WO2012170640A1 - Procédés et compositions pour traitement par une combinaison trail-médicament - Google Patents

Procédés et compositions pour traitement par une combinaison trail-médicament Download PDF

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WO2012170640A1
WO2012170640A1 PCT/US2012/041267 US2012041267W WO2012170640A1 WO 2012170640 A1 WO2012170640 A1 WO 2012170640A1 US 2012041267 W US2012041267 W US 2012041267W WO 2012170640 A1 WO2012170640 A1 WO 2012170640A1
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trail
decoy receptor
deletion
methylation
cell
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Murty VUNDAVALLI
Dongxu XIE
Govind BHAGAT
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The Trustees Of Columbia University In The City Of New York
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • Sequence Listing which is a part of the present disclosure, includes a computer readable form comprising nucleotide and/or amino acid sequences of the present invention.
  • the subject matter of the Sequence Listing is incorporated herein by reference in its entirety.
  • Cervical Cancer affects over 0.5 million women worldwide. When invasive cancer is diagnosed the cure rate is low resulting in high mortality and the treatment response remains unpredictable.
  • HPV human papilloma virus
  • hrHPV high-risk HPV type E6 and E7 proteins interact with critical cell cycle checkpoint genes p53 and pRb, respectively, interfere with the DNA repair mechanisms, as a consequence accelerate the accumulation of genetic alterations and immortalization (1 ).
  • Conventional treatment of cervical cancer often employs chemotherapy using platinum based derivatives followed by radiation.
  • cervical cancer as a single diagnostic entity exhibits differences in clinical behavior and response to therapy, where advanced tumors remain unresponsive to chemo-radiotherapy.
  • Cisplatin is used as the most effective agent in advanced stages of cervical cancer where most patients exhibit acquired resistance or progressive disease after initial response (2).
  • B-cell Non-Hodgkin Lymphoma (B-NHL) subtypes exhibit complex genetic changes that include characteristic chromosome translocations.
  • DLBCL diffuse large-B-cell lymphoma
  • DLBCL diffuse large-B-cell lymphoma
  • the yield of potential therapeutic targets discovered to date has been limited.
  • overall survival has been significantly improved for B-cell lymphomas by the addition of rituximab, a large proportion of B-NHL patients still exhibit resistance to the existing treatments [34]. Therefore, there is a need for developing new therapies and biomarker of response to stratify patients that benefit specific treatments such as TRAIL combination drugs.
  • breast cancer A majority of breast cancer patients exhibit resistance to either recombinant TRAIL or antibodies targeting TRAIL-R1/TRAIL-R2 despite the expression of death receptors on the cell surface [35].
  • Breast cancer is one of the top 3 cancers that exhibit 8p21.3 region deletions (at a proportion of 0.59) and exhibit frequent promoter methylation of either DcR1 or DcR2 genes [16, 36].
  • breast cancer as a group exhibits resistance to commonly used chemotherapy drugs such as doxorubicin and tomoxifen, patients with ER+ phenotype are the most resistant [37].
  • the underlying genetic determinants of TRAIL resistance/sensitivity in breast cancer cells are not known. We hypothesize that 8p deletion and decoy receptor inactivation may play a role in sensitizing breast cancer cells to TRAIL combination drug therapy. Therefore, these suboptimal outcomes of
  • T-cell acute lymphoblastic leukemia/lymphoma T-ALL/LBL
  • PTCL Peripheral T-cell lymphoma
  • TCL T- cell leukemia/lymphoma
  • ALK+ ALCL ALK-positive anaplastic large cell lymphoma
  • studies using anthracycline-containing regimens PTCL show only 32% cases with 5-year overall survival [5, 8].
  • the suboptimal outcome using intensive therapies for newly diagnosed TCL patients suggest that the current standard therapies are not effective and, therefore there is an urgent need for the development of new therapies. It has been recited in the art that "[m]uch effort has been devoted but failed to identify the
  • biomarker(s) that can predict the sensitivity of human cancers to TRAIL-based therapies and “it may be difficult if not impossible to identify the biomarkers that could predict the drug responsiveness in such genetically diversified [cancer]” (Bellail et al. 2009 4, 34-41 , at 38).
  • TCL cells acquire mechanisms to escape the host immune surveillance [9].
  • CTC cytotoxic T cell
  • NK natural killer
  • Tumor necrosis factor related super family of death receptors DR4 and DR5 express on the cell surface and are critical regulators of the extrinsic apoptotic pathway in TRAIL/AP02L-mediated cell death. Binding of TRAIL to their cognate DRs results in death inducing signaling complex (DISC) formation and DISC initiates activation of proteases caspase 8 and 10, thereby driving downstream effector caspase activation and apoptosis.
  • TRAIL as a soluble zinc -coordinated homotrimeric protein triggers apoptosis only in cancer cells without affecting normal cells. Despite this specificity, many human cancers exhibit resistance to TRAIL [1 1 , 12]. Since decoy receptors DcR1 and
  • DcR2 compete with death receptors DR4 and DR5 in binding with TRAIL, over expression of DcR1 and DcR2 protects tumor cells from TRAIL-induced apoptosis.
  • Efforts to identify differences in TRAIL responsiveness in tumor cells has identified decreased stimulation of TRAILR1 or R2 by DcRs, suppression of the intracellular signaling cascade genes, and post-translational modifications as important predictors [12, 22]. But it has been reported that TRAIL -resistance could not be predicted based on over expression of decoy receptors alone and the molecular mechanisms of resistance remain elusive [1 1-14].
  • chemotherapeutic drugs or radiotherapy can exhibit synergistic tumor cell death when combined with recombinant TRAIL (rTRAIL) or agonistic anti-TRAILR mAbs [12].
  • rTRAIL recombinant TRAIL
  • agonistic anti-TRAILR mAbs [12].
  • TRAIL-drug combination therapy in patients carrying 8p deletion/decoy receptor inactivation in cervical cancer and lymphoid malignancies.
  • the method includes detecting an 8p chromosomal deletion in a sample. In some embodiments, the method includes detecting methylation of a promoter of at least one tumor necrosis factor related super family (TNFRSF) decoy receptor. In some embodiments, the method includes detecting expression level of at least one TNFRSF decoy receptor. In some embodiments, the method includes correlating increased TRAIL-induced apoptosis to presence of the 8p deletion. In some embodiments, the method includes correlating increased TRAIL-induced apoptosis to presence of methylation of the at least one decoy receptor. In some embodiments, the method includes correlating increased TRAIL-induced apoptosis to reduced expression levels of the at least one decoy receptor as compared to a control. In some
  • the method includes correlating increased TRAIL-induced apoptosis to two of (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, or (iii) reduced expression levels of the at least one decoy receptor as compared to a control. In some embodiments, the method includes correlating increased TRAIL- induced apoptosis to (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, and (iii) reduced expression levels of the at least one decoy receptor as compared to a control.
  • the method includes detecting an 8p chromosomal deletion in a sample; and either or both of detecting methylation of a promoter of at least one tumor necrosis factor related super family (TNFRSF) decoy receptor or detecting expression level (e.g., decreased expression level) of at least one TNFRSF decoy receptor.
  • TNFRSF tumor necrosis factor related super family
  • the at least one decoy receptor comprises
  • the at least one decoy receptor comprises DcR2/TNFRSF10D. In some embodiments, the at least one decoy receptor comprises DcR1/TNFRSF10C and DcR2/TNFRSF10D.
  • the method includes detecting methylation of a promoter of at least two decoy receptors. In some embodiments, the method includes detecting expression level of at least two decoy receptors. In some embodiments, the method includes detecting methylation of a promoter of at least two decoy receptors and detecting expression level of at least two decoy receptors. In some embodiments, the first decoy receptor comprises DcR1/TNFRSF10C and the second decoy receptor comprises DcR2/TNFRSF10D.
  • the 8p deletion comprises a deletion at 8p12-p21.3. In some embodiments, the 8p deletion comprises an 8.4 Mb minimal region of deletion (MRD) between 22,941-31 ,338 kb physical interval at 8p12-p21.3.
  • MCD 8.4 Mb minimal region of deletion
  • the sample comprises a tumor sample. In some embodiments, the sample comprises cervical cancer tumor sample, a T-cell
  • the sample comprises a TRAIL-resistant tumor sample.
  • the sample is of a subject.
  • the subject is a mammal.
  • the subject is a human.
  • the subject is diagnosed with, or suspected of having, cervical cancer, T-cell
  • leukemia/lymphoma B-cell NHL, or breast cancer.
  • the method includes selecting or modifying a treatment on the basis of detecting one, two, or three of: (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, or (iii) reduced expression levels of the at least one decoy receptor as compared to a control.
  • the method includes administering to the subject a therapeutically effective amount of a TRAIL agonist and an antineoplastic agent upon detecting one, two, or three of: (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, or (iii) reduced expression levels of the at least one decoy receptor as compared to a control.
  • the method includes administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a TRAIL agonist, an antineoplastic agent, and a pharmaceutically acceptable carrier or excipient upon detecting one, two, or three of (i) presence of the 8p deletion, (ii) presence of methylation of the at least one decoy receptor, or (iii) reduced expression levels of the at least one decoy receptor as compared to a control.
  • the TRAIL agonist is selected from the group consisting of Apo2L/TRAIL, HGS-ETR1 MAb, HGS-ETR2 MAb, HGS-TR2J MAb, CS-1008 (TRA-8) MAb, AMG 655 MAb, Apomab MAb, and LBY135 MAb.
  • the antineoplastic agent is selected from the group consisting of an alkylating agent, an antimetabolite, a plant alkaloid, a cytotoxic antibiotic, a platinum compound, a methylhydrazine, a monoclonal antibody, a
  • the antineoplastic agent is selected from the group consisting of Cyclophosphamide, Chlorambucil, Melphalan, Chlormethine, Ifosfamide, Trofosfamide, Prednimustine, Bendamustine, Busulfan, Treosulfan, Mannosulfan,
  • Pralatrexate Mercaptopurine, Tioguanine, Cladribine, Fludarabine, Clofarabine,
  • Gemtuzumab Cetuximab, Bevacizumab, Panitumumab, Catumaxomab, Ofatumumab, Porfimer sodium, Methyl aminolevulinate, Aminolevulinic acid, Temoporfin, Efaproxiral, Imatinib, Gefitinib, Erlotinib, Sunitinib, Sorafenib, Dasatinib, Lapatinib, Nilotinib,
  • Temsirolimus Everolimus, Pazopanib, Vandetanib, Afatinib, Masitinib, Toceranib, Amsacrine, Asparaginase, Altretamine, Hydroxycarbamide, Lonidamine, Pentostatin,
  • FIG. 1 is a pair of images depicting genomic analysis of 8p deletion in invasive cervical cancer.
  • FIG. 1A shows identification of 8p12-21.3 minimal deletion using 250K Nspl SNP array.
  • Each vertical column represents a sample with genomic regions representing from pter (top) to qter (bottom) on chromosome 8 (G-band ideogram).
  • Prefix "T” indicates primary tumor;
  • CL indicates cell line.
  • the blue-red scale bar (-1 to +1 ) at the bottom represents the copy number relative to mean across the samples. Blue indicates a loss and red indicates a gain. All tumors that exhibited chromosome 8 losses are shown from largest to smallest region of deletion.
  • FIG. 1 B shows gene expression profile using U133A array and supervised analysis of TNFRSF10 genes and NKX3-1 mapped to a minimal deleted region (MDR) in cervical cancer lines and primary tumors.
  • MDR minimal deleted region
  • each row represents the gene expression relative to group mean and each column represents a sample.
  • the scale bar (-2 to +2) on the bottom represents the level of expression with intensities of blue representing decrease and red representing increase in expression.
  • FIG. 2 is a series of box plots and gel images showing analysis of gene expression of decoy receptors in cervical cancer cell lines by RT-PCR.
  • FIG. 2A and FIG. 2C are box plots showing semi-quantitative analysis of transcript levels. Promoter methylation correlated with the down regulated expression of TNFRSF10C and TNFRSF10D.
  • FIG. 2B and FIG. 2D show effect of drug treatment using inhibitors of DNA methyltransferases and HDAC on gene reactivation. Bracketed M and UM indicates methylated and unmethylated tumors, respectively.
  • ACTB beta actin; U, untreated; A, azacytidine; T, TSA; A/T, azacytidine and TSA. Reactivation of expression of TNFRSF10D in ummethylated cell lines is indicative of involvement of other epigenetic/chromatin modifications.
  • FIG. 3 is a series of bar graphs showing apoptosis analysis by flow cytometry in cervical cancer cell lines after exposure to TRAIL-combination drugs to assess apoptosis in relation to decoy receptor status and 8p deletion.
  • FIG. 4 is a series of bar graphs showing promoter methylation and gene expression of decoy receptor genes in hematologic malignancies.
  • FIG. 4A shows frequency of promoter methylation in various lineages.
  • MDS myelodysplasia syndrome
  • AML acute myeloid leukemia
  • CML chronic myelogenous leukemia
  • B-ALL Bcell acute lymphoblastic leukemia
  • RL/FH reactive lymphoid/follicular hyperplasia
  • BL Burkitt lymphoma
  • FL follicular lymphoma
  • DLBCL diffuse large B-cell lymphoma
  • MCL mantle cell lymphoma
  • MZL marginal zone lymphoma
  • MM multiple myeloma
  • HD Hodgkin disease
  • T-ALL T-cell acute lymphoblastic leukemia
  • TCL T-cell lymphoma.
  • FIGS. 4B-C show analysis of gene expression of decoy receptors in promoter methylated T-ALL cell lines by RT-PCR and effect of treatment using inhibitors of DNA methyltransferases and HDAC on gene reactivation.
  • FIG. 4B shows TNFRSF10C.
  • FIG. 4C shows TNFRSF10D.
  • Promoter methylation correlated with the down regulated expression of TNFRSF10C in both cell lines ( FIG. 4B) and TNFRSF10D is down-regulated in Jurkat, but detectable levels of expression seen in Karpas-45 ( FIG. 4C).
  • ACTB beta actin
  • Control untreated
  • Aza azacytidine
  • TSA trichostatin A
  • Aza+TSA azacytidine and TSA.
  • TRAIL 0.5 pg/ml
  • Asparaginase 2 lU/ml
  • Dexamethasone 100 nM/ml
  • Methotrexate 50 nM/ml.
  • Panel A TNFRSF10C
  • Panel B TNFRSF10D
  • -ve no detectable expression
  • +ve Expressed.
  • TNFRSF10C show synergistic affect by >10-fold higher sensitivity to TRAIL -doxorubicin treatment compared to TRAIL alone.
  • Tarn Tamoxifen; Dox, Doxorubicin; TRAIL, 0.5 pg/ml; Doxorubicin, 250 ng/ml; Tamoxifen, 15 mM/ml.
  • M methylated; UM, unmethylated.
  • the present disclosure is based, at least in part, on the discovery that cervical cancer cell lines carrying an 8p deletion and decoy receptor methylation/down-regulated expression result in a high apoptotic response to TRAIL-induced combination therapy.
  • TNFRSF death receptors
  • DRs death receptors
  • DR2/TNFRSF10B two decoy receptors, DcR1/TNFRSF10C and DcR2/TNFRSF10D.
  • DR1 , DR2, DcR1 , and DcR2 receptors express on the cell surface and play a role in extrinsic apoptotic pathway.
  • the DR4 and DR5 genes induce apoptosis by binding to TRAIL/Apo2 ligand through their death domains and decoy receptors DcR1 and DcR2 lacking functional intracellular death domains inhibit TRAIL induced apoptosis.
  • a high frequency of promoter hypermethylation and down-regulated expression of DcR1 and DcR2 decoy receptor genes occurs in cervical cancer cell lines.
  • TNFRSF10C/DcR1 and TNFRSF10D/DcR2 can serve as a biomarker for TRAIL response in cervical cancer. Identification of an 8p deletion or decoy receptor promoter hypermethylation can facilitate the correlation of cervical cancer progression with therapeutic outcome, an improved evaluation of treatment, and approaches for early detection.
  • a method for determining susceptibility of a cancer of a subject to TRAIL-induced apoptosis through, for example, TRAIL-combination therapy is also provided herein. Also provided herein is a method for stratification of subjects for TRAIL-combination therapy.
  • An 8p chromosomal deletion can predict an adverse overall survival in cervical cancer.
  • An 8p chromosomal deletion together with promoter methylation-mediated inactivation of a decoy receptor gene e.g., TNFRSF10D and TNFRSF10C
  • a decoy receptor gene e.g., TNFRSF10D and TNFRSF10C
  • An 8p deletion can be a deletion at 8p12-p21 .3.
  • an 8p deletion can include an 8.4 Mb minimal region of deletion (MRD) between 22,941-31 ,338 kb physical interval at 8p12-p21.3.
  • Inactivation of a decoy receptor can independently predict response in the presence of an 8p deletion to TRAIL-combination treatment.
  • methylated- mediated inactivation of a decoy receptor and an 8p deletion can be a biomarker for increased TRAIL-induced apoptosis.
  • TRAIL decoy receptor genes epigenetically inactivated in T-cell neoplasms can be used as biomarkers of TRAIL-anti-leukemic drug combination therapy response to identify patients that benefit this treatment.
  • decoy receptor inactivation can be used as a biomarker for TRAIL-combination treatment in T-cell malignancies, such as neoplasms of immature T-cells (e.g., T-AA/LBL) and mature T-cells (e.g., PTCL) as occurring in, for example, cervical cancer.
  • T-AA/LBL immature T-cells
  • PTCL mature T-cells
  • Use of such biomarker can provide rationale for selective use of combination agents that activate the TRAIL pathway or provide prognostic value in prediction of TRAIL response in cervical cancer or T-cell malignancies.
  • TRAIL decoy receptor genes epigenetically inactivated in B-cell lymphomas can be used as a biomarkers of TRAIL-combination drug therapy response to identify patients that benefit this treatment.
  • TNFRSF10C inactivation can be used as a biomarker for TRAIL-combination drug treatment in B-cell lymphomas such as lymphomas of germinal centre origin (e.g., Burkitt's lymphoma, follicular lymphoma and diffuse large B-cell lymphoma) as occurring in, for example, cervical cancer and T-ALL.
  • lymphomas of germinal centre origin e.g., Burkitt's lymphoma, follicular lymphoma and diffuse large B-cell lymphoma
  • Use of such biomarker can provide rationale for selective use of combination agents that activated TRAIL pathway or provide prognostic value in prediction of TRAIL-combination drug response in mature B-cell lymphomas.
  • TRAIL decoy receptor genes epigenetically inactivated in breast cancer can be used as biomarkers of TRAIL-combination drug therapy response to identify patients that benefit this treatment.
  • TNFRSF10C inactivation can be used as a biomarker for TRAIL-combination drug treatment in breast cancer patients as occurring in, for example, cervical cancer, T-ALL, and B-cell Non-Hodgkin Lymphoma.
  • Use of such biomarker can provide rationale for selective use of combination agents that activated TRAIL pathway or provide prognostic value in prediction of TRAIL-combination drug response in patients with breast cancer.
  • Decreased expression of a decoy receptor in the presence of an 8p deletion can predict response to TRAIL-combination treatment.
  • methylated-mediated decreased expression of a decoy receptor and an 8p deletion can be a biomarker for increased TRAIL-induced apoptosis.
  • a decoy receptor that is methylated or has reduced expression can be, for example, TNFRSF10C or TNFRSF10D.
  • decoy receptor that is methylated or has reduced expression can be, for example, a methylated promoter of TNFRSF10C.
  • decoy receptor that is methylated or has reduced expression can be, for example, a methylated promoter of TNFRSF10D.
  • decoy receptor that is methylated or has reduced expression can be, for example, a methylated promoter of TNFRSF10C and TNFRSF10D.
  • One aspect provides a diagnostic assay to detect one or more of an 8p deletion, decoy receptor inactivation, or reduced decoy receptor expression in a subject.
  • Each of 8p deletion, decoy receptor inactivation, or reduced decoy receptor expression can be as described above.
  • the diagnostic assay can detect two or more of an 8p deletion, decoy receptor promoter hypermethylation, or lack of or reduced decoy receptor expression.
  • the diagnostic assay can detect all of an 8p deletion, decoy receptor methylation, or lack of or reduced decoy receptor expression.
  • Such a test can be used for clinical specimens of a subject to stratify or assign invasive cervical cancer cases for TRAIL-combination therapy.
  • Detection of an 8p deletion can be according to Fluorescence In Situ Hybridization (FISH). This and other means of detecting an 8p deletion is within the skill of the art.
  • FISH Fluorescence In Situ Hybridization
  • Detection of decoy receptor methylation can be according to methylation specific PCR (MSP).
  • MSP methylation specific PCR
  • TNFRSF10D can be according to MSP. This and other means of detecting decoy receptor methylation (e.g., TNFRSF10C or TNFRSFI OD methylation) is within the skill of the art.
  • decoy receptor methylation e.g., TNFRSF10C or TNFRSFI OD methylation
  • Detection of decoy receptor expression levels can be according to a semiquantitative method of estimation of protein.
  • detection of decoy receptor expression levels can be according an immunohistochemistry assay.
  • detection of decoy receptor expression levels can be according an immunohistochemistry assay.
  • decoy receptor protein e.g., TNFRSF10C or TNFRSFI OD protein
  • a diagnostic assay can also include identification of HPV status and type.
  • HPV typing can be according to conventional protocols in cancer (e.g., cervical cancer) management.
  • a diagnostic assay including one, two, three or more of the detection protocols described herein can provide a robust biomarker-directed test to detect 8p deletion, decoy receptor promoter hypermethylation, or decoy receptor reduced expression that can be applicable in, for example, clinical specimens.
  • TRAIL combinatorial therapy can include administration of one or more TRAIL agonists and one or more antineoplastic agents.
  • An antineoplastic agent can be administered at non-toxic dose in combination with TRAIL therapy so as to increase TRAIL-induced-apoptosis in cancer cells.
  • TRAIL therapy can be as described in Bellail et al. 2009 Reviews on Recent Clinical Trials 4, 34-41 , incorporated herein by reference.
  • TRAIL apoptotic pathway can be targeted by recombinant human TRAIL (rhTRAIL) ligands or its agonistic antibodies against DR4 and DR5.
  • rhTRAIL recombinant human TRAIL
  • TRAIL therapy can comprise administering a TRAIL agonist selected from Apo2L/TRAIL (Genentech, Amgen), HGS-ETR1 MAb (Human Genome Sciences), HGS-ETR2 MAb (Human Genome Sciences), HGS-TR2J MAb (Human Genome
  • CS-1008 MAb (Daiichi Sankyo), AMG 655 MAb (Amgen), Apomab MAb (Genentech), or LBY135 MAb (Novartis).
  • TRAIL therapy can be administered in combination with one or more antineoplastic agent.
  • TRAIL therapy can be administered in combination with one or more chemotherapeutic agents.
  • An antineoplastic agent can be a chemotherapeutic agent.
  • An antineoplastic agent can be an alkylating agent, an anti-metabolite agent, a plant alkoloid or terpenoid, a vinca alkaloid, a podophyllotoxin, a taxane, or a topoisomerase inhibitor.
  • antineoplastic agents include, but are not limited to, alkylating agents
  • cytotoxic antibiotics and related substances e.g., actinomycines, anthracyclines and related substances, or other cytotoxic agents
  • An antineoplastic agent can be a nitrogen mustard analogue selected from
  • An antineoplastic agent can be an alkyl sulfonate selected from Busulfan, Treosulfan, or Mannosulfan.
  • An antineoplastic agent can be an ethylene imine selected from Thiotepa, Triaziquone, or Carboquone.
  • An antineoplastic agent can be a nitrosourea selected from Carmustine, Lomustine, Semustine,
  • An antineoplastic agent can be a folic acid analogue selected from Methotrexate, Raltitrexed, Pemetrexed, or Pralatrexate.
  • An antineoplastic agent can be a purine analogue selected from Mercaptopurine,
  • An antineoplastic agent can be a pyrimidine analogue selected from Cytarabine, Fluorouracil, Tegafur, Carmofur, Gemcitabine, Capecitabine, Azacitidine, Decitabine, Fluorouracil combinations, or Tegafur combinations.
  • An antineoplastic agent can be a vinca alkoloid or analogue selected from Vinblastine, Vincristine, Vindesine, Vinorelbine, or Vinflunine.
  • An antineoplastic agent can be a podophyllotoxin derivative selected from Etoposide or Teniposide.
  • An antineoplastic agent can be a colchicine derivative selected from
  • An antineoplastic agent can be a taxane selected from Paclitaxel,
  • An antineoplastic agent can be a natural product selected from Trabectedin.
  • An antineoplastic agent can be an actinomycines selected from Dactinomycin.
  • An antineoplastic agent can be an anthracycline selected from
  • An antineoplastic agent can be a cytotoxic antibiotic selected from Bleomycin, Plicamycin, Mitomycin, or Ixabepilone.
  • An antineoplastic agent can be a platinum compound selected from Cisplatin, Carboplatin, Oxaliplatin, Satraplatin, or Polyplatillen.
  • An antineoplastic agent can be a
  • An antineoplastic agent can be a monoclonal antibody selected from Edrecolomab, Rituximab, Trastuzumab,
  • An antineoplastic agent can be a sensitizer selected from Porfimer sodium, Methyl aminolevulinate, Aminolevulinic acid, Temoporfin, or Efaproxiral.
  • An antineoplastic agent can be a protein kinase inhibitor selected from Imatinib, Gefitinib, Erlotinib, Sunitinib, Sorafenib, Dasatinib, Lapatinib, Nilotinib, Temsirolimus, Everolimus, Pazopanib, Vandetanib, Afatinib, Masitinib, or Toceranib.
  • An antineoplastic agent can be selected from Amsacrine, Asparaginase, Altretamine, Hydroxycarbamide, Lonidamine, Pentostatin, Miltefosine, Masoprocol, Estramustine, Tretinoin, Mitoguazone, Topotecan, Tiazofurine, Irinotecan, Alitretinoin, Mitotane, Pegaspargase, Bexarotene, Arsenic trioxide, Denileukin diftitox, Bortezomib, Celecoxib, Anagrelide, Oblimersen, Sitimagene ceradenovec, Vorinostat, Romidepsin, Omacetaxine mepesuccinate, Eribulin, or Camptothecin.
  • An antineoplastic agent can include cisplatin.
  • An antineoplastic agent can comprise a combination of hycamtin and cisplatin.
  • a combination of hycamtin and cisplatin is FDA approved for treatment of late-stage (IVB) cervical cancer.
  • antineoplastic agents are exemplary and any anticancer agent can be used in combination with a TRAIL agonist where such combination results in increased apoptosis compared to either agent alone.
  • cells having an 8p deletion, methylated promoter of decoy receptors, or reduced decoy receptor expression can exhibit increased apoptosis in response to combinatorial therapy comprising administration of a TRAIL agonist and an antineoplastic agent.
  • Administration of a TRAIL agonist and an antineoplastic agent can increase TRAIL-induced apoptosis as compared to administration of either alone.
  • TRAIL-induced apoptosis in excess of an additive effect of administration of either alone.
  • TRAIL agonist and an antineoplastic agent can synergistically increase TRAIL-induced apoptosis as compared to individual administration.
  • administration of a TRAIL agonist and an antineoplastic agent can increase TRAIL-induced apoptosis by at least about 10% as compared to administration of either alone.
  • administration of a TRAIL agonist and an antineoplastic agent can increase TRAIL-induced apoptosis by at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 350%, at least about 400%, at least about 450%, at least about 500%, or more, as compared to administration of either alone.
  • nucleotide and/or polypeptide variants having, for example, at least 95-99% identity to the reference sequence described herein and screen such for desired phenotypes according to methods routine in the art.
  • conservative substitutions can be made at any position so long as the required activity is retained.
  • Nucleotide and/or amino acid sequence identity percent is understood as the percentage of nucleotide or amino acid residues that are identical with nucleotide or amino acid residues in a candidate sequence in comparison to a reference sequence when the two sequences are aligned. To determine percent identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum percent sequence identity. Sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or Megalign (DNASTAR) software is used to align sequences. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full-length of the sequences being compared.
  • percent sequence identity X/Y100, where X is the number of residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and Y is the total number of residues in B. If the length of sequence A is not equal to the length of sequence B, the percent sequence identity of A to B will not equal the percent sequence identity of B to A.
  • Highly stringent hybridization conditions are defined as hybridization at 65 °C in a 6 X SSC buffer (i.e., 0.9 M sodium chloride and 0.09 M sodium citrate). Given these conditions, a determination can be made as to whether a given set of sequences will hybridize by calculating the melting temperature (T m ) of a DNA duplex between the two sequences. If a particular duplex has a melting temperature lower than 65°C in the salt conditions of a 6 X SSC, then the two sequences will not hybridize. On the other hand, if the melting temperature is above 65 °C in the same salt conditions, then the sequences will hybridize.
  • T m melting temperature
  • Host cells can be transformed using a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717;
  • transfected cells can be selected and propagated to provide recombinant host cells that comprise the expression vector stably integrated in the host cell genome.
  • Host strains developed according to the approaches described herein can be evaluated by a number of means known in the art (see e.g., Studier (2005) Protein Expr Purif. 41 (1 ), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363;
  • RNA interference e.g., small interfering RNAs (siRNA), short hairpin RNA (shRNA), and micro RNAs
  • RNAi RNAi
  • Fanning and Symonds (2006) Handb Exp Pharmacol. 173, 289-303G describing hammerhead ribozymes and small hairpin RNA
  • Helene, C, et al. (1992) Ann. N.Y. Acad. Sci. 660, 27-36; Maher (1992) Bioassays 14(12): 807-15 describing targeting deoxyribonucleotide sequences; Lee et al. (2006) Curr Opin Chem Biol. 10, 1-8, describing aptamers; Reynolds et al. (2004) Nature Biotechnology 22(3), 326 - 330, describing RNAi; Pushparaj and Melendez (2006) Clinical and Experimental
  • RNAi molecules are commercially available from a variety of sources ⁇ e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen).
  • sources e.g., Ambion, TX; Sigma Aldrich, MO; Invitrogen.
  • siRNA molecule design programs using a variety of algorithms are known to the art (see e.g., Cenix algorithm, Ambion; BLOCK-iTTM RNAi Designer, Invitrogen; siRNA Whitehead Institute Design Tools, Bioinofrmatics & Research
  • Traits influential in defining optimal siRNA sequences include G/C content at the termini of the siRNAs, Tm of specific internal domains of the siRNA, siRNA length, position of the target sequence within the CDS (coding region), and nucleotide content of the 3' overhangs.
  • compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety.
  • Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.
  • the formulation should suit the mode of administration.
  • the agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal.
  • the individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents.
  • Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.
  • Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
  • inducers e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.
  • Agents or compositions described herein can also be used in combination with other therapeutic modalities, as described further below.
  • therapies described herein one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition.
  • a subject in need of the therapeutic methods described herein can be a subject having, diagnosed with, suspected of having, or at risk for developing cervical cancer.
  • a subject in need of the therapeutic methods described herein can be a subject having or diagnosed as having one or more of an 8p chromosomal deletion, a methylation- associated inactivation of a decoy receptor, or reduced decoy receptor expression levels in a tumor of the subject.
  • a determination of the need for treatment can be according to diagnostic assays described herein.
  • a determination of the need for treatment will typically be assessed by a history and physical exam consistent with the disease or condition at issue. Diagnosis of the various conditions treatable by the methods described herein is within the skill of the art.
  • the subject can be an animal subject, preferably a mammal, more preferably horses, cows, dogs, cats, sheep, pigs, mice, rats, monkeys, guinea pigs, and chickens, and most preferably a human.
  • An effective amount of a TRAIL agonist and an antineoplastic agent described herein is generally that which can induce apoptosis in cancer cells or a tumor of the subject.
  • administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
  • a therapeutically effective amount of a TRAIL agonist and an antineoplastic agent can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient.
  • the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to induce apoptosis in cancer cells or a tumor of the subject.
  • a TRAIL agonist and an antineoplastic agent can be administered in the same composition.
  • a TRAIL agonist and an antineoplastic agent can be administered in different compositions.
  • compositions described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.
  • Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD 50 (the dose lethal to 50% of the population) and the ED 50 , (the dose therapeutically effective in 50% of the population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio
  • the specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott
  • the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.
  • a TRAIL agonist and an antineoplastic agent can occur as a single event or over a time course of treatment.
  • a TRAIL agonist and an antineoplastic agent can be administered daily, weekly, bi-weekly, or monthly.
  • the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.
  • Treatment in accord with the methods described herein can be performed prior to, concurrent with, or after conventional treatment modalities for cervical cancer, T-cell hematologic malignancies, B-cell NHL, or breast cancer.
  • a TRAIL agonist or an antineoplastic agent can be administered simultaneously or sequentially.
  • a TRAIL agonist or an antineoplastic agent can be administered simultaneously or sequentially with another agent, such as an antibiotic, an
  • a TRAIL agonist or an antineoplastic agent can be administered simultaneously with another agent, such as an antibiotic or an antiinflammatory. Simultaneous administration can occur through administration of separate compositions, each containing one or more of a TRAIL agonist or an
  • antineoplastic agent an antibiotic, an antiinflammatory, or another agent.
  • Simultaneous administration can occur through administration of one composition containing two or more of a TRAIL agonist, an antineoplastic agent, an antibiotic, an antiinflammatory, or another agent.
  • a TRAIL agonist or an antineoplastic agent can be administered sequentially with an antibiotic, an antiinflammatory, or another agent.
  • a TRAIL agonist or an antineoplastic agent can be administered before or after
  • Agents and compositions described herein can be administered according to methods described herein in a variety of means known to the art.
  • the agents and composition can be used therapeutically either as exogenous materials or as endogenous materials.
  • Exogenous agents are those produced or manufactured outside of the body and administered to the body.
  • Endogenous agents are those produced or manufactured inside the body by some type of device (biologic or other) for delivery within or to other organs in the body.
  • administration can be parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, or rectal administration.
  • Agents and compositions described herein can be administered in a variety of methods well known in the arts. Administration can include, for example, methods involving oral ingestion, direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings,
  • microparticles implantable matrix devices, mini-osmotic pumps, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 ⁇ ), nanospheres (e.g., less than 1 ⁇ ), microspheres (e.g., 1 -100 ⁇ ), reservoir devices, a combination of any of the above, or other suitable delivery vehicles to provide the desired release profile in varying proportions.
  • compositions will be known to the skilled artisan and are within the scope of the present disclosure.
  • Delivery systems may include, for example, an infusion pump which may be used to administer the agent or composition in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors.
  • an agent or composition is administered in combination with a biodegradable, biocompatible polymeric implant that releases the agent over a controlled period of time at a selected site.
  • polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof.
  • a controlled release system can be placed in proximity of a therapeutic target, thus requiring only a fraction of a systemic dosage.
  • Agents can be encapsulated and administered in a variety of carrier delivery systems.
  • carrier delivery systems include microspheres, hydrogels, polymeric implants, smart polymeric carriers, and liposomes (see generally, Uchegbu and Schatzlein, eds. (2006) Polymers in Drug Delivery, CRC, ISBN-10: 0849325331 ).
  • Carrier- based systems for molecular or biomolecular agent delivery can: provide for intracellular delivery; tailor biomolecule/agent release rates; increase the proportion of biomolecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the agent in vivo; prolong the residence time of the agent at its site of action by reducing clearance; decrease the nonspecific delivery of the agent to nontarget tissues; decrease irritation caused by the agent; decrease toxicity due to high initial doses of the agent; alter the immunogenicity of the agent; decrease dosage frequency, improve taste of the product; or improve shelf life of the product.
  • Also provided are methods for screening of agents that in combination with a TRAIL agonist provide for increased TRAIL-induced apoptosis as compared to the TRAIL agonist or antineoplastic agent alone. Also provided are methods for screening of TRAIL agonists that in combination with an antineoplastic agent provide for increased TRAIL- induced apoptosis as compared to the antineoplastic agent or TRAIL agonist alone.
  • Candidate substances for screening according to the methods described herein include, but are not limited to, fractions of tissues or cells, nucleic acids, polypeptides, siRNAs, antisense molecules, aptamers, ribozymes, triple helix compounds, antibodies, and small (e.g., less than about 2000 mw, or less than about 1000 mw, or less than about 800 mw) organic molecules or inorganic molecules including but not limited to salts or metals.
  • Candidate molecules encompass numerous chemical classes, for example, organic molecules, such as small organic compounds having a molecular weight of more than 50 and less than about 2,500 Daltons.
  • Candidate molecules can comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, and usually at least two of the functional chemical groups.
  • the candidate molecules can comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups.
  • a candidate molecule can be a compound in a library database of compounds.
  • Candidate molecules for screening according to the methods described herein include both lead-like compounds and drug-like compounds.
  • a lead-like compound is generally understood to have a relatively smaller scaffold-like structure (e.g., molecular weight of about 150 to about 350 kD) with relatively fewer features (e.g., less than about 3 hydrogen donors and/or less than about 6 hydrogen acceptors; hydrophobicity character xlogP of about -2 to about 4) (see e.g., Angewante (1999) Chemie Int. ed. Engl. 24, 3943- 3948).
  • a drug-like compound is generally understood to have a relatively larger scaffold (e.g., molecular weight of about 150 to about 500 kD) with relatively more numerous features (e.g., less than about 10 hydrogen acceptors and/or less than about 8 rotatable bonds; hydrophobicity character xlogP of less than about 5) (see e.g., Lipinski (2000) J. Pharm. Tox. Methods 44, 235-249).
  • initial screening is performed with lead-like compounds.
  • characterization can be based on a set of empirically recognized qualities derived by comparing similarities across the breadth of known drugs within the pharmacopoeia.
  • a candidate drug-like compound should have at least three of the following characteristics: (i) a weight less than 500 Daltons; (ii) a log of P less than 5; (iii) no more than 5 hydrogen bond donors (expressed as the sum of OH and NH groups); and (iv) no more than 10 hydrogen bond acceptors (the sum of N and O atoms).
  • drug-like molecules typically have a span (breadth) of between about 8A to about 15A.
  • kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein.
  • the different components of the composition can be packaged in separate containers and admixed immediately before use.
  • Components include, but are not limited to reagents for detection assays described herein, a TRAIL agonist, or an antineoplastic agent.
  • Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition.
  • the pack may, for example, comprise metal or plastic foil such as a blister pack.
  • Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.
  • Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately.
  • sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen.
  • Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents.
  • suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy.
  • Other containers include test tubes, vials, flasks, bottles, syringes, and the like.
  • Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
  • Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.
  • Removable membranes may be glass, plastic, rubber, and the like.
  • kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.
  • compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g.,
  • numbers expressing quantities of ingredients, properties such as molecular weight, reaction conditions, and so forth, used to describe and claim certain embodiments of the present disclosure are to be understood as being modified in some instances by the term "about.”
  • the term “about” is used to indicate that a value includes the standard deviation of the mean for the device or method being employed to determine the value.
  • the numerical parameters set forth in the written description and attached claims are approximations that can vary depending upon the desired properties sought to be obtained by a particular embodiment.
  • the numerical parameters should be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.
  • the terms “a” and “an” and “the” and similar references used in the context of describing a particular embodiment (especially in the context of certain of the following claims) can be construed to cover both the singular and the plural, unless specifically noted otherwise.
  • the term “or” as used herein, including the claims, is used to mean “and/or” unless explicitly indicated to refer to alternatives only or the alternatives are mutually exclusive.
  • Cells lines for Example 8 include nine CC cell lines (C-4I, C-33A, HT-3, Ca Ski, ME-180, MS751 , SW756, HeLa, and SiHa) from ATCC and 3 other CC cell lines (MARQ, Goerke F12, and MRI-H-186).
  • Cell lines for Example 6 include nine T-ALL (CUTTL1 , HPB-ALL1 , JURKAT, Karpas-45, MOLT-4, MOLT-15,
  • Cell lines for Example 1 1 include ten B-cell NHL cell lines (Daudi, Raji, SUDHL-4, SU-DHL-4, SU-DHL-5, SU-DHL-8, SU-DHL-10, LY- 1 , LY-3, WSU, and Farage) and three breast cancer cell lines (MCF7, Mx-1 , and MDA- MB-231 ). These cell lines have been characterized by molecular cytogenetic methods, and used for generating data on TRAIL-induced apoptosis presented here [31].
  • Example 6 cells grown in culture are treated with defined drug concentrations and periods (e.g. 5-Aza-2'deoxycytidine, 5 ⁇ ; trichostatin, 200 nM; rhTRAIL, 0.5-1 pg; dexamethasone, 50-150 nM; doxorubicin, 10-25 ng).
  • defined drug concentrations and periods e.g. 5-Aza-2'deoxycytidine, 5 ⁇ ; trichostatin, 200 nM; rhTRAIL, 0.5-1 pg; dexamethasone, 50-150 nM; doxorubicin, 10-25 ng).
  • Example 8 cells grown in culture are treated with defined drug concentrations and periods (5-Aza-2'deoxycytidine, 5 ⁇ ; trichostatin, 200 nM; rhTRAIL, 0.5-1 pg; actinomycin D, 0.5-1 pg; cisplatin, 1 -5 pg; doxorubicin, 100-200 nM; and tamoxifen, 100-200 nM).
  • the agonistic antibodies include for TRAILRI (mapatumumab) and TRAILR2 (lexatumumab) (Human Genome Sciences). Cells in culture are exposed to gamma radiation using Gammacell 40 Cesium Unit.
  • FISH Fluorescence in situ hybridization
  • MSP is performed using the standard methods by converting genomic DNA using EpiTect bisulphite kit (Qiagen, CA) with appropriate primer sets. Quantitative methylation analysis will be performed using Sequenom MALDI-TOI mass spectrometry platform as per manufacturer specifications (Sequenom, San Diego, CA). Briefly, 50 ng of bisulphite converted genomic DNA is PCR amplified using T7-promoter tagged primers spanning the CpG island. Using the manufacturer's protocols, the samples are analyzed with the Sequenom MALDI-TOF MS Compact Unit and the Sequenom kit.
  • the quantitative fragment mass data generated by the MALDI-FOF MS and EpiTYPER software is further subjected to quality control analysis on duplicate reactions for each primer set.
  • the final data output consists of average methylation measurements of each informative CpG of duplicate experiments.
  • the software estimates the relative methylation status by the sum of the intensities of the methylated and unmethylated components.
  • the data is further filtered to exclude the cases that yielded less than 80% for all informative CpG units within an amplicon/sample pair.
  • the methylation data generated after QC is subjected to one- dimensional hierarchical clustering analysis to identify relative methylation of the test sample compared to controls.
  • Bisulphite sequencing of MSP products after cloning into pCR2.1 TOPO vector is by standard PCR methods [32].
  • Semi-quantitative PCR or real-time PCR is performed on reversed transcribed RNA isolated from cell lines using Applied Biosystems 7500 PCR system and by standard methods (Foster City, CA).
  • siRNA, shRNA, lentiviral, and plasmids transfections are commercially obtained for siRNA experiments along with negative controls.
  • Transfections are carried out, serially or simultaneously if two genes targeted, using reagents such as Oligofectamine
  • RNA/protein is incomplete in the first transfection.
  • Full-length ORF is cloned in pcDNA and transfected along with vector controls by standard methods and stably selected clones are used in both in vitro and in vivo testing of TRAIL-combination treatments.
  • predesigned inducible HuSH 29mer shRNA constructs against the in pGFP-V-RS expression vectors and/or lentiviral vectors along with the controls are obtained from commercial sources. Alternatively, lentiviral constructs are generated using appropriate vectors.
  • TetR-expression cell lines are generated or selected for stable cell line. Lentiviral production and infections is performed using standard methods and titers are monitored using GFP visualization [33]. Stable cell lines generated by these transfections and transductions are used for TRAIL combination treatment experiments in vitro and in vivo.
  • Tissue microarrays of formalin-fixed, paraffin embedded tissue specimens were constructed on 75 invasive cervical cancers. Immunohistochemistry is performed on paraffin tissue section after antigen retrieval and antigen detection using appropriate antibodies by standard methods using Ventana staining platform. If appropriate, densitometric measurements of color intensity is performed on CAS2000 image analysis system. Immunofluoresence according to conventional protocols is an alternative approach to examine the specificity compared to IHC.
  • Cytotoxicity and flow cytometry Cell viability and cytotoxicity against TRAIL and combination of other drugs is assessed by standard colorimetric MTT (3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay (Invitrogen, Carlsbad, CA). The absorbance is measured at 490 nm with a microtiter plate reader. All treatments are performed in four replicate wells and in 3 repeats.
  • Apoptosis is measured using Pacific BlueTM Annexin V SYTOX® AADvancedTM Apoptosis Kit (Invitrogen, Carlsbad), which detects the externalization of phosphatidylserine in apoptotic cells using recombinant annexin V conjugated to violetfluorescent Pacific Blue dye and dead cell using SYTOX AADvanced stain.
  • LSR II flow cytometry instrument (BD Biosciences, San Jose) is used. All experiments are done in duplicate for each drug combination.
  • Western blot analysis Western blot analysis is performed by standard methods and detection by chemiluminescence reagent. Immunoblotting is performed using relevant antibodies including TRAIL R1 , TRAIL R2, TRAIL R3, TRAIL R4, c-FLIP, caspase 8, caspase 9, p53, HPV E6 and E7.
  • Tumor xenograft and evaluation of drug response Cell lines are genetically modified to express ffluc gene by transfecting the vector containing the luciferase
  • in vivo tumor growth is monitored once a week by the luciferase activity by using bioluminescent image on anesthetized animals.
  • the imaging is performed at Confocal and Specialized Microscopy.
  • Assessment of in vivo efficacy of TRAIL combination therapy of xenografts is performed by tumor-derived biolumenesence using CCD camera (I VIS Spectrum System, Xenogen, Caliper Lifesciences) after injecting D- luciferin. Photons from the bioluminescence signal are measured as total photon flux normalized to exposure time and surface areas and expressed in units of
  • This example shows a complex pattern of genomic alterations at 8p21.3 including large deletions, methylation of DcR genes and their associated down-regulated expression, and reactivation of DcR expression upon inhibiting DNA methyltransferases.
  • NKX3-1 gene expression analysis was performed on 92 probe sets comprising of 53 known genes mapped to MRD obtained from U133A arrays analyzed on 33 primary tumors, 9 cell lines, and 20 normal cervical epithelia. Utilizing the criteria of 1.75-fold change at 90% confidence interval between the group means of normal and tumor, loss of expression in NKX3-1 gene was found in over 90% tumors (see e.g., FIG. 1 B) (28). But no correlation was found between NKX3-1 expression levels and 8p genomic deletion and no mutations/promoter methylation modifications were found in the remaining allele, suggesting that NKX3-1 silenced state is not directly linked to 8p MRD. These data, therefore, suggested potential involvement of other critical tumor suppressor genes at 8p deletion in CC transformation or progression.
  • TNFRSF10B, TNFRSF10C and TNFRSF10D transcript levels of 3 (TNFRSF10B, TNFRSF10C and TNFRSF10D) of the 4 TNFRSF10 gene cluster mapped to 8p MRD that were present on U133A array.
  • TNFRSF10D down-regulation was found in 69% of invasive CC (see e.g., FIG. 1 B), while the pattern of expression of the other decoy receptor gene TNFRSF10C was not consistently down-regulated and the proapoptotic death receptor TNFRSF10B showed higher levels of expression in tumors compared to normal (see e.g., FIG 1 B).
  • TNFRSF family of decoy receptor genes is known to be epigenetically inactivated in multiple human tumors.
  • Cisplatin treatment results in DNA damage by intercalating, which activates apoptotic pathway when p53 mediated DNA damage repair fails and also acts as a radio-sensitizer. But the response rates of cisplatin as a single agent are low in recurrent and metastatic CC and 5-year survival rates remain low. Thus, it is important to develop effective therapeutic interventions and the host genomic alterations may play critical roles in treatment response.
  • TNFRSF genes involved in 8p deletion and inactivation of DcRs likely promote apoptosis, these alterations were thought to play a role in TRAIL response.
  • Various cervical cancer cell lines with 8p deletions and TNFRSF10C or TNFRSFIOD inactivation were exposed to TRAIL alone and in combination with cisplatin, actinomycin D, or radiation to assess cytotoxicity and apoptosis.
  • TNFRSF10D TNFRSF10D methylated with decreased expression in combination of TRAIL with cisplatin or actinomycin D or radiation.
  • a similar affect was observed in cell lines exhibiting TNFRSF10C down regulation associated with promoter hypermethylation (Fig. C). Therefore, in these in vitro experiments, we demonstrated that the tumor cells that carry a combination of methylation and down-regulated transcription of one or both decoy receptors in the presence of 8p deletion elicit efficient antitumor effects to TRAIL-combination treatment. Based on this data, we speculate that CC patients exhibiting 8p deletion and decoy receptor inactivation benefit from TRAIL combination therapy and may serve as biomarkers for treatment response. EXAMPLE 4
  • TNFRSF family of decoy receptors are known to be epigenetically inactivated by promoter hypermethylation in multiple human tumors [16] but their status in TCL is unknown, a wide-variety of hematologic malignancies were examined. Methods are according to Example 1 unless otherwise described.
  • DcR2 A complete lack of transcription of DcR2 was found in 5 of 8 methylated cell lines.
  • the remaining 3 cell lines (Karpas-45, MOLT-15 and T-ALL1 ) showed detectable levels of expression (see e.g., FIG. 4C). All the 8 methylated cell lines showed evidence of reactivation after inhibiting methyltransferases.
  • T-ALL/LBL and T-cell lymphomas are low using the existing treatments of TCL using standard anti-leukemic agents (such as dexamethasone, asparaginase, methotrexate, nelarabine, bortezomib) targeting different pathways [2, 10, 17-20].
  • standard anti-leukemic agents such as dexamethasone, asparaginase, methotrexate, nelarabine, bortezomib
  • Host genomic alterations and the associated pathways can provide targets to test treatment response [21].
  • TCL cases exhibiting decoy receptor inactivation may respond to TRAIL combination anti-leukemic drug therapies.
  • TRAIL combination anti-leukemic drug therapies.
  • nine T-ALL cell lines harboring TNFRSF10C and/or TNFRSF10D methylation and transcriptional inactivation were exposed to TRAIL alone and in combination with Asparaginase (2 lU/ml); Dexamethasone (100 nM/ml); or Methotrexate (50 nM/ml) to assess cytotoxicity and apoptosis.
  • Dexamethasone, or Methotrexatealone showed slightly increased apoptosis in T-ALL cell lines, synergistic affect was seen in combination of TRAIL with Asparaginase,
  • Dexamethasone, or Methotrexatealone see e.g., FIG. 5
  • cell lines carrying methylation with residual expression of TNFRSF10C or 10D were less sensitive to TRAIL-combination treatment (see e.g., FIG. 5).
  • the T-ALL cells that carry a combination of methylation and low levels of transcription of one or both decoy receptors can determine the anti-tumor effects to TRAIL combination treatment.
  • TCL exhibiting decoy receptor inactivation can benefit from TRAIL-combination therapy.
  • MSP bisulphite sequencing and expression by RT- PCR or real-time RT-PCR.
  • Established MTT and flow cytometry methods are used to identify the TRAIL and anti-leukemic drug response in relation to decoy receptor methylation/inactivation status.
  • a number of commonly used drugs namely
  • dexamethasone, doxorubicin, methotrexate, L-asparaginase, nelarabine, bortezomib) clinically relevant for TCL are tested.
  • Gene depletion and over-expression approaches are employed to determine the relative contribution of each of the genes and their synergistic affect in mediating apoptotic response using the following strategies.
  • siRNA and shRNA approaches are employed to generate transient and stable transfectants, respectively, of one or both genes.
  • Two unmethylated/normally expressed cell lines that exhibit resistance to TRAIL-drug induced apoptosis are chosen to transfect either TNFRSF10D or TNFRSFI OC alone or together.
  • siRNA approach recombinant human TRAIL (rhTRAIL) in combination with one or two of the most effectively responded drugs are tested for assessing cytotoxicity and apoptosis.
  • rhTRAIL recombinant human TRAIL
  • TNFRSF10C alone is sufficient to sensitize the cells to TRAIL therapy, or inactivation of both decoy receptors have synergistic affect.
  • This example shows gene depletion and over-expression approaches to demonstrate contribution of DcR genes and synergistic effect in mediating apoptotic response. Function of DcR genes are restored to confirm that over expression results in resistance to TRAIL treatment.
  • TNFRSF10D and TNFRSF10C are targeted by transfecting full length ORF clones either or both using pcDNA vectors and lentiviral ORF vectors to obtain high expression.
  • Two cell lines that exhibit promoter methylation of TNFRSF10D and/or TNFRSF10C and down-regulated expression of respective transcripts are chosen (e.g., MS751 and SW756, both having 8p deletion and resistance to TRAIL-cisplatin induced apoptosis).
  • MS751 showed no evidence of methylation or down-regulated expression in the DcRs.
  • the SW756 cell line exhibited TNFRSF10C methylation and down-regulated expression, but TNFRSF10D is unmethylated and transcription is at the normal level.
  • TNFRSF10D or TNFRSF10C are transfected alone or together.
  • siRNA approach recombinant human TRAIL (rhTRAIL) is tested in combination with cisplatin, actinomycin D or radiation for assessing cytotoxicity and apoptosis.
  • TNFRSF10D and TNFRSF10C alone is sufficient to sensitize the cells to TRAIL therapy, (ii) inactivation of both DcRs will have synergistic affect, and (iii) 8p deletion is essential for the TRAIL response.
  • TNFRSF10D and TNFRSF10C are targeted by transfecting full length ORF clone either or both using pcDNA vectors and/or lentiviral ORF vectors with the aim to obtain high expression of these genes.
  • Three cell lines ME-180, C-4I, and C-33A are used. Both ME-180 and C-4I cell lines carry 8p deletion, while C-33A is disomy for 8p. All three cell lines exhibited promoter methylation and down regulated expression of TNFRSF10C.
  • the cell lines ME-180 and C-33A also showed methylation and down regulated expression of TNFRSF10D, while C- 4I is unmethylated and expressed at normal levels.
  • ME180 showed highest apoptotic response to TRAIL-combination therapy and the C-33A cell line was resistant (see e.g., TABLE 2).
  • the C-4I cell line showed moderate apoptotic response (see e.g., TABLE 2).
  • TCL cell lines e.g. Jurkat and MOLT-16
  • TRAIL- drug resistance two additional cell lines that exhibit highest resistance in vitro are selected.
  • Tumor cells are subcutaneously seeded in immunocompromised nu/nu mice for their potential to develop tumors after implantation and response to TRAIL combination chemotherapy to suppress tumor growth.
  • TRAIL alone, selected drug alone, or combination of both, along with controls are administered. Tumor size is monitored by measuring tumor volume and imaging methods.
  • TNFRSF10D potentiates the tumoricidal affects with TRAIL-combination therapy in vivo as it does in vitro.
  • FISH Fluorescence In Situ Hybridization
  • Multiplex methylation test for decoy receptors Commercially available platforms to assess the promoter hypermethylation qualitatively and quantitatively are used. CpG methylation is assayed as a characteristic tumor-related genomic change and the test includes detection of any level of methylation as a correlative marker. A highly specific and robust qualitative assay (e.g., MSP, bisulphite sequencing, and Sequenom quantitative methylation assays) is used different types of tissues with tumor
  • Multiplex MSP is designed with products under 150 bp in the dense methylated regions, which is determined by bisulphite cloning and sequencing of the cell lines and primary tumors, to amplify both TNFRSF10C and TNFRSF10D promoters.
  • multiple MSP is applied on 50 tumors each with 8p deletion positive and negative by FISH.
  • Comparison of the single and multiplex MSP establishes the sensitivity and specificity of the multiplex test. This establishes "yes or no" for scoring methylation and its relationship with 8p deletion.
  • Sequenom DNA methylation assay based on matrix- assisted laser desorption/ionization of flight mass spectrometry is also used. This approach provides an accurate test for methylation of DcR genes.
  • Identification of expression of DcR genes in CC tissues Because promoter hypermethylation is associated with loss of expression in cancer, the identification of down-regulated transcription or protein provides a means to identify inactivation. This is examined in the entire specimen by RT-PCR/western blot analysis or at individual cell level by in situ methods of detection of mRNA or protein.
  • the IHC is one available method. Several commercially available antibodies are tested against TNFRSF10C and TNFRSF10D on cell lines with known status of methylation and expression on paraformaldehyde fixed cytospin or paraffin-embedded tissue sections. Once the specificity of antibodies is optimized, antibodies are examined on tissues that used in FISH and MSP. Use of DcR1 and DcR2 antibodies in IHC can be according to conventional methods. A standard scoring system of 0-3+ is used. Sensitivity and specificity are established to quantify the levels of expression and correlation with MSP status.
  • Cytotoxicity analysis by MTT assay in B-cell lymphoma cell lines [N 10] was measured after exposure to TRAIL-doxorubicin combination treatment (TRAIL, 0.5 pg/ml; Doxorubicin, 15 ng/ml) in relation to TNFRSF10C gene expression.
  • B-cell lymphoma cell lines with decreased expression of TNFRSF10C showed synergistic affect by >10-fold higher sensitivity to TRAIL -doxorubicin treatment compared to TRAIL alone.
  • TRAIL TRAIL-combination drugs
  • Cell lines included three breast cancer cell lines (MCF7, Mx-1 , and MDA-MB-231 ).
  • FIG. 7 Breast cancer cell lines harboring TNFRSF10C methylation (Mx-1 and MDA-MB-231 ) were highly sensitive to TRAIL combined with either Tamoxifen or Doxorubicin compared to unmethylated breast cancer cell line (MCF7).
  • lymphoblastic leukemia from thymocyte to lymphoblast. Leukemia, 2006. 20(9): p. 1496- 510.
  • KILLER/DR5 is a DNA damage-inducible p53-regulated death receptor gene. Nat Genet, 1997. 17(2): p. 141-3.
  • TRAIL Tumor necrosis factor-related apoptosis-inducing ligand

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Abstract

L'invention concerne des procédés de diagnostic de pathologies, comme le cancer du col de l'utérus, des malignités hématologiques à cellules T, un LNH à cellules B ou un cancer du sein, sensibles à une apoptose médiée par TRAIL accrue. Le diagnostic peut comprendre la présence d'une ou plusieurs parmi une délétion chromosomique 8p, l'inactivation par un récepteur leurre associé à la méthylation, ou une expression de récepteur leurre réduite. Elle concerne également des procédés de traitement et de criblage de composés associés à l'apoptose médiée par TRAIL. Elle concerne également des procédés de traitement et de criblage de composés associés au cancer du col de l'utérus, à des malignités hématologiques à cellules T, au LNH à cellules B ou au cancer du sein.
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WO2014064526A2 (fr) * 2012-10-25 2014-05-01 Mdxhealth Sa Marqueurs de méthylation pouvant prédire la réponse à un médicament
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WO2015107105A1 (fr) * 2014-01-15 2015-07-23 Apogenix Gmbh Procédé de prévision de la sensibilité d'une maladie cancéreuse à un traitement basé sur la méthylation de l'adn
RU2620165C2 (ru) * 2015-08-31 2017-05-23 Федеральное государственное бюджетное образовательное учреждение высшего образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) Способ индукции гибели опухолевых клеток
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2014053650A1 (fr) * 2012-10-04 2014-04-10 Ab Science Utilisation de masitinib pour le traitement du cancer chez des sous-populations de patients identifiées à l'aide de facteurs de prédiction
EA037368B1 (ru) * 2012-10-04 2021-03-19 Аб Сьянс Способ лечения рака поджелудочной железы у пациента, страдающего от связанной с болезнью интенсивной боли, и применение набора, содержащего маситиниб или его фармацевтически приемлемую соль и гемцитаниб, для лечения рака поджелудочной железы
US10238649B2 (en) 2012-10-04 2019-03-26 Ab Science Use of masitinib for treatment of cancer in patient subpopulations identified using predictor factors
WO2014064526A2 (fr) * 2012-10-25 2014-05-01 Mdxhealth Sa Marqueurs de méthylation pouvant prédire la réponse à un médicament
WO2014064526A3 (fr) * 2012-10-25 2014-06-26 Mdxhealth Sa Marqueurs de méthylation pouvant prédire la réponse à un médicament
US20140377274A1 (en) * 2013-06-25 2014-12-25 The Board Of Trustees Of The University Of Illinois Use of 2-deoxy-d-glucose to sensitize cancer cells to an agent that activates the extrinsic apoptotic pathway
US9757457B2 (en) * 2013-06-25 2017-09-12 The Board Of Trustees Of The University Of Illinois Use of 2-deoxy-D-glucose to sensitize cancer cells to an agent that activates the extrinsic apoptotic pathway
WO2015107105A1 (fr) * 2014-01-15 2015-07-23 Apogenix Gmbh Procédé de prévision de la sensibilité d'une maladie cancéreuse à un traitement basé sur la méthylation de l'adn
RU2620165C2 (ru) * 2015-08-31 2017-05-23 Федеральное государственное бюджетное образовательное учреждение высшего образования "Московский государственный университет имени М.В. Ломоносова" (МГУ) Способ индукции гибели опухолевых клеток
WO2017151707A1 (fr) 2016-03-01 2017-09-08 The Board Of Trustees Of The University Of Illinois Variants et protéines de fusion de l-asparaginase ayant une activité l-glutaminase réduite et une stabilité améliorée
US10821160B2 (en) 2016-03-01 2020-11-03 The Board Of Trustees Of The University Of Illinois L-asparaginase variants and fusion proteins with reduced L-glutaminase activity and enhanced stability
US10322192B2 (en) 2016-03-02 2019-06-18 Eisai R&D Management Co., Ltd. Eribulin-based antibody-drug conjugates and methods of use
US10548986B2 (en) 2016-03-02 2020-02-04 Eisai R&D Management Co., Ltd. Eribulin-based antibody-drug conjugates and methods of use
WO2019032952A1 (fr) 2017-08-11 2019-02-14 The Board Of Trustees Of The University Of Illinois Variantes tronquées de la l-asparaginase de cochon d'inde et procédés d'utilisation
CN111050784A (zh) * 2017-08-11 2020-04-21 伊利诺伊大学理事会 截短的豚鼠l-天冬酰胺酶变体及其使用方法
US11578315B2 (en) 2017-08-11 2023-02-14 The Board Of Trustees Of The University Of Illinois Truncated guinea pig L-asparaginase variants and methods of use
CN111050784B (zh) * 2017-08-11 2024-03-19 伊利诺伊大学理事会 截短的豚鼠l-天冬酰胺酶变体及其使用方法

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