COMPOSITIONS AND METHODS FOR CHARACTERIZING SOLID TUMORS RESPONSIVENESS TO ANTI-PD-L1 ANTIBODY MONOTHERAPY

BACKGROUND OF THE INVENTION

Cancer continues to be a major global health burden. Despite progress in the treatment of cancer, there continues to be an unmet medical need for more effective and less toxic therapies, especially for those patients with advanced disease or cancers that are resistant to existing therapeutics.

Lung cancer is among the most common forms of cancer and is the leading cause of cancer deaths among men and women. More people die of lung cancer annually than of colon, breast, and prostate cancers combined. Non-small cell lung cancer is the most common form of lung cancer. While the risk of acquiring lung cancer is higher among patients with a history of smoking, lung cancer also affects non-smokers. Improving survival of lung cancer patients remains difficult despite improved medical therapies. Most lung cancer is detected only in advanced stages when therapy options are limited. There is a growing recognition that lung cancer and other malignancies arise from a variety of pathogenic mechanisms. Methods of characterizing these malignancies at a molecular level are useful for stratifying patients, thereby quickly directing them to effective therapies. Improved methods for predicting the

responsiveness of subjects having lung cancer, including NSCLC, are urgently required.

Bladder cancer is among the five most common malignancies in North America and Europe. The overall incidence of bladder cancer appears to be rising, particularly among patients more than 55 years of age. More than 70% of the bladder cancers are non-muscle-invasive while about 20-30% are muscle-invasive which have a much less favorable prognosis.

The role of the immune system, in particular T cell-mediated cytotoxicity, in tumor control is well recognized. There is mounting evidence that T cells control tumor growth and survival in cancer patients, both in early and late stages of the disease. However, tumor-specific T-cell responses are difficult to mount and sustain in cancer patients.

PD-L1 is part of a complex system of receptors and ligands that are involved in controlling T-cell activation. In normal tissue, PD-L1 is expressed on T cells, B cells, dendritic cells, macrophages, mesenchymal stem cells, bone marrow-derived mast cells, as well as various non-hematopoietic cells. Its normal function is to regulate the balance between T-cell activation and tolerance through interaction with its two receptors: programmed death 1 (also known as PD-1 or CD279) and CD80 (also known as B7-1 or B7.1). PD-L1 is also expressed by tumors and acts at multiple sites to help tumors evade detection and elimination by the host immune system. PD-L1 is expressed in a broad range of cancers with a high frequency. In some cancers, expression of PD-L1 has been associated with reduced survival and unfavorable prognosis. Antibodies that block the interaction between B7-H1 and its receptors are able to relieve PD-L1 - dependent immunosuppressive effects and enhance the cytotoxic activity of antitumor T cells in vitro. Durvalumab, is a human monoclonal antibody directed against human PD-L1 that is capable of blocking the binding of PD-L1 to both the PD-1 and CD80 receptors.

Despite the significant progress made over the past decade in developing strategies for combatting cancer and other diseases, patients with advanced, refractory and metastatic disease have limited clinical options. Chemotherapy, irradiation, and high dose chemotherapy have become dose limiting. There remains a substantial unmet need for new less-toxic methods and therapeutics that have better therapeutic efficacy, longer clinical benefit, and improved safety profiles, particularly for those patients with advanced disease or cancers that are resistant to existing therapeutics.

SUMMARY OF THE INVENTION

The present invention provides methods for selecting patients as having a solid tumor (e.g., bladder cancer, ovarian cancer, colorectal cancer, head and neck cancer, cervical cancer, renal cell carcinoma, and non-small cell lung cancer (NSCLC)) that is responsive to treatment with an anti-PD-Ll antibody, and methods of treating such patients. The method involves detecting expression of an IFNG polynucleotide and/or one or more of polynucleotide markers: CXCL9, CD274, LAG3, in a biological sample of the patient.

In one aspect, the present invention provides a method of treating a solid tumor in a subject, the method comprising administering an anti-PD-Ll antibody, or an antigen binding fragment thereof, to a patient identified as having increased levels of two or more polynucleotide markers selected from the group consisting of CXCL9, CD274, LAG3, and IFNG in a biological sample derived from the patient relative to a reference.

In another aspect, the present invention provides a method of treating lung cancer or bladder cancer in a subject, the method comprising administering an anti-PD-Ll antibody, or an antigen binding fragment thereof, to a patient identified by detecting increased levels of two or more polynucleotide markers selected from the group consisting of CXCL9, CD274, LAG3, and IFNG in a biological sample derived from the patient relative to a reference.

In a further aspect, the present invention provides a method of identifying a subject having a solid tumor that is responsive to an anti-PD-Ll antibody, or an antigen binding fragment thereof, the method comprising detecting an increase in the expression levels of two or more polynucleotide markers selected from the group consisting of CXCL9, CD274, LAG3, and IFNG in a biological sample obtained from the subject relative to a reference, thereby identifying the solid tumor as responsive to anti-PD-Ll therapy.

In an additional aspect, the present invention provides a method of identifying a subject having a solid tumor that is responsive to anti-PD-Ll therapy, the method comprising detecting an increase in the expression levels of two or more polynucleotide markers selected from the group consisting of CXCL9, CD274, LAG3, and IFNG in a biological sample obtained from the subject, relative to a reference, thereby identifying the solid tumor as responsive to anti-PD-Ll therapy.

In another aspect, the present invention provides a method of identifying a subject having a lung cancer or bladder cancer that is responsive to anti-PD-Ll therapy, the method comprising detecting an increase in the expression levels of two or more polynucleotide markers selected from the group consisting of CXCL9, CD274, LAG3, and IFNG in a biological sample obtained from the subject, relative to a reference, thereby identifying the solid tumor as responsive to anti- PD-Ll therapy.

Definitions

Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. The following references provide one of skill with a general definition of many of the terms used in this invention: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless specified otherwise. By "Durvalumab" is meant an antibody or antigen binding fragment thereof that selectively binds a PD-L1 polypeptide and comprises at least a portion of a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and/or at least a portion of a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2.

Information regarding Durvalumab (or antigen-binding fragments thereof) for use in the methods provided herein can be found in US Patent No. 8,779,108, the disclosure of which is incorporated herein by reference in its entirety. The fragment crystallizable (Fc) domain of durvalumab contains a triple mutation in the constant domain of the IgGl heavy chain that reduces binding to the complement component Clq and the Fey receptors responsible for mediating antibody-dependent cell-mediated cytotoxicity (ADCC). Durvalumab is selective for PD-L1 and blocks the binding of PD-L1 to the PD-1 and CD80 receptors. Durvalumab can relieve PD-L1 -mediated suppression of human T-cell activation in vitro and inhibits tumor growth in a xenograft model via a T-cell dependent mechanism.

Durvalumab for use in the methods provided herein comprises a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. In a specific aspect, Durvalumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In a specific aspect, durvalumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the Kabat-defined CDRl, CDR2, and CDR3 sequences of SEQ ID NOs: 3-5, and wherein the light chain variable region comprises the Kabat- defined CDRl, CDR2, and CDR3 sequences of SEQ ID NOs: 6-8. Those of ordinary skill in the art would easily be able to identify Chothia-defined, Abm-defined or other CDR definitions known to those of ordinary skill in the art. In a specific aspect, Durvalumab or an antigen- binding fragment thereof for use in the methods provided herein comprises the variable heavy chain and variable light chain CDR sequences of the 2.14H90PT antibody as disclosed in US Patent No. 8,779,108, which is herein incorporated by reference in its entirety.

The term "antigen binding fragment" refers to a portion of an intact antibody and/or refers to the antigenic determining variable regions of an intact antibody. It is known that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of antibody fragments include, but are not limited to, Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, single chain antibodies, diabodies, and multispecific antibodies formed from antibody fragments.

By "Interferon gamma (IFNG) polypeptide" is meant a protein or fragment thereof having at least about 85% amino acid sequence identity to NCBI Accession No. P01579 and having immunomodulatory activity. An exemplary IFNG amino acid sequence (Uniprot Accession No. P01579) is provided as SEQ ID NO: 9.

By "IFNG polynucleotide" is meant a nucleic acid molecule encoding an IFNG protein. The sequence of an exemplary IFNG polynucleotide is provided at NCBI Accession No.

NM_000619, which is reproduced as SEQ ID NO: 10.

By "anti-PD-Ll antibody" is meant an antibody or antigen binding fragment thereof that selectively binds a PD-L1 polypeptide. Exemplary anti-PD-Ll antibodies are described for example at U.S. Patent No. 8,779,108, which is herein incorporated by reference. Durvalumab is an exemplary PD-L1 antibody. Following treatment with Durvalumab, a patient achieves disease control (DC). Disease control can be a complete response (CR), partial response (PR), or stable disease (SD). Sequences of Durvalumab are provided in a sequence listing herein below.

By "PD-L1 polypeptide" is meant a polypeptide or fragment thereof having at least about 85%, 95% or 100% amino acid identity to NCBI Accession No. NP_001254635 (SEQ ID NO: 11) and having PD-1 and CD80 binding activity.

By "PD-L1 nucleic acid molecule" is meant a polynucleotide encoding a PD-L1 polypeptide. An exemplary PD-L1 nucleic acid molecule sequence is provided at NCBI

Accession No. NM_001267706 (SEQ ID NO: 12).

By "C-X-C motif chemokine 9 precursor (CXCL9) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid sequence identity to NCBI Accession No. NP_002407.1 and having chemokine activity. The sequence of an exemplary CXCL9 polypeptide is provided below.

DEFINITION C-X-C motif chemokine 9 precursor [Homo sapiens] .

ACCESSION NP_002407

1 mkksgvlfll giillvligv qgtp vrkgr cscistnqgt ihlqslkdlk qfapspscek 61 ieiiatlkng vqtclnpdsa dvkelikkwe kqvsqkkkqk ngkkhqkkkv lkvrksqrsr

121 qkktt (SEQ ID NO: 13) By "CXCL9 polynucleotide" is meant a polynucleotide encoding a CXCL9 polypeptide. The sequence of an exemplary CXCL9 polynucleotide (NCBI Accession No. NM_002416.2) is provided below:

1 aaaatgtgtt ctctaaagaa tttctcaggc tcaaaatcca atacaggagt gacttggaac

61 tccattctat cactatgaag aaaagtggtg ttcttttcct cttgggcatc atcttgctgg

121 ttctgattgg agtgcaagga accccagtag tgagaaaggg tcgctgttcc tgcatcagca

181 ccaaccaagg gactatccac ctacaatcct tgaaagacct taaacaattt gccccaagcc

241 cttcctgcga gaaaattgaa atcattgcta cactgaagaa tggagttcaa acatgtctaa

301 acccagattc agcagatgtg aaggaactga ttaaaaagtg ggagaaacag gtcagccaaa

361 agaaaaagca aaagaatggg aaaaaacatc aaaaaaagaa agttctgaaa gttcgaaaat

421 ctcaacgttc tcgtcaaaag aagactacat aagagaccac ttcaccaata agtattctgt

481 gttaaaaatg ttctatttta attataccgc tatcattcca aaggaggatg gcatataata

541 caaaggctta ttaatttgac tagaaaattt aaaacattac tctgaaattg taactaaagt

601 tagaaagttg attttaagaa tccaaacgtt aagaattgtt aaaggctatg attgtctttg

661 ttcttctacc acccaccagt tgaatttcat catgcttaag gccatgattt tagcaatacc

721 catgtctaca cagatgttca cccaaccaca tcccactcac aacagctgcc tggaagagca

781 gccctaggct tccacgtact gcagcctcca gagagtatct gaggcacatg tcagcaagtc

841 ctaagcctgt tagcatgctg gtgagccaag cagtttgaaa ttgagctgga cctcaccaag

901 ctgctgtggc catcaacctc tgtatttgaa tcagcctaca ggcctcacac acaatgtgtc

961 tgagagattc atgctgattg ttattgggta tcaccactgg agatcaccag tgtgtggctt

1021 tcagagcctc ctttctggct ttggaagcca tgtgattcca tcttgcccgc tcaggctgac

1081 cactttattt ctttttgttc ccctttgctt cattcaagtc agctcttctc catcctacca

1141 caatgcagtg cctttcttct ctccagtgca cctgtcatat gctctgattt atctgagtca

1201 actcctttct catcttgtcc ccaacacccc acagaagtgc tttcttctcc caattcatcc

1261 tcactcagtc cagcttagtt caagtcctgc ctcttaaata aacctttttg gacacacaaa

1321 ttatcttaaa actcctgttt cacttggttc agtaccacat gggtgaacac tcaatggtta

1381 actaattctt gggtgtttat cctatctctc caaccagatt gtcagctcct tgagggcaag

1441 agccacagta tatttccctg tttcttccac agtgcctaat aatactgtgg aactaggttt

1501 taataatttt ttaattgatg ttgttatggg caggatggca accagaccat tgtctcagag

1561 caggtgctgg ctctttcctg gctactccat gttggctagc ctctggtaac ctcttactta

1621 ttatcttcag gacactcact acagggacca gggatgatgc aacatccttg tctttttatg

1681 acaggatgtt tgctcagctt ctccaacaat aagaagcacg tggtaaaaca cttgcggata

1741 ttctggactg tttttaaaaa atatacagtt taccgaaaat catataatct tacaatgaaa

1801 aggactttat agatcagcca gtgaccaacc ttttcccaac catacaaaaa ttccttttcc

1861 cgaaggaaaa gggctttctc aataagcctc agctttctaa gatctaacaa gatagccacc

1921 gagatcctta tcgaaactca ttttaggcaa atatgagttt tattgtccgt ttacttgttt

1981 cagagtttgt attgtgatta tcaattacca caccatctcc catgaagaaa gggaacggtg 2041 aagtactaag cgctagagga agcagccaag tcggttagtg gaagcatgat tggtgcccag

2101 ttagcctctg caggatgtgg aaacctcctt ccaggggagg ttcagtgaat tgtgtaggag

2161 aggttgtctg tggccagaat ttaaacctat actcactttc ccaaattgaa tcactgctca

2221 cactgctgat gatttagagt gctgtccggt ggagatccca cccgaacgtc ttatctaatc

2281 atgaaactcc ctagttcctt catgtaactt ccctgaaaaa tctaagtgtt tcataaattt

2341 gagagtctgt gacccactta ccttgcatct cacaggtaga cagtatataa ctaacaacca

2401 aagactacat attgtcactg acacacacgt tataatcatt tatcatatat atacatacat

2461 gcatacactc tcaaagcaaa taatttttca cttcaaaaca gtattgactt gtataccttg

2521 taatttgaaa tattttcttt gttaaaatag aatggtatca ataaatagac cattaatcag

2581 aaaacagatc ttgatttttt ttctcttgaa tgtacccttc aactgttgaa tgtttaatag

2641 taaatcttat atgtccttat ttacttttta gctttctctc aaataaagtg taacactagt

2701 tgagataaaa aaaaaaaaaa aaa (SEQ ID NO: 14)

By "CD274 polypeptide", also known as "PD-Ll polypeptide", is meant a polypeptide or fragment thereof having at least about 85% amino acid sequence identity to GenBank:

EAW58763. The sequence of an exemplary CD274 or PD-Ll polypeptide is provided below:

1 mrifavfifm tywhllnaft vtvpkdly v eygsnmtiec kfpvekqldl aalivyweme 61 dkniiqfvhg eedlkvqhss yrqrarllkd qlslgnaalq itdvklqdag vyrcmisygg 121 adykritvkv napynkinqr il vdpvtse heltcqaegy pkaeviwtss dhqvlsgktt 181 ttnskreekl fnvtstlrin tttneifyct frrldpeenh taelvipelp lahppnerth 241 lvilgaillc lgvaltfifr lrkgrmmdvk kcgiqdtnsk kqsdthleet (SEQ ID NO:

15)

By "CD274 polynucleotide", also known as "PD-Ll polynucleotide", is meant a polynucleotide encoding a CD274 or PD-Ll polypeptide. The sequence of an exemplary CD274 polynucleotide is provided at NCBI Accession No. NM_014143, which is reproduced below:

1 ggcgcaacgc tgagcagctg gcgcgtcccg cgcggcccca gttctgcgca gcttcccgag

61 gctccgcacc agccgcgctt ctgtccgcct gcagggcatt ccagaaagat gaggatattt

121 gctgtcttta tattcatgac ctactggcat ttgctgaacg catttactgt cacggttccc

181 aaggacctat atgtggtaga gtatggtagc aatatgacaa ttgaatgcaa attcccagta

241 gaaaaacaat tagacctggc tgcactaatt gtctattggg aaatggagga taagaacatt

301 attcaatttg tgcatggaga ggaagacctg aaggttcagc atagtagcta cagacagagg

361 gcccggctgt tgaaggacca gctctccctg ggaaatgctg cacttcagat cacagatgtg

421 aaattgcagg atgcaggggt gtaccgctgc atgatcagct atggtggtgc cgactacaag

481 cgaattactg tgaaagtcaa tgccccatac aacaaaatca accaaagaat tttggttgtg

541 gatccagtca cctctgaaca tgaactgaca tgtcaggctg agggctaccc caaggccgaa

601 gtcatctgga caagcagtga ccatcaagtc ctgagtggta agaccaccac caccaattcc 661 aagagagagg agaagctttt caatgtgacc agcacactga gaatcaacac aacaactaat

721 gagattttct actgcacttt taggagatta gatcctgagg aaaaccatac agctgaattg

781 gtcatcccag aactacctct ggcacatcct ccaaatgaaa ggactcactt ggtaattctg

841 ggagccatct tattatgcct tggtgtagca ctgacattca tcttccgttt aagaaaaggg

901 agaatgatgg atgtgaaaaa atgtggcatc caagatacaa actcaaagaa gcaaagtgat

961 acacatttgg aggagacgta atccagcatt ggaacttctg atcttcaagc agggattctc

1021 aacctgtggt ttaggggttc atcggggctg agcgtgacaa gaggaaggaa tgggcccgtg

1081 ggatgcaggc aatgtgggac ttaaaaggcc caagcactga aaatggaacc tggcgaaagc

1141 agaggaggag aatgaagaaa gatggagtca aacagggagc ctggagggag accttgatac

1201 tttcaaatgc ctgaggggct catcgacgcc tgtgacaggg agaaaggata cttctgaaca

1261 aggagcctcc aagcaaatca tccattgctc atcctaggaa gacgggttga gaatccctaa

1321 tttgagggtc agttcctgca gaagtgccct ttgcctccac tcaatgcctc aatttgtttt

1381 ctgcatgact gagagtctca gtgttggaac gggacagtat ttatgtatga gtttttccta

1441 tttattttga gtctgtgagg tcttcttgtc atgtgagtgt ggttgtgaat gatttctttt

1501 gaagatatat tgtagtagat gttacaattt tgtcgccaaa ctaaacttgc tgcttaatga

1561 tttgctcaca tctagtaaaa catggagtat ttgtaaggtg cttggtctcc tctataacta

1621 caagtataca ttggaagcat aaagatcaaa ccgttggttg cataggatgt cacctttatt

1681 taacccatta atactctggt tgacctaatc ttattctcag acctcaagtg tctgtgcagt

1741 atctgttcca tttaaatatc agctttacaa ttatgtggta gcctacacac ataatctcat

1801 ttcatcgctg taaccaccct gttgtgataa ccactattat tttacccatc gtacagctga

1861 ggaagcaaac agattaagta acttgcccaa accagtaaat agcagacctc agactgccac

1921 ccactgtcct tttataatac aatttacagc tatattttac tttaagcaat tcttttattc

1981 aaaaaccatt tattaagtgc ccttgcaata tcaatcgctg tgccaggcat tgaatctaca

2041 gatgtgagca agacaaagta cctgtcctca aggagctcat agtataatga ggagattaac

2101 aagaaaatgt attattacaa tttagtccag tgtcatagca taaggatgat gcgaggggaa

2161 aacccgagca gtgttgccaa gaggaggaaa taggccaatg tggtctggga cggttggata

2221 tacttaaaca tcttaataat cagagtaatt ttcatttaca aagagaggtc ggtacttaaa

2281 ataaccctga aaaataacac tggaattcct tttctagcat tatatttatt cctgatttgc

2341 ctttgccata taatctaatg cttgtttata tagtgtctgg tattgtttaa cagttctgtc

2401 ttttctattt aaatgccact aaattttaaa ttcatacctt tccatgattc aaaattcaaa

2461 agatcccatg ggagatggtt ggaaaatctc cacttcatcc tccaagccat tcaagtttcc

2521 tttccagaag caactgctac tgcctttcat tcatatgttc ttctaaagat agtctacatt

2581 tggaaatgta tgttaaaagc acgtattttt aaaatttttt tcctaaatag taacacattg

2641 tatgtctgct gtgtactttg ctatttttat ttattttagt gtttcttata tagcagatgg

2701 aatgaatttg aagttcccag ggctgaggat ccatgccttc tttgtttcta agttatcttt

2761 cccatagctt ttcattatct ttcatatgat ccagtatatg ttaaatatgt cctacatata

2821 catttagaca accaccattt gttaagtatt tgctctagga cagagtttgg atttgtttat

2881 gtttgctcaa aaggagaccc atgggctctc cagggtgcac tgagtcaatc tagtcctaaa 2941 aagcaatctt attattaact ctgtatgaca gaatcatgtc tggaactttt gttttctgct 3001 ttctgtcaag tataaacttc actttgatgc tgtacttgca aaatcacatt ttctttctgg 3061 aaattccggc agtgtacctt gactgctagc taccctgtgc cagaaaagcc tcattcgttg 3121 tgcttgaacc cttgaatgcc accagctgtc atcactacac agccctccta agaggcttcc 3181 tggaggtttc gagattcaga tgccctggga gatcccagag tttcctttcc ctcttggcca 3241 tattctggtg tcaatgacaa ggagtacctt ggctttgcca catgtcaagg ctgaagaaac 3301 agtgtctcca acagagctcc ttgtgttatc tgtttgtaca tgtgcatttg tacagtaatt 3361 ggtgtgacag tgttctttgt gtgaattaca ggcaagaatt gtggctgagc aaggcacata 3421 gtctactcag tctattccta agtcctaact cctccttgtg gtgttggatt tgtaaggcac 3481 tttatccctt ttgtctcatg tttcatcgta aatggcatag gcagagatga tacctaattc 3541 tgcatttgat tgtcactttt tgtacctgca ttaatttaat aaaatattct tatttatttt 3601 gttacttggt acaccagcat gtccattttc ttgtttattt tgtgtttaat aaaatgttca 3661 gtttaacatc ccagtggaga aagttaaaaa a (SEQ ID NO: 16)

By "Lymphocyte activating 3 (LAG3) polypeptide" is meant a polypeptide or fragment thereof having at least about 85% amino acid sequence identity to NCBI Accession No.

AAH52589 (LAG3 polypeptide). The sequence of an exemplary LAG3 polypeptide is provided below.

DEFINITION LAG3 protein [Homo sapiens] .

ACCESSION AAH52589

1 mweaqflgll flqplwvapv kplqpgaevp vwaqegapa qlpcsptipl qdlsllrrag

61 vtwqhqpdsg ppaaapghpl apgphpaaps swgprprryt vlsvgpgglr sgrlplqprv

121 qldergrqrg dfslwlrpar radageyraa vhlrdralsc rlrlrlgqas mtasppgslr

181 asdwvilncs fsrpdrpasv hwfrnrgqgr vpvresphhh laesflflpq vspmdsgpwg

241 ciltyrdgfn vsimynltvl glepptpltv yagagsrvgl pcrlpagvgt rsfltakwtp

301 pgggpdllvt gdngdftlrl edvsqaqagt ytchihlqeq qlnatvtlai itgqpqvgke

(SEQ ID NO: 17)

By "LAG3 polynucleotide" is meant a polynucleotide encoding a LAG3 polypeptide. The sequence of an exemplary LAG3 polynucleotide is provided at NCBI Accession No.

NM_002286, which is reproduced below:

1 acaggggtga aggcccagag accagcagaa cggcatccca gccacgacgg ccactttgct 61 ctgtctgctc tccgccacgg ccctgctctg ttccctggga cacccccgcc cccacctcct 121 caggctgcct gatctgccca gctttccagc tttcctctgg attccggcct ctggtcatcc 181 ctccccaccc tctctccaag gccctctcct ggtctccctt cttctagaac cccttcctcc 241 acctccctct ctgcagaact tctcctttac cccccacccc ccaccactgc cccctttcct 301 tttctgacct ccttttggag ggctcagcgc tgcccagacc ataggagaga tgtgggaggc 361 tcagttcctg ggcttgctgt ttctgcagcc gctttgggtg gctccagtga agcctctcca 421 gccaggggct gaggtcccgg tggtgtgggc ccaggagggg gctcctgccc agctcccctg

481 cagccccaca atccccctcc aggatctcag ccttctgcga agagcagggg tcacttggca

541 gcatcagcca gacagtggcc cgcccgctgc cgcccccggc catcccctgg cccccggccc

601 tcacccggcg gcgccctcct cctgggggcc caggccccgc cgctacacgg tgctgagcgt

661 gggtcccgga ggcctgcgca gcgggaggct gcccctgcag ccccgcgtcc agctggatga

721 gcgcggccgg cagcgcgggg acttctcgct atggctgcgc ccagcccggc gcgcggacgc

781 cggcgagtac cgcgccgcgg tgcacctcag ggaccgcgcc ctctcctgcc gcctccgtct

841 gcgcctgggc caggcctcga tgactgccag ccccccagga tctctcagag cctccgactg

901 ggtcattttg aactgctcct tcagccgccc tgaccgccca gcctctgtgc attggttccg

961 gaaccggggc cagggccgag tccctgtccg ggagtccccc catcaccact tagcggaaag

1021 cttcctcttc ctgccccaag tcagccccat ggactctggg ccctggggct gcatcctcac

1081 ctacagagat ggcttcaacg tctccatcat gtataacctc actgttctgg gtctggagcc

1141 cccaactccc ttgacagtgt acgctggagc aggttccagg gtggggctgc cctgccgcct

1201 gcctgctggt gtggggaccc ggtctttcct cactgccaag tggactcctc ctgggggagg

1261 ccctgacctc ctggtgactg gagacaatgg cgactttacc cttcgactag aggatgtgag

1321 ccaggcccag gctgggacct acacctgcca tatccatctg caggaacagc agctcaatgc

1381 cactgtcaca ttggcaatca tcacagtgac tcccaaatcc tttgggtcac ctggatccct

1441 ggggaagctg ctttgtgagg tgactccagt atctggacaa gaacgctttg tgtggagctc

1501 tctggacacc ccatcccaga ggagtttctc aggaccttgg ctggaggcac aggaggccca

1561 gctcctttcc cagccttggc aatgccagct gtaccagggg gagaggcttc ttggagcagc

1621 agtgtacttc acagagctgt ctagcccagg tgcccaacgc tctgggagag ccccaggtgc

1681 cctcccagca ggccacctcc tgctgtttct catccttggt gtcctttctc tgctcctttt

1741 ggtgactgga gcctttggct ttcacctttg gagaagacag tggcgaccaa gacgattttc

1801 tgccttagag caagggattc accctccgca ggctcagagc aagatagagg agctggagca

1861 agaaccggag ccggagccgg agccggaacc ggagcccgag cccgagcccg agccggagca

1921 gctctgacct ggagctgagg cagccagcag atctcagcag cccagtccaa ataaactccc 1981 tgtcagcagc aaaaa (SEQ ID NO: 18)

The term "antibody," as used in this disclosure, refers to an immunoglobulin or a fragment or a derivative thereof, and encompasses any polypeptide comprising an antigen- binding site, regardless whether it is produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, polyspecific, non-specific, humanized, single- chain, chimeric, synthetic, recombinant, hybrid, mutated, and grafted antibodies. Unless otherwise modified by the term "intact," as in "intact antibodies," for the purposes of this disclosure, the term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that retain antigen-binding function, i.e., the ability to bind PD-Ll specifically. Typically, such fragments would comprise an antigen-binding domain. The terms "antigen-binding domain," "antigen-binding fragment," and "binding fragment" refer to a part of an antibody molecule that comprises amino acids responsible for the specific binding between the antibody and the antigen. In instances, where an antigen is large, the antigen-binding domain may only bind to a part of the antigen. A portion of the antigen molecule that is responsible for specific interactions with the antigen-binding domain is referred to as "epitope" or "antigenic determinant." An antigen-binding domain typically comprises an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH), however, it does not necessarily have to comprise both. For example, a so-called Fd antibody fragment consists only of a V _H domain, but still retains some antigen-binding function of the intact antibody.

Binding fragments of an antibody are produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact antibodies. Binding fragments include Fab, Fab', F(ab')2, Fv, and single-chain antibodies. An antibody other than a "bispecific" or "bifunctional" antibody is understood to have each of its binding sites identical. Digestion of antibodies with the enzyme, papain, results in two identical antigen-binding fragments, known also as "Fab" fragments, and a "Fc" fragment, having no antigen-binding activity but having the ability to crystallize. Digestion of antibodies with the enzyme, pepsin, results in an F(ab')2 fragment in which the two arms of the antibody molecule remain linked and comprise two-antigen binding sites. The F(ab')2 fragment has the ability to crosslink antigen. "Fv" when used herein refers to the minimum fragment of an antibody that retains both antigen-recognition and antigen-binding sites. "Fab" when used herein refers to a fragment of an antibody that comprises the constant domain of the light chain and the CHI domain of the heavy chain.

The term "mAb" refers to monoclonal antibody. Antibodies of the invention comprise without limitation whole native antibodies, bispecific antibodies; chimeric antibodies; Fab, Fab', single chain V region fragments (scFv), fusion polypeptides, and unconventional antibodies.

By "biologic sample" is meant any tissue, cell, fluid, or other material derived from an organism. In one embodiment, a biological sample is a tumor biopsy sample.

A "biomarker" or "marker" as used herein generally refers to a protein, nucleic acid molecule, clinical indicator, or other analyte that is associated with a disease. In one

embodiment, a marker is differentially present in a biological sample obtained from a subject having a disease (e.g., lung cancer) relative to the level present in a control sample or reference. 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.

"Detect" refers to identifying the presence, absence or amount of the analyte to be detected.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ.

Lung cancer includes small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC). There are three main subtypes of NSCLC: squamous cell carcinoma,

adenocarcinoma, and large cell (undifferentiated) carcinoma. Other subtypes include

adenosquamous carcinoma and sarcomatoid carcinoma.

The terms "isolated," "purified," or "biologically pure" refer to material that is free to varying degrees from components which normally accompany it as found in its native state. "Isolate" denotes a degree of separation from original source or surroundings. "Purify" denotes a degree of separation that is higher than isolation. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not materially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of this invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, for example, polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can denote that a nucleic acid or protein gives rise to essentially one band in an electrophoretic gel. For a protein that can be subjected to modifications, for example, phosphorylation or glycosylation, different

modifications may give rise to different isolated proteins, which can be separately purified.

By "reference" is meant a standard of comparison. In one embodiment, the level of interferon gamma present in a sample from a patient that is partially responsive to a therapy of the invention is compared to the level present in a corresponding sample obtained from a patient having progressive disease.

By "responsive" in the context of therapy is meant susceptible to treatment.

By "specifically binds" is meant a compound (e.g., antibody) that recognizes and binds a molecule (e.g., polypeptide), but which does not substantially recognize and bind other molecules in a sample, for example, a biological sample. For example, two molecules that specifically bind form a complex that is relatively stable under physiologic conditions. Specific binding is characterized by a high affinity and a low to moderate capacity as distinguished from nonspecific binding which usually has a low affinity with a moderate to high capacity. Typically, binding is considered specific when the affinity constant KA IS higher than 10 ⁶ M ^_1 , or more preferably higher than 10 ^s M ^-1 . If necessary, non-specific binding can be reduced without substantially affecting specific binding by varying the binding conditions. The appropriate binding conditions such as concentration of antibodies, ionic strength of the solution,

temperature, time allowed for binding, concentration of a blocking agent (e.g., serum albumin, milk casein), etc., may be optimized by a skilled artisan using routine techniques.

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 of 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. 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-D are Kaplan-Meier plots of Overall Response (OS) for CXCL9, IFNG, LAG3, and CD274 (PD-Ll), respectively, in NSCLC patients. Each plot is cut into high (dashed line) or low (solid line) expression using the receiver operator characteristic (ROC)-identified cut points.

Figures 1E-H are Kaplan-Meier plots of progression-free response (PFS) for CXCL9, IFNG, LAG3, and CD274 (PD-Ll) in NSCLC patients. Each plot is cut into high (dashed line) or low (solid line) expression using the ROC-identified cut points.

Figure 2 provides correlation scatter plots of the four genes CXCL9, IFNG, LAG3, and CD274 (Spearman's coefficient) in non-small cell lung cancer (NSCLC).

Figure 3A is a Kaplan-Meier plot of Overall Response (OS) for the four genes CXCL9, IFNG, LAG3, and CD274 signature in NSCLC patients. Each plot is cut into high (dashed line) or low (solid line) expression using the ROC-identified cut point.

Figure 3B is a Kaplan-Meier plot of progression-free response (PFS) for the four genes CXCL9, IFNG, LAG3, and CD274 signature in NSCLC patients. Each plot is cut into high (dashed line) or low (solid line) expression using the ROC-identified cut point.

Figure 4 shows the on-treatment effects by durvalumab for gene signature in NSCLC before and after dosing.

Figure 5A shows the progression-free response of the four gene signature in bladder cancer patients. Each plot is cut into high (dashed line) or low (solid line) expression using the upper tertile cut point.

Figure 5B shows the progression-free response of IFNG gene signature in bladder cancer patients. Each plot is cut into high (dashed line) or low (solid line) expression using the upper tertile cut point. DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods for selecting patients as having a solid tumor (e.g., bladder cancer, ovarian cancer, colorectal cancer, head and neck cancer, cervical cancer, renal cell carcinoma, and non-small cell lung cancer (NSCLC)) that is responsive to treatment with an anti-PD-Ll antibody, and methods of treating such patients. The method involves detecting expression of an IFNG polynucleotide and/or or more polynucleotide markers:

CXCL9, CD274, LAG3, in a biological sample of the patient.

The invention is based, at least in part, on the discovery that patients having lung cancer (e.g., non-squamous cell or squamous cell non-small cell lung cancer) or bladder cancer that is responsive to treatment with an anti-PD-Ll antibody may be identified by detecting increased levels of CXCL9, CD274, LAG3, and IFNG mRNA in a tumor or blood sample of a subject. In some embodiments, the patients may be identified by detecting increased levels of one or more of CXCL9, CD274, LAG3, and IFNG mRNA in a tumor or blood sample of the patient. In some embodiments, the patient is identified by detecting increased levels of the combination of CXCL9, CD274, LAG3, and IFNG mRNA in a tumor or blood sample of the patient.

Accordingly, the invention provides methods for identifying subjects that have a solid tumor (e.g., breast, colon, colorectal, lung, renal, including renal cell carcinoma, gastric, bladder, non-small cell lung cancer (NSCLC), hepatocellular cancer (HCC), pancreatic cancer, melanoma) that is likely to respond to anti-PD-Ll antibody treatment based on an increase in the level of one or more of CXCL9, CD274, LAG3, and IFNG mRNA in a subject tumor or blood sample. In some embodiments, the invention provides methods for identifying subjects that have lung cancer or bladder cancer that is likely to respond to anti-PD-Ll antibody treatment based on an increase in the level of the combination of CXCL9, CD274, LAG3, and IFNG mRNAs in a subject tumor or blood sample.

Aspects of the present invention provide methods for treating lung cancer (e.g., non- squamous cell or squamous cell non- small cell lung cancer) with an anti-PD-Ll antibody in a patient identified by detecting an increase in the level of one or more of CXCL9, CD274, LAG3, and IFNG polynucleotides in a tumor or blood sample of the patient. In some embodiments, the patient is identified by detecting an increase in the levels of a combination of CXCL9, CD274, LAG3, and IFNG polynucleotides in a tumor or blood sample of the patient. Aspects of the present invention provide methods for treating bladder cancer with an anti- PD-L1 antibody in a patient identified by detecting an increase in the level of one or more of CXCL9, CD274, LAG3, and IFNG polynucleotides in a tumor or blood sample of the patient. In some embodiments, the patient is identified by detecting an increase in the levels of a

combination of CXCL9, CD274, LAG3, and IFNG polynucleotides in a tumor or blood sample of the patient.

B7-H1/CD274/PD-L1

B7-H1, also known as CD274 and PD-L1, is a type I transmembrane protein of approximately 53kDa in size. In humans B7-H1 is expressed on a number of immune cell types including activated and anergic/exhausted T cells, on naive and activated B cells, as well as on myeloid dendritic cells (DC), monocytes and mast cells. It is also expressed on non-immune cells including islets of the pancreas, Kupffer cells of the liver, vascular endothelium and selected epithelia, for example airway epithelia and renal tubule epithelia, where its expression is enhanced during inflammatory episodes. B7-H1 expression is also found at increased levels on a number of tumors including, but not limited to breast, colon, colorectal, lung, renal, including renal cell carcinoma, gastric, bladder, non-small cell lung cancer (NSCLC), hepatocellular cancer (HCC), and pancreatic cancer, as well as melanoma.

B7-H1 is known to bind two alternative ligands, the first of these, PD-1, is a 50-55 kDa type I transmembrane receptor that was originally identified in a T cell line undergoing activation-induced apoptosis. PD-1 is expressed on activated T cells, B cells, and monocytes, as well as other cells of the immune system and binds both B7-H1 (PD-L1) and the related B7-DC (PD-L2). The second is the B7 family member B7-1, which is expressed on activated T cells, B cells, monocytes and antigen presenting cells.

Signaling via the PD-1/B7-H1 axis is believed to serve important, non-redundant functions within the immune system, by negatively regulating T cell responses. B7-H1 expression on tumor cells is believed to aid tumors in evading detection and elimination by the immune system. B7-H1 functions in this respect via several alternative mechanisms including driving exhaustion and anergy of tumor infiltrating T lymphocytes, stimulating secretion of immune repressive cytokines into the tumor micro-environment, stimulating repressive regulatory T cell function and protecting B7-H1 expressing tumor cells from lysis by tumor cell specific cytotoxic T cells.

Anti-PD-Ll Antibodies

Antibodies that specifically bind and inhibit PD-Ll activity (e.g., binding to PD-1 and/or

CD80) are useful for the treatment of lung cancer (e.g., non-small cell lung cancer). Durvalumab is an exemplary anti-PD-Ll antibody that is selective for B7-H1 and blocks the binding of B7- Hl to the PD-1 and CD80 receptors. Durvalumab can relieve B7-H1 -mediated suppression of human T-cell activation in vitro and inhibits tumor growth in a xenograft model via a T-cell dependent mechanism. Other agents that could be used include agents that inhibit PD-Ll and/or PD-1 (AB or other).

Information regarding Durvalumab (or fragments thereof) for use in the methods provided herein can be found in U.S. Patent No. 8,779,108, the disclosure of which is incorporated herein by reference in its entirety. The fragment crystallizable (Fc) domain of durvalumab contains a triple mutation in the constant domain of the IgGl heavy chain that reduces binding to the complement component Clq and the Fey receptors responsible for mediating antibody-dependent cell-mediated cytotoxicity (ADCC).

Durvalumab and antigen-binding fragments thereof for use in the methods provided herein comprises a heavy chain and a light chain or a heavy chain variable region and a light chain variable region. In a specific aspect, Durvalumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a light chain variable region comprising the amino acid sequence of SEQ ID NO: 1 and a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2. In a specific aspect, Durvalumab or an antigen-binding fragment thereof for use in the methods provided herein comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the Kabat- defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 3-5, and wherein the light chain variable region comprises the Kabat-defined CDR1, CDR2, and CDR3 sequences of SEQ ID NOs: 6-8. Those of ordinary skill in the art would easily be able to identify Chothia-defined, Abm-defined or other CDR definitions known to those of ordinary skill in the art. In a specific aspect, Durvalumab or an antigen-binding fragment thereof for use in the methods provided herein comprises the variable heavy chain and variable light chain CDR sequences of the 2.14H90PT antibody as disclosed in U.S. Patent No. 8,779, 108, which is herein incorporated by reference in its entirety.

Characterizing Responsiveness to Anti-PD-Ll Antibody

In characterizing the responsiveness of a solid tumor (e.g., breast, colon, colorectal, lung, renal, including renal cell carcinoma, gastric, bladder, non-small cell lung cancer (NSCLC), hepatocellular cancer (HCC), pancreatic cancer, melanoma) to anti-PD-Ll antibody treatment, the level of expression of one or more of CXCL9, CD274, LAG3 and IFNG polynucleotides is measured in different types of biologic samples (e.g., tumor, blood, and/or lymph node samples).

In some embodiments, the polynucleotide expression of one or more of polynucleotide markers CXCL9, CD274, LAG3 and IFNG is higher in a tumor or blood sample obtained from a subject that is responsive to anti-PD-Ll antibody than the level of expression in a non-responsive subject (e.g., a subject with progressive disease). In one embodiment, levels of polynucleotide markers IFNG and CXCL9 are measured. In one embodiment, levels of polynucleotide markers IFNG and CD274 are measured. In one embodiment, levels of polynucleotide markers IFNG and LAG3 are measured. In one embodiment, levels of polynucleotide markers IFNG, CXCL9, and either LAG3 or CD274 are measured. In one embodiment, an alteration in expression is calculated using cycle threshold (Ct) values. For example, the Ct value of an IFNG gene is obtained and from that value the Ct value of a reference gene (e.g., B2M, ACTB, GAPDH) is subtracted from the mean Ct value for each gene to obtain a Delta-Ct value. The final gene expression score is defined as (20 - ACt). In other embodiments, expression of a marker of the invention is increased by at least about 2, 3, 4, 5 or 10-fold in a responsive patient relative to the level in a non-responsive subject (e.g., a subject with progressive disease). CXCL9, CD274, LAG3 and IFNG polynucleotide fold change values are determined using any method known in the art, including but not limited to quantitative PCR, RT-PCR, Northern blotting, in situ hybridization, fluorescence in situ hybridization (FISH), microarray, and/or RN A- sequencing.

Selection of a Treatment Method

Subjects suffering a solid tumor (e.g., breast, colon, colorectal, lung, renal, including renal cell carcinoma, gastric, bladder, non-small cell lung cancer (NSCLC), hepatocellular cancer (HCC), pancreatic cancer, melanoma) may be tested for one or more of CXCL9, CD274, LAG3 and IFNG polynucleotide expression in the course of selecting a treatment method.

Patients characterized as having high expression (e.g., as defined by Ct score) or increased expression relative to a reference level are identified as responsive to anti-PD-Ll treatment. Treatment with an Anti-PD-Ll Antibody

Patients identified as having tumors or blood samples that express one or more of CXCL9, CD274, LAG3 and IFNG polynucleotides particularly at high levels, are likely to be responsive to anti-PD-Ll antibody therapy. Such patients are administered an anti-PD-Ll antibody, such as Durvalumab, or an antigen-binding fragment thereof. In some embodiments, the anti-PD-Ll antibody, such as Durvalumab, or an antigen-binding fragment thereof can be administered only once or infrequently while still providing benefit to the patient. In further aspects the patient is administered additional follow-on doses. Follow-on doses can be administered at various time intervals depending on the patient's age, weight, clinical assessment, tumor burden, and/or other factors, including the judgment of the attending physician.

In some embodiments, at least two doses of Durvalumab, or an antigen-binding fragment thereof, are administered to the patient. In some embodiments, at least three doses, at least four doses, at least five doses, at least six doses, at least seven doses, at least eight doses, at least nine doses, at least ten doses, or at least fifteen doses or more can be administered to the patient. In some embodiments, Durvalumab or an antigen-binding fragment thereof is administered over a two-week treatment period, over a four-week treatment period, over a six-week treatment period, over an eight-week treatment period, over a twelve-week treatment period, over a twenty-four- week treatment period, or over a one-year or more treatment period. In some embodiments, Durvalumab or an antigen-binding fragment thereof is administered over a three-week treatment period, a six-week treatment period, over a nine-week treatment period, over a twelve-week treatment period, over a twenty-four-week treatment period, or over a one-year or more treatment period. In some embodiments, Durvalumab or an antigen-binding fragment thereof is administered over a two-month treatment period, over a four-month treatment period, or over a six-month or more treatment period (e.g., during a maintenance phase).

The amount of Durvalumab, or an antigen-binding fragment thereof, to be administered to the patient will depend on various parameters, such as the patient's age, weight, clinical assessment, tumor burden and/or other factors, including the judgment of the attending physician.

In certain aspects, administration of Durvalumab, or an antigen-binding fragment thereof, according to the methods provided herein is through parenteral administration. For example, Durvalumab or an antigen-binding fragment thereof, can be administered by intravenous infusion or by subcutaneous injection. In some embodiments, the administration is by intravenous infusion.

The methods provided herein can decrease tumor size, retard tumor growth or maintain a steady state. In certain aspects the reduction in tumor size can be significant based on appropriate statistical analyses. A reduction in tumor size can be measured by comparison to the size of patient's tumor at baseline, against an expected tumor size, against an expected tumor size based on a large patient population, or against the tumor size of a control population. In certain aspects provided herein, the administration of Durvalumab can reduce a tumor size by at least 25%. In certain aspects provided herein, the administration of Durvalumab can reduce a tumor size by at least 25% within about 6 weeks of the first treatment. In certain aspects provided herein, the administration of Durvalumab can reduce a tumor size by at least 50%. In certain aspects provided herein, the administration of Durvalumab can reduce a tumor size by at least 50% within about 10 weeks of the first treatment. In certain aspects provided herein, the administration of Durvalumab can reduce a tumor size by at least 75%. In certain aspects provided herein, the administration of Durvalumab can reduce a tumor size by at least 75% within about 10 weeks of the first treatment.

In certain aspects, use of the methods provided herein, i.e., administration of

Durvalumab, or an antigen-binding fragment thereof, can decrease tumor size within 6 weeks, within 7 weeks, within 8 weeks, within 9 weeks, within 10 weeks, within 12 weeks, within 16 weeks, within 20 weeks, within 24 weeks, within 28 weeks, within 32 weeks, within 36 weeks, within 40 weeks, within 44 weeks, within 48 weeks, or within 52 weeks of the first treatment.

The methods provided herein can decrease or retard tumor growth. In some aspects the reduction or retardation can be statistically significant. A reduction in tumor growth can be measured by comparison to the growth of patient's tumor at baseline, against an expected tumor growth, against an expected tumor growth based on a large patient population, or against the tumor growth of a control population. In certain aspects, a patient achieves disease control (DC). Disease control can be a complete response (CR), partial response (PR), or stable disease (SD).

A "complete response" (CR) refers to the disappearance of all lesions, whether measurable or not, and no new lesions. Confirmation can be obtained using a repeat, consecutive assessment no less than four weeks from the date of first documentation. New, non-measurable lesions preclude CR.

A "partial response" (PR) refers to a decrease in tumor burden > 50% relative to baseline. Confirmation can be obtained using a consecutive repeat assessment at least 4 weeks from the date of first documentation

"Progressive disease" (PD) refers to an increase in tumor burden > 25% relative to the minimum recorded (nadir). Confirmation can be obtained by a consecutive repeat assessment at least 4 weeks from the date of first documentation. New, non-measurable lesions do not define PD.

"Stable disease" (SD) refers to not meeting the criteria for CR, PR, or PD.

In certain aspects, administration of Durvalumab or an antigen-binding fragment thereof can increase progression-free survival (PFS).

In certain aspects, administration of Durvalumab or an antigen-binding fragment thereof can increase overall survival (OS).

As provided herein, Durvalumab or an antigen-binding fragment thereof can also decrease free B7-H1 levels. Free B7-H1 refers to B7-H1 that is not bound (e.g., by

Durvalumab). In some embodiments, B7-H1 levels are reduced by at least 80%. In some embodiments, B7-H1 levels are reduced by at least 90%. In some embodiments, B7-H1 levels are reduced by at least 95%. In some embodiments, B7-H1 levels are reduced by at least 99%. In some embodiments, B7-H1 levels are eliminated following administration of Durvalumab or an antigen-binding fragment thereof. In some embodiments, administration of Durvalumab or an antigen-binding fragment thereof reduces the rate of increase of B7-H1 levels as compared, e.g., to the rate of increase of B7-H1 levels prior to the administration of Durvalumab or an antigen- binding fragment thereof. Kits

The invention provides kits for characterizing the responsiveness of a subject to anti-PD- Ll antibody treatment. In one embodiment, the kit includes a therapeutic composition containing an effective amount of an antibody that specifically binds a PD-Ll polypeptide in unit dosage form.

A diagnostic kit of the invention provides a reagent (e.g., TaqMan primers/ probes for one or more of CXCL9, CD274, LAG3 and IFNG polynucleotide and housekeeping reference genes) for measuring relative expression of CXCL9, CD274, LAG3 and IFNG polynucleotides.

In some embodiments, the kit comprises a sterile container which contains a therapeutic and/or diagnostic composition; 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.

In one embodiment, a kit of the invention comprises reagents for measuring

polynucleotide expression of one or more of CXCL9, CD274, LAG3 and IFNG and a therapeutic anti-PD-Ll antibody. If desired, the kit further comprises instructions for measuring

polynucleotide expression of one or more of CXCL9, CD274, LAG3 and IFNG and/or instructions for administering the anti-PD-Ll antibody to a subject having a solid tumor, lung cancer (e.g., squamous cell or non-squamous cell carcinoma non-small cell lung cancer) or bladder cancer selected as responsive to anti-PD-Ll antibody treatment. In particular

embodiments, the instructions include at least one of the following: description of the therapeutic agent; dosage schedule and administration for treatment or prevention of a solid tumor, bladder cancer or lung cancer (e.g., non-small cell lung cancer, small cell lung cancer) or symptoms thereof; precautions; warnings; indications; counter- indications; over dosage 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.

The practice of the present invention 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 invention, and are not intended to limit the scope of what the inventors regard as their invention.

EXAMPLES

Example 1: CXCL9, CD274, LAG3 and IFNG correlate with the objective response rate (ORR) in NSCLC

The safety and clinical activity of durvalumab on advanced solid tumors was assessed. RNA sequencing data was generated on patient tumors with available clinical data, where 30 had pre/post NSCLC patient tumor pair as well as 30 bladder patient tumors pre-treatment with available clinical data and subjected to bioinformatics analysis. The following genes were evaluated: CXCL9, PD-Ll (CD274), LAG-3, IFNG, PD-1 (PDCDl), NKG7, CD8A, SLAMF7, PD-L2 (PDCD1LG2), TIM-3 (HAVCR2), CD80, CD86, CTLA-4, CD2, TLR8, GZMK,

TNFRSF4, FOXP3, CD276, B7-H4 (VTCN1), and CD37.

Among the 21 candidate genes evaluated, each was cut into low or high groups using receiver operator characteristic (ROC) calculations with area under the curve (AUC), logistic regression, time-to-event analysis such as Kaplan-Meiers and Cox proportional hazards (PH) models, paired Student's t-tests for pre/post comparisons, and correlation analysis using a Spearman's rank coefficient.

The 21 candidate genes were subsequently correlated with objective response rate (ORR), both without and with adjustment for age, gender, previous therapy lines, smoking status and histology (squamous or non-squamous). Results are presented in Tables 1 and 2, where the top 4 genes in each (ranked by area under the curve (AUC) or p-value) include CXCL9, CD274,

LAG3, and IFNG.

Table 1. ROC analysis results for candidate 21 genes correlating with ORR in NSCLC pre treatment

Table 2. Linear model analysis results for candidate 21 genes correlating with ORR in NSCLC treatment adjusting for age, gender, previous treatment lines, histology, and smoking status

Example 2: CXCL9, CD274, LAG3 and IFNG are correlated with the overall response (OS) or progression-free response (PFS) in NSCLC The same cut points for each of the top four genes identified from the ORR analysis (CXCL9, CD274, LAG3 and IFNG) were used to partition the patients into high or low expressing groups. Then Kaplan Meier estimator (KM) analysis was conducted using overall response (OS) or progression-free response (PFS) as an endpoint for each gene individually (Figures 1A-H). Figures 1A-D show the overall response plots for CXCL9, IFNG, LAG3, and CD274 (PDLl) in NSCLC patients. Each plot is cut into high or low expression using the ROC- identified cut points. Figures 1E-H show the progression free survival plots for CXCL9, IFNG, LAG3, and CD274 (PDLl) in NSCLC patients. Each plot is cut into high or low expression using the ROC -identified cut points.

Using a log-rank test, all four genes showed a statistical difference between high expressing and low expressing groups associating with outcome at an alpha=0.05. The y-axis indicates the outcome, which is probability of either overall (Figures 1A-1D) or progression - free (Figures 1E-1H) survival and the x-axis indicates days. NSCLC Patients are stratified into high or low groups using the upper tertile of expression.

Example 3: CD274 (PDLl) showed no significant induction post treatment

Each of the four genes (CXCL9, CD274, LAG3 and IFNG) was evaluated for significant induction post treatment with durvalumab. A paired t-test was calculated between the post and pre- treatment time points for each gene individually. Results are reported in Table 3. Only CD274 (PDLl) showed no significant induction post treatment at an alpha=0.05. These results indicate that three of four genes are induced by durvalumab in the tumor, suggesting immune activation of relevant immune-specific genes caused by treatment of this molecule.

Table 3. On-treatment effects by durvalumab for four genes in NSCLC

Example 4: IFNG and CXCL9 showed the highest correlation Pretreatment levels of each of the four genes, CXCL9, CD274, LAG3 and IFNG, were correlated with each other to show shared information.

Figure 2 shows Correlation scatter plot of four genes (Spearman's coefficient) in NSCLC. The Spearman's coefficient is a nonparametric measure of statistical dependence between two variables. As evident in Figure 2, the highest correlated genes were IFNG and CXCL9 (r=0.717), which is biologically relevant as IFNG induced the chemokine CXCL9, thus one expects these two genes to be most correlated. Each x and y axis for a plot indicates the specific gene regressed against one of the four other genes. Each data point is a NSCLC patient tumor sample at baseline.

Example 5: A composite signature from the four genes CXCL9, CD274, LAG3 and IFNG in NSCLC

A gene signature was calculated using the median expression of the four genes CXCL9, CD274, LAG3 and IFNG and the previous analyses were repeated using this signature as a predictor of objective response rate (ORR) and overall response/progression free survival.

Results are shown in Tables 4 and 5 and Figure 3. The gene signature is shown to be highly correlated with both objective response as well as overall (Figure 3A) and progression- free (Figure 3B) survival in NSCLC. In the survival figure, the y-axis indicates probability of overall survival while the x-axis indicates time in days. The dashed line indicates patients with expression of the gene signature at the upper tertile of expression.

Table 4. ROC analysis results for gene signature correlating with ORR in NSCLC pre treatment

Table 5. Linear model analysis results gene signature correlating with ORR in NSCLC pretreatment adjusting for age, gender, previous treatment lines, histology, and smoking status The on-treatment effects by durvalumab are illustrated in Figure 4. There is an increase in expression of the gene signature post durvalumab treatment, which indicates immune activation with treatment in NSCLC patients. Example 6: Correlation of the gene signature with objective response rate (ORR) in bladder cancer

The four gene signature was cut into low or high groups using the upper tertile of expression (top 33%) and subsequently correlated with objective response rate. Due to the small sample size, the adjusted model was not calculated. Results for all comers, IFNG alone, and the four gene signature are presented in Table 6. The enrichment of responders is apparent in both the IFNG group and the gene signature positive group in bladder cancer patients treated with durvalumab, as was demonstrated in the NSCLC patient cohort.

Table 6. Correlation results for all comers, 4-gene signature, and IFNG with ORR in bladder cancer pre treatment

Example 7: Correlation of gene signature and IFNG with progression free survival in bladder cancer

The four gene signature was used to partition the patients into high or low expressing groups at the upper tertile of expression. Then Kaplan-Meier analysis was conducted using progression-free survival with the gene signature (Figure 5A) or the IFNG mRNA (Figure 5B) in bladder cancer patients. There was a higher correlation between the gene signature high patients and overall or progression-free survival as compared to IFNG high patients, suggesting that bladder cancer patients with high gene signature may have improved survival when treated with durvalumab, compared to only those with high IFNG mRNA. OS data was not mature, indicating that the follow up time for certain patients had not reached a mature time point to allow comparisons. Using a log-rank test, all gene signature showed a statistical difference between high and low groups at an alpha=0.05.

The results were obtained using the following materials and methods

RNA extraction and RNA-sequencing

Tissues were homogenized in 600 μΐ RLT lysis buffer (Qiagen, Valencia, CA) supplemented with β-mercaptoethanol (β-me) using PowerGen 500 cryogenic homogenizer (Fisher Scientific, Pittsburgh, PA). Homogenized tissue lysates were centrifuged for 3 min at 13,000 x g at room temperature to remove insoluble tissue debris. The supernatant was transferred into a fresh microcentrifuge tube and 1 volume of 100% ethanol was added to the supernatant. The supernatant-ethanol mixture was applied to a Zymo-Spin IC column (Zymo Research, Irvine, CA) for the binding of RNA. On-column DNase digestion was performed with RNase-free DNase I (Qiagen, Valencia, CA) for 15 min at room temperature. RNA was eluted in 15 - 20 μΐ RNase-free water, and the quantity and quality of RNA samples were determined using Nanodrop-1000 Spectrophotometer (Thermo Fisher Scientific, Waltham MA) and 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).

RNA-seq libraries were prepared using the TruSeq Stranded Total RNA Prep Kit according to the manufacturer's instructions (Illumina, San Diego, CA). Ribosomal RNAs were depleted from total RNA (150 ng per sample) using the Ribo-Zero Gold rRNA removal solution containing biotinylated probes targeting eukaryotic cytoplasmic and mitochondrial rRNAs. The rRNA-probe complexes were removed from the sample using streptavidin-coated magnetic beads and the resulting rRNA-depleted sample was subjected to thermal RNA fragmentation prior to cDNA synthesis. The first strand cDNA was synthesized using Superscript III reverse transcriptase (Thermo Fisher Scientific, Waltham MA) and random primers, followed by second strand cDNA synthesis. Next, a single adenylate (A) residue was added to 3' end of the double- stranded cDNA molecules and the resulting A-tailed double stranded cDNAs were ligated to 3'- dT-tailed Illumina TruSeq paired-end adaptors containing unique index barcode sequences. The ligated products were purified and enriched with 15 cycles of Polymerase Chain Reaction (PCR) using the primers provided in the Illumina library prep kit. The size distribution of the RNA-seq libraries was analyzed on a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA) and the library concentrations were determined using Kapa Library Quantification kit (Kapa Biosystems, Woburn, MA). Each library was normalized to 4 nM in 10 mM Tris-HCl, pH 8.0 and denatured in 0. IN NaOH for 5 min prior to the final dilution. Paired-end sequencing runs (2 x 100 cycles) were performed on either HiSeq 2000 or

NextSeq 500 sequencing instruments (Illumina, San Diego, CA). For HiSeq 2000 sequencing runs, denatured libraries (12 pM final concentration) were clustered on a TruSeq v3 Paired-End with a cBot system and sequenced using TruSeq v3 SBS reagents (Illumina, San Diego, CA). Sequencing runs on NextSeq 500 system was carried out with 1.8 pM denatured libraries loaded onto a NextSeq 500/550 v2 flowcell and reagents (Illumina, San Diego, CA).

Bioinformatics analysis

CP1108/NCT01693562 is a nonrandomized, open-label, multicenter phase 1/2 clinical trial evaluating safety and clinical activity of durvalumab in multiple advanced solid tumors. RNA sequencing data was generated on 97 NSCLC patient tumors with available clinical data, where 30 had pre/post NSCLC patient tumor pair as well as 30 bladder patient tumors pre- treatment with available clinical data. Candidate genes evaluated included CXCL9, PD-L1 (CD274), LAG-3, IFNG, PD-1 (PDCD1), NKG7, CD8A, SLAMF7, PD-L2 (PDCD1LG2), TIM- 3 (HAVCR2), CD80, CD86, CTLA-4, CD2, TLR8, GZMK, TNFRSF4, FOXP3, CD276, B7-H4 (VTCNl), and CD37. Analyses included receiver operator characteristic (ROC) calculations with area under the curve (AUC), logistic regression, time-to-event analysis such as Kaplan-Meiers and Cox proportional hazards (PH) models, paired Student's t-tests for pre/post comparisons, and correlation analysis using a Spearman's rank coefficient. Other Embodiments

From the foregoing description, it will be apparent that variations and modifications may be made to the invention 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 subcombination) 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.

SEQUENCE LISTING

SEQ ID NO: 2

> PCT/US2010/058007_72 Sequence 72 from PCT/US2010/058007 Organism: Homo sapiens

SEQ ID NO: 3 - VH CDR1

> PCT/US2010/058007_73 Sequence 73 from PCT/US2010/058007 Organism: Homo sapiens

GFTFSRYWMS SEQ ID NO: 4 - VH CDR2

> PCT/US2010/058007_74 Sequence 74 from PCT/US2010/058007 Organism: Homo sapiens NIKQDGSEKYYVDSVKG

SEQ ID NO: 5 - VH CDR3

> PCT/US2010/058007_75 Sequence 75 from PCT/US2010/058007 Organism: Homo sapiens

EGGWFGELAFDY

SEQ ID NO: 6 - VL CDR1

> PCT/US2010/058007_78 Sequence 78 from PCT/US2010/058007 Organism: Homo sapiens

RASQRVSSSYLA

SEQ ID NO: 7 - VL CDR2

> PCT/US2010/058007_79 Sequence 79 from PCT/US2010/058007 Organism: Homo sapiens

DASSRAT

SEQ ID NO: 8 - VL CDR3

> PCT/US2010/058007_80 Sequence 80 from PCT/US2010/058007 Organism: Homo sapiens

QQYGSLPWT

SEQ ID NO: 9

>sp I P01579 I IFNG_HUMAN Interferon gamma OS=Homo sapiens GN=IFNG PE=1 SV=1 MKYTSYILAFQLCIVLGSLGCYCQDPYVKEAENLKKYFNAGHSDVADNGTLFLGILKNWK EESDRKIMQSQIVSFYFKLFKNFKDDQS IQKSVETIKEDMNVKFFNSNKKKRDDFEKLTN YSVTDLNVQRKAIHELIQVMAELSPAAKTGKRKRSQMLFRGRRASQ

SEQ ID NO: 10

1 cacattgttc tgatcatctg aagatcagct attagaagag aaagatcagt taagtccttt

61 ggacctgatc agcttgatac aagaactact gatttcaact tctttggctt aattctctcg

121 gaaacgatga aatatacaag ttatatcttg gcttttcagc tctgcatcgt tttgggttct

181 cttggctgtt actgccagga cccatatgta aaagaagcag aaaaccttaa gaaatatttt

241 aatgcaggtc attcagatgt agcggataat ggaactcttt tcttaggcat tttgaagaat

301 tggaaagagg agagtgacag aaaaataatg cagagccaaa ttgtctcctt ttacttcaaa

361 ctttttaaaa actttaaaga tgaccagagc atccaaaaga gtgtggagac catcaaggaa

421 gacatgaatg tcaagttttt caatagcaac aaaaagaaac gagatgactt cgaaaagctg

481 actaattatt cggtaactga cttgaatgtc caacgcaaag caatacatga actcatccaa

541 gtgatggctg aactgtcgcc agcagctaaa acagggaagc gaaaaaggag tcagatgctg

601 tttcgaggtc gaagagcatc ccagtaatgg ttgtcctgcc tgcaatattt gaattttaaa

661 tctaaatcta tttattaata tttaacatta tttatatggg gaatatattt ttagactcat

721 caatcaaata agtatttata atagcaactt ttgtgtaatg aaaatgaata tctattaata

781 tatgtattat ttataattcc tatatcctgt gactgtctca cttaatcctt tgttttctga

841 ctaattaggc aaggctatgt gattacaagg ctttatctca ggggccaact aggcagccaa

901 cctaagcaag atcccatggg ttgtgtgttt atttcacttg atgatacaat gaacacttat

961 aagtgaagtg atactatcca gttactgccg gtttgaaaat atgcctgcaa tctgagccag

1021 tgctttaatg gcatgtcaga cagaacttga atgtgtcagg tgaccctgat gaaaacatag

1081 catctcagga gatttcatgc ctggtgcttc caaatattgt tgacaactgt gactgtaccc

1141 aaatggaaag taactcattt gttaaaatta tcaatatcta atatatatga ataaagtgta 1201 tcacaac aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa

SSQ ID NO: II

1 mrifavfifm tywhllnapy nkinqril v dpvtsehelt cqaegypkae viwtssdhqv

61 lsgkttttns kreeklfnvt stlrintttn eifyctfrrl dpeenhtael vipelplahp 121 pnerthlvil gaillclgva ltfifrlrkg rmmdvkkcgi qdtnskkqsd thleet

SEQ ID NO: 12

1 ggcgcaacgc tgagcagctg gcgcgtcccg cgcggcccca gttctgcgca gcttcccgag 61 gctccgcacc agccgcgctt ctgtccgcct gcagggcatt ccagaaagat gaggatattt

121 gctgtcttta tattcatgac ctactggcat ttgctgaacg ccccatacaa caaaatcaac

181 caaagaattt tggttgtgga tccagtcacc tctgaacatg aactgacatg tcaggctgag

241 ggctacccca aggccgaagt catctggaca agcagtgacc atcaagtcct gagtggtaag

301 accaccacca ccaattccaa gagagaggag aagcttttca atgtgaccag cacactgaga 361 atcaacacaa caactaatga gattttctac tgcactttta ggagattaga tcctgaggaa

421 aaccatacag ctgaattggt catcccagaa ctacctctgg cacatcctcc aaatgaaagg

481 actcacttgg taattctggg agccatctta ttatgccttg gtgtagcact gacattcatc

541 ttccgtttaa gaaaagggag aatgatggat gtgaaaaaat gtggcatcca agatacaaac

601 tcaaagaagc aaagtgatac acatttggag gagacgtaat ccagcattgg aacttctgat 661 cttcaagcag ggattctcaa cctgtggttt aggggttcat cggggctgag cgtgacaaga

721 ggaaggaatg ggcccgtggg atgcaggcaa tgtgggactt aaaaggccca agcactgaaa

781 atggaacctg gcgaaagcag aggaggagaa tgaagaaaga tggagtcaaa cagggagcct

841 ggagggagac cttgatactt tcaaatgcct gaggggctca tcgacgcctg tgacagggag

901 aaaggatact tctgaacaag gagcctccaa gcaaatcatc cattgctcat cctaggaaga 961 cgggttgaga atccctaatt tgagggtcag ttcctgcaga agtgcccttt gcctccactc

1021 aatgcctcaa tttgttttct gcatgactga gagtctcagt gttggaacgg gacagtattt

1081 atgtatgagt ttttcctatt tattttgagt ctgtgaggtc ttcttgtcat gtgagtgtgg

1141 ttgtgaatga tttcttttga agatatattg tagtagatgt tacaattttg tcgccaaact

1201 aaacttgctg cttaatgatt tgctcacatc tagtaaaaca tggagtattt gtaaggtgct 1261 tggtctcctc tataactaca agtatacatt ggaagcataa agatcaaacc gttggttgca

1321 taggatgtca cctttattta acccattaat actctggttg acctaatctt attctcagac

1381 ctcaagtgtc tgtgcagtat ctgttccatt taaatatcag ctttacaatt atgtggtagc

1441 ctacacacat aatctcattt catcgctgta accaccctgt tgtgataacc actattattt 1501 tacccatcgt acagctgagg aagcaaacag attaagtaac ttgcccaaac cagtaaatag

1561 cagacctcag actgccaccc actgtccttt tataatacaa tttacagcta tattttactt

1621 taagcaattc ttttattcaa aaaccattta ttaagtgccc ttgcaatatc aatcgctgtg

1681 ccaggcattg aatctacaga tgtgagcaag acaaagtacc tgtcctcaag gagctcatag 1741 tataatgagg agattaacaa gaaaatgtat tattacaatt tagtccagtg tcatagcata

1801 aggatgatgc gaggggaaaa cccgagcagt gttgccaaga ggaggaaata ggccaatgtg

1861 gtctgggacg gttggatata cttaaacatc ttaataatca gagtaatttt catttacaaa

1921 gagaggtcgg tacttaaaat aaccctgaaa aataacactg gaattccttt tctagcatta

1981 tatttattcc tgatttgcct ttgccatata atctaatgct tgtttatata gtgtctggta 2041 ttgtttaaca gttctgtctt ttctatttaa atgccactaa attttaaatt catacctttc

2101 catgattcaa aattcaaaag atcccatggg agatggttgg aaaatctcca cttcatcctc

2161 caagccattc aagtttcctt tccagaagca actgctactg cctttcattc atatgttctt

2221 ctaaagatag tctacatttg gaaatgtatg ttaaaagcac gtatttttaa aatttttttc

2281 ctaaatagta acacattgta tgtctgctgt gtactttgct atttttattt attttagtgt 2341 ttcttatata gcagatggaa tgaatttgaa gttcccaggg ctgaggatcc atgccttctt

2401 tgtttctaag ttatctttcc catagctttt cattatcttt catatgatcc agtatatgtt

2461 aaatatgtcc tacatataca tttagacaac caccatttgt taagtatttg ctctaggaca

2521 gagtttggat ttgtttatgt ttgctcaaaa ggagacccat gggctctcca gggtgcactg

2581 agtcaatcta gtcctaaaaa gcaatcttat tattaactct gtatgacaga atcatgtctg 2641 gaacttttgt tttctgcttt ctgtcaagta taaacttcac tttgatgctg tacttgcaaa

2701 atcacatttt ctttctggaa attccggcag tgtaccttga ctgctagcta ccctgtgcca

2761 gaaaagcctc attcgttgtg cttgaaccct tgaatgccac cagctgtcat cactacacag

2821 ccctcctaag aggcttcctg gaggtttcga gattcagatg ccctgggaga tcccagagtt

2881 tcctttccct cttggccata ttctggtgtc aatgacaagg agtaccttgg ctttgccaca 2941 tgtcaaggct gaagaaacag tgtctccaac agagctcctt gtgttatctg tttgtacatg

3001 tgcatttgta cagtaattgg tgtgacagtg ttctttgtgt gaattacagg caagaattgt

3061 ggctgagcaa ggcacatagt ctactcagtc tattcctaag tcctaactcc tccttgtggt

3121 gttggatttg taaggcactt tatccctttt gtctcatgtt tcatcgtaaa tggcataggc

3181 agagatgata cctaattctg catttgattg tcactttttg tacctgcatt aatttaataa 3241 aatattctta tttattttgt tacttggtac accagcatgt ccattttctt gtttattttg 3301 tgtttaataa aatgttcagt ttaacatccc agtggagaaa gttaaaaaa

SEQ ID NO: 13

DEFINITION C-X-C motif chemokine 9 precursor [Homo sapiens] .

ACCESSION NP_002407

1 mkksgvlfll giillvligv qgtp vrkgr cscistnqgt ihlqslkdlk qfapspscek

61 ieiiatlkng vqtclnpdsa dvkelikkwe kqvsqkkkqk ngkkhqkkkv lkvrksqrsr

121 qkktt

SEQ ID NO: 14

1 aaaatgtgtt ctctaaagaa tttctcaggc tcaaaatcca atacaggagt gacttggaac

61 tccattctat cactatgaag aaaagtggtg ttcttttcct cttgggcatc atcttgctgg

121 ttctgattgg agtgcaagga accccagtag tgagaaaggg tcgctgttcc tgcatcagca

181 ccaaccaagg gactatccac ctacaatcct tgaaagacct taaacaattt gccccaagcc

241 cttcctgcga gaaaattgaa atcattgcta cactgaagaa tggagttcaa acatgtctaa

301 acccagattc agcagatgtg aaggaactga ttaaaaagtg ggagaaacag gtcagccaaa

361 agaaaaagca aaagaatggg aaaaaacatc aaaaaaagaa agttctgaaa gttcgaaaat

421 ctcaacgttc tcgtcaaaag aagactacat aagagaccac ttcaccaata agtattctgt

481 gttaaaaatg ttctatttta attataccgc tatcattcca aaggaggatg gcatataata

541 caaaggctta ttaatttgac tagaaaattt aaaacattac tctgaaattg taactaaagt

601 tagaaagttg attttaagaa tccaaacgtt aagaattgtt aaaggctatg attgtctttg

661 ttcttctacc acccaccagt tgaatttcat catgcttaag gccatgattt tagcaatacc

721 catgtctaca cagatgttca cccaaccaca tcccactcac aacagctgcc tggaagagca

781 gccctaggct tccacgtact gcagcctcca gagagtatct gaggcacatg tcagcaagtc

841 ctaagcctgt tagcatgctg gtgagccaag cagtttgaaa ttgagctgga cctcaccaag

901 ctgctgtggc catcaacctc tgtatttgaa tcagcctaca ggcctcacac acaatgtgtc

961 tgagagattc atgctgattg ttattgggta tcaccactgg agatcaccag tgtgtggctt

1021 tcagagcctc ctttctggct ttggaagcca tgtgattcca tcttgcccgc tcaggctgac

1081 cactttattt ctttttgttc ccctttgctt cattcaagtc agctcttctc catcctacca

1141 caatgcagtg cctttcttct ctccagtgca cctgtcatat gctctgattt atctgagtca

1201 actcctttct catcttgtcc ccaacacccc acagaagtgc tttcttctcc caattcatcc

1261 tcactcagtc cagcttagtt caagtcctgc ctcttaaata aacctttttg gacacacaaa

1321 ttatcttaaa actcctgttt cacttggttc agtaccacat gggtgaacac tcaatggtta

1381 actaattctt gggtgtttat cctatctctc caaccagatt gtcagctcct tgagggcaag 1441 agccacagta tatttccctg tttcttccac agtgcctaat aatactgtgg aactaggttt

1501 taataatttt ttaattgatg ttgttatggg caggatggca accagaccat tgtctcagag

1561 caggtgctgg ctctttcctg gctactccat gttggctagc ctctggtaac ctcttactta

1621 ttatcttcag gacactcact acagggacca gggatgatgc aacatccttg tctttttatg

1681 acaggatgtt tgctcagctt ctccaacaat aagaagcacg tggtaaaaca cttgcggata

1741 ttctggactg tttttaaaaa atatacagtt taccgaaaat catataatct tacaatgaaa

1801 aggactttat agatcagcca gtgaccaacc ttttcccaac catacaaaaa ttccttttcc

1861 cgaaggaaaa gggctttctc aataagcctc agctttctaa gatctaacaa gatagccacc

1921 gagatcctta tcgaaactca ttttaggcaa atatgagttt tattgtccgt ttacttgttt

1981 cagagtttgt attgtgatta tcaattacca caccatctcc catgaagaaa gggaacggtg

2041 aagtactaag cgctagagga agcagccaag tcggttagtg gaagcatgat tggtgcccag

2101 ttagcctctg caggatgtgg aaacctcctt ccaggggagg ttcagtgaat tgtgtaggag

2161 aggttgtctg tggccagaat ttaaacctat actcactttc ccaaattgaa tcactgctca

2221 cactgctgat gatttagagt gctgtccggt ggagatccca cccgaacgtc ttatctaatc

2281 atgaaactcc ctagttcctt catgtaactt ccctgaaaaa tctaagtgtt tcataaattt

2341 gagagtctgt gacccactta ccttgcatct cacaggtaga cagtatataa ctaacaacca

2401 aagactacat attgtcactg acacacacgt tataatcatt tatcatatat atacatacat

2461 gcatacactc tcaaagcaaa taatttttca cttcaaaaca gtattgactt gtataccttg

2521 taatttgaaa tattttcttt gttaaaatag aatggtatca ataaatagac cattaatcag

2581 aaaacagatc ttgatttttt ttctcttgaa tgtacccttc aactgttgaa tgtttaatag

2641 taaatcttat atgtccttat ttacttttta gctttctctc aaataaagtg taacactagt

2701 tgagataaaa aaaaaaaaaa aaa

SEQ ID NO: 15

1 mrifavfifm tywhllnaft vtvpkdlyvv eygsnmtiec kfpvekqldl aalivyweme

61 dkniiqfvhg eedlkvqhss yrqrarllkd qlslgnaalq itdvklqdag vyrcmisygg 121 adykritvkv napynkinqr ilvvdpvtse heltcqaegy pkaeviwtss dhqvlsgktt 181 ttnskreekl fnvtstlrin tttneifyct frrldpeenh taelvipelp lahppnerth 241 lvilgaillc lgvaltfifr lrkgrmmdvk kcgiqdtnsk kqsdthleet

SEQ ID NO: 16

1 ggcgcaacgc tgagcagctg gcgcgtcccg cgcggcccca gttctgcgca gcttcccgag

61 gctccgcacc agccgcgctt ctgtccgcct gcagggcatt ccagaaagat gaggatattt 121 gctgtcttta tattcatgac ctactggcat ttgctgaacg catttactgt cacggttccc 181 aaggacctat atgtggtaga gtatggtagc aatatgacaa ttgaatgcaa attcccagta 241 gaaaaacaat tagacctggc tgcactaatt gtctattggg aaatggagga taagaacatt

301 attcaatttg tgcatggaga ggaagacctg aaggttcagc atagtagcta cagacagagg

361 gcccggctgt tgaaggacca gctctccctg ggaaatgctg cacttcagat cacagatgtg

421 aaattgcagg atgcaggggt gtaccgctgc atgatcagct atggtggtgc cgactacaag

481 cgaattactg tgaaagtcaa tgccccatac aacaaaatca accaaagaat tttggttgtg

541 gatccagtca cctctgaaca tgaactgaca tgtcaggctg agggctaccc caaggccgaa

601 gtcatctgga caagcagtga ccatcaagtc ctgagtggta agaccaccac caccaattcc

661 aagagagagg agaagctttt caatgtgacc agcacactga gaatcaacac aacaactaat

721 gagattttct actgcacttt taggagatta gatcctgagg aaaaccatac agctgaattg

781 gtcatcccag aactacctct ggcacatcct ccaaatgaaa ggactcactt ggtaattctg

841 ggagccatct tattatgcct tggtgtagca ctgacattca tcttccgttt aagaaaaggg

901 agaatgatgg atgtgaaaaa atgtggcatc caagatacaa actcaaagaa gcaaagtgat

961 acacatttgg aggagacgta atccagcatt ggaacttctg atcttcaagc agggattctc

1021 aacctgtggt ttaggggttc atcggggctg agcgtgacaa gaggaaggaa tgggcccgtg

1081 ggatgcaggc aatgtgggac ttaaaaggcc caagcactga aaatggaacc tggcgaaagc

1141 agaggaggag aatgaagaaa gatggagtca aacagggagc ctggagggag accttgatac

1201 tttcaaatgc ctgaggggct catcgacgcc tgtgacaggg agaaaggata cttctgaaca

1261 aggagcctcc aagcaaatca tccattgctc atcctaggaa gacgggttga gaatccctaa

1321 tttgagggtc agttcctgca gaagtgccct ttgcctccac tcaatgcctc aatttgtttt

1381 ctgcatgact gagagtctca gtgttggaac gggacagtat ttatgtatga gtttttccta

1441 tttattttga gtctgtgagg tcttcttgtc atgtgagtgt ggttgtgaat gatttctttt

1501 gaagatatat tgtagtagat gttacaattt tgtcgccaaa ctaaacttgc tgcttaatga

1561 tttgctcaca tctagtaaaa catggagtat ttgtaaggtg cttggtctcc tctataacta

1621 caagtataca ttggaagcat aaagatcaaa ccgttggttg cataggatgt cacctttatt

1681 taacccatta atactctggt tgacctaatc ttattctcag acctcaagtg tctgtgcagt

1741 atctgttcca tttaaatatc agctttacaa ttatgtggta gcctacacac ataatctcat

1801 ttcatcgctg taaccaccct gttgtgataa ccactattat tttacccatc gtacagctga

1861 ggaagcaaac agattaagta acttgcccaa accagtaaat agcagacctc agactgccac

1921 ccactgtcct tttataatac aatttacagc tatattttac tttaagcaat tcttttattc

1981 aaaaaccatt tattaagtgc ccttgcaata tcaatcgctg tgccaggcat tgaatctaca

2041 gatgtgagca agacaaagta cctgtcctca aggagctcat agtataatga ggagattaac

2101 aagaaaatgt attattacaa tttagtccag tgtcatagca taaggatgat gcgaggggaa

2161 aacccgagca gtgttgccaa gaggaggaaa taggccaatg tggtctggga cggttggata

2221 tacttaaaca tcttaataat cagagtaatt ttcatttaca aagagaggtc ggtacttaaa

2281 ataaccctga aaaataacac tggaattcct tttctagcat tatatttatt cctgatttgc

2341 ctttgccata taatctaatg cttgtttata tagtgtctgg tattgtttaa cagttctgtc

2401 ttttctattt aaatgccact aaattttaaa ttcatacctt tccatgattc aaaattcaaa

2461 agatcccatg ggagatggtt ggaaaatctc cacttcatcc tccaagccat tcaagtttcc 2521 tttccagaag caactgctac tgcctttcat tcatatgttc ttctaaagat agtctacatt

2581 tggaaatgta tgttaaaagc acgtattttt aaaatttttt tcctaaatag taacacattg

2641 tatgtctgct gtgtactttg ctatttttat ttattttagt gtttcttata tagcagatgg

2701 aatgaatttg aagttcccag ggctgaggat ccatgccttc tttgtttcta agttatcttt

2761 cccatagctt ttcattatct ttcatatgat ccagtatatg ttaaatatgt cctacatata

2821 catttagaca accaccattt gttaagtatt tgctctagga cagagtttgg atttgtttat

2881 gtttgctcaa aaggagaccc atgggctctc cagggtgcac tgagtcaatc tagtcctaaa

2941 aagcaatctt attattaact ctgtatgaca gaatcatgtc tggaactttt gttttctgct

3001 ttctgtcaag tataaacttc actttgatgc tgtacttgca aaatcacatt ttctttctgg

3061 aaattccggc agtgtacctt gactgctagc taccctgtgc cagaaaagcc tcattcgttg

3121 tgcttgaacc cttgaatgcc accagctgtc atcactacac agccctccta agaggcttcc

3181 tggaggtttc gagattcaga tgccctggga gatcccagag tttcctttcc ctcttggcca

3241 tattctggtg tcaatgacaa ggagtacctt ggctttgcca catgtcaagg ctgaagaaac

3301 agtgtctcca acagagctcc ttgtgttatc tgtttgtaca tgtgcatttg tacagtaatt

3361 ggtgtgacag tgttctttgt gtgaattaca ggcaagaatt gtggctgagc aaggcacata

3421 gtctactcag tctattccta agtcctaact cctccttgtg gtgttggatt tgtaaggcac

3481 tttatccctt ttgtctcatg tttcatcgta aatggcatag gcagagatga tacctaattc

3541 tgcatttgat tgtcactttt tgtacctgca ttaatttaat aaaatattct tatttatttt

3601 gttacttggt acaccagcat gtccattttc ttgtttattt tgtgtttaat aaaatgttca

3661 gtttaacatc ccagtggaga aagttaaaaa a

SEQ ID NO: 17

1 mweaqflgll flqplwvapv kplqpgaevp vwaqegapa qlpcsptipl qdlsllrrag

61 vtwqhqpdsg ppaaapghpl apgphpaaps swgprprryt vlsvgpgglr sgrlplqprv

121 qldergrqrg dfslwlrpar radageyraa vhlrdralsc rlrlrlgqas mtasppgslr

181 asdwvilncs fsrpdrpasv hwfrnrgqgr vpvresphhh laesflflpq vspmdsgpwg

241 ciltyrdgfn vsimynltvl glepptpltv yagagsrvgl pcrlpagvgt rsfltakwtp

301 pgggpdllvt gdngdftlrl edvsqaqagt ytchihlqeq qlnatvtlai itgqpqvgke

SEQ ID NO: 18

1 acaggggtga aggcccagag accagcagaa cggcatccca gccacgacgg ccactttgct

61 ctgtctgctc tccgccacgg ccctgctctg ttccctggga cacccccgcc cccacctcct

121 caggctgcct gatctgccca gctttccagc tttcctctgg attccggcct ctggtcatcc

181 ctccccaccc tctctccaag gccctctcct ggtctccctt cttctagaac cccttcctcc

241 acctccctct ctgcagaact tctcctttac cccccacccc ccaccactgc cccctttcct

301 tttctgacct ccttttggag ggctcagcgc tgcccagacc ataggagaga tgtgggaggc

361 tcagttcctg ggcttgctgt ttctgcagcc gctttgggtg gctccagtga agcctctcca 421 gccaggggct gaggtcccgg tggtgtgggc ccaggagggg gctcctgccc agctcccctg

481 cagccccaca atccccctcc aggatctcag ccttctgcga agagcagggg tcacttggca

541 gcatcagcca gacagtggcc cgcccgctgc cgcccccggc catcccctgg cccccggccc

601 tcacccggcg gcgccctcct cctgggggcc caggccccgc cgctacacgg tgctgagcgt

661 gggtcccgga ggcctgcgca gcgggaggct gcccctgcag ccccgcgtcc agctggatga

721 gcgcggccgg cagcgcgggg acttctcgct atggctgcgc ccagcccggc gcgcggacgc

781 cggcgagtac cgcgccgcgg tgcacctcag ggaccgcgcc ctctcctgcc gcctccgtct

841 gcgcctgggc caggcctcga tgactgccag ccccccagga tctctcagag cctccgactg

901 ggtcattttg aactgctcct tcagccgccc tgaccgccca gcctctgtgc attggttccg

961 gaaccggggc cagggccgag tccctgtccg ggagtccccc catcaccact tagcggaaag

1021 cttcctcttc ctgccccaag tcagccccat ggactctggg ccctggggct gcatcctcac

1081 ctacagagat ggcttcaacg tctccatcat gtataacctc actgttctgg gtctggagcc

1141 cccaactccc ttgacagtgt acgctggagc aggttccagg gtggggctgc cctgccgcct

1201 gcctgctggt gtggggaccc ggtctttcct cactgccaag tggactcctc ctgggggagg

1261 ccctgacctc ctggtgactg gagacaatgg cgactttacc cttcgactag aggatgtgag

1321 ccaggcccag gctgggacct acacctgcca tatccatctg caggaacagc agctcaatgc

1381 cactgtcaca ttggcaatca tcacagtgac tcccaaatcc tttgggtcac ctggatccct

1441 ggggaagctg ctttgtgagg tgactccagt atctggacaa gaacgctttg tgtggagctc

1501 tctggacacc ccatcccaga ggagtttctc aggaccttgg ctggaggcac aggaggccca

1561 gctcctttcc cagccttggc aatgccagct gtaccagggg gagaggcttc ttggagcagc

1621 agtgtacttc acagagctgt ctagcccagg tgcccaacgc tctgggagag ccccaggtgc

1681 cctcccagca ggccacctcc tgctgtttct catccttggt gtcctttctc tgctcctttt

1741 ggtgactgga gcctttggct ttcacctttg gagaagacag tggcgaccaa gacgattttc

1801 tgccttagag caagggattc accctccgca ggctcagagc aagatagagg agctggagca

1861 agaaccggag ccggagccgg agccggaacc ggagcccgag cccgagcccg agccggagca

1921 gctctgacct ggagctgagg cagccagcag atctcagcag cccagtccaa ataaactccc 1981 tgtcagcagc aaaaa