WO2015116902A1 - G-protein coupled receptors in hedgehog signaling - Google Patents

G-protein coupled receptors in hedgehog signaling Download PDF

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Publication number
WO2015116902A1
WO2015116902A1 PCT/US2015/013697 US2015013697W WO2015116902A1 WO 2015116902 A1 WO2015116902 A1 WO 2015116902A1 US 2015013697 W US2015013697 W US 2015013697W WO 2015116902 A1 WO2015116902 A1 WO 2015116902A1
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tpra40
cell
method
agent
hedgehog
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PCT/US2015/013697
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French (fr)
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Jaskirat SINGH
Susanna J. SCALES
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Genentech, Inc.
Curis, Inc.
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Priority to US201461934620P priority Critical
Priority to US61/934,620 priority
Application filed by Genentech, Inc., Curis, Inc. filed Critical Genentech, Inc.
Publication of WO2015116902A1 publication Critical patent/WO2015116902A1/en

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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/566Immunoassay; Biospecific binding assay; Materials therefor using specific carrier or receptor proteins as ligand binding reagents where possible specific carrier or receptor proteins are classified with their target compounds
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/72Assays involving receptors, cell surface antigens or cell surface determinants for hormones
    • G01N2333/726G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2500/00Screening for compounds of potential therapeutic value

Abstract

The disclosure provides methods for identifying TPRA40 antagonists and agonists. The method further provides methods for inhibiting hedgehog signaling and/or inhibiting unwanted cell proliferation, such as unwanted cell proliferation caused, in whole or in part, by hyperactive hedgehog signaling, using a TPRA40 antagonist.

Description

G-Protein Coupled Receptors in Hedgehog Signaling

RELATED APPLICATIONS

This application claims the benefit of priority to United States provisional application

65/934,620, filed January 31, 2014. The foregoing disclosure is hereby incorporated by reference in its entirety,

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on January 22, 2015, is named CIBT-224-W01 ___SL.txt and is 37,643 bytes in size. BACKGROUND OF THE DISCLOSURE

Members of the Hedgehog (Hh) family of signaling molecules mediate many important short- and long-range patterning processes during invertebrate and vertebrate embryonic, fetal, and adult development. Hedgehog activity exerts its effects on cells and tissues via the hedgehog signaling pathway.

Hedgehog signaling occurs through the interaction of a hedgehog protein (e.g., in mammals, Slih, Dhh, or Ihh) with the hedgehog receptor, patched (Ptch), and the co-receptor Smoothened (Smo). There are two mammalian homoiogs of Ptch, Ptch-1 and Ptch-2, both of which are 12 transmembrane proteins containing a sterol sensing domain (Motoyama et al, Nature Genetics JL8: 104-106 (1998), Carpenter et al, P.N.A.S. (U.S.A.) 95(23): 13630-40 (1998). The interaction of Hh with Ptch triggers a signaling cascade that, results in the regulation of transcription by zinc-finger transcriptions factors of the Gli family.

In humans, the Hh signaling cascade is initiated in the target cell by the Hh ligand binding to the 12-span transmembrane protein Patched 1 , In the absence of a Hh ligand, Patched inhibits the activity of the seven-transmembrane-span receptor-like protein, Smoothened.

Binding of Hh to Patched results in the loss of Patched activity and the consequent activation of Smoothened, which transduces the Hh signal to the cytoplasm (Taipale et al,, 2002, Nature 418: 892-897). The Hh signal is transmitted via an alteration of the balance between the activator and repressor forms of the Gii family of zinc-finger transcription factors. Hh signaling occurs in the nonmotile cilia to which the Smoothened protein and other downstream pathway components transit to activate the Gli transcription factors (Rubin and de Sauvage, 2006, Nat Rev Drug Discov 5: 1026-1033: Corbit et ai, 2005, Nature 437: 1018-1021: Huangfu and Anderson, 2005, Proc Natl Acad Sci U S A 102: 11325-11330: Huangfu et al., 2003, Nature 426: 83-87). The Gli transcription factors exist as three separate zinc-finger proteins, Glil and Gii2 functioning as transcriptional activators and Gii3 functioning mainly as a transcriptional repressor ( Ruiz I Altaba, 1997, Cell 90: 193-196). The expression of Glil is highly dependent upon active Hh signaling and thus Glil expression is often used as a readout of pathway activation. In the absence of a Hh ligand, Patched blocks Smoothened activity and full length Gli proteins are proteolytically processed to generate the repressor GLIR, largely derived from Gli3, which represses Hh target genes. Hh binding to Patched relieves Smoothened inhibition, promotes generation of the activator GLI", largely contributed by Gli 2 and the subsequent expression of the Hh target genes, including positive feedback by Gli 1. Gli activation is regulated at several different, levels via phosphorylation by inhibitors such as SuFu, Ren, protein kinase A (PKA), glycogen synthase kinase 3β (G8K3p) and activators such as Dyrkl , Ras and Akt (Varjosalo and Taipale, 2007, J Cell Sci 120: 3-6; Ferretti et al., 2005, Trends Mol Med 5 5 : 537-545; For Review See, Gupta, 2010, Ther Adv Med Oncol. 2(4): 237-250), The Hedgehog signaling pathway has been extensively studied and one of skill in the art readily understands what is meant by a component or gene implicated in the hedgehog pathway and what is meant by hedgehog signaling, and hedgehog signal transduction. Numerous components of the hedgehog pathway that participate in the transduction of signal or regulation of signaling are known.

Exemplary components include: Sonic hedgehog. Patched!, Glil , Gli2, Gli3, protein kinase A (PKA), Suppressor of Fused (SuFu), and Smoothened.

Malignant tumors (cancers) are the second leading cause of death in the United States, after heart disease (Boring et ai., CA Cancel J. Clin. 43:7 ( 1993)). Cancer is an example of unwanted cell proliferation and is characterized by the increase in the number of abnormal, or neoplastic, cells derived from a normal tissue which proliferate to form a tumor mass or otherwise proliferate unchecked by proper control. Cancer may be further characterized by the invasion of adjacent tissues by these neoplastic tumor ceils, and the generation of malignant ceils which eventually spread via the blood or lymphatic system to regional lymph nodes and to distant sites via a process called metastasis. In a cancerous state, a ceil proliferates under conditions in which normal cells would not grow. Cancer manifests itself in a wide variety of forms, characterized by different degrees of invasiveness and aggressiveness.

Hedgehog signaling has been implicated in a. wide variety of cancers and carcinogenesis.

Specifically, increased or misregulated hedgehog signaling activity has been implicated in cancer, and a current product approved for the treatment of locally advanced or metastatic basal cell carcinoma, vismodigib (Erivedge®), is an inhibitor of hedgehog signaling. Vismodigib is a smoothened inhibitor, and thus, inhibits hedgehog signaling at the level of smoothened. For example, mutations in components of the hedgehog signaling pathway (e.g., mutations in hedgehog signaling pathway genes) that lead to misregulated hedgehog signaling activity, such as signaling in the absence of hedgehog ligand, have been identified in certai cancers. For example, loss-of-function mutations in Patched!, gain-of-function mutations in Smoothened, and loss-of-function mutations in Suppressor of Fused lead to misregulated hedgehog signaling and have been identified in cancers. These are exemplary of mutations in components of the hedgehog pathway (e.g., are exemplary of mutations in a hedgehog signaling pathway gene).

TPRA40 (also known as TPRA1 and GPR175) is an orphan G-protein coupled receptor whose physiological functions were previously unknown. TPRA40 is a 40 kDa protein having seven transmembrane domains (Fujimoto et al., 2001, Biochirn Biophys Acta, 1518(1 -2): 173-7) and an 84-amino acid cytoplasmic region (Fujimoto et al.). TPRA40 has been shown to be expressed during oxidative stress, aging and under certain pathophysiological conditions (AM et al., 2008, J Cell Physiol, 217(1): 194-206). in addition, it has been proposed that Sjogren syndrome Nuclear Autoantigen of 14 kDa (Ssnal , also known as NA14), an autoantigen of Sjogren's syndrome, co-immunoprecipitates with TPRA40 in an immunoprecipitation assay, suggesting a direct or indirect interaction between these two proteins, at least under conditions of overexpression (AM et al.). To date, however, the role of TPRA40 in any particular signaling pathway was unknown. In addition, it was previously unknown whether TPRA40 was associated with any disease conditions, such as cancer.

Given the role of hedghog signaling in a variety of cancers, identification of components of the hedgehog signaling pathway suitable as targets for drag development represents a. significant advance. The present disclosure provides a novel component of the hedgehog signaling pathway suitable as a target for drag development.

SUMMARY OF THE DISCLOSURE

The disclosure provides methods for inhibiting cell proliferation, such as unwanted cell proliferation, in a cell in vitro or in vivo. The disclosure is based on appreciation and

understanding of the role of TPRA40 as a novel component of the hedgehog signaling pathway that endogenously functions as a positive regulator of hedgehog signaling. As such, the disclosure provides methods for inhibiting hedgehog signaling in cells, such as any of the cells described herein, using a TPRA40 antagonist. In certain embodiments, the disclosure provides methods for inhibiting cell proliferation, such as unwanted cell proliferation, in a cell in vitro or in vivo using a TPRA40 antagonist. Suitable cells in which proliferation may be inhibited include cells that are responsive to hedgehog protein and/or in which hedgehog signaling is active or hyperactive, as well as cells comprising one or more mutations in a hedgehog pathway gene (e.g., comprising one or more mutations in a component of the hedgehog signaling pathway) or otherwise determined to exhibit hedgehog signaling activity.

Moreover, the disclosure provides various methods for screening for and/or identifying agents suitable as TPRA40 antagonists. Identified agents may then be used in vitro or in vivo.

Moreover, the disclosure provides various methods for screening for and/or identifying agents suitable as TPRA40 agonists. Identified agents may then be used in vitro or in vivo.

In one aspect, the disclosure provides for a method of reducing hedgehog signaling in a ceil, for example, in a ceil that is responsive to hedgehog protein or exhibits active or hyperactive hedgehog signaling activity. In certain embodiments, the ceil is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene (e.g., a mutation in a component of the hedgehog pathway), wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, wherein the method comprises the step of contacting the cell with an effective amount of a TPRA40 antagonist. In certain embodiments, the cell exhibits active or hyperactive hedgehog signaling.

In some aspects, the disclosure provides for a method of inhibiting unwanted growth, proliferation or survival of a cell, for example, in a cell that is responsive to hedgehog protein or exhibits active or hyperactive hedgehog signaling activity. In certain embodiments, the cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene (e.g., a mutation in a. component of the hedgehog pathway), wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, wherein the method comprises the step of contacting the cell with an effective amount of a TPRA40 antagonist. In certain embodiments, the cell exhibits active or hyperactive hedgehog signaling.

In some aspects, the disclosure provides for a method of inhibiting growth, proliferation or survival of a tumor ceil, for example, in a cell that is responsive to hedgehog protein or exhibits active or hyperactive hedgehog signaling activity. In certain embodiments, the cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, wherein the method comprises the step of contacting the cell with an effective amount of a TPRA40 antagonist. In certain embodiments, the cell exhibits active or hyperactive hedgehog signaling. In certain embodiments, the cell exhibits hyperactive hedgehog signaling.

In some aspects, the disclosure pro vides for a method of inhibiting unwanted growth, proliferation or survival of a cell, wherein the cell comprises one or more mutations in

suppressor-of-fused, in which one or more mutations result in the cell having suppressor-of- fused loss-of-function, wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand (e.g., the absence of hedgehog protein), wherein the method comprises the step of contacting the cell with an effective amount of a TP A40 antagonist.

In some aspects, the disclosure provides for a method of inhibiting growth, proliferation or survival of a tumor cell, wherein the cell comprises one or more mutations in suppressor-of- fused resulting in the cell having suppressor-of-fused loss-of-function, wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, wherein the method comprises the step of contacting the cell with an effective amount of a TPRA40 antagonist.

The following may apply, in certain embodiments, to any of the methods disclosed herein.

In certain embodiments of any of the methods disclosed herein, hedgehog signaling is hyperactive in the cell. In certain embodiments, the hedgehog signaling is overactive because the cell or an adjacent cell overexpressed hedgehog protein (e.g., where the cell is a cancer cell or a. cell in a tumor, the tumor cell or stroma overexpress a hedgehog protein). In certain embodiments, the cell comprises one or more mutations in a hedgehog signaling pathway gene (e.g., one or more mutations in a component of the hedgehog a signaling pathway). In certain embodiments, the cell comprises one or more mutations in a component of the hedgehog signaling pathway. In certain embodiments, the one or more mutations are in smoothened, and the cell has a smoothened gain-of-function. In certain embodiments, the gain-of-function smoothened mutation results in a coiistitutively active Smoothened protein. In certain embodiments, the one or more mutations are in .patched], and the cell has a patched loss-of- function. In certain embodiments, the one or more mutations result in overexpression of a hedgehog protein or, as noted above, hedgehog protein is overexpressed in the cell or in an adjacent cell, despite the fact that the cell does not have a mutation in a hedgehog gene and/or in a component of the hedgehog pathway. In certain embodiments, the overexpressed hedgehog protein is Sonic hedgehog protein. In certain embodiments, the overexpressed hedgehog protein is Indian hedgehog protein. In certain embodiments, the overexpressed hedgehog protein is Desert hedgehog protein. In certain embodiments, the one or more mutations are in suppressor- of-fitsed, and the cell has suppressor-of-fused loss-of-function. In certain embodiments, prior to contacting the cell with the TPRA40 antagonist, the cell is determined to have one or more mutations in a hedgehog signaling pathway gene or otherwise determined to exhibit hedgehog signaling activity, such as determined to have active or hyperactive hedgehog signaling. In certain embodiments, the cell is determined to exhibit hedgehog signaling activity by measuring GUI or Patched] levels.

The following may apply, in certain embodiments, to any of the methods disclosed herein. In certain embodiments of any of the methods described herein, the cell is a cell in culture. In certain embodiments of any of the methods described herein, the method comprises contacting a culture comprising a plurality of cells. In certain embodiments, the cell is in a vertebrate, and contacting the cell comprises administering the TPRA40 antagonist to the vertebrate. In certain embodiments, the vertebrate is a human subject. In certain embodiments, the cell is a vertebrate cell, such as a mammalian ceil. In certain embodiments, the cell is a cancer ceil or cancer cell line. The following may apply, in certain embodiments, to any of the methods disclosed herein. In certain embodiments of any of the methods described herein, the cell is a cancer cell and/or the vertebrate is a vertebrate diagnosed with cancer. In certain embodiments, the cancer cell is a cancer cell selected from the group consisting of: a colon, lung, prostate, skin, blood, liver, kidney, breast, bladder, bone, brain, meduUoblastoma, sarcoma, rhabdomyosarcoma, basal cell carcinoma, gastric, ovarian, esophageal, pancreatic, or testicular cancer cell. In certain embodiments, the cancer cell is a cancer ceil selected from the group consisting of: a

meduUoblastoma, meningioma, adenoid cystic carcinoma, basal cell carcinoma, and

rhabdomyosarcoma cancer cell.

The following may apply, in certain embodiments, to any of the methods disclosed herein.

In certain embodiments of the disclosure, the TPRA40 antagonist is a polynucleotide molecule that inhibits the expression of TPRA40. For example, in certain embodiments the TPRA40 antagonist is a polynucleotide that binds to (e.g., targets) TPRA40. In certain embodiments, the polynucleotide molecule is an antisense oligonucleotide that hybridizes to a TPRA40 transcript to inhibit expression of TPRA40. In certain embodiments, the TPRA40 antagonist is an RNAi (e.g., an RNAi molecule) that targets the TPRA40 mRNA transcript. In certain embodiments, the RNAi molecule comprises an siRNA. In certain embodiments, the siRNA is 19-23 nucleotides in length. In certain embodiments, the siRNA is double stranded, and includes short overhang(s) at one or both ends. In certain embodiments, the RNAi comprises an shRNA. In certain embodiments, the siRNA targets TPRA40 mRNA transcript. In certain embodiments, the siRNA comprises one or more of the nucleotide sequences selected from: SEQ ID NOs: 16-23. In certain embodiments, the TPRA40 antagonist is a small molecule that binds to TPRA40, such as a small molecule that binds to and inhibits an activity of TP A40. In certain embodiments, the TPRA40 antagonist is an antibody that binds to TPRA40 protein. In certain embodiments, the antibody is a monoclonal antibody. In certain embodiments, the TPRA40 antagonist is a polypeptide antagonist.

The following may apply, in certain embodiments, to any of the methods disclosed herein. In certain embodiments, regardless of the method or the particular TPRA40 antagonist used, the method comprises contacting the cell (at the same or a differing time) with an additional antagonist of the hedgehog signaling pathway (e.g., generically a hedgehog pathway inhibitor - HPI). In certain embodiments, the additional antagonist of the hedgehog signaling pathway is a veratrum-type steroidal alkaloid. In certain enibodiments, the veratrum-type steroidal alkaloid is cy dopamine or KAAD-cyclopamine or a derivative thereof (e.g., IPI-269609 or ΙΡΪ- 926/saridegib), In certain embodiments, the veratrum-type steroidal alkaloid is jervine, IPI- 269609 or IPI-926. In certain embodiments, the antagonist is a non-veratrum-type synthetic small molecule inhibitor of Smoothened (e.g., the HPI is a small molecule inhibitor of

Smoothened). In certain embodiments, the antagonist is Erivedge (vismodegib), BMS-833923 (XL319), LDE225 (Erismodegib), PF-04449913, VP-LDE225, VP-LEQ506, TAK-441 , XL- 319, LY-2940680, SEN450, Itraconazole, MRT- 10, MRT-83, or PF-04449913. In certain embodiments, the additional antagonist of the hedgehog signaling pathway is an antibody. In certain embodiments, the antibody is an antibody that specifically binds Sonic, Indian or Desert hedgehog protein. In certain embodiments, the additional antagonist of the hedgehog pathway is a hedgehog inhibitor (e.g., the HPI is a hedgehog inhibitor - an inhibitor of hedgehog protein). In certain embodiments, the hedgehog inhibitor is robotkinin. In certain embodiments, the HPI (e.g., hedgehog pathway inhibitor; antagonist of the hedgehog signaling pathway) is selected from the group consisting of: vismodegib, sonidegib, BMS-833923, PF-04449913, and

LY2940680. In certain embodiments, the additional antagonist of the hedgehog signaling pathway is an RNAi antagonist.

In another aspect, the disclosure provides for a method of screening for a TPRA40 antagonist, wherein the method comprises: a) contacting a cell that expresses TPRA40, adenylvl cyclase and a reporter (e.g., a report construct, such as a reporter that indicates adenylyl cyclase activity) with an adenylyl cyclase activator and an agent; b) determining, as compared to an untreated control, whether the agent rescues the adenylyl cyclase activity suppressed by TPRA40 expression, wherein if the agent increases adenylyl cyclase activity relative to the non-agent treated TPRA40 expressing cells, then the agent is identified as a TPRA40 antagonist. In certain embodiments, the untreated control is the same type of cell (e.g., expressing TPRA40, adenylyl cyclase, and a reporter), but the untreated control is not contacted with the agent. In certain embodiments, TPRA40 is expressed in the cell exogenously, such as by transfecting or transforming the cell with a vector expressing TPRA40. Optionally, the ceil may also endogenously express TPRA40. In certain embodiments, the cell is contacted with the activator and the agent simultaneously, concurrently, or consecutively. In another aspect, the disclosure provides for a method of identifying a TPRA40 antagonist, comprising: a) providing a cell that expresses TPRA40 and that expresses a reporter gene capable of indicating adenylyl cyclase activity; b) contacting the cell with an activator of adenylyl cyclase and with an agent, wherein the cells are contacted with the activator and the agent simultaneously, concurrently, or consecutively: and c) determining, as compared to a control, whether the agent rescues adenylyl cyclase activity induced by the activator, wherein if the agent increases the adenylyl cyclase activity relative to the control, then the agent is identified as a TPRA40 antagonist. In certain embodiments, the untreated control is the same type of ceil (e.g., expressing TPRA40 and the reporter), but the untreated control is not contacted with the agent. In certain embodiments, TPRA40 is expressed in the cell exogenously, such as by transfecting or transforming the cell with a vector expressing TPRA40. Optionally, the cell may also endogenously express TPRA40. In certain embodiments, the cell is contacted with the activator and the agent simultaneously, concurrently, or consecutively.

In another aspect, the disclosure provides for a method of screening for an agent for inhibiting the proliferation, growth or survival of a cancer cell. For example, the method comprises: a) screening for an agent that binds to TPRA40 protein, reduces expression of TPRA40, inhibits transport of TPRA40 protein to the plasma membrane or to primary cilia, prevents activation of TPRA40 or uncouples TPRA40 from God; b) contacting a cancer cell with an amount of the agent identified in step a), wherein the cancer cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, and c) determining, as compared to a control, whether the agent inhibits the proliferation or growth of the cancer cell, wherein if the agent inhibits cell proliferation or growth relative to the control, then an agent that, inhibits the proliferation or growth of the cancer cell is identified. Methods in which any one of the specific activities recited in step (a) are recited as the screen are expressly contemplated.

In another aspect, the disclosure provides for a method of screening for an agent for inhibiting hedgehog signaling in a cell, wherein the method comprises: a) screening for an agent that binds to TPRA40 protein, reduces expression of TPRA40, inhibits transport of TPRA40 protein to the plasma membrane or to primary cilia, prevents activation of TPRA40 or uncouples TPRA40 from God; b) contacting a cell with an amount of the agent identified in step a), wherein the cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, and e) determining, as compared to a control, whether the agent inhibits hedgehog signaling in the cell, wherein if the agent inhibits hedgehog signaling in the cell relative to the control, then an agent that inhibits hedgehog signaling is identified. Methods in which any one of the specific activities recited in step (a.) are recited as the screen are expressly contemplated.

In another aspect, the disclosure provides for a method of identifying a TPRA40 antagonist, wherein the method comprises: a) screening for an agent that binds to TPRA40 protein; b) contacting a cell with an amount of the agent identified in step a), wherein the cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, and c) determining, as compared to a control, whether the agent that binds to TPRA40 protein also inhibits hedgehog signaling in the cell, wherein if the agent inhibits hedgehog signaling in the cell relative to the control, then the agent is identified as a TPRA40 antagonist.

The following may apply, in certain embodiments, to any of the screening methods disclosed herein, as well as any other method where context indicates. In certain embodiments of any of the screening methods described herein, the cell is in culture. In certain embodiments, the cell is in an animal. In certain embodiments, the cel l is a vertebrate cell, such as a rodent, hamster, or human cell. In certain embodiments, the cell is a cancer cell, such as from a primary tumor or a cancer cell line, in certain embodiments, the hedgehog signaling is overactive because the cell or an adjacent ceil overexpressed hedgehog protein (e.g., where the cell is a cancer cell or a cell in a tumor, the tumor cell or stroma overexpress a hedgehog protein). In certain embodiments, the cell comprises one or more mutations in a hedgehog signaling pathway gene (e.g., a mutation in a component of the hedgehog signaling pathway). In certain embodiments, the one or more mutations are in smoothened, and the cell has a smoothened gain- of-function. In certain embodiments, the gain-of-function smoothened mutation results in a constitutiveiy active smoothened protein. In certain embodiments, the one or more mutations are in patched!, and the cell has a patched loss-of- function. In certain embodiments, the tumor overexpresses a hedgehog protein, in certain embodiments, the one or more mutations are in suppressor-of-fused, and the ceil has suppressor-of-fused loss-of- function.

The following may apply, in certain embodiments, to any of the screening methods disclosed herein, as well as any other method where context indicate. In certain embodiments, the agent for use in any of the screening methods described herein is a polypeptide or an antibody. In certain embodiments, the agent is a small molecule. In certain embodiments, the agent is an siKNA or shRNA that decreases TPRA40 transcription. In certain embodiments, the agent binds TPRA40 protein. In other words, the methods are suitable for use in screening to identify TPRA40 antagonists that are, for example, polypeptides, including antibodies, or small molecules, or polynucleotides. In certain embodiments, the agent is identified in step a) using a yeast, two-hybrid screen. In certain embodiments, the agent is identified in step a) using a high throughput binding or activity screen of a small molecule library. In certain embodiments, the agent inhibits transport of the TPRA40 protein to the plasma membrane or to primary cilia. In certain embodiments, the agent, is by a method comprising the steps of: i) contacting a cell expressing TPR.A40 with an agent, and ii) determining the localization of TPRA40 in the first cell expressing TPRA40 using immunohistochemistry. In certain embodiments, the agent is identified by a method comprising the steps of: i) contacting a cell expressing TPRA40 with an agent; and ii) determining the levels of TPRA40 in a plasma membrane or ciliary membrane fraction. In certain embodiments, the agent reduces expression of TPRA40 protein or RNA. In certain embodiments, the agent is identified by a method comprising: i) contacting a cell expressing TPRA40 with an agent; and ii) determining activity of TPRA40 in the cell using a &7/-luciferase reporter or adenylate cyclase reporter assay. In certain embodiments, the agent is identified in step a) by a method comprising: i) contacting a cell expressing TPRA40 with an agent; and ii) determining the expression of TPRA40 in the cell by RT-PCR. In certain embodiments, the agent is identified in step a) by a method comprising: i) contacting a cell expressing TPRA40 with an agent; and ii) determining the expression of TPRA40 in the cell using Northern Blot analysis of TPRA40 RNA or Western Blot, flow cytometry,

immunofluorescence or immunohistochemistry analysis of TPRA40 protein.

In certain embodiments of any of the screening methods disclosed herein, the cell is treated with a compound that induces adenyiyl cyclase activity (e.g., an activator of adenylyl cyclase or an adenylate cyclase activator) prior to step a). In some embodiments of any of the screening methods disclosed herein, the reporter gene is used in order to determine whether adenylyl cyclase activity has been rescued by an agent and/or to evaluate an increase in adenylyl cyclase activity. In some embodiments, the reporter gene is a luciferase gene controlled by a cAMP response element.

In some embodiments of any of the screening methods disclosed herein, the compound that induces adenylyl cyclase activity is forskolin, 8-bromo-cAMP or dibutyryl-cAMP.

The following may apply, in certain embodiments, to any of the screening methods disclosed herein, as well as any other method where context indicate. In some embodiments of any of the screening methods disclosed herein, the agent is further assessed in an assay for hedgehog signaling. In some embodiments, the assay for hedgehog signaling comprises the steps of: i. contacting a cell with an amount of the agent, wherein the cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, and ii. determining, as compared to a control, whether the agent inhibits hedgehog signaling in the cell, wherein if the agent inhibits hedgehog signaling in the cell relative to the control, then an agent that inhibits hedgehog signaling is identified.

The following may apply, in certain embodiments, to any of the screening methods disclosed herein, as well as any other method where context indicate. In certain embodiments of any of the screening methods disclosed herein, TPRA40 is expressed in a cell exogenously (e.g., by transfecting or transforming the cell with a vector expressing TPR40). In certain

embodiments, TPRA40 is stably expressed in the cell. In certain embodiments, TPRA4Q is transiently expressed in the cell. In certain embodiments, the ceil is transformed with a vector expressing TPRA40 protein. In certain embodiments, the reporter gene is a luciferase gene controlled by a cAMP response element. In certain embodiments, the activator is forskolin, 8- bromo-cAMP or dibutyryl-cAMP. Even when TPRA40 is expressed using exogenous means (e.g., introducing TPRA40) it is also contemplated that the cell may optionally express endogenous TPRA40. When providing TPRA40 using exogenous means, the TPRA40 may be from the same species as the cell or from a different species (e.g., use a vector to express human TPRA40 in a murine cell or use a vector to express human TPRA40 in a human cell). The disclosure specifically contemplates that any of the embodiments described above may be combined with any other embodiment, as well as with any aspect of the disclosure. Moreover, these aspects and embodiments may be combined with each other, as well as with embodiments described in the detailed descriptio .

Also contemplated are methods for screening for TPRA40 agonists. The disclosure contemplates that any of the foregoing assays for identifying agents that act as TPRA40 antagonists and/or inhibit hedgehog signaling may be used, similarly but evaluating for the opposite effect or read-out, to identify agents that act as TPRA40 agonists and/or promote hedgehog signaling. Such assays are explicitly contemplated and are summarized below.

Embodiments described above and herein that describe appropriate cells, assay reagents, reporters, activators and the like are applicable to agonist assays and are expressly contemplated.

In certain aspects, the disclosure provides for a method of screening for a TPRA40 agonist, wherein the method comprises: a) contacting a cell that expresses TPRA40, adenylyl cyclase and a reporter with an agent; b) determining, as compared to an untreated control, whether the agent suppresses the adenylyl cyclase activity, wherein if the agent suppresses adenylyl cyclase activity relative to the non-agent treated TPRA40 expressing cells, then the agent is identified as a TPRA40 agonist.

In another aspect, the disclosure provides for a method of identifying a TPRA40 agonist, comprising: a) providing a cell that expresses TPRA40 and that expresses a reporter gene capable of indicating adenylyl cyclase activity; b) contacting the cell with an activator of adenylyl cyclase and with an agent, wherein the cells are contacted with the activator and the agent simultaneously, concurrently, or consecutively: and c) determining, as compared to a control, whether the agent suppresses adenylyl cyclase activity induced by the activator, wherein if the agent suppresses the adenylyl cyclase activity relative to the control, then the agent is identified as a TPRA40 agonist.

In another aspect, the disclosure provides for a method of screening for an agent for inducing hedgehog signaling in a cell, wherein said method comprises: a) screening for an agent that binds to TPRA40 protein, induces expression of TPRA40, facilitates transport of TPRA40 protein to the plasma membrane or to primary cilia., induces activation of TPRA40 or couples it with Gai; b) contacting a cell with an amount of the agent identified in step a), and c ) determining, as compared to a control, whether said agent induces hedgehog signaling in said cell, wherein if said agent induces hedgehog signaling in said cell relative to the control, then an agent that induces hedgehog signaling is identified.

In another aspect, the disclosure provides for a method of identifying a TPRA40 agonist, wherein said method comprises: a) screening for an agent that binds to TPRA40 protein; b) contacting a cell with an amount of the agent identified in step a), and c) determining, as compared to a control, whether said agent that binds to T'PRA40 protein also induces hedgehog signaling in said cell, wherein if said agent induces hedgehog signaling in said cell relative to the control, then the agent is identified as a TPRA40 agonist. In certain embodiments, the agent is a small molecule. In certain embodiments, the agent is a polypeptide. In certain embodiments, the agent is a polynucl eoti de .

In another aspect, a TPRA40 agonist may be used in methods of promoting hedgehog signaling in a cell, and such methods are contemplated.

The disclosure contemplates all combinations of any of the foregoing aspects and embodiments, as well as combinations with any of the embodiments set forth in the detailed description and examples. For example, any of the TPRA40 antagonists described generally or specifically herein (e.g., TPRA40 antagonists of the disclosure) may be used in any of the methods described herein.

BRIEF DESCRIPTION OF TFIE DRAWINGS

Figures 1 A and 1 B show that TPRA40 depletion inhibited Hh- and Snio-stimulated 67/- luciferase activity in S52 cells. (A) S12 cells were depleted of murine I tSS (by 72%), Ssnal (by 75%) and TPRA40 (by 80%) by 50nM siRNA transfection (with pools of four siRNAs per target) for 72 hours, the last 24 hours of which they were incubated in serum-free media with (black; right bar in each set of bars) or without (grey; left bar in each set of bars) 200ng/ml octyl-Shh to stimulate Hh signaling. &7/-iuciferase activity was measured versus renilla-luciferase (as a measure of viability) and the data were expressed as a % of the non-targeting control siRNA (siNTC) +Hh. The mean and standard deviation of four independent experiments are shown. *, p <0.05; **, p < 0.01 ; ***, p < 0.001 (student's unpaired t-test). (B) S12 cells were treated as in (A) except treated with DMSO control or stimulated with lOOnM of the Smoothened, small molecule agonist Hhl .2 (HhAgl .2) instead of octyl-Shh (black; right bar in each set of bars). Results are expressed as a percentage of non-targeting control (siNTC), agonist (HhAgl .2) treated cells (black; right bar in each set of bars). TPRA40 depletion using siRNA inhibited both Hh and Smoothened agonist stimulated G f-luciferase activity by approximately 40%.

Figures 2A and 2B show that TPRA40 depletio inhibited Hh-stimulated GUI induction in S 12 cells. (A) S 12 cells were depleted of TPRA40 by siRNA treatment and stimulated with 200ng/ml octylated Sonic Hedgehog protein (black; Hh; right bar in each set) or without Hedgehog protein (grey; left bar in each set) as in Figure 1 , but analyzed by qRT-PCR for endogenous murine GUI expression instead of G/Mueiferase activity. The mean and standard deviation of three independent experiments was plotted and showed an approximately 50% reduction in Hedgehog pathway stimulation in TPRA40 siRNA treated cells (i.e., TPRA40- deficient cells). (B) Cells were treated as in (A) but lysed and analyzed by western blotting with a Gl.il -specific monoclonal antibody, then reprobed for tubulin (55kDa) as a loading control. The Hh-induced upregulation of Gli l protein (-150 kDa) was also diminished by TPRA40 knockdown (a representative blot of two independent experiments is shown).

Figures 3A-C show that three of the four individual siRNA. components of the siTPRA40 pool are active and reduce TPRA40 expression. (A) The four siRNAs to TPRA40 that make up the pool (siRNAs #9-12) were transfected individually into S 12 cells at 25nM and the Gli- luciferase activity measured as in Figure 1 A except the data were normalized to siNTC +Hh as 1 . Mean and standard deviation of three experiments are shown. siRNAs 1 0-12 inhibited Gli- luciferase stimulation similarly to the !OOnM poo! (by 34-40%), while siRNA #9 did not. (B) S I 2 cells were treated as in (A) but endogenous Glil levels were measured by qRT-PCR, 25nM siRNA #9 again had no effect, while siRNAs 10-12 inhibited Glil induction, albeit less effectively than ΙΟΟηΜ of the 4 siRNA pool. Data from a single experiment is shown. (C) TPRA40 gene expression levels by qRT-PCR were decreased by more than 80% by the siRNA pool and individual siRNAs 9 and 10; by about 60% by siRNA #1 1 and by about 70% by siRNA #12. Hedgehog stimulation had no effect on TPRA40 levels (in the non-targeting control

(NTC)-transfected cells), consistent with the conclusion that TPRA40 is not itself a hedgehog pathw ay target gene. Mean and standard deviations of triplicate samples from a single experiment is shown. In Figure 3B and 3C, results in the presence of hedgehog treatment are shown in black (the right bar in each set) and the results in the absence of hedgehog treatment are shown in grey (the left bar in each set). Figure 4 shows that depletion of TPRA40 inhibited hedgehog signaling in Daoy medullablastoma ceils. Daoy nieduilablastoma cells exhibit constitutiveiy active hedgehog signaling (in the absence of Hh ligand) as monitored by hGlil niRNA levels. This constitutive signaling can be decreased by treatment with 0.5 or 2 μΜ Cy dopamine, a well-known

Smoothened inhibitor. TPRA40 depletion by transfection of 50nM siRNA pool to hTPRA40 also reduced hGUI levels and, when combined with cy dopamine, further reduced hGlil levels. Mean and standard deviations of triplicate samples from a single experiment is shown. For each of the DMSO or cyclopamine treatment groups, data for cells treated with the TPRA40 siRNA pool is showrn by the right-hand bar (black).

Figures 5A-C show that TPRA40 acts downstream of Ptch l and SuFu. (A) Schematic of key hedgehog pathway components, with positive regulators Shh (Sonic Hedgehog), Srao (Smoothened) and Gli activators (Gli-A) in normal font and negative regulators Ptc (Patched 1), SuFu (Suppressor of Fused), PKA (Protein Kinase A) and Gli repressors (Gli-R) in bold type. (B-C) Depletion of the negative regulator Ptchl (B, data normalized to siPtchl ) or depletion of the negative regulator SuFu (C, data normalized to siSuFu) stimulated G i-luciferase activity in SI 2 cells in the absence of ligand (Sonic hedgehog protein). This stimulation of hedgehog signaling activity was partially rescued by co-depletion of TPRA40. Mean and standard deviation of 3 independent experiments is shown.

Figure 6 shows that TPRA40 does not act downstream of PKA, S 12 cells were treated with DMSO vehicle control or 80 μ.Μ cell permeable 14-22 amide (a PKA inhibitor) for 24 hours in the absence of stimulation with Hedgehog (e.g., ligand) following 48 hours of treatment with siR As to TPRA40 (black/right bar) or non-targeting control (grey/left bar). TPRA40 depletion does not rescue PKA inhibition, suggesting that TPRA40 acts at the level of, or upstream of, PKA.

Figures 7 A and 7B show that TPRA40 knockdown increased levels of Gli3 repressor. (A)

Western blot of Gli3 using monoclonal antibody 6F5. Data is shown for the following: culture in the presence or absence of 24 hour Hedgehog treatment and TPRA40 or Ift88 depletion, with tubulin 1A2 as a loading control. (B) Quantitation of 3 westerns of Gii3FL and G113R

normalized to tubulin (mean and standard deviation normalized to NTC -Hh). Hedgehog stimulation for 24 hours inhibits PKA activity and attenuates GH3R production, which requires primary cilia (as evidenced by the increase in Gli3R levels following Ift88 depletion). TPRA40 knockdown increased both the baseline level of Gli3R (by about 25%) and the level remaining after Hh stimulation (by 2 fold).

Figures 8A-8B show the characterization of antibodies to endogenous TPRA40 by western blotting of S12 cells. S12 cells transfected with NTC or TPRA40 siRNAs for 48 hours were serum starved in the presence or absence of Hedgehog protein for 24 hours (72 hours total knockdown), then lysed and subjected to western blotting. (A) Custom-made rabbit anti- TPRA40 C-terminal antibody 12569B (generated by YenZym) was the antibody used in this experiment. This antibody detects a single band of about 55kDa. that is not affected by Hh treatment (i.e., is obsen'ed regardless of whether the cells are cultured in the presence or absence of Hedgehog), but disappears following TPRA40 depletion using siRNAs. These results indicate that this antibody is specific for TPRA40. (B) A mouse anti-TPRA40 antibody 6H2

(commercially available from Santa Cruz) was the antibody used in this experiment. This antibody also detects TPRA40. However, this antibody appears less clean and also recognizes a couple of smaller non-TPRA40 proteins, albeit to a lesser extent.

Figures 9A-9B show that endogenous TPRA40 localizes to primary cilia of 812 cells in a

Hedgehog-dependent, fashion. (A) SI 2 cells were fixed and processed for immunofluorescence using anti-TPRA40 monoclonal antibody 6H2 (left panels and red channel) and rabbit anti- Aril 3b antibody (a marker for primary cilia in the middle and green channel) following 24 hours serum starvation alone (upper panel) or with Hedgehog stimulation (lower panel). Nuclei stained with DAPI are in blue, (B) Counting the number of Arll3b-positive cilia for TPRA40 staining revealed TPRA40 was found in only about 20% of cilia in the absence of Hedgehog stimulation, but accumulated in 60% of cilia following overnight Hedgehog stimulation. This level of accumulation to cilia is very similar to the extent of accumulation of Smoothened (mean and SD of 2 experiments is shown).

Figure 10 shows that TPRA40 expression inhibited cAMP production in a CRE- luciferase reporter assay. (A) Schematic of a model of TPRA40 as a modulator of c AMP levels in a cell. (B) 293T cells expressing CRE (cAMP Response ElementVluciferase along with GFP (negative control, white diamonds) or a TPRA40 expression construct (black squares) were treated with varying doses of Forskolin, a potent activator of Adenylyl Cyclase, thus increasing cAMP levels inside the cells. The graph shows that exogenous expression of TPRA40 suppressed the CRE-reporter activity in a dose dependent manner, suggesting that this GPCR is coupled to Galpha(i), which inhibits cAMP production. The mean and standard deviation of four independent experiments normalized to 20μΜ forskolin in GFP-transfected ceils is shown.

Figure 11A shows that S 12 cells trans feeted with siRNAs to Galpha (i)l show reduced Hedgehog signaling compared to NTC treated cells. Knockdown of Galpha(i)l with siRNAs decreased G/j-luciferase activity in Hedgehog-treated S 12 cells by about 50%, consistent with increased cAMP production stimulating more PKA activity and Gli3R production. Co expression of siTPRA40 along with siGalpha (i)l did not rescue reporter activity compared to siGalpha(i)i alone, suggesting that TPRA40 functions at the level of or upstream of Galpha (i)l . Mean and SD of 3 independent experiments is shown. Figure 1 IB shows that Gli3 depletion is partially rescued by TPRA40 knockdown in S12 cells. S 2 cells depleted of Gli3 by siRNA show active Hedgehog signaling in the absence of ligand due to loss of GH3 repressor (the Gli- luciferase signal is initiated by Gli2 activator). TPRA40 depletion partially inhibits the signal in GH3-depleted cells. Mean and SD of Gli-luciferase signals from. 5 independent experiments (in the absence of Hedgehog ligand) were expressed as a percentage of siGH3 alone. Figure 11C shows that TPRA40 depletion does not prevent Gli3 accumulation at cilia tips. The images on the left show a representative immunofluorescence analysis of S 12 cells serum starved overnight and treated for one hour (lower two panels) or not (upper two panels) with Hedgehog prior to fixation. Cells were co-stained for the ciliary marker acetylated tubulin (left column and green channel in merged right column) and Gli3 (middle column and red channel in merge). Depletion of TPRA40 by siRNA treatment does not prevent the Hedgehog-dependent accumulation of GH3 at cilia tips (bottom row). Arrows show the tips of the primary cilia. The number of cilia with and without Gli3 at the tips as counted for each condition and plotted as a percentage of total cilia in the graph (the mean and SD of three independent experiments is shown). The grey bars (the left of each pair of bars) show that less than 10% of cilia have Gli3 at the tips in the absence of Hedgehog stimulation, while the black bars (the right of each pair of bars) show

approximately 80% of cilia have Gli3 at their tips irrespective of the presence of TPRA40.

Figure 12 provides a working model for TPRA40 function as a positive regulator of Hedgehog signaling.

Figure 13 shows an alignment of the amino acid sequences of mouse and human TPRA40. Human TPRA40 (top, 373 amino acids, predicted MW 41034 Da; Swissprot Q86W33) is aligned with mouse TPRA40 (middle, 369 amino acids, predicted MW 40560 Da; Swissprot Q99MU1) and zebrafisli TPRA l (bottom, 378 amino acids, predicted MW 41685 Da; Swissprot Q4V8X0) using the Align program in GSeqWeb. Identical amino acids are colored, and the positions (predicted by Swissprot) of the 7 transmembrane (tni) domains typical of GPCRs are underlined in blue. Mouse and human TPRA share 91 ,4% identity and 94.1% similarity at the protein level. Zebrafish TPRAl is 70.0% and 68.7% identical (79.3% and 78.0% similar) to human and mouse TPRA40, respectively, suggesting an evolutionarily conserved function. As expected for a GPCR, the N-terminus is luminal/extracellular and the C-terminus is cytoplasmic. This topography was verified by FACS with epitope tags at each end of the protein (data not shown). DETAILED DESCRIPTION OF THE DISCLOSURE

I. Overview

Hedgehog (Hh) signaling plays an essential role in vertebrate embryonic development, affecting tissue patterning of many organs. All key components of this signaling pathway traffic through primary cilia, with the GPCR. GPR161 and Hedgehog receptor Patched exiting cilia, while the GPCR-like protein Smoothened and the SuFu/Gli complex accumulate in cilia in response to Hedgehog ligand. Here we identify a novel orphan G-protein-coupled receptor, TPRA40 (also known as TPRAl and GPR175), that also localizes to primary cilia upon Hh stimulation and positively regulates Hh signaling downstream of Smoothened. TPRA40 knockdown decreases Hh signaling activity as evidenced by decreased Hh-stimulated activity in a (7/i-luciferase reporter (murine S 32 cell) assay and decreased elevation of the endogenous early transcriptional target GUI. Furthermore, TPRA40 depletion reduces constitutive (cyclopamine- sensitive) GUI expression in human meduilobiastoma (Daoy) ceils. Epistasis experiments in S 12 ceils indicate that TPRA40 acts at the level of Protein Kinase A downstream of SuFu and may be coupled to G alpha(i) since its overexpression inhibits forskolin-mediated cAMP production in CRE-iueiferase reporter 293 cells. These data support a role for TPRA40 as a novel positive regulator of the Hh signaling pathway.

The present disclosure is based upon the identification of TPRA40 as a novel component of the hedgehog signaling pathway and as positive regulator of Hedgehog signaling. Based on this identification, the present disclosure provides assays for screening to identify agents that antagonize TPRA40 expression and/or activity and/or localization (e.g., TPRA40 antagonists). TPRA40 antagonists, such as those so identified, may be used to inhibit hedgehog signaling in any of a number of in vivo and in vitro settings, such as in cells with hyperproliferation or otherwise characterized by unwanted cell proliferation and/or in cells with hyperactive hedgehog signaling (e.g., such as due to a mutation in a component of the hedgehog signaling pathway or due to stimulation by hedgehog protein overexpressed by the cell or by an adjacent cell).

Moreover, the present disclosure provides examples of TPRA40 antagonists, including working examples of specific antagonists and generic and specific examples of other antagonists and classes of antagonists (collectively, "TPRA40 anta gonists of the disclosure" or "TPRA40 antagonists described herein"), and provides numerous methods for using TPRA40 antagonists of the disclosure. The disclosure provides numerous methods of using TPRA40 antagonists in vitro and/or in vivo. In certain embodiments, an agent identified as a TPRA40 antagonist, using any of the screening assays provided herein may be used in any of the methods of inhibiting cell proliferation and/or hedgehog signaling provided herein.

Thus, it is specifically contemplated that the TPRA40 antagonists of the present disclosure will not only interfere with aspects of hedgehog signal transduction activity (e.g., inhibit hedgehog signaling or decreasing hedgehog signaling), but will likewise be capable of changing the fate of a cell or tissue that is affected by hedgehog signaling, such as cells undergoing norma] development or disease states that are characterized by aberrant (e.g., o ver- expressing) hedgehog signaling. More specifically, the TPRA40 antagonists described herein may be used for inhibiting hedgehog signaling that can occur either (i) as active, wild-type hedgehog signaling or (ii) as a result of hyperactivation of the hedgehog pathway, such as due to mutation or excess hedgehog protein. Disorders resulting from hyperacti vation of the hedgehog pathway can be attributed to mutations arising in hedgehog signaling components or

inappropriate activation or stimulation that does not result from a mutation or lesion in a hedgehog signaling component such as overexpression of hedgehog ligand(s) (e.g.,

overexpression of Sonic Hedgehog). One of skill in the art will readily recognize that TPRA40 antagonists are suitable for the treatment of conditions or disorders characterized by hyperactive hedgehog signaling as well as modifying the cell fate during development by suppression of hedgehog signaling.

'The present disclosure also provides assays for screening to identify agents that agonize TPRA40 expression and/or activity and/or localization (e.g., TPRA40 agonists). TPRA40 agonists so identified may be used to promote hedgehog signaling in any of a. number of in vivo and/or in vitro settings. It is also specifically contemplated that TPRA40 agonists of the present disclosure can he used to promote hedgehog signal transduction and be capable of changing the fate of a cell or tissue that is affected by hedgehog signaling, such as cells undergoing normal development or disease states that are characterized by aberrant (i.e., under-expressing) hedgehog signaling. Agonists may be useful in promoting hair growth and/or studying hair growth. Merely by way of example, agonists are also useful as reagents for stem cell biology, cell proliferation, cell differentiation, and to study hedgehog signaling.

II. Defiiiitioiis

Before continuing to describe the present disclosure in further detail, it is to be understood that this disclosure is not limited to specific compositions or process steps, as such may vary. It must be noted that, as used in this specification and the appended claims, the singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, the Concise Dictionary of Biomedicine and Molecular Biology, Juo, Pei- Show, 2nd ed., 2002, CRC Press; The Dictionary of Cell and Molecular Biology, 3rd ed., 1999, Academic Press; and the Oxford Dictionary Of Biochemistry And Molecular Biology, Re vised, 2000, Oxford Uni versity Press, pro vide one of skill with a general dictionary of many of the terms used in this disclosure.

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical

Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.

It is convenient to point out here that "and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

A "TPRA40 polypeptide," includes both "native sequence TPRA40 polypeptides" and a

TPRA40 polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%», 99% or 100% identical to the amino acid sequence of any of SEQ ID NOs: 1-3, but which retains a biological activity of a native TPRA40 polypeptide. In certain embodiments, TPRA40 polypeptides of the disclosure retain a biological activity of native TPRA40, such as the ability to positively regulate hedgehog signaling, the ability to localize to cilia in response to stimulation with hedgehog ligand, and the ability to modulate adenylate cyclase activity. In certain embodiments, the TPRA40 polypeptide varies by about 1 , 2, 3, 4, 5, 6, 7, 8, 9, or 10, or about 20 amino acid residues in length from the corresponding native sequence polypeptide (e.g., SEQ ID NO: 1 , 2, or 3). Alternatively, in certain

embodiments, the TPRA40 polypeptide has no more than one amino acid substitution, such as a. conservative substitution, as compared to the corresponding native polypeptide sequence, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, such as

conservative substitutions, as compared to the native polypeptide sequence.

A "native sequence TPRA40 polypeptide" comprises a polypeptide having the same amino acid sequence as the corresponding TPRA40 polypeptide derived from, nature. Such native sequence TPRA40 polypeptides can be isolated from nature or can be produced by recombinant or synthetic means. The term "native sequence TPRA40 polypeptide" specifically encompasses naturally-occurring truncated or secreted forms of the specific TPRA40

polypeptide (e.g., an extracellular domain sequence), naturally-occurring variant forms (e.g., alternatively spliced forms) and naturally-occurring allelic variants of the polypeptide. In one embodiment, the native sequence TPRA40 polypeptides disclosed herein are mature or full- length native sequence polypeptides corresponding to the sequence of any of SEQ ID NOs: 1-3, in the presence or absence of the N-terminal methionine.

"TPRA40 polypeptide variant" means a TPRA40 polypeptide, preferably active forms thereof, as defined herein, having at least about 80%) amino acid sequence identity with a fuil- length native TPRA40 polypeptide sequence, respectively, as disclosed herein, and variant forms thereof lacking one or more of the C-terminal domain, an extracellular domain, a cytoplasmic domain, an N-terminal domain or any other fragment of a full length native sequence TPRA40 polypeptide, such as those referenced herein. Such variant polypeptides include, for instance, polypeptides wherein one or more amino acid residues are added, or deleted, at the N- or C- terminus of the full-length native amino acid sequence. In a specific aspect, such variant polypeptides will have at least about 80% amino acid sequence identity, alternatively at least

7 ? about 81%, 82%, 83%, 84%, 85%, 86%, 87%», 88%, 89%, 90%, 91%, 92%, 93%», 94%, 95%, 96%), 97%, 98%, or 99% amino acid sequence identity, to a full-length native sequence TPRA40 polypeptide (e.g., any of SEQ ID NOsi 1-3), as disclosed herein, and variant forms thereof lacking an extracellular domain, or any other fragment of a full length native sequence TPRA40 polypeptide, such as those disclosed herein. In certain embodiments, such variant polypeptides will vary at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 amino acids in length from the corresponding native sequence. Alternatively, in certain embodiments, the TPRA40 polypeptide has no more than one amino acid substitution, such as a conservative substitution, as compared to the corresponding native polypeptide sequence, alternatively no more than 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions, such as conservative substitutions, as compared to the native polypeptide sequence.

"Percent, (%) amino acid sequence identity" with respect to the TPRA40 polypeptide sequences identified herein is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in the specific TPRA4Q polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Alignment for purposes of determining percent amino acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign

(DNASTAR.) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared. For purposes herein, however, %> amino acid sequence identity values are generated using the sequence comparison computer program ALIGN-2 sequence comparison computer program was authored by Genentech, Inc. and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco, California. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D. All sequence comparison parameters used with ALIGN-2 are set by the ALIGN-2 program and do not vary. "TPRA40 variant polynucleotide" or "TPRA40 variant nucleic acid sequence" means a nucleic acid molecule which encodes a TPRA40 polypeptide, preferably active forms thereof, as defined herein, and which have at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native sequence TPRA40 polypeptide sequence identified herein, or any other fragment of the respective full-length TPRA40 polypeptide sequence as identified herein (such as those encoded by a nucleic acid that represents only a portion of the complete coding sequence for a full-length TPRA40 polypeptide).

Ordinarily, TPRA40 variant polynucleotide will have at least about 80% nucleic acid sequence identity, alternatively at least about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% nucleic acid sequence identity with a nucleic acid sequence encoding the respective full-length native sequence TPRA40, or any other fragment of the respective full-length TPRA40 polypeptide sequence identified herein. Such variant polynucleotides do not encompass the native nucleotide sequence. Ordinarily, such variant polynucleotides vary at least about 50 nucleotides in length from the native sequence polypeptide, alternatively the variance can be at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 1 10, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 550, 520, 530, 540, 550, 560, 570, 580, 590, 605 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length.

"Percent (%) nucleic acid sequence identity" with respect to TPRA40 polypeptide - encoding nucleic acid sequences identified herein, is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the TPRA40 nucleic acid sequence of interest, respectively, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. For purposes herein, however, % nucleic acid sequence identity values are generated using the sequence comparison computer program ALlGN-2. The ALIGN -2 sequence comparison computer program was authored by Genentech, Inc. and the source code has been filed with user documentation in the U.S. Copyright Office, Washington D.C., 20559, where it is registered under U.S. Copyright Registration No.TXUS 10087. The ALIGN-2 program is publicly available through Genentech, Inc., South San Francisco,

California. The ALIGN-2 program should be compiled for use on a UNIX operating system, preferably digital UNIX V4.0D, All sequence comparison parameters used with ALIGN-2 are set by the ALIGN-2 program and do not vary. In situations where ALIGN-2 is employed for nucleic acid sequence comparisons, the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) is calculated as follows: 100 times the fraction W/Z where W is the number of nucleotides scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of C and D, and where Z is the total number of nucleotides in D. It will be appreciated that where the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the % nucleic acid sequence identit of C to D will not equal the % nucleic acid sequence identity of D to C.

In other embodiments, TPRA40 variant polynucleotides are nucleic acid molecules that encode TPRA40 polypeptides, and which are capable of hybridizing, preferably under stringent hybridization and wash conditions, to nucleotide sequences encoding a full-length TPRA40 polypeptide, as disclosed herein. Such variant polypeptides may be those that are encoded by such variant polynucleotides.

"Isolated" means identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that would typically interfere with diagnostic or therapeutic uses for an agent.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a. presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

"Stringency" of hybridization reactions is readily determinable by one of ordinary skill in the art, and generally is an empirical calculation dependent upo probe length, washing temperature, and salt concentration. In general, longer probes require higher temperatures for proper annealing, while shorter probes need lower temperatures. Hybridization generally depends on the ability of denatured DNA to reanneal when complementary strands are present in an environment, below their melting temperature. The higher the degree of desired homology between the probe and hybridizable sequence, the higher the relative temperature which can be used. As a result, it follows that higher relative temperatures would tend to make the reaction conditions more stringent, while lower temperatures less so. For additional details and explanation of stringency of hybridization reactions, see Ausubel et al., Current Protocols in Molecular Biology, Wiley Tnterscience Publishers, (1995).

"Stringent conditions" or "high stringency conditions", as defined herein, may be identified by those that: (1) employ low ionic strength and high temperature for washing, for example 0.015 M sodium chloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50°C; (2) employ during hybridization a denaturing agent, such as formamide, for example, 50% (v/v) formamide with 0.1 % bovine serum albumin/0.1 % Ficoli 0.1 % polyvinylpyrrolidone/50mM sodium phosphate buffer at pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or (3) overnight hybridization in a solution that employs 50% formamide, 5 x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with a 10 minute wash at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) followed by a 10 minute high-stringency wash consisting of 0.1 x SSC containing EDTA at 55°C. "Moderately stringent conditions" may be identified as described by Sambrook et ai., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press, 1989, and include the use of washing solution and hybridization conditions (e.g., temperature, ionic strength and %SDS) less stringent that those described above. An example of moderately stringent conditions is overnight incubation at 37°C in a solution comprising: 20% formamide, 5 x SSC (150 niM NaCl, 15 niM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 x Denhardt's solution, 10% dextran sulfate, and 20 mg/mi denatured sheared salmon sperm DNA, followed by washing the filters in 1 x SSC at about 37-50°C. The ordinarily skilled artisan will recognize how to adjust the temperature, ionic strength, etc. as necessary to accommodate factors such as probe length and the like.

"Active" or "activity", when context indicates such term is used to refer to a TPRA40 polypeptide, refers to form(s) of a TPRA40 polypeptide which retain a biological and/or an immunological activity of native or naturally-occurring TPRA40 polypeptide, wherein

"biological" activity refers to a biological function caused by a native or naturally-occurring TPRA40 polypeptide, other than the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring TPRA40 polypeptide, and an "immunological" acti vity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native or naturally-occurring TPRA40 polypeptide. Exemplary biological activities of TPRA40 are described herein. "Carriers" as used herein include pharmaceutically acceptable carriers, excipients, or stabilizers which are nontoxic to the cell or mammal being exposed thereto at the dosages and concentrations employed. Often the physiologically acceptable carrier is an aqueous pH buffered solution. Examples of

physiologically acceptable carriers include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterforts such as sodium; and/or nonionic surfactants such as 'TWEEN*', polyethylene glycol (PEG), and PLURONICS*. Pharmaceutical compositions or formulations, such as compositions or formulations of the disclosure comprising a TPRA40 antagonist of the disclosure, may be formulated with one or more carriers and/or excipients.

By "solid phase" or "solid support" is meant a non-aqueous matrix to which a molecule that binds TPRA40 polypeptide of the present disclosure, or to which a TPRA40 polypeptide fragment (e.g., the C-terminal region of the TPRA40 polypeptide) can adhere or attach.

Examples of solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones. In certain embodiments, depending on the context, the solid phase can comprise the well of an assay plate: in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles.

An "effective amount" of a TPRA40 antagonist agent is an amount sufficient to inhibit, partially or entirely, hedgehog signaling that is dependent upon stimulation from hedgehog or that is due to one or more mutations in a gene in the hedgehog signaling pathway (e.g.

smoothened or patched). Alternatively of additionally, an effective amount of TPRA40 antagonist is an amount sufficient to reduce the rate of proliferation of a cell and/or rate of survival of a cell and/or the rate of growth of a cell that is expressing or overexpressing hedgehog or that has active or hyperactive hedgehog signaling. Alternative or additionally, an effective amount of TPRA40 antagonist is an amount sufficient to decrease or halt the growth, proliferation, and/or survival of a tumor, such as a tumor responsive to hedgehog signaling, characterized by hyperactive hedgehog signaling, or comprising a mutation in one or more components of the hedgehog signaling pathway. An "effective amount" may be determined empirically and in a routine manner, in relation to this purpose, in some embodiments, the effective amount is determined with respect to the amount of a TPRA40 antagonist sufficient to inhibit, partially or entirely, hedgehog signaling in at least 10%, 15%, 20%), 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells in a cell culture and/or to inhibit hedgehog signaling in a cell by at least 10%, 15%, 20%, 25%, 30%, 40%», 50%, 60%, 70%, 80%, 90%, or 100%. In some embodiments, the effective amount is determined with respect to the amount of a TPRA40 antagonist sufficient to reduce the rate of proliferation of a cell and/or rate of survival and/or rate of growth of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells in a ceil culture, wherein the cells are expressing or overexpressing hedgehog or have active hedgehog signaling. In some embodiments, the effective amount is determined with respect to the amount of a TPRA40 antagonist sufficient to reduce GUI expression in at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells in a cell culture and/or to inhibit hedgehog signaling in a ceil by at 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%), 90%), or 100%. The term "effective amount" also refers to a TPRA40 antagonist or other drug effective to "treat" a disease or disorder in a subject or mammal. In the case of hedgehog signaling, the effective amount of the drug will improve aberrant hedgehog signaling such that it is closer to normal physiological levels; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) the infiltration of tumor cells into peripheral tissue or organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and/or relieve to some extent one or more of the symptoms associated with the tumor or cancer. See the definition herein of "treating". To the extent the drug may prevent growth and/or kill existing cancer cells, it may be cytostatic and/or cytotoxic.

A "growth inhibitory amount" of a TPRA40 antagonist, is an amount capable of inhibiting the growth of a cell, especially tumor, e.g., cancer cell, either in vitro or in vivo. For purposes of inhibiting neoplastic cell growth, such an amount may be determined empirically and in a routine manner,

A "cytotoxic amount" of a TPRA40 antagonist is an amount capable of causing the destruction of a cell, especially a tumor cell, e.g., cancer cell, either in vitro or in vivo. For purposes of inhibiting neoplastic cell growth may be determined empirically and in a routine manner.

The term "TPRA40 antagonist" refers to an agent that inhibits the expression and/or activity and/or localization of TPRA40 by: (i) binding to TPRA40 DNA, R A, or protein, and/or (ii) disrupting the interaction between TPRA40 and a TPRA40 target or binding partner, and/or (iii) modulating TPRA40 activity downstream of Suppressor of Fused (SuFu) or Smoothened (Smo). in certain embodiments, the activity of TPRA40 that is decreased or inhibited by the TPRA40 antagonist is the activity of TP A40 as a positive regulator of hedgehog signaling (e.g., the TPRA40 antagonist inhibits or antagonizes hedgehog signaling), in some embodiments, the TPRA40 antagonist inhibits the transport of TPRA40 polypeptide to the plasma, membrane and/or cilia. In some embodiments, the TPRA40 antagonist inhibits a biological function of the TPRA40 polypeptide. In some embodiments, the TPRA40 antagonist inhibits expression of TPRA40 RNA or protein. In some embodiments, the TPRA40 antagonist prevents interaction between TPRA40 polypeptide with the Gaipha-i protein. In some embodiments, the TPRA40 antagonist inhibits TPRA40-mediated inhibition of Protein Kinase A. In some embodiments, the TPRA40 antagonist induces an increase in cAMP levels in a cell. The disclosure provides working examples of agents that are TPRA40 antagonists, as well as numerous other specific and generic examples of such TPRA40 antagonist agents and categories of agents (collectively, "TPRA40 antagonists of the disclosure" or "a TPRA40 antagonists of the disclosure").

The term "TPRA40 antagonist" expressly includes, in certain embodiments, TPRA40 polypeptide variants (e.g. TPRA40 polypeptide variants that bind the ligand bound by TPRA40 or that interact with Ssnal but do not promote hedgehog signaling), anti-TPRA40 antibodies

(e.g., antibodies that, bind to the C-terminal 84 amino acids of TPRA40 polypeptide or to any of the extracellular portions of the TPRA40 polypeptide), TPRA40-binding antibody fragments thereof, TPRA40 antigen binding fragments, TPRA40-binding oligopeptides (e.g., oligopeptides that bind to the ligand binding site of TPRA40 to prevent ligand binding, or that bind to the C- terminal region of TPRA40), polynucleotides that inhibit TPRA40 expression (e.g. , TPRA40 sense/antisense nucleic acid and/or TPRA40 RNAi), and /or TPRA40 binding small organic molecules (e.g., small organic molecules that bind to the ligand binding site of TPRA40 and prevent ligand binding or that bind to TPRA40 and interfere with protei -protein interactions or trafficking). A "TPRA40 antagonist polypeptide" includes an anti-TPRA40 antibody, an antagonist TPRA40 chimeric polypeptide and a TPRA40 binding oligopeptide. Methods for identifying TPRA40 antagonists may comprise contacting the TPRA40 polypeptide, including a ceil expressing it, with a candidate agonist or antagonist molecule and measuring a detectable change in one or more biological activities normally associated with the TPRA40 polypeptide, e.g., ability to inhibit adenylyl cyclase activity (e.g., also known in the art and referred to interchangeably as adenylate cyclase activity) and'Or to promote hedgehog signaling. In certain embodiments, the TPRA40 antagonist is an antagonist of hedgehog signaling.

The term "TPRA40 agonist" refers to an agent that activates, maintains or potentiates the expression and'Or activity of TPRA40 and'Or facilitates the transport of TPRA40 to the plasma membrane or cilia by: (i) binding to TPRA40 DNA, RNA, or protein, and/ or (ii) facilitating the interaction between TPRA40 and a TPRA40 target or binding partner, and/or (iii) modulating TPRA40 activity downstream of Suppressor of Fused (SuFu) or Smoothened (Smo). In certain embodiments, the T PA40 agonist promotes (e.g., agonizes) hedgehog signaling.

A TPRA40 antagonist "which binds" a target of interest, e.g. TPRA40, is one that binds the target with sufficient affinity so as to be a useful diagnostic, prognostic and/or therapeutic agent. In certain embodiments, the antagonist does not significantly cross-react with other proteins. Moreover, the term "specific binding" or "specifically binds to" or is "specific for" or "specifically targets", means binding that is measurably different from a non-specific interaction. This concept may be similarly used when referring to binding or targeting of a nucleic acid antagonist to its target. Specific binding can be measured, for example, by determining binding of a molecule compared to binding of a control molecule, which generally is a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition with a control molecule that is similar to the target, for example, an excess of non-labeled target. In this case, specific binding is indicated if the binding of the labeled target to a probe is competitively inhibited by excess unlabeled target. In one embodiment, such terms refer to binding where a molecule binds to a particular polypeptide or epitope on a particular polypeptide without substantially binding to any other polypeptide or polypeptide epitope. Alternatively, such terms can be described by a molecule having a d for the target of at least about 10"4 , 1 0 * M, 1 0"6 M, 1 0 ~ VI . 50"s M, 1 0 M, 10"10 M, 1 0 ; ; , I 0" 12 M, or greater.

A ' PRA40- hyperactive tumor or cancer" produces excessive levels of TPRA40, such that hedgehog signaling is active or hyperactive, such that a TPRA40 hedgehog antagonist can bind thereto or otherwise target and have a therapeutic effect with respect to the tumor.

A tumor that "overexpresses" hedgehog or in which hedgehog signaling is "hyperactive" is one which has significantly higher levels of hedgehog at the ceil surface thereof, or that produces and secretes, compared to a noncancerous cell of the same tissue type, or that has more active hedgehog signaling and/or has hedgehog signaling that is not dependent on the presence of Hedgehog protein, as compared to a noncancerous cel l of the same tissue type. Such

overexpression or hyperactivity may result from gene amplification or by increased transcription or translation of certain hedgehog pathway genes or by mutation in a component of the hedgehog signaling pathway. In some embodiments, the hyperactive hedgehog signaling is because the ceil or an adjacent cell overexpresses hedgehog protein, such as Sonic hedgehog protein. In

^ ί some embodiments, the hyperactive hedgehog signaling is due to a mutation in a gene in the hedgehog pathway (e.g., a component). In some embodiments, the mutated gene is in any of the patched, smoothened, or SuFu, genes. In certain embodiments, hyperactive hedgehog signaling is because the cell comprises a smoothened gain-of- function mutation. In certain embodiments, hyperactive hedgehog signaling is because the cell comprises a Suppressor of Fused (SuFu) loss- of-function mutation. In certain embodiments, the cell comprises one or more mutations in a hedgehog pathway gene, and hyperactive hedgehog signaling is signaling that is not dependent on the presence and/or concentration of hedgehog ligand.

The "growth state" of a cell refers to the rate of proliferation of the cell and/or the state of differentiation of the cell. An "altered growth state" is a growth state characterized by an abnormal rate of proliferation, e.g., a cell exhibiting an increased or decreased rate of proliferation relative to a normal cell.

The term "hedgehog" or "hedgehog polypeptide" (Hh) is used herein to refer generic-ally to any of the mammalian homologs of the Drosophila hedgehog, i.e. , sonic hedgehog (SHh), desert hedgehog (SHh) or Indian hedgehog (IHh). The term may be used to describe protein or nucleic acid.

The terms "hedgehog signaling pathway", "hedgehog pathway", "hedgehog signaling" and "hedgehog signal transduction pathway" as used herein, interchangeably to refer to the signaling cascade mediated by hedgehog and its receptors (e.g., patched, patched-2) and which results in changes of gene expression and other phenotypic changes typical of hedgehog activity. The hedgehog pathway may, in certain embodiments, be activated or potentiated in the absence of hedgehog through activation of a downstream component (e.g., overexpression of

Smoothened, loss of function of patched, loss of function of Suppressor of fused, gain of function of smoothened, or transfections with Smoothened or Patched mutants to result in constitutive activation with activate hedgehog signaling in the absence of hedgehog). The transcription factors of the Gli family are often used as markers or indicators of hedgehog pathway activation. Hedgehog signaling is dysregulated in some cancers, sometimes due to mutations in a hedgehog pathway gene.

The term "Hh signaling component" or "component of the hedgehog signaling pathway" refers to gene and/or protein products that participate in the Hh signaling pathway (e.g., Shh, Smo, Ptch, Gli, and SuFu). A Hh signaling component frequently materially or substantially affects the transmission of the Hh signal in cel ls or tissues, thereby affecting the downstream gene expression levels and/or other phenotypic changes associated with hedgehog pathway activation.

Each Hh signaling component, depending on their biological function and effects on the final outcome of the downstream gene activation or expression, can be classified as either positive or negative regulators, A positive regulator is a Hh signaling component that positively affects the transmission of the Hh signal, e.g., stimulates downstream biological events when Hh is present (e.g., stimulates or promotes GUI expression), A negative regulator is a Hh signaling component that negatively affects the transmission of the Hh signal, e.g., inhibits downstream biological events when Hh is present (e.g., inhibits or decreases G I expression). In some embodiments, a Hh signaling component may be determined to be a positive or negative regulator of Hh signaling by inhibiting or expressing/overexpressing/activating the signaling component and monitoring GUI transcription. In these embodiments, if inhibition of the signaling component results in increased GUI transcription, then the signaling component is a negative regulator of Hh signaling, and if inhibition of the signaling component results in decreased GUI transcription, then the signaling component is a positive regulator of Hh signaling. And, if expression/overexpressi on/activation of a signaling component results in increased GUI transcription, then the signaling component is a positive regulator of Hh signaling, and if expression/overexpression/activation of a signaling component results in decreased GUI transcription, then the signaling component is a negati ve regulator of Hh signaling.

The term "patched loss-of- function" refers to an aberrant modification or mutation of a Ptch gene, or a decreased expression level of the gene, which results in a phenotype that resembles contacting the ceil with a hedgehog protein, e.g., aberrant activation of a hedgehog pathway. The loss-of- function may include a loss of the ability of the ptch gene product to regulate the expression l evel of the transcription factors Glil , Gli2 and/or Gli3.

The term "proliferating" and "proliferation" refer to a cell or cells undergoing mitosis. The term "smoothened gain-of-function" refers to an aberrant modification or mutation of a Smo gene, or in the ability of a Ptch gene product to bind to Smo and thereby suppress hedgehog signaling, which results in a phenotype that resembles activating the hedgehog pathway with hedgehog, e.g., aberrant activation of a hedgehog pathway. III. TPRA 0 Antagonists

i- TPRA40 and TPRA40 Antagonists

TPRA40 (also known as TPRA1 orGPR175) is an orphan G-protein coupled receptor whose physiological functions were previously unknown. TPRA40 is a 40 kDa protein having seven transmembrane domains (Fujimoto et ai., 2001, Biochim Biophys Acta, 1518(1 -2): 173-7) and an 84-amino acid cytoplasmic region (Fujimoto et al.). TPRA40 has been shown to be expressed during oxidative stress, aging and under certain pathophysiological conditions (A et al., 2008, J Cell Physiol, 217(1): 194-206). The role of TPRA40 in any particular signaling pathway was previously unknown. In addition, it was previously unknown whether TPRA40 was associated with any disease conditions, such as cancer, although it may be elevated in obese and diabetic mice models (Yang et, al., 1 999, Endocrinol 140: 2859). The present disclosure demonstrates for the first time a role for TPRA40 as a component of the hedgehog signaling pathway. Specifically, the disclosure demonstrates that TPRA40 is a positive regulator of hedgehog signaling, and that inhibition of TPRA40 activity results in inhibition of the hedgehog signaling pathway. In addition, the present disclosure provides data showing that TPRA40 acts downstream of both Smoothened and SuFu, and that TPRA40 interacts with a Galpha-i protein to inhibit adenylyl cyclase. As aberrant hedgehog signaling is involved in a number of disorders (e.g., cancer, psoriasis and trichosis), TPRA40 antagonists would be useful in treating these diseases, in modulating hedgehog signaling in vitro and/or in vivo, and in modulating cell growth, proliferation, and or survival in. vitro and/or in vivo.

The present disclosure provides TPRA40 antagonists. As defined herein, TPRA40 antagonist refers to an agent that inhibits the expression and/or activity and/or localization of TPRA40 by: (i) binding to TPRA40 DNA, RNA, or protein, and/or (ii) disrupting the interaction between TPRA40 and a TPRA40 target or binding partner, and/or (iii) modulating TPRA40 activity downstream of Suppressor of Fused (SuFu) or Smoothened (Smo). In other words, a

TPRA40 antagonist of the disclosure inhibits the expression and/or activity and'or localization of TPRA40 to antagonize one or more biological activities of native TP A4Q. In certain

embodiments, the activity of TPRA40 that is decreased or inhibited by the TPRA40 antagonist is the activity of TPRA40 as a positive regulator of hedgehog signaling (e.g., the TPRA40 antagonist inhibits or antagonizes hedgehog signaling). In some embodiments, the TPRA40 antagonist inhibits the transport of TPRA40 polypeptide to the plasma membrane and'or cilia. In some embodiments, the TPRA40 antagonist inhibits a biological function of the TPRA40 polypeptide. In some embodiments, the TPRA40 antagonist inhibits expression of TPRA40 RNA or protein. In some embodiments, the TPRA40 antagonist prevents interaction between TPRA40 polypeptide with the Galpha-i protein. In some embodiments, the TPRA40 antagonist inhibits TPRA40-mediated inhibition of Protein Kinase A. In some embodiments, the TPRA40 antagonist induces an increase in cAMP levels in a ceil. The disclosure provides working examples of agents that are TPRA40 antagonists, as well as numerous other specific and generic examples of such TPRA40 antagonist agents and categories of agents (collectively, "TPRA40 antagonists of the disclosure" or "a TPRA40 antagonist of the disclosure"),

The term expressly includes TPRA40 polypeptides variants (e.g. TPRA40 polypeptide variants that bind the ligand bound by TPRA40 but do not promote downstream hedgehog signaling), anti~TPRA40 antibodies (e.g., antibodies that bind to an epitope within the C-terminal 84 amino acids of TPRA40 polypeptide or to an epitope that includes a portion of the C-terminal 84 amino acids of TPRA40, or an antibody to any extracel lular portion of the TPRA40 polypeptide), TPRA40-binding antibody fragments thereof, TPRA40~binding antigen binding fragments, TPRA40-binding oligopeptides (e.g., oligopeptides that bind to the ligand binding site of TPRA40 to prevent ligand binding, or that bind to the C-terminal region of TPRA40), TPRA40 sense/an tisense nucleic acid, TPRA40 binding small molecules (e.g., small organic molecules that bind to TPRA40 and inhibit TPRA40 activity), and/or polynucleotides that inhibit TPRA40 expression (e.g., RNAi, an tisense oligonucleotides).

In some embodiments, the TPRA40 antagonist inhibits TPRA40 bioactivity. In some embodiments, TPRA40 bioactivity refers to its role in facilitating hedgehog signaling. In some embodiments, TPRA40 bioactivity refers to the ability to promote GUI expression. In some embodiments, TPRA40 bioactivity is inhibition of adenylyi cyclase. In some embodiments, TPRA40 bioactivity is inhibition of Protein Kinase A. As such, in some embodiments, the TPRA40 antagonists inhibit the hedgehog signaling pathway, inhibit GUI expression and/or release the inhibition of adenylyi cyclase and/or Protein Kinase A,

In some embodiments, the TPRA40 antagonist is for use in treating a cell. In some embodiments, the cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene (e.g., one or more mutations in a component of the hedgehog signaling pathway), wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand. In some embodiments, the TPRA40 antagonist is contacted with the ceil in an amount effective to inhibit hedgehog signaling, inhibit GUI expression and/or release the inhibition of adenylyl cyclase and/or Protein Kinase A. In some embodiments, the effective amount of the TPRA40 antagonist needed to inhibit hedgehog signaling or inhibit GUI expression is determined by assessing the amount of TPRA40 antagonist needed to reduce Glil protein levels {e.g., by SDS-PAGE) and/or Glil mRNA transcript levels (e.g., by RT-PCR or Northern Blot) in the treated cell or cells. In some embodiments, the effective amount of the TPRA40 antagonist inhibits Glil protein levels and/or Glil mRNA transcript by at least 10%, at least 20%>, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% as compared to an untreated control cell or cells. In some embodiments, the effective amount of the TPRA40 antagonist needed to release the inhibition of adenylyl cyclase and/or Protein Kinase A activity is monitored by assessing cAMP levels in the treated cell or cells. In some embodiments, the effective amount of the TPRA40 antagonist increases cAMP levels in the treated cell or cells by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%, at least 150%, or at least 200% as compared to an untreated control cell or cells. In some embodiments, the effective amount of the TPRA40 antagonist needed to release the inhibition of adenylyl cyclase and or Protein Kinase A activity is monitored by assessing the expression levels of a gene under the control of a cAMP Response Element (CRE) in the treated cell or cells. In some embodiments, the effective amount of the TPRA40 antagonist increases expression levels of the CRE-controlled gene by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100%), at least 150%, or at least 200% as compared to an untreated control cell or cells. In some embodiments, the gene under the control of the cAMP Response Element is a transgenic reporter gene. In some embodiments, the reporter gene is luciferase. In some embodiments, the cell or cell contacted with the TPRA40 antagonist is treated with an agent to induce adenylyl cyclase (e.g., forskolin, 8-bromo-cAMP or dibutyryl-cAMP) as well as contacting the cell or cells with the TPRA40 antagonist.

In some embodiments, the TPRA40 antagonist inhibits processes associated with active hedgehog signaling, e.g., cell proliferation. In some embodiments, the effects of the TPRA40 antagonist on cell proliferation may be monitored by using any of the standard cell proliferation assays known in the art. in some embodiments, the assay measures the rate of DNA synthesis in ceil populations (e.g. , using Ή-TdR Proliferation or BrDU incorporation assays). In some embodiments, the assay measures cell viability (e.g., by using an MI , XTT, CellT iterGlo or WST- 1 assay). In some embodiments, the assay measures plasma membrane damage/leakage (e.g., by means of a trypan blue exclusion assay or propidium idodide exclusion assay).

In some embodiments, the TPRA40 antagonist inhibits the growth and/or proliferation of tumor cells. In some embodiments, compositions for use in treatment comprise growth inhibitory amounts of at least one type of TPRA40 antagonist (e.g., anti-TPRA antibody), so as to inhibit growth of tumor cells by greater than 20%, preferably from about 20% to about 50%, and even more preferably, by greater than 50% (e.g., from about 50% to about 100%) as compared to the appropriate control. A "growth inhibitory" amount of a TPRA40 antagonist is one which results in measurable growth inhibition of cancer cells expressing/overexpressing hedgehog and/or expressing/overexpressing the TPRA40 polypeptide. In one embodiment, growth inhibition can be measured at a molecule concentration of about 0.1 to 30 μ¾/ιη1 or about 0.5 iiM to 200 tiM in cell culture, where the growth inhibition is determined 1 -10 days after exposure of the tumor cells to the antibody. Growth inhibition of tumor cells in vivo can be determined in various ways such as is described in the Experimental Examples section below. An amount of any of the TPRA40 antagonists disclosed herein is growth inhibitory in vivo if administration of such molecule at about 5 jig/kg to about 100 mg/kg body weight results in reduction in tumor size or tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody, preferably within about 5 to 30 days.

In one embodiment, the TPRA40 antagonists disclosed herein induce apoptosis. A TPRA40 antagonist which "induces apoptosis" is one which induces programmed cell death of a cell (e.g. , a tumor cell) as determined by binding of annexin V, fragmentation of DNA, cell shrinkage, dialation of endoplasmic reticulum, cell fragmentation, and/or formation of membrane vesicles (called apoptotic bodies) or by monitoring caspase activity or cleavage. The cell is usually one which overexpresses a hedgehog polypeptide and/or that has active hedgehog signaling. Various methods are available for evaluating the cellular events associated with apoptosis. For example, phosphatidyl serine (PS) translocation can be measured by annexin binding; DNA fragmentation can be evaluated through DNA laddering; and nuclear/chroniatin condensation along with DNA fragmentation can be evaluated by any increase in hypodiploid cells; caspase activity can be assayed using caspase substrate kits or by western blotting for cleaved caspases or PARI5. In some embodiments, the TPRA40 antagonist which induces apoptosis is one which results in about 2 to 50 fold, preferably about 5 to 50 fold, and most preferably about 10 to 50 fold, induction of annexi bindi g relative to untreated cells in an annexin binding assay.

A TPRA40 antagonist which "induces cell death" is one which causes a viable cell (e.g., a tumor or cancer cell) to become nonviable. Such a ceil is one which has active hedgehog signaling and/or which expresses a. hedgehog polypeptide (and in some eases overexpresses it) and which expresses a. TPRA40 polypeptide (and in some cases overexpresses it) as compared to a non-diseased cell. The ability to induce cell death can be assessed, for example, relative to untreated cells by suitable techniques, such as loss of membrane integrity as evaluated by uptake ofpropidium iodide (PI), trypan blue (see Moore et al. Cytotechnology 17: 1-1 1 (1995)) or 7AAD. In some embodiments, cell death-inducing TPRA40 antagonists are those which induce PI uptake in the PI uptake assay.

In some embodiments, the TPRA40 antagonist may be used to treat a subject in need thereof, comprising the step of administering the TPRA40 antagonist to the subject. In some embodiments, the TPRA40 antagonists may be used to treat a subject suffering from any of the diseases or disorders described herein. In some embodiments, any of the TPRA40 antagonists described herein may be used for inhibiting hedgehog signaling or inhibiting cell proliferation (particularly unwanted cell proliferation), growth, or increasing survival in a subject in need thereof. In some embodiments, the TPRA40 is for use in treating unwanted cell proliferation in a subject. In some embodiments, the unwanted cell proliferation (or unwanted ceil growth) is cancer. In some embodiments, the TPRA40 antagonist is for use in inhibiting unwanted angiogenesis in a subject. In some embodiments, the unwanted angiogenesis is associated with a tumor. In some embodiments, the TPRA40 antagonist is for use in treating a skin disease (e.g. psoriasis, acne) in a subject. In some embodiments, the TPRA40 antagonist is an antibody (e.g., an antibody, antigen binding fragment or an immunoconjugate), a TPRA40-binding oligopeptide, a TPRA40 polypeptide variant, a polynucleotide antagonist (e.g., a sense/antisense nucleic acid or an RNAi molecule), or a small molecule. The disclosure contemplates, in certain

embodiments, that any of the TPRA40 antago ists described herein (TPRA40 antagonists of the disclosure) may be suitable for use in any of the in vitro or in vivo methods of the disclosure. The disclosure provides numerous examples of specific agents and categories of agents that are TPRA40 antagonists (e.g., TPRA40 antagonists of the disclosure). The disclosure contemplates numerous methods for using TPRA40 antagonists in vitro or in vivo, as well as using TPRA40 antagonists in assays and as reagents to identify other components of the hedgehog signaling pathway and/or natural ligands or binders of TPRA40. The disclosure contemplates that any of the TPRA40 antagonists of the disclosure (e.g., specific antagonists or categories of antagonists) may be used in any of the methods described herein. The disclosure contemplates that any of the structural and functional features of TPRA40 antagonists may be combined and used in combination with any of the features of the methods described herein. ii. Screening for TPRA40 Hedgehog Antagonists

In some embodiments, the disclosure provides for a method of screening for a TPRA40 antagonist. In some embodiments the screen is of single agents or a discrete number of agents. In some embodiments, the screen is of pools of agents. In some embodiments, the screen is of candidate agents. In some embodiments, the screen is high-throughput screening. In some embodiments, the screen is of a library or libraries of compounds (e.g., libraries of small molecules, libraries of antisense oligonucleotides, or libraries of antibodies or peptides). In some embodiments, screening may involve a primary assay alone or a primary assay and one or more secondary assays. In some embodiments, any of the agents identified as TPRA40 antagonists in the screening methods described herein can be further assessed in an additional assay (e.g., a hedgehog signaling assay (e.g., by using any of the GUI expression assays described herein or known in the art to examine Glil nucleic acid or protein expression in response to an agent), a TPRA40 activity assay (e.g., by using any of the adenyiyl cyclase assays described herein or known in the art), a TPRA40 binding assay (e.g., by using any of the TPRA40 binding assays described herein, such as with FACS antibody screen), a cell proliferation assay (e.g., by using any of the cell proliferation assays described herein or known in the art ),

In some embodiments, the disclosure provides for a method of screening for a TPRA40 antagonist, wherein the method comprises: a) contacting a cell that expresses TPRA40 and adenyiyl cyclase with an agent; b) determining, as compared to an untreated control, whether the agent rescues (e.g., increases: relieves inhibition) adenyiyl cyclase activity, wherein if the agent rescues (e.g., increases) adenyiyl cyclase activity relative to the control, then an agent is identified as a TPRA40 antagonist. In some embodiments, the disclosure provides for a method of identifying a TPRA40 inhibitor or antagonist, comprising: a) providing a cell that expresses TPRA40 and that expresses a reporter construct to indicate adenylyi cyclase activity; b) contacting the ceil with an activator of adenylyi cyclase and with an agent, wherein the cells are contacted with the activator and the agent simultaneously, concurrently, or

consecutively; and c) determining, as compared to a control, whether the agent rescues (e.g., increases: relieves inhibition) the adenylyi cyclase activity induced by the activator, wherein if the agent increases the adenylyi cyclase activity relative to the non-agent treated control, then the agent is identified as a TPRA40 inhibitor or antagonist. In some embodiments, the cell or cells used in this method are contacted with a compound that induces adenylyi cyclase activity (e.g., forskolin, 8-bromo-cAMP or dibutyryl-cAMP) prior to step a). In some embodiments, a reporter gene is used in order to determine whether adenylyi cyclase activity has been inhibited by an agent. In some embodiments, the reporter gene is a luciferase gene controlled by a cAMP response element, (CRE). In these embodiments, inhibition of adenylyi cyclase by active

TPRA40 will result in a reduction in cAMP levels and, therefore, a corresponding reduction in luciferase expression. An agent that inhibits TPRA40 would permit adenylyi cyclase to regain activity (e.g., in the presence of forskolin, 8-bromo-cAMP or dibutyryl-cAMP), thereby resulting in an increase in cAMP levels and luciferase expression. In some embodiments, the agent identified in step b) is further tested to determine whether or not it binds to TPRA40. In some embodiments, the identified in step b) is further tested to determine whether or not it inhibits hedgehog signaling. In some embodiments, the agent identified in step b) is further tested to determine whether or not it inhibits proliferation, growth or survival of a cancer cell.

In some embodiments, the disclosure provides for a method of identifying a TPRA40 inhibitor, wherein the method comprises: a) screening for an agent that binds to TPRA40 protein; b) contacting a cell with an amount of the agent identified in step a ), wherein the cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, and c) determining, as compared to a control, whether the agent that binds to TPRA40 protein also inhibits hedgehog signaling in the cell, wherein if the agent inhibits hedgehog signaling in the cell relative to the control, then the agent is identified as a TPRA40 inhibitor. In some embodiments, the disclosure provides for a method of screening for an agent for inhibiting the proliferation, growth or survival of a cancer cell, wherein the method comprises: a) screening for an agent that binds to TPRA40 protein, reduces expression of TPRA40, inhibits transport of TPRA40 protein to the plasma membrane or to primary cilia, prevents activation of TPRA40 or uncouples TPRA40 from Gai, or inhibits TPRA40 inhibition of adenylyl cyclase: b) contacting a cancer cell with an amount of the agent identified in step a), wherein the cancer cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, and c) determining, as compared to a control, whether the agent inhibits the proliferation or growth of the cancer cell, wherein if the agent inhibits cell proliferation or growth relative to the control, then an agent that inhibits the proliferation or growth of the cancer cell is identified.

In some embodiments, the disclosure provides for a. method of screening for an agent for inhibiting hedgehog signaling in a cell, wherein the method comprises: a) screening for an agent that binds to TPRA40 protein, reduces expression of TPRA40, inhibits transport of TPRA40 protein to the plasma membrane or to primary cilia, prevents activation of TPRA40 or uncouples TPRA40 from Gal, or inhibits TPRA40 inhibition of adenylyl cyclase; b) contacting a cell with an amount of the agent identified in step a), wherein the cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein the one or more mutations resul ts in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, and c) determining, as compared to a control, whether the agent inhibits hedgehog signaling in the cell, wherein if the agent inhibits hedgehog signaling in the cell relative to the control, then an agent that inhibits hedgehog signaling is identified.

In some embodiments, the cell used in the screening methods described herein is in culture. In some embodiments, the agent contacted with the cells in the culture is sufficient to inhibit, partially or entirely, hedgehog signaling in at least 10%, 15%, 2G%», 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%), or 100% of cells in a cell culture. In some embodiments, the agent contacted with the cells in the culture is sufficient to reduce the rate of proliferation of a cell and/or rate of survival of at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% of cells in a ceil culture, wherein the cells are expressing or overexpressing hedgehog or have active hedgehog signaling.

In other embodiments, the ceil is in an animal. In some embodiments, the animal is a mammal or other vertebrate. In some embodiments, the animal is post-natal. In some embodiments, the animal is pediatric. In some embodiments, the animal is adult. When referring to ceils in vitro, the cells may be of any vertebrate species, such as a mammal, such as rodent, hamster, or human. In vitro or in vivo, a cell may be a cancer cell, such as a primary cancer cell, a metastatis cancer cell, or a cancer cell line.

In some embodiments, the cell comprises one or more mutations in a hedgehog signaling pathway gene. In some embodiments, the one or more mutations are in TPRA.40. In some embodiments, the one or more mutations are in smoothened. In some embodiments, the smoothened mutation is a smoothened gain-of-function mutation. In some embodiments, the gain-of-function smoothened mutation results in a constitutively active smoothened protein. In some embodiments, the smoothened mutation is a mutation at a position corresponding to position 535 of SEQ ID NO: 42. In certain embodiments, the mutation is a mutation at a position corresponding to position 562 of SEQ ID NO: 42. In certain embodiments, the mutation is W535L at position 535 or at that corresponding position in SEQ ID NO: 42. In some embodiments, the smoothened mutation is a mutation corresponding to position R562Q of SEQ ID NO: 42 (a R562Q mutation at position 562 or at a position corresponding to position 562 of SEQ ID NO: 42. In some embodiments, the smoothened mutation is a mutation at a position corresponding to position 412 of SEQ ID NO: 42, such as a L412F at such a position of SEQ ID NO: 42. In some embodiments, the smoothened mutation has a mutation that renders it resistant to certain smoothened inhibitors. In some embodiments, the smoothened protein comprises an amino acid alteration at amino acid position 518 of SEQ ID NO: 42 or at a position

corresponding to position 518 of SEQ ID NO: 42. In some embodiments, the amino acid alteration is E518 or E518A substitution at the amino acid position corresponding to amino acid position 518 of SEQ ID NO: 42. In some embodiments, the smoothened protein comprises an amino acid alteration at amino acid position 473 of SEQ ID NO: 42 or at a position corresponding to position 473 of SEQ ID NO: 42. In some embodiments, the amino acid alteration is the substitution of aspartic acid with any of histidine, glycine, phenylalanine, tyrosine, leucine, isoleucine, proline, serine threonine, methionine, glutamiiie, or asparagine at the amino acid position corresponding to amino acid position 473 of SEQ ID NO: 42. See, e.g., WO 201 1/028950 and WO2012047968, eacli of which is incorporated by reference.

In some embodiments, unknown events result in overexpression of a hedgehog protein. For example, hedgehog protein may be overexpressed in the cell or in an adjacent cell. In some embodiments, the overexpressed hedgehog protein is Sonic hedgehog protein. In some embodiments, the overexpressed hedgehog protein is Indian hedgehog protein. In some embodiments, the overexpressed hedgehog protein is Desert hedgehog protein.

In some embodiments, the one or more mutations are in suppressor-of-fused, and the ceil has suppressor-of-fused (SuFu) loss-of-function. In some embodiments, the SuFu mutation results in a loss-of-function in SuFu activity. In some embodiments, the SuFu mutation is in a medulioblastoma, meningioma, adenoid cystic carcinoma, basal cell carcinoma and

rhabdomyosarcoma cancer cell. In some embodiments, the SuFu mutation is any of the mutations described in Tables 1 or 2 or any of the mutations described in Brugieres et al, 2012, JCO, 30(17):2087-2093 , which is incorporated herein in its entirety.

Table 1 : Germline SUFU Mutations

Age at ~t listologic Subtype Associated

>iagnosis Symptoms of

4 years Desmoplastic Developmental Loss of

delay contiguous

genes at

lOq

Frontal IVS1--1A

bossing, →T

hypertelorism

NA Desmoplastic None N,A 143 ins A

NA Desmoplastic Meningioma in NA

radiation field

8 months MBEN Macrocrania, Inherited c. i 022

palmar and 1G>A

pla tar pits

<1 month MBEN None inherited c.72delC

<3 MBEN None Inherited c.72delC

months None Inherited c.72insC

months

6-12 Desmoplastic/nodular None Inherited c.72insC

months

<6 Desmoplastic/nodular None Inherited c,72insC

months

12-24 MB NOS None Inherited c.72insC

months

22 months Desmoplastic/nodular N NA c.846insC

23 months Desmoplastic/nodular N NA c. i 022

1G>A

Abbreviations: MB, medulloblastonia; MBEN, MB with extensive nodularity; N A, not available; NOS, not otherwise specified.

Table 2, Germline Pathogenic SUFU Mutations

Exoa-'l ron Type of Nucleotide Change Consequence (In SEQ Tumor

Mutation (In SEQ ID NO: ID NO: 43) Analysis

44)

1 Intron 1 Splice→ C.182 + 3A>T p.ThrSSfs Not available frameshift

2 Exon 2 Frameshift ,294__295dupCT p,Tyr99fs Not available

3 Intron 2 Splice -→ .31 8-l OdelT p.Phel 07fs Loss of wild- frameshift type allele

4 Exon 3 Large .318-? 454 -'d up p.Glul06- UV (c. l022 +

duplication ? Glul 52- 5G>A)

5 Exon 3 Missense .422T>G p.Metl41Arg Not available

6 Exon 9 Nonsense p.Gln375X Not available

7 Exo 9 Frameshift c 1 149 ! 1 SOdupCT p.Cys384fs Loss of wild- type allele

8 Intron 10 Splice -* C.1297-1 G>C p..' Not available frameshift

Abbreviation: LJV, unknown variant. n some embodiments, the SuFU mutation is any of the mutations corresponding to c.l 022+1 G>A (IVS8-1G>T), c.72delC, c.72insC, 143insA, c.846insC, or IVS1 -1A->T of SEQ ID NO: 44. In some embodiments, the SuFu mutation is any of the mutations described in Taylor et al (2002) Nat Genet 31 :306-310 (e.g., IVS8-1 G>T (=c. l022 +1G>A), 1 129del, P15L and Ng's two (all +LOH)); Slade et al (201 1) Fam Cancer 10:337-342, 201 1 (e.g. , c. l 022 +1G>A; c.848insC); Pastorino et al (2009) Am J Med Genet A 149A: 1539-1543 (e.g., c.1022 · ! (..! A): Ng et al (2005) Am J Med Genet A 134:399-403 (e.g., 143 ins A; IV S 1 -1A>T); Kijima et al (2012) Fam Cancer 1 1 : 565-70 (e.g., C.550OT (Q184X)); Aavikko et al (2012) Am J Hum Genet 91 : 520-526 (e.g., C.3670T (R123Q); Stephens et al (2013) J Clin Invest 123: 2965-2968 (e.g., x881_882insG (L295fs)); or Reifenberger et al (2005) Brit J Dermatology 152: 43-51 (e.g., C560OT (PI 871,)).

In some embodiments, the agent, tested in any of the screening methods described herein is a small molecule. In other embodiments, the agent is a polypeptide. In other embodiments, the agent is an siRNA antagonist.

In some embodiments, the agent tested in any of the screening methods described herein binds a TPRA40 protein. In some embodiments, the agent that binds TPRA40 is identified or confirmed using a yeast two-hybrid screen. In some embodiments, the agent that binds TPRA40 is identified or confirmed using high throughput binding screen of a small molecule library. In some embodiments, the agent that binds TPRA40 is identified or confirmed using a co- immunoprecipitation assay. In some embodiments, the agent that binds TPRA40 is identified or confirmed by labeling the agent (e.g., with a fluorescent label or radiolabel) and detecting whether the labeled agent binds to TPRA40.

In some embodiments, the agent tested in any of the screening methods described herein inhibits transport of the TPRA40 protein to the plasma membrane or to primary cilia, prevents activation of TPRA40 or uncouples TPRA40 from God. In some embodiments, an agent that inhibits transport of the TPRA40 protein is identified or confirmed by utilizing a method comprising the steps of: i) contacting a cell expressing TPRA40 with an agent, ii) determining the localization of TPRA40 in the first cell expressing TPRA40 using immunofluorescence. In some embodiments, the TPRA40 is tagged or labeled (e.g., fliiorescently or radiolabeled) in order to monitor its localization in a cell. In some embodiments, an agent that inhibits transport of the TPRA40 protein is identified or confirmed by utilizing a method comprising the steps of: i ) contacting a cell expressing TPRA40 with an agent; and ii) determining the levels of TPRA40 in a plasma membrane or ciliary membrane fraction. In some embodiments, the levels of TPRA40 are determined by fractionation of ceil components and determining the levels of TPRA40 in each component (e.g., by SDS PAGE analysis).

In some embodiments, the agent tested in any of the screening methods described herein reduces expression of TPRA40 protein or RNA. In some embodiments, an agent that reduces expression of TPRA40 protein or RNA is identified or confirmed by a method comprising the steps of: i) contacting a. cell expressing TPRA40 with an agent; and ii) determining activity of TPRA40 in the cell using a CRE-luciferase or G/z-iuciferase reporter assay. In some

embodiments, an agent that reduces expression of TPRA40 protein or RNA is identified or confirmed by a method comprising the steps of: i) contacting a cell expressing TPRA40 with an agent; and ii) determining the expression of TPRA40 in the cell by RT-PCR. In some embodiments, an agent that reduces expression of TPRA40 protein or RNA is identified or confirmed by a method comprising the steps of: i) contacting a cell expressing TPRA40 with an agent; and ii) determining the expression of TPRA40 in the cell using Northern Blot analysis of TPRA40 RNA. or Western Blot or immunofluorescence analysis of TPRA40 protein.

In some embodiments, the agent tested in any of the screening methods described herein inhibits TPRA40 inhibition of adenyly l cyclase. In some embodiments, an agent that reduces expression of TPRA40 protein or RNA is identified or confirmed by using a reporter gene assay. In some embodiments, the reporter gene is a lueiferase gene controlled by a c AMP response element (CRE) and is stimulated by compounds that increase cAMP levels, such as forskolin, 8- bromo-cAMP or dibutyryl-cAMP. In these embodiments, inhibition of adenylyl cyclase by active TPRA40 (in the presence of forskolin) will result in a reduction in cAMP levels and, therefore, a corresponding reduction in lueiferase expression. An agent that inhibits TPRA40 would permit adenylyl cyclase to regain activity (e.g., in the presence of forskolin), thereby resulting in an increase in cAMP levels and lueiferase expression.

In some embodiments, the agent identified in any of the screening methods described herein is further assessed in an assay for hedgehog signaling. In some embodiments, wherein the assay for hedgehog signaling comprises the steps of: i. contacting a cell with an amount of the agent, wherein the cell is responsive to hedgehog protein or comprises one or more mutations in a. hedgehog signaling pathway gene, wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, and ii. determining, as compared to a control, whether the agent inhibits hedgehog signaling in the cell, wherein if the agent inhibits hedgehog signaling in the cell relative to the control, then an agent that inhibits hedgehog signaling is identified.

In some embodiments of any of the screening methods described herein, the TPRA40

DNA is exogenously expressed in a cell. In some embodiments, the TPRA40 DNA is stably expressed in the cell. In some embodiments, the TPRA40 DNA is transiently expressed in the ceil. In some embodiments, the ceil is an S 12 ceil. In some embodiments, the ceil is a 293T cell.

The growth inhibitory effects of the various TPRA40 antagonists useable in the disclosure may be assessed by methods known in the art, e.g., using cells which express a

TPRA40 polypeptide either endogenously or following transfection with the respective TPRA40 gene. For example, appropriate tumor cell lines and cells transfected with TPRA40-encodmg DNA may be treated with the TPRA40 antagonists of the disclosure at various concentrations for a few days (e.g., 2-7 days) and stained with crystal violet, MTT or analyzed by some other colorimetric or luciferase-based (eg CellTiterGio) assay. Another method of measuring proliferation would be by comparing 3H~thymidine uptake by the cells treated in the presence or absence of such TPRA40 antagonists. After treatment, the cells are harvested and the amount of radioactivity incorporated into the DNA quantitated in a scintillation counter. Appropriate positive controls include treatment of a selected cell line with a growth inhibitory antibody or small molecule known to inhibit growth of that cell line. Growth inhibition of tumor cells in vivo can be determined in various ways known in the art. Preferably, the tumor cell is one that has one or more mutations in a hedgehog pathway signaling gene. Preferably, such TPRA40 antagonists will inhibit cell proliferation of a hedgehog-expressing tumor cell in vitro or in vivo by about 10-25% , by about 25- 100%, by about 30-100%, by about 50-100%, or by about or 70- 100% compared to the untreated tumor ceil. Growth inhibition can be measured at a TPRA40 antagonist concentration of about 0.5 to 30 ug/ml, about 0.5 nM to 200 nM, about 200 nM to Ι Μ, about 1 μΜ to 5 μΜ, or about 5 μΜ to 10 uM, in cell culture, where the growth inhibition is determined 1-10 days after exposure of the tumor cells to the antagonist. The antagonist is growth inhibitory in vivo if administration of antagonist and/or agonist at about I ag/kg to about 100 mg/kg body weight results in reduction in tumor size or reduction of tumor cell proliferation within about 5 days to 3 months from the first administration of the antibody or small mol ecule antagonist, preferably within about 5 to 30 days.

In some embodiments, to select for TPRA40 antagonists which induce cell death, loss of membrane integrity as indicated by, e.g., propidium iodide (PI), trypan blue or 7AAD uptake may be assessed relative to control. A PI uptake assay can be performed in the absence of complement and immune effector cells. TPRA40 polypeptide-expressing expressing tumor cells are incubated with medium alone or medium containing the appropriate TPRA40 antagonist. The ceils are incubated for a 3 day time period. Following each treatment, cells are washed and aliquoted a into 35 mm strainer-capped 12 x 75 tubes (1 ml per tube, 3 tubes per treatment group) for removal of cell clumps. Tubes then receive PI (10 μ¾/πι1). Samples may be analyzed using a FACSCAN® flow cytometer and FACSCO VERT® CeUQuest software (Becton Dickinson), or any other device used by the skilled worker for analyses. Those TPRA40 antagonists that induce statistically significant levels of cell death as determined by PI uptake may then be selected.

In some embodiments, to screen for TPRA40 hedgehog antagonists which bind to an epitope on a TPRA40 polypeptide, a routine cross-blocking assay such as that described in

Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. This assay can be used to determine if a test antibody, polypeptide, oligopeptide or other organic molecule binds the same site or epitope as a known T RA40 antagonist. Alternatively, or additionally, epitope mapping can be performed by methods known in the art. For example, the TPRA40 sequence can be mutagenized such as by alanine scanning or by making chimerae with immunologically distinct GPCR proteins, to identify contact residues. The mutant antigen is initially tested for binding with polyclonal antibody to ensure proper folding. In a different method, peptides corresponding to different regions of a TPRA40 polypeptide can be used in competition assays with the test antibodies or with a test antibody and an antibody with a characterized or known epitope.

In some embodiments, the TPRA40 polypeptide or the candidate TPRA40 hedgehog antagonist agent is immobilized on a solid phase, e.g., on a microliter plate, by covalent or non- covalent attachments. Non-covalent attachment generally is accomplished by coating the solid surface with a solution of the TPRA40 or candidate agent and drying. Alternatively, an immobilized antibody, e.g., a monoclonal antibody, specific for the target portion of TPRA40 to be immobilized can be used to anchor it to a solid surface. The assay may be performed by adding the non-immobilized component, which may be labeled by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component. When the reaction is complete, the non-reacted components may be removed, e.g., by washing, and complexes anchored on the solid surface are detected. When the originally non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred. Where the originally non-immobilized component does not carry a label, complexing can be detected, for example, by using a labeled antibody specifically binding the immobilized complex.

If the candidate TPRA40 antagonist interacts with but does not bind directly to a.

TPRA40 polypeptide identified herein, its interaction with that polypeptide can be assayed by methods well known for detecting protein-protein interactions. Such assays include traditional approaches, such as, e.g., cross-linking, co-immunopreeipitation, and co-purification through gradients or chromatographic columns. In addition, protein- protein interactions can be monitored by using a yeast-based genetic system described by Fields and co-workers (Fields and Song, Nature (London). 340:245-246 (1989); Chien et al, Proc. Natl. Acad. Sci. USA, 88:9578- 9582 (1991)) as disclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA. 89: 5789-5793 (1991), Many transcriptional acti vators, such as yeast GAL4, consist of two physically discrete modular domains, one acting as the DNA -binding domain, the other one functioning as the transcription- activation domain. The yeast expression system described in the foregoing publications (generally referred to as the "two-hybrid system") takes ad vantage of this property , and employs two hybrid proteins, one in which the target protein is fused to the DNA-binding domain of GAL4, and another, in which candidate activating proteins are fused to the activation domain. The expression of a GALl-LacZ reporter gene under control of a GAL4-activated promoter depends on reconstitution of GAL4 activity via protein-protein interaction. Colonies containing interacting polypeptides are detected with a chromogenic substrate for β-gaiactosiclase. A complete kit (MATCHMAKER 1M) for identifying protein-protein interactions between two specific proteins using the two- hybrid technique is commercially available from Clontech. This system can also be extended to map protein domains involved in specific protein interactions as well as to pinpoint amino acid residues that are crucial for these interactions. The assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays, and cell-based assays, which are well characterized in the art.

Agents that interfere with the interaction of TPRA40 polypeptide and other intra- or extracellular components (e.g., Ssnal NA14) can be tested by means well-known by the skilled worker. In some embodiments, a reaction mixture is prepared containing the TPRA40 polypeptide and an intra- or extracellular component under conditions and for a. time allowing for the interaction and binding of the two products. In some embodiments, to test the ability of a candidate compound to inhibit binding, the reaction is run in the absence and in the presence of the test compound. In addition, a placebo may be added to a third reaction mixture, to serve as positive control. The binding (complex formation) between the test compound a d the intra- or extracellular component present, in the mixture is monitored as described hereinabove. The formation of a complex in the control reaction(s) but not in the reaction mixture containing the test agent indicates that the test agent interferes with the interaction of the test compound and its reaction partner.

The disclosure contemplates methods for identifying TPRA40 antagonists using any one or combination of the foregoing assay steps. In other words various screening assays can be combined to identify antagonists having, for example, a particular acti vity or to confirm that an agent that antagonizes TPRA40 in one assay also inhibits hedgehog signaling in an independent assay. For any assay or method of identification, results may be compared to one or more appropriate controls, including positive and/or negative controls.

For any of the foregoing assay methods for screening and/or identifying TPRA40 antagonists, agents may be screened singly or in pools. Agents may be screened from a library of agents or a set of candidate agents. Suitable agents that may be screened include, but are not limited to, antibodies, antibody fragments, peptides, antisense oligonucleotides, RN Ai and small molecules.

In certain embodiments, once an agent is identified as a TPRA40 antagonist, the agent can then be formulated and further evaluated in a ceil or animal-based assay. For example, the agent can be tested in a ceil or animal-based cancer model to evaluate efficacy as an anti-cancer agent. The foregoing systems for screening for TPRA40 antagonists can also be used to screen for TPRA40 agonists, such as small molecules that bind to TPRA40 and agonize its activity. The disclosure contemplates that any of the assays described herein to screen for antagonists can be adapted for screening for agonists by changing the relevant read-out (e.g., identifying as agonists an agent that promotes hedgehog signaling rather than identifying as an antagonist an agent that inhibits hedgehog signaling).

In certain embodiments, the disclosure provides for a method of screening for a TPRA40 agonist, wherein the method comprises: a) contacting a cell that expresses TPRA40, adenylyl cyclase and a reporter with an agent; b) determining, as compared to an untreated control, whether the agent suppresses (e.g., decreases) the adenylyl cyclase activity, wherein if the agent suppresses adenylyl cyclase activity relative to the non-agent treated TPRA40 expressing cells, then the agent is identified as a TPRA40 agonist. In certain embodiments, the disclosure provides for a method of identifying a TPRA40 agonist, comprising: a) providing a cell that expresses TPRA40 and that expresses a reporter gene capable of indicating adenylyl cyclase activity; b) contacting the cell with an activator of adenylyl cyclase and with an agent, wherein the cells are contacted with the activator and the agent simultaneously, concurrently, or consecutively; and c) determining, as compared to a control, whether the agent suppresses (e.g., decreases) adenylyl cyclase acti vity induced by the acti vator, wherein if the agent suppresses (e.g., decreases) the adenylyl cyclase activity relative to the control, then the agent is identified as a TPRA40 agonist. In certain embodiments, the disclosure provides for a method of screening for an agent for inducing hedgehog signaling in a cell, wherein said method comprises: a) screening for an agent that binds to TPRA40 protein, induces expression of TPRA40, facilitates transport of TPRA40 protein to the plasma membrane or to primary cilia, induces activation of TPRA40 or couples it with God; b) contacting a cell with an amount of the agent identified in step a), and c) determining, as compared to a control, whether said agent induces (e.g., increases) hedgehog signaling in said cell, wherein if said agent induces (e.g., increases) hedgehog signaling in said cell relative to the control, then an agent that induces (e.g., increases) hedgehog signaling is identified. In certain embodiments, the disclosure provides for a method of identifying a TPRA40 agonist, wherein said method comprises: a.) screening for an agent that binds to TPRA40 protein; b) contacting a cell with an amount of the agent identified in step a), and c) determining, as compared to a control, whether said agent that binds to TPRA40 protein also induces (e.g., increases) hedgehog signaling in said ceil, wherein if said agent induces (e.g., increases) hedgehog signaling in said cell relative to the control, then the agent is identified as a TPRA40 agonist. In certain embodiments, the agent is a small molecule. In certain

embodiments, the agent is a polypeptide. In certain embodiments, the agent is a polynucleotide. ϋί· Exemplary TPRA Antagonists

Below is provided a description of exemplary TPRA40 antagonists and categories of TPRA40 antagonists. Such antagonists, as well as any of the TPRA40 antagonists described generally or specifically herein may be used in any of the methods of the disclosure (e.g., TPRA40 antagonists of the disclosure). The disclosure contemplate that any of the TPRA40 antagonists of the disclosure can be described using any combination of functional and structural features described herein.

A. Anti-TPRA40 antibodies.

In one embodiment, the present, disclosure provides the use of anti-TPRA40 antibodies, which may find use herein as therapeutic, diagnostic and/or prognostic agents (e.g. for determining the severity of and/or prognosing the disease course of a hedgehog pathway- hyperactive tumor or cancer). Antibodies that may be used for such puiposes include polyclonal, monoclonal, humanized, bispecific, and heteroconjugate antibodies. In some embodiments, the term "antibodies" may refer to antigen- binding fragments. Thus, in certain embodiments, a TPRA40 antagonist of the disclosure comprises an anti-TPRA40 antibody. Exemplary antibodies that function as TPRA40 antagonists include: (i) an antibody that binds to TPRA40 and prevents binding to ligancl; (ii) an antibody that binds to TPRA40 and prevents transport to cilia; (iii) an antibody that binds to TPRA40 and prevents binding to Ssnal/NA- 14. In other embodiments, a TPRA40 antagonist is an antibody that does not bind directly to TPRA40 but prevents transport of TPRA40 to cilia or blocks a protein-protein interaction necessary for native TPRA40 function, expression or localization.

The term "anti-TPRA40 antibody" is used in the broadest sense and covers, for example, anti-TPRA40 monoclonal antibodies, anti-TPRA40 antibody compositions with poiyepitopic specificity, polyclonal antibodies, single chain anti-TPRA40 antibodies, multispecific antibodies (e.g., bispecific) and antigen binding fragments (see below) of all of the above enumerated antibodies as long as they exhibit the desired biological or immunological activity. Such antibodies bind to TPRA40. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein. In the case of TPRA40 antagonists which are anti-TPRA40 antibodies, such antibodies bind to TPRA40 to inhibit an activity (e.g., to antagonize a function.

An "isolated" antibody is one which has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment that are purified away from the isolated antibody are materials which would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. In certain embodiments, the antibody will be purified (1) to greater than 95% by weight of antibody as determined by the Lowry method, and most preferably more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of a spinning cup sequenator, or (3) to homogeneity by SDS- PAGE under reducing or nonredueing conditions using Coomassie blue or silver stain. Isolated antibody includes the antibody in situ within recombinant cells since at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step. The basic 4-chain antibody unit is a heterotetrarneric glycoprotein composed of two identical light (L) chains and two identical heav (H) chains (an IgM antibody consists of 5 of the basic heterotetramer unit along with an additional polypeptide called J chain, and therefore contain 10 antigen binding sites, while secreted IgA antibodies can polymerize to form polyvalent assemblages comprising 2-5 of the basic 4-chain units along with J chain). In the case of IgGs, the 4-chain unit is generally about 150,000 daltons. Each L chain is linked to an H chain by one covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has at the N-terminus, a variable domain (VH) followed by three constant domains (CH) for each of the a and γ chains and four CH domains for μ and c isotypes. Each L chain has at the N-terminus, a variable domain (VL) followed by a constant domain (CL) at its other end. The VL is aligned with the VH and the CL is aligned with the first constant domain of the heavy chain (CHI). Particular amino acid residues are believed to form an interface between the light chain and heavy chain variable domains. The pairing of a VH and VL together forms a single antigen-binding site. For the structure and properties of the different classes of antibodies, see, e.g., Basic and Clinical Immunology, 8th edition, Daniel P. States, Abba I. Terr and Tristram G. Pare low (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and Chapter 6.

The L chain from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains (CH), immunoglobulins can be assigned to different classes or isotypes. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, having heavy chains designated α, δ, ε, y, and μ, respectively. The γ and a classes are further divided into subclasses on the basis of relatively minor differences in CH sequence and function, e.g., humans express the following subclasses: IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2. In some embodiments, the antibody disclosed herein is any of the classes or subclasses of immunoglobulins described herein.

The term "variable", with respect to domains of an immunoglobulin, refers to the fact that certain segments of the variable domains differ extensively in sequence among antibodies. The V domain mediates antigen binding and define specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed across the approximately 110-amino acid span of the variable domains. Instead, the V regions consist of relatively invariant stretches called framework regions (FRs) of 15-30 amino acids separated by shorter regions of extreme variability called "hypervariable regions" that are each 9-12 amino acids long. The variable domains of native heavy and light chains each comprise four FRs, largely adopting a β-sheet configuration, connected by three hypervariable regions, which form loops connecting, and in some cases forming part of, the β-sheet stnicture. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen- binding site of antibodies (see abat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g. if the Kabat system is used, around about residues 24-34 (LI), 50-56 (L2) and 89-97 (L3) in the VL, and around about residues 31-35B (HI), 50-65 (H2) and 95-102 (H3) in the VH ( abat et al, Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)) and/or those residues from a "hypervariable loop" (e.g. if the Chothia system is used, around about residues 26-32 (LI), 50-52 (L2) and 91-96 (L3) in the VL, and 26- 32 (HI), 52A-55 (H2) and 96-101 (H3) in the VH (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

Antibodies, such as monoclonal antibodies, may be made by a variety of techniques, including, for example, the hybridoma method (e.g., Kohler et al, Nature, 256:495 (1975);

Harlow et at, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling et al, in: Monoclonal Antibodies and T-Cel! Hybridomas 563-681 , (Elsevier, N.Y., 1981 )), recombinant DNA methods (see, e.g., U.S. Patent No. 4,816,567), phage display technologies (see, e.g., Clackson et al. Nature, 352:624-628 (1991); Marks et al., J. MoL Biol, 222:581 -597 (1991); Sidhu et al, J. MoL Biol. 338(2):299-310 (2004); Lee et ah, J.Mol.Biol 340(5)1013-1093 (2004); Fellouse, Proc. Nat. Acad. Sci USA 101 (34): 12467- 12472 (2004); and Lee et al. J. Immunol. Methods 284(1- 2): 1 19-132 (2004), and technologies for producing human or human-like antibodies in animals that have parts or all of the human immunoglobulin loci or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 5996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al., Proc, Natl. Acad. ScL USA, 90:2551 (1993); Jakobovits et al, Nature, 362:255-258 ( 1993); Bruggemann et al, Year in Immuno., 7:33 (1993); U.S. Patent Nos. 5,545,806; 5,569,825; 5,591 ,669 (all of GenPharm); 5,545,807; WO 1997/17852; U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126;

5,633,425; and 5,661,016; Marks et al., Bio/Technoloev. K): 779-783 (1992); Lonberg et al., Nature. 368: 856-859 (1994); Morrison, Nature, 368: 812-813 (1994); Fishwild et al, Nature Biotechnology, 14: 845-851 (1996); Neuberger, Nature Biotechnology. J4: 826 (1996); and Lonberg and Huszar, intern. Rev. Immunol. 13: 65-93 (1995). Moreover, once an antibody of a desired specificity is identified, it can be made recomhinantly based on the sequence of, for example, the VH and VL domains.

"Chimeric" antibodies (immunoglobulins) have a portion of the heavy and/or light chain identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al, Proc. Natl. Acad. ScL USA 81 :6851-6855 (1984)). Humanized antibody as used herein is a subset of chimeric antibodies.

"Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies which contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient or acceptor antibody) in which hypervariable region residues of the recipient are replaced by hypervariable region residues from a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues.

Furthermore, humanized antibodies may comprise residues which are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance such as binding affinity. Generally, the humanized antibody will comprise substantially all of at least, one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin sequence although the FR regions may include one or more amino acid substitutions that improve binding affinity. The number of these amino acid substitutions in the FR are typically no more than 6 in the H chain, and in the L chain, no more than 3. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human

immunoglobulin. For further details, see Jones et al., Nature 321 :522-525 (1986); Reichmann et at., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

"Antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab,

Fab', F(ab')2, and Fv fragments (including scFv); diabodies; linear antibodies (see U.S. Patent No. 5,641 ,870, Example 2; Zapata et al., Protein Eng. 8(10): 1057-1062 [1995]); single-chain antibody molecules; and niulti specific antibodies formed from antibody fragments.

In certain embodiments, the TPRA40 antagonist is an anti-TPRA40 antibody. Regardless of the specific type of antibody (e.g., chimeric, murine, humanized, human, a human antibody made in non-human ceils, etc.) or the method used to make it, suitable TPRA40 antagonists can be readily identified by generating a panel of antibodies that bind TPRA40 and testing to identify the antibodies having the desired function (e.g., antagonist activity).

In some embodiments, the anti-TPRA40 antibodies for use as TPRA40 antagonists bind to the putative extracellular N-terminal portion of the TPRA40 polypeptide (e.g., any of the amino acids corresponding to amino acids 1-47 of SEQ ID NO: 1), In some embodiments, the anti-TPRA4Q antibodies bind to the putative intracellular C-terminal portion of the TPRA40 protein (e.g., any of the amino acids corresponding to amino acids 286-373 of SEQ ID NO: 1). In some embodiments, the anti-TPRA40 antibodies bind to any of the putative extracellular portions of TPRA40 (e.g., any of the amino acids corresponding to amino acids 97-122, 172-191 , or 261-264 of SEQ ID NO: 1). In some embodiments, the anti-TPRA40 antibodies bind to any of the putative intracellular portions of TPRA40 (e.g., any of the amino acids corresponding to amino acids 70-74, 144-150, and 213-239). In certain embodiments, the anti-TPRA40 antibodies bind to the C-terminal region of a TPRA40 protein and prevents TPRA40 from interacting with another protein, e.g., Ssnal /NA 14. In some embodiments, the anti-TPRA40 antibodies bind to a li gaml-binding site of TPRA40 and does not activate hedgehog signaling downstream of

TPRA40, but rather, sterically blocks a ligand from binding to TPRA40. In some embodiments, the anti-TPRA40 antibodies bind to the TPRA40 to sterically block an interaction between TPRA40 and a G protein (e.g., a G-alpha-i protein).

Suitable antibodies may be polyclonal antibodies or monoclonal antibodies.

1 . Polyclonal Antibodies

In some embodiments, the antibodies of the present disclosure are polyclonal antibodies. In some embodiments, polyclonal antibodies are raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen (especially when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized. For example, the antigen can be conjugated to keyhole limpet hemocyanin ( LH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional or derivatizing agent, e.g., maieimidobenzoyi sulfosuccinimide ester (conjugation through cysteine residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl2, or R!N=C=NR, where R and R! are different alkyl groups. In particular embodiments, animals are immunized against the antigen, immunogenic conjugates, or derivatives by combining, e.g., 100 μg or 5 ,ug of the protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermaily at multiple sites. One month later, the animals are boosted with 1/5 to 1/10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. Seven to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. Conjugates also can be made in recombinant cell culture as protein fusions. Also, aggregating agents such as alum are suitably- used to enhance the immune response.

2. Monoclonal Antibodies

In some embodiments of the present, disclosure, th e antibodies described herein are monoclonal antibodies. Monoclonal antibodies may be made using the hybridoma method first, described by Kohler et ah, Nature. 256:495 (1975), or may be made by recombinant DMA methods (U.S. Patent No. 4,816,567). In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized as described above to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Given the high homology (94.1 %) between human and mouse TPRA40, it may be useful to use TPRA40 knockout mice for immunization in order to increase the chances of an immune response. Alternatively, lymphocytes may be immunized in vitro. In some

embodiments, after immunization, lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell (Coding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). There are numerous other approaches for screening to identify antibodies of a desired specificity known in the art, including phage display.

In certain embodiments, the hybridoma cells described herein are seeded and grown in a suitable culture medium which medium preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells (also referred to as fusion partner). For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine

phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas typically will include hypoxanthine, aniinopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. In certain embodiments, fusion partner myeloma cells are those that fuse efficiently, support stable high-level production of antibody by the selected antibody-producing cells, and are sensitive' to a selective medium that selects against the unfused parental cells. Preferred myeloma cell lines are murine myeloma lines, such as those derived from MOPC-21 and MPC- 11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego,

California USA, and SP-2 and derivatives e.g., X63-Ag8-653 cells available from the American Type Culture Collection, Manassas, Virginia, USA. Human myeloma and mouse-human heteromyeloma. cell lines also ha ve been described for the production of human monoclonal antibodies (Kozbor, J. Immunol.. 133:3001 (1984); and Brodeur et at, Monoclonal Antibody Production Techniques and Applications, pp. 51 -63 (Marcel Dekker, Inc., New York, 1987)). Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis described in Munson et al., Anal. Biochem.. 107:220 (1980).

Once hybridoma cells that produce antibodies of the desired specificity, affinity, and/or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Coding, Monoclonal Antibodies: Principles and Practice, pp.59- 103

(Academic Press, 5986)). Suitable culture media for this purpose include, for example, D-MEM or RPM1- 1640 medium. In addition, the hybridoma cells may be grown in vivo as ascites tumors in an animal e.g, by i.p. injection of the cells into mice. The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum' by conventional antibody purification procedures such as, for example, affinity chromatography (e.g., using protein A or protein G-Sepharose) or ion-exchange chromatography, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (.e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies),

The hybridoma method is one way to make antibodies which can be readily tested for the desired activity, and hybridomas expressing an antibody having the desired activity are a source of DNA encoding that antibody. However, many other methods are known in the art. Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as E. coli cells, simian COS ceils, Chinese Hamster Ovary (CHO) cells, or myeloma ceils that do not otherwise produce antibody protein, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra ex al, Curr. Opinion in Immunol., 5:256-262 (1993) and Pluckthun, Immunol. Revs. 130: 151-188 (1992). In a further embodiment, monoclonal antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al, Nature, 348:552-554 (1990).

Clackson et al, Nature. 352:624-628 (1991) and Marks et al, J. Mol. Biol. 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries.

S ubsequent, publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al, Bio/Technology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as a strategy for constructing very large phage libraries (Waterhouse et al, Nuc. Acids. Res. 21 :2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolation of monoclonal antibodies. The DNA that encodes the antibody may be modified to produce chimeric or fusion antibody polypeptides, for example, by substituting human heavy chain and light chain constant domain (CH and QJ sequences for the homologous murine sequences (U.S. Patent No. 4,816,567; and Morrison, et al, Proc. Natl Acad. ScL USA, 81 :6851 ( 1984)), or by fusing the

immunoglobulin coding sequence with all or part of the coding sequence for a nori- immunoglobulin polypeptide (heterologous polypeptide). The non- immunoglobulin polypeptide sequences can substitute for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-combining site of an antibody to create a chimeric bivalent antibody comprising one antigen-combining site having specificity for an antigen and another antigen-combining site having specificity for a different antigen.

3. Human and Humanized Antibodies

In some embodiments of the present disclosure, the antibodies for use as TPRA40 antagonists are human or humanized antibodies. N umerous methods exist in the art for the making of human and humanized antibodies.

4. Antibody fragments In some embodiments, the present disclosure provides for TPRA40 antagonists that are antibody fragments. In certain circumstances, there are advantages of using antibody fragments, rather than whole antibodies. The smaller size of the fragments allows for rapid clearance, while retaining similar antigen binding specificity of the corresponding full length molecule, and may lead to improved access to target tissues, e.g., solid tumors.

Various techniques have been developed for the production of antibody fragments.

Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and Biophysical Methods 24: 107-117 (1992); and Brennan et al, Science, 229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and scFv antibody fragments can all be expressed in and secreted from E. coli, thus all owing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries discussed above.

Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et aL, Bio/Technology 10: 163-167 (1992)). According to another approach, F(ab')2 fragments can be isolated directly from recombinant host cell culture. Fab and F(ab')2 fragment with increased in vivo half-life comprising a salvage receptor binding epitope residues are described in U.S. Patent No. 5,869,046. Other techniques for the production of antibody fragments will be apparent to the skilled practitioner. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93/16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. Fv and sFv are the only species with intact combining sites that are devoid of constant regions; thus, they are suitable for reduced

nonspecific binding during in vivo use. sFv fusion proteins may be constructed to yield fusion of an effector protein at either the amino or the carboxy terminus of an sFv. See Antibody

Engineering, ed. Borrebaeck, supra. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Patent 5,641 ,870 for example. Such linear antibody fragments may be monospecific or bispecific.

B. TPRA40 Binding Oligopeptides

In some embodiments, TPRA40 binding oligopeptides are examples of TPRA40 antagonists of the disclosure. TPRA40 binding oligopeptides of the present disclosure are oligopeptides that bind, preferably specifically, to a. TPRA40 polypeptide, as described herein. In some embodiments, the TPRA40 binding oligopeptide binds to the putative extracellular N- terminal portion of the TPRA40 polypeptide (e.g., any of the amino acids corresponding to amino acids 1 -47 of SEQ ID NO: 1 ). In some embodiments, the TPRA40 binding oligopeptides bind to the putative intracellular C-terminal portion of the TPRA40 protein (e.g., any of the amino acids corresponding to amino acids 286-373 of SEQ ID NO: 1). In some embodiments, the TPRA40 binding oligopeptide binds to any of the putative extracellular portions of TPRA40 (e.g., any of the amino acids corresponding to amino acids 97-122, 172-191 , or 261-264 of SEQ ID NO: 1). In some embodiments, the TPRA40 binding oligopeptide binds to any of the putative intracellular portions of TPRA40 (e.g., any of the amino acids corresponding to amino acids 70- 74, 144-150, and 213-239). In certain embodiments, the TPRA40 binding oligopeptide binds to the C-terminal region of a TPRA40 protein and prevents TPRA40 from interacting with another protein, e.g., Ssnal/NA14, In some embodiments, the TPRA40 binding oligopeptide binds to a ligand-binding site of TPRA40 and does not activate hedgehog signaling downstream of

TPRA40, but rather, stericaliy blocks a ligand from binding to TPRA40. In some embodiments, the TPRA40 binding oligopeptide binds to the TPRA40 to stericaliy block an interaction between TPRA40 and a G protein (e.g., a G-a!pha-i protein).

In some embodiments, the TPRA40 binding oligopeptides disclosed herein may be chemically synthesized using known oligopeptide synthesis methodology or may be prepared and purified using recombinant technology, TPRA40 binding oligopeptides may be about 5 amino acids in length, alternatively at least about 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 55, 16, 17, 1 8, 1 9, 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, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids in length or more, and such oligopeptides are capable of binding, preferably specifically, to a TPRA40 polypeptide, respectively, as described herein. TPRA40 binding oligopeptides may be identified without undue experimentation using well known techniques. In this regard, it is noted that techniques for screening oligopeptide libraries for oligopeptides that are capable of specifically binding to a polypeptide target are well known in the art (see, e.g., U.S. Patent Nos. 5,556,762, 5,750,373, 4,708,871 , 4,833,092, 5,223,409, 5,403,484, 5,571 ,689, 5,663,143; PCX Publication Nos. WO 84/03506 and WO84/03564;

Gevsen et al, Proc. Natl. Acad. Sci. U.S.A., 81 :3998-4002 (1984); Geysen et al, Proc. Natl. Acad. Sci. U.S.A., 82: 178-182 (1985); Geysen et al, in Synthetic Peptides as Antigens. 130- 149 (1986); Geysen et al, J. Immunol. Meth.. 102:259-274 (1987); Schoofs et al, J. Immunol, 140:61 1-616 (1988), Cwiria, S. E. et al. (1990) Proc. Natl. Acad. Sci. USA. 87:6378; Lowman, H.B. et al. (1991) Biochemistry. 30: 10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al (1991), I Mol. Biol, 222:581 ; Kang, A.S. et al. (1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G, P. (1991) Current Opin. Biotechnol..2:668).In this regard, bacteriophage (phage) display is one well known technique which allows one to screen large oligopeptide libraries to identify member(s) of those libraries which are capable of specifically binding to a polypeptide target. Phage display is a technique by which variant polypeptides are displayed as fusion proteins to the coat protein on the surface of bacteriophage particles (Scott, J. . and Smith, G. P. (1990) Science 249: 386). The utility of phage display lies in the fact that large libraries of selectively randomized protein variants (or randomly cloned cDNAs) can be rapidly and efficiently sorted for those sequences that bind to a target molecule with high affinity. Display of peptide (Cwiria, S. F, et al. (1990) Proc. Natl. Acad. Sci. USA, 87:6378) or protein (Lowman, H.B. et al (1991) Biochemistry. 30: 10832; Clackson, T. et al. (1991) Nature, 352: 624; Marks, J. D. et al. (1991 ), J. Mol. Biol. 222:581 ; Kang, A.S. et al (1991) Proc. Natl. Acad. Sci. USA. 88:8363) libraries on phage have been used for screening niiilions of polypeptides or

oligopeptides for ones with specific binding properties (Smith, G. P. (5991) Current Opin.

BiotechnoL, 2:668). Sorting phage libraries of random mutants requires a strategy for constructing 'and propagating a large number of variants, a procedure for affinity purification using the target receptor, and a means of evaluating the results of binding enrichments. U.S. Patent Nos. 5,223,409, 5,403,484, 5,571 ,689, and 5,663,143.

Although most phage display methods have used filamentous phage, lambdoid phage display systems (WO 95/34683; U.S. 5,627,024), T4 phage display systems (Ren et al, Gene, 215: 439 (1998); Zhu et al, Cancer Research. 58(15): 3209-3214 (1998); Jiang et al, Infection & Immunity, 65(11): 4770-4777 (1997); Ren et al., Gene, 195(2):303~311 (1997); Ren, Protein Sci.. 5: 1833 (1996); Efimov et al, Virus Genes. 10: 173 (1995)) and T7 phage display systems (Smith and Scott, Methods in Enzymology, 217: 228-257 (1993); U.S. 5,766,905) are also known.

Many other improvements and variations of the basic phage display concept ha ve now been developed. These improvements enhance the ability of display systems to screen peptide libraries for binding to selected target molecules and to display functional proteins with the potential of screening these proteins for desired properties. Combinatorial reaction devices for phage display reactions have been developed (WO 98/14277) and phage display libraries have been used to analyze and control bimolecular interactions (WO 98/20169; WO 98/20159) and properties of constrained helical peptides ( WO 98/20036). WO 97/35196 describes a method of isolating an affinity ligand in which a phage display library is contacted with one solution in which the ligand will bind to a target molecule and a second solution in which the affinity ligand will not bind to the target molecule, to selectively isolate binding ligands, WO 97/46251 describes a method of biopanning a random phage display library with an affinity purified antibody and then isolating binding phage, followed by a niicropanning process using micropiate wells to isolate high affinity binding phage. The use of Staphylococcus aureus protein A as an affinity tag has also been reported (Li et al. (1998) MoT Biotech., 9: 187). WO 97/47314 describes the use of substrate subtraction libraries to distinguish enzyme specificities using a combinatorial library which may be a phage display library. A method for selecting enzymes suitable for use in detergents using phage display is described in WO 97/09446. Additional methods of selecting specific binding proteins are described in U.S. Patent Nos. 5,498,538, 5,432,018, and WO 98/15833.

Methods of generating peptide libraries and screening these libraries are also disclosed in U.S. Patent Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, 5,498,530, 5,770,434, 5,734,018, 5,698,426, 5,763,592, and 5,723,323.

The disclosure contemplates that, in certain embodiments, a TPRA40 antagonist for use in the methods of the disclosure comprises a TPRA40 oligopeptide, such as any of the TPRA40 oligopeptides described herein. TPRA40 oligopeptides having the desired activity as a TPRA40 antagonist may be readily selected using, for example, any of the assays described herein to confirm that a TPRA40 oligopeptide has the desired function of a TPRA40 antagonist of the disclosure.

C. TPRA40 Polypeptide Variants

In some embodiments, the TPRA40 antagonists are TPRA40 polypeptide variants. In certain embodiments the polypeptide variant is a dominant negative. Such variants can be prepared by introducing appropriate nucleotide changes into the encoding DNA, and/or by synthesis of the desired polypeptide. Those skilled in the art will appreciate that amino acid changes may alter post-transiational processes of these molecules, such as changing the number or position of glycosylation sites or altering the membrane anchoring characteristics. Variations in amino acid sequence can be made, for example, using any of the techniques and guidelines for conservative and non-conservative mutations set forth, for instance, in U.S. Patent No. 5,364,934. Variations may be a. substitution, deletion or insertion of one or more codons encoding the amino acid sequence that results in a change in the amino acid sequence as compared with the native sequence. Optionally the variation is by substitution of at least one amino acid with any other amino acid in one or more of the domains of the amino acid sequence of interest. Guidance in determining which amino acid residue may be inserted, substituted or deleted without adversely affecting the desired activity may be found by comparing the sequence of the amino acid sequence of interest with homologous known protein molecules and minimizing the number of amino acid sequence changes made in regions of high homology. Amino acid substitutions can be the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, such as the replacement of a leucine with a serine, i.e., conservative amino acid replacements. Insertions or deletions may optionally be in the range of about 1 to 5 amino acids. The variation allowed may be determined by systematically making insertions, deletions or substitutions of amino acids in the sequence and testing the resulting variants for activity exhibited by the full-length or mature native sequence.

Fragments of the various TPRA.40 polypeptides are provided herein. Such fragments may be truncated at the N-terminus or C-termmus, or may lack internal residues, for example, when compared with a full length native protein. Such fragments which lack amino acid residues that are not essential for a desired biological activity are also useful with the disclosed methods.

In some embodiments, the TPRA40 fragments may be used as inhibitors of hedgehog signaling. For example, a fragment of TPRA40 that interacts with a TPRA40 binding partner, but which does not inhibit adenyiyl cyclase, may be used as a TPRA antagonist. In some embodiments, the fragment of TPRA40 is a soluble C-terminal portion of the TPRA40 polypeptide (e.g., a soluble polypeptide comprising a portion of the amino acids corresponding to amino acids 286-373 of SEQ ID NO: 1. In some embodiments, the fragment of TPRA40 is a soluble N-terminal portion of the TPRA40 polypeptide (e.g. , a. soluble polypeptide comprising a portion of the amino acids corresponding to amino acids 1-47 of SEQ ID NO: 1). In some embodiments, the fragment of TPRA40 comprises a soluble portion of any of the putative extracellular portions of TPRA40 (e.g., a soluble polypeptide comprising at least a portion of any of the amino acids corresponding to amino acids 70-74, 97-122 , 144-150, 172-191 , 213-239 or 261 -264 of SEQ ID NO: 1). In some embodiments, the fragments comprise at least 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or 90 amino acids of the full-length TPRA40 amino acid sequence.

The above polypeptide fragments may be prepared by any of a number of conventional techniques. In some embodiments, desired peptide fragments may be chemically synthesized. An alternative approach involves generating such fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues, or by digesting the DNA with suitable restriction enzymes and isolating the desired fragment. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding the desired fragment by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed at the 5' and 3' primers in the PCR. Preferably, such fragments share at least one biological and/or immunological activity with the corresponding full length molecule.

In particular embodiments, conservative substitutions of interest are shown in Table 3 under the heading of preferred substitutions. If such substitutions result in a change in biological activity, then more substantial changes, denominated exemplary substitutions in Table 3, or as further described below in reference to amino acid classes, are introduced and the products screened in order to identify the desired variant.

TABLE 3

Original Bxensplaxy Preferred

Residue S«b it¾t|s.l 8 S¾bsiily i

Figure imgf000068_0001

Substantial modifications in function or immunological identity of the TPRA40 polypeptides are accomplished by selecting substitutions that, differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues are divided into groups based on common side-chain properties:

(1) hydrophobic: Norieucine, Met, Ala, Val, Leu, He;

(2) neutral hydropbilic: Cys, Ser, Thr; Asn; Gin (3) acidic: Asp, Giu;

(4) basic: His, Lys, Arg;

(5) residues that influence chain orientation: Giy, Pro; and

(6) aromatic: Trp, Tyr, Phe.

In some embodiments, non-conservative substitutions will entail exchanging a member of one of these classes for another class. Such substituted residues also may be introduced into the conservative substitution sites or, more preferably, into the remaining (non-conserved) sites.

In some embodiments, the variations can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PGR mutagenesis. Site-directed mutagenesis [Carter et ah, Nucl. Acids Res:, 13:4331 (1986); Zoller et al., Nucl. Acids Res...10:6487 (1987)], cassette mutagenesis [Weils et al, Gene, 34:315 (1985)], restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc. London SerA, 317:415 (1986)] or other known techniques can be performed on the cloned DNA to produce the anti-TPRA40 molecule.

In some embodiments, scanning amino acid analysis can also be employed to identify one or more amino acids along a contiguous sequence. Among the preferred scanning amino acids are relatively small, neutral amino acids. Such amino acids include alanine, glycine, serine, and cysteine. Alanine is typically a. preferred scanning amino acid among this group because it eliminates the side-chain beyond the beta-carbon and is less likely to alter the main-chain conformation of the variant [Cunningham and Wells, Science, 244: 1081-1085 (1989)]. Alanine is also typically preferred because it is the most, common amino acid. Further, it is frequently found in both buried and exposed positions [Creighton, The Proteins. (W.H. Freeman & Co., N. Y.); Chothia, J. MoT. Biol.. 150: 1 (1976)]. If alanine substitution does not yield adequate amounts of variant, an isoteric amino acid can be used.

Any cysteine residue not, involved in maintaining the proper conformation of the

TPRA40 polypeptides also may be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine bond(s) may be added to such a molecule to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

In one embodiment, the substi utional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized or human antibody).

Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient way for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino substitutions at each site. The antibody variants thus generated are displayed in a monovalent fashion from filamentous phage particles as fusions to the gene DI product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as herein disclosed. In order to identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues contributing significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal stmcture of the antigen- antibody complex to identify contact points between the antibody and target polypeptide. Such contact residues and neighboring residues are candidates for substitution according to the techniques elaborated herein. Once such variants are generated, the panel of variants is subjected to screening as described herein and antibodies with superior properties in one or more relevant assays may be selected for further development.

Nucleic acid molecules encoding amino acid sequence variants of TPRA40 polypeptides may be prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of naturally occurring amino acid sequence variants) or preparation by oligonucleotide-mediated (or site-directed) mutagenesis, PGR mutagenesis, and cassette mutagenesis of a native sequence or an earlier prepared variant.

D. Modifications of TPRA40 polypeptides

In one embodiment, the TPRA40 antagonist comprises a fusion of any of the TPRA40 polypeptides disclosed herein (e.g., TPRA40 chimeric polypeptides) with a tag polypeptide which provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally placed at the amino- or carboxyl- terminus of such antibody or polypeptide. The presence of such epitope-tagged forms of such antibodies or polypeptides can be detected using an antibody against the tag polypeptide. Also, pro vision of the epitope tag enabl es such antibodies or polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag. Various tag polypeptides and their respective antibodies are well loiown in the art. Examples include poly-histidine (poly-his) or poly-histidine-giycine (poly-his-gly) tags; the flu HA tag polypeptide and its antibody 12CA5 [Field et al, MoT Cell. Biol. 8:2159-2165 (1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10 antibodies thereto [Evan et al, Molecular and Cellular Biology, 5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al., Protein Engineering, 3(6): 547-553 (1990)]. Other tag polypeptides include the Flag-peptide [Hopp et al., BioTechnoiogy, 6: 1204-1210 (1988)]; the T3 epitope peptide [Martin et al, Science. 255: 192-194 (1992)]; an a-tubulin epitope peptide [Skinner et al, J. Biol. Cheiru 266: 15163-15166 (1991)]; and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al, Proc. Natl. Acad. Sci. USA. 87:6393- 6397 (1990)]. In an alternative embodiment, the TPRA40 antagonist may comprise a fusion of the TPRA40 polypeptides with an immunoglobulin or a particular region of an immunoglobulin (e.g., Fc domain). For a bivalent form of the TPRA40 antagonist (also referred to as an

"imrnunoadhesin"), such a fusion could be to the Fc region of an IgG molecule. The Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a preceding antibody or polypeptide in the place of at least one variable region within an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion includes the hinge, CH2 and CHS, or the hinge, CHI , CH2 and CHS regions of an IgGl molecule. For the production of immunoglobulin fusions see also US Patent No. 5,428,130 issued June 27, 1995.

E. Preparation of TPRA40 polypeptides

The disclosure herein provides for the production of TPRA40 polypeptides (e.g.,

TPRA40 antagonist polypeptides) in some embodiments, by culturing cells transformed or iransfected with a vector containing nucleic acid such antibodies, polypeptides and oligopeptides. It is, of course, contemplated that alternative methods, which are well known in the art, may be employed to prepare any of the antibodies, polypeptides and oligopeptides disclosed herein. For instance, the appropriate amino acid sequence, or portions thereof, may be produced by direct peptide synthesis using solid-phase techniques [see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, CA (1969); errifield, J. Am. Chem. Soc, 85:2149-2154 (1963)] . In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be accomplished, for instance, using an Applied

Biosystems Peptide Synthesizer (Foster City, CA) using the manufacturer's instructions. Various portions of any of the antibodies, polypeptides or oligopeptides described herein may be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired product.

1. Isolation of DNA Encoding TPRA40 polypeptides

In some embodiments, DNA encoding a TPRA40 polypeptide may be obtained from a cDNA library prepared from tissue believed to possess such antibody, polypeptide or

oligopeptide mRNA and to express it at a detectable level. Accordingly, D A encoding such polypeptides can be conveniently obtained from a cDNA library prepared from human tissue, a genomic library or by known synthetic procedures (e.g., automated nucleic acid synthesis).

Libraries can be screened with probes (such as oligonucleotides of at least about 20-80 bases) designed to identify the gene of interest or the protein encoded by it. Screening the cD A or genomic library with the selected probe may be conducted using standard procedures, such as described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), Alternatively, PCR methodology may be used. [Sambrook et al, supra: Dieffenbach et al,, PCR Primer: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1995)]. Alternatively, TPRA40 cDNA can simply be purchased from commercial sources (e.g. from Open Biosystems' mammalian gene collection (ThermoScientific)),

Techniques for screening a cDNA library are well known in the art. In some

embodiments, the oligonucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized. In some embodiments, the oligonucleotide is labeled such that it can be detected upon hybridization to DNA in the library being screened. Methods of labeling are well known in the art, and include the use of radiolabels like 32P~labeled ATP, biotinylation or enzyme labeling. Hybridization conditions, including moderate stringency and high stringency, are provided in Sambrook et al., supra.

Sequences identified in such library screening methods can be compared and aligned to other known sequences deposited and available in public databases such as GenBank or other private sequence databases. Sequence identity (at either the amino acid or nucleotide level) within defined regions of the molecule or across the full-length sequence can be determined using methods known in the art and as described herein.

In some embodiments, nucleic acid having protein coding sequence may be obtained by screening selected cDNA or genomic libraries using the deduced amino acid sequence disclosed herein for the first time, and, if necessary, using conventional primer extension procedures as described in Sambrook et al,, supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcribed into cDNA.

2. Selection and Transformation of Host Cells

In some embodiments, host cells are transfected or transformed with expression or cloning vectors described herein for TPRA40 polypeptide production and cultured in

conventional nutrient media modified as appropriate for inducing promoters, selecting

transformants, or amplifying the genes encoding the desired sequences. The culture conditions, such as media, temperature, pH and the like, can be selected by the skilled artisan without undue experimentation. In general, principles, protocols, and practical techniques for maximizing the productivity of cell cultures can be found in Mammalian Cell Biotechnology: A Practical Approach. M. Butler, ed. (ERL Press, 1991) and Sambrook et al., supra.

Methods of eukaryotic cell transfection and prokaryotic cell transformation are known to the ordinarily skilled artisan, for example, CaCl2, CaP04, liposome-mediated and electroporation. Depending on the host cell used, transformation is performed using standard techniques appropriate to such cells. The calcium treatment employing calcium chloride, as described in Sambrook et al., supra, or electroporation is generally used for prokaryotes. Infection with Agrobacterium tumefaciens is used for transformation of certain plant cells, as described by Shaw et al, Gene, 23:315 (1983) and WO 89/05859 published 29 June 1989. For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology, 52:456-457 (1978) can be employed. General aspects of mammalian cell host system transfections have been described in U.S. Patent No. 4,399,216. Transformations into yeast, are typically carried out according to the method of Van Solingen et, al, J. B act,., 130:946 (1977) and Hsiao et al. Proc. Natl. Acad. Sci. (USA). 76:3829 (1979). However, other methods for introducing DNA into cells, such as by nuclear microinjection, electroporation, bacterial protoplast fusion with intact cells, or polycations, e.g., polybrene, polyornithine, or any other methods available to the skilled worker may also be used. For various techniques for

transforming mammalian cells, see Keown et ah, Methods in Enzymology. 185:527-537 (5990) and Mansour et al, Nature, 336:348-352 (1988).

Suitable host cells for cloning or expressing the DNA in the vectors herein include prokaryote, yeast, or higher eukaryote cells. Suitable prokaryotes include but are not limited to eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E, coli XI 776 (ATCC 31 ,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as

Escherichia, e.g., E. coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacilli such as B.

subtilis and B. iicheniformis {e.g., B. licheniformis 41P disclosed in DD 266,710 published 12 April 1989), Pseudomonas such as P. aeruginosa, and Streptomyces. These examples are illustrative rather than limiting. Strain W3110 is one particularly preferred host or parent host because it is a common host strain for recombinant DNA product fermentations. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W31 10 may be modified to effect a genetic mutation in the genes encoding proteins endogenous to the host, with examples of such hosts including E. coli W3110 strain 1 A2, which has the complete genotype tonA : E. coli W3110 strain 9E4, which has the complete genotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptrSphoA El 5 (argF- lac)169 degP ompTkan ; E, coli W3110 strain 37D6, which has the complete genotype tonA ptrS phoA El 5 (argF-lac)169 degP ompTrbs? UvG kan ; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistant degP deletion mutation; and an E. coli strain having mutant peripiasmie protease disclosed in U.S. Patent No. 4,946,783 issued 7 August 1990.

Alternatively, in vitro methods of cloning, e.g., PGR or other nucleic acid polymerase reactions, are suitable. Full length antibody, antibody fragments, and antibody fusion proteins can be produced in bacteria, in particular when glycosylation and Fc effector function are not needed, such as when the therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin) and the immunoconjugate by itself shows effectiveness in tumor cell destruction. Full length antibodies have greater half life in circulation. Production in E. coli is faster and more cost efficient. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. 5,648,237 (Carter et. al), U.S. 5,789,199 ( y et al), and U.S. 5,840,523 (Simmons et al.) which describes translation initiation region (TIR) and signal sequences for optimizing expression and secretion, these patents incorporated herein by reference. After expression, the antibody is isolated from the E, coli cell paste in a soluble fraction and can be purified through, e.g., a protein A or G column depending on the isotype. Final purification can be carried out similar to the process for purifying antibody expressed in suitable ceils (e.g., CHO cells).

In some embodiments, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding TPRA40 polypeptides. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Others include

Schizosaccharomvces pombe (Beach and Nurse, Nature. 290: 140 [ 1981]; EP 139,383 published 2 May 1985); Kiuyveromyces hosts (U.S. Patent No. 4,943,529; Fleer et al, Bio/Technology. 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al, J. Bacterid.. 154(2):737-742 [1983]), .fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24, 178), K. waitii (ATCC 56,500), K. drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technology, 8: 135 (1990)), . thermotolerans, and K. marxianus; yarrowia

~! "3 (EP 402,226); Pichia pastoris (EP 183,070; Sreekrishna et al, J. Basic Microbiol., 28:265- 278 [1988]); Candida; Trichoderma reesia (EP 244,234); Neurospora crassa (Case et al, Proc. Natl. Acad. ScL USA, 76:5259-5263 [1979]); Schwanniomyces such as Schwanniomyces occideiitalis (EP 394,538 published 31 October 1990); and filamentous fungi such as, e.g., Neurospora, Peniciliium, Tolypocladium (WO 91/00357 published 10 January 1991), and Aspergillus hosts such as A. nidulans (Ballance et al, Biochem. Biophvs, Res. Commun.. 112:284-289 [1983]; Tilburn et al, Gene. 26:205-221 [1983]; Yelton et al, Proc. Natl. Acad. Sci. USA. 81 : 1470-1474 [1984]) and A. niger (Kelly and Hynes, EMBO J., 4:475-479 [1985]). Methylotropic yeasts are suitable herein and include, but are not limited to, yeast capable of growth on methanol selected from the genera consisting of Hansenula, Candida, Kloeckera, Pichia, Saccharomyces,

Tomlopsis, and Rhodotorula. A list of specific species that are exemplary of this class of yeasts may be found in C. Anthony, The Biochemistry of Methylotrophs. 269 (1982).

In some embodiments, suitable host, cells for the expression of glycosylated TPRA40 polypeptide production are derived from multicellular organisms. Examples of invertebrate cells include insect cells such as Drosophila S2 and Spodoptera Sf9, as well as plant cells, such as cell cultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco. Numerous baculoviral strains and variants and corresponding permissive insect host cells from hosts such as

Spodoptera fugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyx mod have been identified. A variety of viral strains for transfection are publicly available, e.g., the L-I variant of Autographa cal ifornica NP V and the Bm-5 strain of Bombyx mori NPV, and such viruses may be used as the virus herein according to the present disclosure, particularly for transfection of Spodoptera frugiperda cells.

However, interest has been greatest in vertebrate cells, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure, in some embodiments, the TPRA40 polypeptides are produced in vertebrate cells. Examples of useful mammalian host cell lines are monkey kidney CVl line transformed by SV40 (COS-7, ATCC CRL 1651 ); human embryonic kidney line (293 or 293 ceils subcloned for growth in suspension culture, Graham et al, J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BH , ATCC CCL 10); Chinese hamster ovary ceils/'-DHFR (CHO, Urlaub et ai, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse Sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980)); monkey kidney cells (CVl ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI ceils (Mather et ai, Annals N. Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells: and a human hepatoma line (Hep G2).

In some embodiments, host cells are transformed with the above-described expression or cloning vectors for TPRA40 polypeptide production and cultured in conventional nutrient media modified as appropriate for inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences.

3. Selection and Use of a Repli cable Vector

In some embodiments, the nucleic acid (e.g., cDNA or genomic DNA) encoding any of the TPRA40 polypeptides disclosed herein may be inserted into a repli cable vector for cloning (amplification of the DNA) or for expression. Various vectors are publicly available. The vector may, for example, be in the form of a plasmid, cosmid, viral particle, or phage. The appropriate nucleic acid sequence may be inserted into the vector by a variety ofprocedures. In general, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques which are known to the skilled artisan. The TPRA40 polypeptide may be produced recombinantly not only directly, but also as a fusion polypeptide with a heterologous polypeptide, which may be an epitope tag or affinity tag to enable purification, but should not contain an -terminal signal sequence because TPRA40 does not have one of its own. Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells. Such sequences are well known for a variety of bacteria, yeast, and viruses. The origin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteria, the 2μ plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells.

In some embodiments, expression and cloning vectors will contain a selection gene, also termed a selectable marker. Typical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply critical nutrients not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli.

An example of suitable selectable markers for mammalian cells are those that enable the identification of ceils competent to take up nucleic acid encoding the desire protein, such as DHFR or thymidine kinase. In some embodiments, an appropriate host cell when wild-type DHFR is employed is the CHO cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et ai, Proe. Natl. Acad, Sci. USA, 77:4216 (1980). A suitable selection gene for use in yeast is the trp gene present in the yeast plasmid YRp7 [Stinchcomb et al, Nature. 282:39 (1979); Kingsman et al, Gene. 7: 141 (1979); Tschemper et al, Gene, 10: 157 (1980)]. The trp J gene provides a selection marker for a mutant strain of yeast lacking the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 [Jones, Genetics, 85: 12 (1977)].

Expression and cloning vectors usually contain a promoter operably linked to the nucleic acid sequence encoding the desired amino acid sequence, in order to direct mRNA synthesis. Promoters recognized by a variety of potential host cells are well known. Promoters suitable for use with prokaryotic hosts include the β-lactamase and lactose promoter systems [Chang et al, Nature, 275:615 (1978); Goeddel et al, Nature. 281 :544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res.. 8:4057 (1980); EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al, Proc. Natl. Acad. Sci. USA. 80:21 -25 (5983)] . Promoters for use in bacterial systems also will contain a Shine-Dalgarno (S.D.) sequence operably linked to the DNA encoding the desired protein sequence.

Examples of suitable promoting sequences for use with yeast hosts include the promoters for 3-phosphoglycerate kinase [Hitzeman et al, J. Biol. Chem.. 255:2073 (1980)] or other glycolytic enzymes [Hess et al, J. Adv. Enzyme Reg., 7: 149 (1968); Holland, Biochemistry. 17:4900 (1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glueose-6- phosphate isomerase, 3- phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other yeast promoters, which are inducible promoters having the additional advantage of transcription controlled by growth conditions, are the promoter regions for alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metaliothionein, glyceraldehyde-3 -phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for use in yeast expression are further described in EP 73,657.

DNA Transcription in mammalian host cells is controlled, for example, by promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 1,504 published 5 July 1989), adenovirus (such as Adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-B virus and Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g., the actin promoter or an immunoglobulin promoter, and from heat-shock promoters, provided such promoters are compatible with the host cell systems. Transcription of a DNA encoding the TPRA40 polypeptide may be increased by inserting an enhancer sequence into the vector. Enhancers are cis- acting elements of DNA, usually about from 10 to 300 bp, that act on a promoter to increase its transcription. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, a- fetoprotein, and insulin). In some embodiments, an enhancer from, a eukaryotic cell virus will be used. Examples include the SV40 enhancer on the late side of the replication origin (bp 100- 270), the cytomegalovirus early promoter enhancer; the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. The enhancer may be spliced into the vector at a position 5' or 3' to the coding sequence of the preceding amino acid sequences, but is preferably located at a site 5' from the promoter.

In some embodiments, expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. Such sequences are commonly available from the 5' and, occasionally 3', untranslated regions of eukaryotic or viral DNAs or cDNAs. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the respective antibody, polypeptide or oligopeptide described in this section.

Still other methods, vectors, and host cells suitable for adaptation to the synthesis of the respective antibody, polypeptide or oligopeptide in recombinant vertebrate cell culture are described in Gething et al, Nature. 293:620-625 (1981); Mantel et al, Nature, 281 :40-46 (1979); EP 117,060; and EP 1 17,058. 4. Culturing the Host Cells

In some embodiments, the host cells used to produce the TPRA40 polypeptides may be cultured in a variety of media. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbeeeo's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing the host cells. In addition, any of the media described in Ham et al, Meth. Enz. 58:44 (1979), Barnes et al, Anal. Biocliem.102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762; 4,560,655: or 5,122,469: WO 90/03430; WO 87/00195: or U.S. Patent Re. 30,985 may be used as culture media for the host cells. Any of these media may be supplemented as necessary with hormones and/or other growth factors (such as insulin, transferrin, or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium, and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENT AMY CIN(TM) drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements may also be included at appropriate concentrations that would be known to those skilled in the art. The culture conditions, such as temperature, pH, and the like, are those previously used with the host, cell selected for expression, and will be apparent to the ordinarily skilled artisan.

5. Detecting Gene Amplification/Expression

In some embodiments, gene amplification and/or expression may be measured in a sample directly, for example, by conventional Southern blotting, Northern blotting to quantitate the transcription of mRNA [Thomas, Proc. Natl. Acad. Sci. USA. 77:5201 -5205 (1 80)], dot blotting (DNA analysis), or in situ hybridization, using an appropriately labeled probe, based on the sequences provided herein. Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes, RNA duplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. The antibodies in turn may be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.

In some embodiments, gene expression, alternatively, may be measured by

immunological methods, such as imniunohistochemical staining of ceils or tissue sections and assay of cell culture or body fluids, to quantitate directly the expression of gene product.

Antibodies useful for imniunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal, and may be prepared in any mammal. Conveniently, the antibodies suitable for the present method may be prepared against a native sequence polypeptide or oligopeptide, or against exogenous sequence fused to DNA and encoding a specific antibody epitope of such a polypeptide or oligopeptide.

6. Protein Purification

In some embodiments, any of the TPRA40 polypeptides disclosed herein may be recovered from culture medium or from host cell lysates. If membrane-bound, it can be released from the membrane using a. suitable detergent solution (e.g. Triton-X 100) or by enzymatic cleavage. In some embodiments, ceils employed in expression of the preceding can be disrupted by various physical or chemical means, such as freeze-thaw cycling, soni cation, mechanical disruption, or cell lysing agents.

In some embodiments, it may be desirable to purify any of the TPRA polypeptides described herein produced by a host cell. The following procedures are examples of suitable purification procedures: by fractionation on an ion-exchange column; ethanol precipitation; reverse phase HPLC; chromatography on silica or on a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammonium sulfate precipitation; gel filtration using, for example, Sephadex G-75; protein A Seph arose columns to remove contaminants such as IgG; and metal chelating columns to bind epitope-tagged forms of the desired molecules. Various methods of protein purification may be employed and such methods are known in the art and described for example in Deutscher, Methods in Enzymologv, 182 (1990); Scopes, Protein Purification: Principles and Practice. Springer- Verlag, New York (1982). The purification step(s) selected will depend, for example, on the nature of the production process used and the particular antibody, polypeptide or oligopeptide produced for the claimed methods.

In some embodiments, when using recombinant techniques, the TPRA40 polypeptide can be produced intraceiluiarly, in the periplasmic space, or directly secreted into the medium. If TPRA40 polypeptides are produced intraceiluiarly, as a first step, the particulate debris, either host cells or iysed fragments, are removed, for example, by centrifugation or ultrafiltration. Carter et al, Bio/Technology 10: 163-167 (1992) describe a procedure for isolating antibodies which are secreted to the periplasmic space of E: coli. Briefly, cell paste is thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyifluoride (PMSF) over about 30 mi . Cell debris can be removed by centrifugation. Where the antibody is secreted into the medium, superaatants from such expression systems are generally first concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the foregoing steps to inhibit proteolysis and antibiotics may be included to prevent the growth of adventitious contaminants.

In some embodiments, purification can occur using, for example, hydroxyiapatite chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being the preferred purification technique. The suitability of protein A as an affinity liga d depends on the species and isotype of any immunoglobulin Fc domain that is present in the antibody. Protein A can be used to purify antibodies that are based on human γ! , γ2 or γ4 heavy chains (Lmdmark et al., J. Immunol. Meth. 62: 1 -13 (1983)). Protein G is recommended for all mouse isotypes and for human γ3 (Guss et al., EMBO J. 5: 15671575 (1986)). In some embodiments, the matrix to which the affinity ligand is attached is agarose, but other matrices are available. Mechanically stable matrices such as controlled pore glass or poly(styrenedivinyl)benzene allow for faster flow rates and shorter processing times than can be achieved with agarose. Where the antibody comprises a CHS domain, the Bakerbond

ABX(TM)resin (J. T. Baker, Phillipsburg, NJ) is useful for purification. Other techniques for protein purification such as fractionation on an ion- exchange column, ethanol precipitation, Reverse Phase HPLC, chromatography on silica, chromatography on heparin SEPHAROSE(TM) chromatography on an anion or cation exchange resin (such as a polyaspartic acid column), chromatofocusmg, SDS-PAGE, and ammonium sulfate precipitation are also available depending on the antibody to be recovered.

In some embodiments, following any preliminary purification step(s), the mixture comprising the antibody of interest and contaminants may be subjected to low pH hydrophobic interaction chromatography using an elution buffer at a pH between about 2.5-4.5. in some embodiments, the chromatography step is performed at low salt concentrations (e.g., from about 0-0.25M salt).

F. Polynucleotide TPRA40 Antagonists

In some embodiments, the TPRA40 antagonist is a. polynucleotide that inhibits expression of TPRA40. In some embodiments, the TPRA40 antagonist is a sense/antisense oligonucleotide. Molecules that would be expected to inhibit TPRA40, and therefor inhibit or antagonize hedgehog signaling, include fragments of the respective TPRA40-encoding nucleic acids such as antisense or sense oligonucleotides ("TPRA40 sense/antisense NA"). Such nucleic acids comprise a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to the respective target (a) TPRA40 mRNA (sense) or (b) TPRA40 DNA (antisense) sequences.

TPRA40 sense/antisense NA comprise a fragment of the coding region of the TPRA40 RNA or DNA. The ability to derive an antisense or a sense oligonucleotide, based upon a. cDNA sequence encoding a given protein is described in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988) and van der Kxol et al. (BioTechniques 6:958, 1988).

Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. Such methods are encompassed by the present disclosure. The TPRA40 sense/antisense NA may be used to block the respective expression of: TPRA40 polypeptides, wherein those TPRA40 polypeptides may play a role in the activation or amplification of hedgehog signaling. In some embodiments, the TPRA40 sense/antisense NA may further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleases; Nucleic acid with such resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences. The TPRA40 sense/antisense NA used in accordance with this disclosure may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, CA). Any other means for such synthesis laiown in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives. The compounds of the disclosure may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include, but are not limited to, U.S. Fat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932;

5,583,020; 5,591,721 ; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295;

5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herei incorporated by reference.

Other examples of TPRA40 sense/antisense NA suitable for use in the present disclosure include those oligonucleotides which are covalentiy linked to organic moieties, such as those described in WO 90/10048, and other moieties that increases the affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense

oligonucleotide for the target nucleotide sequence.

Although antisense oligonucleotides may function via any of a number of mechanisms, in certain embodiments, the antisense conjugates promote degradation of the TPRA40 mRNA transcript. In certain embodiments, the antisense conjugates promote RNaseH-mediated degradation of the TPRA40 mRNA transcript. Note, however, that the disclosure contemplates that a given antisense conjugate may have multiple effects. Thus, as long as an antisense conjugate promotes RNaseH-mediated degradation, it may also have other effects (e.g., identification of a mechanism does not imply that such mechanism is the sole mechanism by which the antisense conjugate impacts a cell or transcript)

One type of antisense oligonucleotide contemplated when some level of RNaseH- mediated degradation is desired, are antisense oligonucleotides sometimes termed "gapmers". The oligonucleotides have a central portion that is flanked by two wing portions (e.g., wing- central portion- wing). The central portion has nucleotide content and chemistry capable of promoting RNaseH-mediated degradation when hybridized to RNA. For example, the central portion comprises at least 7 nucleotides of D'NA and/or modified nucleotides that retain the ability to promote RNaseH-mediated degradation when hybridized to RNA, such as

phosphorothioate nucleotides. The central portion may also contain a mixture of DNA and modified nucleotides, including mixtures of different modified nucleotides. Alternatively, the central portion may contain only DNA nucleotides or only modified nucleotides. In contrast to the central portion, the wing portions are not intended to mediate RNaseH- mediated degradation. Rather, the wing portions are intended to improve the stability, half-life, or specificity of the oligonucleotides. Wing portions may include, for example, one or more modified nucleotides (including combinations) selected from: locked nucleic acid (LNA) nucleotides, 2'-0-methoxyefhyi nucleotides, 2-O-methyl nucleotides, peptide nucleic acids, and the like. For any of these modified nucleotides provided in the wing portion, the modified nucleotides may be modified DNA or modified RNA nucleotides.

In some embodiments, the antisense oligonucleotide is a morpholino molecule that stericaily blocks the binding of a protein or nucleic acid to a target RNA or DNA sequence. In some embodiments, the morpholino also triggers degradation of the target RN A or DN A sequence. In some embodiments, the morpholino molecule binds to TPRA40 RNA. In some embodiments, the morpholino molecule comprises 20-30 nucleotides. In other embodiments, the morpholino molecule comprises 23-27 nucleotides. In other embodiments, the morpholino molecule comprises 25 nucleotides.

In some embodiments, the antisense oligonucleotides of the present disclosure include a nucleotide analog having a constrained furanose ring conformation, such as Locked Nucleic Acids (LNAs). In LNAs, a 2'-hydroxyl group is linked to the 3' or 4' carbon atom of the sugar ring thereby forming a bieyelie sugar moiety.

In some embodiments, in modified oligonucleotide, both the sugar and the

internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic that has been shown to have excellent hybridization properties, is referred to as a peptide nucleic acid (FNA). In PNA compounds, the sugar- backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucieobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone.

Representative United States patents that teach the preparation of FNA nucleotides include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331 ; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA nucleotides can be found in Nielsen et al, Science, 1991, 254, 14974500. Antisense or sense oligonucleotides suitable for use in the present disclosure may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaP04-mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus. In a preferred procedure, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector: A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovims M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

In some embodiments, any of the sense or antisense oligonucleotides disclosed herein may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. In some embodiments, conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.

In some embodiments, any of the sense or antisense oligonucleotides disclosed herein may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448, The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase. Antisense or sense RNA or DNA molecules are generally at least about 5 nucleotides in length, alternatively 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, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 700, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800, 810, 820, 830, 840, 850, 860, 870, 880, 890, 900, 910, 920, 930, 940, 950, 960, 970, 980, 990, or 1000 nucleotides in length, wherein in this context the term "about" means the referenced nucleotide sequence length plus or minus 10% of that referenced length. In some embodiments, the polynucleotide TPRA40 antagonist is an interfering RNA or RNAi molecule.

An RNAi is RNA of 10 to 50 nucleotides in length which reduces expression of a target gene, wherein portions of the strand are sufficiently complementary (e.g. having at least 80% identity to the target gene). The method of RNA interference refers to the target-specific suppression of gene expression (i.e., "gene silencing"), occurring at a post- transcriptional level (e.g., translation), and includes all posttranscriptional and transcriptional mechanisms of RNA mediated inhibition of gene expression, such as those described in P.D. Zamore, Science 296: 1265 (2002) and Hannan and Rossi, Nature 431; 371-378 (2004). As used herein, RNAi can be in the form of small interfering RNA (siRNA), short hairpin RN A (shRNA), and/or micro RNA. (mi RNA).

Such RNAi molecules are often a double stranded RNA complexes that may be expressed in the form of separate complementary or partially complementary RN A strands. Methods are well known in the art for designing double-stranded RNA complexes. For example, the design and synthesis of suitable shRNA and siRNA may be found in Sandy et at,

BioTechmques 39: 215-224 (2005). An "RNA coding region" is a nucleic acid that, can serve as a template for the synthesis of an RNA molecule, such as a double-stranded RNA complex. Preferably, the RNA coding region is a DNA sequence.

A "small interfering RNA" or siRNA is a double stranded RNA (dsRNA) duplex of 10 to 50 nucleotides in length which reduces expression of a target gene, wherein portions of the first strand is sufficiently complementary (e.g. having at least 80% identity to the target gene), siRNAs are designed specifically to avoid the anti-viral response characterized by elevated interferon synthesis, nonspecific protein synthesis inhibition and RNA degradation that often results in suicide or death of the cell associated with the use of RNAi in mammalian cells.

Paddison et ai., Proc Natl Acad Sci USA 99(3): 1443-8. (2002). In some embodiments, the siRNA molecule comprises the nucleotide sequence of any SEQ ID NOs: 16-23.

The term "hairpin" refers to a looping RNA structure of 7-20 nucleotides. A "short hairpin RNA" or shRN A is a single stranded RNA 10 to 50 nucleotides in length characterized by a hairpin turn which reduces expression of a target gene, wherein portions of the RNA strand are sufficiently complementary (e.g. having at least 80% identity to the target gene). The term "stem-loop" refers to a pairing between two regions of the same molecule base- pair to form a double helix that ends in a short unpaired loop, giving a lollipop-shaped structure.

A "micro RNA" (previously known as stRNA) is a single stranded RNA of about 10 to 70 nucleotides in length that are initially transcribed as pre-miR A characterized by a "stem- loop" structure, which are subsequently processed into mature miRNA after further processing through the RNA-induced silencing complex (RISC).

An "RN A coding region" is a nucleic acid that can serve as a template for the synthesis of an RN A molecule, such as a double-stranded RNA complex. In some embodiments, the RNA coding region is a DNA sequence.

In some embodiments, the RNA coding region encodes a double-stranded RNA complex

(e.g., siRNA, miRNA, shRNA) that is capable of down-regulating the expression of a particular gene or genes. In some embodiments, a double-stranded RNA comple is expressed in the form of an RN A molecule having a stem- loop or a so- called "hairpin" structure. As used herein, "hairpin" structure encompasses shRNAs and miRNAs. In some embodiments, a double- stranded RN A complex is expressed in the form of separate complementary or partially complementary RNA strands. Methods are well-known in the art for designing double-stranded RNA complexes, eg, siRNA, miRNA, and shRNAs. For example, resources and citations describing the design of effective shRNA and siRNA are found in Sandy et al, BioTechniques 39:215-224 (2005). It is understood that the sequences of a double- stranded RNA complex may be of natural origin or may be synthetic.

In some embodiments, the RNA complex comprises a double-stranded region

corresponding to a region of a gene to be down-regulated is expressed in the ceil . One strand of the RNA double- stranded region is substantially identical (typically at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) in sequence to the sequence of the coding region targeted for down regulation. The other strand of the double-stranded region (interchangeably termed "RNA double-stranded region) is complementary to the sequence of the coding region targeted for down regulation, or partially complementary to the coding region targeted for down regulation (typically at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to the complement of the coding region targeted). It is understood that the double-stranded region can be formed by two separate RNA stranded, or by the self-complementary portions of a single RNA having a hairpin structure. In some embodiments, the double-stranded region is generally at least about 15 nucleotides in length and, in some embodiments, is about 15 to about 30 nucleotides in length. However, a significantly longer double-stranded region can be used effectively in some organisms. In some embodiments, the double-stranded region is between about 19 and 22 nucleotides in length. The double- stranded region is preferably identical to the target nucleotide sequence over this region. When the coding region to be down regulated is in a family of highly conserved genes, the sequence of the RNA double-stranded region can be chosen with the aid of sequence comparison to target only the desired gene. On the other hand, if there is sufficient identity among a family of homologous genes within an organism, a double- stranded can be designed that would down regulate a plurality of genes simultaneously. In some embodiments, a single RNA. coding region in the construct serves as a template for the expression of a self- complementary hairpin RN A, comprising a sense region, a loop region and an antisense region. The sense and anti sense regions are each preferably about 15 to about 30 nucleotides in length. The loop region preferably is about 2 to about 15 nucleotides in length, more preferably from about 4 to about 9 nucleotides in length. Following expression the sense and antisense regions form a duplex.

In some embodiments, the disclosure provides for siRNA molecules comprising a nucleotide sequence that is at least 85, 90, 95, 96, 97, 98, 99 or 100% identical to any of the nucleotide sequences of S EQ ID NOs: 16-23. In some embodiments, the siRNA molecule does not comprise the nucleotide sequence of SEQ ID NO: 16,

In another embodiment, the vector comprises two RNA coding regions. The first coding region is a template for the expression of a first RNA and the second coding region is a template for the expression of a second RNA. Following expression, the first and second RNAs form a duplex. The retroviral construct preferably also comprises a first Pbl III promoter operably linked to the first RNA coding region and a second Pol HI promoter operably linked to the second RNA coding region.

It is understood that, in certain embodiments, a vector of the disclosure can encompass nucleic acid sequences sufficient to form more than RNA coding region that inhibit expression of distinct target genes. In this embodiment, simultaneous inhibition of distinct target genes can be accomplished with a single vector of the disclosure. The number of different RNA complex transcripts that can be expressed simultaneously is limited only by the packaging capacity of the vector (if a viral vector is used) and adjacent promoters, including any of the promoters described below, can be selected to eliminate or minimize interference and allow for efficient simultaneous inhibition of multiple target genes. The inhibition of multiple RNA construct transcripts of adjacent promoters, for example, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, or 10 or more adjacent promoters allows the user to generate a desire phenotype that develops only when several coding regions (eg,- genes) are targeted simultaneously and enables manipulation and elucidation of complex genetic systems.

G. Small Molecules

In some embodiments, the TPRA40 antagonist is a. small molecule, such as a small organic molecule. A "small molecule" or "small organic molecule" is defined herein to have a molecular weight below about 1000, below about 900, below about 800, below about 700, below about, 600 or below about 500 Daltons. In some embodiments, the TPRA40 antagonist, is a small molecule that, binds to TPRA40 and inhibits hedgehog signaling. In some embodiments, the TPRA40 antagonist is a small molecule that binds to TPRA40 and prevents TPRA40 from interacting with a binding partner. In some embodiments, the TPRA40 antagonist is a small molecule that binds to TPRA40 and inhibits TPR.A40 localization to the plasma membrane or to cilia.

It should be noted that the disclosure also provides TPRA40 agonists. In certain embodiments, the TPRA40 agonist is a small molecule that binds to TPRA40 and promotes hedgehog signaling.

IV. Diagnostic Methods mid Methods of Using TPRA4Q Antagonists

In some embodiments, the present disclosure relates to methods of modulating a differentiation state, survival, and/'or proliferation of a cell. In some embodiments, the cell is in a subject (e.g., a human patient). In some embodiments, the cell is in culture, and the method comprises an in vitro method. In certain embodiments, the cell is a cancer cell. In certain embodiments, the cell is characterized by unwanted or abnormal cell proliferation.

In some embodiments, the disclosure provides for a method of reducing hedgehog signaling in a cell, wherein the cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene (e.g., a component of the hedgehog signaling pathway), wherein the one or more mutations results in increased hedgehog signaling and/'or activation of the hedgehog signaling pathway in the absence of ligand, wherein the method comprises the step of contacting the cell with an effective amount of a TPRA40 antagonist.

In some embodiments, the disclosure provides for a method of inhibiting unwanted growth, proliferation or survival of a cell, wherein the cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, wherein the method comprises the step of contacting the cell with an effective amount of a TPRA40 inhibitor.

In some embodiments, the disclosure provides for a method of inhibiting growth, proliferation or survival of a tumor cell, wherein the cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein the one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, wherein the method comprises the step of contacting the cell with an effective amount of a TPRA40 inhibitor.

In some embodiments, the cell treated with any of the methods disclosed herein comprises one or more mutations in a hedgehog signaling pathway gene. In some embodiments, the one or more mutations are in the TPRA40 gene.

In some embodiments, the one or more mutations are in smoothened, and the cell has a smoothened mutation. In some embodiments, the smoothened mutation is a smoothened gain-of- function mutation. In some embodiments, the gain-of-function smoothened mutation results in a consilium i active smoothened protein. In some embodiments, the smoothened mutation comprises a mutation corresponding to position W535L of SEQ ID NO: 42. In some

embodiments, the smoothened mutation comprises a mutation corresponding to position R562Q of SEQ ID NO: 42. In some embodiments, the smoothened mutation comprises a mutation corresponding to position L412F of SEQ ID NO: 42. In some embodiments, the smoothened mutation has a mutation that renders it resistant to certain smoothened inhibitors. In some embodiments, the smoothened protein comprises an amino acid alteration at amino acid position 518 of SEQ ID NO: 42. In some embodiments, the amino acid alteration is E518 or E518A substitution at the amino acid position corresponding to amino acid position 518 of SEQ ID NO: 42. In some embodiments, the smoothened protein comprises an amino acid alteration at amino acid position 473 of SEQ ID NO: 42. In some embodiments, the amino acid alteration is the substitution of aspartic acid with any of histidine, glycine, phenylalanine, tyrosine, leucine, isoleueine, proline, serine threonine, methionine, glutamine, or asparagine at the amino acid position corresponding to amino acid position 473 of SEQ ID NO: 42, In certain embodiments, the mutation in Smoothened comprises a mutation at any of the specific positions, such as position corresponding to a particular position in SEQ ID NO: 42, as set forth above with respect to the screening assay. See, e.g., WO 201 1/028950 and WO2012047968, each of which is incorporated by reference. In some embodiments, the smoothened mutation is a mutation at a. position corresponding to position 535 of SEQ ID NO: 42. In certain embodiments, the mutation is a mutation at a position corresponding to position 562 of SEQ ID NO: 42. In certain embodiments, the mutation is W535L at position 535 or at that corresponding position in SEQ ID NO: 42. In some embodiments, the smoothened mutation is a mutation corresponding to position R562Q of SEQ ID NO: 42 (a R562Q mutation at position 562 or at a position

corresponding to position 562 of SEQ ID NO: 42. In some embodiments, the smoothened mutation is a mutation at a position corresponding to position 412 of SEQ ID NO: 42, such as a 1,412F at such a position of SEQ ID NO: 42. In some embodiments, the smoothened mutation has a mutation that renders it resistant to certain smoothened inhibitors. In some embodiments, the smoothened protein comprises an amino acid alteration at amino acid position 518 of SEQ ID NO: 42 or at a position corresponding to position 558 of SEQ ID NO: 42. In some embodiments, the amino acid alteration is E518 or E518A substitution at the amino acid position

corresponding to amino acid position 518 of SEQ ID NO: 42. In some embodiments, the smoothened protein comprises an amino acid alteration at amino acid position 473 of SEQ I D NO: 42 or at a position corresponding to position 473 of SEQ ID NO: 42.

In some embodiments, the one or more mutations are in patched!, and the ceil has a patched loss-of-function. In some embodiments, the one or more mutations are in a hedgehog gene and result in overexpression of a hedgehog protein. In some embodiments, the

overexpressed hedgehog protein is Sonic hedgehog protein. In some embodiments, the overexpressed hedgehog protein is Indian hedgehog protein. In some embodiments, the overexpressed hedgehog protein is Desert hedgehog protein.

In some embodiments, the one or more mutations are in suppressor-of-fused, and the cell has suppressor-of-fused (SuFu) loss-of-function. In some embodiments, the results in a loss-of- function in SuFu activity. In some embodiments, the SuFu mutation is in a medulloblastoma, meningioma, adenoid cystic carcinoma, basal cell carcinoma and rhabdomyosarcoma cancer cell. In some embodiments, the SuFu mutation is any of the mutations described in Brugieres et al., 2012, JCO, 30(17):2087-2093, which is incorporated herein in its entirety. In some

embodiments, the SuFu mutation comprises any of the mutations indicated in Tables 1 and 2. In some embodiments, the SuFu mutation comprises a mutation at a position corresponding to any of the following amino acid position in SEQ ID NO: 43: position 15, 184, 123, 295, 187. In certain embodiments, the SuFu mutation comprises any one or more of: P15L, Q184X, R123C, L295fs, or P 187L, where the mutation is at that position or at the position corresponding to the stated position in SEQ ID NO: 43. In some embodiments, the SuFU mutation is any of the mutations corresponding to e. l022+! G>A (TVS8-1G>T), e.72delC, c.72insC, 143insA, e.846insC, or IVS l -1 A->T of SEQ ID NO: 44. In some embodiments, the SuFu mutation is any of the mutations described in Taylor et al (2002) Nat Genet 31 :306-310 (e.g., IVS8- 1G---T (=c. l 022 + 1 G>A), 1 129del, P15L and Ng's two (all +LOH)); Slade et al (201 1) Fam Cancer 10:337-342, 201 1 (e.g., c.1022 +1G>A; c.848insC); Pastorino et al (2009) Am J Med Genet A 149 A: 1539- 1543 (e.g. , c.1022 + 1 G>A.) ; Ng et al (2005) Am J Med Genet A 134:399-403 (e.g. , 143insA; IVS 1 - IA>T); Kijima et al (2012) Fam Cancer 1 1 : 565-70 (e.g., C.550OT (Q184X)); Aavikko et al (2012) Am J Hum Genet 91 : 520-526 (e.g., c.367C>T (R123C)); Stephens et al (2053) J Cli Invest 123: 2965-2968 (e.g., x881_882insG (L295fs)); or Reifenberger et al (2005) Brit J Dermatology 152: 43-51 (e.g., c560C>T (P187L)).

In some embodiments, prior to contacting the cell with the TPRA40 inhibitor, the cell is determined to have one or more mutations in a hedgehog signaling pathway gene.

In some embodiments, the cell treated with any of the methods described herein is a cell in which the hedgehog signaling pathway is active. In some embodiments, the cell is a ceil in which the hedgehog signaling pathway is constitutively active. In some embodiments, the cell is a cell that has been stimulated with hedgehog protein or hedgehog agonist. In some

embodiments, the activity of the hedgehog pathway in a cell is determined by monitoring Giil levels or activity in a Gli-luciferase reporter assay.

In some embodiments, the cell treated with any of the methods described herein is a cell in culture. In some embodiments, the disclosure provides for a method comprises contacting a culture comprising a plurality of cells. In some embodiments, the ceil is in a vertebrate. In some embodiments, the cell is in a mammal, and contacting the cell comprises administering the TPRA40 inhibitor to the mammal. In some embodiments, the mammal is a human subject. In some embodiments, the ceil is a cancer cell and/or the mammal is a mammal diagnosed with cancer. In some embodiments, the cancer cell is a cancer cell selected from the group consisting of: a colon, lung, prostate, skin, blood, liver, kidney, breast, bladder, bone, brain,

meduUobiastoma, sarcoma, basal cell carcinoma, gastric, ovarian, esophageal, pancreatic, or testicular cancer cell.

In some embodiments, the TPRA40 antagonist used in any of the methods disclosed herein is a polynucleotide molecule that inhibits the expression of SsnaI NA14 or TPRA40. In some embodiments, the polynucleotide molecule is an antisense oligonucleotide that hybridizes to a NA14 transcript to inhibit expression of NA.14. In some embodiments, the TPRA40 antagonist is a RNAi antagonist that targets the NA14 or TPRA40 mRNA transcript. In some embodiments, the TPRA40 antagonist is not an RNAi antagonist that does not target NA I4 transcript. In some embodiments, the RNAi antagonist is an siRNA.. In some embodiments, the siRNA. is 19-23 nucleotides in length. In some embodiments, the siRNA is double stranded, and includes short overhang(s) at one or both ends. In some embodiments, the siRNA targets NA 14 mRNA transcript. In some embodiments, the siRNA does not target NA 14 mRNA transcript. In some embodiments, the siRNA targets TPRA40 mRNA transcript. In some embodiments, the siRNA comprises one or more of the nucleotide sequences selected from: SEQ ID NOs: 16-23. In some embodiments, the siRNA comprises one or more of the nucleotide sequences selected from SEQ ID NOs: 17-23. In some embodiments, the RNAi comprises an shRNA.

In some embodiments, the TPRA40 antagonist used in any of the methods disclosed herein is a small molecule that binds to TPRA40.

In some embodiments, the TPRA40 antagonist used in any of the methods disclosed herein is an antibody that binds to TPRA40 protein. In some embodiments, the antibody is a monoclonal antibody.

In some embodiments, the TPRA4Q antagonist used in any of the methods disclosed herein is a polypeptide antagonist. In some embodiments, the polypeptide antagonist is a dominant negative Ssnal/NA14 protein. In some embodiments, the dominant negative NA14 protein is capable of binding TPRA40 but is incapable of binding microtubules. In some embodiments, the dominant negative NA14 protein lacks an N- terminal coiled-coil motif. In some embodiments, the TPRA40 antagonist is not a dominant negative N A14 protein. In some embodiments, the cell contacted with an agent according to any of the methods described herein is also contacted with an additional inhibitor of the hedgehog signaling pathway (e.g., a HPI). In some embodiments, the additional inhibitor of the hedgehog signaling pathway is a veratrum-type steroidal alkaloid. In some embodiments, the veratrum-type steroidal alkaloid is cyclopamine, or KAAD-cyclopamine or any functional derivatives thereof (e.g., IPI-269609 or IPI-926). In some embodiments, the veratrum-type steroidal alkaloid is jervine, or any functional derivatives thereof. In some embodiments, the additional inhibitor is vismodegib, sonidegib, BMS-833923, PF-04449913, or LY2940680, or any functional derivatives thereof. In some embodiments the additional inhibitor is smoothened inhibitor chemically unrelated to veratrum alkaloids or vismodegib, including but not limited to: sonidegib, BMS-833923, PF- 04449913, LY2940680, Erivedge, BMS-833923 (XL319), LDE225 (Erismodegib), PF- 04449913, VP-LDE225, VP-LEQ506, TAK-441 , XL-31 9, LY-2940680, SEN450,

Itraconazole, M T-10, MRT-83, or PF-04449913.). In some embodiments, the additional inhibitor is any of the compo unds disclosed in Amakye, et al., Nature Medicine, 19(1 1 ): 1410- 1422 (which is incorporated herein in its entirety). In some embodiments, the additional inhibitor of the hedgehog signaling pathway is an antibody. In some embodiments, the antibody is an antibody that binds, such as specifically binds, hedgehog proteins. In some embodiments, the additional inhibitor of the hedgehog signaling pathway is an RNAi antagonist.

In some embodiments, the present disclosure provides for methods of diagnosing a disease based on increased expression of TPRA40. In some embodiments, the present disclosure provides for methods of treating a subject (e.g., a human) in need thereof by administering to the subject a TPRA40 antagonist.

In some embodiments, a determination of Glil expression and/or TPRA40

expression/overexpression or of increased TPRA40 activity in a cell is indicative that the cell is a neoplastic cell. In some embodiments, a determination of TPRA40 expression/overexpression or of increased TPRA40 activity in a biological sample obtained from a subject is indicative that the subject comprises a neoplasm. To determine TPRA40 expression in a tumor or cancer, various diagnostic assays are available. In one embodiment, TPRA40 polypeptide overexpression, may be analyzed by immunohistochemistry (IHC). Parraffin embedded tissue sections from a. tumor biopsy may be subjected to an IHC assay and accorded a TPRA40 protein staining intensity criteria. In one embodiment, the staining intensity criteria is set up as follows: Score O - no staining is observed or membrane staining is observed in less than 10% of tumor ceils.

Score 1+ - a faint/barely perceptible membrane staining is detected in more than 10% of the tumor cells. The cells are only stained in part of their membrane.

Score 2+ - a weak to moderate complete membrane staining is observed in more than 10% of the tumor cells.

Score 3+ - a moderate to strong complete membrane staining is observed in more than 10% of the tumor cells.

In this embodiment, those tumors with 0 or 1+ scores for TPRA40 polypeptide expression may be characterized as underexpressing, or not overexpressing TPRA40, whereas those tumors with 2+ or 3+ scores may be characterized as overexpressing TPRA40. In some embodiments, FISH assays such as the INFORM* (sold by Ventana, Arizona) or

PATHVISION™ (Vysis, Illinois) may be carried out on formalin- fixed, paraffin-embedded tumor tissue to determine the extent (if any) of TPRA40 overexpression in the tumor. TPRA40 overexpression or amplification may be evaluated using an in vivo diagnostic assay, e.g., by administering a molecule (such as an antibody, oligopeptide or organic molecule) which binds the molecule to be detected and is tagged with a detectable label (e.g., a radioactive isotope or a fluorescent label) and externally scanning the patient for localization of the label.

"Treating" or "treatment" or "alleviation" refers to improving, alleviating, and/or decreasing the severity of one or more symptoms of a condition being treated. By way of example, treating cancer refers to improving (improving the patient's condition), alleviating, delaying or slowing progression or onset, decreasing the severity of one or more symptoms of cancer. For example, treating cancer includes any one or more of: decreasing tumor size, decreasing rate of tumor size increase, halting increase in size, decreasing the number of metasteses, decreasing pain, increasing survival, and increasing progression free survival.

"Diagnosing" refers to the process of identifying or determining the distinguishing characteristics of a disease or tumor. In the case of cancer, the process of diagnosing is sometimes also expressed as staging or tumor classification based on severity or disease progression.

Subjects in need of treatment or diagnosis include those already with aberrant hedgehog signaling as well as those prone to having or those in whom aberrant hedgehog signaling is to be prevented. For example, a. subject or mammal is successfully "treated" for aberrant hedgehog signaling if, according to the method of the present disclosure, after receiving a therapeutic amount of a TPRA40 antagonist, the patient shows observable and/'or measurable reduction in or absence of one or more of the following: reduction in the number of tumor cells or absence of such cells: reduction in the tumor size; inhibition (i.e., slow to some extent and preferably stop) of tumor ceil infiltration into peripheral organs including the spread of cancer into soft tissue and bone: inhibition (i.e., slow to some extent and preferably stop) of tumor metastasis: inhibition, to some extent, of tumor growth; and/or relief to some extent, of one or more of the symptoms associated with the specific cancer; reduced morbidity and mortality, and improvement in quality of life issues. To the extent such TPRA40 hedgehog antagonists may prevent growth and/'or kill existing cancer cells, it may be cytostatic and/or cytotoxic. Reduction of these signs or symptoms may also be felt by the patient. Additionally, successful exposure to the TPRA40 antagonist, (particularly in cases where no tumor response is measurable) can be monitored by GUI expression either in skin punch biopsies or hair follicles (as done for vismodegib).

The above parameters for assessing successful treatment and improvement in the disease are readily measurable by routine procedures familiar to a physician. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and/or determining the response rate (RR). Metastasis can be determined by staging tests and tests for calcium level and other enzymes to determine the extent of metastasis. CT scans can also be done to look for spread to regions outside of the tumor or cancer. The disclosure described herein relating to the process of prognosing, diagnosing and/or treating involves the

determination and evaluation of TPRA40 and hedgehog amplification and expression.

"Mammal" for purposes of the treatment of, alleviating the symptoms of or diagnosis of a disease (e.g., cancer) refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, ferrets, etc. in some embodiments, the mammal is human. In some embodiments, the mammal is post-natal. In some embodiments, the mammal is pediatric. In some

embodiments, the mammal is adult.

Administration "in combination with" one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order.

In some embodiments, the TPRA40 antagonists described herein are for use in treating cancer in a subject. In some embodiments, depending on the stage of the cancer, cancer treatment involves one or a combination of the following therapies: surgery to remove the cancerous tissue, radiation therapy, and chemotherapy. In some embodiments, therapy comprising of administering TPRA40 antagonists may be especially desirable in elderly patients who do not tolerate the toxicity and side effects of chemotherapy well and in metastatic disease where radiation therapy has limited usefulness. In some embodiments, the TPRA40 antagonists of the present inventive method may also be used to alleviate hedgehog and/or TPRA40 overexpressing cancers upon initial diagnosis of the disease or during relapse. For therapeutic applications, such TPRA40 antagonists can, in some embodiments, be used in combination with, before or after application of other conventional agents and/or methods for the treatment of a tumor, e.g., hormones, antiangiogens, or radiolabeled compounds, or with surgery, cryotherapy, radiotherapy and/or chemotherapy. In some embodiments, the TPRA40 antagonist is administered to a subject in combination with a chemotherapeutic agent and/or a growth inhibitory agent and/or a HPL

Exemplary HPIs are described herein and include, for example, smoothened inhibitors and hedgehog inhibitors. Exemplary chemotherapeutic agents are known in the art and include, for example, hydroxyureataxanes (such as paclitaxel and doxetaxel) and/or anthracycline antibiotics; alkylating agents such as diiotepa and CYTOXAN* cyclophosphamide; alky] sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylarnelamines including altretamine, triethyienemelamine, trietylenephosphoramide, triethiylenethiophosphoramide and trimethylolomelamine; acetogeains (especially bullatacin and bullatacinone); delta-9- tetrahydrocannabinoi (dronabinol, MARINOL*'); beta-iapachone; lapachol; colchicines; betulinie acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN*), CP 1 -11 (irinotecan, CAMPTOSAR*), acetyicamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; cailystatin; CC-1065 (including its adozeiesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, W-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, cholophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, iiovembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosureas such as earmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially caiicheamicin gammali and calicheamicin omegall (see, e.g., Ag[eta]ew, Chem lntl, Ed. Engl., 33: 183-186 (1994)): dynemicin, including dynemicin A: an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinoniyein, carabiciii, ca[pi]ninomycin, carzinophilin, chromomyciiiis, dactinomycin, daunorubicin, detombicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN*' doxorubicin (including rnorpholino-doxorubiein, cyanomorpholino-doxorubicin, 2-pyn lino-doxorubiciii and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marceilomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifiuridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testo!actone; anti- adrenals such as aminoglutethimide, mitotane, tri!ostane; folic acid replenisher such as frolmic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;

diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate;

hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins;

mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;

losoxantrone; 2-ethylhydrazide; procarbazine; PSK(R) polysaccharide complex (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid;

triaziquone; 2,2,,2"-trichiorotriethyiamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDIS1NE*', FELDESIN^); dacarbazine;

mannomustine; mitobronitol; mitolactol; pipobroman; gacv osine; arabinoside ("Ara-C");

thiotepa; taxoids, e.g., TAXOL^ paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NX), ABRAXANE1Jvl Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, Illinois), and TAXOTERE® doxetaxel (Rli[delta]iie-Poulenc Rorer, Antony, France); chloranbucii; gemcitabine (GEMZAR^); 6- thioguanine; niercaptopurine; methotrexate; platinum analogs such as cisplatin and carboplatin; vinblastine (VELBAN®); platinum; etoposide (VP- 16); ifosfamide; mitoxantrone; vincristine (ONCOVIN*'); oxaliplatin; leucovovin; vinorelbine (NAVELBINE^); novantrone; edatrexate; daunomyein; aminopterin; ibandronate; topoisomera.se inhibitor RFS 2000;

difluorometlhylornithine (DMFO); retinoids such as retinoic acid; eapecitabine (XELODA^); pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone, and FOLFOX, an abbreviation for a. treatment regimen with oxaliplatin (ELOXATINllvI) combined with 5-FU and leucovovin.

Also included in the definition of chemo therapeutic agent are anti-hormonal agents that act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer, and are often in the form of systemic, or whole-body treatment. They may be hormones themselves. Examples include anti-estrogens and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (including NOLVADEX* tamoxifen), EVI8TA® raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LYl 17018, onapristone, and FARESTON®toremifene; anti-progesterones; estrogen receptor down -regulators (ERDs); agents that function to suppress or shut down the ovaries, for example, leutinizing hormone-releasing hormone (LHRH) agonists such as LUPRON* and ELIGARD* leuprolide acetate, goserelin acetate, buserelin acetate and tripterelin; other anti-androgens such as flutamide, mlutamide and bicahrtamide; and aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as, for example, 4(5)-imidazoles,

aminoglutethimide, MEGASE® megestrol acetate, AROMASIN® exemestane, formestanie, fadrozole, RFVISOR® vorozole, FEMARA® ietrozole, and ARIMDDEX® anastrozole. In addition, such definition of chemotherapeutic agents includes bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), DIDROCAL® etidronate, NE-58095, ZOMETA*' zoledronic acid/zoledronate, FOSAMAX® alendronate, AREOLA® pamidronate, SKELiD® tiludronate, or ACTONEL® risedronate; as well as troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in abherant cell proliferation, such as, for example, P C-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN* vaccine, LEUVECTTN® vaccine, and VAXID® vaccine; LURTOTECAN'^ topoisomerase 1 inhibitor; ABARELDC* rmRH; lapatinib ditosylate (an ErbB-2 and EGFR dual tyrosi e kinase small- molecule inhibitor also known as GW572016); and pharmaceutically acceptable salts, acids or derivatives of any of the above.

A "growth inhibitory agent" when used herein refers to a compound or composition which inhibits growth of a cell, especially a hedgehog responsive, a hedgehog expressing (and in some cases overexpressing) or TPRA40-expressing (and in some cases overexpressing) cell, either in vitro or in vivo. Thus, the growth inhibitory agent may be one which significantly reduces the percentage of hedgehog-expressing cells in S phase. Examples of growth inhibitory agents include agents that block cell cycle progression (at a place other than S phase), such as agents that induce G l arrest and M-phase arrest. Classical M-phase blockers include the vincas (vincristine and vinblastine), taxanes, and topoisomerase H inhibitors such as doxorubicin, epirubicin, daunorubicin, etoposide, and bleomycin. Those agents that arrest Gl also spill over into S-phase arrest, for example, DNA alkylating agents such as tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin, methotrexate, 5-fluorouracil, and ara-C. Further information can be found in The Molecular Basis of Cancer, Mendelsohn and Israel, eds., Chapter 1, entitled "Cell cycle regulation, oncogenes, and antineoplastic drugs" by Murakami et al. (WB Saunders: Philadelphia, 1995). The taxanes or hydroxyureataxanes (paclitaxel and docetaxel) are anticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE*,

Rhone-Poulenc Rorer), derived from the European yew, is a semisynthetic analogue of paclitaxel (lAXO L*, Bristol-Myers Squibb). These molecules promote the assembly of microtubules from tubulin dimers and stabilize microtubules by preventing depolymerization, which results in the inhibition of mitosis in ceils.

Enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytoiaca americana proteins (PAP1, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. A variety of radionuclides are available for the production of radioconjugated antibodies. Examples include " Bi, , ,1, 13 'in, 90Y, and ,86 e. Conjugates of the antibody and cytotoxic agent are made using a variety of Afunctional protein-coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of iniidoesters (such as dimethyl adipimidate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), bis-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), bis- diazonium derivatives (such as bis-(p-diazoiiiumbenzoyl)- ethylenediamine), diisocyapates (such as tolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as l,5-difluoiO-2,4- dinitrobenzene). For example, a ricin imniunotoxin can be prepared as described in Vitetta et a.L, Science, 238: 1098 (1987), Carbon- 14-labeled -isothioeyanatobenzyl-S-methy dietiiy ene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugation of radionucleotide to the antibody. See W094/11026. Conjugates of an antibody and one or more small molecule toxins, such as a calicheamicm, maytansinoids, a trichothene, and CC 1065, and the derivatives of these toxins that have toxin activity, are also contemplated herein.

Chemotherapeutic drugs such as TAXGTERE* (docetaxel), TAXGL* (palictaxel), estramustine and mitoxantrone are used in treating cancer, in particular, in good risk patients. In particular, combination therapy with palictaxel and modified derivatives (see, e.g., EP0600517) is contemplated. In some embodiments, the TPRA40 antagonist is administered with a therapeutically effective dose of any of the chemotherapeutic agents disclosed herein (see definition above). In another embodiment, the TPRA40 antagonist is administered in

conjunction with chemotherapy to enhance the activity and efficacy of the chemotherapeutic agent, e.g., paclitaxel. The Physicians' Desk Reference (PDR) discloses dosages of these agents that have been used in treatment- of various cancers. The dosing regimen and dosages of these aforementioned chemotherapeutic drugs that are therapeutically effective will depend on the particular cancer being treated, the extent of the disease and other factors familiar to the physician of skill in the art and can be determined by the physician.

In some embodiments, the TPRA40 antagonists described herein are for used in treating an adenocarcinoma. "Adenocarcinoma" refers to a malignant tumor originating in the glandular epithelium.

In some embodiments, the TPRA40 antagonists described herein are for used in treating a carcinoma.. "Carcinoma" refers to a. malignant growth derived from epithelial cells that tends to metastasize to other areas of the body. Examples include "basal cell carcinoma" - an epithelial tumor of the skin that, while seldom metastasizing, can result in local invasion and destruction; "squamous cell carcinoma" - tumors arising from squamous epithelium and having cuboid ceils; "carcinosarcoma" - malignant tumors comprising both carcinomatous and sarcomatous tissues; "adenocystic carcinoma"- tumors characterized by large epithelial masses containing round gland-like spaces or cysts, frequently containing mucus, that are bordered by layers of epithelial ceils; - "epidermoid carcinoma"- see squamous ceil carcinoma; "nasopharyngeal carcinoma" - malignant tumor arising in the epithelial lining of the space behind the nose; "renal cell carcinoma"- tumor in the renal parenchyma composed of tubular cells in varying arrangements. Additional carcinomatous epithelial growth include "papillomas", which are benign tumors derived from the epithelium and having papillomavirus as a causative agent; and

"epidermoidomas" , which are cerebral of meningeal tumors formed by inclusion of ectodermal elements at the time of closure of the neural groove.

In some embodiments, any of the TPRA40 antagonists disclosed herein are administered to a human patient, in accord with known methods, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intracranial, intracerobrospinal, intraarticular, intrathecal, intravenous, intraarterial, subcutaneous, oral, topical, or inhalation routes. Other therapeutic regimens may be combined with the administration of the foregoing TPRA40 antagonists. In some embodiments, the combined administration includes coadministration, using separate formulations or a single pharmaceutical formulation, and consecutive

administration in either order, wherein preferably there is a time period while both (or all) active agents simultaneously exert their biological activities, in some embodiments, combined therapy results in a synergistic therapeutic effect. In some embodiments, the TPRA40 antagonist is coadministered (simultaneously or consecutively) with a second hedgehog pathway inhibitor, such as a smoothenecl inhibitor or a hedgehog inhibitor (e.g., robotkinin). In some embodiments, the TPRA40 antagonist is coadministered with a steroidal alkaloid. In some embodiments, the steroidal alkaloid is cyclopamine, or KAAD-cyclopamine or jervine or any functional derivative thereof (e.g., IPI-269609 or IPI-926). In some embodiments, the TPRA40 antagonist is coadministered with vismodegib, so idegib, BMS-833923, PF-04449913, or LY2940680 or any derivative thereof. In some embodiments, the TPRA40 antagonist is coadministered with any of the compounds disclosed in Amakye, et al.. Nature Medicine, 19(1 1): 1410-1422 (wliichi is incorporated herein in its entirety). In some embodiments the antagonist is coadministered with another smooth ened inhibitor chemically unrelated to veratrum alkaloids or vismodegib, including but not limited to: sonidegib, BMS-833923, PF-04449913, LY2940680, BMS-833923 (XL319), LDE225 (Erismodegib), PF-04449913, VP-LDE225, VP-LEQ506, TAK-441 , XL- 319, LY-2940680, SEN450, Itraconazole, MRT-10, MRT-83, or PF-04449913).

In some embodiments, the therapeutic treatment methods of the present disclosure involves the combined administration of the preceding TPRA40 antagonist and one or more of the chemotherapeutic agents or growth inhibitory agents described herein, including coadministration of cocktails of different chemotherapeutic agents. Example chemotherapeutic agents are disclosed herein. Preparation and dosing schedules for such chemotherapeutic agents may be used according to manufacturers' instructions or as determined empirically by the skilled practitioner. Preparation and dosing schedules for such chemotherapy are also described in Chemotherapy Service Ed,, M.C. Perry, Williams & Wilkms, Baltimore, MD (1992), For the prevention or treatment of disease, the dosage and mode of administration will be chosen by the physician according to known criteria. The appropriate dosage of TPRA40 antagonists will depend on the type of disease to be treated, the severity and course of the disease, whether administration is for preventive or therapeutic purposes, previous therapy (including) the patient's clinical history and response, and the discretion of the attending physician. The preceding TPRA40 antagonists may be suitably administered to the patient at one time or over a series of treatments. Administration may occur by intravenous infusion or by subcutaneous injections or orally in the case of certain small molecule inhibitors. Suitable dose will depend on the agent and the particular use and is determined. For repeated administrations over several days or longer, depending on the condition, the treatment is sustained until a desired suppression of disease symptoms occurs. The progress of this therapy can be readily monitored by conventional methods and assays and based on criteria known to the physician or other persons of skill in the art. For any of the methods of the disclosure, it is contemplated that a TPRA40 antagonist can be administered as a monotherapy or in combination with one or more other therapeutic agents.

Aside from administration of TPRA40 polypeptide antagonists to a patient, the present disclosure contemplates administration of the TPRA40 antagonist by gene therapy. Such administration of a nucleic acid encoding the TPRA40 hedgehog polypeptide antagonists is encompassed by the expression "administering a therapeutically effective amount of a TPRA40 antagonist". See, for example, WQ96/07321 published March 14, 1996 concerning the use of gene therapy to generate intracellular antibodies.

There are two major approaches to getting such nucleic acid (optionally contained in a vector) into the patient's cells; in vivo and ex vivo. In some embodiments, in vivo delivery of the nucleic acid involves injection directly into the patient, usually at the site where the antibody is required. In some embodiments, for ex vivo treatment, the patient's cells are removed, the nucleic acid is introduced into these isolated cells and the modified ceils are administered to the patient either directly or, for example, encapsulated within porous membranes which are implanted into the patient (see, e.g., U.S. Patent Nos. 4,892,538 and 5,283, 187). There are a variety of techniques available for introducing nucleic acids into viable cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured cells in vitro, or in vivo in the cells of the intended host. Techniques suitable for the transfer of nucleic acid into mammalian cells in vitro include the use of liposomes, electroporation, microinjection, cell fusion, DEAE-dextran, the calcium, phosphate precipitation method, etc. A commonly used vector for ex vivo delivery of the gene is a retroviral vector. The currently preferred in vivo nucleic acid transfer techniques include transfection with viral vectors (such as adenovirus. Herpes simplex I virus, or adeno- associated virus) and lipid-based systems (useful lipids for lipid-mediated transfer of the gene are DOTMA, DOPE and DC-Choi, for example). For review of the currently known gene marking and gene therapy protocols see Anderson et al., Science 256:808- 813 ( 1992). See also WO 93/25673 and the references cited therein.

As hedgehog is known to stimulate angiogenesis, it follows based on the teachings herein that TPRA40, which antagonizes hedgehog signaling activity, would antagonize hedgehog mediated processes, (e.g., angiogenesis) particularly when some level of hedgehog activity is necessary for angiogenesis. in some embodiments, the disclosure provides for a method of modulating angiogenesis in a subject by administering to the subject a therapeutically effective amount of a TPRA40 antagonist. In some embodiments, the disclosure provides a method of treating any of the angiogenesis-related conditions, disorders and/or diseases disclosed herein.

In some embodiments, any of the TPRA40 antagonists described herein inhibits angiogenesis. Angiogenesis is fundamental to many disorders. Persistent, unregulated angiogenesis occurs in a range of disease states, tumor metastases and abnormal growths by endothelial ceils. The vasculature created as a result of angiogenic processes supports the pathological damage seen in these diseases.

In some embodiments, any of the TPRA40 antagonists described herein are used for treating a disease associated with or resulting from angiogenesis by inhibiting angiogenesis. Diseases associated with or resulting from angiogenesis include: ocular neovasscular disease, age-related macular degeneration, diabetic retinopathy, retinopathy of prematurity, corneal graft rejection, neovascuiar glaucoma, retroiental fibroplasias, epidemic keratoconjuctivitis, Vitamin A deficiency, contact lens overwear, atopic keratitis, superior limbic keratitis, pterygium keratitis sicca, Sjogren's syndrome, acne rosacea, phylectenulosis, syphilis, Mycobacteria infections, lipid degeneration, chemical burns, bacterial ulcers, fungal ulcers, Herpes simplex infections, Herpes zoster infections, protozoan infections, Kaposi's sarcoma, Mooren's ulcer, Terrien's marginal degeneration, marginal keratolysis, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener's granulomatosis, sacroidosis, scleritis, Stevens-Johnson syndrome, pemphigoid radial keratotomy, corneal graph rejection, rheumatoid arthritis, systemic lupus, polyarteritis, trauma, Wegener's granulomatosis, sarcoidosis, scleritis, Stevens-Johnson syndrome, pemphigoid radial keratotomy, corneal graph rejection, rheumatoid arthritis, osteoarthritis chronic inflammation (e.g., ulcerative colitis or Crohn's disease), hemangioma. Osier- Weber Rendu disease, and hereditary hemorrhagic telangiectasis.

In some embodiments, any of the TPRA.40 antagonists described herein may be used for treating cancer, such as by inhibiting angiogenesis, as angiogenesis is known to play a critical role in cancer. A tumor cannot expand without a blood supply to provide nutrients and remove cellular wastes. Tumors in which angiogenesis is important include solid tumors such as rhabdomyosarcomas, retinoblastoma, Ewing sarcoma, neuroblastoma, osteosarcoma, and benign tumors such as acoustic neuroma, neurofibroma, trachoma and pyogenic granulomas.

Angiogenic factors have been found associated with several solid tumors, and preventing angiogenesis could halt the growth of these tumors and the resultant damage to the animal due to the presence of the tumor. Angiogenesis is also associated with blood-born tumors such as leukemias, any of various acute or chronic neoplastic diseases of the bone marrow in which unrestrained proliferation of white blood cells occurs, usually accompanied by anemia., impaired blood clotting, and enlargement of the lymph nodes, liver, and spleen. It is believed that angiogenesis plays a role in the abnormalities in the bone marrow that give rise to leukemia-like tumors.

In some embodiments, any of the TPRA40 antagonists described herein may be used for inhibiting metastasis, such as by inhibiting angiogenesis. Initially, angiogenesis is important in the vascularization of the tumor which allows cancerous cells to enter the blood stream and to circulate throughout the body. After the tumor cells have left the primary site, and have settled into the secondary, metastatic site, angiogenesis must occur before the new tumor can grow and expand. Therefore, prevention of angiogenesis could lead to the prevention of metastasis of tumors and possibly contain the neoplastic growth at the primary site. In other embodiments, the TPRA40 antagonists are useful for inhibiting metastasis, regardless of mechanism of action (e.g., regardless of whether that inhibition is due to inhibition of angiogenesis).

Angiogenesis is also involved in normal physiological processes such as reproduction and wound healing. Angiogenesis is an important step in ovulation and also in implantation of the blastula after fertilization. In some embodiments, prevention of angiogenesis using any of the TPRA. antagonists described herein could be used to induce amenorrhea, to block ovulation or to prevent implantation by the blastula.

In certain embodiments, a TPRA40 antagonist is used in the treatment of a cancer selected from any of the cancers described herein or a cancer in which one or more cells of a tumor comprises a mutation in a hedgehog pathway component, such as any of the mutations described herein. It should be generally appreciated and is specifically noted herein that tumors comprise cells that may have a level of heterogeneity. Accordingly, not all cells in a tumor necessarily comprise a particular deleterious mutation. Accordingly, the disclosure contemplates methods in which a cancer or tumor being treated comprises cells having a mutation in a component of the hedgehog pathway, such as any of the mutations described herein, even if such a mutation is not present in every cell of the tumor.

It is further contemplated that use of TPRA40 antagonists may be specifically targeted to disorders where the affected tissue and/or cells exhibit high hedgehog pathway activation.

Expression of Gli genes activated by the hedgehog signaling pathway, including Glil and Gli2, most consistently correlate with hedgehog signaling across a wide range or tissues and disorders, while Gli3 is somewhat less so. The Gli genes encode transcription factors that activate expression of many genes needed to elicit the full effects of hedgehog signaling. However, the G1I3 transcription factors can also act as a repressor of hedgehog effector genes, and therefore, expression of Gii3 can cause a decreased effect of the hedgehog signaling pathway. Whether Gli3 acts as a transcriptional activator or repressor depends on post-transiational events, and therefore it is expected that methods for detecting the activating form (versus the repressing form) of Gli3 protein (such as western blotting) would also be a reliable measure of hedgehog pathway activation. The Glil gene is strongly expressed in a wide array of cancers, hyperplasias and immature lungs, and serves as a. marker for the relative activation of the hedgehog pathway. In addition, tissues such as immature lung, that have high Gli gene expression, are strongly affected by hedgehog inhibitors. Accordingly, it is contemplated that the detection of Gli gene expression may be used as a powerful predictive tool to identity tissues and disorders that will particularly benefit from treatment with a hedgehog antagonist. In some embodiments, GUI expression levels are detected, either by direct detection of the transcript or by detection of protein levels or activity. Transcripts may be detected using any of a wide range of techniques that depend primarily on hybridization or probes to the Glil transcripts or to cDNAs synthesized therefrom. Well known techniques include Northern blotting, reverse-transcriptase PGR and microarray analysis of transcript levels. Methods for detecting Gli protein levels include Western blotting, immunoprecipitation, two-dimensional polyacry] amide gel electrophoresis (2D SDS- PAGE - preferably compared against a standard wherein the position of the Gli proteins has been determined), and mass spectroscopy. Mass spectroscopy may be coupled with a series of purification steps to allow high-throughput identification of many different protein levels in a particular sample. Mass spectroscopy and 2D SDS-PAGE can also be used to identify post- transcriptionai modifications to proteins including proteolytic events, ubiquitination,

phosphorylation, lipid modification, etc. Gli activity may also be assessed by analyzing binding to substrate DNA or in vitro transcriptional activation of target promoters. Gel shift assay, DNA footprinting assays and DNA-protein crosslinking assays are all methods that may be used to assess the presence of a protein capable of binding to GIJ binding sites on DNA. J Mol. Med 77(6):459-68 (1999); Cell 100(4): 423-34 (2000); Development 127(19): 4923-4301 (2000).

Because Gli 1 is so ubiquitously expressed during hedgehog activation, any degree of Gli I overexpression should be useful in determining that a TPRA40 antagonist will be an effective therapeutic. In some embodiments, Glil should be expressed at a level at least twice as high as in a normal control cell/tissue/subject. In some embodiments, Glil expression is four, six, eight or ten times as high as in a normal cell/tissue/subject.

In certain embodiments, Glil transcript levels are measured, and diseased or disordered tissues showing abnormally high Glil levels are treated with a TPRA40 antagonist. In other embodiments, the condition being treated is known to have a significant correlation with aberrant activation of the hedgehog pathway, even though a measurement of Glil expression levels is not made in the tissue being treated. Premature lung tissue, lung cancers (e.g., adeno carcinomas, bronco-alveolar adenocarcinoma, small cell carcinomas), breast cancers (e.g., inferior ductal carcinomas, inferior lobular carcinomas, tubular carcinomas), prostate cancers (e.g.,

adenocarcinomas), and benign prostatic hyperplasias all show strongly elevated Glil expression levels in certain cases. Accordingly, Glil expression levels are a powerful diagnostic device to determine which of these tissues should be treated with a TPRA40 antagonist. In addition, there is substantial correlative evidence that cancers of the urothelial cells (e.g., bladder cancer, other urogenital cancers) will also have elevated gli-1 levels in certain cases. For example, it, is known that loss of heterozygosity on chromosome 9q22 is common in bladder cancers. The Ptchl gene is located at, this position and Ptchl loss of function is probably a partial cause of

hyperproliferation, as in many other cancer types. Accordingly, such cancers would also show high Glil expression and would be particularly amenable to treatment with a hedgehog antagonist.

In certain embodime ts, any of the TPRA40 antagonists described herein are used for treating a subject having a tumor having a ptch-1 and/or ptch-2 mutation, e.g., a patch- 5 or patch- 2 loss of function mutation. Expression of ptch-1 and ptch-2 is also activated by the hedgehog signaling pathway, but not typically to the same extent as gli genes, and as a result are inferior to the gli genes as markers of hedgehog pathway activation. In certain tissues, only one of ptch-1 or ptch-2 is expressed although the hedgehog pathway is highly active. For example, in testicular development, desert hedgehog plays an important role and the hedgehog pathway is activated, but only ptc-2 is expressed. Accordingly, these genes may be individually unreliable as markers for hedgehog pathway activation, although simultaneous measurement of both genes is contemplated as a more useful indicator for tissues to be treated with a hedgehog antagonist.

In certain embodiments, any of the TPRA40 antagonists described herein may be used for treating a cell, tumor or subject having a smoothened mutation. In some embodiments, the smoothened mutation results in a constitutively active smoothened protein, in some embodiments, the smoothened mutation is a mutation corresponding to position W535L of SEQ ID NO: 42. In some embodiments, the smoothened mutation is a mutation corresponding to position R562Q of SEQ ID NO: 42. In some embodiments, the smoothened mutation has a mutation that renders it resistant to certain smoothened inhibitors. In some embodiments, the smoothened protein comprises an amino acid alteration at amino acid position 518 of SEQ ID NO: 42. In some embodiments, the amino acid alteration is E5 I 8 or E518A substitution at the amino acid position corresponding to amino acid position 518 of SEQ ID NO: 42. In some embodiments, the smoothened protein comprises an amino acid alteration at amino acid position 473 of SEQ ID NO: 42. In some embodiments, the amino acid alteration is the substitution of aspartic acid with any of histidine, glycine, phenylalanine, tyrosine, leucine, isoleueine, proline, serine threonine, methionine, glutamine, or asparagine at the amino acid position corresponding to amino acid position 473 of SEQ ID NO: 42. See, e.g. , WO 2011/028950 and WO2012047968, each of which is incorporated by reference.

In certain embodiments, any of the TPRA40 antagonists described herein may be used for treating a cell, tumor or subject, having a SuFu mutation . In some embodiments, the SuFu mutation results in a loss-of-function in SuFu activity. In some embodiments, the SuFu mutation is in a medulloblastoma, meningioma, adenoid cystic carcinoma, basal cell carcinoma or rhabdomyosarcoma cancer cell. In some embodiments, the SuFu mutation is any of the mutations described in Brugieres et al, 2012, JCO, 30(17):2087-2093, which is incorporated herein in its entirety. In some embodiments, the SuFu mutation is any of the mutations indicated in Tables 1 and 2. In some embodiments, the SuFu mutation is any of the mutations

corresponding to P15L, Q184X, R123C, L295fs, or P187L of SEQ ID NO: 43. In some embodiments, the SuFU mutation is any of the mutations corresponding to c.1022+1 G>A (IVS8-10T), c.72delC, c.72insC, 143insA, c.846insC, or IVS1-1A->T of SEQ ID NO: 44. In some embodiments, the SuFu mutation is any of the mutations described in Taylor et al (2002) Nat Genet 31 :306-310 (e.g., IVS8-1G>T ( c. ! 022 +IG>A), 1 129del, P15L and g's two (all +LOH)); Slade et al (201 1) Fam Cancer 10:337-342, 201 1 (e.g., c.1022 +1G>A; c.848insC); Pastorino et al (2009) Am J Med Genet A I49A: 1539-1543 (e.g., c.1022 +1 G>A); Ng et al (2005) Am J Med Genet A 134:399-403 (e.g., 143insA; IVS I-IA>T); Kijima et al (2012) Fam Cancer 1 1 : 565-70 (e.g., C.550OT (QI 84X)); Aavikko et al (2012) Am J Hum Genet 91 : 520-526 (e.g., C.3670T (R123C)); Stephens et al (2013) J Clin Invest 123: 2965-2968 (e.g., x881__882insG (L295fs)); or Reifenberger et al (2005) Brit j Dermatology 152: 43-51 (e.g., c560C>T (P187L)).

In light of the broad involvement of hedgehog signaling in the formation of ordered spatial arrangements of differentiated tissues in vertebrates, the TPRA40 antagonists of the present disclosure could be used in a process for generating and/or maintaining an array of different vertebrate tissue both in vitro and in vivo. The TPRA40 antagonist, can be, as appropriate, any of the preparations described above.

In some embodiments, the TPRA40 antagonists of the present disclosure are further applicable to cell culture techniques wherein reduction in hedgehog signaling is desirable. Use of the present method may he in culture of, for example, neuronal stem cells, such as in the use of such cultures for the generation of new neurons and glia. In some embodiments, these cultures can be contacted with TPRA40 antagonists in order to alter the rate of proliferation or neuronal stem cells in the culture and/or alter the rate of differentiation, or to maintain the integrity of a culture of certain terminally differentiated neuronal cells. In one embodiment, the TPRA40 antagonists can be used in culture of certain neuron types (e.g., sensory neurons, motor neurons). Such neuronal cultures can be used as convenient assay systems as well as sources of implantable cells for therapeutic treatments.

Stern cells useful for use in any of the methods of the present disclosure are generally known. For example, several neural crest cells have been identified, some of which are multipotent and likely represent uncommitted neural crest cells, and others of which can generate only one type of cell, such as sensor}' neurons, and likely represent committed progenitor cells. The role of TPRA40 antagonists employed in the present method to culture such stem cells can be to regulate differentiation of the uncommitted progenitor, or to regulate further restriction of the developmental fate of a committed progenitor, or to regulate further restriction of the developmental fate of a committed progenitor cell towards becoming a terminally differentiated neuronal cell. For example, the present method can be used in vitro to regulate the

differentiation of neural crest cells into glial ceils, Schwann ceils, chromaffin cells, cholinergic, sympathetic or parasympathetic neurons, as well as peptinergic and serotonergic neurons. The TPRA40 antagonist can be used alone, or in combination with other neurotrophic factors that act to more particularly enhance a particular differentiation fate of the neuronal progenitor cell. In addition to use of the TPRA40 antagonists in combination with implantation of cell cultures, another aspect of the present disclosure relates to the therapeutic application of TPRA40 antagonists to regulate the growth state of neurons and other neuronal ceils in both the central nervous system and the peripheral nervous system.

In some embodiments, the TPRA40 antagonists can be used in the treatment of neoplastic or hyperplastic transformation such as may occur in the central nervous system. For instance, the TPRA40 antagonists can be utilized to cause such transformed cells to become either postmitotic or apoptotic. The TPRA40 antagonists may, therefore, be used as part of a treatment for, e.g., malignant gliomas, meningiomas, medulloblastomas, neuroectodermal tumors, and ependymomas,

In some embodiments, the TPRA40 antagonists can be used as part of a treatment, regimen for malignant meduUoblastoma and other primary CNS malignant neuroectodermal tumors, MeduUoblastoma, a primary brain tumor, is the most common brain tumor in children. A. meduUoblastoma is a primitive neuroectodermal (PNET) tumor arising in the posterior fossa. They account for approximately 25% of all pediatric brain tumors. Histologically, they are small round cell tumors commonly arranged in a true rosette, but may display some differentiation to astrocytes, ependymal cells or neurons. PNETs may arise in other areas of the brain including the pineal gland (pineobiastoma) and cerebrum. Those arising in the supratentorial region general ly have a worsened prognosis.

Medulloblastom/PNETs are known to recur anywhere in the CNS after resection, and can even metastasize to bone. Pretreatment evaluation should therefore include and examination of the spinal cord to exclude the possibility of "dropped metastases". Gadolinium-enhanced MR] has largely replaced myelography for this purpose, and CSF cytology is obtained postoperatively as a routine procedure.

In some embodiments, the TPRA40 antagonists are used as part of a treatment program for ependymomas. Ependymomoas account for approximately 10% of the pediatric brain tumors in children. Grossly, they are tumors that arise from the ependymal lining of the ventricles and microscopically form rosettes, canals, and perivascular rosettes. In the CHOP series of 51 children reported with epenymomas, ¾ were histologically benign, approximately 2/3 arose from the region of the 4th ventricule, and one third presented in the supratentorial region. Age at presentation peaks between birth and 4 years. The median age is about 5 years. Because so many children with this disease are babies, they often require multimodal therapy. In some embodiments, the TPRA40 antagonists can be used in cell culture and therapeutic method relating to the generation and maintenance of non-neuronai tissue. Such uses are contemplated as a result of the involvement of hedgehog signaling components (e.g., ptc, hedgehog, smo, fused, SuFu, Cos-2, etc.) in niorphogenic signals of other vertebrate organogenic pathways, such as endodermal patterning, and mesodermal and endodermal differentiation.

As hedgehog signaling, especially ptc, hedgehog, and smoothened, are involved in controlling the development of stem cells responsible for formation of the digestive tract, liver, lungs, and other organs derived from the primitive gut. Shh is the inductive signal from the endoderrn to the mesoderm, which is critical to gut morphogenesis. Therefore, in some embodiments, the TPRA40 antagonists can be employed for regulating the development and maintenance of an artificial liver that can have multiple metabolic functions of a nonnal liver. In one embodiment, the TPRA40 antagonists can be used to regulate functions of a normal liver. In one embodiment, the TPRA40 antagonists can be used to regulate the proliferation and differentiation of digestive tube stem cells to form hepatocyte cultures which can be used to populate extracellular matrices, or which can be encapsulated in biocompatible polymers, to form both implantable and extracorporeal artificial livers.

In some embodiments, the TPRA40 antagonists can be employed therapeutically to regulate such organs after physical, chemical or pathological insult. For instance, therapeutics comprising any of the TPRA40 antagonists described herein can be used in liver repair subsequent to a partial hepactectomy.

In some embodiments, the TPRA40 antagonists of the present disclosure can be used in the treatment of hyperplastic and neoplastic disorders affecting pancreatic tissue, especially those characterized by aberrant proliferation of pancreatic cells. For instance, pancreatic cancers are marked by abnormal proliferation of pancreatic cells, which can result in alterations of insulin secretory capacity of the pancreas. For example, certain pancreatic hyperplasias, such as pancreatic carcinomas, can result in hypoinsulinemia due to dysfunction of β-cells or decreased islet cell mass. Moreover, manipulation of hedgehog signaling properties at different points may be useful as part of a strategy for reshaping/repairing pancreatic tissue both in vivo and in vitro. In one embodiment, the present disclosure makes use of the apparent involvement of ptc, hedgehog and smoothened in regulating the development of pancreatic tissue. In general, the TPRA40 antagonists can be employed therapeutically to regulate the pancreas after physical, chemical or pathological insult. In some embodiments, the TPRA40 antagonists can be applied to cell culture techniques, and in particular, may be employed to enhance the initial generation of prosthetic pancreatic tissue devices. Manipulation of proliferation and differentiation of pancreatic tissue, such as through using TPRA40 antagonists, can provide a means for more carefully controlling the characteristics of a cultured tissue. In an exemplary embodiment, the TPRA40 antagonists can be used to augment production of prosthetic devise which require β- islet cells, such as may be used in the encapsulation devices described in, for example, as described in U.8.P. 4,892,538, 5,106,627, 4,391 ,909 and 4,353,888. Early progenitor cells to the pancreatic islets are multipotential, and apparently eoactivate ail the islet-specific genes from the time they first appear. As development proceeds, expression of islet-specific hormones, such as insulin, becomes restricted to the pattern of expression characteristic of mature islet cells. The phenotype of mature islet, cells, however, is not, stable in culture, as reappearance of embryonal traits in mature β -cells can be observed. By utilizing any of the TPRA40 antagonists described herein, the differentiation path or proliferative index of the cells can be regulated.

In some embodiments, the TPRA40 antagonists of the present disclosure may also be used as part of a treatment of lung carcinoma and adenocarcinoma, and other proliferative disorders involving the lung epithelia. It has been shown that Shh is expressed in human lung squamous carcinoma and adenocarcinoma cells. Fujita et al., Biochem. Biophys. Res.Commun. 238: 658 (1997), The expression of Shh was also detected in the human lung squamous carcinoma tissues, but not in the normal lung tissue of the same patient. They also obsen'ed that Shh stimulates the incorporation of BrdU into the carcinoma cells and stimulates their cell growth, while anti-Shh-H inhibited their cell growth. These results suggest that ptch, hedgehog, and'Or smoothenecl is involved in cell growth of such transformed lung tissue and therefore indicates that the subject can be used as part of a treatment of lung carcinoma and

adenocarcinomas, and other proliferative disorders involving the lung epithelia.

In some embodiments, the TPRA40 antagonists of the present disclosure, based on the involvement of hedgehog signaling in various tumors, or expression of hedgehog or its receptors in such tissues during development, can be used to inhibit growth of a tumor having dysregulated hedgehog activity. Such tumors include, but are not limited to: tumors related to Gorlin's syndrome (e.g., medulloblastoma, meningioma, etc.), tumors associated with a ptch mutation (e.g., hemangiona, rhabdomyosarcoma, etc.), tumors resulting from Glil amplification (e.g.. glioblastoma, sarcoma, etc.), tumors resulting from Smo dysfunction (e.g., basal cell carcinoma, etc.), tumors connected with TRC8, a ptc homoiog (e.g., renal carcinoma, thyroid carcinoma, etc.), Ext-1 related tumors (e.g., bone cancer, etc.), Sft/x-induced tumors (e.g., lung cancer, chondrosarcomas, etc.), tumors overexpressing a hedgehog protein, and other tumors (e.g., breast cancer, urogenital cancer (e.g.,, kidney, bladder, ureter, prostate, etc.), adrenal cancer, gastrointestinal cancer (e.g., stomach, intestine, etc.).

In some embodiments, the TPRA40 antagonists of the present disclosure may also be used to treat several forms of cancer. These cancers include, but are not limited to: prostate cancer, bladder cancer, lung cancer (including small cell and non-small cell), colon cancer, kidney cancer, liver cancer, breast cancer, cervical cancer, endometrial or other uterine cancer, ovarian cancer, testicular cancer, cancer of the penis, cancer of the vagina, cancer of the urethra, gall bladder cancer, esophageal cancer, or pancreatic cancer. Additional cancer types include cancer of skeletal or smooth muscle, stomach cancer, cancer of the small intestine, cancer of the salivary gland, anal cancer, rectal cancer, thyroid cancer, parathyroid cancer, pituitary cancer, and nasopharyngeal cancer. Further exemplary forms of cancer which can be treated with the hedgehog antagonists of the present disclosure include cancers comprising hedgehog expressing cells. Still further exemplary forms of cancer which can be treated with the hedgehog antagonists of the present disclosure include cancers comprising Gli expressing cells. In one embodiment, the cancer is not characterized by a mutation in patched- 1 . In some embodiments, the cancer is characterized by a smoothened and/or SuFu mutation.

For example, the pharmaceutical preparations of the TPRA40 antagonists of the disclosure are intended for the treatment of hyperplastic conditions, such as keratosis, as well as for the treatment of neoplastic epidermal conditions such as those characterized by a high proliferation rate for various skin cancers, e.g., squamous cell carcinoma. In some embodiments, the TPRA40 antagonists of the disclosure can also be used in the treatment of autoimmune diseases affecting the skin, in particular, of clermatoiogical diseases involving morbid

proliferation and/or keratinization of the epidermis, as for example, caused by psoriasis or atopic dermatosis. Many common diseases of the skin, such as psoriasis, squamous cell carcinoma, keratoacantlioma and actinic keratosis are characterized by localized abnormal proliferation and growth. For example, in psoriasis, which is characterized by scaly, red, elevated plaques on the skin, the keratinocytes are known to proliferate much more rapidly than normal and to differentiate less completely. In one embodiment, the preparations of the TPRA40 antagonists of the present disclosure are suitable for the treatment of dermatological ailments linked to keratinization disorders causing abnormal proliferation of skin cells, which disorder may be marked by either inflammatory or non- inflammatory components. In some embodiments, the TPRA40 antagonists that promote quiescence or differentiation can be used to treat varying forms of psoriasis, e.g., cutaneous, mucosal or ungual. Psoriasis, as described above, is typically characterized by epidermal keratinocytes that display marked proliferative activation and differentiation along a "regenerative" pathway. Treatment with such TPRA40 antagonists according to the present disclosure can be used to reverse the pathological epidermal activation and can provide a basis for sustained remission of the disease.

A variety of other keratotic lesions are also candidates for treatment with the TPRA40 antagonists of the present, disclosure. Actinic keratoses, for example, are superficial

inflammatory premalignant tumors arising on sun-exposed and irradiated skin. The lesions are erythematous to brown with variable scaling. Current therapies include excisional and cryosurgery. These treatments are painful, however, and often produce cosmetically

unacceptable scarring. In some embodiments, treatment of keratosis, such as actinic keratosis, can include application, preferably topical, of a TPRA40 antagonist composition in amounts sufficient to inhibit hyperproliferation of epidermal/epidermoid cells of the lesion.

Acne represents yet another dermatologic ailment which may be treated by the TPRA40 antagonists of the present disclosure. Acne vulgaris, a multi factor disease most commonly occurring in teenagers and young adults, is characterized by the appearance of inflammatory and noninflammatory lesions on the face and upper trunk. The basic defect which gives rise to acne vulgaris is hypercornification of the duct of a hyperactive sebaceous gland. Hypercornification blocks the normal mobility of skin and follicle microorganisms, and in so doing, stimulates the release of lipases by Propinobacterian acnes and Staphylococcus epidermidis bacteria and

Pitrosporum ovale, a yeast. In some embodiments, treatment with an antiproliferative TPRA40 antagonist, particularly topical preparations, may be useful for preventing the transitional features of the ducts, e.g., hypercornification, which lead to lesion formation. The subject treatment may further include, for example, antibiotics, retinoids and antiandrogens.

In some embodiments, the TPRA40 antagonists of the present disclosure may also be used in a method treating various forms of dermatitis. Dermatitis is a descriptive term referring to poorly demarcated lesions that are either pruritic, erythematous, scaly, blistered, weeping, fissured or crusted. These lesions arise from any of a wide variety' of causes. The most common types of dermatitis are atopic, contact and diaper dermatitis. For example, seborrheic dermatitis is a chronic, usually pruritic, dermatitis with erythema, dry, moist, or greasy scaling, and yeliow- crusted patches on various areas, especially the scalp, with exfoliation of an excessive amount of dry scales. In some embodiments, the TPRA40 antagonists may also be used in the treatment of stasis dermatitis, an often chronic, usually eczematous dermatitis. Actinic dermatitis is a dermatitis that due to exposure to actinic radiation such as that from the sun, ultraviolet waves, or x- or gamma-radiation. According to the present disclosure, the TPRA40 antagonists can be used in the treatment and/or prevention of certain symptoms of dermatitis caused by unwanted proliferation of epithelial cells. Such therapies for these various forms of dermatitis can also include topical and systemic corticosteroids, antipruritics, and antibiotics. Additional skin ailments that may be treated with the TPRA40 antagonists of the present disclosure include disorders specific to non-humans, such as mange.

The foregoing are merely exemplary of in vitro and in vivo uses for TPRA40 antagonists of the disclosure. TPRA40 antagonists are also suitable for use in identifying natural targets or binding partners for TPRA40, to study TPRA40 bioactivity, to purify TPRA40 and its binding partners from various cells and tissues, and to identify additional components of the hedgehog signaling pathway.

As detailed herein, the discl osure also provides for methods of screening to identify

TPRA40 agonists. TPRA40 agonists can be used in methods of promoting hedgehog signaling in vitro and/or in vivo. TPRA40 agonists are also suitable in subjects in needs thereof and can be administered as described herein. In some embodiments, the disclosure provides for a method of administering any of the TPRA40 agonists described herein, such as a small molecule agonist, to a subject in need thereof. In some embodiments, the subject has Down's Syndrome, ischemic heart disease, or alopecia. See, e.g., Sci Transl Med. 2013 Sep 4;5(201):201ral20. dot:

10.1 126/scitranslmed.3005983; Pharmazie. 2012 Jun;67(6):475-81 ; Cardiovasc Res. 2012 Sep 1;95(4):507-16. doi: 10.1093/cvr/cvs216; and J Invest Dermatol. 2005 Oct; 125(4):638-46. In some embodiments, the subject is a pre-term baby that has been or is being treated with gluco- corticoids, and the subject is administered the TPRA40 agonist in order to prevent nerve damage. See, e.g., Sci Transl Med. 2011 Oct 19:3(105): 105ral04. doi: 10.1126/scitranslmed.3002731. Exemplary TPRA40 agonists are small molecule that hind to TPRA40. Also contemplated is overexpression of TPRA40 as a TPRA40 agonist.

V. Methods of ,A^^

Various delivery systems are known and can be used to administer TPRA40 antagonists of the disclosure to cells or subjects. Such delivery systems are similarly applicable to TPRA40 agonists. Any such methods of administration may be used in the context of any of the methods of use described herein and/or in the context of any of the TPRA40 antagonists of the disclosure, such as the TPRA40 antagonists described herein. Methods of introduction can be enteral or parenteral, including but not limited to, intradermal, intramuscular, intraperitoneal,

intramyocardial, intravenous, subcutaneous, pulmonary, intranasal, intraocular, epidural, superficial/topical (e.g., a cream) and oral routes. The route of introduction may be selected based on, for example, the particular TPRA40 antagonist used (e.g., small molecule versus protein) and the intended use/purpose of administration. When administering to subjects (such as to cells in a subject), a TPRA40 antagonist, may be administered by any convenient route, for example, by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local. When administered to cells in culture, TPRA40 antagonist may be added to culture media, delivered into cells via a viral vector, delivered into cells by transfection, electroporation, or

transformation, optionally, as part of a vector, and the like. Similarly, delivery via a viral or other vector may be used to facilitate delivering into cells in vivo.

In certain embodiments, a TPRA40 antagonist is administered intravenously. In certain embodiments, it may be desirable to administer the TPRA40 antagonist locally to the area in need of treatment (e.g., to the site of a tumor); this may be achieved, for example, and not by way of limitation, by local infusion or injection during surgery, by means of a catheter, or by means of an implant, the implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, fibers, or commercial skin substitutes.

Note that the disclosure contemplates methods in which a TPRA40 antagonist of the disclosure is administered, at the same or different times, with a. second TPRA40 antagonist and/or a. second therapeutic agent. Administration may be by the same of differing routes of administration. For example, the disclosure contemplates a regimen in which a TPRA40 antagonist of the disclosure is administered intravenously and a second therapeutic agent is administering orally.

The foregoing applies to any of the TPRA40 antagonists, compositions, and methods described herein. The disclosure specifically contemplates any combination of the features of such TPRA40 antagonists, compositions, and methods (alone or in combination) with the features described for the various pharmaceutical compositions and routes of administration described in this section and in the section provided below. The formulations provided below are merely exemplary, and TPRA40 antagonists for use in the subject methods can be formulated as appropriate for the intended use and route of administration.

VI. Pharmaceutical Formulations

In some embodiments, any of the TPRA40 antagonists described herein or TPRA40 a agonists in accordance with the disclosure may be formulated in a pharmaceutical

composition. Similarly, pharmaceutical formulations as described herein as applicable to

TPRA40 agonists.

Pharmaceutical compositions of the TPRA40 antagonists used in accordance with the present disclosure may be prepared for storage by mixing the agent(s) having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science of Practice of Pharmacy. 20th edition, Gennaro, A. et al., Ed., Phi ladelphia College of Pharmacy and Science (2000)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyklimethylbenzyi ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, giutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; to icifiers such as trehalose and sodium chloride; sugars such as sucrose, mannitol, trehalose or sorbitol; surfactant such as polysorbate; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as 'T WEEN®,

PLURONICS® or polyethylene glycol (PEG).

In some embodiments, any of the formulations of TPRA antagonists in accordance with the present disclosure and/or described herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other. It should be recognized that, in certain embodiments, a TPRA40 antagonist and a second active agent are formulated together (e.g., a formulation or composition contains both agents). In other embodiments, the two (or more) active agents are formulated separately such that the separate formulations can be marketed, sold, stored, and used together or separately. When formulated, separately, the disclosure

contemplates that they can be administered at the same or differing times and, in certain embodiments, may be combined and administered simultaneously.

For example, in addition to the preceding therapeutic agent(s), it may be desirable to include in the formulation, an additional antibody, e.g., a second such therapeutic agent, or an antibody to some other target (e.g., a growth factor that affects the growth of a tumor). In some embodiments, it may be desirable to include in the fonnulation a hedgehog inhibitor (e.g., robotkinin). Alternatively, or additionally, the composition may further comprise a

chemotherapeutic agent, cytotoxic agent, cytokine, growth inhibitory agent, anti- hormonal agent, and/or cardioprotectant. Such molecules are suitably present in combination in amounts that are effective for the purpose intended. In some embodiments, the additional active compound is a steroidal alkaloid. In some embodiments, the steroidal alkaloid is cyclopamine, or KAAD- cyclopamine or jervine or any functional derivative thereof (e.g., IPI-269609 or IPI-926). In some embodiments, the additional active compound is vismodegib, sonidegib, BMS-833923, PF- 04449913, or LY2940680 or any derivative thereof. In some embodiments, the additional active compound is any of the compounds disclosed in Amakye, et al., Nature Medicine, 19(1 1): 1410- 1422 (whichi is incorporated herein in its entirely). In some embodiments the additional active compound is another smoofhened inhibitor chemically unrelated to veratrum alkaloids or vismodegib, including but not limited to: Erivedge, BMS-833923 (XL319), LDE225

(Erismodegib), PF-04449913, NVP-LDE225, NVP-LEQ506, TAK-441 , XL-319, LY-2940680, SEN450, Itraconazole, MRT-10, MRT-83, or PF-04449913). As noted above, the disclosure contemplates formulations in which a second active agent is formulated together with a TPRA40 antagonist (e.g., as a single formulation comprising two active agents), as well as embodiments in which the two active agents are present in two separate formulations or compositions.

In some embodiments, any of the TPRA antagonists of the disclosure, such a s those described herein, may also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin- microcapsules and poly-(methylmethacylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, naiio- particles and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington: The Science and Practice of Pharmacy, supra.

In some embodiments, any of the TPRA40 antagonists of the disclosure are formulated in sustained-release preparations. Suitable examples of sustained- release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vmylalcoho!)), poiylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y ethyl-L-glutarnate, non- degradable ethyl .en e- vinyl acetate, degradable lactic acid-glycolic acid copolymers such as the LUPRON DEPOT*' (injectable microspheres composed of lactic acid- glyceric acid copolymer and leupro!ide acetate), and poiy~D(-)-3-hydroxybutyric acid.

The amount of the compositions of the disclosure for use in the methods of the present disclosure can be determined by standard clinical techniques and may vary depending on the particular indication or use. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

In certain embodiments, compositions of the disclosure, including pharmaceutical preparations, are non-pyro genie. In other words, in certain embodiments, the compositions are substantially pyrogen free. In one embodiment the formulations of the disclosure are pyro gen- free formulations that are substantially free of endotoxins and/or related pyrogenic substances. Endotoxins include toxins that are confined inside a microorganism and are released only when the microorganisms are broken down or die. Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, even low amounts of endotoxins must be removed from intravenously administered pharmaceutical drug solutions. The Food & Drug Administration ("FDA") has set an upper limit of 5 endotoxin units (EU) per dose per kilogram body weight in a single one hour period for intravenous drug applications (The United States Pharmacopeia! Convention, Pharmacopeia! Forum 26 (1):223 (2000)). When therapeutic proteins are administered in relatively large dosages and/or over an extended period of time (e.g., such as for the patient's entire life), even small amounts of harmful and dangerous endotoxin could be dangerous. In certain specific embodiments, the endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.

In some embodiments, the TPRA40 antagonists are formulated in sterile formulations. This is readily accomplished by filtration through sterile filtration membranes. VII. Articles of Manufacture and Kits

In some embodiments, the TPRA40 antagonists of the present disclosure, such as the TPRA40 antagonists disclosed herein are prepared in an article of manufacture. In some embodiments, the article of manufacture comprises a container and a label or package insert on or associated with the container indicating a use for the inhibition in whole or in part of hedgehog signaling, or alternatively for the treatment of a disorder or condition resulting from activation of the hedgehog signaling pathway. Suitable containers include, for example, bottles, vials, syringes, etc. The containers may be formed from a variety of materials such as glass or plastic. In some embodiments, the container holds a composition which is effective for treating the cancer condition and may have a sterile access port (for example the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a TPRA40 antagonist. The label or package insert will further comprise instructions for administering the TPRA40 antagonist. Additionally, the article of manufacture may further comprise a second container comprising a pharmaceutically- acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution and dextrose solution. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

In some embodiments, kits are provided that are useful for various other purposes, e.g., for TPRA40-expressing cell killing assays, for purification or immunoprecipitation of TPRA40 polypeptide from ceils. For isolation and purification of TPRA40, the kit can contain the respective TPRA40-binding reagent coupled to beads (e.g., sepharose beads). Kits can be provided which contain such molecules for detection and quantitation of TPRA40 polypeptide in vitro, e.g., in an ELISA or a Western blot. In some embodiments, as with the article of manufacture, the kit comprises a container and a label or package insert on or associated with the container. In some embodiments, the container holds a composition comprising at least one such TPRA40 antagonist reagent useable with the disclosure. In some embodiments, additional containers may be included that contain, e.g., diluents and buffers, control antibodies. In some embodiments, the label or package insert may provide a description of the composition as well as instructions for the intended in vitro or diagnostic use.

Exemplification

The disclosure now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present disclosure, and are not intended to limit the disclosure.

Example 1 : siRNA Knockdown of TPRA40 Partially Inhibits Hedgehog Signaling

Pools of siRNAs were used to knockdown expression of TPRA40 or Ssnal (a gene previously shown to be involved in the Hedgehog signaling pathway; Lai et al., 201 1 , Moi Biol Cell 22: 1104) in the presence or absence of an agent that stimulates hedgehog signaling, and the impact on hedgehog signaling was assessed in S12 cells comprising a 6 -luciferase reporter. TPRA40 depletion inhibited, by approximately 40%, Hedgehog signaling stimulated by either Sonic hedgehog protein or by a small molecule Smoothened agonist. This supports the conclusion that TPRA40 is a positive regulator of the hedgehog signaling pathway, and that it acts downstream of Smoothened. Briefly, for the experiments summarized in Figure 1A, S12 cells were depleted of murine lft88 (by 72%), Ssnal (by 75%) and TPRA40 (by 80%) using siRNAs. Cells were transfected with 50nM siRNA (with pools of four siRNAs per target, per standard practice) for 72 hours, the last 24 hours of which they were incubated in serum-free media in the presence or absence of 200ng/ml octyl-Shh to stimulate hedgehog signaling, (In Figure 1A, the results following incubation with Shh are shown in black - the right bar in each set of bars; the results following incubation without Shh are shown in grey - the left bar in each set of bars). G/ -lueiferase activity was measured versus renilla-iuciferase (as a measure of viability) and the data were expressed as a % of the non-targeting control siRN A (siNTC) +Hh. The mean and standard deviation of four independent experiments are shown, *, p <0.05; **, p < 0,01 ; ***, p < 0.001 (student's unpaired t-test). The efficiency of each siRNA was measured by qRT-PCR analysis of the corresponding gene.

For the experiments summarized in Figure B, SI 2 cells were treated as in (A) except hedgehog signaling was stimulated with Ι ΟΟηΜ of the Smootbened, small molecule agonist, Hhl.2 instead of octyl-Shh (black; right bar in each set of bars) or vehicle control (DMSO) (grey; left bar in each set of bars). The results were expressed as a percentage of stimulated control (NTC) cells.

Example 2: siRNA Knockdown of TPRA40 Reduced Hedgehog Induced Gli mRNA and Protein Expression in 812 Cells

TPRA40 depletion using siRNA inhibited hedgehog-stimulated Glil induction in S I 2 ceils. The inhibition in Glil induction was observed at both the mRNA and protein level, as both Glil mRNA and Glil protein expression were reduced following TPRA40 depletion. Figure 2A summarizes the results of experiments in which Glil mRNA was evaluated. Briefly, S12 cells were depleted of TPRA40 by siRNA treatment and stimulated with 200ng/ml Sonic hedgehog protein (black; Hh; right bar in each set) or serum-starved in the absence of hedgehog protein stimulation (grey; left bar in each set) as in Example 1, but analyzed by qRT-PCR for

endogenous murine Glil expression (instead of 6 -luciferase activity, as was done in Example 1). The mean and standard deviation of three independent experiments was plotted and showed an approximately 50% reduction in hedgehog pathway stimulation, as measured by change in endogenous Glil expression, in TPRA40 siRNA treated (i.e., TPRA40-deficient) cells. Figure 2B summarizes the results of experiments in which Gli l protein levels were evaluated. Briefly, ceils were treated as in 2A but lysed and analyzed by western blotting with a Glil -specific monoclonal antibody, then reprobed for tubulin (55kDa) as a loading control. The Hh-induced upregulation of Glil protein (-150 kDa) was also diminished by TPRA40 knockdown (a representative blot of two independent experiments is shown).

Example 3: Analysis of the Individual Components of the TPRA40 siRNA Pool Confirmed that the Observed Effects on TPRA40 Expression and Activity are Not Off-Target Effects

Figures 3A-C show that three of the four individual siRNA components of the siTPRA40 pool are active and reduce TPRA40 expression. Moreover, the active, individual siRNAs cause approximately the same level of inhibition as the pool, consistent with the conclusion that the observed effects on TPRA40 and hedgehog signaling are specific and don't represent off-target effects.

Figure 3A summarizes experiments in which the four siRNAs to TPRA40 that make up the pool (siRNAs #9- 12) were transfected individually into S12 cells at 25nM and the Gli- luciferase activity was measured, as described in Example 1 , except the data were normalized to siNTC -'-Hh as 1 . As in the experiments depicted in Figure 1 A, Sonic hedgehog protein was added to stimulate hedgehog signaling. Mean and standard deviation of three experiments are shown. siRNAs #10-12 inhibited Gli-luciferase stimulation similarly to the Ι ΟΟηΜ pool (by 34- 40%), while siRNA #9 did not.

Figure 3B shows the results of an experiment in which S I 2 cells were treated as in (A) but endogenous GUI (murine Gli l ; niGli expression in S 12 cells) levels were measured by qRT- PCR. 25nM siRNA #9 again had no effect, while siRNAs #10-12 inhibited Glil induction, albeit less effectively than l OOnM of the 4 siRNA pool. Data from a single experiment is shown.

Figure 3C shows the results of an experiment in which TPRA40 gene expression levels (murine TPRA40; mTPRA40 expression in S 12 cells), assayed by qRT-PCR, were decreased by more than 80% by the siRNA pool and individual siRNAs #s 9 and 10; by about 60% by siRN A #1 1 and by about 70% by siRNA #12. in addition, we observed that hedgehog stimulation had no effect on TPRA40 levels (in the non-targeting control (NTC)-transfected cells), consistent with the conclusion that TPRA40 is not itself a hedgehog pathway target gene. Data from a single experiment is shown. In Figures 3B and 3C, results in the presence of hedgehog treatment are shown in black (the right bar in each set) and the results in the absence of hedgehog treatment are shown in grey (the left bar in each set).

Example 4: siRNA Knockdown of TPRA40 Inhibited Endogenous Hedgehog Signaling in Medulioblastoma Cells.

Figure 4 summarizes results showing that depletion of TPRA40 using siRNA inhibited hedgehog signaling in Daoy medulioblastoma ceils. These results are consistent with the conclusion that TPRA40 acts downstream of the activating mutation in Daoy cells. Moreover, this experiment demonstrated that modulation of TPRA40 regulates Hedgehog signaling not only in normal cells (such as S 12 cells), but also in cancer cells.

Daoy medulioblastoma cells are a human cell type that exhibits constitutively active Hh signaling (in the absence of Hh ligand), as monitored by hGlil mRNA levels. This constitutive signaling can be decreased by treatment with 0.5 or 2 μΜ Cyclopamine, a well-known Smoothened inhibitor. TPRA40 depletion (depletion of endogenous, human TPRA40 expressed in these medulioblastoma cells) by transfection of 50nM of the siRNA pool to hTPRAAO also reduced hGlil levels and, when combined with cyclopamine, further reduced hGlil levels (See Figure 4). Data from a single experiment is shown. For each of the DMSO or cyclopamine treatment groups, data for cells treated with the TPRA40 siRNA pool is shown by the right-hand bar (black).

Example 5: Ordering TPRA40 in the Hedgehog Signaling Pathway

Figure 5A provides a schematic representation of certain key Hedgehog pathway components, with positive regulators shown in normal font and negative regulators shown in bold font. The following positive regulators are depicted: Shh (Sonic hedgehog), Smo (Smoothened) and Gli activators (i.e. Giil , Gli2 Mi length or Gli3 full length). The following negative regulators (in bold font) are depicted: Ptc (Patched 1), SuFu (Suppressor of Fused) and PKA (Protein Kinase A) and Gli repressors (truncated Gli2 or truncated Gli3). Arrows denote a positive effect of the upstream gene, while bars denote an inhibitory effect of the upstream gene on the downstream one. PKA phosphorylates (depicted by the letter P) Gli2 and Gli3-fuli length (FL), which initiates their cleavage into repressor forms (Gli-R). We conducted a series of experiments to orient TPRA40 in the Hedgehog signaling pathway, relative to other pathway players.

Figures 5B and C summarize experiments designed to order TPRA40 in the hedgehog signaling pathway and show that TPRA40 acts downstream of Ptchl and SuFu. The experiments were conducted in S12 cells in the absence of stimulation with Hedgehog (ligand). As shown, depletion using siRNAs of the negative regulator Ptchl (B, data normalized to siPtchl) or depletion of the negative regulator SuFu (C, data, normalized to siSuFu) stimulated G/ -lueiferase activity in S12 ceils in the absence of ligand. This stimulation of hedgehog signaling activity was partially rescued by co-depletion of TPRA40 using the siR A pool in both cases. These experiments are consistent with the conclusion that TPRA40 acts downstream of Ptchl and SuFu in the Hh pathway.

Figure 6 shows that TPRA40 does not act downstream of PKA; but rather, acts upstream of or at the same level as PKA (protein kinase A). This experiment was conducted in SI 2 cells in the absence of stimulation with hedgehog. SI 2 cells were treated with either DMSO vehicle control or 80 μΜ cell permeable 14-22 amide (inhibitor of PKA) for 24 hours in the absence of Hedgehog (ligand) following 48 hours treatment with siRNAs to TPRA40 (depicted by black/right bar for each set of bars in Figure 6) or non-targeting control (depicted by grey/left bar). TPRA40 depletion using the siRNA pool to murine TPRA40 described above did not rescue the 14-22 amide induced G/Muciferase activity. In other words, TPRA40 depletion did not rescue PKA inhibitor-mediated stimulation of the pathway. This is consistent with the conclusion that TPRA40 acts upstream of or at the level of PKA.

Example 6: siRNA Knockdown of TPRA40 increased Gli3 Processing

Figure 7 shows that TPRA40 knockdown modestly increased levels of Gii3 repressor. To help interpret the results of these experiments, it is useful to note what typically occurs as part of the Hedgehog signaling pathway. Briefly, full length Gli3 (GH3-FL, ~ 190kDa) is cleaved into a repressor form (GH3R, -80 kDa) in the absence of Hedgehog via PKA phosphorylation

(followed by 08Κ3β and C 1 phosphorylation, which recruits pTRCP and SCF/Cull to degrade the C-tenninal half of Gli3).

Figure 7A (left hand side of figure) presents Western blot analysis of Gli3 using monoclonal antibody 6F5 with and without 24 hour Hedgehog treatment and in the presence or absence of siR A depletion of TPRA40 or ift88. The tubulin antibody 1A2 was used as a loading control. Figure 7B (two panels on right hand side of figure ) depicts quantitation of three westerns of GH3FL and GH3R normalized to tubulin. Hedgehog stimulation for 24 hours inhibits P A activity and attenuates Gli3R production, which requires primary cilia, (as evidenced by the increase in GH3R levels following Ift88 depletion). TPRA40 knockdown increased both the baseline level of GH3R (by about 25%) and the level remaining after

Hedgehog stimulation (by 2 fold). These results are consistent with less Hh pathway induction in the presence of Hh, as well as more PKA activity in the absence of Hh. In Figure 7B, for each set of bars indicative of results observed following treatment with a given pool of siRNAs, the results observed in the presence of Hedgehog treatment are shown in black (right bar) and the results observed in the absence of Hedgehog are shown in grey (left bar), normalized to NTC in the absence of Hh.

Ex ample 7: Characterization of Anti-TPRA 40 Antibodies in S 12 Cells

Two TPRA40 antibodies were obtained and characterized. Figures 8 A and 8B show the characterization of these antibodies to endogenous TPRA40 by western blotting of S12 cells. 812 cells transfected with NTC or TPRA40 siRNAs for 48 hours were serum starved in the presence or absence of Hedgehog for 24 hours (72 hours total knockdown), then !ysed and subjected to western blotting.

Pane! A shows the results obtained using a custom-made rabbit anti~TPRA40 C-terminal antibody 12569B (generated by YenZym; see material and methods below for additional information). This antibody detected a single band of about 55kDa that is not affected by Hh treatment (i.e., the band is observed regardless of whether the ceils are cultured in the presence or absence of Hedgehog protein), but disappears following TPRA40 depletion using siRNAs. These results indicate that this antibody is specific for TPRA40.

Panel B shows results obtained using the mouse anti-TPRA40 antibody 6H2

(commercially available from Santa Cruz). This antibody also detected TPRA40. However, this antibody appeared less clean and also recognized a couple of smaller non-TPRA40 proteins, albeit to a lesser extent. The predicted molecular weight from the sequence is 40,560 Da plus N- glycosyiation (molecular weight decreases to -35,000 Da following PNGaseF treatment [data not shown]). Example 8: TPRA40 Localizes to Cilia in a Hedgehog-Dependent Manner

Figures 9 A and 9B show that endogenous TPRA40 localizes to primary cilia of S12 cells in a Hedgehog-dependent fashion, (A) S12 cells were fixed and processed for

immunofluorescence using anti-TPRA40 monoclonal antibody 6H2 (left panel and red channel in merge) and rabbit anti-Aril 3b antibody (a marker for primary cilia in the middle panel and green channel in merge) following 24 hours serum starvation alone (upper panel: in the absence of Hedgehog stimulation) or with Hedgehog stimulation (lower panel: +Hh). Nuclei stained with DAPI are in blue in the merged right panel. (B) Counting the number of Arll3b-positive cilia, for TPRA40 staining revealed that TPRA40 was found in only about 20% of cilia in the absence of Hedgehog stimulation, but accumulated in 60% of cilia following overnight Hedgehog stimulation. This level of accumulation to cilia is very similar to the extent of accumulation of Smoothened (mean and SD of 2 experiments is shown). In Figure 9B, for each of the TPRA40 and Smo data sets, data obtained in the presence of Hedgehog stimulation is shown in black (right, bar in each set of bars) and data obtained in the absence of Hedgehog stimulation is shown in grey (left bar in each set of bars).

Example 9: TPRA40 is a GPCR

Figure 10 summarizes experiments designed to characterize this orphan GPCR. TPRA40 expression inhibited cAMP production in a CRE-luciferase reporter assay. The diagram on the left depicts the assay set up, comprising 293T cells stably expressing CRE (cAMP Response Elementj-luciferase transiently transfected with GFP (negative control, not shown) or a TPRA40 (squiggle traversing the plasma membrane 7 times) expression construct were treated with varying doses of Forskolin, a potent activator of Adenyiyl Cyclase, thus increasing cAMP levels inside the cells. If the transfected GPCR is coupled to Galpha(s, stimulatory), the CRE- luciferase activity should increase more than in control GFP-transfected cells upon Forskolin treatment, whereas it if it coupled to Galpha(i, inhibitory), the CRE-luciferase activity should increase less than in GFP-transfected cells. The graph on the right shows that exogenous expression of TPRA40 suppressed the CRE-reporter activity in a dose dependent manner, suggesting that this GPCR is coupled to Galpha(i), which inhibits cAMP production. The mean and standard deviation of four independent experiments normalized to 20μΜ forskolin in GFP- transfeeted cells is shown.

Example 10: Epistasis Analysis of TPRA40 Relative to Galpha(i)

Figure 11A shows that S12 cells trans fected with siRNAs to Galpha (i) show reduced Hh signaling compared to NTC treated cells. Knockdown of Galpha(i)l with siRNAs decreased Gli- luciferase activity in Hh-treated S12 cells by about 50%, consistent with increased cAMP production (due to relief of inhibition by Galpha(i) depletion) stimulating more PKA activity and Gli3R production. Co expression of siTPRA40 along with siGalpha (i)l did not rescue reporter activity compared to siGalpha(i)! alone, suggesting that TPRA40 functions at the level of or upstream of Galpha (i) l . Mean and SD of 3 independent experiments are shown.

Figure 1 IB shows that GH3 depletion is partially rescued by TPRA40 knockdown in S 12 cells. S12 cells depleted of Gli3 by siRNA show active Hedgehog signaling in the absence of ligand due to loss of Gli3 repressor (the Gli-luciferase signal is initiated by Gli2 activator).

TPRA40 depletion partially inhibits the signal in Gli3-depleted cells. The sequences of the murine Gli3 siRNAs in the pool are:

G A AC A A C CCIJ A GUC A AG G A (SEQ ID NO: 45)

GCUCUACCGUGCAGAAUUA (SEQ ID NO: 46)

GCUUAGUGCUGCAAAAUUA (SEQ ID NO: 47)

CCUCACAUAUCAACAUCUA (SEQ ID NO: 48)

Figure 1 1 C shows that TPRA40 depletion does not prevent GH3 accumulation at cilia tips. Depletion of TPRA40 by siRNA treatment does not prevent the Hedgehog-dependent

accumulation of Gli3 at cilia tips (bottom row). Arrows show the tips of the primary cilia.

Figure 1 1 C shows that less than 10% of cilia have Gli3 at the tips in the absence of Hedgehog stimulation, while the black bars (the right of each pair of bars) show approximately 80%) of cilia have Gii3 at their tips irrespective of the presence of TPRA40. This suggests that the regulation of GH3 processing by TPRA40 and PKA occurs after the initial translocation of Gli3 to primary cilia. Example 1 1 : Working Model for TPRA40 in Hedgehog Signaling Pathway Figure 12 provides a working model for TPRA40 function as a positive regulator of Hedgehog signaling. This model is provided merely to facilitate discussion of the pathway, and the inventors do not intend to be bound by theory. The claimed disclosure is fully operative and does not rely on the accuracy of the details of this model. Left panel: in the absence of

Hedgehog protein (ligand), Patched 1 (Ptchl , red multipass protein) in the primary ciiium suppresses the activity of extraciiiary Smoothened (Smo, green GPCR) and prevents its localization to cilia. TPRA40 (brown GPCR) is not present in cilia (its depiction on the plasma membrane is speculative) and so does not interact with ciliary Galpha(i) (Gai, blue circle), leading to high local concentrations of cyclic AMP (cAMP). cAMP stimulates protein kinase A (PKA, yellow oval; asterisks denote active forms of all proteins) to phosphorylate full length Gli3 and Gli2 (Gli-FL, red/green rectangle), which primes further GU3FL phosphorylation by CK1 (white oval) and 08 3β (purple oval). Phospho-Gli3 becomes a substrate for PTRCP binding, which recruits 8CF ubiquitin ligase (not shown) to promote proteasome-dependent cleavage of Gli3 into its repressor form (Gii-R, red rectangle). Gli3-R represses transcriptional activation of Hh pathway target genes, thus the pathway is off". Right panel: in the presence of Hedgehog stimulation, Ptchl is removed from cilia, allowing Smo to enter cilia and become activated. This leads to accumulation of TPRA40 in cilia, where it interacts with Gai to inhibit local cAMP production, preventing PKA activity and Gli.3 cleavage. The SuFu-GliFL complex accumulates at the tips of primary cilia, dissociates, and activated Gli-FL (Gli-A) exits cilia and enters the nucleus to promote transcription of Hh target genes such as GUI. Optionally, TPRA40 undergoes an activation step (like Smoothened) in addition to ciliary translocation in order to exert its activity.

Example 12: TPRA40 Amino Acid Sequence information

Figure 13 shows an alignment of the amino acid sequences of mouse and human TPRA40.

Human TPRA40 (top, 373 amino acids, predicted MW 41034 Da; Swissprot Q86W33) is aligned with mouse TPRA40 (middle, 369 amino acids, predicted MW 40560 Da; Swissprot Q99MU 1) and zebrafish TPRA l (bottom, 378 amino acids, predicted MW 41685 Da; Swissprot Q4V8X0) using the Align program in GSeqWeb. Identical amino acids are colored, and the positions (predicted by Swissprot ) of the 7 transmembrane (tm) domains typical of GPCRs are underlined in blue. Mouse and human TPRA share 91.4% identity and 94.1 % similarity at the protein level. Zebrafish TPRA1 is 70.0% and 68.7% identical (79.3% and 78.0% similar) to human and mouse TPRA40, respectively, suggesting an evolutionarily conserved function. As expected for a GPCR, the N-terminus is luminal/extracellular and the C-terminus is cytoplasmic. This topography was verified by FACS with epitope tags at each end of the protein (data not shown).

Materials and Methods:

The following methods were used in the experiments described in the Examples.

siPvNA transfection.

Pools of pre-designed ON-TARGETplus siRNAs for murine TPRA40 (GPR175 siRNAs 09-12), Smo, G-alpha(i) ! , 1FT88, or siGenome for murine Ssnal were purchased from

Dharmacon Inc. (Lafayette, CO). S 12 cells were seeded into 96-well white wall clear-bottom plates at 6 xl( cells/well; 8-well LabTekJl microscope slides at 2xl04 cells/well; or 60mm plates at 4xl05 cells/well and reverse-transfected with 50nM final siRNA pools, following 20 minutes preincubation of siRNA pool with DharmaFECT-2 ( Dharmacon) in Opti-MEM (Gibco) at room temperature. For epistasis experiments, two siRNA pools at 50nM each were used. For testing the four individual components of the pool, each siTPRA40 siRNA was transfected at 25nM. After 48 hours, ceils were shifted into 0.5% FBS media to promote ciliogenesis for an additional 16-24 hours to promote ciiiation (± Hh for indicated times).

The following siR As were used in these examples (either individually or as a member of a pool of siRNAs). These siRNAs were double-stranded and prepared in accordance with manufacturer's protocols. For each of these examples, the sequence for one strand is provided below:

Mouse TPRA40 siRNA #9:GGAGGAGUUUCUACGUGUA (SEQ ID NO: 16) Mouse TPRA40 siRNA #10:CGGCAGUUCUGGCUCGUCA (SEQ ID NO: 17) Mouse TPRA40 siRNA #1 1 :CGACAGUUGCUGACAAGAU (SEQ ID NO: 18) Mouse TPRA40 siRNA #12:GCCAUUGAGCUGAGUCUGA (SEQ ID NO: 19) Human TPRA40 siRNA #6:GGUCAGCUCCUGCUUCUUC (SEQ ID NO: 20) Human ! PR A 40 siRNA #7:GAGAGUAAGUCCAGCAUCA (SEQ ID NO: 21) Human TPRA40 siRNA #8:GGAGGAGCUUCUACGUGUA (SEQ ID NO: 22) Human TPRA40 siRNA #9:CCACAACCUUCCUGUACUU (SEQ ID NO: 23) Mouse Galpha(i)i siRNA #1 : G AAUAGC ACAGCCAAAUUA (SEQ ID NO: 24) Mouse Galpha(i)l si NA #2:GGAUGAUGCUCGCCAACUU (SEQ ID NO: 25)

Mouse Gaipha(i)l siRNA #3:UAACAGACGUCAUCAUAAA (SEQ ID NO: 26) Mouse Galpha(i)l siRNA #4: GAAGAGGAGUGUAAGCAGU (SEQ ID NO: 27) Mouse Smo si N A #1 : CAAUUGGCCUGGUGCUUAU (SEQ ID NO: 28)

Mouse Smo siRNA #2:GAGCGUAGCUUCCGGGACU (SEQ ID NO: 29)

Mouse Smo siRNA #3:GGAGUAGUCUGGUUCGUGG (SEQ ID NO: 30)

Mouse Smo siRNA #4:GCUACAAGAACUAUCGGUA (SEQ ID NO: 31)

Mouse Ift88 siRNA#5: GUAGCUAGCUGCUUUAGAA (SEQ ID NO: 32)

Mouse Ift88 siRNA#6:CGUCAGCUCUCACUAAUAA (SEQ ID NO: 33)

Mouse Ift88 siRNA#7:GCUUGGAGCUUAUUACAUU (SEQ ID NO: 34)

Mouse Ift88 siRNA.#8 : CGG AGAAUGUUGAA.UGUUU (SEQ ID NO: 35)

Mouse Ssnal siRNA #1 : GAACCUGACUAAAGCCACA (SEQ ID NO: 36)

Mouse Ssnal siRNA #2: GCAACGAGUUUGACCGGAC (SEQ ID NO: 37)

Mouse Ssnal siRNA #3: GG AGUUGUGUCAG AAG CG A (SEQ ID NO: 38)

Mouse Ssnal siRNA #4: ACGAGUUGGUCAAGUGCAU (SEQ ID NO: 39)

SI 2 Gli-luciferase assay.

S12 cells, which are lOTl /2 fibroblasts stably transfected with 8x Gli -binding sites fused to a luciferase reporter (Frank-Kamenetsky et al., 2002), were plated at 6,000 cells/well of a white-walled clear-bottomed 96-well plate (Costar 3610) in regular growth medium (HG (high glucose)-DMEM, 10% FBS, 1% glutamine) and reverse transfected with siRNAs. After 48 hours, the medium was changed to 0.5% serum HG-DMEM ± 200 ng/'ml octyl-Shh (Genentech) and incubated for another 24 hours to stimulate hedgehog signaling. PKA was inhibited using 80 μΜ cell permeable 14-22 amide (Tocris Biosciences). G/ -luciferase activity was measured using an HTS-Steady Lite luciferase detection kit (Perkin Elmer) and a TopCount luminometer; multiple assays were carried out, each in triplicate. Data were fit to a 4-parameter sigmoidal equation, from which the IC50 was derived using Kaleidagraph (Synergy Software) and were corrected for cell viability using ceil Titer Glo (Promega). Real-Time quantitative PGR (qPCR). Total RNA was extracted from cells using the RNeasy Protect Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's protocol. On-column genomic DNA digestion was performed with RNAse-Free DNAse Set (Qiagen). cDNA synthesis from total RNA was conducted using the High Capacity Reverse Transcription Kit (Applied Biosystems, Foster City, CA) with random hexamer primers. Quantitative PGR reactions were performed in triplicate on an ABI PRISAd^ 7500 Sequence detection system (Applied Biosystems) using murine ribosomal protein L19 (mRPL19) as the endogenous control. Gene expression was calculated using the relative quantification (2"AAU) method. PGR primers and Taqman probes (5' FAM and 3' TAMRA-labeled) are as follows:

Murine Glil

mGlil 5' primer: GCA GTG GGT AAC ATG AGT GTC T (SEQ ID NO: 4)

mGlil 3' primer: AGG CAC TAG AGT TGA GGA ATT GT (SEQ ID NO: 5) mGlil probe: CTC TCC AGG CAG AGA CCC CAG C (SEQ ID NO: 6) Human Glil

hGlil 5 ' primer: CGC TGC GAA AAC ATG TCA AG (SEQ ID NO: 7)

hGlil 3 ' primer: CCA CGG TGC CGT TTG GT (SEQ ID NO: 8)

hGlil probe: CAG TGC ATG GTC CTG ACG CCC A (SEQ ID NO: 9) Murine TPRA40

mTPRA40 5 ' primer: TGC AGG AGG CCA ATG GAA (SEQ ID NO: 10)

mTPRA40 3 ' primer: GGG CTC ACT GAT ATT GGA TGC T (SEQ ID NO: 1 1) mTPRA40 probe: ACA GCG TGG CCA CCG CCC (SEQ ID NO: 12) Human TPRA40

hTPRA40 5' primer: CCT GGT CTA CTC TCT GGT GGT CAT (SEQ ID NO: 13) KTPRA40 3' primer: CCG AGA AGG CAG GGA GAT G (SEQ ID NO: 14)

KTPRA40 probe: CCC AAG ACC CCG CTG AAG GAG C (SEQ ID NO: 15) Antibodies.

Purchased antibodies were anti-G!ii mouse monoclonal L42B10 (Cell Signaling

Technology cat #2643); anti~TPRA40 mouse monoclonal 6H2 (Santa Cruz Sc- 134350); rabbit anti-Aril 3b (Protemtech 1 171 1 -1 -AP); mouse anti-acetylated tubulin 6-1 I B- 1 (Sigma T6793); mouse anti-tyrosinated tubulin 1 A2 (Sigma T9028); mouse anti-gamma tubulin GTU-88 (Sigma T6657). Anti-GH3 mouse monoclonal 6F5 (Wen et al, 2010) was made at Genentech. Rabbit polyclonals 10322B to the C-terminal 1 10 aa of murine Smoothened and 12965B to a peptide comprising the C-terminal 19 aa of murine TPRA40 (CHTGSINSTDSERW A1NA-COOH) were raised at YenZym (South San Francisco, CA).

Western blotting.

Cells were lysed in RIPA buffer (50 mM Tris [pH 7,5], 150 mM NaCL 1 mM EDTA, 1%

Triton X-100, 0, 1% SDS, 1% sodium deoxycholate) containing freshly added phosphatase inhibitor cocktails Ϊ and ii (Sigma), Complete protease inhibitor cocktail (Roche), and 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma). Equal amounts of protein (as measured using the BCA kit (Pierce)) were separated on 4-12% reducing Tris-Glycine gels prior to transfer to nitrocellulose membranes (Invitrogen) and blocking in 5% w/v milk in TBST (Tris-buffered saline with 0.05% Tween 20). Anti-Gli3 6F5 (Wen et al. 2010) was used at 51ug/rnl, mouse anti- TPRA40 at 1 : 1000 and rabbit anti-TPRA40 at 1 :1000 overnight at 4 °C, Detection was accomplished using HRP-conj ugated anti-rabbit (Jackson Immunoresearch) or anti-mouse secondary antibodies (GE Healthcare) and ChemiGlow (alpha-Innotech). Blots were reprobed for protein loading using mouse anti tubulin 1A2 at, 1 : 10,000 followed by HRP-anti-mouse and ECL detection (GE Healthcare). Blots were exposed to BioMax light film (Kodak).

Immunofluorescence microscopy.

S12 cells were plated on 8-well slides at 3x l 04 cells/well and reverse transfeeted with siRNAs as above. After 72 hours knockdown, with the final 16-24 hours in serum- free media ± 200ng/ml octyl-Shh (Genentech), cells were fixed with 3% PFA for 20 minutes at room temperature and quenched for 10 minutes in 50 mM H CI. Cells were then penneabilized with 0.1% Triton-X-100 (Sigma) in PBS for 10 minutes, blocked in 1 % BSA for 30 minutes and subsequently incubated with the following primary antibodies: rabbit anti-Smo l .ug/ml, mouse a.nti-TPRA40 (1 :50), in conjunction with 1 :3000 mouse anti-acetylated tubulin and/or 1 :2300 mouse anti-gamma tubulin or rabbit anti-Aril 3b (1 :400). After 3x 10 minute washes, Cy3 or FITC-conj ugated secondary anti-rabbit or anti-mouse antibodies (Jackson Immunoresearch) were applied for at least 20 minutes at room temperature, washed 3x and coverslips were mounted using ProLong Gold with DAPI ( Invitrogen). Slides were imaged using Axio Imager M2 microscope with a 60X objective and SlideBook 5,5 software. The percentage of cilia (as identified by acetylated tubulin or Aril 3b staining) that stained for TPRA40 or Smo in the presence or absence of Hh stimulation was counted manually.

293 CRE-luciferase assay for cyclic AMP.

HEK293 cells were plated at 10,000 cells/well in 96-well white walled plates. Ceils were reverse transfected with 50ng Cre-luciferase, 10 ng Renilla Luciferase (Proniega) and 90 ng of murine TPRA40 with an N-terminai HA tag (pRK-nHA-TPRA40) with 0.45 μί of Fugene 6. After 36 hours, different amounts of Forskolin (Cell Signaling) were added for 4 hours and the Firefly and Renilla luciferase were measured using Dual Glow Luciferase Substrate (Promega) and a TopCount or EnVision himinometer.

Statistics. Comparison of TPRA40-depleted versus non-depleted cells was performed using the student's unpaired t-test ( w.graphpad.com). *, p <0.05; **, p < 0.01 ; ***, p < 0.001

S equence Information

SEQ ID NO: 1- Human TPRA40 amino acid sequence (GenBank Accession No. AAH50711.1) MDTLEEVTWANGSTALPPPLAPNISVPHRCLLLLYEDIGTSRVRYWDLLLLIPNVLFLIF LLW LPSARAKIRITSSPIFITFYILVFWALVGIARA SMTVSTSNAATVAD ILWEl TRFFLLAJELSVIILGLAFGBLESKSSIKRVLAITTVLSLAYSVTQGTLEILYPDAHLSA EDFNIYGHGGRQFWLVSSCFFFLVYSLYVILPKTPL ERISLPSRRSFYVYAGILALLNL LQGLGSVLLCFDIIEGLCCVDATTFLYFSFFAPLIYVAFLRGFFGSEPKILFSYKCQVDE TEEPDVHLPQPYAVARREGLEAAGAAGASAASYSSTQFDSAGGVAYLDDIASMPCHTG

81N8TDSERWKAINA

SEQ ID NO: 2- Mouse TPRA40 amino acid sequence (GenBank Accession No. NP 036036.2) MASLQEAJNGSTAWPPPTASNISEPHQCLLLLYEDIGSSRVRYWDLLLLIPNVLFFIFLLWK LPLARAKIR SSPIFITFYILWVVALVGIARAVVSMTVSASDAATVADKJLWEITRFFLL AIELSVIILGLAFGHLESKSSIKRVLAITTVLSLAYSVTQGTLEILYPDSHLSAEDFNIYGHG GRQFWLVSSCFFFLVYSLWILPKTPLKERVSLPSRRSFYVYAGILATLNLLQGLGSALLC ANUVGLCCVDATTFLYFSFFAPLIYVAFLRGFFGSEPKJLFSY CQVDEAEEPDMHLPQP YAVARREGIESAGPACASAANYSSTQFDSAGVAYLDDIASMPCHTGSINSTDSERWKA1

NA SEQ ID NO: 3- Zebrafish TPRA40 amino acid sequence (GenBank Accession No.

NP..001025422.1)

MLETVTDVASFVHYGNTSVFPTADNSSEIIPDGESNISKPHRCLQVLYDDIGTSRVRYWD VMLLIPNVAFLVFLMWK PSARAKJRLT^

AATLIDKVLWEITRFFLLAIELSVIILGLAFGHLES SSIKRVLAITAVLSLAYSITQGTLEIR FPDKFII,SAKDFNIYGHGGRI^FWL.ASSCFFFL ΎSL.IVILPKTPIRE ISLPS RSFY ΎSGII, ALL LVQGLGSALLCADIIEGLCCvOWTFLWSWAPLIYv FLKGFFGSEPKnLFSYKSQ IΓ)EPEΓ)SDΛ^HLPHTSSSGLGR DLDRGSYSSTQIDGSG YLDDΛ/ /ΓSGPFGGAHSINSΛ )S DRWR.STNT SEQ ID NO: 4- exemplary murine Glil 5' primer

GCA GTG GGT AAC ATG AGT GTC T

SEQ ID NO: 5- exemplary murine Glil 3' primer

AGG CAC TAG AGT TGA GGA ATT GT

SEQ ID NO: 6- exemplary murine Gli l probe

CTC TCC AGG CAG AGA CCC GAG C

SEQ ID NO: 7- exemplary human Glil 5' primer

CGC TGC GAA AAC ATG TGA AG

SEQ ID NO: 8- exemplary human Glil 3' primer

CCA CGG TGC CGT TTG GT SEQ ID NO: 9- exemplary human Glil probe

CAG TGC ATG GTC CTG ACG CCC A SEQ ID NO: 10- exemplary murine TPRA40 5' primer TGC AGG AGG CCA ATG GAA SEQ ID NO: 1 1- exemplary murine TPRA40 3 ' primer GGG CTC ACT GAT ATT GGA TGC T

SEQ ID NO: 12- exemplary murine TPRA40 probe ACA GCG TGG CCA CCG CCC

SEQ ID NO: 13- exemplary human TPRA40 5' primer CCT GGT CTA CTC TCT GGT GGT CAT

SEQ ID NO: 14- exemplary human TPRA40 3' primer CCG AGA AGG C AG GGA GAT G

SEQ ID NO: 15- exemplary human TPRA40 probe CCC AAG ACC CCG CTG AAG GAG C SEQ ID NO: 16- Mouse TPRA40 siRNA #9

GGAGGAGUUUCUACGUGUA

SEQ ID NO: 17- Mouse TPRA40 siRNA #10

CGGC AGUUCU GGCUCGUCA

SEQ ID NO: 18- Mouse TPRA40 siRNA #1 1

CGACAGUUGCUGACAAGAU

SEQ ID NO: 19- Mouse TPRA40 siRNA #12

GCCAUUGAGCUGAGUGUGA SEQ ID NO: 20- Human TPRA40 siRNA #6 GGUCAGCUCCIJGCUUCUUC

SEQ ID NO: 21- Human TPRA40 siRNA #7 GAGAGUAAGUCCAGCAUCA

SEQ ID NO: 22- Human TPRA40 siRNA #8 GGAGGAGCUUCUACGUGUA SEQ ID NO: 23- Human TPRA40 siRNA #9 CCACAACCUUCCUGUACUU

SEQ ID NO: 24- Mouse Galpha(i)! siRNA #1 GAAUAGCACAGCCAAA.UUA

SEQ ID NO: 25- Mouse Galpha(i)l siRNA #2 GGAUGAUGCUCGCCAACUU

SEQ ID NO: 26- Mouse Galpha(i)l siRNA #3 UAACAGACGUCAUCAUAAA

SEQ ID NO: 27- Mouse Galpha(i)l siRNA #4 GAAGAGGAGUGUAAGCAGU SEQ ID NO: 28- Mouse Smo siRNA #1 CAAUUGGCCUGGUGCUUAU

SEQ ID NO: 29- Mouse Smo siRNA #2 GAGCGUAGCUUCCGGGACU

SEQ ID NO: 30- Mouse Smo siRNA #3 GGAGUAGUCUGGUUCGUGG

SEQ ID NO: 31- Mouse Snio siRNA #4 GCUAC AAG AACUAUCG GUA

SEQ ID NO: 32- Mouse Ift88 siRNA#5 GUAGCUAGCUGCUUUAGAA

SEQ ID NO: 33- Mouse Ift88 siRNA#6 CGUC AG CUCUC ACUA AU A

SEQ ID NO: 34- Mouse Ift88 siRNA#7 GCUUGG AGCUUAUU A C AUU SEQ ID NO: 35- Mouse Ift88 siRNA#8 CGGAGAAUGUUGAAUGUUU

SEQ ID NO: 36- Mouse Ssnal siRNA #5 GAACCUGACUAAAGCCAC

Q ID NO: 37- Mouse Ssnal siRNA #2

GCAACGAGUUUGACCGGAC

SEQ ID NO: 38- Mouse Ssnal siRNA #3 GGAGUUGUGUCAGAAGCGA

ID NO: 39- Mouse Ssnal siRNA #4

ACGAGUUGGUCAAGUGCAU

SEQ ID NO: 40- TPRA40 Sense Morpholino ACATCGGTCACCGTCTCCAGCATTC SEQ ID NO: 41- TPRA40 Antisense Morpholino

ACAACCGTGACCCTCTCCACCATTC SEQ ID NO: 42- human Smoothened amino acid sequence (GenBank Aceesion No.

NP_ 005622.1)

MAAARPARGPELPLLGLLLLLLLGDPGRGAASSGNATGPGPRSAGGSARRSAAVTGPPP

PLSHCGRAAPCEPLRYNVCLGSVLPYGATSTLLAGDSDSQEEAHGKLVLWSGLRNAPR

CWAVIQPLLCAVYMP CENDRVELPSRTLCQATRGPCAIVERERGWPDFLRCTPDRP'PE GCTNEVQNIKF^SSGQCE LVRTDNP SWTEDW.GCGIQCQ PLFTEAEI1QDMHSYIA. AFGAVTGI TI.FTI.ATFVADWRNS RYPAVILFY ACFFVGSIGWL.AQFMDGAEEEIV' CRADGTMRL iEPTS ETI,SCVIIF\ YALA/iA.GWWFWI.TYAWHTSF AI,GTTYQPI. SGKTSYFinXTWSLPFVT^TVAn^AVAQVDGDSVSGICFVGYK YRYRAGFVXAPIGLVXI VGGYFLIRGVMTLFSIKSNHPGLLSEKAASKI ETMLRLGIFGFLAFGFVIJTFSCHFYDFF NQAEWERSFRDYΛ CQANΛXIGL T QPIPDCEI NRPSL.IΛX ][NLFAMFGTGIAMSTW \^VTKATLLIWRRTWCRLTGQSDDEP RIKKSKMIAKAFSKRHELLQNPGQELSFSMI-IT VSHDGPVAGLAFDLNEPSADVSSAWAQHVTKMVARRGAILPQDISVTPVATPVPPEEQA NLWLVEAE1SPELQKRLGRKKKRR R KEVCPLAPPPELHPPAPAPSTIPRLPQLPRQKC LVAAGAWGAGDSCRQGAWTLVS PFCPEPSPPQDPFLPSAPAPVAWAHGRRQGLGPIH SRTNLMDTELMDADSDF

SEQ ID NO: 43- human Suppressor of Fused (SuFu) amino acid sequence (GenBank Aceesion No. NM_016169.2)

MAELRPSGAPGPTAPPAPGPTAPPAFASLFPPGLHAIYGECRRLYPDQPNPLQVTAIVKY WLGGPDPLDYVSMYRNVGSPSANIPEHWHYISFGLSDLYGDNRVHEFTGTDGPSGFGFE LTFRLKRETGESAPPTWPAELMQGLARYVFQSENTTCSGDHVSWHSPLDNSESRIQHML LTEDPQMQPYQTPFGVVTFLQIVGVCTEELHSAQQWNGQGILELLRTVPIAGGPWLITD MRRGETIFEIDPHLQERVDKGIETDGSNLSGVSAKCAWDDLSRPPEDDEDSRSICIGTQPR

RLSGKDTEQIRETLRRGLEINSKPVLPPINPQRQ GLAHDRAPSRKDSLESDSSTAIIPHELI RTRQLESVHLKiTSiQESGALIPLCLRGRLLHGRHFTYKSITGDMAITFVSTGVEGAFATEE HPYAAHGPVVLQlLLTEEF MLEDLEDLTSPEEFKXPKEYSWPEKKL VSiLI5D rVFDS PLH

SEQ ID NO: 44- human Suppressor of Fused (SuFu) cDNA sequence (GeiiBank Accession No, NM_016169.2)

CGCCGTGCGCAGGCGCGGAGCTAGACCTCGCTGCAGCCCCCATCGCCTCGGGGAGT

CTCACCCACCGAGTCCGCCCGCTGGCCCGTCAGTGCTCTCCCCGTCGTTTGCCCTCTC

CAGTTCCCCCAGTGCCTGCCCTACGCACCCCGATGGCGGAGCTGCGGCCTAGCGGCG

CCCCCGGCCCCACCGCGCCCCCGGCCCCTGGCCCGACTGCCCCCCCGGCCTTCGCTT CGCTCTTTCCCCCGGGACTGCACGCCATCTACGGAGAGTGCCGCCGCCTTTACCCTG ACCAGCCGAACCCGCTCCAGGTTACCGCTATCGTCAAGTACTGGTTGGGTGGCCCAG ACCCCTTGGACTATGTTAGCATGTACAGGAATGTGGGGAGCCCTTCTGCTAACATCC CCGAGCACTGGCACTACATCAGCTTCGGCCTGAGTGATCTCTATGGTGACAACAGAG TCCATGAGTTTACAGGAACAGATGGACCTAGTGGTTTTGGCTTTGAGTTGACCTTTC GTCTGAAGAGAGAAACTGGGGAGTCTGCCCCACCAACATGGCCCGCAGAGTTAATG CAGGGCTTGGCACGATACGTGTTCCAGTCAGAGAACACCTTCTGCAGTGGGGACCAT GTGTCCTGGCACAGCCCTTTGGATAACAGTGAGTCAAGAATTCAGCACATGCTGCTG

CAGATCGTTGGTGTCTGCACTGAAGAGCTACACTCAGCCCAGCAGTGGAACGGGCA GGGCATCCTGGAGCTGCTGCGGACAGTGCCTATTGCTGGCGGCCCCTGGCTGATAAC TGACATGCGGAGGGGAGAGACCATATTTGAGATCGATCCACACCTGCAAGAGAGAG TTGACAAAGGCATCGAGACAGATGGCTCCAACCTGAGTGGTGTCAGTGCCAAGTGT GCCTGGGATGACCTGAGCCGGCCCCCCGAGGATGACGAGGACAGCCGGAGCATCTG CATCGGCACACAGCCCCGGCGACTCTCTGGCAAAGACACAGAGCAGATCCGGGAGA CCCTGAGGAGAGGACTCGAGATCAACAGCAAACCTGTCCTTCCACCAATCAACCCT CAGCGGCAGAATGGCCTCGCCCACGACCGGGCCCCGAGCCGCAAAGACAGCCTGGA AAGTGACAGCTCCACGGCCATCATTCCCCATGAGCTGATTCGCACGCGGCAGCTTGA GAGCGTACATCTGAAATTCAACCAGGAGTCCGGAGCCCTCATTCCTCTCTGCCTAAG GGGCAGGCTCCTGCATGGACGGCACTTTACATATAAAAGTATCACAGGTGACATGG CCATCACGTTTGTCTCCACGGGAGTGGAAGGCGCCTTTGCCACTGAGGAGCATCCTT ACGCGGCTCATGGACCCTGGTTACAAATTCTGTTGACCGAAGAGTTTGTAGAGA AAATGTTGGAGGATTTAGAAGATTTGACTTCTCCAGAGGAATTCAAACTTCCCAAAG AGTACAGCTGGCCTGAAAAGAAGCTGAAGGTCTCCATCCTGCCTGACGTGGTGTTCG ACAGTCCGCTACACTAGCCTGGGCTGGGCCCTGCAGGGGCCAGCAGGGAGCCCAGC TGCTCCCCAGTGACTTCCAGTGTAACAGTTGTGTCAACGAGATCTCCACAAATAAAA GGACAAGTGTGAGGAAGACTGCGCAGTGCCACCCCGCAGCCCAGTGGGGTGCCATG CACAGGCCACAGGCCCTCCACCTCACCTCCAGCTCAGGGGCCGCACCCCGCCGCTG GCTAAGCCTTGTGACCCATCAGGCCAGTGAGTGGGCAAATGCGGACCCTCCCTGCCT GCAGCCTGCACAGATTCTGGTTTGAGGTTTGACTCTGGACCCTGGCTGTGCCCCTAG GTGGAGACAGCCCTCTTTCTCACCTACCCCCTGCCGCACAGCCCAGCAGGAGGGAG GCGGACAGCCAGATGCAGAGCGAGTGGATGCACTTCCCAGCTCATCTCTGGAAGCC TTTGCTACTCAAGCTCCTCTGGCCGCGGAACAATTCCTCTGATCATGTTTGGTTTTCT TCTTCCTTATTTTATTTTGTAGAAACCGGGTGGTATTTTATTGCTCTGCAAAGATGTC CAGAAGCCATGTATATAATGTTTTTTAAACAGAACTTCATTCCCCGTTGAACTTTCGC ATTCTCTGACAGAGGCCTAGGGCTGTATCTCTCCCTGGGCTGCCACCAGAGAAGGTG CTTGGTGTTCGCCTGCCAGCCCAGAGCCCTGGAGGAGCCGGCTGCACAGAGAGGCT TTTCTTCCCAGCTGGGCCTGATGGAGCCCGGGGCAGGGGGAGAGTAGAGACACTCC CTTGTGCAGCTTTGAGCCTAGTTTAGCTGGGGCCAGGGAGGGGTGCTACTGTTTTCC AAGTGAATGGGTCTCAAAGACTTGGTGACCCCAGCCTCATCTTCTAGGCCTTTTCCA TCCAACCAGGCCTACCTGGGAGAGGGTGAGGTTCAGCACATCACACACCATCCCCA CTGTCATTCAGGGCCTGGGTCTCCAGCTCTGTAACCAGTCCTGTCCCATTTCCTCAGT CCCTGGGCCTCCCAGCCTTCAGGCTGTAGGGCTGCCTTACTAAAATTGAAAAAT CCACCTCTTAACATCTCTTTCACTTTGGTTTTGCTAACACTGCTCTCTGCTGCCCTCCC ATCCTCCCTGTATCCATTCATGCCCTATCTTTCATTCTCCACTCCTAATCCCTCTCC1T TCTGGCATCCTGGCCTCTCGTGGTCCTCAGCCCCTCACCCCCAGTACTGCAGATCTCA CAGTTTGCCTTCCAGAAGCCAGCCTATCTCTAGCCCATGGTTTTGGAGTTCCTCTCGG GTTATCTCCCACGCCTGACCTGGAACCAGCAAGCCCCTTTCCTGCCTTCTTACCCCCA ACTCTAGGGATGGGACTGTTACAATACTTCAAGATCACTCTTTACACCTCTTCAAAG CAAAGTCATGACAATGCAGGGCTCCTCATTGCTCCCATCTGCCTCTGCTGCACACAC AGGCACCAGCAGGGATGCCACAGGAGTGCCCACAGGGTGCAGGACTCCACTGATGA GAGATCCAGCCAAAGAGCTGCCCCCAGGGGTATGAGGGCACCAGCTGGGTTCTCCA GGGAGCAGGAGTTGGACCTCCATGGAGCCACTAGGCCTGGCCTCCTCTACACATCCC CAGGGCTATCTGGTTAATTCCATCAAGCTCAGAGTTAAAAGGCATATCAGCCTGG AGTATTTGGGAGAGACTGGCTGCAGATCCCCGCCAGCCAAGATGCAAGCCACTCGG GACCTGATGTCGGCAGCTGTGCCTCTACTGCCCTGAGGACTTACCAGAGGGAGCCCT ACTGGCCTTCCCCCACCACAGCAGCCCTGCCTGTGAAGCTCTTGTTTCTGACATTTCA CAGGCAGAGAGGTGCCATCAGTTCGCCTCCATTCCTTGCCACCATGACCAGCCTCTC CCTGAACTCTCTCTTGCTCGGGACCTGCCTGAGGGCTCCCTGCTGCAGTTCGCCGTA CTTCCATCTGCTGGGTGCCTCCATCGTTGGTTGGGTGGGGATGGGGCATTTTCTGAG CTAAGCTTTGTCATTAGTTTGTGAAGCACCTGGTCAGCAACCTGCCCCAGACCTGGA GGGTCTTTGTGGACTGAAGGTAGACACCAGCCAGCATGGTGGCCCTGTTCTGGGGG AGCAGGGTAAGGCAGGAGGAAGTGGGTGAGCTCCGAGATGATGAGCACATGAAGC CTGTGGCCCCTTCGTACCTGCAATATGTCAGGAGCCTCACGCTCACCCAAGATCCTG CAGGGGCCAGGCTCCATCTCACTGGCTCTGAGGGCAGGACAGGGTATCACACATTT CTCACCAGGCCTCCTTTCCTATGGGCATTGGTGCCTCCCAGAGGTTTCTTGGGCTGCT GGCTGGTGAGAGAGGACCCTTAAAGAAGATCAAGCCAAGCTGACCTTGGACCCTGT CCAGCACAGCTTCTGGCACAGGATGCTTGGTGAATGTACCCTTTCTTTCCCTCCCTGC AGCTCTGAGGGAGCCCCTGACCTTGTAGTGGGTGGAGGAGGTAAGGGGCCTCCCTC CCTAAATCTGCCTCTTCTGCAAGCTACTTGGAGACTTGCCTAGTTGTACCCACCCCTC CAGGTCCCTGGTGCTAGAGCTTCTGAGAAGGGCCTTTCCCTTTCCTCTTTGCCTGCTA TATAAGGCAGGCTCCTGTGGCTCTGCTGGCTCAGTGTGGGCTGCAGGAGGACTGCAG ACTCAGCTGCAATTCTGAGGGGGGTTTGGGAGGCTTGTGCGAGGTCTCAGGCCTGTG TGGGGAGCTGGTGCCTCTTCCTGCCCGTATCTTTCTCTTCCAAGGGCAGTGCTCCAAG GCAGGGACTGGAGAAGCCAAGGGGAGAGTCTAAAAGGGCTAGAGCATTTTTAAAA ATAGACACAGGGTCTTGGGACTGGGGTTTCGGATTGAGTTGCAAGCAGGGAGAAAA CCTGAAGGTCGGTGCCCCTATGGGGCTGACCAGTAGAGAATTTCCTTTACTGTATTT TTGTGTCTGGTCTTCCCTTTCTGGCTTCTAGGACATCCATGCCAGGTGAGGTGCCTGG GTCCCTGTTACAAGTCAGGAGCCCTGTAGGGAGACCCCTCCT1TTGTACAAGTACCT GAATGCTGCGACAAGCAGATTTTTGTAAAATTTTATATTAGTTTTTAATGTCAGTGGC GACTCGGTTCCTGGGGCTGCAGCCAGCCTGGGACTTTTGTAAGAATTTTTGGGTGAC TCACTTAGATGTCGTTTCCTTCTTGCCCCCTCTTCCTCTCTGTAATCTAAGTGCATTAA ACATCTTTGCAG SEQ ID NO: 45- Mouse Gli3 siRNA #5

GAACAACCCUAGUCAAGGA SEQ ID NO: 46- Mouse Gli3 siRNA #6

GCUCUACCGUGCAGAAUUA

SEQ ID NO: 47- Mouse Gli3 siRNA #7

GCUUAGUGCUGCAAAAUUA

SEQ ID NO: 47- Mouse Gli3 siRNA #8

CCUCACAUAUCAACA.UCUA

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference.

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

Claims

We Claim:
1. A method of screening for a TPRA40 antagonist, wherein the method comprises: a) contacting a cell that expresses TPRA4Q, adenylyl cyclase and a. reporter construct indicative of adenylyl cyclase activity with an activator of adenylyl cyclase and an agent:
b) determining, as compared to an untreated control, whether the agent rescues adenylyl cyclase activity suppressed by TPRA40 expression, wherein if the agent increases adenylyl cyclase activity relative to the untreated control, which untreated control comprises TPRA40 expressing ceils that are contacted with the adenylyl cyclase activator but are not contacted with the agent, then the agent is identified as a TPRA40 antagonist.
2. A method of identifying a TRPA40 antagonist, comprising:
a) providing a cell that expresses TRPA40 and that expresses a reporter gene capable of indicating adenylyl cyclase activity;
b) contacting the cell with an activator of adenylyl cyclase and with an agent, wherein the cells are contacted with the activator and the agent simultaneously, concurrently, or consecutively; and
c) determining, as compared to a control, whether the agent rescues adenylyl cyclase activity induced by the activator, wherein if the agent increases the adenylyl cyclase activity relative to the control, then the agent is identified as a TPRA40 antagonist.
3. A method of screening for an agent for inhibiting the proliferation, growth or survival of a cancer cell, wherein said method comprises:
a) screening for an agent that binds to TPRA40 protein, reduces expression of TPRA40, inhibits transport of TPRA40 protein to the plasma membrane or to primary cilia, prevents activation of TPRA40 or uncouples TPRA40 from God;
b) contacting a cancer cell with an amount of the agent identified in step a), wherein said cancer cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein said one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of liga d, and c) determining, as compared to a control, whether said agent inhibits the proliferation or growth of said cancer ceil, wherein if said agent inhibits cell proliferation or growth relative to the control, then an agent that inhibits the proliferation or growth of said cancer cell is identified.
4. A method of screening for an agent for inhibiting hedgehog signaling in a cell, wherein said method comprises:
a) screening for an agent that binds to TPRA40 protein, reduces expression of TPRA40, inhibits transport of TPRA40 protein to the plasma, membrane or to primary cilia, prevents activation of TPRA40 or uncouples TPRA40 from God;
b) contacting a cell with an amount of the agent identified in step a), wherein said cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein said one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, and
c) determining, as compared to a control, whether said agent inhibits hedgehog signaling in said cell, wherein if said agent inhibits hedgehog signaling in said cell relative to the control, then an agent that inhibits hedgehog signaling is identified.
5. A method of identifying a TPRA40 antagonist, wherein said method comprises: a) screening for an agent that binds to TPRA40 protein;
b) contacting a cell with an amount of the agent identified in step a), wherein said cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein said one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, and
c) determining, as compared to a control, whether said agent that binds to TPRA40 protein also inhibits hedgehog signaling in said cell, wherein if said agent inhibits hedgehog signaling in said cell relative to the control, then the agent is identified as a TPRA40 antagonist.
6. The method of any of claims 1 -5, wherein said cell is in culture.
The method of any of claims 1 -5, wherein said cell is in an animal.
8. The method of any of claims 1-7, wherein said agent is a small molecule.
9. The method of any of claims 1-7, wherein said agent is a polypeptide or an antibody.
10. The method of any of claims 1-7, wherein said agent is an siRNA or shRNA that decreases TPRA40 translation.
11. The method of any of claims 1 -9, wherein said agent binds TPRA40 protein.
12. The method of any of claims 3-11, wherein said agent is identified in step a) using a yeast two-hybrid screen.
13. The method of any of claims 1-11, wherein said agent is identified in step a) using a high throughput binding or activity screen of a small molecule library.
14. The method of any of claims 1 -13, wherein said agent inhibits transport of the TPRA40 protein to the plasma membrane or to primary cilia.
15. The method of any of claims 3-54, wherein said agent is identified in step a) by a method comprising the steps of:
i) contacting a ceil expressing TPRA40 with an agent,
ii) determining the localization of TPRA40 in said first ceil expressing TPRA40 using immunofluorescence.
16. The method of any of claims 3-14, wherein said agent is identified in step a) by a method comprising the steps of:
i) contacting a ceil expressing TPRA40 with an agent; and
ii) determining the levels of TPRA40 in a. plasma membrane or ciliary membrane fraction.
17. The method of any of claims 1 -16, wherein said agent reduces expression of TPRA40 protein or RNA.
18. The method of any of claims 3-17, wherein said agent is identified in step a) by a method comprising:
i) contacting a cell expressing TPRA40 with an agent; and
ii) determining activity of TPRA40 in said cell using a G/i-lueiferase reporter or adenylyl cyclase reporter assay.
19. The method of any of claims 3- 7, wherein said agent is identified in step a) by a method comprising:
i) contacting a cell expressing TPRA40 with an agent; and
ii) determining the expression of GUI in said cell by RT-PCR or Western Blotting.
20. The method of any of claims 3-17, wherein said agent is identified in step a) by a method comprising:
i) contacting a cell expressing TPRA40 with an agent; and
ii) determining the expression of TPRA40 in said cell using RT-PCR or Northern Blot analysis of TPRA40 RNA or Western Blot, flow cytometry, immunofluorescence or
immunohistochemistry analysis of TPRA40 protein.
21. The method of any of claims 1-20, wherein said cell used in step (b) comprises one or more mutations in a hedgehog signaling pathway gene.
22. The method of any of claims 1 -21 , wherein said one or more mutations are in smoothened, and the cell has a smoothened gain-of-function.
23. The method of any of claims 1 -22, wherein said gain-of-function smoothened mutation results in a constitutively active smoothened protein.
24. The method of any of claims 1-23, wherein said one or more mutations are in patched and the ceil has a patched loss-of- function.
25. The method of any of claims 1-24, wherein said tumor overexpresses a hedgehog protein,
26. The method of any of claims 1-25, wherein said one or more mutations are in suppressor-of-fused, and the cell has suppressor-of-fused loss-of-function. 27. The method of any of claims 1 -26, wherein a reporter gene is used in order to determine whether adenylyl cyclase activity has been rescued by an agent.
28. The method of claim 27, wherein the reporter gene is a luciferase gene controlled by a cAMP response element.
29. The method of any of claims 1-28, wherein the activator of adenylyl cyclase is forskolin.
30. The method of any of claims 1 -28, wherein the acti vator of adenylyl cyclase activity is 8-bromo-cAMP.
31. The method of any of claims 1 -30, wherein the agent is further assessed in an assay for hedgehog signaling. 32. The method of claim 31 , wherein the assay for hedgehog signaling comprises the steps of:
i. contacting a ceil with an amount of the agent, wherein said cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein said one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, and ii. determining, as compared to a control, whether said agent inhibits hedgehog signaling in said cell, wherein if said agent inhibits hedgehog signaling in said cell relative to the control, then an agent that inhibits hedgehog signaling is identified. 33. The method of any one of claims 1-32, wherein the TPRA40 is exogenously expressed in a cell,
34. The method of claim 33, wherein the TPRA40 is stably expressed in the cell. 35. The method of claim 33, wherem the TPRA40 is transiently expressed in the cell.
36. The method of claim 33, wherein the cell is transformed with a vector expressing TPRA40 protein. 37. The method of any of claims 1 -36, wherein the cell is an S I 2 cell.
38. The method of any of claims 1 -36, wherein the cell is a 293T cell .
39. The method of any of claims 1-38, wherein the reporter gene is a luciferase gene controlled by a cA P response element.
40. The method of any of claims 1-39, wherein the activator is a combination of one or more of forskolin, 8-bromo-cAMP or dibutyryl-cAMP. 41. A method of screening for a TPRA40 agonist, wherein the method comprises: a) contacting a ceil that expresses TPRA40, adenylyl cyclase and a reporter constmct with an activator of adenylyl cyclase and an agent;
b) determining, as compared to an untreated control, whether the agent suppresses adenylyl cyclase activity, wherein if the agent suppresses adenylyl cyclase activity relative to the untreated control, which untreated control comprises TPRA40 expressing cells that are contacted with the adenvlyl cyclase activator but are not contacted with the agent, then the agent is identified as a TPRA40 agonist.
42. A method of identifying a TRPA40 ago ist, comprising:
a) providing a cell that expresses TRPA40 and that expresses a reporter gene capable of indicating adenvlyl cyclase activity;
b) contacting the cell with an activator of adenvlyl cyclase and with an agent, wherein the cells are contacted with the activator and the agent simultaneously, concurrently, or consecutively; and
c) determining, as compared to a control, whether the agent suppresses adenvlyl cyclase activity induced by the activator, wherein if the agent suppresses the adenvlyl cyclase activity relative to the control, then the agent is identified as a TPRA40 agonist.
43. A method of screening for an agent for inducing hedgehog signaling in a cell, wherein said method comprises:
a) screening for an agent that binds to TPRA40 protein, induces expression of TPRA40, facilitates transport of TPRA40 protein to the plasma membrane or to primary cilia, induces activation of TPRA40 or promotes its coupling with God;
b) contacting a cell with an amount of the agent identified in step a), and
c) determining, as compared to a control, whether said agent induces hedgehog signaling in said cell, wherein if said agent induces hedgehog signaling in said cell relative to the control, then an agent that induces hedgehog signaling is identified.
44. A method of identifying a TPRA40 agonist, wherein said method comprises: a) screening for an agent that binds to TPRA40 protein;
b) contacting a cell with an amount of the agent identified in step a), and
c) determining, as compared to a control, whether said agent that binds to TPRA40 protein also induces hedgehog signaling in said cell, wherein if said agent induces hedgehog signaling in said cell relative to the control, then the agent is identified as a TPRA40 agonist.
45. The method of any of claims 45 -44, wherein the agent is a small molecule.
46. The method of any of claims 41-44, wherein the agent is a polypeptide.
47. The method of any of claims 41-44, wherein the agent is a polynucleotide.
48. The method of any of claims 41-47, wherein said cell is in culture.
49. The method of any of claims 41-47, wherein said cell is in an animal.
50. The method of any of claims 4 -47, wherein said agent hinds TPRA40 protein.
51. The method of any of claims 4 -50, wherein said agent is identified in step a) using a yeast two-hybrid screen.
52. The method of any of claims 41-51 , wherein said agent is identified in step a) using a high throughput binding or activity screen of a small molecule library.
53. The method of any of claims 45-52, wherein said agent facilitates transport of the TPRA40 protein to the plasma membrane or to primary cilia.
54. The method of any of claims 43-53, wherein said agent is identified in step a) by a method comprising the steps of:
i) contacting a ceil expressing TPRA40 with an agent,
ii) determining the localization of TPRA40 in said first cell expressing TPRA40 using immuno fiuorescenc e .
55. The method of any of claims 41-54, wherein said agent is identified in step a) by a method comprising the steps of:
i) contacting a cell expressing TPRA40 with an agent; and
ii) determining the levels of TPRA40 in a plasma membrane or ciliary membrane fraction.
56. The method of any of claims 41 -55, wherein said agent increases expression of TPRA40 protein or 'NA.
57. The method of any of claims 41 -56, wherein said agent is identified in step a) by a method comprising:
i) contacting a cell expressing TPRA40 with an agent; and
ii) determining activity of TPRA40 in said cell using a G/ -luciferase reporter or adenylyl cyclase reporter assay. 58. The method of any of claims 41-56, wherein said agent is identified in step a) by a method comprising;
i) contacting a cell expressing TPRA40 with an agent; and
ii) determining the expression of TPRA40 in said cell by RT-PCR or using Northern Blot analysis of TPRA40 RNA.
59. The method of any of claims 41-56, wherein said agent is identified in step a) by a method comprising:
i) contacting a ceil expressing TPRA40 with an agent; and
ii) determining the expression of TPRA40 in said cell by Western Blot, flow cytometry, immunofluorescence or immunohistochemistry analysis of TPRA40 protein.
60. The method of claim 59, wherein a reporter gene is used in order to determine whether adenylyl cyclase activity has been suppressed by an agent. 61. The method of claim 60, wherein the reporter gene is a iuciferase gene controlled by a cAMP response element.
62. The method of any of claims 41 -61 , wherein the activator of adenylyl cyclase activity is forskolin.
63. The method of any of claims 41-61 , wherein the activator of adenylyl cyclase is 8-bromo-cAMP or dibutyryl-cAMP.
64. The method of any of claims 41-63, wherein the agent is further assessed in an assay for hedgehog signaling.
65. The method of any one of claims 41-64, wherein the TPRA40 is exogenouslv expressed in a cell, 66. The method of claim 65, wherem the TPRA40 is stably expressed in the cell.
67. The method of claim 65, wherein the TPRA40 is transiently expressed in the cell.
68. The method of claim 65, wherein the cell is transformed with a vector expressing TPRA40 protein.
69. The method of any of claims 41 -68, wherein the cell is an 812 cell.
70. The method of any of claims 41-68, wherein the cell is a 293T cell.
71. The method of claim 41 -70, wherein the reporter gene is a luciferase gene controlled by a cAMP response element.
72. A method of reducing hedgehog signaling in a cell, wherein said cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein said one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, wherein said method comprises the step of contacting said ceil with an effective amount of a TPRA40 antagonist. 73. A method of inhibiting unwanted growth, proliferation or survival of a cell, wherein said cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein said one or more mutations results in increased hedgehog signaling and/'or activation of the hedgehog signaling pathway in the absence of ligand, wherein said method comprises the step of contacting said cell with an effective amount of a TPRA40 antagonist.
74. A method of inhibiting growth, proliferation or survival of a tumor cell, wherein said cell is responsive to hedgehog protein or comprises one or more mutations in a hedgehog signaling pathway gene, wherein said one or more mutations results in increased hedgehog signaling and'or activation of the hedgehog signaling pathway in the absence of ligand, wherein said method comprises the step of contacting said cell with an effective amount, of a TPRA40 antagonist.
75. The method of any of claims 72-74, wherein the TRPA40 antagonist comprises an agent that inhibits the expression and/or activity and/or localization of TPRA40 by: (i) binding to TPRA40 DNA, RNA, or protein.
76. The method of any of claims 72-75, wherein hedgehog signaling is hyperactive in the cell. 77. The method of any one of claims 72-76, wherein said cell comprises one or more mutations in a hedgehog signaling pathway gene.
78. The method of claim 77, wherein said one or more mutations are in smoothened, and the cell has a smoothened gain-of-function.
79. The method of claim 78, wherein said gain-of-function smoothened mutation results in a constitutively active smoothened protein.
80. The method of any of claims 76-79, wherein said one or more mutations are in patched L and the ceil has a patched loss-of- function.
81. The method of any of claims 72-76, wherein said cell or an adjacent ceil overexpresses a hedgehog protein.
82. The method of claim 81 , wherein said overexpressed hedgehog protein is Sonic hedgehog protein.
83. The method of claim 81 , wherein said overexpressed hedgehog protein is Indian hedgehog protein. 84. The method of claim 81 , wherein said overexpressed hedgehog protein is Desert hedgehog protein.
85. The method of claim 77, wherein said one or more mutations are in suppressor- of-fiised, and the cell has suppressor-of-fused loss-of-fiinction.
86. The method of any of claims 76-85, wherein prior to contacting said cell with said TPRA40 antagonist, said cell is determined to have one or more mutations in a hedgehog signaling pathway gene or otherwise determined to exhibit hedgehog signaling activity. 87. The method of claim 86, wherein said cell is determined to exhibit hedgehog signaling activity by measuring GUI or Patched 1 levels.
88. The method of any of claims 72-87, wherein said cell is a cell in culture. 89. The method of claim 88, wherein the method comprises contacting a culture comprising a plurality of cells.
90. The method of any of claims 72-89, wherein said ceil is in a vertebrate, and contacting said cell comprises administering the TPRA40 antagonist to the vertebrate.
91. The method of claim 90, wherein said vertebrate is a human subject.
92. The method of claims 90 or 91, wherein said cell is a cancer cell and/or said vertebrate is a vertebrate diagnosed with cancer. 93. The method of claim 92, wherein said cancer cell is a cancer cell selected from the group consisting of: a. colon, lung, prostate, skin, blood, liver, kidney, breast, bladder, bone, brain, mediilloblastoma, meningioma, glioma, chondrosarcoma, gall bladder, sarcoma, rhabdomyosarcoma, basal ceil carcinoma, gastric, ovarian, esophageal, pancreatic, leukemia, lymphoma, multiple myeloma or testicular cancer cell.
94. A method of inhibiting unwanted growth, proliferation or survival of a cell, wherein said cell comprises one or more mutations in suppressor-of-jused, in which one or more mutations result in the cell having suppressor-of-fused loss-of-function, wherein said one or more mutations results in increased hedgehog signaling and/or activation of the hedgehog signaling pathway in the absence of ligand, wherein said method comprises the step of contacting said cell with an effective amount, of a TPRA40 antagonist.
95. A method of inhibiting growth, proliferation or survival of a rumor cell, wherein said cell comprises one or more mutations in suppressor-of-fused resulting in the cell having suppressor-of-fused loss-of-function, wherein said one or more mutations results in increased hedgehog signaling and/or acti vation of the hedgehog signaling pathway in the absence of ligand, wherein said method comprises the step of contacting said cell with an effective amount of a TPRA40 antagonist. 96. The method of any of claims 94 or 95, wherein said cell is a cell in culture.
97. The method of claim 96, wherein the method comprises contacting a culture comprising a plurality of cells. 98. The method of any of claims 94-97, wherein said ceil is in a vertebrate, and contacting said cell comprises administering the TPRA40 antagonist to the vertebrate.
99. The method of claim 98, wherein said vertebrate is a human subject.
100. The method of any of claims 98 or 99, wherein said cell is a cancer cell and/or said vertebrate is a vertebrate diagnosed with cancer.
101. The method of claim 100, wherein said cancer cell is a cancer cell selected from the group consisting of: a medulloblastoma, meningioma, adenoid cystic carcinoma, basal cell carcinoma, and rhabdomyosarcoma cancer cell.
102. The method of any of claims 72-101, wherein said TPRA40 antagonist comprises a polynucleotide molecule that inhibits the expression of TPRA40.
103. The method of claim 102, wherein the polynucleotide molecule is an antisense oligonucleotide that hybridizes to a TPRA40 transcript to inhibit expression of TPRA40.
104. The method of any of claims 72-103, wherein said TPRA40 antagonist comprises an RNAi that targets the TPRA40 mRNA transcript. 105. The method of claim 104, wherein the RNAi comprises an siRNA.
106. The method of claim 105, wherein said siRNA is 19-23 nucleotides in length.
107. The method of claim 105 or 106, wherein said siRNA is double stranded, and includes short overhang(s) at one or both ends.
108. The method of claim 104, wherein the RNAi comprises an shRNA.
109. The method of claim 105 or 108, wherein said siRNA or shRNA targets TPRA40 niRNA transcript.
1 10. The method of claim 104 or 109, wherein said siRNA comprises one or more of the nucleotide sequences selected from: SEQ ID NOs: 16-23.
111. The method of any of claims 72-101, wherein said TPRA40 antagonist is a small molecule that binds to TPRA40.
112. The method of any of claims 72-101, wherein said TPRA40 antagonist comprises an antibody that binds to TPRA40 protein. 1 13. The method of claim 1 12, wherein said antibody is a monoclonal antibody.
1 14. The method of any of claims 72-101, wherein said TPRA4G antagonist comprises a polypeptide antagonist. 115. The method of any one of claims 72-1 14, wherein said cell is also contacted with an additional antagonist of the hedgehog signaling pathway.
1 16. The method of claim 1 15, wherein said additional antagonist of the hedgehog signaling pathway is a veratrum-type steroidal alkaloid.
117. The method of claim 1 16, wherein said veratrum-type steroidal alkaloid is cyclopamine or AAD-cyclopamine or a derivative thereof.
1 18. The method of claim 1 16, wherein said veratrum-type steroidal alkaloid is jervine, IPI-269609 or IPl-926.
119. The method of claim 1 15, wherein said antagonist is a non- veratrum-type synthetic small molecule inhibitor of Smoothened.
120. The method of claim 1 19, wherein said antagonist is Erivedge (vismodegib), BMS-833923 (XLS 19), LDE225 (Erismodegib), PF-04449913, NVP-LDE225, NVP-LEQ506, TA -441 , XL-319, LY-2940680, SEN450, Itraconazole, MRT- 10, MRT-83, or PF-04449913.
121. The method of claim 1 15, wherein said additional antagonist of the hedgehog sig aling pathway is an antibody.
122. The method of claim 121 , wherein said antibody is an antibody that binds Sonic, Indian and/or Desert hedgehog protein.
123. The method of claim 1 15, wherein said additional antagonist of the hedgehog pathway is a small molecule hedgehog inhibitor.
124. The method of claim 123, wherein said hedgehog inhibitor is robotkinin.
125. The method of claim 1 15, wherein said additional antagonist of the hedgehog signaling pathway is selected from the group consisting of: vismodegib, sonidegib, BMS-833923, PF-04449913, and LY2940680.
126. The method of claim 1 15, wherein said additional antagonist of the hedgehog signaling pathway is an RNAi antagonist.
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