibodies in accordance with the present invention and/or pharmaceutical compositions thereof may be administered via portal vein catheter. However, the invention encompasses the delivery of ETV1 antibodies in accordance with the present invention and/or pharmaceutical compositions thereof by any appropriate route taking into consideration likely advances in the sciences of drug delivery.

[0196] In certain embodiments, ETV1 antibodies in accordance with the present invention and/or pharmaceutical compositions thereof in accordance with the invention may be administered at dosage levels sufficient to deliver from about 0.001 mg/kg to about 100 mg/kg, from about 0.01 mg/kg to about 50 mg/kg, from about 0.1 mg/kg to about 40 mg/kg, from about 0.5 mg/kg to about 30 mg/kg, from about 0.01 mg/kg to about 10 mg/kg, from about 0.1 mg/kg to about 10 mg/kg, or from about 1 mg/kg to about 25 mg/kg of subject body weight per day to obtain the desired therapeutic effect. The desired dosage may be delivered more than three times per day, three times per day, two times per day, once per day, every other day, every third day, every week, every two weeks, every three weeks, every four weeks, every two months, every six months, or every twelve months. In certain embodiments, the desired dosage may be delivered using multiple administrations (e.g., two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or more administrations).

Prophylactic Applications

[0197] In some embodiments, ETVl antibodies in accordance with the invention may be utilized for prophylactic applications. In some embodiments, prophylactic applications involve systems and methods for preventing, inhibiting progression of, and/or delaying the onset of GIST, prostate cancer, and/or any other ETVl -associated condition in individuals susceptible to and/or displaying symptoms of GIST, prostate cancer, and/or any other ETVl -associated condition. In some embodiments, prophylactic applications involve systems and methods for preventing, inhibiting progression of, and/or delaying the onset of GIST formation. In some embodiments, prophylactic applications involve systems and methods for preventing, inhibiting progression of, and/or delaying the onset of GIST disease. In some embodiments, prophylactic applications involve systems and methods for preventing, inhibiting progression of, and/or delaying the onset of gastrointestinal failure.

Combination Therapy

[0198] It will be appreciated that ETVl antibodies in accordance with the present invention and/or pharmaceutical compositions thereof can be employed in combination therapies. By "in combination with," it is not intended to imply that the agents must be administered at the same time and/or formulated for delivery together, although these methods of delivery are within the scope of the invention. Compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutics or medical procedures. In will be appreciated that therapeutically active agents utilized in combination may be

administered together in a single composition or administered separately in different

compositions. In general, each agent will be administered at a dose and/or on a time schedule determined for that agent. [0199] The particular combination of therapies (e.g., therapeutics or procedures) to employ in a combination regimen will take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that pharmaceutical compositions of the provided antibodies can be employed in combination therapies (e.g., combination chemotherapeutic therapies), that is, the pharmaceutical

compositions can be administered concurrently with, prior to, or subsequent to, one or more other desired therapeutic and/or chemotherapeutic procedures.

[0200] Therapeutically effective amounts of antibodies in accordance with the invention combined with for use in combination with a provided pharmaceutical composition and at least one other active ingredient. For example, provided antibodies, or a pharmaceutically acceptable composition thereof, are administered in combination with chemotherapeutic agents to treat proliferative diseases and cancer such as GIST. In some embodiments, an active ingredient is a chemotherapeutic agent, such as, but not limited to, Adriamycin, dexamethasone, vincristine, cyclophosphamide, fluorouracil, topotecan, taxol, interferons, platinum derivatives, taxane (e.g., paclitaxel), vinca alkaloids (e.g., vinblastine), anthracyclines (e.g., doxorubicin),

epipodophyllotoxins (e.g., etoposide), cisplatin, methotrexate, actinomycin D, actinomycin D, dolastatin 10, colchicine, emetine, trimetrexate, metoprine, cyclosporine, daunorubicin, teniposide, amphotericin, alkylating agents (e.g., chlorambucil), 5 -fluorouracil, campthothecin, cisplatin, metronidazole, imatinib, Gleevec™, sunitinib and Sutent® and combinations thereof.

[0201] In certain embodiments, the provided antibodies, or a pharmaceutically acceptable composition thereof, are administered in combination with an antiproliferative or

chemotherapeutic agent selected from any one or more of Abarelix, aldesleukin, Aldesleukin, Alemtuzumab, Alitretinoin, Allopurinol, Altretamine, Amifostine, Anastrozole, Arsenic trioxide, Asparaginase, Azacitidine, BCG Live, Bevacuzimab, Avastin, Fluorouracil, Bexarotene, Bleomycin, Bortezomib, Busulfan, Calusterone, Capecitabine, Camptothecin, Carboplatin, Carmustine, Celecoxib, Cetuximab, Chlorambucil, Cisplatin, Cladribine, Clofarabine,

Cyclophosphamide, Cytarabine, Dactinomycin, Darbepoetin alfa, Daunorubicin, Denileukin, Dexrazoxane, Docetaxel, Doxorubicin (neutral), Doxorubicin hydrochloride, Dromostanolone Propionate, Epirubicin, Epoetin alfa, Erlotinib, Estramustine, Etoposide Phosphate, Etoposide, Exemestane, Filgrastim, floxuridine fludarabine, Fulvestrant, Gefitinib, Gemcitabine, Gemtuzumab, Goserelin Acetate, Histrelin Acetate, Hydroxyurea, Ibritumomab, Idarubicin, Ifosfamide, Imatinib Mesylate, Interferon Alfa-2a, Interferon Alfa-2b, Irinotecan, Lenalidomide, Letrozole, Leucovorin, Leuprolide Acetate, Levamisole, Lomustine, Megestrol Acetate,

Melphalan, Mercaptopurine, 6-MP, Mesna, Methotrexate, Methoxsalen, Mitomycin C, Mitotane, Mitoxantrone, Nandrolone, Nelarabine, Nofetumomab, Oprelvekin, Oxaliplatin, Paclitaxel, Palifermin, Pamidronate, Pegademase, Pegaspargase, Pegfilgrastim, Pemetrexed Disodium, Pentostatin, Pipobroman, Plicamycin, Porfimer Sodium, Procarbazine, Quinacrine, Rasburicase, Rituximab, Sargramostim, Sorafenib, Streptozocin, Sunitinib Maleate, Talc, Tamoxifen,

Temozolomide, Teniposide, VM-26, Testolactone, Thioguanine, 6-TG, Thiotepa, Topotecan, Toremifene, Tositumomab, Trastuzumab, Tretinoin, ATRA, Uracil Mustard, Valrubicin, Vinblastine, Vincristine, Vinorelbine, Zoledronate, or Zoledronic acid.

[0202] The particular combination of therapies (e.g., imatinib, sunitinib, sutent,

GLEEVEC™, therapeutic antibodies to ETV1, etc.) to employ in a combination regimen will generally take into account compatibility of the desired therapeutics and/or procedures and the desired therapeutic effect to be achieved. It will also be appreciated that the therapies and/or chemotherapeutics employed may achieve a desired effect for the same disorder (for example, an inventive antigen may be administered concurrently with another chemotherapeutic), or they may achieve different effects.

[0203] It will be appreciated that the therapies employed may achieve a desired effect for the same purpose (for example, ETV1 antibodies useful for treating, preventing, and/or delaying the onset of GIST may be administered concurrently with another agent useful for treating, preventing, and/or delaying the onset of GIST), or they may achieve different effects (e.g., control of any adverse effects). The invention encompasses the delivery of pharmaceutical compositions in combination with agents that may improve their bioavailability, reduce and/or modify their metabolism, inhibit their excretion, and/or modify their distribution within the body.

[0204] In some embodiments, agents utilized in combination with be utilized at levels that do not exceed the levels at which they are utilized individually. In some embodiments, the levels utilized in combination will be lower than those utilized individually. [0205] In some embodiments, provided antibodies in accordance with the reference antibodies (e.g., a hybridoma cell line that secretes human monoclonal antibody ETVl, deposited in the American Type Culture Collection (ATCC) as set forth in Table 1 may be administered with imatinib, with sunitinib, with SUTENT®, and/or with GLEEVEC™, or any combination thereof (e.g., with both SUTENT® and GLEEVEC™).

[0206] In some embodiments, combination therapy may involve administrations of a plurality of antibodies directed to a single epitope (e.g., a single conformational epitope). In some embodiments, combination therapy can comprise a plurality of antibodies that recognize distinct epitopes.

[0207] In certain embodiments, compositions in accordance with the provided antibodies comprise exactly one antibody to ETVl (e.g., Mab 10D12C5B4D4, Mab 2F11G11D10D11, Mab 14D1F8C4E6, Mab 8E6B7C9D10, Mab 13A7E12G5A5, Mab 2F11G11D10B4, and/or Mab 13A7E12G5B8). In certain embodiments, compositions include and/or combination therapy utilize exactly two, exactly three, exactly four, exactly five, exactly six, exactly seven, or more than seven ETVl antibodies. In some embodiments, compositions comprise and/or combination therapy utilize all possible permutations and combinations of antibodies Mab 10D12C5B4D4, Mab 2F11G11D10D11, Mab 14D1F8C4E6, Mab 8E6B7C9D10, Mab 13A7E12G5A5, Mab 2F11G11D10B4, and/or Mab 13A7E12G5B8. Exemplary compositions comprising and/or combination therapy requirements utilize 1, 2, 3, 4, 5, 6 or 7 antibodies selected from the group consisting of Mab 10D12C5B4D4, Mab 2F11G11D10D11, Mab 14D1F8C4E6, Mab

8E6B7C9D10, Mab 13A7E12G5A5, Mab 2F11G11D10B4, and Mab 13A7E12G5B8 are shown in Table 3 :

Exemplary Compositions Comprising ETVl Antibodies

One Antibody Two Antibodies Three Antibodies Four Antibodies Five Antibodies Six Antibodies Seven Antibodies

Mab Mab Mab Mab Mab Mab

2F11G11D10B4 2F11G11D10D11 2F11G11D10D11, 2F11G11D10D11, 2F11G11D10D11, 2F11G11D10D11,

and Mab Mab Mab Mab Mab

13A7E12G5B8 10D12C5B4D4 10D12C5B4D4, 8E6B7C9D10, 10D12C5B4D4,

and Mab Mab 14D1F8C4E6 Mab Mab

14D1F8C4E6 and Mab 10D12C5B4D4, 14D1F8C4E6,

2F11G11D10B4 Mab Mab

2F11G11D10B4 13A7E12G5A5,

and Mab Mab

13A7E12G5B8 2F11G11D10B4

and Mab

13A7E12G5B8

Mab Mab Mab Mab Mab Mab

13A7E12G5B8 8E6B7C9D10 and 2F11G11D10D11, 2F11G11D10D11, 2F11G11D10D11, 8E6B7C9D10,

Mab Mab Mab Mab Mab

10D12C5B4D4 10D12C5B4D4 10D12C5B4D4, 8E6B7C9D10, 10D12C5B4D4,

and Mab Mab 14D1F8C4E6 Mab Mab

13A7E12G5A5 and Mab 14D1F8C4E6, 14D1F8C4E6,

13A7E12G5B8 Mab Mab

13A7E12G5A5 13A7E12G5A5,

and Mab Mab

2F11G11D10B4 2F11G11D10B4

and Mab

13A7E12G5B8

Mab Mab Mab Mab

8E6B7C9D10 and 2F11G11D10D11, 2F11G11D10D11, 2F11G11D10D11,

Mab 14D1F8C4E6 Mab Mab Mab

10D12C5B4D4 14D1F8C4E6, 8E6B7C9D10,

and Mab Mab Mab

2F11G11D10B4 13A7E12G5A5 14D1F8C4E6,

and Mab Mab

2F11G11D10B4 13A7E12G5A5

and Mab

13A7E12G5B8

Mab Mab Mab Mab

8E6B7C9D10 and 2F11G11D10D11, 2F11G11D10D11, 2F11G11D10D11,

Mab Mab Mab Mab

13A7E12G5A5 10D12C5B4D4 14D1F8C4E6, 8E6B7C9D10,

and Mab Mab Mab

13A7E12G5B8 13A7E12G5A5 13A7E12G5A5,

and Mab Mab

13A7E12G5B8 2F11G11D10B4

and Mab

13A7E12G5B8

Mab Mab Mab Mab

8E6B7C9D10 and 2F11G11D10D11, 2F11G11D10D11, 2F11G11D10D11,

Mab Mab 14D1F8C4E6 Mab Mab

2F11G11D10B4 and Mab 13A7E12G5A5, 10D12C5B4D4,

13A7E12G5A5 Mab Mab

2F11G11D10B4 14D1F8C4E6,

and Mab Mab

13A7E12G5B8 13A7E12G5A5

and Mab

2F11G11D10B4

Mab Mab Mab Mab

8E6B7C9D10 and 2F11G11D10D11, 2F11G11D10D11, 2F11G11D10D11,

Mab Mab 14D1F8C4E6 Mab Mab

13A7E12G5B8 and Mab 8E6B7C9D10, 10D12C5B4D4,

2F11G11D10B4 Mab 14D1F8C4E6 Mab

and Mab 14D1F8C4E6,

13A7E12G5A5 Mab

13A7E12G5A5

and Mab

13A7E12G5B8 One Antibody Two Antibodies Three Antibodies Four Antibodies Five Antibodies Six Antibodies Seven Antibodies

Mab Mab Mab Mab

10D12C5B4D4 2F11G11D10D11, 2F11G11D10D11, 2F11G11D10D11,

and Mab Mab 14D1F8C4E6 Mab Mab

14D1F8C4E6 and Mab 8E6B7C9D10, 14D1F8C4E6,

13A7E12G5B8 Mab 14D1F8C4E6 Mab

and Mab 13A7E12G5A5,

2F11G11D10B4 Mab

2F11G11D10B4

and Mab

13A7E12G5B8

Mab Mab Mab Mab

10D12C5B4D4 2F11G11D10D11, 2F11G11D10D11, 8E6B7C9D10,

and Mab Mab Mab Mab

13A7E12G5A5 13A7E12G5A5 8E6B7C9D10, 10D12C5B4D4,

and Mab Mab 14D1F8C4E6 Mab

2F11G11D10B4 and Mab 14D1F8C4E6,

13A7E12G5B8 Mab

13A7E12G5A5

and Mab

2F11G11D10B4

Mab Mab Mab Mab

10D12C5B4D4 2F11G11D10D11, 2F11G11D10D11, 8E6B7C9D10,

and Mab Mab Mab Mab

2F11G11D10B4 13A7E12G5A5 8E6B7C9D10, 10D12C5B4D4,

and Mab Mab Mab

13A7E12G5B8 13A7E12G5A5 14D1F8C4E6,

and Mab Mab

2F11G11D10B4 13A7E12G5A5

and Mab

13A7E12G5B8

Mab Mab Mab Mab

10D12C5B4D4 2F11G11D10D11, 2F11G11D10D11, 8E6B7C9D10,

and Mab Mab Mab Mab

13A7E12G5B8 2F11G11D10B4 8E6B7C9D10, 10D12C5B4D4,

and Mab Mab Mab

13A7E12G5B8 13A7E12G5A5 14D1F8C4E6,

and Mab Mab

13A7E12G5B8 2F11G11D10B4

and Mab

13A7E12G5B8

Mab 14D1F8C4E6 Mab Mab Mab

and Mab 8E6B7C9D10, 2F11G11D10D11, 8E6B7C9D10,

13A7E12G5A5 Mab Mab Mab

10D12C5B4D4 14D1F8C4E6, 10D12C5B4D4,

and Mab Mab Mab

14D1F8C4E6 13A7E12G5A5 13A7E12G5A5,

and Mab Mab

2F11G11D10B4 2F11G11D10B4

and Mab

13A7E12G5B8

Mab 14D1F8C4E6 Mab Mab Mab

and Mab 8E6B7C9D10, 2F11G11D10D11, 8E6B7C9D10,

2F11G11D10B4 Mab Mab Mab

10D12C5B4D4 14D1F8C4E6, 14D1F8C4E6,

and Mab Mab Mab

13A7E12G5A5 2F11G11D10B4 13A7E12G5A5,

and Mab Mab

13A7E12G5B8 2F11G11D10B4

and Mab

13A7E12G5B8 One Antibody Two Antibodies Three Antibodies Four Antibodies Five Antibodies Six Antibodies Seven Antibodies

Mab 14D1F8C4E6 Mab Mab Mab

and Mab 8E6B7C9D10, 2F11G11D10D11, 10D12C5B4D4,

13A7E12G5B8 Mab Mab Mab

10D12C5B4D4 13A7E12G5A5, 14D1F8C4E6,

and Mab Mab Mab

2F11G11D10B4 2F11G11D10B4 13A7E12G5A5,

and Mab Mab

13A7E12G5B8 2F11G11D10B4

and Mab

13A7E12G5B8

Mab Mab Mab

13A7E12G5A5 8E6B7C9D10, 8E6B7C9D10,

and Mab Mab Mab

2F11G11D10B4 10D12C5B4D4 10D12C5B4D4,

and Mab Mab 14D1F8C4E6

13A7E12G5B8 and Mab

13A7E12G5A5

Mab Mab Mab

13A7E12G5A5 8E6B7C9D10, 8E6B7C9D10,

and Mab Mab 14D1F8C4E6 Mab

13A7E12G5B8 and Mab 10D12C5B4D4,

13A7E12G5A5 Mab 14D1F8C4E6

and Mab

2F11G11D10B4

Mab Mab Mab

2F11G11D10B4 8E6B7C9D10, 8E6B7C9D10,

and Mab Mab 14D1F8C4E6 Mab

13A7E12G5B8 and Mab 10D12C5B4D4,

2F11G11D10B4 Mab 14D1F8C4E6

and Mab

13A7E12G5B8

Mab Mab

8E6B7C9D10, 8E6B7C9D10,

Mab 14D1F8C4E6 Mab

and Mab 10D12C5B4D4,

13A7E12G5B8 Mab

13A7E12G5A5

and Mab

2F11G11D10B4

Mab Mab

8E6B7C9D10, 8E6B7C9D10,

Mab Mab

13A7E12G5A5 10D12C5B4D4,

and Mab Mab

2F11G11D10B4 13A7E12G5A5

and Mab

13A7E12G5B8

Mab Mab

8E6B7C9D10, 8E6B7C9D10,

Mab Mab

13A7E12G5A5 10D12C5B4D4,

and Mab Mab

13A7E12G5B8 2F11G11D10B4

and Mab

13A7E12G5B8

Mab Mab

8E6B7C9D10, 8E6B7C9D10,

Mab Mab

2F11G11D10B4 14D1F8C4E6,

and Mab Mab

13A7E12G5B8 13A7E12G5A5

and Mab

2F11G11D10B4 One Antibody Two Antibodies Three Antibodies Four Antibodies Five Antibodies Six Antibodies Seven Antibodies

Mab Mab

10D12C5B4D4, 8E6B7C9D10,

Mab 14D1F8C4E6 Mab

and Mab 14D1F8C4E6,

13A7E12G5A5 Mab

13A7E12G5A5

and Mab

13A7E12G5B8

Mab Mab

10D12C5B4D4, 8E6B7C9D10,

Mab 14D1F8C4E6 Mab

and Mab 13A7E12G5A5,

2F11G11D10B4 Mab

2F11G11D10B4

and Mab

13A7E12G5B8

Mab Mab

10D12C5B4D4, 10D12C5B4D4,

Mab 14D1F8C4E6 Mab

and Mab 14D1F8C4E6,

13A7E12G5B8 Mab

13A7E12G5A5

and Mab

2F11G11D10B4

Mab Mab

10D12C5B4D4, 10D12C5B4D4,

Mab Mab

13A7E12G5A5 14D1F8C4E6,

and Mab Mab

2F11G11D10B4 13A7E12G5A5

and Mab

13A7E12G5B8

Mab Mab

10D12C5B4D4, 10D12C5B4D4,

Mab Mab

13A7E12G5A5 13A7E12G5A5,

and Mab Mab

13A7E12G5B8 2F11G11D10B4

and Mab

13A7E12G5B8

Mab Mab

10D12C5B4D4, 14D1F8C4E6,

Mab Mab

2F11G11D10B4 13A7E12G5A5,

and Mab Mab

13A7E12G5B8 2F11G11D10B4

and Mab

13A7E12G5B8

Mab

14D1F8C4E6,

Mab

13A7E12G5A5

and Mab

2F11G11D10B4

Mab

14D1F8C4E6,

Mab

13A7E12G5A5

and Mab

13A7E12G5B8

Mab

13A7E12G5A5,

Mab

2F11G11D10B4

and Mab

13A7E12G5B8 [0208] It will be appreciated by one of skill in the art that any permutation or combination of antibodies Mab 10D12C5B4D4, Mab 2F11G11D10D11, Mab 14D1F8C4E6, Mab 8E6B7C9D10, Mab 13A7E12G5A5, Mab 2F11G11D10B4, and/or Mab 13A7E12G5B8 can be combined with any other antibody (e.g., antibodies that recognize ETV1) to formulate compositions and/or combination therapy regimens comprising a plurality of different antibodies.

Diagnostic Applications

[0209] In some embodiments, the provided antibodies are used for diagnostic

applications. For example, by virtue of the variety of binding profiles of ETV1 antibodies, diagnostic assays may be employed which will detect a plurality of tumor class and/or subclass, so as to provide a pan-ETVl antibody, while at the same time being able to dissect individual genotypes and/or subtypes by subtractive analysis.

[0210] The function of ETV1 as a master regulator facilitates diagnostic uses of ETV1 expression and activity levels. It is well known in the art that the gene expression profile of a tumor or subclass of tumor may be associated with different prognoses. Such information may include, but is not limited to, the average life expectancy of a patient, the likelihood that a patient will survive for a given amount of time (e.g., 6 months, 1 year, 5 years, etc.), the likelihood that a patient will be cured of a disease, the likelihood that a patient's disease will respond to a particular therapy (wherein response may be defined in any of a variety of ways). For example, differences in the prognosis of patients with KIT positive and PDGFRA positive GISTs have been described.

[0211] In certain embodiments, methods of the present invention utilize a novel parameter for diagnosis and prognosis of GIST cancers: ETV1 expression and/or activity. In a particular embodiment, a method is provided for determining the likelihood of gastrointestinal stromal tissue being cancerous and/or afflicted by a GIST. The method comprises determining a level of expression or activity of ETV1 in a gastrointestinal stromal tissue sample obtained from a subject; comparing the determined ETV1 level with that of a reference correlated with predetermined probablity of being cancerous and/or afflicted by a GIST; and based on the comparing, determining that the tissue sample has an increased or decrease probability, relative to the reference, of being cancerous and/or afflicted by a GIST. Moreover, the present invention offers the possibility of providing additional diagnostic, prognostic, or predictive information based on modification of classification methods to include ETV1.

[0212] The present invention also offers the possibility of analyzing tumor sample archives containing tissue samples that were obtained from patients and stored with information regarding the progress of the patient's disease. In general such archives consist of tumor samples embedded in paraffin blocks. These tumor samples can be analyzed for their expression of polypeptides encoded by the marker genes of the present invention, particularly ETV1. For example, immunohisto chemistry can be performed using antibodies that bind to the

polypeptides. Tumors with elevated, mutated or otherwise aberrant expression may then be identified and correlated with the severity of the tumor, responses to therapy and other avaible clincial information, e.g., age at death, length of survival, etc. ETV1 expression in the samples may be quantitated or semi-quantitated relative to a control. The information derived from these samples may be used to construct a reference database. In particular, the average expression across a population or subpopulation of normal and afflicted individuals may be determined, thereby creating a reference correlated with a predetermined probability of being cancerous. Once suitable prognostic or predictive correlations are identified, a patient's likely outcome can be predicted based on the level and type of ETV1 expression in his or her tumor.

[0213] In some embodiments, ETV1 expression analysis comprises identifying whether

ETV1 is upregulated or downregulated relative to a reference. The reference may be derived from average expression across a population of individuals or may be single prior sample derived from the subject. Differential expression relative to the reference can then be determined. The identification can be performed using a statistical test to determine statistical significance of any differential expression observed. In some embodiments, statistical significance is determined using a parametric statistical test. The parametric statistical test can comprise, for example, a fractional factorial design, analysis of variance (ANOVA), a t-test, least squares, a Pearson correlation, simple linear regression, nonlinear regression, multiple linear regression, or multiple nonlinear regression. Alternatively, the parametric statistical test can comprise a one-way analysis of variance, two-way analysis of variance, or repeated measures analysis of variance. In other embodiments, statistical significance is determined using a nonparametric statistical test. Examples include, but are not limited to, a Wilcoxon signed-rank test, a Mann- Whitney test, a Kruskal-Wallis test, a Friedman test, a Spearman ranked order correlation coefficient, a Kendall Tau analysis, and a nonparametric regression test. In some embodiments, statistical significance is determined at a p-value of less than about 0.05, 0.01, 0.005, 0.001, 0.0005, or 0.0001.

[0214] The degree of differential expression can also be taken into account. For example, ETV1 can be considered as differentially expressed when the fold-change in expression compared to control level is at least 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.2, 2.5, 2.7, 3.0, 4, 5, 6, 7, 8, 9 or 10-fold different in the sample versus the control. The differential expression takes into account both overexpression and underexpression. ETV1 can be considered up or downregulated if the differential expression meets a statistical threshold, a fold-change threshold, or both. For example, the criteria for identifying differential expression can comprise both a p- value of 0.001 and fold change of at least 2.0-fold (up or down).

[0215] In some embodiments, the reference to which differential expression of ETV1 is determined is a normal gene product. For example, the reference may be a housekeeping gene product such as 18S RNA or GAPDH. In alternative embodiments, differential expression may be determined relative to ETV1 expression in noncancerous gastrointestinal ICCs derived from healthy subjects. In other embodiments, differential expression may be determined relative to ETV1 expression in cancerous ICCs cells or GIST cell lines. Fold changes in ETV1 expression may be calculated by the delta-delta Ct method (M.W. Pfaffl, A new mathematical model for relative quantification in real-time RT-PCR, Nucleic Acids Res. 29 (2001) e45) or other methods known to those of skill in the art.

[0216] In one aspect, the invention provides a method of determining ETV1 levels or activity and correlating the results obtained therein with a diagnosis or prognostic assessement of GIST cancer and severity. In certain embodiments it may prove advantageous to combine detection of the ETV1 marker with detection of the KIT and/or PDGFRA markers. [0217] As is well known in the art, a polypeptide may be detected using any of a variety of techniques. Antibody detection methods are well known in the art including, but are not limited to, enzyme-linked immunosorbent assays (ELISAs) and Western blots. Some such methods are amenable to being performed in an array format.

[0218] In certain embodiments of the inventive methods, a single provided antibody is used whereas in other embodiments of the invention multiple provided antibodies, directed either against the same or against different target can be used to increase the sensitivity or specificity of the detection technique or to provide more detailed information than that provided by a single provided antibody. Thus the invention encompasses the use of a battery of provided antibodies. Of course these agents can also be used in conjunction with antibodies against polypeptides encoded by other useful marker genes (e.g., CD34 when used with GISTs).

[0219] In general, the inventive polypeptides are detected within a sample that has been obtained from a subject, e.g., a tissue sample, cell sample, cell extract, body fluid sample, etc. The invention encompasses the recognition that ETV1 (or portions thereof) may be present in serum, enabling detection through a blood test rather than requiring a biopsy specimen. Similar methods may be applied to other body fluid samples, e.g., ascites, urine, saliva, etc.

[0220] In certain embodiments, binding can be detected by adding a detection entity to a provided antibody as discussed in the the following section. In certain embodiments, the detection techniques of the present invention will include a negative control, which can involve applying the test to a control sample (e.g., from a normal non-cancerous tissue) so that the signal obtained thereby can be compared with the signal obtained from the sample being tested.

[0221] In general, the results of the inventive detection techniques can be presented in any of a variety of formats. The results can be presented in a qualitative fashion. For example, the test report may indicate only whether or not ETV1 was detected, perhaps also with an indication of the limits of detection. The results may be presented in a semi-quantitative fashion. For example, various ranges may be defined, and the ranges may be assigned a score (e.g., 0 to 3 where 0 means no binding detected and 3 means strong binding detected) that provides a certain degree of quantitative information. Such a score may reflect various factors, e.g., the number of cells in which ETV1 is detected, the intensity of the signal (which may indicate the level of expression of ETV1), etc. The results may be presented in a quantitative fashion, e.g., as a percentage of cells in which ETV1 is detected, as a protein concentration, etc. As will be appreciated by one of ordinary skill in the art, the type of output provided by a test will vary depending upon the technical limitations of the test and the biological significance associated with detection of the polypeptide.

Detection entities

[0222] In some embodiments, the provided antibodies are used for detection applications.

Multifunctional agents described herein may be used which comprise at least one detection entity, in addition to a provided antibody as described herein.

[0223] A detection entity may be any entity that allows detection of ETV1 protein after binding to a tissue or system of interest. Any of a wide variety of detectable agents can be used as detection entity (e.g., labeling moieties) in multifunctional antibody agents of the provided antibodies. A detection entity may be directly detectable or indirectly detectable. Examples of detection entity include, but are not limited to: various ligands, radionuclides (e.g., 3H, 14C, 18F, 19F, 32P, 35S, 1351, 1251, 1231, 64Cu, 187Re, l l lln, 90Y, 99mTc, 177Lu, etc.), fluorescent dyes (for specific exemplary fluorescent dyes, see below), chemiluminescent agents (such as, for example, acridinum esters, stabilized dioxetanes, and the like), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductors nanocrystals (i.e., quantum dots), metal nanoparticles (e.g., gold, silver, copper, platinum, etc.) nanoclusters, paramagnetic metal ions, enzymes (for specific examples of enzymes, see below), colorimetric labels (such as, for example, dyes, colloidal gold, and the like), biotin, dioxigenin, haptens, and proteins for which antisera or monoclonal antibodies are available.

[0224] In certain embodiments, a detection entity comprises a fluorescent label.

Numerous known fluorescent labeling moieties of a wide variety of chemical structures and physical characteristics are suitable for use in the practice of methods of diagnosis of the present invention. Suitable fluorescent dyes include, but are not limited to, fluorescein and fluorescein dyes (e.g., fluorescein isothiocyanine or FITC, naphthofluorescein, 4',5'-dichloro-2',7'- dimethoxyfluorescein, β carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryl dyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes (e.g., carboxytetramethyl- rhodamine or TAMRA, carboxyrhodamine 6G, carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G, rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.), coumarin and coumarin dyes (e.g. , methoxycoumarin, dialkylaminocoumarin,

hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Oregon Green Dyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514., etc.), Texas Red, Texas Red-X, Spectrum Red™, Spectrum Green™, cyanine dyes (e.g., Cy-3™, Cy-5™, Cy-3.5™, Cy-5.5™ etc.), Alexa Fluor dyes (e.g., Alexa Fluor 350, Alexa Fluor 488, Alexa Fluor 532, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680, etc.), BODIPY dyes (e.g., BODIPY FL, BODIPY R6G, BODIPY TMR, BODIPY TR, BODIPY 530/550, BODIPY 558/568, BODIPY 564/570, BODIPY 576/589, BODIPY 581/591, BODIPY 630/650, BODIPY 650/665, etc.), IRDyes (e.g., IRD40, IRD 700, IRD 800, etc.), and the like. For more examples of suitable fluorescent dyes and methods for coupling fluorescent dyes to other chemical entities such as proteins and peptides, see, for example, "The Handbook of Fluorescent Probes and Research Products", 911 lEd., Molecular Probes, Inc., Eugene, OR.

[0225] Favorable properties of fluorescent labeling agents include high molar absorption coefficient, high fluorescence quantum yield, and photostability. In certain embodiments, labeling fluorophores desirably exhibit absorption and emission wavelengths in the visible (i.e., between 400 and 750 nm) rather than in the ultraviolet range of the spectrum (i.e., lower than 400 nm).

[0226] In certain embodiments, a detection entity comprises an enzyme. Examples of suitable enzymes include, but are not limited to, those used in an ELISA, e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase, etc. Other examples include beta-glucuronidase, beta-D-glucosidase, urease, glucose oxidase, etc. An enzyme may be conjugated to a targeting entity (e.g., chlorotoxin moiety) using a linker group such as a carbodiimide, a diisocyanate, a glutaraldehyde, and the like. More detailed description of suitable linkers is provided elsewhere herein.

[0227] In certain embodiments, a detection entity comprises a radioisotope that is detectable by Single Photon Emission Computed Tomography (SPECT) or Position Emission Tomography (PET). Examples of such radionuclides include, but are not limited to, iodine-131 (1311), iodine-125 (1251), bismuth-212 (212Bi), bismuth-213 (213Bi), astatine-221 (211At), copper-67 (67Cu), copper-64 (64Cu), rhenium-186 (186Re), rhenium-186 (188Re), phosphorus- 32 (32P), samarium- 153 (153Sm), lutetium-177 (117Lu), technetium-99m (99mTc), gallium-67 (67Ga), indium-1 1 1 (11 lln), and thallium-201 (201T1).

[0228] In certain embodiments, a labeling moiety comprises a radioisotope that is detectable by Gamma camera. Examples of such radioisotopes include, but are not limited to, iodine-131 (1311), and technetium-99m (99mTc).

[0229] In certain embodiments, a detection entity comprises a paramagnetic metal ion that is a good contrast enhancer in Magnetic Resonance Imaging (MRI). Examples of such paramagnetic metal ions include, but are not limited to, gadolinium III (Gd3+), chromium III (Cr3+), dysprosium III (Dy3+), iron III (Fe3+), manganese II (Mn2+), and ytterbium III (Yb3+). In certain embodiments, the detection entity comprises gadolinium III (Gd3+). Gadolinium is an FDA-approved contrast agent for MRI, which accumulates in abnormal tissues causing these abnormal areas to become very bright (enhanced) on the magnetic resonance image. Gadolinium is known to provide great contrast between normal and abnormal tissues in different areas of the body, in particular in the brain.

[0230] In certain embodiments, a labeling moiety comprises a stable paramagnetic isotope detectable by nuclear magnetic resonance spectroscopy (MRS). Examples of suitable stable paramagnetic isotopes include, but are not limited to, carbon- 13 (13C) and fluorine- 19 (19F).

Therapy Selection

[0231] Another aspect of the invention relates to the selection of a treatment regimen based on ETV1 over-expression or particular activating mutations. For example, it is known in the art that certain anti-cancer theraperies, particularly chemotherapy and small-molecule pharmaceutical therapies, produce superior result in cancer cells that are positive for a particular mutant allele or over-/under-expression of a particular protein or enzyme. The present invention provides methods of identifying patients with aberrant ETVl expression who may benefit from one therapy over another. For example, therapy may be personalized based on over-expression versus mutation, and sub-categorized based on the type of ETVl mutation and/or other abberations with known GIST-related proteins such as KIT or PDGFRA.

Characterization and/or Identification of ETVl-related Reagents

[0232] In some embodiments, the present invention provides antibodies that can be used to identify and/or characterize one or more agents that mimic an ETVl epitope or agent and/or induce a strong antibody response to ETVl . In some embodiments, a vaccine may be designed to induce production of antibodies that have been found to be lacking in the patient. In some embodiments, it is desirable for vaccine compositions to comprise antigens that have a native conformation, mediate a protective response (e.g., complement activation, virus neutralization, etc.), and/or can induce a strong antibody response. In some embodiments, a vaccine contains an epitope or mimotope thereof to which antibodies are not being produced naturally in the individual. Administration of such a vaccine might induce a patient's immune system to start producing a set of antibodies directed against the administered epitope. It will be appreciated that the mimotopes (or epitopes) in accordance with the invention can be used alone or in combination with recombinant proteins, inhibitors, and/or as a cocktail of several different mimotopes.

Kits

[0233] The invention provides a variety of kits for conveniently and/or effectively carrying out methods in accordance with the present invention. Kits typically comprise one or more ETVl antibodies in accordance with the invention (e.g., Mab 10D12C5B4D4, Mab 2F11G11D10D11, Mab 14D1F8C4E6, Mab 8E6B7C9D10, Mab 13A7E12G5A5, Mab

2F11G11D10B4, and/or Mab 13A7E12G5B8). In some embodiments, kits comprise a collection of different ETVl antibodies to be used for different purposes (e.g., diagnostics, treatment, and/or prophylaxis). Typically kits will comprise sufficient amounts of ETVl antibodies to allow a user to perform multiple administrations to a subject(s) and/or to perform multiple experiments. In some embodiments, kits are supplied with or include one or more ETV1 antibodies that have been specified by the purchaser.

[0234] In certain embodiments, kits for use in accordance with the present invention may include one or more reference samples; instructions (e.g., for processing samples, for performing tests, for interpreting results, for solubilizing ETV1 antibodies, for storage of ETV1 antibodies, etc.); buffers; and/or other reagents necessary for performing tests. In certain embodiments kits can comprise panels of antibodies. Other components of kits may include cells, cell culture media, tissue, and/or tissue culture media.

[0235] Kits may comprise instructions for use. For example, instructions may inform the user of the proper procedure by which to prepare a pharmaceutical composition comprising ETV1 antibodies and/or the proper procedure for administering pharmaceutical compositions to a subject.

[0236] In some embodiments, kits include a number of unit dosages of a pharmaceutical composition comprising ETV1 antibodies. A memory aid may be provided, for example in the form of numbers, letters, and/or other markings and/or with a calendar insert, designating the days/times in the treatment schedule in which dosages can be administered. Placebo dosages, and/or calcium dietary supplements, either in a form similar to or distinct from the dosages of the pharmaceutical compositions, may be included to provide a kit in which a dosage is taken every day.

[0237] Kits may comprise one or more vessels or containers so that certain of the individual components or reagents may be separately housed. Kits may comprise a means for enclosing the individual containers in relatively close confinement for commercial sale, e.g., a plastic box, in which instructions, packaging materials such as styrofoam, etc., may be enclosed.

[0238] In some embodiments, kits are used in the treatment, diagnosis, and/or prophylaxis of a subject suffering from and/or susceptible to GIST. In some embodiments, such kits comprise (i) at least one ETV1 antibody; (ii) a syringe, needle, applicator, etc. for administration of the at least one ETV1 antibody to a subject; and (iii) instructions for use. [0239] In some embodiments, kits are used in the treatment, diagnosis, and/or prophylaxis of a subject suffering from and/or susceptible to GIST. In some embodiments, such kits comprise (i) at least one HCV antibody provided as a lyophilized powder; and (ii) a diluent for reconstituting the lyophilized powder. Such kits may optionally comprise a syringe, needle, applicator, etc. for administration of the at least one ETV1 antibody to a subject; and/or instructions for use.

[0240] The present invention provides kits containing reagents for the generation of vaccines comprising at least one ETV1 antibody. In some embodiments, such kits may include cells expressing ETV1 antibodies, characteristic portions thereof, and/or biologically active portions thereof; (ii) media for growing the cells; and (iii) columns, resin, buffers, tubes, and other tools useful for antibody purification. In some embodiments, such kits may include (i) plasmids containing nucleotides encoding ETV1 antibodies, characteristic portions thereof, and/or biologically active portions thereof; (ii) cells capable of being transformed with the plasmids, such as mammalian cell lines, including but not limited to, Vera and MDCK cell lines; (iii) media for growing the cells; (iv) expression plasmids containing no nucleotides encoding ETV1 antibodies as negative controls; (v) columns, resin, buffers, tubes, and other tools useful for antibody purification; and (vi) instructions for use.

[0241] In some embodiments, kits are used to detect the presence of ETV1 in one or more samples. Such samples may be pathological samples, including, but not limited to, blood, serum/plasma, peripheral blood mononuclear cells/peripheral blood lymphocytes (PBMC/PBL), sputum, urine, feces, throat swabs, dermal lesion swabs, cerebrospinal fluids, cervical smears, pus samples, food matrices, and tissues from various parts of the body such as brain, spleen, intestine, prostate and liver. Such samples may be environmental samples, including, but not limited to, soil, water, and flora. Other samples that have not been listed may also be applicable. In some embodiments, such kits comprise (i) at least one ETV1 antibody; (ii) a sample known to contain ETV1, as a positive control; and (iii) a sample known not to contain ETV1, as a negative control; and (iv) instructions for use. [0242] These and other aspects of the present invention will be further appreciated upon consideration of the following Examples, which are intended to illustrate certain particular embodiments of the invention but are not intended to limit its scope, as defined by the claims.

Examples

Example 1: Identification and Characterization of human ETV1 monoclonal antibodies

[0243] The present Example describes identification and characterization of various monoclonal antibodies provided in accordance with the present invention.

[0244] Among other things, the present Example describes a set of monoclonal antibodies related in sequence to the provided reference antibodies.

[0245] Among other things, the present Example describes certain ETV1 monoclonal antibodies that bind human ETV1 upregulated in gastrointestinal stromal tumors (GIST).

Materials and Methods

Antibody Production and Isolation

[0246] The peptide sequence CNPHP YNEQ YVY (SEQ ID NO : 31 ) was used as the immunizing antigen for ETV1 antibody production. The antigen was conjugated to KLH using Imject Maleimide -Activated mcKLH kit from Pierce. One 8-week old Wistar rat was

immunized with 100 μg peptide-KLH conjugate along with Complete Frauds Adjuvant. On days 28 and 48 after initial immunization, boosts one and two were performed with 100 μg peptide- KLH conjugate along with Incomplete Frauds Adjuvant. On day 59 after initial immunization, the rat was bled for ELISA against the immunization peptide. A ELISA >1 :40,000 was observed. On day 72, the final boost was performed with 100 μg peptide-KLH conjugated without adjuvant. On day 76, spleen was harvested and spenocytes were fused with myeloma cell (Sp2/0) using polyethylene glycol (PEG). The myeloma-to-spleen cell ratio was 1 : 10. Cells were plated into wells of 96-well plate in HAT media. On day 89, the wells were screened using ELISA and clones in the positive wells were subcloned. On day 103, the subclones were screened and a limited dilution performed to identify and isolate a second subclone. On day 117, the second subclone was screened by ELISA. Positive subclones were then expanded, frozen, and tested for ETVl specificity by Western Blot. Clones that were positive by Western Blot were further expanded.

Results

[0247] FIG. 1 depicts an exemplary Western Blot result illustrating binding of provided monoclonal antibodies to ETVl in a G882 cell line with and without imatinib (IMAT) added. 400ug of protein were loaded per lane in an 11% SDS-polyacrylamide gel. The secondary antibody used was Jackson Catalog #: 115-035-003 in a 1 :5000 dilution. Primary isolated ETVl monoclonal antibodies were loaded as follows - 10D12C5B4D4 (lane 1), 2F11G11D10D11 (lane 2), 14D1F8C4E6 (lane 3), 8E6B7C9D10 (lane 4), 13A7E12G5A5 (lane 5),

2F11G11D10B4 (lane 6), 13A7E12G5B8 (lane 7). Lane 8 was loaded with serum in a 1 :500 dilution.

Example 2: ETVl as a diagnostic, prognostic and therapeutic target for Gastrointestinal stromal tumors or GIST

[0248] The present Examples describes how the ETS family member ETVl is highly expressed in the subtypes of ICCs sensitive to oncogenic KIT mediated transformation (Kwon, J. G. et al, 2009, Gastroenterology, 136: 630-639; incorporated herein by reference), and is required for their development.

[0249] Among other things, the present Example describes how ETVl expression can be used as a diagnostic, prognostic or therapeutic target for GIST.

Materials and Methods Cell lines, Antibodies, and Reagents

[0250] The GIST882 cell line obtained from an imatinib naive patient, harbours a homozygous exon 13 KIT mutation (K642E) and is maintained in RPMI supplemented with 15% FBS, lOmM HEPES pH 7.5. The GIST48 cell line, obtained from an imatinib-resistant patient, harbour a homozygous exon 11 KIT mutation (V560D) and a secondary heterozygous Exon 17 KIT mutation (D820A), is maintained in Ham's F10 media supplemented with 15% FBS, 0.5% MITO+ Serum Extender (BD Biosciences), and 30 mg/L bovine pituitary extract (BD

Biosciences)( Bauer, S., et al, 2006, Cancer Res, 66: 9153-9161; incorporated herein by reference). The U20S osteosarcoma, LNCaP prostate cancer, and NIH-3T3 mouse embryonic fibroblast cells were obtained from ATCC and cultured as recommended.

[0251] GIST48 and GIST882 cells were established in the Fletcher laboratory (DFCI).

All other cells were obtained from ATCC. Etvl-/- mice, with targeted deletion of the ETS domain, was obtained from the Jessell laboratory (Columbia) and CB17-SCID mice was from Taconic. Antibody sources are: ETV1, ANOl, PGP9.5 (Abeam), KIT for WB, P-Tyr703-KIT (Cell Signaling), P-Tyr204-ERK, GAPDH (Santa Cruz), and anti-mouse Kit for IF (clone ACK2, E-Biosciences).

[0252] The following antibodies are used: rabbit anti-ETVl WB, IHC, and ChIP

(Abeam), mouse anti-KIT for WB (Cell Signaling), rabbit anti-Phospho-tyr703 KIT for WB (Cell Signalling), mouse anti-phospho-tyr204 ERK for WB (Santa Cruz Biotechnologies), mouse anti-GAPDH for WB (Santa Cruz Biotechnologies), ACK2 rat anti-mouse Kit for IF (E- Biosceinces), rabbit anti-PGP9.5 for IF (Abeam), rabbit anti-ANOl for IF (Abeam). Secondary antibodies were Alexa-488 labelled anti-rabbit antibody and Alexa-594 labelled anti-rat antibody (Invitrogen). PD0325901 was synthesized in the MSKCC Organic Synthesis Core Facility by O. Ouerfelli and imatinib was a gift from Novartis.

Expression Data Mining

[0253] Five publically deposited database containing expression of GIST samples are as follows: Expression Project for Oncology (ExpO, GSE2109) containing 7 GIST samples among over 2,000 tumors of all types, NIELSEN SARCOMA (GSE3443) containing 10 GIST samples among 44 sarcoma samples (Nielsen, T. O. et al, 2002, Lancet, 359: 1301-1307; incorporated herein by reference), SEGAL SARCOMA (GSE2719) containing 5 GIST samples among 50 sarcoma samples (Segal, N. H. et al, 2003, Am J Pathol, 163: 691-700; incorporated herein by reference), YAMAGUCHI GIST (GSE8167) with 32 GIST samples (Yamaguchi, U. et al, 2008, J Clin Oncol, 26: 4100-4108; incorporated herein by reference), and OSTROWSKI GIST (GSE 17743) with 29 GIST samples (Ostrowski, J. et al, 2009, BMC Cancer, 9: 413;

incorporated herein by reference). Normalized expression of ETV1 of a given sample was calculated as the Z-score (i.e., standard deviations from median) of ETV1 probe signal among all probes. GIST-signature gene sets derived from three datasets (ExpO, Nielsen, and Segal) containing both GIST and non-GIST malignancies included genes that met the following two criteria: 1) multiple-hypothesis testing significance of q<0.05, and 2) a mean expression that is 1.5 Z-score higher in GIST than non-GIST tumors. The three gene sets, EXPO GIST SIG, NIELSEN GIST SIG, and SEGAL GIST SIG contain 102, 156, and 30 genes respectively.

[0254] For mouse ICC gene expression, we analyzed an expression dataset of FACS sorted ICC-MY, ICC-DMP, and smooth muscle cells from the mouse small intestine (GSE7809)( Chen, H. et al, 2007, Physiol Genomics, 31 : 492-509; incorporated herein by reference). ICC- MY and ICC-DMP signature genes sets were defined as genes expressed higher in ICC-MY and ICC-DMP compared to muscle by 1.5 Z-score with p<0.05 and contain 61 and 81 genes respectively.

Tumor Samples

[0255] Clinical samples from patients with GIST and other sarcomas were obtained according to MSKCC IRB protocol and frozen and paraffin embedded tissue samples were banked. All GIST and non-GIST tumors have been pathologically reviewed and confirmed by a sarcoma expert (CRA). All GIST tumors used have been sequenced for KIT and PDGFA mutations. For tumors in FIG. 2, G1-G8 are GISTs: Gl, G2 and G4 (KIT exonl 1 mutation), G3 (KIT exon 9 mutation), G5 and G6 (KIT and PDGFRA wild-type), G7 and G8 (PDGFRA D842V mutation); S1-S5 are leiomyosarcoma, GI leiomyoma, GI leiomyoma, intra-abdominal desmoid tumor, and GI schwannoma respectively.

FISH of tumor samples

[0256] Two BACs, one covering ETV1 gene body and one on the 3' side (RP11-138H16,

RP11-124L22, CHORI) were labelled with Spectrum Green-d-UTP (Vysis) and two BACs covering 5' upstream region of ETV1 (RP11-703 A4, RP11-115D14) were labelled with

Spectrum Orange-d-UTP. Paraffin-embedded slides were de-waxed with xylene (Fisher), and then heated in lOmM sodium citrate (PH 6) by microwaving for 5 to 10 minutes in a tender cooker (Nordic Ware). Slides were co-denatured with the probes for 6 minutes at 75°C using HYbrite (Vysis), and kept in a dark, moist chamber at 37°C for 16 hours. Slides were then washed with 50% formamide (Fisher)/2XSSC at 45°C according to standard procedure. Nuclei were stained with DAPI, and slides were mounted with Vectashield (Vector). Results were viewed under a Zeiss microscope, and images were captured with an ISIS camera.

RNA isolation and qRT-PCR

[0257] For tissue culture cells, RNA was isolated using RNeasy (Qiagen). For frozen samples of clinical specimens and for explanted xenografts, tissue samples were grounded in 500 μΐ Trizol (Invitrogen) using PowerGen homogenizer (Fisher Scientific), followed by addition of 100 μΐ chloroform. The samples were then centrifuged at 10,000g x 15 minutes. The upper phase was mixed with equal volume of 70% ethanol, and the RNA was further purified using an RNeasy column.

[0258] For qRT-PCR, RNA was reverse transcribed using High-Capacity cDNA Reverse

Transcription Kit (ABI) and PCR was run using Power SYBR master mix (ABI) on a Realplex machine (Eppendorf). Expression was normalized to the ribosomal protein RPL27 (de Jonge, H. J. et al, 2007, PLoS One, 2: e898; incorporated herein by reference). Primers were designed using primer blast (www.ncbi.nlm.nih.gov/tools/primer-blast) except when cited and purchased from Operon. The following primers pairs were used:

CTSL 1 : F : AGGCGCGTGACTGGTTGAGC (SEQ ID NO : 2)

R: TGCATCGCCTTCCACTTGGTCC (SEQ ID NO: 3)

DUSP6 : F : TGCCGGGCGTTCTACCTGGA (SEQ ID NO : 4)

R: GGCGAGCTGCTGCTACACGA (SEQ ID NO: 5)

ETV 1 -Exon23 : F : AAC AGAGATCTGGCTC ATG ATTC A (SEQ ID NO : 6)

R: CTTCTGCAAGCCATGTTTCCTGTA9 (SEQ ID NO: 7)

ETVl-Exon67: F: CTACCCCATGGACCACAGATTT (SEQ ID NO: 8)

R: CTTAAAGCCTTGTGGTGGGAAG9 (SEQ ID NO: 9)

KIT: F: GGGATTTTCTCTGCGTTCTG (SEQ ID NO: 10)

R: GATGGATGGATGGTGGAGAC (SEQ ID NO: 11)

PLAT : F : C ACTGGGCCTGGGC AAAC ATA (SEQ ID NO : 12)

R: CACGTCAGCCTGCGGTTCTTC (SEQ ID NO: 13)

PR0M1 : F: GCCACCGCTCTAGATACTGC (SEQ ID NO: 14)

R: GCTTTTC CT ATGC C AAAC C A (SEQ ID NO: 15)

PTPRE : F : TGCACGCGGAGC AGAAGGTG (SEQ ID NO : 16)

R: GCCGTGCATGGTCTGCAGGT (SEQ ID NO: 17)

RPL27 : F : C ATGGGC AAGAAGAAGATCG (SEQ ID NO : 18)

R: TCCAAGGGGATATCCACAGA (SEQ ID NO: 19)

TIMP3 : F : CCAGCGCAAGGGGCTGAACT (SEQ ID NO : 20)

R: TAGCCGCCCTTCTGCCGGAT (SEQ ID NO: 21)

Lentiviral knockdown

[0259] Two pLKO.l constructs against ETVl (ETVlshl : TRCN0000013923, targeting

GTGGGAGTAATCTAAACATTT (SEQ ID NO: 22) in 3' UTR and ETVlsh2:

TRCN0000013925, targeting CGACCCAGTGTATGAACACAA (SEQ ID NO: 23 in exon 7) were purchased from Open Biosystems and pLKO.l shScr (targeting CCTAAGGTTAAGTCGCCCTCG (SEQ ID NO: 24) was purchased from Addgene (Sarbassov, D. D., et al, 2005, Science, 307: 1098-1101; incorporated herein by reference). Lentiviruses were generated by co-transfecting the shETVl hairpin constructs with psPax2 and pVSVG (Addgene) into 293FT cells (Invitrogen) using Lipofectamine 2000 (Invitrogen). Viruses were harvested at 48 and 72 hours after transfection and concentrated 100-fold using Lenti-X concentrator (Clontech) and stored at -80 in aliquots. Infection was performed in 12-well dishes in the presence of 7.5 μg/ml polybrene (Sigma) by a 1 hour centrifugation at 500 x g. To determine virus titre in GIST48 and GIST882 cells, two steps were taken. First, the relative titre of shScr, ETVlshl, and ETVlsh2 was obtained by counting puromycin resistant colonies after infection of U20S cells, whose growth is insensitive to ETVl knockdown. Next, the absolute titre of the shScr was calculated by counting puromyicn resistant colonies after infection into GIST48 and GIST882 cells.

Stable gene expression

[0260] ETVl cDNA (Open Biosystems) and EGFP were cloned into MSCV-puro.

Human wild-type KIT in MSCV-IRES-EGFP vector was obtained from Gary Gilliland and generation of Δ560 mutation was performed using QuikChange II XL site-directed mutagenesis kit (Stratagene). Retrovirus was produced in 293FT cells by standard methods using ecotropic packaging vector and infected into NIH-3T3 cells at MOI=5. For ETVl expression, cells were selected with puromycin (1 μg/ml). For KIT expression, GFP FACS demonstrated >90% infection and therefore, cells were not further enriched.

Cell growth and cell cycle analysis after shRNA-mediated knockdown

[0261] For GIST48 and GIST882 cells, cells were plated at 2 x 105 cells per well in a 24- well plate on day 0 and infected with pLKO.l hairpin viruses (MOI=5) on day 1. Cells were not further puromycin selected. Triplicate wells were counted every three days until day 13. For U20S cells, cells were plated at 1 x 105 cells per well of 24-well plate on day 0 and infected at MOI=5 in 24-well plates on Day 1. On day 3, they were split into 96-well plates (2,500/well). Viability was assessed using Cell Titer Glo (Promega). For cell cycle analysis, cells were trypsinized on day 4 after infection, fixed in 70% ethanol and incubated with 20 μg/ml propidium iodide (Sigma) and 0.2 mg/ml RNAse A (Invitrogen) for 2 hours prior to FACS analysis on a FACSCalibur™ (BD Biosciences). Fluorescence intensity of Gl and G2 peaks were normalized between samples and cell cycle analysis was performed using Flow Jo software.

Xenograft Growth

[0262] For GIST882, cells were infected at MOI=l on day 0 and briefly selected with 5 μg/ml puromycin between days 2-4. On day 4, 5x106 cells resuspended in 100 μΐ of 1 : 1 mix of growth media and Matrigel™ (BD Biosciences) were subcutaneously injected into CB17-SCID mice (Taconic). Tumor sizes were measured weekly staring 6 weeks after xenografting. After 10 weeks, xenografts were explanted and RNA isolated for qRT-PCR analysis. For NIH-3T3 cells stably expressing combinations of vector, ETVl, KIT, and ΚΙΤΔ560, 1x106 cells were implanted as above and tumor sizes were measured starting 2 weeks after xenografting.

Anchorage independent growth

[0263] Transduced NIH-3T3 cells were suspended in soft agar with 20% calf-serum at

5,000 cells per well in a6-well plate in triplicate. After 3 weeks, each well was stained with 0.5%> crystal violet, imaged and counted using GelCount (Oxford Optronix).

Immunofluorescence

[0264] For ETVl immunofluorescence of paraffin sections of tumor samples, heat- mediated antigen retrieval was performed followed by staining with ETVl antibody at 2.5 μg/ml and Alexa-594 labeled anti-rabbit secondary antibody. [0265] For immunofluorescence of cryostat sections of the mouse GI tract, mouse stomach, small intestine, cecum, and large intestine were dissected and fixed in 4%

paraformaldehyde for 2 hours followed by an overnight incubation in 30% sucrose. They were then embedded in OCT, flash frozen, and cut into 5 μιη sections using a Cryostat. Etvl-/- and Etvl+/+ littermate controls were embedded onto the same block to ensure identical processing. Tissue sections were blocked overnight using 5% goat serum, incubated with primary antibodies at 4° C overnight, and secondary antibody for 2 hours at room temperature. Slides were mounted using Prolong® Gold (Invitrogen) and images were taken on a Nikon Eclipse TE2000-E microscope using a Photometric Coolsnap HQ camera. Images were taken with at 20X (N.A. 0.75) or 60X (N.A. 1.4) objectives. Monochrome images taken with DAPI, FITC, and Texas Red filter sets were pseudo-colored blue, green, and red respectively and merged using Image J (rsbweb.nih.gov/ij/). The exposure, threshold, and maximum were identical between Etvl-/- and Etvl+/+ images and the gamma was 1.0.

[0266] For whole-mount staining, mouse GI tissue was fixed in ice-cold acetone for >1 hours. They were then rehydrated in PBS, stretched, and the mucosa of these GI tissues was blunt dissected away. Samples were then blocked in 5% goat serum, incubated with ACK2 rat- anti-KIT antibody or PGP9.5 antibody, followed by secondary antibody. Images were taken with at 20X (N.A. 0.75) or 60X (N.A. 1.4) objectives with Z-step of 1 μιη through the muscle layer. The Z-stack of images was deconvoluted using Autoquant X2 software (Media Cybernetics). Movies were generated from deconvolued stacks using ImageJ software.

[0267] To quantify ICC subsets, immunofluorescence images from at least four fields each from two Etvl+/+ and Etvl-/- mice (at least eight fields total) of the stomach, small intestine, cecum, and large intestine were examined. A percentage of ICCs were obtained by dividing the total number of KIT-positive immunostaining cells with the number of DAPI- positive nuclei. The results are shown as percentage of residual KIT-positive immunostaining in Etvl-/- mice compared to Etvl+/+ controls.

Expression Profiling [0268] For profiling of ETV1 knockdown, GIST48 and GIST882 cells plated in 12-well plates were infected in triplicates with shScr, and ETVlshl and ETVlsh2 viruses at MOI=5. Four days after infection, R As were isolated from duplicate samples and protein lysates were obtained from the third for confirmatory ETV1 Western Blot. The duplicate samples were expression profiled using an Illumina Human HT-12 array. Raw data was imported into

Genespring 10, quartile normalized and log 2 transformed. The log transformed expression value of each probe was averaged across duplicates. For each probe, the average change by ETV1 knockdown is calculated as:

((ETVlshl GIST48-ShScrGIST48)+(ETVlsh2GIST48-ShScrGIST48)+(ETVlshlGIST882-

ShScr_GisT882)+(ETVlsh2GisT882-ShScr_GisT882))/4

[0269] We next generated a ranked list of genes based on the averaged log2 transformed fold change. The top forty-eight genes showed an averaged decrease of >1.7-fold with ETV1 knockdown and shown in FIG. 3. We defined a "shETVl DN" gene set as the 410 genes with average decrease > 1.4-fold and a "shETVl UP" gene set as the 380 genes with average increase of > 1.4-fold.

[0270] For profiling of imatinib treatment, GIST882 cells were treated with vehicle or

ΙμΜ imatinib for 8 hours in triplicate and profiled as above.

Gene set enrichment analysis

[0271] Given a ranked list of genes, GSEA determines whether predefined sets of genes

(e.g., genes within a Gene Ontology category) are disproportionally overrepresented in the top or the bottom of the list instead of randomly across the list (Subramanian, A. et al., 2005, Proc Natl Acad Sci USA, 102, 15545-15550; incorporated herein by reference). To assess the phenotypic association with ETV1 knockdown, we used the ranked list of genes generated above from the most upregulated by ETV1 knockdown to most downregulated by ETV1 knockdown. For genes with multiple probes by the Illumina HT-12 array, median signal was used. We used gene sets from the Molecular Signatures Database C2v2.5 with 1892 curated gene sets of chemical and genetic perturbations and canonical pathways and C5v2.5 with 1454 Gene Ontology gene sets. In addition, we added five custom gene sets including the three GIST signature gene sets

EXPO GIST SIG, NIELSEN GIST SIG, and SEGAL GIST SIG and two ICC signature gene sets ICC MY SIG, and ICC DMP SIG defined above.

ChlP-Seq

[0272] ChlP-Seq was performed as previously described (Goldberg, A. D. et al, 2010,

Cell, 140: 678-691; incorporated herein by reference). Briefly, 108 GIST48 cells were crosslinked for 15 -minutes in 1% paraformaldehyde, washed, and lysed. Chromatin was sheared using Bioruptor (Diagenode) to fragments of -150 bp and was incubated with anti-rabbit IgG dynabeads (Invitrogen) pre-conjugated with anti-ETVl antibody, washed, and eluted. The eluted chromatin was reverse-cross-linked, and DNA was column purified. The purified ChIP DNA was blunt-ended, ligated to Solexa adaptors, amplified with 18-cycles of PCR, and sequenced on Solexa Genome Analyzer. We performed independent qPCR validation of separate ChIP samples using the following primers:

DUSP6 Promoter: F: GCCCGCTGTTGCAGCTTGTT (SEQ ID NO: 25)

R: GCCGGCTGGAACAGGTTGTG (SEQ ID NO: 26)

GPR20 Enhancer: F: CCCTCCCAGGCTCTCCCCAC (SEQ ID NO: 27)

R: TCCGGGCCTGCTCTCTGTCC (SEQ ID NO: 28)

GAPDH promoter: F: TCCCAAAGTCCTCCTGTTTCA (SEQ ID NO: 29)

R: CAGCAGGACACTAGGGAGTCAA (SEQ ID NO: 30)

[0273] All reads (~36-bp) were aligned to the human genome (hgl 8) using the ELAND alignment software within the Illumina Analysis Pipeline. Unique reads mapped to a single best- matching location with no more than two mismatches were kept and used to generate genome- wide distribution of ETVl -binding and for peak identification. Redundant reads were considered once. The software MACS (Zhang, Y. et al., 2008, Genome Biol, 9: R137; incorporated herein by reference) was applied to the ChlP-Seq data with sequencing data from input DNA as control for identifying genomic regions enriched with ETVl ChIP signals (i.e., peaks). For each peak, we subsequently calculated the number of reads (extending 200-nt in 3 '-direction) covering each nucleotide position and then defined the maximal count as peak height.

Results and Discussion

[0274] All mined datasets were downloaded Gene Expression Omnibus (GSE2109,

GSE7809, GSE2719, GSE3443, GSE8167, GSE17743) and were analyzed by Oncomine™ or using Genespring 10. GIST-signature genes from three datasets containing both GIST and non- GIST malignancies met the following two criteria: 1) q<0.05, and 2) a Z-score expression difference >1.5 between GIST and non-GIST tumors. Expression profiling of GIST cell lines with different shRNA conditions was performed in duplicate on Illumina Human HT-12 array. GSEA was performed using MSigDB C2, MSigDB C4, and the GIST and ICC signature gene sets. For ChlP-Seq, sheared chromatin enriched by ETVl IP was sequenced on Solexa Genome Analyzer, aligned using ELAND alignment software. Peaks were identified by MACS using input DNA as control using a FDR <1%.

[0275] Using FDR < 1 % (as defined by MACS) and a height of > 15 reads, we defined

14,741 peaks. These peaks were classified as promoter peaks (within +/- 2kb of transcription start sites), enhancer peaks (from -50 kb of transcription start sites to + 5kb of transcription end sites, but excluding promoter regions), or otherwise intergenic peaks based on their genomic distances to known Refseq gene annotation. To associate an ETVl peak with a specific gene, we used the following protocol. A promoter peak was assigned to all genes if it was in the promoter regions of multiple genes. An enhancer peak within a gene body was assigned to the host gene, but otherwise was assigned to the nearest gene. Nucleotide sequences of all ETVl peaks were retrieved from the reference human genome and subjected to motif analysis by the program MEME (Bailey, T. L., et al, 2006, Nucleic Acids Res, 34: W369-373; incorporated herein by reference). [0276] To correlate gene expression with ETVl binding sites, we first limited the analysis the genes with peaks to those that were also represented on the mRNA microarray (Illumina HT-12). Overlapping analysis of genes whose expression was perturbed by ETVl knockdown and those with ETVl binding peaks was performed using Excel macros and significance was calculated using Fisher's exact test.

[0277] Reasoning that transcription factors are likely to play critical roles in defining the cellular context, we utilized three expression datasets (Nielsen, T. O. et al, 2002, Lancet, 359: 1301-1307; Segal, N. H. et al, 2003, Am J Pathol, 163: 691-700; each of which is incorporated herein by reference) to search for GIST specific genes that might provide new molecular insights. We identified an eleven-gene signature exclusively associated with GIST in all three datasets that included the ETS family transcription factor ETVl (FIG. 2A). Examination of individual tumor samples revealed that ETVl is highly expressed in all GISTs and at

significantly higher levels than any other tumor type (FIG. 2B, FIG. 6). ETVl was of immediate interest since ETS family transcription factors are well established oncogenes in Ewing sarcoma, melanoma, and prostate cancer (Tomlins, S. A. et al, 2005, Science, 310: 644-648; Mertens, F. et al, 2009, Semin Oncol, 36: 312-323; Jane-Valbuena, J. et al, 2010, Cancer Res, 70: 2075- 2084; each of which is incorporated herein by reference).

[0278] Next, we assessed mRNA and protein levels of ETVl in GIST and other sarcomas in clinical samples, GIST cell lines (imatinib-resistant GIST48 and imatinib-sensitive GIST882), the U20S osteosarcoma cell line, and the LNCaP prostate cancer cell line known to overexpress ETVl due to translocation (Tomlins, S. A. et al, 2007, Nature, 448: 595-599; incorporated herein by reference) (FIG 2C, D). ETVl mRNA and protein were highly and exclusively expressed in all GISTs and GIST cell lines, and in LNCaP cells. As expected, KIT mRNA and protein were highly expressed in all GIST tumors and GIST cell lines, but not in other sarcomas or non-GIST cell lines, and phospho-KIT was only seen in GIST samples with activating KIT mutations. Four additional GIST samples amenable to immunohistochemical analysis all showed strong nuclear ETVl staining whereas a leiomyosarcoma control sample did not (FIG. 7). These data show that ETVl is universally highly expressed in all GISTs both at transcript and protein levels. [0279] To explore the requirement of ETVl in GIST pathogenesis, we performed RNAi experiments using two ETVl -specific hairpins validated for both protein and mRNA suppression (FIG. 8A). Infection with either hairpin resulted in growth inhibition of both GIST cell lines, but did not affect the growth of U20S cells. Consistent with the level of ETVl knockdown, ETVlsh2 was more growth suppressive than ETVlshl in both GIST cell lines (FIG. 2E). Cell cycle analysis showed that ETVl knockdown resulted in both decreased cell cycle progression and increased apoptosis (FIG. 8B). ETVl knockdown also impaired the tumorigenicity of GIST882 cells in SCID mouse xenografts, and those tumors that did grow had escaped

ETVl suppression (FIG. 2F). Collectively, these observations indicate that ETVl is required for GIST growth and survival.

[0280] Next, we addressed the mode of high ETVl expression in GIST. FISH on 4 GIST samples and 2 GIST cell lines showed no evidence of amplification or "breakaway" between the 3' and 5' ends of ETVl locus. qRT-PCR showed no evidence of differential exon expression, which is expected with ETVl translocation (FIG. 9). Furthermore, no focal ETVl amplification was found in 40 GIST tumors and 6 GIST cell lines in a recent 250K SNP array study

(Beroukhim, R. et al., 2010, Nature, 463: 899-905; incorporated herein by reference). The fact that high levels of ETVl expression are consistently observed in the absence of obvious genomic alterations raises the possibility that the ICCs that give rise to GIST may endogenously express ETVl .

[0281] The musculature of the GI tract is organized into two principal layers— the inner circular muscle (CM) layer beneath the mucosa (M) and the outer longitudinal muscle (LM) layer (Ward, S. M., et al, 2001, Am J Physiol Gastrointest Liver Physiol, 281 : G602-611;

incorporated herein by reference). In the large intestine, myenteric ICCs (ICC-MY) form a network between the CM and LM layers surrounding the neuronal myenteric plexus, intramuscular ICCs (ICC-IM) are singly dispersed in the CM, and submucosal ICCs (ICC-SMP) form network surrounding the submucosal plexus (FIG. 3A). In the small intestine, ICC-IMs and ICC-SMPs are absent and ICC-DMPs form a network around the deep muscular plexus in the CM close to the mucosa (FIG. 10A). All four ICC subtypes are identified by positive membrane expression of Kit (Ward, S. M., et al, 2001, Am J Physiol Gastrointest Liver Physiol, 281 :

G602-611; incorporated herein by reference) (FIG. 3B and FIG. 10B). In the large intestine, ICC-MYs and ICC-IMs but not ICC-SMPs stain with nuclear Etvl (FIG. 3B). In the small intestine, ICC-MYs but not ICC-DMPs stain with nuclear Etvl (FIG. 10B). This finding is further supported by our analysis of a published ICC expression dataset from mouse small intestine (Chen, H. et al, 2007, Physiol Genomics, 31 : 492-509; incorporated herein by reference) showing that Etvl is only highly expressed in ICC-MYs (FIG. IOC). Notably, in the K 558 mutant mice only ICC-MY and ICC-IM develop hyperplasia while ICC-SMP and ICC- DMP do not (Kwon, J. G. et al, 2009, Gastroenterology, 136: 630-639; incorporated herein by reference). These data suggest that ETVl is a lineage-specific transcription factor for the ICCs that give rise to GIST.

[0282] We therefore asked if Etvl is required for the normal development of ICCs by examining the GI tracts of Etvl-/- mice (Arber, S., et al, 2000, Cell, 101 : 485-498; incorporated herein by reference). Cross section and reconstructed whole-mount images from Etvl-/- mice showed significant loss of Kit-positive ICC-IMs and ICC-MYs in the large intestine (FIG. 3C-D, FIG. 14), small intestine, stomach, and cecum (FIGS. 11-14). In contrast, ICC-DMPs and ICC- SMPs in the small and large intestine respectively were preserved, consistent with absent Etvl expression in these ICC subtypes. These results were confirmed with a second ICC marker Anol (Gomez-Pinilla, P. J. et al, 2009, Am J Physiol Gastrointest Liver Physiol, 296: G1370-1381; incorporated herein by reference) (FIG. 15). Immunostaining with the neuronal marker PGP9.5 confirmed the integrity of the myenteric plexus in Etvl-/- mice (FIG. 3C, FIGS. 11-13, FIG. 16). Collectively, these data indicate that Etvl is selectively required for development of ICC-MY and ICC-IM and, by implication, a lineage-specific survival factor for the ICC-GIST lineage.

[0283] To identify ETVl target genes in GIST, we analyzed the effect of shRNA- mediated ETVl suppression on the transcriptomes of GIST48 and GIST882 cells. The overlap of genes perturbed by both ETVl -specific hairpins and across both cell lines was highly statistically significant, suggesting that ETVl regulates a core set of genes in GIST (FIG. 17). To minimize cell line-specific and off-target effects, we generated a ranked gene list based on the average change in gene expression induced by the two ETVl -specific hairpins in both GIST cell lines (FIG. 4A, B). We independently confirmed downregulation of 5 of these genes using real-time RT-PCR (FIG. 18). Among the 48 genes suppressed >1.7-fold by ETVl knockdown, 32 were expressed at higher levels in human GIST samples relative to other tumor types in the ExpO expression dataset (FIG. 4B). We performed gene set enrichment analysis (GSEA)( Subramanian, A. et al, 2005, Proc Natl Acad Sci U S A, 102, 15545-15550; incorporated herein by reference) of the "shETVl" ranked list using >3,000 gene sets in the Molecular Signature Database along with 5 custom gene sets defined by GIST-signature genes from the Segal, Nielsen, and ExpO datasets and by ICC-MY- and ICC-DMP-signature genes. All three GIST sets along with the ICC-MY set were among the most negatively enriched gene sets while the ICC-DMP set was not (FIG. 4C, FIG. 19). These data suggest that ETVl is a master regulator of a transcriptional program conserved in ICC-IM/MYs and GISTs.

[0284] To define the direct transcriptional targets of ETVl in GIST, we performed genome-wide analyses of ETVl -binding sites using ChlP-Seq in GIST48 cells. We identified 14,741 ETVl -binding sites (ETVl peaks) which are enriched in promoter regions (FIG. 4D). Motif analysis of the peaks identified the ETS core consensus motif, GGAA, in -90% of peaks (FIG. 4F). Integrative analyses of the ETVl ChlP-Seq data with the transcriptomes from shRNA-mediated ETVl suppression in GIST cells showed that 38 of the top 48 shETVl downregulated genes contain ETVl peaks (FIG. 4B, E, FIG. 20). Analysis of genes with 1.4-fold change by shETVl knockdown revealed that both shETVl upregulated and shETVl

downregulated genes are enriched for ETVl peaks. Furthermore, enhancer binding and in particular enhancer and promoter binding is highly predicative of transcriptional activation by ETVl (FIG. 4H). Since enhancers are in general cell-lineage specific (Heintzman, N. D. et al., 2009, Nature, 459: 108-112; Visel, A. et al, 2009, Nature, 457: 854-858; each of which is incorporated herein by reference), our data suggest that these ICC-GIST-lineage specific genes are likely directly regulated by ETVl binding to their enhancer regulatory elements.

[0285] The dual requirement of KIT and ETVl in normal ICC development and GIST survival raise the possibility that KIT and ETVl cooperate in GIST oncogenesis. Inhibition of KIT signalling by imatinib in imatinib-sensitive GIST882 cells resulted in rapid loss of ETVl protein, without significant effect on ETVl mRNA levels (FIG. 5A, B, FIG. 21). Similar results were observed with the MEK inhibitor PD325901. This loss of ETVl protein was faster than the natural degradation rate, as revealed by cyclohexamide experiments to inhibit protein synthesis, and was rescued from proteosomal degradation by MG132 (FIG. 5B). Therefore, KIT-MEK signalling stabilizes ETVl protein. Consistent with this KIT-MEK-ETV1 signalling pathway model, the overlap between genes transcriptionally altered by imatinib treatment (KIT-regulated) and by ETVl knockdown in GIST882 cells is highly significant (FIG. 5C). Furthermore, these ETVl transcriptional targets preferentially contain ETVl enhancer peaks (FIG. 5D), indicating that KIT signalling influences the ETVl transcriptional output of the tissue and lineage-specific genes in GIST.

[0286] Having established a signalling pathway from KIT to ETVl, we explored their potential cooperativity in tumorigenesis by expressing ETVl, wild-type KIT, KIT harbouring a common GIST mutation (ΚΙΤΔ560) and control vectors in combination in NIH3T3 cells. KIT- dependent stabilization of ETVl protein was recapitulated in this system (FIG. 5E). In anchorage independent colony formation assays, ETVl significantly increased the number and size of colonies in ΚΙΤΔ560 expressing cells but was insufficient to confer anchorage-independent growth on its own (FIG. 22). Furthermore, ΚΙΤΔ560 and ETVl strongly cooperated in conferring tumorigenic growth in SCID mice (FIG. 5F, G).

[0287] Taken together, these findings establish an oncogenic role for ETVl in GIST that differs from classical models of ETS-driven malignancies where structural alterations (e.g., TMPRSS2-ETV1 translocation in prostate cancer, ETVl amplification in melanoma) lead to aberrant expression and promote tumorigenesis (Tomlins, S. A. et al, 2005, Science, 310: 644- 648; Jane-Valbuena, J. et al, 2010, Cancer Res, 70: 2075-2084; each of which is incorporated herein by reference). Rather, ETVl expression in GIST is inherited from ICC-MY/IM cells, where ETVl is also a survival factor. We further established that KIT activity, through MEK, stabilizes ETVl, providing a mechanism for KIT-ETV1 cooperativity (FIG. 5H). These observations provide an explanation for why patients and mice with germline activating KIT mutations develop neoplasia in only the ICC-MY/IM lineage. While the mechanism of ETV1- mediated oncogenesis in GIST differs from other ETS-driven cancers, we anticipate that the ETVl -dependent transcriptional program defined here may serve as a valuable resource for further understanding of other ETVl - and other ETS-driven transcriptional programs in various cellular contexts such as prostate cancer.

[0288] The fact that ETVl is universally highly expressed in all GISTs makes it immediately useful as a candidate diagnostic biomarker, since the current standard of KIT immunoreactivity is negative in about 5% of all GISTs (Miettinen, M. & Lasota, 2006, Arch Pathol Lab Med, 130: 1466-1478; incorporated herein by reference). While transcription factors has classically been considered "undruggable", reports of successful inhibition of the NOTCH transcription factor complex and AR activity by blocking coactivator binding have challenged this paradigm (Andersen, R. J. et al.,2010, Cancer Cell, 17: 535-546; Moellering, R. E. et al, 2009, Nature, 462: 182-188; each of which is incorporated herein by reference). Due to established requirements of ETV1 in subsets of prostate cancer and melanoma, efforts to find ETV1 inhibitors are underway and may yield novel therapeutic agents for imatinib-resistant GIST.

Equivalents and Scope

[0289] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above Description, but rather is as set forth in the appended claims.

[0290] In the claims articles such as "a", "an" and "the" may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Thus, for example, reference to "an antibody" includes a plurality of such antibodies, and reference to "the cell" includes reference to one or more cells known to those skilled in the art, and so forth. Claims or descriptions that include "or" between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention includes embodiments in which more than one, or all of the group members are presenting, employed in, or otherwise relevant to a given product or process. Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitation, elements, clauses, descriptive terms, etc., from one or more of the listed claims is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for anyone of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

[0291] Where elements are presented as lists, e.g., in Markush group format, it is to be understood that each subgroup of the elements is also disclosed, and any element(s) can be removed from the group. It should be understood that, in general, where the invention, or aspects of the invention, is/are referred to as comprising particular elements, features, etc., certain embodiments of the invention or aspects of the invention consist, or consist essentially of, such elements, features, etc. For purposes of simplicity those embodiments have not been specifically set forth in haec verba herein. It is noted that the term "comprising" is intended to be open and permits the inclusion of additional elements or steps.

[0292] Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and understand of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or sub-range within the state ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0293] In addition, it is to be understood that any particular embodiment of the present invention that falls within the prior art may be explicitly excluded from any one or more of the claims. Since such embodiments are deemed to be known to one of ordinary skill in the art, they may be excluded even if the exclusion is not set forth explicitly herein. Any particular embodiment of the compositions of the invention (e.g., any HCV genotype/subtype, any HCV antibody, any epitope, any pharmaceutical composition, any method of administration, any therapeutic application, etc.) can be excluded from any one or more claims, for any reason, whether or not related to the existence of prior art.

[0294] The publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure.

Other Embodiments

[0295] Those of ordinary skill in the art will readily appreciate that the foregoing represents merely certain preferred embodiments of the invention. Various changes and modifications to the procedures and compositions described above can be made without departing from the spirit or scope of the present invention, as set forth in the following claims.

Claims

Claims What is claimed is:

1. A hybridoma cell line deposited with the American Type Culture Collection (ATCC) and selected from the group consisting of:

2. A monoclonal antibody produced by a hybridoma cell line according to claim 1.

3. A monoclonal antibody that binds to an epitope of ETV1 recognized by an antibody of claim 2.

4. An antibody with an amino acid sequence that is at least 80% identical to the amino acid sequence of an antibody of claim 2.

5. An antibody with an amino acid sequence that is at least 90% identical to the amino acid sequence of an antibody of claim 2.

6. An antibody with an amino acid sequence that is at least 95% identical to the amino acid sequence of an antibody of claim 2.

7. An antibody with an amino acid sequence that is at least 99% identical to the amino acid sequence of an antibody of claim 2.

8. An antibody with an amino acid sequence that includes a heavy chain variable domain having at least 80% sequence identity to the heavy chain variable domain of an antibody of claim 2.

9. An antibody with an amino acid sequence that includes a light chain variable domain having at least 80% sequence identity to the light chain variable domain of an antibody of claim 2.

10. An antibody with an amino acid sequence that includes a heavy chain variable domain having at least 80% sequence identity to the heavy chain variable domain of an antibody of claim 2 and a light chain variable domain having at least 80% sequence identity to the light chain variable domain of the same antibody of claim 2.

11. An antibody with an amino acid sequence that includes a light chain variable domain with the same complementary determining regions (CDRs) as the light chain variable domain of an antibody of claim 2.

12. An antibody with an amino acid sequence that includes a heavy chain variable domain with the same complementary determining regions (CDRs) as the heavy chain variable domain of an antibody of claim 2.

13. An antibody with an amino acid sequence that includes a light chain variable domain with the same complementary determining regions (CDRs) as the light chain variable domain of an antibody of claim 2 and a heavy chain variable domain with the same complementary determining regions (CDRs) as the heavy chain variable domain of the same antibody of claim 2.

14. An antibody of any one of claims 3-13, wherein the antibody is monoclonal.

15. An antibody of any one of claims 3-13, wherein the antibody is humanized.

16. An antibody of any one of claims 4-13 that binds a peptide sequence of SEQ ID NO: 1.

17. An antibody of any one of claims 4-13 that binds to an epitope of ETV1 recognized by an antibody of claim 2.

18. An antibody of any one of claims 3-17 that binds a peptide sequence of SEQ ID NO: 31.

19. An ETV1 -binding fragment of an antibody of any one of claims 2-17.

20. A pharmaceutical composition comprising an antibody of any one of claims 2-19 and a pharmaceutically acceptable excipient.

21. The pharmaceutical composition of claim 20, further comprising at least one additional chemotherapeutic agent.

22. An isolated nucleic acid encoding an antibody of any one of claims 2-19.

23. A cell line capable of expressing an antibody of any one of claims 2-19.

24. A method comprising culturing a hybridoma cell line of claim 1 under appropriate conditions and isolating a monoclonal antibody therefrom.

25. A kit comprising an antibody of any one of claims 2-19 or an ETVl -binding fragment thereof, a device for administering the at least one antibody or ETVl -binding fragment to a subject, and instructions for use.

26. A method for determining the likelihood of gastrointestinal stromal tissue being cancerous or gastrointestinal stromal tumor (GIST), comprising:

(a) determining a level of expression or activity of ETVl in a gastrointestinal stromal tissue sample obtained from a subject using an antibody of any one of claims 2-19 or an ETV1- binding fragment thereof;

(b) comparing the determined ETVl level with that of a reference correlated with a predetermined probability of being cancerous or GIST; and

(c) based on the comparing, determining that the tissue sample has an increased or decreased probability, relative to the reference, of being cancerous or GIST.

27. The method of claim 26, wherein the step (b) of comparing comprises establishing that the determined level in the tissue sample is higher than the reference level, and the reference level correlates with that observed in normal tissue, so that step (c) of determining comprises detemining that the tissue sample has an elevated likelihood of being cancerous or GIST.

28. The method of claim 26, wherein the step (a) of determining comprises performing an analysis selected from the group consisting of Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, ELISA, immunohistochemistry, and combinations thereof, so that the ETV1 level of expression or activity is determined.'

29. The method of claim 26, wherein the step (a) of determining comprises performing immunohistochemistry on the tissue sample.

30. A method for determining the likelihood of cancerous gastrointestinal stromal tissue responding to therapy, comprising:

(a) determining a level of expression or activity of ETV1 in a cancerous

gastrointestinal stromal tissue sample obtained from a subject using an antibody of any one of claims 2-19 or an ETV1 -binding fragment thereof;

(b) comparing the determined ETV1 level with that of a reference correlated with a predetermined probability of responding to a therapeutic regimen; and

(c) based on the comparing, determining that the subject from which the tissue sample was obtained has an increased or decreased probability, relative to the reference, of responding to the therapeutic regimen.

31. The method of claim 30, wherein the reference is an average of responses derived from a population of individuals.

32. A method for identifying or characterizing an agent that affects GIST phenotype comprising:

(a) contacting cultured GIST tissue or cells with an agent to be characterized; and

(b) determining that ETV1 level of expression or activity is altered when the agent is present as compared with when it is absent, wherein determining uses an antibody of any one of claims 2-19 or an ETV1 -binding fragment thereof.

33. The method in claim 32, wherein an increase in the level of ETVl correlates with the onset of or progression of a GIST phenotype.

34. The method in claim 32, wherein a decrease in the level of ETVl correlates with the regression of a GIST phenotype.

35. The method of claim 32, wherein the step (b) of determining comprises performing an analysis selected from the group consisting of Northern blotting, Western blotting, protein gel electrophoresis, immunoprecipitation, immunohistochemistry, and combinations thereof, so that the ETVl level of expression or activity is determined.

36. A method of treating GIST by administering to a subject suffering from or susceptible to GIST an antibody of any one of claims 2-19 or an ETVl -binding fragment thereof that alters ETVl level of expression or activity.

37. A method of treating GIST in a subject in need thereof comprising administering a therapeutically effective amount of pharmaceutical composition comprising an agent identified by the method of claim 32.

38. The method of claim 37, wherein the pharmaceutical composition inhibits EVT1 expression.

39. The method of claim 37, wherein the pharmaceutical composition promotes the degradation of ETVl protein.

40. A kit for use in detecting ETVl expression comprising:

(a) an antibody of any one of claims 2-19 or an ETVl -binding fragment thereof capable of detecting the level of ETVl expression or activity,

(b) a container comprising the antibody or ETVl -binding fragment therof;

(c) a control; and

(d) instructions to provide guidance for carrying out an assay embodied by the kit and for making a determination of ETVl based upon that assay.

41. The kit of claim 40, wherein the antibody or ETVl -binding fragment thereof is attached to a substrate, which substrate is applied to a sample from a patient or to a surface that may contain ETVl and the surface of the substrate is then processed to assess whether specific binding occurs between the antibody or ETVl -binding fragment thereof and ETVl in the sample.

42. The kit of claim 41, wherein the control is a control slide comprising tumor samples.

43. A method comprising steps of:

(a) monitoring the level of ETVl expression or activity in a subject suffering from GIST using an antibody of any one of claims 2-19 or an ETVl -binding fragment thereof;

(b) detecting a change in the monitored level of ETVl expression or activity over time; and

(c) initiating or altering therapy administered to the subject after detecting the change.

44. The method of claim 43, wherein the subject is undergoing a first therapy and the step of initiating or altering comprises ceasing the first therapy, modifying the first therapy, adding a second therapy, or combinations thereof.

45. A method of inhibiting the growth of ICC cancerous cell with aberrant ETVl expression or activity comprising contacting the cell with an antibody of any one of claims 2-19 or an ETVl -binding fragment thereof so as to decrease the expression or activity of ETVl, thereby inhibiting the growth of the ICC cancerous cell.