Compositions and methods for identification, assessment, prevention, and treatment of melanoma using pd-l1 isoforms

Abstract

The present invention relates to compositions and methods for identifying, assessing, preventing, and treating melanoma, A variety of PD-L1 isoform biomarkers are provided, wherein alterations in the copy number of one or more of the biomarkers and/or alterations in the amount, structure, and/or activity of one or more of the biomarkers is associated with melanoma status.

What is claimed: 
     
         1 . An isolated polypeptide selected from the group consisting of polypeptides comprising an amino acid sequence listed in Table 2, and fragments thereof, and polypeptides comprising an amino acid sequence having at least 80% identity across their full length with a nucleic acid sequence listed in Table 2, and fragments thereof. 
     
     
         2 . The isolated polypeptide of  claim 1 , wherein the polypeptide has the ability to promote immunoinhibitory function, promote cytokine expression, inhibit T cell activation, inhibit cellular proliferation, bind to PD-1, or bind B7-1. 
     
     
         3 . The isolated polypeptide of  claim 1 , wherein the polypeptide is expressed by melanoma cells. 
     
     
         4 . The isolated polypeptide of  claim 1 , further comprising a heterologous polypeptide. 
     
     
         5 . A pharmaceutical composition comprising a polypeptide of  claim 1  and a pharmaceutically acceptable agent selected from the group consisting of excipients, diluents, and carriers. 
     
     
         6 . An isolated nucleic acid molecule selected from the group consisting of nucleic acid molecules comprising a nucleic acid sequence listed in Table 2 and nucleic acid molecules comprising a nucleic acid sequence having, at least 80% identity across their full length with a nucleic acid sequence listed in Table 2. 
     
     
         7 . The isolated nucleic acid molecule of  claim 6  that encodes a polypeptide of  claim 1 . 
     
     
         8 . An isolated nucleic acid molecule comprising, a nucleotide sequence which is complementary to the nucleotide sequence of a nucleic acid molecule of  claim 6 . 
     
     
         9 . The isolated nucleic acid molecule of  claim 6 , further comprising a nucleic acid sequence encoding a heterologous polypeptide. 
     
     
         10 . The isolated nucleic acid molecule of  claim 9 , wherein the heterologous polypeptide is selected from the group consisting of a signal peptide, a peptide tag, a dimerization domain, an oligomerization domain, an antibody, or an antibody fragment. 
     
     
         11 . A pharmaceutical composition comprising a nucleic acid molecule of  claim 6 , and a pharmaceutically acceptable agent selected from the group consisting of excipients, diluents, and carriers. 
     
     
         12 . A vector comprising a nucleic acid molecule of  claim 6  or a nucleic acid that encodes the polypeptide of  claim 1 . 
     
     
         13 . The vector of  claim 12 , which is an expression vector. 
     
     
         14 . A host cell transfected with the expression vector of  claim 13 . 
     
     
         15 . A method of producing a polypeptide comprising culturing the host cell of  claim 14  in an appropriate culture medium to, thereby, produce the polypeptide. 
     
     
         16 . The method of  claim 15 , wherein the host cell is a bacterial cell or a eukaryotic cell. 
     
     
         17 . The method of  claim 15 , further comprising the step of isolating the polypeptide from the medium or host cell. 
     
     
         18 . An antibody which selectively binds to a polypeptide of  claim 1 . 
     
     
         19 . The antibody of  claim 18 , wherein the antibody is a monoclonal antibody or antigen binding portion thereof. 
     
     
         20 . A non-human animal model engineered to express a polypeptide of  claim 1 . 
     
     
         21 . A method of prognosing melanoma progression in a subject, the method comprising:
 a) determining the copy number, level of expression, or level of activity of one or more biomarkers listed in Table 2 or a fragment thereof in a subject sample;   b) determining the copy number, level of expression, or level of activity of the one or more biomarkers in a control sample or a predetermined reference; and   c) comparing the copy number, level of expression, or level of activity of said one or more biomarkers detected in steps a) and b);   wherein a significant modulation in the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample relative to the copy number, level of expression, or level of activity of the one or more biomarkers in the control sample or predetermined reference prognoses melanoma progression in the subject.   
     
     
         22 . The method of  claim 21 , wherein the subject sample and/or the control sample has not been contacted with any melanoma treatment or inhibitor of an immune checkpoint inhibitor. 
     
     
         23 . The method of  claim 21  or  22 , wherein the subject has not been administered any melanoma treatment or inhibitor of an immune checkpoint inhibitor. 
     
     
         24 . The method of any one of  claims 21 - 23 , wherein a significant increase in the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample relative to the copy number, level of expression, or level of activity of the one or more biomarkers in the control sample or predetermined reference indicates that subject is likely to have melanoma progression. 
     
     
         25 . The method of any one of  claims 21 - 23 , wherein a significant decrease in the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample relative to the copy number, level of expression, or level of activity of the one or more biomarkers in the control sample or predetermined reference indicates that the subject is unlikely to have melanoma progression. 
     
     
         26 . The method of any one of  claims 21 - 25 , wherein the melanoma progression is (a) shorter survival time, (b) increased metastasis, (c) increased cellular proliferation, (d) increased tumor burden, or (e) increased m-stage. 
     
     
         27 . The method of any one of  claims 21 - 26 , further comprising recommending, prescribing, or administering a therapeutic agent to the subject that specifically modulates the copy number, level of expression, or level of activity of the one or more biomarkers. 
     
     
         28 . The method of any one of  claims 21 - 27 , further comprising recommending, prescribing, or administering a therapeutic agent to the subject an inhibitor of one or more immune checkpoint inhibitors. 
     
     
         29 . A method of prognosing subjects afflicted with melanoma according to predicted clinical outcome of treatment with one or more inhibitors of an immune checkpoint inhibitor, the method comprising:
 a) determining the copy number, level of expression, or level of activity of one or more biomarkers listed in Table 2 or a fragment thereof in a first subject sample at a first point in time;   b) repeating step a) during at least one subsequent point in time and after administration to the subject of one or more inhibitors of an immune checkpoint inhibitor; and   c) comparing the copy number, level of expression, or level of activity of said one or more biomarkers detected in steps a) and b);   wherein a significant modulation in the copy number, level of expression, or level of activity of the one or more biomarkers in the first subject sample relative to at least one subsequent subject sample indicates the predicted clinical outcome of treatment with the one or more inhibitors of an immune checkpoint inhibitor.   
     
     
         30 . The method of  claim 29 , wherein the first subject sample is obtained from the subject prior to, concurrently with, or after administration of one or more inhibitors of an immune checkpoint inhibitor. 
     
     
         31 . The method of  claim 29  or  30 , wherein between the first point in time and the subsequent point in time, the subject has undergone treatment, completed treatment, and/or is in remission for the cancer. 
     
     
         32 . The method of any one of  claims 29 - 31 , wherein the first and/or at least one subsequent sample is selected from the group consisting of ex vivo and in vivo samples. 
     
     
         33 . The method of any one of  claims 29 - 32 , when the first and/or at least one subsequent sample is obtained from an animal model of the cancer. 
     
     
         34 . The method of any one of  claims 29 - 33 , wherein the first and/or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject. 
     
     
         35 . The method of any one of  claims 29 - 34 , wherein a significant increase in the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample relative to the copy number, level of expression, or level of activity of the one or more biomarkers in the control sample or predetermined reference indicates that subject is likely to have a beneficial outcome from treatment with the one or more inhibitors of an immune checkpoint inhibitor. 
     
     
         36 . The method of any one of  claims 29 - 34 , wherein a significant decrease in the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample relative to the copy number, level of expression, or level of activity of the one or more biomarkers in the control sample or predetermined reference indicates that the subject is unlikely to have a beneficial outcome from treatment with the one or more inhibitors of an immune checkpoint inhibitor. 
     
     
         37 . The method of any one of  claims 29 - 36 , wherein the beneficial outcome is (a) increased survival time, (b) decreased metastasis, (c) decreased cellular proliferation, (d) decreased tumor burden, or (e) increased m-stage. 
     
     
         38 . The method of any one of  claims 29 - 37 , further comprising recommending, prescribing, or administering a therapeutic agent to the subject that specifically modulates the copy number, level of expression, or level of activity of the one or more biomarkers. 
     
     
         39 . The method of any one of  claims 29 - 38 , further comprising recommending, prescribing, or administering a therapeutic agent to the subject an inhibitor of one or more immune checkpoint inhibitors if the subject is likely to have a beneficial outcome from treatment with the one or more inhibitors of an immune checkpoint inhibitor. 
     
     
         40 . A method of diagnosing a subject afflicted with melanoma, the method comprising:
 a) determining the copy number, level of expression, or level of activity of one or more biomarkers listed in Table 2 or a fragment thereof in a subject sample;   b) determining the copy number, level of expression, or level of activity of the one or more biomarkers in a control sample or a predetermined reference; and   c) comparing the copy number, level of expression, or level of activity of said one or more biomarkers detected in steps a) and b);   wherein a significant modulation in the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample relative to the copy number, level of expression, or level of activity of the one or more biomarkers in the control sample or predetermined reference indicates melanoma.   
     
     
         41 . The method of  claim 40 , wherein the subject sample and/or the control sample has not been contacted with any melanoma treatment or inhibitor of an immune checkpoint inhibitor. 
     
     
         42 . The method of  claim 40  or  41 , wherein the subject has not been administered any melanoma treatment or inhibitor of an immune checkpoint inhibitor. 
     
     
         43 . The method of any one of  claims 40 - 42 , wherein a significant increase in the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample relative to the copy number, level of expression, or level of activity of the one or more biomarkers in the control sample or predetermined reference indicates that the subject likely has melanoma. 
     
     
         44 . The method of any one of  claims 40 - 42 , wherein a significant decrease in the copy number, level of expression, or level of activity of the one or more biomarkers in the subject sample relative to the copy number, level of expression, or level of activity of the one or more biomarkers in the control sample or predetermined reference indicates that the subject is not likely to have melanoma. 
     
     
         45 . The method of any one of  claims 40 - 44 , wherein the melanoma progression is (a) shorter survival time, (b) increased metastasis, (c) increased cellular proliferation, (d) increased tumor burden, or (e) increased m-stage. 
     
     
         46 . The method of any one of  claims 40 - 45 , further comprising recommending, prescribing, or administering a therapeutic agent to the subject that specifically modulates the copy number, level of expression, or level of activity of the one or more biomarkers. 
     
     
         47 . The method of any one of  claims 40 - 46 , further comprising recommending, prescribing, or administering a therapeutic agent to the subject one or more Braf inhibitors, MEK inhibitors, and/or inhibitors of an immune checkpoint inhibitor. 
     
     
         48 . A method of assessing the efficacy of an agent for treating melanoma in a subject, comprising:
 a) determining in a first subject sample contacted with the agent or maintained in the presence of the agent the copy number, level of expression, or level of activity of one or more biomarkers listed in Table 2;   b) determining the copy number, level of expression, or level of activity of one or more biomarkers listed in Table 2 in at least one subsequent subject sample maintained in the absence of the test compound; and   c) comparing the copy number, level of expression, or level of activity of one or more biomarkers listed in Table 2 from steps a) and b), wherein a significantly increased copy number, level of expression, or level of activity of the one or more biomarkers listed in Table 1 in the first subject sample relative to at least one subsequent subject sample, indicates that the agent treats the melanoma in the subject.   
     
     
         49 . A method of assessing the efficacy of an agent for treating melanoma in a subject, comprising:
 a) determining in a first subject sample the agent the copy number, level of expression, or level of activity of one or more biomarkers listed in Table 2;   b) repeating step a) during at least one subsequent point in time after administration of the agent; and   c) comparing the copy number, level of expression, or level of activity of the one or more biomarkers listed in Table 2 determined in steps a) and b), wherein a significantly increased copy number, level of expression, or level of activity of the at least one biomarker listed in Table 1 in the first subject sample relative to the at least one subsequent subject sample, indicates that the agent treats the cancer in the subject.   
     
     
         50 . The method of  claim 49 , wherein between the first point in time and the subsequent point in time, the subject has undergone treatment, completed treatment, and/or is in remission for the cancer. 
     
     
         51 . The method of  claim 49  or  50 , wherein the first and/or at least one subsequent sample is selected from the group consisting of ex vivo and in vivo samples. 
     
     
         52 . The method of any one of  claims 49 - 51 , wherein the first and/or at least one subsequent sample is obtained from an animal model of the cancer. 
     
     
         53 . The method of any one of  claims 49 - 52 , wherein the first and/or at least one subsequent sample is a portion of a single sample or pooled samples obtained from the subject. 
     
     
         54 . A cell-based assay for screening for cytotoxic or cytostatic agents comprising contacting, a melanoma cell with a test agent, and determining the ability of the test agent to decrease the amount or activity of one or more biomarkers listed in Table 2. 
     
     
         55 . The cell-based assay of  claim 54 , wherein the step of contacting occurs in vivo, ex vivo, or in vitro. 
     
     
         56 . A cell-based assay for screening for agents that have a cytotoxic or cytostatic effect on a melanoma cell comprising, contacting the melanoma cell with a test agent, and determining the ability of the test agent to decrease the amount or activity of one or more biomarkers listed in Table 2. 
     
     
         57 . The cell-based assay of  claim 56 , wherein the step of contacting occurs in vivo, ex vivo, or in vitro. 
     
     
         58 . A method for preventing or treating melanoma, comprising contacting a melanoma cell with an agent that inhibits the expression and/or activity of one or more polypeptides of  claim 1  or one or more nucleic acids of  claim 6  to thereby modulate the metabolic response. 
     
     
         59 . The method of  claim 58 , wherein the agent is selected from the group consisting of an antisense nucleic acid molecule, an RNA interference molecule, a blocking antibody, and a non-activating form of the biomarker polypeptide or fragment thereof. 
     
     
         60 . The method of  claim 58  or  59 , further comprising contacting the cell with an additional agent that prevents or treats melanoma. 
     
     
         61 . The method of any one of  claims 58 - 60 , wherein the step of contacting occurs in vivo. 
     
     
         62 . The method of any one of  claims 58 - 60 , wherein the step of contacting occurs in vitro. 
     
     
         63 . A method for preventing or treating melanoma in a subject, comprising administering to the subject an agent that inhibits the expression and/or activity of one or more polypeptides of  claim 1  or one or more nucleic acids of  claim 6  in the subject, thereby preventing or treating the metabolic disorder in the subject. 
     
     
         64 . The method of  claim 63 , wherein the agent is selected from the group consisting of an antisense nucleic acid molecule, an RNA interference molecule, a blocking antibody, and a non-activating form of the biomarker polypeptide or fragment thereof. 
     
     
         65 . The method of  claim 63  or  64 , wherein the agent is administered by intravenous or subcutaneous injection. 
     
     
         66 . The method of any one of  claims 63 - 65 , the agent is administered in a pharmaceutically acceptable formulation. 
     
     
         67 . A method of identifying a binding partner to a polypeptide of  claim 1  or biologically active portion thereof comprising:
 a) contacting the polypeptide or biologically active portion thereof, or a cell expressing the polypeptide or biologically active portion thereof, with a test compound; and 
 b) determining whether the polypeptide or biologically active portion thereof binds to the test compound. 
 
     
     
         68 . A cell-based assay for screening for compounds which modulate the expression and/or activity of a polypeptide of  claim 1  or biologically active portion thereof comprising contacting a cell expressing the polypeptide or biologically active portion thereof with a test compound and determining the ability of the test compound to modulate the expression and/or activity of the polypeptide or biologically active portion thereof. 
     
     
         69 . A method for identifying a compound which modulates the expression and/or activity of a polypeptide of  claim 1  or biologically active portion thereof comprising:
 a) contacting the polypeptide or biologically active portion thereof with a test compound; and 
 b) determining the effect of the test compound on the expression and/or activity of the polypeptide or biologically active portion thereof to thereby identify a compound which modulates the activity of the polypeptide or biologically active portion thereof. 
 
     
     
         70 . The method of any one of  claims 21 - 47 , wherein the control sample is determined from a cancerous or non-cancerous sample from either the patient or a member of the same species to which the patient belongs. 
     
     
         71 . The method of any one of  claims 21 - 47  and  70 , wherein the control sample comprises cancer cells known to be responsive or non-responsive to the anti-immune checkpoint inhibitor therapy. 
     
     
         72 . The method of any one of  claims 21 - 53  and  70 - 71 , wherein the subject sample is selected from the group consisting of whole blood, serum, and plasma. 
     
     
         73 . The method of any one of  claims 21 - 53  and  70 - 72 , wherein the copy number is assessed by microarray, quantitative PCR (qPCR), high-throughput sequencing, comparative genomic hybridization (CGH), or fluorescent in situ hybridization (FISH). 
     
     
         74 . The method or assay of any one of  claims 21 - 72 , wherein the amount of the at least one biomarker listed in Table 2 is detected using a reagent which specifically binds with the protein. 
     
     
         75 . The method or assay of  claim 74 , wherein the reagent is selected from the group consisting of an antibody, an antibody derivative, and an antibody fragment. 
     
     
         76 . The method or assay of any one of  claims 21 - 72 , wherein the at least one biomarker listed in Table 2 is assessed by detecting the presence in the sample of a transcribed polynucleotide or portion thereof. 
     
     
         77 . The method or assay of  claim 76 , wherein the transcribed polynucleotide is an mRNA or a cDNA. 
     
     
         78 . The method or assay of  claim 76 , wherein the step of detecting further comprises amplifying the transcribed polynucleotide. 
     
     
         79 . The method or assay of  claim 76 , wherein the transcribed polynucleotide is detected by identifying a nucleic acid that anneals with the biomarker nucleic acid, or a portion thereof, under stringent hybridization conditions. 
     
     
         80 . The method or assay of any one of  claims 21 - 79 , wherein the anti-immune checkpoint inhibitor therapy is selected from the group consisting of inhibitors of PD-L1, PD-1, CTLA-4, and combinations thereof. 
     
     
         81 . The method or assay of  claim 80 , wherein the anti-immune checkpoint inhibitor therapy is selected from the group consisting of anti-PD-L1 antibodies, anti-PD-antibodies, anti-CTLA-4 antibodies, and combinations thereof. 
     
     
         82 . The method or assay of any one of  claims 21 - 81 , wherein responsiveness to anti-immune checkpoint inhibitor therapy is measured by at least one criteria selected from the group consisting of clinical benefit rate, survival until mortality, pathological complete response, semi-quantitative measures of pathologic response, clinical complete remission, clinical partial remission, clinical stable disease, recurrence-free survival, metastasis free survival, disease free survival, circulating tumor cell decrease, circulating marker response, and RECIST criteria. 
     
     
         83 . The method or assay of any one of  claims 21 - 82 , wherein the melanoma is a BRAF inhibitor-resistant melanoma or a MEK inhibitor-resistant melanoma. 
     
     
         84 . The method or assay of any one of  claims 1 - 83 , wherein the subject does not have renal cell carcinoma, head and neck cancer, and/or lung cancer. 
     
     
         85 . The method or assay of any one of  claims 1 - 84 , wherein the subject is a mammal. 
     
     
         86 . The method or assay of  claim 85 , wherein the mammal is an animal model of melanoma. 
     
     
         87 . The method or assay of  claim 85 , wherein the mammal is a human.