Method for detecting replication or colonization of a biological therapeutic

Abstract

Methods for detecting replication in or colonization of a host by a biological therapeutic, such as an oncolytic virus, cells administered for cell therapy and gene therapy vectors, are provided. In the methods, a product produced by the biological therapeutic is detected in a sample of tissue or body fluid distinct from the administered therapy or locus thereof, thereby permitting assessment of the therapy and/or monitoring its progress.

1 . A method of detecting replication in or colonization of a target locus in a subject by a therapeutic bacterium or detecting replication of the therapeutic bacterium in a non-target cell in the subject, comprising:
 obtaining a sample from a subject to whom the therapeutic has been administered;   testing the sample to detect a shed protein encoded by the bacterium, wherein:
 the therapeutic bacterium accumulates in tumors, tumor tissues, metastases, areas of inflammation, immunoprivileged sites or tissues, wounds or infections; 
 a target locus is tumor, metastasis, inflamed tissue or wounded tissue; and 
 detection of a shed protein encoded by the bacterium indicates that the bacterium is replicating in and/or colonizing a target locus. 
   
     
     
         2 . The method of  claim 1 , wherein the bacterium is a non-pathogenic mutual, commensual or probiotic strain of bacteria. 
     
     
         3 . The method of  claim 1 , wherein the bacterium is selected from among  Escherichia coli, Bacteroides, Eubacterium, Streptococcus, Actinomyces, Veillonella, Nesseria, Prevotella, Campylobacter, Fusobacterium, Eikenella, Porphyromonas, Priopionibacteria, Clostridia, Salmonella, Shigella, Bifidobacteria  and  Staphylococcus  species. 
     
     
         4 . The method of  claim 1 , wherein the bacterium is an  Escherichia coli  Nissle strain bacterium. 
     
     
         5 . A method for monitoring therapeutic progress in a subject having tumors, comprising:
 (a) administering to the subject a bacterium encoding a β-glucuronidase;   (b) obtaining a sample from the subject, wherein the sample is a body fluid or tissue sample that is not a tumor sample;   (c) detecting β-glucuronidase activity in the sample, wherein the detection is by the addition of a substrate for β-glucuronidase; and   (d) determining the presence of a product catalyzed by the reaction of the β-glucuronidase with the substrate,   wherein the detection of the product indicates that the bacterium has colonized or is replicating in a tumor tissue or cell in the subject and is treating the tumor such that therapy should be continued.   
     
     
         6 . The method of  claim 5 , wherein the bacterium is a non-pathogenic mutual, commensual or probiotic strain of bacteria. 
     
     
         7 . The method of  claim 5 , wherein the bacterium is selected from among  Escherichia coli, Bacteroides, Eubacterium, Streptococcus, Actinomyces, Veillonella, Nesseria, Prevotella, Campylobacter, Fusobacterium, Eikenella, Porphyromonas, Priopionibacteria, Clostridia, Salmonella, Shigella, Bifidobacteria  and  Staphylococcus  species. 
     
     
         8 . The method of  claim 5 , wherein the bacterium is an  Escherichia coli  Nissle strain bacterium. 
     
     
         9 . The method of  claim 5 , wherein the β-glucuronidase is a human or bacterial β-glucuronidase. 
     
     
         10 . The method of  claim 9 , wherein the β-glucuronidase comprises the sequence of amino acids set forth in SEQ ID NO:121, or a catalytically active portion thereof, or a sequence of amino acids having at least 85% sequence identity with the sequence set forth in SEQ ID NO:121. 
     
     
         11 . The method of  claim 9 , wherein the β-glucuronidase comprises the sequence of amino acids set forth in SEQ ID NO:4, or a catalytically active portion thereof, or a sequence of amino acids having at least 85% sequence identity with the sequence set forth in SEQ ID NO:4. 
     
     
         12 . The method of  claim 9 , wherein the β-glucuronidase comprises the sequence of amino acids set forth in SEQ ID NO:4, or a catalytically active portion thereof, or a sequence of amino acids having at least 90% sequence identity with the sequence set forth in SEQ ID NO:4. 
     
     
         13 . The method of  claim 9 , wherein the β-glucuronidase comprises the sequence of amino acids set forth in any of SEQ ID NOS:4, 114-121, 128, 130, 132, 134, 136, 138, 140, 142, 144 and 146, or a catalytically active portion thereof, or a sequence of amino acids having at least 85% sequence identity to the sequence set forth in any of SEQ ID NOS:4, 114-121, 128, 130, 132, 134, 136, 138, 140, 142, 144 and 146. 
     
     
         14 . The method of  claim 5 , wherein the substrate is selected from the group consisting of fluorescein di-β-D-glucuronide (FDGlcU), 4-methylumbelliferyl-β-D-glucuronide (4-MUG), carboxyumbelliferyl β-D-glucuronide (CUGlcU), 5-(pentafluorobenzoylamino)-fluorescein di-β-D-glucuronide (PFB-FDGlcU), C 12 -fluorescein β-D-glucuronidase, 5-bromo-4-chloro-3-indolyl β-D-glucuronide (X-GlcU or BCIG), p-nitrophenyl-β-D-glucuronide, red-β-D-GlcU,CHA (magenta-β-D-GlcA; 5-bromo-6-chloro-3-indolyl-β-D-glucuronide, cyclohexylammonium salt), rose-β-D-GlcU,CHA (salmon-β-D-GlcUA; 5-bromo-6-chloro-3-indolyl-β-D-glucuronide, cyclohexylammonium salt), phenyl-β-D-glucuronide, and pharmaceutically acceptable salts thereof. 
     
     
         15 . The method of  claim 14 , wherein the substrate is selected from among fluorescein di-β-D-glucuronide (FDGlcU) and 4-methylumbelliferyl-β-D-glucuronide (4-MUG). 
     
     
         16 . The method of  claim 5 , wherein the sample is a body fluid that is selected from among blood, plasma, serum, lymph, ascetic fluid, cystic fluid, urine, nipple exudates, sweat, tears, saliva, mouth gargle, peritoneal fluid, cerebrospinal fluid (CSF), synovial fluid, aqueous humour, vitreous humour, amniotic fluid, bile, cerumen (earwax), Cowper's fluid (pre-ejaculatory fluid), Chyle, Chyme, female ejaculate, interstitial fluid, lymph fluid, menses, breast milk, mucus, snot, phlegm, pleural fluid, pus, sebum, semen, vaginal lubrication, and feces. 
     
     
         17 . The method of  claim 16 , wherein the sample is collected between or between about 12 hours and 1 month after treatment with the bacterium. 
     
     
         18 . The method of  claim 16 , wherein the sample is obtained within 1 week of treatment with the virus, and detection of the β-glucuronidase activity in the sample indicates that the virus is replicating in tumor cells and is effective for treatment. 
     
     
         19 . The method of  claim 16 , wherein the sample is obtained periodically following administration of the virus to monitor the progress of treatment by detecting an increase in the amount of β-glucuronidase in the sample, indicating replication of the virus in tumors, followed by a decrease indicating that tumors are shrinking. 
     
     
         20 . The method of  claim 5 , wherein the cancer comprises a bladder tumor, breast tumor, prostate tumor, glioma tumor, adenocarcinoma, ovarian carcinoma, pancreatic carcinoma, liver tumor, skin tumor, pancreatic cancer, non-small cell lung cancer, multiple myeloma, leukemia, lung and bronchus tumor, breast tumor, colon and rectum tumor, kidney tumor, stomach tumor, esophagus tumor, liver and intrahepatic bile duct tumor, urinary bladder tumor, brain tumor and other nervous system tumor, head and neck tumor, oral cavity tumor, pharynx tumor, cervix tumor, uterine corpus tumor, thyroid tumor, ovary tumor, testes tumor, prostate tumor, malignant melanoma, cholangiocarcinoma, thymoma, non-melanoma skin cancer, hematologic tumor, malignancy, childhood leukemia and lymphoma, multiple myeloma, Hodgkin's disease, lymphomas of lymphocytic and cutaneous origin, acute and chronic leukemia, acute lymphoblastic leukemia, acute myelocytic leukemia, chronic myelocytic leukemia, plasma cell neoplasm, lymphoid neoplasm or is a cancer associated with HIV infection. 
     
     
         21 . The method of  claim 5 , wherein the cancer is a solid tumor. 
     
     
         22 . The method of  claim 5 , wherein the β-glucuronidase is a secreted protein. 
     
     
         23 . The method of  claim 5 , wherein the β-glucuronidase is shed from infected cells. 
     
     
         24 . The method of  claim 5 , wherein the β-glucuronidase is heterologous to the bacterium. 
     
     
         25 . The method of  claim 1 , wherein the shed protein is an endogenous protein. 
     
     
         26 . The method of  claim 1 , wherein the shed protein is β-glucuronidase. 
     
     
         27 . The method of  claim 1 , wherein the shed protein is heterologous to the bacterium.