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Title:
COMPOSITIONS AND METHODS FOR TREATING NEOPLASIA
Document Type and Number:
WIPO Patent Application WO/2012/074842
Kind Code:
A2
Abstract:
The invention features compositions comprising agent that inhibits thymic stromal lymphopoietin (TSLP) or TSLP Receptor expression or biological activity, and related methods of using the compositions for treating or preventing cancer progression or metastasis.

Inventors:
BIRAGYN ARYA (US)
LEONARD WARREN J (US)
Application Number:
PCT/US2011/061872
Publication Date:
June 07, 2012
Filing Date:
November 22, 2011
Export Citation:
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Assignee:
US OF AMERICA AS REPRESENTED BY THE SECRETARY NAT INST OF HEALTH (US)
BIRAGYN ARYA (US)
LEONARD WARREN J (US)
Domestic Patent References:
WO2010086445A12010-08-05
Other References:
VALERIE I. BROWN ET AL.: 'Thymic stromal-derived lymphopoietin induces proliferation of pre-B leukemia and antagonizes mTOR inhibitors, suggesting a role for interleukin-7R a signaling' CANCER RESEARCH vol. 67, no. 20, 2007, ISSN 0008-5472 pages 9963 - 9970
ALEXANDER PEDROZA-GONZALEZ ET AL.: 'Thymic stromal lymphopoietin fosters human breast tumor growth by promoting type 2 inflammation' J. EXP. MED. vol. 208, no. 3, 21 February 2011, ISSN 0022-1007 pages 479 - 490
PUREVDORJ B. OLKHANUD ET AL.: 'Thymic stromal lymphopoietin is a key mediator of breast cancer progression' THE JOURNAL OF IMMUNOLOGY vol. 186, no. 10, 13 April 2011, ISSN 0022-1767 pages 5656 - 5662
Attorney, Agent or Firm:
CORLESS, Peter F et al. (P.O. Box 55874Boston, MA, US)
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Claims:
What is claimed is:

1. A method of reducing the invasiveness of a neoplastic cell, the method comprising contacting a neoplastic cell with an agent that inhibits thymic stromal lymphopoietin (TSLP) or TSLP Receptor expression or biological activity, thereby reducing the invasiveness of the neoplastic cell.

2. A method of treating a subject having a solid tumor, the method comprising

administering to a subject a therapeutically effective amount of an agent that inhibits TSLP or TSLP Receptor expression or biological activity, thereby treating the subject.

3. A method of treating or preventing metastasis in a subject having a neoplasm, the method comprising administering to a subject a therapeutically effective amount of an agent that inhibits TSLP or TSLP Receptor expression or biological activity, thereby treating or preventing metastasis in the subject.

4. A method of treating or preventing tumor progression or metastasis in a subject having a solid tumor, the method comprising administering to a subject a therapeutically effective amount of an agent that inhibits TSLP or TSLP Receptor expression or biological activity, thereby treating or preventing tumor progression or metastasis in the subject.

5. The method of any of claims 1-4, wherein the agent is selected from the group consisting of antibodies, polypeptides, nucleic acid molecule, inhibitory nucleic acid molecules, and small molecules.

6. The method of any of claims 1-4, wherein the agent is an antibody that binds TSLP.

7. The method of claim 6, wherein the antibody is a monoclonal antibody.

8. The method of claim 8, wherein the monoclonal antibody is a human or humanized antibody.

9. The method of any one of claims 1-8, further comprising the step of co-administering one or more chemo therapeutics.

10. The method of claim 9, wherein the one or more chemotherapeutics is selected from the group consisting of abiraterone acetate, altretamine, anhydrovinblastine, auristatin, azacitidine, bendamustine, bevacizumab, bexarotene, bicalutamide, BMS-184476, 2,3,4,5,6-pentafluoro-N- (3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, bortezomib, N,N-dimethyl-L- valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide, cachectin, capecitabine, cemadotin, cetuximab, chlorambucil, 3',4'-didehydro-4'-deoxy-8'-norvin-caleukoblastine, docetaxol, doxetaxel, carboplatin, carmustine (BCNU), cisplatin, cryptophycin,

cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, dasatinib, daunorubicin, dolastatin, doxorubicin (adriamycin), erlotinib, etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyurea taxanes, ifosfamide, imatinib, irinotecan, lenalidomide, liarozole, lonidamine, lomustine (CCNU), mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, 5-fluorouracil, nilutamide, onapristone, paclitaxel, panitumumab, pazopanib, prednimustine, procarbazine, rituximab, RPR109881, sorafenib, estramustine phosphate, sunitinib, tamoxifen, tasonermin, taxol, temozolomide, trastuzumab, tretinoin, vinblastine, vincristine, vindesine sulfate, vinflunine, and vorinostat.

11. The method of claim 5, wherein the inhibitory nucleic acid molecule is an siRNA, antisense nucleic acid molecule, or shRNA.

12. The method of any of the preceding claims, wherein the neoplastic cell is a cancer cell which is present in a solid tumor.

13. The method of claim 12, wherein the cancer is selected from the group consisting of breast cancer, prostate cancer, melanoma, glioblastomas, colon cancer, ovarian cancer, and non- small cell lung cancer.

14. A pharmaceutical composition for the treatment of a solid tumor comprising a therapeutically effective amount of an agent that inhibits TSLP or TSLP Receptor expression or biological activity. 15. A pharmaceutical composition for the treatment or prevention of metastasis comprising a therapeutically effective amount of an agent that inhibits TSLP or TSLP Receptor expression or biological activity.

16. The pharmaceutical composition of claim 14 or 15, further comprising one or more chemotherapeutics.

17. The pharmaceutical composition of claim 16, wherein the one or more chemotherapeutics is selected from the group consisting of abiraterone acetate, altretamine, anhydrovinblastine, auristatin, azacitidine, bendamustine, bevacizumab, bexarotene, bicalutamide, BMS- 184476, 2,3,4,5, 6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, bortezomib, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylamide, cachectin, capecitabine, cemadotin, cetuximab, chlorambucil, cyclophosphamide, 3',4'- didehydro-4'-deoxy-8'-norvin-caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine (BCNU), cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, dasatinib, daunorubicin, dolastatin, doxorubicin

(adriamycin), erlotinib, etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyurea taxanes, ifosfamide, imatinib, irinotecan, lenalidomide, liarozole, lonidamine, lomustine (CCNU), mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, 5-fluorouracil, nilutamide, onapristone, paclitaxel, panitumumab, pazopanib, prednimustine, procarbazine, rituximab, RPR109881, sorafenib, estramustine phosphate, sunitinib, tamoxifen, tasonermin, taxol, temozolomide, trastuzumab, tretinoin, vinblastine, vincristine, vindesine sulfate, vinflunine, and vorinostat. 18. A kit for the treatment of a neoplasm, the kit comprising an effective amount of an agent that inhibits TSLP or TSLP Receptor expression or biological activity and directions for using the kit for the treatment of a neoplasm.

19. A method of characterizing the aggressiveness of a neoplasm, comprising determining the

level of expression of TSLP in a subject sample, wherein an increased level of expression relative to a reference indicates that the neoplasm is aggressive.

20. The method of claim 19, wherein the reference is a cancer-free subject.

21. A method of monitoring a subject diagnosed as having a neoplasm, the method comprising determining the level of expression of TSLP in a subject sample, wherein an alteration in the level of expression relative to the level of expression in a reference indicates the severity of neoplasm in a subject.

22. The method of claim 21, wherein an increase in the level of TSLP indicates that the neoplasm is aggressive.

23. A method of monitoring a subject being treated for a neoplasm, the method comprising determining the level of expression of TSLP in a subject sample, wherein an alteration in the level of expression relative to the level of expression in a reference indicates the efficacy of the treatment in the subject.

24. A method of selecting a treatment regimen for a subject diagnosed as having a neoplasm, the method comprising determining the level of expression of TSLP in a subject sample relative to a reference, wherein the level of expression of TSLP indicates an appropriate treatment regimen for the subject.

25. The method of claim 24, wherein a decreased level of TSLP indicates that conservative treatment is appropriate. 26. The method of claim 25, wherein conservative treatment is selected from the group consisting of continued monitoring of the patient's condition, less aggressive surgery, less aggressive chemotherapy, radiotherapy, radiofrequency ablation, thermoablation via focused ultrasound, and intraarterial embolisation techniques.

27. The method of claim 24, wherein an increased level of TSLP indicates that aggressive treatment is appropriate.

28. The method of claim 27, wherein aggressive treatment is selected from the group consisting of high dose chemotherapy, surgery, radiotherapy, radiofrequency ablation, thermoablation via focused ultrasound, and intraarterial embolisation techniques.

29. A diagnostic kit for the diagnosis of a neoplasm in a subject comprising an antibody capable of detecting TSLP and written instructions for use of the kit for diagnosis of a neoplasm.

Description:
COMPOSITIONS AND METHODS FOR TREATING NEOPLASIA

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser. No. 61/416,619, filed November 23, 2010 the entire contents of which are hereby incorporated herein by reference.

STATEMENT OF RIGHTS TO INVENTIONS MADE UNDER FEDERALLY

SPONSORED RESEARCH

Research supporting this application was carried out by the United States of America as represented by the Secretary, Department of Health and Human Services. The Government has certain rights in this invention.

BACKGROUND OF THE DISCLOSURE

The spread of cancer cells from a primary tumor site to distant organs is known as metastasis. Metastasis is considered one of the most problematic aspects underlying the pathophysiology of cancer because conventional treatment methods such as surgery and radiotherapy are ineffective in treating or preventing the dispersion of tumor cells. Indeed, metastasis is the leading cause of therapeutic failure in cancer patients and long term patient survival correlates inversely with tumor progression. Novel therapeutic and diagnostic strategies are urgently required for treating cancer progression and metastasis.

SUMMARY OF THE DISCLOSURE

As described below, the present disclosure features compositions and methods for the treatment of neoplasias progression and metastasis.

In one aspect, the present disclosure generally features a method of reducing the invasiveness of a neoplastic cell, the method involving contacting a neoplastic cell with an ag

BOS2 897671.1 that inhibits thymic stromal lymphopoietin (TSLP) or TSLP Receptor expression or biological activity, thereby reducing the invasiveness of the neoplastic cell.

In another aspect, the present disclosure features a method of treating a subject having a solid tumor, the method involving administering to a subject a therapeutically effective amount of an agent that inhibits TSLP or TSLP Receptor expression or biological activity, thereby treating the subject.

In yet another aspect, the present disclosure features a method of treating or preventing metastasis in a subject having a neoplasia, the method involving administering to a subject a therapeutically effective amount of an agent that inhibits TSLP or TSLP Receptor expression or biological activity, thereby treating or preventing metastasis in the subject.

In a further aspect, the present disclosure features a method of treating or preventing tumor progression or metastasis in a subject having a solid tumor, the method involving administering to a subject a therapeutically effective amount of an agent that inhibits TSLP or TSLP Receptor expression or biological activity, thereby treating or preventing tumor progression or metastasis in the subject.

In an additional aspect, the present disclosure features a pharmaceutical composition for the treatment of a solid tumor containing a therapeutically effective amount of an agent that inhibits TSLP or TSLP Receptor expression or biological activity.

In yet another aspect, the present disclosure features a pharmaceutical composition for the treatment or prevention of metastasis containing a therapeutically effective amount of an agent that inhibits TSLP or TSLP Receptor expression or biological activity.

In a further aspect, the present disclosure features a kit for the treatment of a neoplasia, the kit containing an effective amount of an agent that inhibits TSLP or TSLP Receptor expression or biological activity and directions for using the kit for the treatment of a neoplasia.

In another aspect, the present disclosure features a method of characterizing the aggressiveness of a neoplasia, involving determining the level of expression of TSLP in a subject sample, where an increased level of expression relative to a reference indicates that the neoplasia is aggressive.

In another aspect, the present disclosure features a method of monitoring a subject diagnosed as having a neoplasia, the method involving determining the level of expression of TSLP in a subject sample, wherein an alteration in the level of expression relative to the level of expression in a reference indicates the severity of neoplasia in a subject. In another aspect, the present disclosure features a method of selecting a treatment regimen for a subject diagnosed as having a neoplasia, the method involving determining the level of expression of TSLP in a subject sample relative to a reference, where the level of expression of TSLP indicates an appropriate treatment regimen for the subject.

In yet another aspect, the present disclosure features a diagnostic kit for the diagnosis of a neoplasia in a subject containing an antibody capable of detecting TSLP and written instructions for use of the kit for diagnosis of a neoplasia.

In various embodiments of any of the above aspects or any other aspect of the disclosure delineated herein, the agent is selected from antibodies, polypeptides, nucleic acid molecule, inhibitory nucleic acid molecules, and small molecules. In another embodiment the agent is an antibody that binds TSLP. In yet another embodiment the antibody is a monoclonal antibody. In a further embodiment the monoclonal antibody is a human or humanized antibody. In additional embodiments the methods involve the step of co-administering one or more chemotherapeutics.

In other embodiments the one or more chemotherapeutics is selected from the group consisting of abiraterone acetate, altretamine, anhydro vinblastine, auristatin, azacitidine, bendamustine, bevacizumab, bexarotene, bicalutamide, BMS-184476, 2,3,4,5,6-pentafluoro-N- (3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, bortezomib, N,N-dimethyl-L- valyl-L-valyl-N-methyl-L-valyl-L-prolyl-L-proline-t-butylami de, cachectin, capecitabine, cemadotin, cetuximab, chlorambucil, cyclophosphamide, 3',4'-didehydro-4'-deoxy-8'-norvin- caleukoblastine, docetaxol, doxetaxel, carboplatin, carmustine (BCNU), cisplatin, cryptophycin, cytarabine, dacarbazine (DTIC), dactinomycin, dasatinib, daunorubicin, dolastatin, doxorubicin (adriamycin), erlotinib, etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyurea taxanes, ifosfamide, imatinib, irinotecan, lenalidomide, liarozole, lonidamine, lomustine (CCNU), mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, 5-fluorouracil, nilutamide, onapristone, paclitaxel, panitumumab, pazopanib, prednimustine, procarbazine, rituximab, RPR109881, sorafenib, estramustine phosphate, sunitinib, tamoxifen, tasonermin, taxol, temozolomide, trastuzumab, tretinoin, vinblastine, vincristine, vindesine sulfate, vinflunine, and vorinostat. In certain embodiments the inhibitory nucleic acid molecule is an siRNA, antisense nucleic acid molecule, or shRNA. In additional embodiments the neoplastic cell is a cancer cell which is present in a solid tumor. In further embodiments the cancer is selected from breast cancer, prostate cancer, melanoma, glioblastomas, colon cancer, ovarian cancer and non-small cell lung cancer. In yet additional embodiments the reference is a cancer-free subject. In certain embodiments the level of TSLP indicates that the neoplasia is aggressive. In additional embodiments a decreased level of TSLP indicates that conservative treatment is appropriate. In further embodiments conservative treatment is selected from the group consisting of continued monitoring of the patient's condition, less aggressive surgery, less aggressive chemotherapy, radiotherapy, radiofrequency ablation, thermoablation via focused ultrasound, and intraarterial embolisation techniques. In yet other embodiments an increased level of TSLP indicates that aggressive treatment is appropriate. In additional embodiments aggressive treatment is selected from high dose chemotherapy, surgery, radiotherapy, radiofrequency ablation, thermoablation via focused ultrasound, and intraarterial embolisation techniques.

Definitions

By "thymic stromal lymphopoietin" or "TSLP" is meant an interleukin ("IL")-7-like type 1 inflammatory cytokine having at least about 85% sequence identity to NCBI Accession No. NP_ 149024 or a fragment thereof that regulates tumor progression and metastasis. An exemplary sequence of human TSLP is:

1 mfpfallyvl svsfrkifil qlvglvltyd ftncdfekik aaylstiskd litymsgtks

61 tefnntvscs nrphclteiq sltfnptagc aslakemfam ktkaalaiwc pgysetqina

121 tqamkkrrkr kvttnkcleq vsqlqglwrr fnrpllkqq

By a "nucleic acid encoding TSLP" is meant a nucleic acid having at least about 85% sequence identity to NCBI Accession No. NM_033035.4. An exemplary nucleic acid encoding TSLP is:

1 gcagccagaa agctctggag catcagggag actccaactt aaggcaacag catgggtgaa 61 taagggcttc ctgtggactg gcaatgagag gcaaaacctg gtgcttgagc actggcccct

121 aaggcaggcc ttacagatct cttacactcg tggtgggaag agtttagtgt gaaactgggg 181 tggaattggg tgtccacgta tgttcccttt tgccttacta tatgttctgt cagtttcttt 241 caggaaaatc ttcatcttac aacttgtagg gctggtgtta acttacgact tcactaactg 301 tgactttgag aagattaaag cagcctatct cagtactatt tctaaagacc tgattacata 361 tatgagtggg accaaaagta ccgagttcaa caacaccgtc tcttgtagca atcggccaca

421 ttgccttact gaaatccaga gcctaacctt caatcccacc gccggctgcg cgtcgctcgc 481 caaagaaatg ttcgccatga aaactaaggc tgccttagct atctggtgcc caggctattc 541 ggaaactcag ataaatgcta ctcaggcaat gaagaagagg agaaaaagga aagtcacaac 601 caataaatgt ctggaacaag tgtcacaatt acaaggattg tggcgtcgct tcaatcgacc 661 tttactgaaa caacagtaaa ccatctttat tatggtcata tttcacagca ccaaaataaa 721 tcatctttat taagtagatg aaacattaac tctaactgtg acaaagaaga ccacaaatag

781 ttatctttta attacagaag agtttcttaa cttacttttg taagttttta ttgtgtaagt 841 ttataatgca ggggaagtac tactcctcaa atgttgaggg aagcttccat aacattgatg 901 actggcttca tggcagtaat tctcggctgt agttgcataa gcattgctca agaggaaaat 961 ccaaaagtgc agcaggagaa ctcttttccc tgaaaaagga aaaatattga actcaatgat 1021 agcacctaaa cttacattta aaagacagac attccttcta catgtaatga cacttcttgt

1081 gttaaactaa aaatttacaa gagaagaaag tgaaagcaaa tggggtttca caaatagttg 1141 taaatatagt gaagcaattt gaaataattt tcaagcaaag tattgtgaaa gtattctaag 1201 ccaagtttta aatattatct aacagacaag agtggtatat acaagtagat cctgagaagt 1261 acctttgtta cagctactat aaatatacat ataaattata gaatctactt taatttattt 1321 tgtgaacact tttgaaaatg tacatgttcc tttgtaattg acactatata tttcttaata

1381 aaataattct caaatttgtt tcttatgaat catctctcaa atctagttag acaatttgca 1441 cacatacttt tctaagggac attatcttcc ttcaggtttt tacctccact catccttaga 1501 gcccactgac tgctcccctt tatacctgtt ggccctgcct ataggagaga atatttggag 1561 ataggcagct tcaggatgca ttgcaatcat ccttttctta aattatgtca ctagtctttt 1621 attttttccc ctcttgaact ttcctcacac ctggaagaaa caaagtagga aaaagtgaac

1681 aggggatgtc aaatcgattc ttgaattccc gctgcaagct agagccgcag gcaccctctc 1741 actcaatttc cactcagaac cctataaaca ccagtgggaa gggcaaccca ctgcacgtgg 1801 gaatgcactg atttttccta ggagtagaca tgttcctcta attactccct gagggttagt 1861 tggggctaaa ccatgacaga agtggggaag ttcaatgtcc ttaaatccat cttacttgcc 1921 aacaggtaag aggaagctta cattacatgt ccagtccaca tttaaagagc acttactgtg

1981 gaacaagcct tcagccaaac aatggggata gaaaagtagg taagactcag cctttgtcca 2041 gagaagctca gggtatagct gaataggcag tttcttttgt cctgaggaaa atcaggacat 2101 gcctgctttc taaaaatctt cctctgaaga cctgacccaa gctcttaaat gctattgtaa 2161 gagaaatttc tttgtctatt aactccattt tagtagggat tcactgacta gattttactg 2221 aactatgaaa ataaatacac ataatttttc acaaaatttt gggcccaatt cccctaaaag 2281 aattgaggat tagggagaaa ggagacaact caaagtcatc ccattaagtg cagtttcttt 2341 gaatcttctg ctttatcttt aaaaatttgt ataatttata tattttattc tatgtgttcc 2401 atagatatct taatgtaaaa ttagtcattt aaattacact gtcaattaaa agtaatgggc 2461 aagagattgc atcatactaa tttagtaaga acgttcccaa atgttgtaac aatgtggatc 2521 atacatctct ggttttttaa atgtattgag gctttcttgg tggactagta tagtatacgg 2581 tcagttatgt caatgtttca tggtcaataa aaaggaagtt gcaaattgt

By "TSLP Receptor" or "thymic stromal lymphopoietin receptor" or "TSLPR" is meant a polypeptide having at least about 85% sequence identity to NCBI Accession No. AAK60618.1, and that forms a heterodimeric receptor complex with IL-7 receptor alpha ("IL-7Rcc"). The heterodimeric receptor complex mediates TSLP signaling. An exemplary sequence of human TSLP Receptor is:

1 mgrlvllwga avfllggwma lgqggaaegv qiqiiyfnle tvqvtwnask ysrtnltfhy

61 rfngdeaydq ctnyllqegh tsgclldaeq rddilyfsir ngthpvftas rwmvyylkps

121 spkhvrfswh qdavtvtcsd lsygdllyev qyrspfdtew qskqentcnv tiegldaekc

181 ysfwvrvkam edvygpdtyp sdwsevtcwq rgeirdacae tptppkpkls kfilisslai

241 llmvslllls lwklwrvkkf lipsvpdpks ifpglfeihq gnfqewitdt qnvahlhkma

301 gaeqesgpee plvvqlakte aesprmldpq teekeasggs lqlphqplqg gdvvtiggft

361 fvmndrsyva 1

By a "nucleic acid encoding TSLP Receptor" is meant a nucleic acid having at least about 85% sequence identity to NCBI Accession No. AF338733.1. An exemplary nucleic acid encoding TSLP Receptor is:

1 atggggcggc tggttctgct gtggggagct gccgtctttc tgctgggagg ctggatggct 61 ttggggcaag gaggagcagc agaaggagta cagattcaga tcatctactt caatttagaa 121 accgtgcagg tgacatggaa tgccagcaaa tactccagga ccaacctgac tttccactac 181 agattcaacg gtgatgaggc ctatgaccag tgcaccaact accttctcca ggaaggtcac 241 acttcggggt gcctcctaga cgcagagcag cgagacgaca ttctctattt ctccatcagg 301 aatgggacgc accccgtttt caccgcaagt cgctggatgg tttattacct gaaacccagt 361 tccccgaagc acgtgagatt ttcgtggcat caggatgcag tgacggtgac gtgttctgac 421 ctgtcctacg gggatctcct ctatgaggtt cagtaccgga gccccttcga caccgagtgg 481 cagtccaaac aggaaaatac ctgcaacgtc accatagaag gcttggatgc cgagaagtgt

541 tactctttct gggtcagggt gaaggctatg gaggatgtat atgggccaga cacataccca

601 agcgactggt cagaggtgac atgctggcag agaggcgaga ttcgggacgc ctgtgcagag

661 acaccaacgc ctcccaaacc aaagctgtcc aaatttattt taatttccag cctggccatc

721 cttctgatgg tgtctctcct ccttctgtct ttatggaaat tatggagagt gaagaagttt

781 ctcattccca gcgtgccaga cccgaaatcc atcttccccg ggctctttga gatacaccaa

841 gggaacttcc aggagtggat cacagacacc cagaacgtgg cccacctcca caagatggca

901 ggtgcagagc aagaaagtgg ccccgaggag cccctggtag tccagttggc caagactgaa

961 gccgagtctc ccaggatgct ggacccacag accgaggaga aagaggcctc tgggggatcc

1021 ctccagcttc cccaccagcc cctccaaggc ggtgatgtgg tcacaatcgg gggcttcacc

1081 tttgtgatga atgaccgctc ctacgtggcg ttgtga

By "TSLP antagonist" is meant any agent that inhibits TSLP or TSLP Receptor expression or biological activity.

By "metastasis," "metastatic disease," or "invasiveness" is meant the spread of cancer cells from a tissue or organ of origin to an another tissue or organ.

By "solid tumor" is meant an abnormal mass of tissue that usually does not contain cysts or liquid areas.

By "agent" is meant any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.

By "aggressive" and "aggressiveness" relative to neoplasia is meant that an aggressive neoplasia is more likely to metastasize compared to a non-aggressive neoplasia.

By "ameliorate" is meant decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.

By "alteration" is meant a change (increase or decrease) in the expression levels or activity of a gene or polypeptide as detected by standard art known methods such as those described herein. As used herein, an alteration includes a 10% change in expression levels, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression levels. "

By "analog" is meant a molecule that is not identical, but has analogous functional or structural features. For example, a polypeptide analog retains the biological activity of a corresponding naturally- occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such

biochemical modifications could increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding. An analog may include an unnatural amino acid.

In this disclosure, "comprises," "comprising," "containing" and "having" and the like can have the meaning ascribed to them in U.S. Patent law and can mean " includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise has the meaning ascribed in U.S. Patent law and the term is open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited is not changed by the presence of more than that which is recited, but excludes prior art embodiments.

"Chemotherapeutic" means any agent useful for treating neoplasia in a subject.

Chemotherapeutic includes but is not limited to abiraterone acetate, altretamine,

anhydrovinblastine, auristatin, azacitidin, bendamustin, bevacizumab, bexarotene, bicalutamide, BMS184476, 2,3,4,5,6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, bortezomib, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly- 1-Lproline-t- butylamide, cachectin, capecitabin, cemadotin, cetuximab, chlorambucil, cyclophosphamide, 3',4'-didehydro-4'-deoxy-8'-norvin- caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine (BCNU),cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, dasatinib, daunorubicin, dolastatin, doxorubicin

(adriamycin), erlotinib, etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyureataxanes, ifosfamide, imatinib, irinotecan, lenalidomid, liarozole, lonidamine, lomustine (CCNU), mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, 5-fluorouracil, nilutamide, onapristone, paclitaxel, panitumumab, pazopanib, prednimustine, procarbazine, rituximab, RPR109881, sorafinib, stramustine phosphate, sunitinib, tamoxifen, tasonermin, taxol, temozolomide, transtuzumab, tretinoin, vinblastine, vincristine, vindesine sulfate, vinflunine, and vorinostat.

The phrase "in combination with" is intended to refer to all forms of administration that provide a molecule such as a TSLP antagonist together with a second agent, such as a chemotherapeutic agent, where the two are administered concurrently or sequentially or in any order. "Detect" refers to identifying the presence, absence or amount of the analyte to be detected.

By "detectable label" is meant a composition that when linked to a molecule of interest renders the latter detectable, via spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Non-limiting examples of diseases include breast cancer, prostate cancer, glioblastoma, osteosarcoma, colon cancer, non-small cell lung cancer, ovarian cancer, and melanoma

By "effective amount" is meant the amount of a required to ameliorate the symptoms of a disease relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of a disease varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an "effective" amount.

The invention provides a number of targets that are useful for the development of highly specific drugs to treat or a disorder characterized by the methods delineated herein. In addition, the methods of the invention provide a facile means to identify therapies that are safe for use in subjects. In addition, the methods of the invention provide a route for analyzing virtually any number of compounds for effects on a disease described herein with high- volume throughput, high sensitivity, and low complexity.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide. A fragment may contain 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

"Hybridization" means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds. By "inhibitory nucleic acid" is meant a double- stranded RNA, siRNA, shRNA, or antisense RNA, or a portion thereof, or a mimetic thereof, that when administered to a mammalian cell results in a decrease (e.g., by 10%, 25%, 50%, 75%, or even 90-100%) in the expression of a target gene. Typically, a nucleic acid inhibitor comprises at least a portion of a target nucleic acid molecule, or an ortholog thereof, or comprises at least a portion of the complementary strand of a target nucleic acid molecule. For example, an inhibitory nucleic acid molecule comprises at least a portion of any or all of the nucleic acids delineated herein.

By "isolated polynucleotide" is meant a nucleic acid (e.g., a DNA) that is free of the genes which, in the naturally-occurring genome of the organism from which the nucleic acid molecule of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA that is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or that exists as a separate molecule (for example, a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes an RNA molecule that is transcribed from a DNA molecule, as well as a recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequence.

By an "isolated polypeptide" is meant a polypeptide of the present disclosure that has been separated from components that naturally accompany it. Typically, the polypeptide is isolated when it is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated. Preferably, the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, a polypeptide of the present disclosure. An isolated polypeptide of the invention may be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

By "marker" is meant any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or disorder.

As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise acquiring the agent.

"Primer set" means a set of oligonucleotides that may be used, for example, for PCR. A primer set would consist of at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 30, 40, 50, 60, 80, 100, 200, 250, 300, 400, 500, 600, or more primers. By "reduces" is meant a negative alteration of at least 10%, 25%, 50%, 75%, or 100%. By "reference" is meant a standard or control condition.

A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of or the entirety of a specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence. For polypeptides, the length of the reference polypeptide sequence will generally be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides or any integer thereabout or there between.

By "siRNA" is meant a double stranded RNA. Optimally, an siRNA is 18, 19, 20, 21, 22, 23 or 24 nucleotides in length and has a 2 base overhang at its 3' end. These dsRNAs can be introduced to an individual cell or to a whole animal; for example, they may be introduced systemically via the bloodstream. Such siRNAs are used to down-regulate mRNA levels or promoter activity.

By "specifically binds" is meant a compound or antibody that recognizes and binds a polypeptide of the present disclosure, but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample, which naturally includes a polypeptide of the present disclosure.

Nucleic acid molecules useful in the methods of the present disclosure include any nucleic acid molecule that encodes a polypeptide of the present disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity. Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present disclosure include any nucleic acid molecule that encodes a polypeptide of the present disclosure or a fragment thereof. Such nucleic acid molecules need not be 100% identical with an endogenous nucleic acid sequence, but will typically exhibit substantial identity.

Polynucleotides having "substantial identity" to an endogenous sequence are typically capable of hybridizing with at least one strand of a double- stranded nucleic acid molecule. By "hybridize" is meant pair to form a double- stranded molecule between complementary polynucleotide sequences (e.g., a gene described herein), or portions thereof, under various conditions of stringency. (See, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399;

Kimmel, A. R. (1987) Methods Enzymol. 152:507).

By "substantially identical" is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein). Preferably, such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.

Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary approach to determining the degree of identity, a BLAST program may be used, with a probability score between e "3 and e "100 indicating a closely related sequence.

By "subject" is meant a mammal, including, but not limited to, a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

Ranges provided herein are understood to be shorthand for all of the values within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated. Unless specifically stated or obvious from context, as used herein, the term "or" is understood to be inclusive. Unless specifically stated or obvious from context, as used herein, the terms "a", "an", and "the" are understood to be singular or plural.

Unless specifically stated or obvious from context, as used herein, the term "about" is understood as within a range of normal tolerance in the art, for example within 2 standard deviations of the mean. About can be understood as within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, or 0.01% of the stated value. Unless otherwise clear from context, all numerical values provided herein are modified by the term about.

The recitation of a listing of chemical groups in any definition of a variable herein includes definitions of that variable as any single group or combination of listed groups. The recitation of an embodiment for a variable or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

Any compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A- 1F show that TSLP expression and TSLP Receptor signaling correlate with metastatic potential. Figure 1A is a graph showing that metastatic 4T1 cells and 4T1.2 cells, but not non-metastatic 4T1-PE and 4T1.2-NM cells, express TSLP. Shown is secreted TSLP (mean + SEM of triplicates, pg/ml). Figure IB is a western blot showing that human cancer cells express TSLP. The western blot includes lysates of breast cancer (MCF-7 and MDA-MB-231 cells), melanoma (4ACC1273 and 938 cells), and ovarian cancer lines (OVCAR 433, 2008, BG1 and HOSEB). Figure IC is a pair of graphs that show that unlike shRNA control K5 clone or C7 clone (high level TSLP expressers), a TSLP-low clone B7 poorly progressed (left panel) and metastasized (right panel) in BALB/c mice. TSLPR deficient mice (Tslpr A ) do not efficiently support progression of 4T1 cells (left panel, Figure ID) and B 16 melanoma (Figure IE) and metastasis (right panel, Figure ID). Shown are mean cross section area+ SEM (left panel) and mean of lung metastatic foci + SEM (right panel) of four mice/group experiment in Tslp A mice (Figures ID and IE) and HLA-matched BALB/C (Figures IC and ID) and C57BL/6 (Figure IE) mice. Figure IE is a plot showing that serum levels of TSLP correlated with cancer burden (R =0.996). Tumor data of individual mice were plotted on the basis of low (close to background) and high levels of serum TSLP levels. All experiments were performed at least three times. *P < 0.05.

Figures 2A-2D show that TSLP signaling promotes cancer progression. Figure 2A is a graph showing that the diminished ability of Tslp A mice to support 4T1 tumor growth was partially reversed by adoptive transfer of T cells depleted of CD25 + cells (Olkhanud, P.B. et al. (2009) Cancer Res. 69:5996-6004). Figure 2B is a graph showing that depletion of CD4 + T cells inhibits 4T1 tumor progression in WT BALB/c mice. Compared with control IgG, GK1.5 anti- CD4-treated mice had >90 CD4 + T cell depletion in the blood at day 10 post tumor challenge. Unlike WT mice, Tslpf 1' mice transferred with DCs had reduced ability to support 4T1 cancer growth (Figure 2C) and metastasis (Figure 2D). Figure 2E is a graph showing that lung metastases are not augmented in Tslp ' ' mice transferred with T cells. All data shown here were reproduced at least twice. *P<0.05.

Figures 3 A is a photomicrograph that shows that primary 4T1.2 cancer cells growing in the mammary gland induce TSLP expression in lung epithelium of BALB/c mice. Shown is a representative lung section stained with anti-TSLP and control antibody of tumor-bearing and naive mice. Figure 3B is a graph that shows that metastatic 4T1 cancer cells, but not their non- metastatic cell subsets, induce systemic expression of Th2 cytokines. Naive BALB/c mice were injected subcutaneously with CM from metastatic 4T1 cancer cells (CM-4T1) or non-metastatic 4T1-PE cells (CM-4T1PE) and the expression of IL-5, IL13, and TSLP was measured in BAL fluid and blood by ELISA after 24 hours. All data shown here were reproduced at least three times. *P<0.05. Figure 3C is a photomicrograph that shows that TSLP is expressed in metastatic foci of the lungs of breast cancer patients. Shown is a representative lung section stained with anti-TSLP and control antibody. DETAILED DESCRIPTION OF THE DISCLOSURE

The present disclosure features compositions and methods that are useful for the treatment of neoplasia progression and metastasis (e.g. breast cancer, prostate cancer, glioblastoma, osteosarcoma, colon cancer, non-small cell lung cancer, ovarian cancer, and melanoma).

The present disclosure is based, at least in part, on the discovery that thymic stromal lymphopoietin (TSLP) is expressed by aggressive tumors and the observation that TSLP plays an important functional role in the progression and metastasis of neoplasias. The inhibition of TSLP signaling was observed to significantly reduce cancer progression and metastasis.

Antagonists of TSLP signaling were shown to be effective in inhibiting cancer progression and metastasis.

The present disclosure provides methods of treating metastatic cancer which comprise administering a therapeutically effective amount of an agent that inhibits TSLP or TSLP

Receptor expression or biological activity to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to a metastatic cancer or symptom thereof. The method includes the step of administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to treat the disease or disorder or symptom thereof, under conditions such that the disease or disorder is treated. Another aspect of the disclosure provides for measuring the levels of TSLP in a subject having cancer and thereby diagnosing the aggressiveness of the cancer. In additional aspects, the disclosure provides a method of monitoring a patient' s response to an anti-cancer therapy by measuring the level of TSLP before and after treatment whereby a decrease in TSLP following treatment is indicative of a beneficial response to therapy.

Inflammation is a double-edged sword that can promote or suppress cancer progression. Thymic stromal lymphopoietin (TSLP), an IL-7-like type 1 inflammatory cytokine that is often associated with the induction of Th2-type allergic responses in the lungs, was shown to be expressed in human and murine cancers. Studies with highly metastatic breast cancer cells indicated that TSLP is required for cancer escape and metastasis, as its inactivation in cancer cells alone was sufficient to almost completely abrogate cancer progression and lung metastasis. The cancer-promoting activity of TSLP primarily required signaling through the TSLP Receptor on CD4 + T cells promoting Th2- skewed immune responses and production of immune suppressive factors such as IL-13. Expression of TSLP therefore is a useful prognostic marker and reducing TSLP or TSLP Receptor expression or activity would have therapeutic potential.

Cancer progression and metastasis is a multifaceted process that involves inflammation and the active participation of immune cells, such as myeloid suppressive cells (MSCs) and regulatory T cells (Tregs). For example, in the mouse mammary adenocarcinoma 4T1 cancer model, which represents a highly aggressive model of human breast carcinoma, cancer-produced GM-CSF, IL-Ιβ, and TGFP promote the generation of MSCs and M2 macrophages to impair antitumor immune responses and promote metastases. Consistent with a link between allergic respiratory inflammation and metastases, it was shown that metastasis of 4T1 adenocarcinoma from the mammary gland to the lung requires lung inflammation, with the production of Th2- type chemokines recruiting CCR4 + 4T1 cell subsets and Tregs.

The demonstration that TSLP expression correlates with tumor metastasis coupled with the observation that the inhibition of TSLP signaling severely reduced cancer metastasis established the importance of TSLP in cancer progression and metastasis. For example, TSLP was found to be predominantly expressed by metastatic clones, specifically by 4T1.2 cells (Fig.lA) that exhibited enhanced lung metastasis. Considering the importance of TSLP in mediating allergic inflammation ( Liu, Y.J. et al. (2007) Annu Rev Immunol 25: 193-219), TSLP produced/induced by cancers explains a recent association of asthma with enhanced lung metastases in patients with breast cancer (Taranova, A.G. et al. (2008) Cancer Res 68:8582- 8589). Consistent with this, TSLP was found to be expressed in metastatic cancer cells in the lungs of breast cancer patients (Fig.3C). The enhanced tumor size when purified WT CD4 + T cells were adoptively transferred into Tslp A mice, leading to Th2-type immune responses and local production of IL-13, a critical mediator of allergic inflammation (Miyata, M. et al. (2009) Eur J Immunol 39:3078-3083) that blocks cancer immune surveillance by activating MSCs

(Terabe, M. et al. (2000) Nat Immunol 1:515-520) and M2 macrophages ( Sinha, P. et al. (2005) Cancer Res 65: 11743-11751) identified the importance of TSLP Receptors on T cells. Thus, breast cancer uses TSLP as a means to escape from antitumor adaptive immune responses (Terabe, M. et al. (2000) Nat Immunol 1:515-520; Gallina, G. et al. (2006) / Clin Invest

116:2777-2790). The fact that this did not lead to enhanced metastasis is consistent with the requirement for Tregs to protect metastasizing tumor cells from NK cells ( Olkhanud, P.B. et al. (2009) Cancer Res. 69:5996-6004). Cancer-produced and/or-induced TSLP was found to induce a multi-faceted chain of events that lead to cancer escape and metastasis. As such, TSLP can serve as a metastasis prognostic marker, and targeting TSLP signaling is a way to treat or control cancers, given that TSLP inactivation in tumors or disabling its signaling by using Tslp A mice was sufficient to diminish both cancer progression and metastasis.

The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of an agent that inhibits thymic stromal lymphopoeitin (TSLP) or TSLP Receptor expression or biological activity. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method). As used herein, the terms "treat," treating," "treatment," and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.

As used herein, the terms "prevent," "preventing," "prevention," "prophylactic treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject, who does not have, but is at risk of or susceptible to developing a disorder or condition.

The therapeutic methods of the disclosure (which include prophylactic treatment) in general comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).

In one embodiment, the disclosure provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with cancer progression or metastasis, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this invention; this pre-treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment. Antibodies

Antibodies that inhibit TSLP or TSLP Receptor expression or biological activity are useful in the methods of the present disclosure. In one embodiment, antibodies that bind TSLP inhibit TSLP biological activity and are useful for the treatment of cancer metastasis. In another embodiment, antibodies that bind the TSLP Receptor inhibit TSLP signaling and are useful agents in the treatment of cancer. Methods of preparing antibodies are well known to those of ordinary skill in the science of immunology. As used herein, the term "antibody" means not only intact antibody molecules, but also fragments of antibody molecules that retain immunogen- binding ability. Such fragments are also well known in the art and are regularly employed both in vitro and in vivo. Accordingly, as used herein, the term "antibody" means not only intact immunoglobulin molecules but also the well-known active fragments F(ab') 2 , and Fab. F(ab') 2 , and Fab fragments that lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983). The antibodies of the disclosure comprise whole native antibodies, bi-specific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies.

Unconventional antibodies include, but are not limited to, nanobodies, linear antibodies (Zapata et al., Protein Eng. 8(10): 1057-1062,1995), single domain antibodies, single chain antibodies, and antibodies having multiple valencies (e.g., diabodies, tribodies, tetrabodies, and pentabodies). Nanobodies are the smallest fragments of naturally occurring heavy-chain antibodies that have evolved to be fully functional in the absence of a light chain. Nanobodies have the affinity and specificity of conventional antibodies although they are only half of the size of a single chain Fv fragment. The consequence of this unique structure, combined with their extreme stability and a high degree of homology with human antibody frameworks, is that nanobodies can bind therapeutic targets not accessible to conventional antibodies. Recombinant antibody fragments with multiple valencies provide high binding avidity and unique targeting specificity to cancer cells. These multimeric scFvs (e.g., diabodies, tetrabodies) offer an improvement over the parent antibody since small molecules of ~60-100kDa in size provide faster blood clearance and rapid tissue uptake See Power et al., (Generation of recombinant multimeric antibody fragments for tumor diagnosis and therapy. Methods Mol Biol, 207, 335-50, 2003); and Wu et al. (Anti-carcinoembryonic antigen (CEA) diabody for rapid tumor targeting and imaging. Tumor Targeting, 4, 47-58, 1999). Various techniques for making unconventional antibodies have been described. Bi- specific antibodies produced using leucine zippers are described by Kostelny et al. {J Immunol 148(5): 1547-1553, 1992). Diabody technology is described by Hollinger et al. (Proc Natl Acad Sci USA 90:6444-6448, 1993). Another strategy for making bi-specific antibody fragments by the use of single-chain Fv (sFv) diners is described by Gruber et al. (J Immunol 152:5368, 1994). Tri-specific antibodies are described by Tutt et al. (J Immunol 147:60, 1991). Single chain Fv polypeptide antibodies include a covalently linked VH::VL heterodimer which can be expressed from a nucleic acid including VR- and VL-encoding sequences either joined directly or joined by a peptide-encoding linker as described by Huston, et al. (Proc Nat Acad Sci USA, 85:5879-5883, 1988). See, also, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778; and U.S. Patent

Publication Nos. 20050196754 and 20050196754.

In one embodiment, an antibody that inhibits TSLP or TSLP Receptor expression or biological activity is a monoclonal antibody. Alternatively, the anti- TSLP or anti-TSLP

Receptor antibody is a polyclonal antibody. The preparation and use of polyclonal antibodies are also known the skilled artisan. The disclosure also encompasses hybrid antibodies, in which one pair of heavy and light chains is obtained from a first antibody, while the other pair of heavy and light chains is obtained from a different second antibody. Such hybrids may also be formed using humanized heavy and light chains. Such antibodies are often referred to as "chimeric" antibodies.

In general, intact antibodies are said to contain "Fc" and "Fab" regions. The Fc regions are involved in complement activation and are not involved in antigen binding. An antibody from which the Fc' region has been enzymatically cleaved, or which has been produced without the Fc' region, designated an "F(ab') 2 " fragment, retains both of the antigen binding sites of the intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an "Fab"' fragment, retains one of the antigen binding sites of the intact antibody. Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain, denoted "Fd." The Fd fragments are the major determinants of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity). Isolated Fd fragments retain the ability to specifically bind to immunogenic epitopes.

Antibodies can be made by any of the methods known in the art utilizing chemokines, or immunogenic fragments thereof, as an immunogen. One method of obtaining antibodies is to immunize suitable host animals with an immunogen and to follow standard procedures for polyclonal or monoclonal antibody production. The immunogen will facilitate presentation of the immunogen on the cell surface. Immunization of a suitable host can be carried out in a number of ways. Nucleic acid sequences encoding TSLP, TSLP Receptor, or immunogenic fragments thereof, can be provided to the host in a delivery vehicle that is taken up by immune cells of the host. The cells will in turn express the receptor on the cell surface generating an immunogenic response in the host. Alternatively, nucleic acid sequences encoding TSLP, TSLP Receptor, or immunogenic fragments thereof, can be expressed in cells in vitro, followed by isolation of the receptor and administration of the receptor to a suitable host in which antibodies are raised.

Alternatively, antibodies that inhibit TSLP or TSLP Receptor expression or biological activity may, if desired, be derived from an antibody phage display library. A bacteriophage is capable of infecting and reproducing within bacteria, which can be engineered, when combined with human antibody genes, to display human antibody proteins. Phage display is the process by which the phage is made to 'display' the human antibody proteins on its surface. Genes from the human antibody gene libraries are inserted into a population of phage. Each phage carries the genes for a different antibody and thus displays a different antibody on its surface.

Antibodies made by any method known in the art can then be purified from the host. Antibody purification methods may include salt precipitation (for example, with ammonium sulfate), ion exchange chromatography (for example, on a cationic or anionic exchange column preferably run at neutral pH and eluted with step gradients of increasing ionic strength), gel filtration chromatography (including gel filtration HPLC), and chromatography on affinity resins such as protein A, protein G, hydroxyapatite, and anti-immunoglobulin.

Antibodies can be conveniently produced from hybridoma cells engineered to express the antibody. Methods of making hybridomas are well known in the art. The hybridoma cells can be cultured in a suitable medium, and spent medium can be used as an antibody source.

Polynucleotides encoding the antibody of interest can in turn be obtained from the hybridoma that produces the antibody, and then the antibody may be produced synthetically or

recombinantly from these DNA sequences. For the production of large amounts of antibody, it is generally more convenient to obtain an ascites fluid. The method of raising ascites generally comprises injecting hybridoma cells into an immunologically naive histocompatible or immunotolerant mammal, especially a mouse. The mammal may be primed for ascites production by prior administration of a suitable composition (e.g., Pristane).

Monoclonal antibodies (Mabs) produced by methods of the invention can be "humanized" by methods known in the art. "Humanized" antibodies are antibodies in which at least part of the sequence has been altered from its initial form to render it more like human immunoglobulins. Techniques to humanize antibodies are particularly useful when non-human animal (e.g., murine) antibodies are generated. Examples of methods for humanizing a murine antibody are provided in U.S. patents 4,816,567, 5,530,101, 5,225,539, 5,585,089, 5,693,762 and 5,859,205. Pharmaceutical Therapeutics

The present disclosure provides agents that decrease the expression or activity of TSLP or TSLP Receptor for the treatment of cancer. In one embodiment, the disclosure provides pharmaceutical compositions comprising an expression vector encoding a TSLP inhibitor polypeptide. In another embodiment, a chemical entity discovered to have medicinal value using the methods described herein is useful as a drug or as information for structural modification of existing agent, e.g., by rational drug design. For therapeutic uses, the compositions or agents identified using the methods disclosed herein may be administered systemically, for example, formulated in a pharmaceutically- acceptable carrier. Preferable routes of administration include, for example, subcutaneous, intravenous, interperitoneally, intramuscular, or intradermal injections that provide continuous, sustained levels of the drug in the patient. Treatment of human patients or other animals will be carried out using a therapeutically effective amount of a cancer therapeutic in a physiologically-acceptable carrier. Suitable carriers and their formulation are described, for example, in Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner of administration, the age and body weight of the patient, and the clinical symptoms of cancer progression or metastasis. Generally, amounts will be in the range of those used for other agents used in the treatment of cancer progression or metastasis, although in certain instances lower amounts will be needed because of the increased specificity of the compound. A compound is administered at a dosage that controls the clinical or physiological symptoms of cancer progression or metastasis as determined by a diagnostic method known to one skilled in the art, or using any that assay that measures the transcriptional activation of a gene associated with cancer progression or metastasis. Formulation of Pharmaceutical Compositions

The administration of an agent of the disclosure or analog thereof for the treatment of cancer progression or metastasis may be by any suitable means that results in a concentration of the therapeutic that, combined with other components, is effective in ameliorating, reducing, or stabilizing neoplasia or a symptom thereof. In one embodiment, administration of the agent reduces the binding of TSLP to TSLP Receptor. In another embodiment, the agent is

administered to a subject for the prevention or treatment of a disease associated with cancer.

Methods of administering such agents are known in the art. The disclosure provides for the therapeutic administration of an agent by any means known in the art. The compound may be contained in any appropriate amount in any suitable carrier substance, and is generally present in an amount of 1-95% by weight of the total weight of the composition. The composition may be provided in a dosage form that is suitable for parenteral (e.g., subcutaneously, intravenously, intramuscularly, or intraperitoneally) administration route. The pharmaceutical compositions may be formulated according to conventional pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988- 1999, Marcel Dekker, New York). Suitable formulations include forms for oral administration, depot formulations, formulations for delivery by a patch, and semi-solid dosage forms to be topically or trans-dermally delivered.

Pharmaceutical compositions according to the disclosure may be formulated to release the active compound substantially immediately upon administration or at any predetermined time or time period after administration. The latter types of compositions are generally known as controlled release formulations, which include (i) formulations that create a substantially constant concentration of the drug within the body over an extended period of time; (ii) formulations that after a predetermined lag time create a substantially constant concentration of the drug within the body over an extended period of time; (iii) formulations that sustain action during a predetermined time period by maintaining a relatively, constant, effective level in the body with concomitant minimization of undesirable side effects associated with fluctuations in the plasma level of the active substance (saw-tooth kinetic pattern); (iv) formulations that localize action by, e.g., spatial placement of a controlled release composition adjacent to or in the central nervous system or cerebrospinal fluid; (v) formulations that allow for convenient dosing, such that doses are administered, for example, once every one or two weeks; and (vi) formulations that target tumor cells by using carriers or chemical derivatives to deliver the therapeutic agent to a particular cell type whose function is perturbed in cancer. For some applications, controlled release formulations obviate the need for frequent dosing during the day to sustain the plasma level at a therapeutic level.

Any of a number of strategies can be pursued in order to obtain controlled release in which the rate of release outweighs the rate of metabolism of the compound in question. In one example, controlled release is obtained by appropriate selection of various formulation parameters and ingredients, including, e.g., various types of controlled release compositions and coatings. Thus, the therapeutic is formulated with appropriate excipients into a pharmaceutical composition that, upon administration, releases the therapeutic in a controlled manner.

Examples include single or multiple unit tablet or capsule compositions, oil solutions, suspensions, emulsions, microcapsules, microspheres, molecular complexes, nanoparticles, patches, and liposomes.

Parenteral Compositions

The pharmaceutical composition may be administered parenterally by injection, infusion or implantation (subcutaneous, intravenous, intramuscular, intraperitoneal, or the like) in dosage forms, formulations, or via suitable delivery devices or implants containing conventional, non- toxic pharmaceutically acceptable carriers and adjuvants. The formulation and preparation of such compositions are well known to those skilled in the art of pharmaceutical formulation. Formulations can be found in Remington: The Science and Practice of Pharmacy, supra.

Compositions for parenteral use may be provided in unit dosage forms (e.g., in single-dose ampoules), or in vials containing several doses and in which a suitable preservative may be added (see below). The composition may be in the form of a solution, a suspension, an emulsion, an infusion device, or a delivery device for implantation, or it may be presented as a dry powder to be reconstituted with water or another suitable vehicle before use. Apart from the active therapeutic (s), the composition may include suitable parenterally acceptable carriers and/or excipients. The active therapeutic (s) may be incorporated into microspheres, microcapsules, nanoparticles, liposomes, or the like for controlled release. Furthermore, the composition may include suspending, solubilizing, stabilizing, pH-adjusting agents, tonicity adjusting agents, and/or dispersing, agents. As indicated above, the pharmaceutical compositions according to the disclosure may be in the form suitable for sterile injection. To prepare such a composition, the suitable active therapeutic(s) are dissolved or suspended in a parenterally acceptable liquid vehicle.

Controlled Release Parenteral Compositions

Controlled release parenteral compositions may be in the form of suspensions, microspheres, microcapsules, magnetic microspheres, oil solutions, oil suspensions, or emulsions. Alternatively, the active drug may be incorporated in biocompatible carriers, liposomes, nanoparticles, implants, or infusion devices. Materials for use in the preparation of microspheres and/or microcapsules are, e.g., biodegradable/bioerodible polymers such as polygalactia poly-(isobutyl cyanoacrylate), poly(2-hydroxyethyl-L-glutam- nine) and, poly(lactic acid). Biocompatible carriers that may be used when formulating a controlled release parenteral formulation are carbohydrates (e.g., dextrans), proteins (e.g., albumin), lipoproteins, or antibodies. Materials for use in implants can be non-biodegradable (e.g., polydimethyl siloxane) or biodegradable (e.g., poly(caprolactone), poly(lactic acid), poly(glycolic acid) or poly(ortho esters) or combinations thereof).

Solid Dosage Forms For Oral Use

Formulations for oral use include tablets containing an active ingredient(s) in a mixture with non-toxic pharmaceutically acceptable excipients. Such formulations are known to the skilled artisan. Excipients may be, for example, inert diluents or fillers (e.g., sucrose, sorbitol, sugar, mannitol, microcrystalline cellulose, starches including potato starch, calcium carbonate, sodium chloride, lactose, calcium phosphate, calcium sulfate, or sodium phosphate); granulating and disintegrating agents (e.g., cellulose derivatives including microcrystalline cellulose, starches including potato starch, croscarmellose sodium, alginates, or alginic acid); binding agents (e.g., sucrose, glucose, sorbitol, acacia, alginic acid, sodium alginate, gelatin, starch, pregelatinized starch, microcrystalline cellulose, magnesium aluminum silicate,

carboxymethylcellulose sodium, methylcellulose, hydroxypropyl methylcellulose, ethylcellulose, polyvinylpyrrolidone, or polyethylene glycol); and lubricating agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc stearate, stearic acid, silicas, hydrogenated vegetable oils, or talc). Other pharmaceutically acceptable excipients can be colorants, flavoring agents, plasticizers, humectants, buffering agents, and the like. The tablets may be uncoated or they may be coated by known techniques, optionally to delay disintegration and absorption in the gastrointestinal tract and thereby providing a sustained action over a longer period. The coating may be adapted to release the active drug in a predetermined pattern (e.g., in order to achieve a controlled release formulation) or it may be adapted not to release the active drug until after passage of the stomach (enteric coating). The coating may be a sugar coating, a film coating (e.g., based on hydroxypropyl methylcellulose, methylcellulose, methyl hydroxyethylcellulose, hydroxypropylcellulose,

carboxymethylcellulose, acrylate copolymers, polyethylene glycols and/or

polyvinylpyrrolidone), or an enteric coating (e.g., based on methacrylic acid copolymer, cellulose acetate phthalate, hydroxypropyl methylcellulose phthalate, hydroxypropyl

methylcellulose acetate succinate, polyvinyl acetate phthalate, shellac, and/or ethylcellulose). Furthermore, a time delay material such as, e.g., glyceryl monostearate or glyceryl distearate may be employed.

The solid tablet compositions may include a coating adapted to protect the composition from unwanted chemical changes, (e.g., chemical degradation prior to the release of the active cancer progression or metastasis therapeutic substance). The coating may be applied on the solid dosage form in a similar manner as that described in Encyclopedia of Pharmaceutical

Technology, supra.

At least two active cancer progression or metastasis therapeutics may be mixed together in the tablet, or may be partitioned. In one example, the first active therapeutic is contained on the inside of the tablet, and the second active therapeutic is on the outside, such that a substantial portion of the second active therapeutic is released prior to the release of the first active therapeutic.

Formulations for oral use may also be presented as chewable tablets, or as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent (e.g., potato starch, lactose, microcrystalline cellulose, calcium carbonate, calcium phosphate or kaolin), or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin, or olive oil. Powders and granulates may be prepared using the ingredients mentioned above under tablets and capsules in a conventional manner using, e.g., a mixer, a fluid bed apparatus or a spray drying equipment.

Controlled Release Oral Dosage Forms Controlled release compositions for oral use may be constructed to release the active cancer progression or metastasis therapeutic by controlling the dissolution and/or the diffusion of the active substance. Dissolution or diffusion controlled release can be achieved by appropriate coating of a tablet, capsule, pellet, or granulate formulation of agent, or by incorporating the compound into an appropriate matrix. A controlled release coating may include one or more of the coating substances mentioned above and/or, e.g., shellac, beeswax, glycowax, castor wax, carnauba wax, stearyl alcohol, glyceryl monostearate, glyceryl distearate, glycerol

palmitostearate, ethylcellulose, acrylic resins, dl-polylactic acid, cellulose acetate butyrate, polyvinyl chloride, polyvinyl acetate, vinyl pyrrolidone, polyethylene, polymethacrylate, methylmethacrylate, 2-hydroxymethacrylate, methacrylate hydro gels, 1,3 butylene glycol, ethylene glycol methacrylate, and/or polyethylene glycols. In a controlled release matrix formulation, the matrix material may also include, e.g., hydrated metylcellulose, carnauba wax and stearyl alcohol, carbopol 934, silicone, glyceryl tristearate, methyl acrylate-methyl methacrylate, polyvinyl chloride, polyethylene, and/or halogenated fluorocarbon.

A controlled release composition containing one or more therapeutic agent may also be in the form of a buoyant tablet or capsule (i.e., a tablet or capsule that, upon oral administration, floats on top of the gastric content for a certain period of time). A buoyant tablet formulation of the compound(s) can be prepared by granulating a mixture of the compound(s) with excipients and 20-75% w/w of hydrocolloids, such as hydroxyethylcellulose, hydroxypropylcellulose, or hydroxypropylmethylcellulose. The obtained granules can then be compressed into tablets. On contact with the gastric juice, the tablet forms a substantially water-impermeable gel barrier around its surface. This gel barrier takes part in maintaining a density of less than one, thereby allowing the tablet to remain buoyant in the gastric juice. Inhibitory Nucleic Acids

Inhibitory nucleic acid molecules are those oligonucleotides that inhibit the expression or activity of TSLP or TSLP Receptor for the treatment of cancer progression and metastasis. Such oligonucleotides include single and double stranded nucleic acid molecules (e.g., DNA, RNA, and analogs thereof) that bind a nucleic acid molecule that encodes TSLP or TSLP Receptor (e.g., antisense oligonucleotide molecules, siRNA, shRNA) as well as nucleic acid molecules that bind directly to a TSLP polypeptide or TSLP Receptor polypeptide to modulate its biological activity (e.g., aptamers). Ribozymes

Catalytic RNA molecules or ribozymes that include an antisense TSLP or TSLP Receptor sequence of the present disclosure can be used to inhibit expression of a TSLP or TSLP Receptor nucleic acid molecule in vivo. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334:585- 591. 1988, and U.S. Patent Application Publication No. 2003/0003469 Al, each of which is incorporated by reference.

Accordingly, the disclosure also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases. In preferred embodiments of this disclosure, the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif. Examples of such hammerhead motifs are described by Rossi et al., Aids Research and Human Retroviruses, 8: 183, 1992. Example of hairpin motifs are described by Hampel et al., "RNA Catalyst for Cleaving Specific RNA Sequences," filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988,

Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al., Nucleic Acids Research, 18: 299, 1990. These specific motifs are not limiting in the disclosure and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this disclosure is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.

Small hairpin RNAs consist of a stem-loop structure with optional 3' UU-overhangs. While there may be variation, stems can range from 21 to 31 bp (desirably 25 to 29 bp), and the loops can range from 4 to 30 bp (desirably 4 to 23 bp). For expression of shRNAs within cells, plasmid vectors containing either the polymerase III Hl-RNA or U6 promoter, a cloning site for the stem-looped RNA insert, and a 4-5-thymidine transcription termination signal can be employed. The Polymerase III promoters generally have well-defined initiation and stop sites and their transcripts lack poly(A) tails. The termination signal for these promoters is defined by the polythymidine tract, and the transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3' UU overhang in the expressed shRNA, which is similar to the 3' overhangs of synthetic siRNAs. Additional methods for expressing the shRNA in mammalian cells are described in the references cited above. siRNA

Short twenty-one to twenty-five nucleotide double- stranded RNAs are effective at down- regulating gene expression (Zamore et al., Cell 101: 25-33; Elbashir et al., Nature 411: 494-498, 2001, hereby incorporated by reference). The therapeutic effectiveness of an siRNA approach in mammals was demonstrated in vivo by McCaffrey et al. (Nature 418: 38-39,2002).

Given the sequence of a target gene, siRNAs may be designed to inactivate that gene. Such siRNAs, for example, could be administered directly to an affected tissue, or administered systemically. The nucleic acid sequence of an Pari gene can be used to design small interfering RNAs (siRNAs). The 21 to 25 nucleotide siRNAs may be used, for example, as therapeutics to treat cancer progression or metastasis.

The inhibitory nucleic acid molecules of the present disclosure may be employed as double- stranded RNAs for RNA interference (RNAi) -mediated knock-down of TSLP or TSLP Receptor expression. In one embodiment, TSLP expression is reduced in a tumor cell. In another embodiment, TSLP Receptor expression is reduced in a T cell. RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl,

ChemBioChem 2:239-245, 2001; Sharp, Gene Dev 15:485-490, 2000; Hutvagner and Zamore, Curr Opin Genet Devel 12:225-232, 2002; and Hannon, Nature 418:244-251, 2002). The introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of- function phenotypes in mammalian cells.

In one embodiment of the disclosure, a double- stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the disclosure. The dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired. dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550- 553, 2002; Paddison et al. Gene Dev 16:948-958, 2002. Paul et al. Nat Biotechnol 20:505-508, 2002; Sui et al. Proc Natl Acad Sci USA 99:5515-5520, 2002; Yu et al. Proc Natl Acad Sci USA 99:6047-6052, 2002; Miyagishi et al. Nat Biotechnol 20:497-500, 2002; and Lee et al. Nat Biotechnol 20:500-505, 2002, each of which is hereby incorporated by reference. Small hairpin RNAs consist of a stem-loop structure with optional 3' UU-overhangs. While there may be variation, stems can range from 21 to 31 bp (desirably 25 to 29 bp), and the loops can range from 4 to 30 bp (desirably 4 to 23 bp). For expression of shRNAs within cells, plasmid vectors containing either the polymerase III Hl-RNA or U6 promoter, a cloning site for the stem-looped RNA insert, and a 4-5-thymidine transcription termination signal can be employed. The Polymerase III promoters generally have well-defined initiation and stop sites and their transcripts lack poly(A) tails. The termination signal for these promoters is defined by the polythymidine tract, and the transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3' UU overhang in the expressed shRNA, which is similar to the 3' overhangs of synthetic siRNAs. Additional methods for expressing the shRNA in mammalian cells are described in the references cited above.

Delivery of Nucleotide-base Oligomers

Naked inhibitory nucleic acid molecules, or analogs thereof, are capable of entering mammalian cells and inhibiting expression of a gene of interest. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798,

6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference). Dosage

Human dosage amounts can initially be determined by extrapolating from the amount of compound used in mice, as a skilled artisan recognizes it is routine in the art to modify the dosage for humans compared to animal models. In certain embodiments it is envisioned that the dosage may vary from between about 1 mg compound/Kg body weight to about 5000 mg compound/Kg body weight; or from about 5 mg/Kg body weight to about 4000 mg/Kg body weight or from about 10 mg/Kg body weight to about 3000 mg/Kg body weight; or from about 50 mg/Kg body weight to about 2000 mg/Kg body weight; or from about 100 mg/Kg body weight to about 1000 mg/Kg body weight; or from about 150 mg/Kg body weight to about 500 mg/Kg body weight. In other embodiments this dose may be about 1, 5, 10, 25, 50, 75, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000 mg/Kg body weight. In other embodiments, it is envisaged that higher does may be used, such doses may be in the range of about 5 mg compound/Kg body to about 20 mg compound/Kg body. In other embodiments the doses may be about 8, 10, 12, 14, 16 or 18 mg/Kg body weight. Of course, this dosage amount may be adjusted upward or downward, as is routinely done in such treatment protocols, depending on the results of the initial clinical trials and the needs of a particular patient.

Therapeutic Methods

The present disclosure provides methods of treating cancer progression and metastasis by inhibiting or reducing TSLP, TSLP Receptor, or TSLP signaling. The methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a compound that antagonizes TSLP signaling by the methods described herein to a subject (e.g., a mammal such as a human). Thus, one embodiment is a method of treating a subject suffering from or susceptible to cancer metastasis. The method includes the step of administering to the subject a therapeutic amount or an amount of a compound herein sufficient to treat the disease or symptom thereof, under conditions such that the disease is treated.

The methods herein include administering to the subject (including a subject identified as in need of such treatment) an effective amount of a compound described herein, or a composition described herein to produce such effect. Identifying a subject in need of such treatment can be in the judgment of a subject or a health care professional and can be subjective (e.g. opinion) or objective (e.g. measurable by a test or diagnostic method).

The therapeutic methods of the disclosure, which include prophylactic treatment, in general comprise administration of a therapeutically effective amount of the agent herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a cancer progression or metastasis or symptom thereof. Determination of those subjects "at risk" can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like). The agent herein may be also used in the treatment of any other disorders in which transcriptional activity may be implicated.

In one embodiment, the disclosure provides a method of monitoring treatment progress. The method includes the step of determining a level of diagnostic marker (Marker) (e.g., any target delineated herein modulated by a compound herein, a protein or indicator thereof, etc.) or diagnostic measurement (e.g., screen, assay) in a subject suffering from or susceptible to a disorder or symptoms thereof associated with cancer progression or metastasis, in which the subject has been administered a therapeutic amount of a compound herein sufficient to treat the disease or symptoms thereof. The level of Marker determined in the method can be compared to known levels of Marker in either healthy normal controls or in other afflicted patients to establish the subject's disease status. In one embodiment, the Marker is TSLP. In preferred embodiments, a second level of Marker in the subject is determined at a time point later than the determination of the first level, and the two levels are compared to monitor the course of disease or the efficacy of the therapy. In certain preferred embodiments, a pre-treatment level of Marker in the subject is determined prior to beginning treatment according to this disclosure; this pre- treatment level of Marker can then be compared to the level of Marker in the subject after the treatment commences, to determine the efficacy of the treatment. Kits

The disclosure provides kits for the treatment or prevention of cancer progression or metastasis. In one embodiment, the kit includes a therapeutic or prophylactic composition containing an effective amount of an agent of the invention (e.g., TSLP antagonist) in unit dosage form. In some embodiments, the kit comprises a sterile container which contains a therapeutic or prophylactic compound; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container forms known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding medicaments.

If desired an agent of the disclosure is provided together with instructions for

administering it to a subject having or at risk of developing cancer progression or metastasis. The instructions will generally include information about the use of the composition for the treatment or prevention of cancer progression or metastasis. In other embodiments, the instructions include at least one of the following: description of the compound; dosage schedule and administration for treatment or prevention of cancer progression or metastasis or symptoms thereof; precautions; warnings; indications; counter-indications; overdosage information; adverse reactions; animal pharmacology; clinical studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

Combination Therapies for the Treatment of a Neoplasm

Compositions and methods of the disclosure may be used in combination with any conventional therapy known in the art. In one embodiment, a composition of the disclosure (e.g., a composition comprising a TSLP antagonist) having anti-neoplastic activity may be used in combination with any anti-neoplastic therapy known in the art. Exemplary anti-neoplastic therapies include, for example, chemotherapy, cryotherapy, hormone therapy, radiotherapy, and surgery. A TSLP antagonist composition of the disclosure may, if desired, include one or more chemotherapeutics typically used in the treatment of a neoplasm, such as abiraterone acetate, altretamine, anhydrovinblastine, auristatin, azacitidin, bendamustin, bevacizumab, bexarotene, bicalutamide, BMS 184476, 2,3,4,5, 6-pentafluoro-N-(3-fluoro-4-methoxyphenyl)benzene sulfonamide, bleomycin, bortezomib, N,N-dimethyl-L-valyl-L-valyl-N-methyl-L-valyl-L-proly- 1-Lproline-t-butylamide, cachectin, capecitabin, cemadotin, cetuximab, chlorambucil,

cyclophosphamide, 3',4'-didehydro-4'-deoxy-8'-norvin- caleukoblastine, docetaxol, doxetaxel, cyclophosphamide, carboplatin, carmustine (BCNU),cisplatin, cryptophycin, cyclophosphamide, cytarabine, dacarbazine (DTIC), dactinomycin, dasatinib, daunorubicin, dolastatin, doxorubicin (adriamycin), erlotinib, etoposide, 5-fluorouracil, finasteride, flutamide, hydroxyurea and hydroxyureataxanes, ifosfamide, imatinib, irinotecan, lenalidomid, liarozole, lonidamine, lomustine (CCNU), mechlorethamine (nitrogen mustard), melphalan, mivobulin isethionate, rhizoxin, sertenef, streptozocin, mitomycin, methotrexate, 5-fluorouracil, nilutamide,

onapristone, paclitaxel, panitumumab, pazopanib, prednimustine, procarbazine, rituximab, RPR109881, sorafinib, stramustine phosphate, sunitinib, tamoxifen, tasonermin, taxol, temozolomide, transtuzumab, tretinoin, vinblastine, vincristine, vindesine sulfate, vinflunine, and vorinostat. Other examples of chemotherapeutic agents can be found in Cancer Principles and Practice of Oncology by V. T. Devita and S. Hellman (editors), 6th edition (Feb. 15, 2001), Lippincott Williams & Wilkins Publishers. Diagnostics

Aggressive tumors (those more likely to progress and metastasize) express higher levels of TSLP than non-aggressive tumors. Accordingly, the expression levels of TSLP are correlated with a particular disease state (e.g., malignant versus non-malignant), and thus are useful in diagnosis. Accordingly, the present disclosure provides a number of diagnostic assays that are useful for the identification or characterization of a neoplasia.

In one embodiment, a patient having a neoplasia will show an alteration in the expression of TSLP. Alterations in gene expression are detected using methods known to the skilled artisan and described herein. Such information can be used to diagnose a neoplasia, to identify dormant neoplasias, or to identify fast-growing neoplasia. In one embodiment, the level of TSLP polypeptide is detected using an antibody based method such as enzyme-linked immunosorbent assay (ELISA). In another embodiment, an alteration in the expression of a TSLP mRNA is detected using real-time quantitative PCR (Q-rt-PCR).

Antibodies used for TSLP detection, including but not limited to those antibodies described herein as useful as TSLP antagonists, are useful in diagnostic methods of the disclosure.

Primers used for amplification of a nucleic acid encoding TSLP, including but not limited to those primer sequences described herein, are useful in diagnostic methods of the disclosure. The primers of the disclosure embrace oligonucleotides of sufficient length and appropriate sequence so as to provide specific initiation of polymerization on a significant number of nucleic acids. Specifically, the term "primer" as used herein refers to a sequence comprising two or more deoxyribonucleotides or ribonucleotides, preferably more than three, and most preferably more than 8, which sequence is capable of initiating synthesis of a primer extension product, which is substantially complementary to a locus strand. The primer must be sufficiently long to prime the synthesis of extension products in the presence of the inducing agent for

polymerization. The exact length of primer will depend on many factors, including temperature, buffer, and nucleotide composition. The oligonucleotide primer typically contains between 12 and 27 or more nucleotides, although it may contain fewer nucleotides. Primers of the disclosure are designed to be "substantially" complementary to each strand of the genomic locus to be amplified and include the appropriate G or C nucleotides as discussed above. This means that the primers must be sufficiently complementary to hybridize with their respective strands under conditions that allow the agent for polymerization to perform. In other words, the primers should have sufficient complementarity with the 5' and 3' flanking sequences to hybridize therewith and permit amplification of the genomic locus. While exemplary primers are provided herein, it is understood that any primer that hybridizes with the target sequences of the disclosure are useful in the method of the invention for detecting nucleic acids encoding TSLP.

In one embodiment, TSLP- specific primers amplify a desired reverse transcribed RNA target using the polymerase chain reaction (PCR). The amplified product is then detected using standard methods known in the art. In one embodiment, a PCR product (i.e., amplicon) or realtime PCR product is detected by probe binding. In one embodiment, probe binding generates a fluorescent signal, for example, by coupling a fluorogenic dye molecule and a quencher moiety to the same or different oligonucleotide substrates (e.g., TaqMan® (Applied Biosystems, Foster City, CA, USA), Molecular Beacons (see, for example, Tyagi et al., Nat Biotechnol 14(3):303-8, 1996), Scorpions® (Molecular Probes Inc., Eugene, OR, USA)). In another example, a PCR product is detected by the binding of a fluorogenic dye that emits a fluorescent signal upon binding (e.g., SYBR® Green (Molecular Probes)). Such detection methods are useful for the detection of a TSLP PCR product.

In another embodiment, hybridization with PCR probes that are capable of detecting a TSLP molecule, or closely related molecules, may be used to hybridize to a nucleic acid sequence derived from a patient having a neoplasia. The specificity of the probe determines whether the probe hybridizes to a naturally occurring sequence, allelic variants, or other related sequences. Hybridization techniques may be used to monitor expression levels of these genes (for example, by Northern analysis (Ausubel et al., supra).

In general, the measurement of a TSLP molecule in a subject sample is compared with a diagnostic amount present in a reference. A diagnostic amount distinguishes between aggressive and non-agressive tumor tissue. The skilled artisan appreciates that the particular diagnostic amount used can be adjusted to increase sensitivity or specificity of the diagnostic assay depending on the preference of the diagnostician. In general, any significant increase or decrease (e.g., at least about 30% - 50%) in the level of a TSLP molecule in the subject sample relative to a reference may be used to diagnose a neoplasia, or to characterize a neoplasia as aggressive or non-agressive. In one embodiment, the reference is the level of TSLP molecule present in a control sample of a corresponding non-agressive tumor. In another embodiment, the reference is the level of TSLP present in a corresponding tissue sample obtained from a patient that does not have a neoplasia. In another embodiment, the reference is a baseline level of TSLP present in a biologic sample derived from a patient prior to, during, or after treatment for a neoplasia. In yet another embodiment, the reference is a standardized curve. Types of biological samples

The level of a TSLP molecule can be measured in different types of biologic samples. In one embodiment, the biologic sample is a tissue sample that includes cells of a tissue or organ. Such tissue is obtained, for example, from a biopsy. In another embodiment, the biologic sample is a biologic fluid sample (e.g., blood, urine, seminal fluids, ascites, or cerebrospinal fluid.

Kits

The disclosure also provides kits for the diagnosis or monitoring of a neoplasia in a biological sample obtained from a subject. In one embodiment, the kit detects an increase in the expression of a TSLP relative to a reference level of expression. In another embodiment, the kit detects an alteration in the sequence of a TSLP molecule derived from a subject relative to a reference sequence. In related embodiments, the kit includes reagents for monitoring the expression of a TSLP molecule, such as antibodies that bind specifically to TSLP.

Optionally, the kit includes directions for monitoring TSLP levels in a biological sample derived from a subject. In other embodiments, the kit comprises a sterile container which contains the primer, probe, antibody, or other detection regents; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container form known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding nucleic acids. The instructions will generally include information about the use of the primers or probes described herein and their use in diagnosing a neoplasia. Preferably, the kit further comprises any one or more of the reagents described in the diagnostic assays described herein. In other embodiments, the instructions include at least one of the following: description of the antibodies; methods for using the enclosed materials for the diagnosis of a neoplasia; precautions; warnings; indications; clinical or research studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

The disclosure also provides kits for the treatment of a neoplasia in a subject. In one embodiment, the kit includes an effective amount of a TSLP antagonist and directions for using the kit for the treatment of neoplasia. In another embodiment, the kit includes an effective amount of two or more TSLP antagonists. In other embodiments, the kit comprises a sterile container which contains the TSLP antagonists; such containers can be boxes, ampoules, bottles, vials, tubes, bags, pouches, blister- packs, or other suitable container form known in the art. Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding nucleic acids. The instructions will generally include information about the use of the TSLP antagonists herein and their use in treating a subject with a neoplasia. In other embodiments, the instructions include at least one of the following: methods for using the enclosed materials for the treatment of a neoplasia; precautions; warnings; indications; clinical or research studies; and/or references. The instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.

Patient Monitoring

The disease state or treatment of a patient having a neoplasia can be monitored using the methods and compositions of the disclosure. In one emobidment, an antibody that binds to TSLP is used to quantify TSLP polypeptide levels. In another embodiment, a probe that hybridizes to a nucleic acid encoding TSLP is used to quantify TSLP mRNA levels, in another embodiment, a microarray is used to assay expression levels of one or more differentially downstream targets of TSLP. Such monitoring may be useful, for example, in assessing the efficacy of a particular drug or therapeutic regimen in a patient. In one embodiment, the expression levels of mRNAs encoding downstream targets of TSLP is monitored in neoplastic cells of a subject being treated for a neoplasia. In one embodiment, a reduction in the levels of TSLP indicates that the subject's treatment is effective, and no change in the levels of TSLP, or an increase in the levels of TSLP indicates the subject's treatment is ineffective. Recombinant Polypeptide Expression

The practice of the present disclosure employs, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are well within the purview of the skilled artisan. Such techniques are explained fully in the literature, such as, "Molecular Cloning: A Laboratory Manual", second edition (Sambrook, 1989); "Oligonucleotide Synthesis" (Gait, 1984); "Animal Cell Culture" (Freshney, 1987); "Methods in Enzymology" "Handbook of Experimental Immunology" (Weir, 1996); "Gene Transfer Vectors for Mammalian Cells" (Miller and Calos, 1987); "Current Protocols in Molecular Biology" (Ausubel, 1987); "PCR: The Polymerase Chain Reaction", (Mullis, 1994); "Current Protocols in Immunology" (Coligan, 1991). These techniques are applicable to the production of the polynucleotides and polypeptides of the invention, and, as such, may be considered in making and practicing the invention. Particularly useful techniques for particular embodiments will be discussed in the sections that follow.

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the assay, screening, and therapeutic methods of the disclosure, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1: TSLP Expression correlates with tumor cell progression and metastasis.

To identify cancer-produced factors that activate lungs to facilitate metastasis of 4T1 breast cancer cells (Olkhanud, P.B. et al. (2009) Cancer Res 69:5996-6004), gene expression profiles between metastatic 4T1 and non-metastatic 4T1-PE cells were compared. Metastatic cells were found to express higher levels of TSLP as compared to poorly metastatic versions. The highest expression was seen in the 4T1.2 clone (Fig. 1A), which exhibits enhanced lung metastasis (Lelekakis, M. et al. (1999) Clin Exp Metastasis 17: 163-170). Moreover, human breast cancer MCF-7 and MDA-MB-231 cells and human melanoma 938 mel cells were also found to expressed TSLP (Fig. IB), indicating that cancer cell TSLP expression is not limited to mouse cancer cells and is not merely a mouse specific phenomenon. To examine whether TSLP plays a functional role in tumor growth, clones of 4T1 cells that express low levels of TSLP (clone B7, 150 + 15 pg/ml) or moderate levels of TSLP(clone C7, 380 + 30 pg/ml), and control shRNA-transduced 4T1 cells (K5) were subcutaneously (s.c.) injected into the mammary gland of syngeneic BALB/c mice. Whereas control clone K5 and clone C7 generated large tumors (left panel, Fig. lC) and many lung metastases (right panel), the low TSLP expressing clone B7 grew poorly and had few metastases (Fig.lC). This data demonstrates that TSLP expression functionally correlates with tumor growth and metastasis.

Example 2: TSLP functions in promoting tumor progression and metastasis, at least in part, through TSLP signaling in non-tumor tissue.

To further characterize the role of TSLP in cancer progression and metastasis, congenic Tslp A mice were challenged with 4T1 cancer cells. 4T1 tumor growth (left panel, Fig. ID) and metastasis (right panel) were severely diminished in Tslp A mice, as compared to identical cancer cell clones in WT mice. This demonstrates that tumor growth and metastasis is promoted by TSLP produced by cancer cells signaling through TSLP Receptors on non-tumor cells. This observation was not restricted to 4T1 cells, because subcutaneous injected B16 melanoma cells were also found to progress significantly slower in congenic Tslp A mice than in WT C57BL/6 mice (Fig. IE). Moreover, serum levels of TSLP positively correlated with tumor growth, with significantly elevated TSLP levels in the sera of Tslpf A mice (Fig. IF), possibly indicative of a lack of consumption of TSLP. Taken together, these results demonstrate the importance of TSLP in malignant cell growth.

Example 3: TSLP signaling through CD4 + T cells but not DC cells resulted in enhanced tumor growth.

Recently, it was reported that OVA-induced allergic pulmonary inflammation in mice promotes CD4 + T cell-dependent recruitment of intravenously-injected cancer cells into the lungs (Taranova, A.G. et al. (2008) Cancer Res 68:8582-8589), indicating the existence of a link between asthma and metastasis. However, in airway allergic inflammation in mice, TSLP activates CD4 + T cells either directly ( Liu, Y.J. et al. (2007) Annu Rev Immunol 25: 193-219; Al- Shami; A. et al. (2005) / Exp Med 202:829-839) or indirectly through DCs (16, 17)( Ito, T. et al. (2005) J Exp Med 202: 1213-1223; Gilliet, M. et al. (2003) / Exp Med 197: 1059-1063).

Therefore, the role of CD4 + T cells and DC cells in tumor progression and metastasis was investigated by adoptively transferring 4T1 tumor-bearing Tslp A mice with CD4 + T cells or bone marrow derived DCs from congenic naive BALB/c mice. Since lung metastasis of 4T1 cancer cells primarily depends on Tregs ( Olkhanud, P.B. et al. (2009) Cancer Res. 69:5996- 6004), CD4 + T cells depleted of CD25 + Tregs were used. In the Tslp A mice receiving these cells, there was significantly enhanced tumor growth (Fig.2A), underscoring the importance of CD4 + T cells as a target for TSLP (Al-Shami, A. et al. (2005) J Exp Med 202:829-839; He, R. et al. (2008) Proc Natl Acad Sci U S A 105: 11875-11880). Consistent with this, 4T1 cancer progression was reduced in WT BALB/c mice that were depleted of CD4 + T cells (Fig.2B). In contrast, the transfer of the DCs did not increase and in fact markedly reduced the already poor ability of Tslp A mice to support tumor growth (Fig.2C). As expected, there was no effect when the DCs were added to WT mice (Fig.2C). Adoptive transfer of DCs also abrogated lung metastases in Tslpf 1' mice (Fig.2D); however, consistent with the importance of Tregs in promoting metastasis of 4T1 cancer cells (Olkhanud, P.B. et al. (2009) Cancer Res. 69:5996- 6004), the transfer of non-Treg CD4 + T cells did not increase the number of lung metastases (Fig.2E).

Example 4: Cancer-produced TSLP induces CD4+ T cells to elicit Th2-type skewed responses that facilitate cancer escape and metastasis.

In allergic responses TSLP conditions the lung immune environment (Headley, M.B. et al. (2009) J Immunol 182: 1641-1647). Whether cancer-produced TSLP supports tumor progression by promoting Th2-type responses from CD4 + T cells was investigated. Indeed, as compared to control BALB/c mouse lungs, TSLP was found to be highly expressed by airway lining cells of mice with primary 4T1 adenocarcinoma in the mammary gland (Fig.3A), indicating that this could promote local expression of Th2-type cytokines. To test this idea, naive BALB/c mice were subcutaneously injected with conditioned medium from metastatic, TSLP expressing 4T1.2 cells (CM-4T1, Fig.3B) or non-metastatic, TSLP non-expressing 4T1-PE cells (CM-4T1PE) )(Fig. lA) ( Olkhanud, P.B. et al. (2009) Cancer Res. 69:5996-6004).

Compared with the mice injected with CM-4T1PE, significantly more IL-5, IL-13, and TSLP were observed in bronchoalveolar lavage fluid of mice that were injected with CM-4T1 (Fig.3B). Significant levels of TSLP and IL-5 were also detected in the blood of CM-4T1 -treated mice (Fig.3B), indicating that systemic Th2-type responses in these animals suppress anti-tumor Thl responses and CD8 + CTLs ( DeNardo, D.G. & Coussens, L.M. (2007) Breast Cancer Res 9:212). Hence, cancer-produced TSLP appears to target CD4 + T cells to elicit Th2-type skewed responses to facilitate cancer escape and metastasis.

The results described above were obtained using the following methods and materials. Experimental procedures Cells and mice. Female BALB/c and C57BL/6 mice were from the Jackson Laboratory (Bar Harbor, ME). Tslp ~ ' ~ mice were described previously (Al-Shami, A. et al. (2004) J Exp Med 200: 159-168) and housed under SPF conditions. The use of 4T1 and 4T1.2 cells and the generation their non-metastatic 4T1-PE clones were described elsewhere (Olkhanud, P.B. et al. (2009) Cancer Res. 69:5996-6004). B16F10 melanoma, MCF-7 and MDA-MB-231,

OVCAR433, 2008, HOSEB and BG1 cells were from American Type Culture Collection.

U4ACC1273 and 938 mel were from Dr. Ashani Weeraratna (NIA/NIH); To generate T cells depleted of Tregs, CD4 + T cells were isolated by mouse T cell CD4 Subset Column Kit and separated from CD25 + cells using CD25 Microbead kit (Miltenyi Biotec, Auburn, CA).

In vivo manipulations. Animal care was provided in accord with procedures outlined in the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, 1985). Experiments were performed using 4-8 weeks old female mice in a pathogen-free environment. Syngeneic mice were s.c. challenged with 4T1 cancer cells or their subsets (lxlO 4 cells, in the 4th mammary gland) or B16F10 melanoma cells (lxlO 5 ) at day 0. To test the role of T cells and DCs, HLA-matched lxl0 7 splenic and lymph node CD4 + T cells (purified from T cells depleted of Tregs) or lxlO 6 BM-derived immature DCs were i.v. and s.c. injected, respectively, at days 0 and 5 after tumor challenge, and tumor growth was measured every other day. Mice were culled 28 days after tumor challenge and lungs were analyzed for metastases as previously described (Olkhanud, P.B. et al. (2009) Cancer Res 69:5996-6004). In vivo CD4 + T cells were depleted by i.p injecting 400 μg anti-CD4 mAb GK1.5 (NCI-FCRDC, Frederick, MD), or normal rat IgG (Sigma) at days - 4, -1, 3 and 7 relative to tumor challenge. Depletion of CD4 + T cells was > 90%, as assessed 3 days after final treatment in the blood of the treated mice. Detection of cytokine expression. Blood (plasma) and broncheoalveolar lavage (BAL in lml PBS) was assessed for TSLP, IL-5 and IL-13 (eBioScience, San Diego, CA) by ELISA. Lungs of naive BALB/c mice were s.c. injected 5 times for five days with 0.1 ml of serum- free tumor conditioned medium to assess cytokine expression by ELISA. Immunohistochemistry staining was performed as described (Olkhanud, P.B. et al. (2009) Cancer Res 69:5996-6004) using paraffin-embedded lung sections of 4T1.2 cells or patients with breast cancer (ETIB, NCI, Bethesda, MD). Anti-mouse TSLP Ab (BAF555, R&D, MN), anti- human TSLP Ab (ab47943, Abeam, Cambridge, MA), biotinylated anti-rabbit IgG (BA1000, Fisher Scientific), and IHS reagents were from Thermo Scientific (Fremont, CA), such as antigen unmasking solution, horse serum (S-2000), Avidin-Biotin blocking kit, goat IgG (T5000), streptavidin-peroxidase and DAB Plus Substrate System. All slides were counterstained with hematoxylin.

Statistical Analysis. Results are presented as the mean of triplicates + SEM of at least three experiments. Differences were tested using Student's t test and a 2-sided p-value less than 0.05 was considered statistically significant.

Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the disclosure described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.

The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or sub-combination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.

All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.