Patent Grant
10087435
The invention relates, in part, to methods and compounds for reducing threonyl-tRNA synthetase (TARS) activity in cells and tissues.
Angiogenesis plays a role in diseases such as cancer and other proliferative disorders. For example, a small solid tumor may be able to survive in the absence of vascularization, but to provide sufficient nutrients and oxygen and to remove waste products from cells that make up larger tumors, vascularization of the tissue is necessary. Triggers and regulators of angiogenesis in cells and tissues are not fully understood, but it is thought that hypoxia and lack of adequate nutritional access in cells in tumors greater than approximately 2 cm3 in size may result in angiogenesis, which supports further tumor growth with increased delivery of oxygen and nutrients. Angiogenesis may be a factor in the progression of a tumor or cancer, not only by providing nutrient support for a tumor to continue to grow in size, but angiogenesis may also play a role in metastatic activity in some cancers.
Angiogenesis has emerged as a target for cancer therapy due to the reliance of many cancers on new vessels and the poor prognosis associated with cancers that have advanced angiogenesis (Folkman, J. (2001) Semin Oncol 28 (6), 536-542). Angiogenesis is normally suppressed by angiopoietin-1 which is secreted by vascular pericytes and inhibits endothelial cell proliferation. There are many factors involved in the tumor angiogenic switch, but initiation of angiogenesis by hypoxic tumor cells is primarily through induction of hypoxia inducible factor-1α (HIF-1α) which stimulates expression of vascular endothelial growth factor (VEGF). VEGF acts in combination with other growth factors and receptors to increase activation of the Ras/MAP kinase and phosphoinositide 3 kinase (PI3 kinase) pathways in endothelial cells. These pathways are involved in induction of genes involved in endothelial cell proliferation and migration. (Bikfalvi, A. and Bicknell, R. (2002) Trends in Pharmacological Sciences 23 (12), 576-582; Liao, D. and Johnson, R. (2007) Cancer and Metastasis Reviews 26 (2), 281-290; and Olsson, A. K. et al. (2006) Nat Rev Mol Cell Biol 7 (5), 359-371).
Cell and tissue growth, for example vascular growth in angiogenesis, are known to involve protein synthesis but processes involved in the initiation, regulation, and modulation of protein synthesis in angiogenesis appear to be quite complex and are not well understood. The lack of understanding of the complex pathways and interactive regulatory events necessary to trigger and support angiogenesis in cells limits approaches to treat disorders that are characterized, in part, by angiogenesis.
The invention includes, in part, methods and compounds for treating diseases and conditions characterized by and/or associated with altered threonyl-tRNA synthetase (TARS) activity, which include, but are not limited to diseases and conditions in which angiogenesis is altered. It has now been shown for the first time that TARS is a potent angiogenic inducer in vitro and in vivo affecting endothelial cell migration and tube formation. TARS is also shown to be secreted by endothelial cells in response to angiogenic or inflammatory signaling, indicating its novel role as a pro-angiogenic chemokine. Furthermore, an association is revealed between TARS levels and both ovarian and prostate cancers in human patient samples.
According to an aspect of the invention, methods decreasing angiogenesis in at least one cell are provided. The methods include contacting a plurality of cells with an effective amount of a threonyl-tRNA synthetase (TARS) activity-inhibiting compound to decrease angiogenesis in at least one cell of the plurality of cells. In some embodiments, the TARS-activity-inhibiting compound includes an anti-threonyl-tRNA synthetase (TARS) antibody or antigen-binding fragment thereof; a fragment of TARS that possesses negative complementation/inhibition activity; a small molecule inhibitor of TARS; a threonyl adenylate mimetic (e.g. threonyl sulfamoyl adenylate analog or a 3′ end portion of the aminoacylated tRNA); or a compound that mimics the transition state of a ThrRS catalyzed aminoacylation reaction. In certain embodiments, the plurality of cells is in culture. In some embodiments, the culture is an organ culture. In some embodiments, the plurality of cells is in a subject and contacting the cells includes administering TARS-activity-inhibiting compound to the subject. In certain embodiments, the TARS-activity-inhibiting compound is an anti-threonyl-tRNA synthetase (TARS) antibody or antigen-binding fragment thereof. In some embodiments, the cells have or are at risk of developing an angiogenesis-associated disease or condition. In some embodiments, the angiogenesis-associated disease or condition is a cancer, a tumor, a hemangioma, vascular overgrowth, venous malformation, arterial malformation, overweight, macular degeneration, inflammatory disease, psoriasis, diabetes, or rheumatoid arthritis. In certain embodiments, the cancer is a metastatic carcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma; androgen independent prostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lung cancer, or mesothelioma. In some embodiments, the method also includes contacting the plurality of cells with one or more additional angiogenesis-inhibiting compounds. In some embodiments, contacting the plurality of cells with the TARS-activity-inhibiting compound and one or more of the additional angiogenesis-inhibiting compounds results in a synergistic decrease in angiogenesis in the plurality of cells. In some embodiments, the TARS-activity-inhibiting compound is administered to the subject as a pharmaceutical composition. In certain embodiments, the pharmaceutical composition includes a TARS-activity-inhibiting compound and a pharmaceutically acceptable carrier. In some embodiments, the TARS-activity-inhibiting compound is in conjunction with a delivery agent. In some embodiments, the delivery agent is a microbead. In some embodiments, the subject is a human. In certain embodiments, the plurality of cells includes one or more pre-vascular cells, angioblasts, vascular cells, immune cells, include T cells; fibroblasts; neuronal cells, glial cells, cells of the lymphatic system, tumor cells, stem cells, progenitor cells, and inflammatory cells. In some embodiments, the vascular cells comprise endothelial cells, adventitial cells, pericytes and/or smooth muscle cells. In some embodiments, the TARS-activity-inhibiting compound reduces secreted TARS activity. In some embodiments, the activity is reduced by reducing TARS secretion. In certain embodiments, the TARS-activity-inhibiting compound reduces non-secreted TARS activity. In some embodiments, the plurality of cells is contacted with an amount of the TARS activity-inhibiting compound that is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of an amount of the TARS activity-inhibiting compound that results in an amino acid starvation response in the at least one cell. In some embodiments, the plurality of cells is contacted with an amount of TARS activity-inhibiting compound that does not result in an amino acid starvation response in the at least one cell. In certain embodiments, the plurality of cells is contacted with TARS activity-inhibiting compound that does not result in an amino acid starvation response in the at least one cell.
According to another aspect of the invention, methods of decreasing angiogenesis in a subject are provided. The methods include administering to the subject in need of such treatment an effective amount of a threonyl-tRNA synthetase (TARS) activity-inhibiting compound to decrease angiogenesis in the subject. In some embodiments, the TARS-activity-inhibiting compound is an anti-threonyl-tRNA synthetase (TARS) antibody or antigen-binding fragment thereof; a fragment of TARS that possesses negative complementation/inhibition activity; a small molecule inhibitor of TARS; a threonyl adenylate mimetic (e.g. threonyl sulfamoyl adenylate analog or a 3′ end portion of the aminoacylated tRNA); or a compound that mimics the transition state of a ThrRS catalyzed aminoacylation reaction. In some embodiments, the subject has or is at risk of having an angiogenesis-associated disease or condition. In some embodiments, the angiogenesis-associated disease or condition is a cancer, (primary or metastatic), a tumor, a hemangioma, vascular overgrowth, venous malformation, arterial malformation, overweight, macular degeneration, inflammatory disease, psoriasis, diabetes, or rheumatoid arthritis. In certain embodiments, the cancer is a metastatic carcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma; androgen independent prostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lung cancer, or mesothelioma. In some embodiments, the method also includes administering one or more additional angiogenesis-inhibiting compounds to the subject. In some embodiments, administering the TARS-activity-inhibiting compound and the one or more of the additional angiogenesis-inhibiting compounds results in a synergistic decrease in angiogenesis in the subject. In some embodiments, the TARS-activity-inhibiting compound is administered to the subject as a pharmaceutical composition including the TARS-activity-inhibiting compound and a pharmaceutically acceptable carrier. In certain embodiments, the TARS-activity-inhibiting compound is administered to the subject in a pharmaceutical composition. In some embodiments, the pharmaceutical composition includes the TARS-activity-inhibiting compound and a pharmaceutically acceptable carrier. In some embodiments, the TARS-activity-inhibiting compound is administered in conjunction with a delivery agent. In some embodiments, the delivery agent is a microbead. In some embodiments, the subject is a human. In certain embodiments, the TARS-activity-inhibiting compound is administered orally, parenterally, intraperitoneally, subcutaneously, intranasally, intravenously, intrathecally, intramuscularly, intracranially, transmucosally, vaginally, via instillation, rectally, or topically. In some embodiments, the subject has been diagnosed with an angiogenesis-associated disease or condition. In some embodiments, the TARS-activity-inhibiting compound reduces a secreted TARS activity. In certain embodiments, the activity is reduced by reducing TARS secretion. In some embodiments, the TARS-activity-inhibiting compound reduces non-secreted TARS activity. In some embodiments, the amount of the TARS activity-inhibiting compound that is administered to the subject is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of an amount of the TARS activity-inhibiting compound that results in an amino acid starvation response in the subject. In some embodiments, the TARS activity-inhibiting compound that is administered to the subject does not result in an amino acid starvation response in the subject.
According to yet another aspect of the invention, methods of decreasing angiogenesis in a plurality of cells are provided. The methods include contacting the plurality of cells with a TARS-activity-inhibiting compound that decreases an interaction of threonyl-tRNA synthetase (TARS) with VHL, or decreases the effect of TARS on VHL function in at least one cell in the plurality of cells, wherein the decrease in the interaction decreases angiogenesis in the plurality of cells. In certain embodiments, the interaction of TARS with VHL includes the formation, maintenance, or activity of a TARS/VHL complex. In some embodiments, decreasing the interaction of the TARS/VHL complex includes decreasing the formation of a TARS/VHL complex in the plurality of cells. In some embodiments, the decrease in the formation of the TARS/VHL complex is sufficient to increase in an ubiquitination function of VHL on HIF-1α. In some embodiments, decreasing the interaction of the TARS/VHL complex includes decreasing the activity of a TARS/VHL complex in the plurality of cells. In some embodiments, decreasing the interaction of the TARS/VHL complex includes decreasing the maintenance of a TARS/VHL complex in the plurality of cells. In certain embodiments, decreasing the maintenance of the TARS/VHL complex activity includes increasing disassociation of the TARS/VHL complex. In some embodiments, the plurality of cells is in culture. In some embodiments, the culture is an organ culture. In certain embodiments, the plurality of cells is in a subject and contacting the cells includes administering a TARS-activity-inhibiting compound or a VHL-activity-inhibiting compound to the subject. In some embodiments, the TARS-activity-inhibiting compound includes an anti-threonyl-tRNA synthetase (TARS) antibody or antigen-binding fragment thereof; a small molecule inhibitor of TARS; a threonyl adenylate mimetic (e.g. threonyl sulfamoyl adenylate analog or a 3′ end portion of the aminoacylated tRNA); or a compound that mimics the transition state of a ThrRS catalyzed aminoacylation reaction. In some embodiments, the VHL-activity-inhibiting compound includes an anti-VHL antibody or antigen-binding fragment thereof; or a small molecule inhibitor of VHL. In certain embodiments, the subject has or is at risk of having an angiogenesis-associated disease or condition. In some embodiments, the angiogenesis-associated disease or condition is a cancer, a tumor, a hemangioma, vascular overgrowth, venous malformation, arterial malformation, overweight, macular degeneration, inflammatory disease, psoriasis, diabetes, or rheumatoid arthritis. In some embodiments, the cancer is a metastatic carcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma; androgen independent prostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lung cancer, or mesothelioma. In some embodiments, the methods also include contacting the plurality of cells with one or more additional angiogenesis-inhibiting compounds. In certain embodiments, contacting the plurality of cells with the compound that decreases an interaction of threonyl-tRNA synthetase (TARS) with VHL, or decreases the effect of TARS on VHL and one or more of the additional angiogenesis-inhibiting compounds results in a synergistic decrease in angiogenesis in the plurality of cells. In some embodiments, the compound that decreases an interaction of threonyl-tRNA synthetase (TARS) with VHL, or decreases the effect of TARS on VHL function TARS-activity-inhibiting compound is administered to the subject as a pharmaceutical composition. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier. In some embodiments, the compound that decreases an interaction of threonyl-tRNA synthetase (TARS) with VHL, or decreases the effect of TARS on VHL is administered in conjunction with a delivery agent. In certain embodiments, the delivery agent is a microbead. In some embodiments, the subject is a human. In some embodiments, the TARS-activity-inhibiting compound is administered to the subject orally, parenterally, intraperitoneally, subcutaneously, intranasally, intravenously, intrathecally, intramuscularly, intracranially, transmucosally, vaginally, via instillation, rectally, or topically. In some embodiments, the compound reduces a secreted TARS activity. In some embodiments, the activity is reduced by reducing TARS secretion. In certain embodiments, the compound reduces non-secreted TARS activity. In some embodiments, the plurality of cells is contacted with an amount of the TARS activity-inhibiting compound that is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of an amount of the TARS activity-inhibiting compound that results in an amino acid starvation response in the at least one cell. In some embodiments, the plurality of cells is contacted with an amount of TARS activity-inhibiting compound that does not result in an amino acid starvation response in the at least one cell. In some embodiments, the plurality of cells is contacted with a TARS activity-inhibiting compound that does not result in an amino acid starvation response in the at least one cell.
According to another aspect of the invention, methods of assisting in the selection of a treatment to inhibit angiogenesis in a subject are provided. The methods include obtaining a cell sample from a subject having or at risk of having an angiogenesis-associated disease or condition, determining the threonyl-tRNA synthetase (TARS)/von Hippel Lindau factor (VHL) interaction in the cell sample; comparing the determined TARS/VHL interaction to a control TARS/VHL interaction, and selecting a treatment for the angiogenesis-associated disease or condition in the subject based at least in part on the difference between the determined TARS/VHL interaction and the control TARS/VHL interaction, wherein if the determined TARS/VHL interaction is greater than the control interaction, the selected treatment is a treatment that inhibits the TARS/VHL interaction in the subject and inhibits angiogenesis in the subject. In certain embodiments, the interaction of TARS with VHL includes the formation, maintenance, or activity of a TARS/VHL complex. In some embodiments, decreasing the interaction of the TARS/VHL complex includes decreasing the formation of a TARS/VHL complex in the subject. In some embodiments, decreasing the interaction of the TARS/VHL complex includes decreasing the activity of a TARS/VHL complex in the subject. In some embodiments, decreasing the interaction of the TARS/VHL complex includes decreasing the maintenance of a TARS/VHL complex in the subject. In certain embodiments, decreasing the maintenance of the TARS/VHL complex activity includes increasing disassociation of the TARS/VHL complex. In some embodiments, the control TARS/VHL interaction is a predetermined standard TARS/VHL. In some embodiments, the control is a normal control. In some embodiments, determining the interaction includes determining the level of the TARS/VHL complex in a tissue sample from the subject. In certain embodiments, selecting the treatment includes comparing the level of the TARS/VHL complex with a control level of TARS/VHL complex and basing the selection at least in part on the comparison; comparing the level of HIF-1α in the subject to a control level of HIF-1α and basing the selection at least in part on the comparison; comparing the level of ubiquitination of HIF-1α in the subject to a control level of ubiquitination and basing the selection at least in part on the comparison; or comparing the level of vascular endothelial growth factor (VEGF) in the subject to a control level of VEGF and basing the selection at least in part on the comparison. In some embodiments, the treatment for the angiogenesis-associated disease or condition includes administering to the subject an effective amount of a TARS-activity-inhibiting compound to decrease angiogenesis in the subject. In some embodiments, the TARS-activity-inhibiting compound includes an anti-threonyl-tRNA synthetase (TARS) antibody or antigen-binding fragment thereof; a fragment of TARS that possesses negative complementation/inhibition activity; a small molecule inhibitor of TARS; a threonyl adenylate mimetic (e.g. threonyl sulfamoyl adenylate analog or a 3′ end portion of the aminoacylated tRNA); or a compound that mimics the transition state of a ThrRS catalyzed aminoacylation, GTPase or Ap4A synthesis reaction. In certain embodiments, the TARS-activity-inhibiting compound reduces a secreted TARS activity. In some embodiments, the activity is reduced by reducing TARS secretion. In some embodiments, the compound reduces non-secreted TARS activity. In certain embodiments, the angiogenesis-associated disease or condition is a cancer, a tumor, a hemangioma, vascular overgrowth, venous malformation, arterial malformation, overweight, macular degeneration, inflammatory disease, psoriasis, diabetes, or rheumatoid arthritis. In some embodiments, the cancer is a metastatic carcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma; androgen independent prostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lung cancer, or mesothelioma. In some embodiments, the amount of the TARS activity-inhibiting compound that is administered to the subject is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of an amount of the TARS activity-inhibiting compound that results in an amino acid starvation response in the subject. In some embodiments, the TARS activity-inhibiting compound that is administered to the subject does not result in an amino acid starvation response in the subject.
According to yet another aspect of the invention, methods of identifying a candidate TARS-activity-inhibiting compound are provided. The methods include contacting a plurality of cells with a candidate compound and determining the effect of the contact on a TARS/VHL interaction in the plurality of cells, wherein a compound that decreases the TARS/VHL interaction in the plurality of cells, is a candidate TARS-activity-inhibiting compound. In certain embodiments, the interaction of TARS with VHL includes the formation, maintenance, or activity of a TARS/VHL complex. In some embodiments, decreasing the interaction of the TARS/VHL complex includes decreasing the formation of TARS/VHL complex in the plurality of cells. In some embodiments, decreasing the interaction of the TARS/VHL complex includes decreasing the activity of a TARS/VHL complex in the plurality of cells. In some embodiments, decreasing the interaction of the TARS/VHL complex includes decreasing the maintenance of a TARS/VHL complex in the plurality of cells. In some embodiments, decreasing the maintenance of the TARS/VHL complex activity includes increasing disassociation of the TARS/VHL complex. In certain embodiments, the method also includes comparing the TARS/VHL interaction in the plurality of cells with a control TARS/VHL interaction in a plurality of cells not contacted with the candidate compound, wherein a decrease in the TARS/VHL interaction in the plurality of cells compared to the control level indicates that the candidate compound is a candidate TARS-activity-inhibiting compound. In some embodiments, determining the TARS/VHL interaction includes determining the level of the TARS/VHL complex in the plurality of cells. In some embodiments, the plurality of cells is in culture. In some embodiments, the plurality of cells is from a sample obtained from a subject. In some embodiments, the subject has or is suspected of having an angiogenesis-associated disease or condition. In certain embodiments, the angiogenesis-associated disease or condition is a cancer, a tumor, a hemangioma, vascular overgrowth, venous malformation, arterial malformation, overweight, macular degeneration, inflammatory disease, psoriasis, diabetes, or rheumatoid arthritis. In some embodiments, the cancer is a metastatic carcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma; androgen independent prostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lung cancer, or mesothelioma. In some embodiments, the compound reduces a secreted TARS activity. In some embodiments, the activity is reduced by reducing TARS secretion. In some embodiments, the compound reduces non-secreted TARS activity. In certain embodiments, the compound contacts the cells in an amount that does not result in an amino acid starvation response in a cell. In some embodiments, the compound does not result in an amino acid starvation response in the contacted cell.
According to another aspect of the invention, pharmaceutical compositions that include a threonyl-tRNA synthetase (TARS)-activity-inhibiting compound and a pharmaceutically acceptable carrier are provided. In some embodiments, the TARS-activity-inhibiting compound is an anti-threonyl-tRNA synthetase (TARS) antibody or antigen-binding fragment thereof; a fragment of TARS that possesses negative complementation/inhibition activity; a small molecule inhibitor of TARS; a threonyl adenylate mimetic (e.g. threonyl sulfamoyl adenylate analog or a 3′ end portion of the aminoacylated tRNA); or a compound that mimics the transition state of a ThrRS catalyzed aminoacylation reaction. In certain embodiments, the TARS-activity-inhibiting compound includes an anti-threonyl-tRNA synthetase (TARS) antibody or antigen-binding fragment thereof. In some embodiments, the pharmaceutical composition also includes one or more additional angiogenesis-inhibiting compounds. In some embodiments, the composition is formulated for oral, parenteral, nasal, intravenous, intrathecal, intramuscular, intracranial, transmucosal, vaginal, instillation, rectal, or topical administration. In some embodiments, the TARS-activity-inhibiting compound reduces a secreted TARS activity. In certain embodiments, the activity is reduced by reducing TARS secretion. In some embodiments, the TARS-activity-inhibiting compound reduces non-secreted TARS activity.
According to yet another aspect of the invention, methods of immune system suppression in a subject are provided. The methods include administering to the subject in need of such treatment an effective amount of a threonyl-tRNA synthetase (TARS)-activity-inhibiting compound to suppress the immune system in the subject. In some embodiments, the TARS-activity-inhibiting compound is an anti-threonyl-tRNA synthetase (TARS) antibody or antigen-binding fragment thereof; a fragment of TARS that possesses negative complementation/inhibition activity; a small molecule inhibitor of TARS; a threonyl adenylate mimetic (e.g. threonyl sulfamoyl adenylate analog or a 3′ end portion of the aminoacylated tRNA); or a compound that mimics the transition state of a ThrRS catalyzed aminoacylation reaction. In some embodiments, the TARS-activity-inhibiting compound is an anti-threonyl-tRNA synthetase (TARS) antibody or antigen-binding fragment thereof. In certain embodiments, the subject has or is at risk of having an immune system-associated disease or condition. In some embodiments, the immune system-associated disease or condition is rheumatoid arthritis, cancer, interstitial lung disease, organ rejection, lupus, asthma, or allergic rhinitis. In some embodiments, suppression of the immune system includes reducing T cell development in the subject. In some embodiments, the method also includes administering one or more additional immune system-suppressing compounds to the subject. In certain embodiments, administering the TARS-activity-inhibiting compound and the one or more of the additional immune system-suppressing compounds results in a synergistic suppression of the immune system in the subject. In some embodiments, the TARS-activity-inhibiting compound is administered to the subject as a pharmaceutical composition including the TARS-activity-inhibiting compound and a pharmaceutically acceptable carrier. In some embodiments, the subject is a human. In certain embodiments, the TARS-activity-inhibiting compound is administered orally, parenterally, intraperitoneally, subcutaneously, intranasally, intravenously, intrathecally, intramuscularly, intracranially, transmucosally, vaginally, via instillation, rectally, or topically. In some embodiments, the TARS-activity-inhibiting compound is administered after diagnosis of an immune system-associated disease or condition. In some embodiments, the TARS-activity-inhibiting compound reduces a secreted TARS activity. In some embodiments, the activity is reduced by reducing TARS secretion. In certain embodiments, the TARS-activity-inhibiting compound reduces non-secreted TARS activity. In some embodiments, the amount of the TARS activity-inhibiting compound that is administered to the subject is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of an amount of the TARS activity-inhibiting compound that results in an amino acid starvation response in the subject. In some embodiments, the TARS activity-inhibiting compound that is administered to the subject does not result in an amino acid starvation response in the subject.
According to another aspect of the invention, methods of decreasing angiogenesis in a first cell are provided. The methods include contacting a second cell with a compound that decreases threonyl-tRNA synthetase (TARS) secretion in the second of cell and/or decreases an extracellular activity of TARS secreted from the second cell, wherein the decrease in the TARS secretion from the second cell or a decrease in the extracellular activity of the TARS secreted from the second cell decreases angiogenesis in the first cell. In some embodiments, the first cell is culture. In certain embodiments, the culture is an organ culture. In some embodiments, the first and second cells are in a subject and contacting the second cell includes administering the compound to subject. In some embodiments, the compound includes an antibody or antigen-binding fragment thereof, or a small molecule inhibitor of TARS secretion or TARS extracellular function. In some embodiments, the subject has or is at risk of having an angiogenesis-associated disease or condition. In certain embodiments, the angiogenesis-associated disease or condition is a cancer, a tumor, a hemangioma, vascular overgrowth, venous malformation, arterial malformation, overweight, macular degeneration, inflammatory disease, psoriasis, diabetes, or rheumatoid arthritis. In some embodiments, the cancer is a metastatic carcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma; androgen independent prostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lung cancer, or mesothelioma. In some embodiments, the method also includes contacting the plurality of cells with one or more additional angiogenesis-inhibiting compounds. In some embodiments, contacting the plurality of cells with the compound that decreases TARS secretion and/or decreases an extracellular function of TARS and one or more of the additional angiogenesis-inhibiting compounds results in a synergistic decrease in angiogenesis in the plurality of cells. In certain embodiments, the compound that decreases TARS secretion and/or decreases an extracellular function of TARS is administered to the subject as a pharmaceutical composition. In some embodiments, the pharmaceutical composition includes a pharmaceutically acceptable carrier. In some embodiments, the compound that decreases TARS secretion and/or decreases an extracellular function of TARS is administered in conjunction with a delivery agent. In some embodiments, the delivery agent is a microbead. In certain embodiments, the subject is a human. In some embodiments, the compound that decreases TARS secretion and/or decreases an extracellular function of TARS is administered to the subject orally, parenterally, intraperitoneally, subcutaneously, intranasally, intravenously, intrathecally, intramuscularly, intracranially, transmucosally, vaginally, via instillation, rectally, or topically. In some embodiments, the second cell is contacted with an amount of the TARS activity-inhibiting compound that is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of an amount of the TARS activity-inhibiting compound that results in an amino acid starvation response in the first or second cell. In certain embodiments, the second cell is contacted with an amount of TARS activity-inhibiting compound that does not result in an amino acid starvation response in the first or second cell. In some embodiments, the second cell is contacted with a TARS activity-inhibiting compound that does not result in an amino acid starvation response in the first or second cell.
According to another aspect of the invention, methods of assisting in the selection of a treatment to inhibit angiogenesis in a subject are provided. The methods include obtaining a cell sample from a subject having or at risk of having an angiogenesis-associated disease or condition, determining a level of an extracellular threonyl-tRNA synthetase (TARS) molecule in the cell sample; comparing the determined level of the extracellular TARS molecule in the sample to a control level of the extracellular TARS molecule, and selecting a treatment for the angiogenesis-associated disease or condition in the subject based at least in part on a difference between the determined extracellular TARS level and the control extracellular TARS level, wherein if the determined extracellular TARS level is greater than the control extracellular TARS level, the selected treatment is a treatment that reduces the extracellular TARS level and inhibits angiogenesis in the subject. In some embodiments, determining the extracellular TARS level includes determining the level of secreted TARS. In certain embodiments, determining the extracellular TARS level includes determining the level of extracellular TARS activity. In some embodiments, the control extracellular TARS level is a predetermined standard extracellular TARS level. In some embodiments, the control is a normal control. In certain embodiments, the treatment for the angiogenesis-associated disease or condition includes administering to the subject an effective amount of an extracellular TARS reducing compound to reduce extracellular TARS secretion and/or activity in amount effective to decrease angiogenesis in the subject. In some embodiments, the angiogenesis-associated disease or condition is a cancer, a tumor, a hemangioma, vascular overgrowth, venous malformation, arterial malformation, overweight, macular degeneration, inflammatory disease, psoriasis, diabetes, or rheumatoid arthritis. In some embodiments, the cancer is a metastatic carcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma; androgen independent prostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lung cancer, or mesothelioma. In some embodiments, the TARS molecule is a TARS polypeptide.
According to another aspect of the invention, methods of identifying a candidate angiogenesis-inhibiting compound are provided. The methods include contacting a plurality of cells with a candidate compound and determining the effect of the contact on at least one of the amount or activity of an extracellular threonyl-tRNA synthetase (TARS) molecule in the plurality of cells, wherein a compound that decreases at least one of the amount or activity of the extracellular TARS molecule in the plurality of cells, and does not result in an amino acid starvation response in the plurality of cells is a candidate angiogenesis-inhibiting compound. In certain embodiments, decreasing the amount or activity of the extracellular TARS molecule includes reducing secretion of the TARS molecule in the plurality of cells. In some embodiments, the method also includes comparing at least one of the amount or activity of the extracellular TARS molecule in the plurality of cells with a control amount or activity level of the extracellular TARS molecule, respectively, in a plurality of cells not contacted with the candidate compound, wherein a decrease in at least one of the amount or activity of the extracellular TARS molecule in the plurality of cells compared to the control amount or activity, respectively, indicates that the candidate compound as a candidate angiogenesis-inhibiting compound. In some embodiments, the plurality of cells is in culture. In some embodiments, the plurality of cells is from a sample obtained from a subject. In certain embodiments, the subject has or is suspected of having an angiogenesis-associated disease or condition. In some embodiments, the angiogenesis-associated disease or condition is a cancer, a tumor, a hemangioma, vascular overgrowth, venous malformation, arterial malformation, overweight, macular degeneration, inflammatory disease, psoriasis, diabetes, or rheumatoid arthritis. In some embodiments, the cancer is a metastatic carcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma; androgen independent prostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lung cancer, or mesothelioma. In certain embodiments, the TARS molecule is a TARS polypeptide.
Mus musculus TARS polypeptide sequence having
C Elegans TARS polypeptide sequence having
S cerevisiae TARS polypeptide sequence having
Homo sapiens TARS cytoplasmic isoform 1 having
Angiogenesis is involved in many cellular functions and processes including in diseases and conditions such as cancer, tumors, hemangiomas, vascular overgrowth, venous malformation, arterial malformation, overweight (fat storage), macular degeneration, inflammatory disease, psoriasis, diabetes, and rheumatoid arthritis that may be characterized by excess angiogenesis and/or for which it may be desirable to limit or reduce angiogenesis for treatment. In addition, TARS activity has now also been found to be associated with immune system activity and methods of the invention, in part, include in some aspects treatments that reduce TARS activity in order to suppress immune system activity and treat an immune system disease or condition. Thus, methods of the invention can be used to treat angiogenic and/or immune system diseases or conditions. As used herein an “angiogenic” disease or condition is also referred to as an “angiogenesis-associated” disease or condition.
It has now been identified that threonyl-tRNA synthetase (TARS) plays a role in angiogenesis and can be used in methods to treat diseases and conditions characterized by abnormal (e.g., increased) levels of TARS. As used herein, with respect to TARS molecule activity and quantitation, the terms: “increased”, “elevated”, and “higher” are used interchangeably. As used herein with respect to TARS molecule activity and quantitation, the terms “decrease”, “reduced”, and “lower” are used interchangeably.
In cancers, angiogenesis signaling can be an early step in invasive cancer growth, ascites formation, and metastasis. Cells that are environmentally stressed by hypoxia and/or starvation respond by expressing genes that support anaerobic metabolism and stimulate angiogenesis. Because of their rapid growth, many cancer cell types continuously express these genes in an effort to continue growing in a nutrient-poor environment. The development of vasculature, e.g., angiogenesis, involves changes in protein synthesis and may be initiated by environmental stress such as hypoxia or starvation in a cell. Aminoacyl tRNA synthetases are believed to function in some aspects of angiogenesis. Cancer treatments that reduce angiogenesis have recently been shown to causes hypoxia, enhancing the ability of cancer stem cells to increase their invasiveness and metastatic potential. Hence, cancer diagnoses and treatments that influence the hypoxic response are significant and novel area of cancer therapeutics.
It is now understood that levels of TARS expression and function can be modulated in methods to treat angiogenic diseases and conditions that are characterized at least in part by increased TARS activity. Diseases and conditions that have increased TARS activity may include angiogenic and/or immune system diseases and conditions, examples of which are provided herein, and include but are not limited to cancer. Thus, the improved understanding the role of TARS in early angiogenesis signaling and immune function have now been used to identify novel treatment targets to recognize and treat angiogenic and immune system conditions, such as cancer, thus improving the likelihood of successful treatment.
Protein synthesis is known to include activities of aminoacyl-tRNA synthetases, which are enzymes that catalyze the aminoacylation of tRNA by their cognate amino acids. Threonyl-tRNA synthetase (TARS) is an aminoacyl tRNA synthestase that is known to charge tRNA with threonine during protein synthesis. Protein synthesis plays a role in many different activities of cells and tissues such as growth and development, differentiation, replication, signaling, etc. and alterations in aminoacyl-tRNA synthetase activities and functions may result in disruption of cell processes and disease.
Cancer cells respond to environmental stress and the tumor microenvironment plays a role in determining cancer cell survival and growth responses. Cancer cells rely on these responses because they rapidly outgrow their blood supply and must survive under conditions of hypoxia, starvation, and metabolic stress. Cells relieve these stresses by decreasing protein translation through the unfolded protein response and increasing blood supply through secretion of angiogenic cytokines and growth factors. A novel connection between these metabolic and angiogenic responses has now been identified and features, in part, the ability of tRNA synthetase inhibitors to alter the angiogenesis signaling pathway through a novel mechanism. In addition to having a role in cancer, angiogenesis also occurs physiologically during fetal development, wound healing, pregnancy, weight gain, and ischemic preconditioning, and is a feature found numerous additional diseases and conditions. In addition, it has now been identified that increased TARS activity is associated with increased immune system activity and that in some immune system diseases and conditions in which activity of the immune system is too high, methods of the invention that include reducing TARS activity may be used to treat such immune system diseases and conditions. Thus, some aspects of the invention include methods to treat an immune system disease or condition. Examples of immune system conditions that can be treated with methods and TARS-activity-inhibiting compounds of the invention, include, but are not limited to rheumatoid arthritis, cancer, interstitial lung disease, organ rejection, lupus, asthma, or allergic rhinitis. In some embodiments of the invention, administering an effective amount of a TARS-activity-inhibiting compound suppresses the immune system, which in some cases includes reducing T cell development in the subject. A TARS-activity-inhibiting compound of the invention may be administered to a subject in conjunction with, or in series with, one or more additional immune system-suppressing compounds, and in some embodiments, the resulting effect in the subject includes a synergistic suppression of the immune system in the subject.
TARS is an aminoacyl-tRNA synthetase that selectively catalyzes the ATP-dependent formation of threonyl-tRNA, a substrate for the protein translation machinery. Aside from their canonical functions in protein synthesis, aminoacyl-tRNA synthetases have been implicated in autoimmune and cytokine function, recovery from hypoxic stress, and angiogenesis. (Brown, M. V. et al. (2010) Vascul Pharmacol 52 (1-2), 21-26). Secretion and cytokine activities of extracellular TARS have now been examined and it has now been identified that TARS is secreted under conditions of exposure to cytokines (e.g., TNF-α and VEGF) and that one or more specific domains of TARS, including the N-terminal domain of TARS (the TGS domain), are regulatory in nature and able to confer cytokine activity.
Threonyl-tRNA synthetase (TARS) is a metabolic workhorse that functions to charge tRNA with threonine during protein synthesis. TARS is ubiquitously expressed in a number of prokaryotic and eukaryotic organisms. TARS is alternatively known as threonine tRNA ligase 1; Threonine-tRNA ligase, threonyl-transfer ribonucleate synthetase, threonyl-transfer RNA synthetase, threonyl-transfer ribonucleic acid synthetase, threonyl ribonucleic synthetase, threonine-transfer ribonucleate synthetase, threonine translase, TRS, and ThrRS. An example of a human TARS protein sequence is provided as GENBANK™ Accession No. P26639. Examples of TARS polypeptide sequences of other species include: Mus musculus: GENBANK™ Accession No. Q9D0R2; C Elegans: GENBANK™ Accession No. P52709; S cerevisiae: GENBANK™ Accession No. P04801. A human TARS nucleic acid sequence is provided as GENBANK™ Accession No. NM_152295.
It has now been discovered that TARS acts in a previously unknown manner to promote angiogenesis. Studies have now shown that inhibition of TARS reduces both the hypoxic response of cancer cells and, unexpectedly, that application of exogenous purified TARS also stimulated angiogenesis in an in vivo angiogenesis assay. Thus, TARS may have dual functions as a metabolic regulator and as an angiogenic cytokine, and may be secreted from cells exposed to ischemic stress. It has also now been found that TARS mRNA and TARS polypeptide may be selectively overexpressed in various cancers, including but not limited to ovarian tumors, which are highly angiogenic. TARS polypeptide expression and activity levels are positively correlated with angiogenesis and with cancer metastases. It has also now also been identified that levels and/or activity of TARS mRNA and TARS polypeptide may be higher than normal in additional diseases and conditions as described herein.
It has now been identified that compounds of the invention that can be administered to reduce TARS activity levels at levels that do not trigger an amino acid starvation response.
Amino acid starvation response (AAS) is a broad-based cellular response to nutrient deprivation (particularly amino acids) and is marked by several distinctive physiological markers, including the induction of eIF2α phosphorylation, and the increased transcription of many stress responses. Markers that may be useful to assess the presence or absence of AAS include, but are not limited to asparagine synthase, eIF4E-BP, etc. AAS can be triggered or induced by starvation for many of the 20 amino acids, including but not limited to proline and essential amino acids such as phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, leucine, lysine, histidine, etc. For example: see Sundrud et al, (2009) Science 324:133 (2009); Keller et al., (2012) Nat. Chem Biol. 8(3):311-317. Surprisingly, the therapeutic effects of compounds useful in treatment methods of the invention may result with administration of amounts of the compounds that are insufficient to cause an amino acid starvation response. Thus, extremely low levels of compounds can be administered in embodiments of the invention as a treatment for a disease or condition associated with elevated TARS polypeptide activity. In addition, it has now been identified that compounds of the invention that can be administered to reduce TARS activity levels at may include compounds that do not trigger or result in an amino acid starvation response in a cell, tissue, or subject contacted with the compound. Thus, a compound of the invention that is effective to treat a disease or condition characterized by elevated TARS activity may be a compound that does not cause an amino acid starvation response in the treated cell or subject. Assessing AAS status in cells, tissues, and subjects can be done using routine assay methods known to those skilled in the art. For example: see Harding H P et al (2000) Mol Cell 6(5):1099-1108, also Harding H P et al (2003) Mol Cell 11(3):619-633.
It has now been shown that an increase in expression and/or activity of TARS is correlated with an increase in angiogenesis of cells and also that an increase in expression and/or activities of TARS (potentially including GTPase and Ap4A synthetic functions) is correlated with metastasis of a cancer compared to a lower level of expression and/or activity of TARS in a poorly metastatic or non-metastatic cancer. It has now been identified that TARS polypeptides and TARS-encoding nucleic acids provide targets for treatments to inhibit angiogenesis and for treatments to inhibit the metastatic process in cancers. Thus, treatment to reduce an elevated level of TARS, which may be a level of TARS polypeptide or level of TARS-encoding nucleic acid, for example, may reduce angiogenesis in a cell, a tissue, and/or a subject. In some embodiments of the invention, an effective treatment to reduce an elevated level of TARS in a cell, tissue, or subject, is a treatment that does not result in an amino acid starvation response in the cell, tissue, or subject. Thus, in some aspects of the invention, a treatment for a disease or condition associated with an elevated TARS activity may be distinguished from other treatments that result in an amino acid starvation response in a cell, tissue, or subject. In some aspects, a treatment that decreases TARS levels and/or function may reduce the likelihood of metastatic activity of a cancer. Further, by monitoring a subject undergoing a treatment of the invention and comparing changes in the level of a TARS polypeptide or TARS-encoding nucleic acid in the subject, one can evaluate changes in metastatic activity of the cancer and can assess the efficacy of a compound that is used to treat an angiogenic or immune disease or condition in the subject.
The present invention provides methods of treating a disease or condition associated with abnormal TARS activity. As used herein, the term “TARS activity” refers to a function of the TARS molecule, such as, but not limited to, aminoacylation of tRNA by threonine, association of a TARS polypeptide with a von Hippel Lindau (VHL) polypeptide to form a complex, association of a TARS polypeptide with elongation factor 1 (eEF1) to form a complex, associate of a TARS polypeptide with an E3 ubiquitin ligase, secretion of TARS protein or fragment of protein, binding of TARS to membrane receptors, or binding of TARS to extracellular matrix proteins.
In some embodiments of the invention, a disease or condition may be characterized by increased TARS activity compared with a control level of TARS activity. It will be understood that a decrease in TARS activity (for example resulting from a treatment of the invention) may be due to a decrease in the amount of TARS expressed in a cell, tissue, or subject, a decrease in the function or activity of TARS that is expressed in a cell, tissue, or subject, and/or a decrease in the secretion of TARS by a cell, tissue, or subject. Thus, in some embodiments of the invention, a reduction in TARS activity may be a result of a reduction in the amount of TARS polypeptide in a cell, tissue, or fluid. In certain embodiments of the invention the amount of TARS polypeptide may be unchanged (e.g., normal compared with a normal control) but the functional activity of the TARS that is present in the cell, tissue, or subject may be reduced. The altered activity may the result of a decrease in availability of a post-translationally modified version of TARS, which may be differentially secreted from one or more relevant cell types (including cancer cells, HUVEC cells, or cells of the innate immune system).
It has been identified that altered TARS activity in cells and/or tissues is correlated with various diseases and conditions. In certain diseases and conditions the level of TARS activity is statistically significantly higher in cells and/or tissues having the disease or condition compared to the level of TARS activity in cells and/or tissues that do not have the disease or condition. A level of TARS activity in a disease or condition characterized by a significantly higher activity compared to a normal control level may have a level of TARS activity that is at least 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 125%, 150%, 175%, 200%, or higher than a normal control level of TARS activity, e.g., a level in an equivalent sample that does not have a disorder or condition characterized by elevated, e.g., higher levels of TARS activity. As used herein, a disease or condition that may be characterized by elevated TARS activity may also be referred to as a disease or condition associated with elevated TARS activity.
Examples of diseases and conditions that may be characterized elevated TARS activity include, but are not limited to cancer, a tumor, a hemangioma, vascular overgrowth, venous malformation, arterial malformation, overweight, macular degeneration, inflammatory disease, psoriasis, diabetes, interstitial lung disease, and rheumatoid arthritis. An increase in angiogenesis may be a characteristic of diseases and conditions in which a higher TARS activity (versus a normal control level) is present. Non-limiting examples of cancers that may be characterized by elevated levels of TARS activity include a metastatic carcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma; androgen independent prostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lung cancer, and mesothelioma. In some aspects of the invention methods are provided to assess a change in the level of TARS activity in a disease or condition characterized by increased TARS activity and methods of the invention may be used to monitor the level of TARS activity over time to assess increases or decreases in TARS activity over time.
Treatment methods of the invention may include administering one or more compounds to a subject in need of such treatment, to modify a level of TARS activity in a cell or tissue sample to treat the disease or condition. Thus, in some aspects the invention includes methods of decreasing angiogenesis and/or an immune system response in a cell, tissue or subject, wherein the method includes contacting the cells, tissues, or subject with an effective amount of a threonyl-tRNA synthetase (TARS) activity-inhibiting compound to decrease angiogenesis in the cell, tissue, or subject. As used herein, a TARS-activity-inhibiting compound means a compound that reduces TARS activity. A TARS activity-inhibiting compound may reduce TARS activity directly, e.g., by interacting directly with a TARS molecule, or may reduce TARS activity indirectly, e.g., by modulating activity of another molecule that in turn is important in TARS activity. Examples of TARS-activity inhibiting compounds include, but are not limited to compounds such as an anti-threonyl-tRNA synthetase (TARS) antibody or antigen-binding fragment thereof; a fragment of TARS that possesses negative complementation/inhibition activity; a small molecule inhibitor of TARS; a threonyl adenylate mimetic (e.g. threonyl sulfamoyl adenylate analog or a 3′ end portion of the aminoacylated tRNA); or a compound that mimics the transition state of a ThrRS catalyzed aminoacylation reaction, etc. In some embodiments, the TARS-activity-inhibiting compound is not borrelidin, BC194, or other therapeutic compound set forth in the U.S. Pat. No. 7,560,252.
Some aspects of the invention include contacting a cell with a TARS-activity-inhibiting compound that decreases an interaction of threonyl-tRNA synthetase (TARS) with VHL, or decreases the effect of TARS on VHL function in the cell. The cell may be one of a plurality of cells, and contact with the TARS-activity-inhibiting compound may decrease angiogenesis in the cell or plurality of cells. In some embodiments of the invention, the interaction of TARS with VHL comprises the formation, maintenance, or activity of a TARS/VHL complex, and decreasing the interaction of the TARS/VHL complex comprises decreasing the formation of a TARS/VHL complex in the plurality of cells. In certain embodiments, the decrease in the formation of the TARS/VHL complex is sufficient to increase an ubiquitination function of VHL on HIF-1α. In certain embodiments of the invention, decreasing the interaction of the TARS/VHL complex comprises decreasing the activity of a TARS/VHL complex in the plurality of cells. In some embodiments of the invention, decreasing the interaction of the TARS/VHL complex comprises decreasing the maintenance of a TARS/VHL complex in the plurality of cells. In some embodiments of the invention decreasing the maintenance of the TARS/VHL complex activity comprises increasing disassociation of the TARS/VHL complex. In certain embodiments of the invention, the plurality of cells is in a subject and is contacted with a TARS-activity-inhibiting compound or a VHL-activity-inhibiting compound that is administered to the subject. In the context of the present invention, a VHL-activity-inhibiting compound may be a compound that indirectly inhibits TARS activity, and thus may also be referred to as a TARS-activity-inhibiting compound.
Identification of Candidate TARS-Activity-Inhibiting Compounds
In some aspects of the invention, methods are provided to identify candidate compounds for treating an angiogenic or immune system disease or condition as are methods to determine the efficacy of a compound for the treatment of the disease or condition. Such methods may include, for example, determining one or more levels of TARS in a cell, tissue or subject and comparing the TARS secondary activity levels (e.g., including but not limited to GTPase and Ap4A synthetic functions) in a cell, tissue, or subject contacted with the compound or treatment of the invention, with a cell, tissue, or subject not contacted with the compound or treatment of the invention.
A TARS level can be determined using methods of the invention to measure the amount and/or activity of a TARS molecule in an in vitro assay of a biological sample that has been obtained from the subject. As used herein, the term “measure” may refer to a determination of the presence or absence of a TARS molecule, may refer to a determination of a quantity a TARS molecule, or may refer to a determination of a functional level of a TARS molecule. Methods of measuring polypeptides or nucleic acids are known in the art, and non-limiting examples of measuring means are provided herein.
Detection methods suitable for use in methods of the present invention can be used to detect TARS polypeptide or nucleic acid molecules in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of TARS mRNA include reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), Northern hybridizations, in situ hybridizations, DNA or oligonucleotide array, and next generation sequencing. In vitro techniques for detection of TARS DNA include polymerase chain reaction (PCR) and Southern hybridizations. In vitro techniques for detection of TARS polypeptide include, but are not limited to enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence, and other known suitable techniques. Alternatively, TARS polypeptide can be detected in vivo in a subject by introducing into the subject a labeled anti-TARS antibody. For example, the antibody can be labeled with a detectable marker such as a colorimetric marker, enzymatic marker, radioactive marker, etc. whose presence and location in a subject can be detected by standard imaging techniques.
Treatments to Decrease TARS Activity
The invention in some aspects relates to methods for modulating angiogenesis or an immune response in a cell, tissue, and/or subject. As used herein the term “modulating” means changing a level of angiogenesis or the immune response. In some embodiments of the invention, changing angiogenesis includes changing a level of angiogenesis in a cell or tissue. As used herein, the term “modulating” used in reference to angiogenesis or an immune response using a treatment of the invention, means decreasing angiogenesis or the immune response, respectively. Thus, methods of the invention may include, in some embodiments, treatments to decrease angiogenesis and/or an immune response in a cell, tissue or subject. In some embodiments of the invention, angiogenesis and/or an immune system response may be decreased by decreasing the level and/or activity of a threonyl-tRNA synthetase (TARS) in the cell, tissue, and/or subject. In some embodiments of the invention methods may include decreasing the level of a TARS polypeptide-encoding nucleic acid in a cell, tissue, or subject, which may result in decreased activity of TARS in the cell, tissue, or subject. Certain embodiments of the invention methods may include directly decreasing the level of TARS polypeptide in a cell, tissue, or subject, for example, by administering to a cell, tissue, or subject an effective amount of an antibody that disrupts activity of a TARS polypeptide. Such methods may be used to treat a disease characterized by an abnormally high level of TARS activity that results in an undesirable level of angiogenesis and/or an immune system response.
In some aspects of the invention, a treatment includes administration of a TARS-activity-inhibiting compound reduces secreted TARS activity. As used herein, “secreted TARS activity” refers to activity of TARS outside its cell of origin. For example, in certain embodiments of treatment methods of the invention, a TARS-activity-reducing compound is administered to a cell or subject and the compound reduces TARS activity by reducing TARS secretion. In some embodiments of treatment methods of the invention, a TARS-activity-reducing compound is administered to a cell or subject and the compound reduces non-secreted TARS activity—for example, reduces the activity of TARS within the cell in which it was produced.
Non-limiting examples of angiogenic diseases or conditions that may be treated with methods and compounds of the invention include, but are not limited to: a cancer, a tumor, a hemangioma, vascular overgrowth, venous malformation, arterial malformation, overweight, macular degeneration, inflammatory disease, psoriasis, diabetes, or rheumatoid arthritis. In some embodiments of the invention, the cancer is a metastatic carcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma; androgen independent prostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lung cancer, or mesothelioma. Examples of immune system diseases and conditions that may be treated with methods and compounds of the invention include, but are not limited to rheumatoid arthritis, cancer, interstitial lung disease, organ rejection, lupus, asthma, or allergic rhinitis.
As used herein, the terms “treat”, “treated”, or “treating” when used with respect to a disorder such as an angiogenic or immune system disease or condition that may be characterized by abnormal TARS polypeptide activity may refer to a prophylactic treatment that decreases the likelihood of a subject developing the angiogenic or immune system disease or condition, and also may refer to a treatment after the subject has developed the disease or condition in order to eliminate or reduce the level of the disease or condition, prevent the disease or condition from becoming more advanced (e.g., more severe), and/or to slow the progression of the disease compared to in the absence of the therapy.
In certain embodiments of the invention, changing a TARS molecule activity may include reducing the activity of a TARS-encoding nucleic acid or reducing the activity of a TARS polypeptide in a cell, tissue, or subject. Thus, as used herein, a TARS molecule may refer to a TARS polypeptide or to a nucleic acid that encodes a TARS polypeptide. In certain embodiments of the invention, changing TARS polypeptide activity includes decreasing functioning of a TARS polypeptide in a cell, tissue, or subject. In some such embodiments, the level of the TARS polypeptide does not change, but the function of one or more of the TARS polypeptides in a cell may be altered, for example, decreased. Examples of methods that may alter the function of a TARS polypeptide may include, but are not limited to contacting the TARS polypeptide with an antibody or functional fragment thereof that binds to the TARS polypeptide and reduces its function. For example, in some embodiments of the invention an antibody that inhibits TARS function may be delivered to a cell as part of a treatment regimen. In some embodiments of the invention, compounds that inhibit TARS function may be administered to a cell or subject and result in a modulation of TARS polypeptide activity. Compounds that inhibit a TARS polypeptide function and/or reduce a TARS polypeptide level may be referred to herein as TARS-modulating compounds or TARS-activity-inhibiting compounds. In some embodiments of the invention, a TARS-modulating compound may include an anti-TARS polypeptide antibody or functional fragment thereof, a small molecule TARS inhibitor.
Compounds that decrease a TARS polypeptide activity may be administered in an effective amount to a subject in need of treatment of an angiogenic or immune system disease or condition. Administering a compound that decreases TARS polypeptide activity to a subject may reduce an angiogenic or immune system disease or condition in the subject.
TARS-Activity-Inhibiting Compounds—Nucleic Acids and Polypeptides
A compound useful to treat an angiogenic or immune system disease or condition characterized by abnormal TARS polypeptide activity may, in some embodiments of the invention be a TARS polypeptide or nucleic acid that encodes a TARS polypeptide.
A method of the invention may include administering an exogenous TARS polypeptide or exogenous TARS polypeptide-encoding nucleic acid to a subject. In some embodiments, the administered exogenous TARS polypeptide may be non-functional or have reduced TARS function, for example may be have a mutation that reduces or eliminates the TARS polypeptide function. In certain embodiments, the administered exogenous TARS polypeptide-encoding nucleic acid may produce a TARS polypeptide that has reduced or absent TARS function, (e.g., encode a mutated TARS molecule).
One aspect of the invention involves isolated nucleic acid molecules that encode TARS or biologically active portions thereof, as well as nucleic acid fragments sufficient for use to administer to a subject as a treatment or to assess candidate compound or efficacy of a treatment regimen of the invention. As used herein, the term “nucleic acid molecule” is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA). A nucleic acid molecule may be single-stranded or double-stranded or may be a double-stranded DNA molecule. An “isolated” nucleic acid molecule is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived. Moreover, an “isolated” nucleic acid molecule, such as a cDNA molecule, may be free of other cellular material.
In some aspects of the invention, an isolated nucleic acid molecule of the invention comprises the nucleotide sequence shown in GENBANK™ Accession No.: NM_152295 The sequence of GENBANK™ Accession No. NM_152295 corresponds to the human TARS cDNA. This cDNA comprises sequences encoding the TARS polypeptide (i.e., “the coding region”, from nucleotides 1 to 2850), and 3′ untranslated sequences (nucleotides 2468-2850). Alternatively, the nucleic acid molecule may comprise only the coding region of GENBANK™ Accession No NP_689508 (e.g., nucleotides 296-2467).
The invention further encompasses nucleic acid molecules that differ from the sequence set forth in GENBANK™ Accession No. NM_152295 (and portions thereof) due to degeneracy of the genetic code and thus encode the same TARS protein as that encoded by the sequence set forth in GENBANK™ Accession No. NM_152295. Accordingly, in another embodiment, an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence as set forth in GENBANK™ Accession No. NM_152295. Moreover, the invention encompasses nucleic acid molecules that encode biologically active portions of the sequence set forth in GENBANK™ Accession No. NM_152295.
A nucleic acid molecule having the nucleotide sequence as set forth in GENBANK™ Accession No. NM_152295, or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein. For example, a human TARS cDNA library using all or portion of the sequence set forth in GENBANK™ Accession No. NM_152295 as a hybridization probe and standard hybridization techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd., ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989). Moreover, a nucleic acid molecule encompassing all or a portion of the sequence set forth as GENBANK™ Accession No.: NM_152295 can be isolated using any suitable method, including as a non-limiting example, use of the polymerase chain reaction using oligonucleotide primers designed based upon the sequence set forth as GENBANK™ Accession No.: NM_152295. For example, TARS mRNA can be isolated from cells using standard, art-known methods and cDNA can be prepared using reverse transcriptase and art-known methods. Synthetic oligonucleotide primers for PCR amplification can be designed based upon the nucleotide sequence set forth in GENBANK™ Accession No. NM_152295 and nucleic acids of the invention can be amplified using cDNA or, alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques. The nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis. Furthermore, oligonucleotides corresponding to TARS nucleotide sequence can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
In addition to the human TARS nucleotide sequence set forth as GENBANK™ Accession No.: NM_152295, it will be appreciated by those skilled in the art that DNA sequence polymorphisms that lead to changes in the amino acid sequences of TARS may exist within a population (e.g., the human population). Such genetic polymorphism in the TARS gene may exist among individuals within a population due to natural allelic variation. Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in TARS that are the result of natural allelic variation and that do not alter the functional activity of TARS are intended to be within the scope of the invention. Moreover, nucleic acid molecules encoding TARS polypeptides from other species, and thus which have a nucleotide sequence that differs from the human sequence set forth as GENBANK™ Accession No.: NM_152295, are intended to be within the scope of the invention. Nucleic acid molecules corresponding to natural allelic variants and nonhuman homologues of the human TARS cDNA of the invention can be isolated based on their similarity/identity to the human TARS nucleic acid disclosed herein using the human cDNA, or a portion thereof, as a hybridization probe—according to standard hybridization techniques under stringent hybridization conditions, which are recognized in the art.
In some aspects of the invention, an isolated nucleic acid molecule of the invention that hybridizes under stringent conditions to the sequence set forth in GENBANK™ Accession No.: NM_152295 corresponds to a naturally-occurring nucleic acid molecule. As used herein, a “naturally-occurring” nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein). In one embodiment, the nucleic acid encodes a natural human TARS polypeptide.
In addition to naturally-occurring allelic variants of the TARS sequence that may exist in the population, the skilled artisan will further appreciate that changes may be introduced by mutation into the nucleotide sequence set forth as GENBANK™ Accession No. NM_152295 thereby leading to changes in the amino acid sequence of the encoded TARS protein, without altering the functional ability of the TARS protein. For example, nucleotide substitutions leading to amino acid substitutions at “non-essential” amino acid residues may be made in the sequence set forth as GENBANK™ Accession No. NM_152295. A “non-essential” amino acid residue is a residue that can be altered from the wild-type sequence of TARS polypeptide (e.g., the sequence set forth as GENBANK™ Accession No. NM_152295) without altering the activity of TARS, whereas an “essential” amino acid residue is required for TARS activity.
Accordingly, another aspect of the invention pertains to nucleic acid molecules encoding TARS polypeptides that contain changes in amino acid residues that are not essential for TARS activity, e.g., residues that are not conserved or only semi-conserved among members of the subfamily. Such TARS polypeptides differ in amino acid sequence from the sequence set forth as GENBANK™ Accession No. NM_152295 yet retain TARS activity. In one embodiment, the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least 70%, 75%, 80%, 85%, 90%, 95%, 97%, or at least 99% similar to the amino acid sequence set forth as GENBANK™ Accession No.: NM_152295 and retains a level of TARS activity or has no TARS activity. In some aspects of the invention, a TARS polypeptide with reduced or no TARS function may be administered to a subject or cell, to compete with functional TARS in the subject or cell respectively, thereby reducing TARS activity in the subject or cell.
To determine the percent identity (similarity) of two amino acid sequences (e.g., GENBANK™ Accession No. NM_152295 and a mutant form thereof), the sequences are aligned for optimal comparison purposes (e.g., gaps may be introduced in the sequence of one protein for optimal alignment with the other protein). The amino acid residues at corresponding amino acid positions are then compared. When a position in one sequence (e.g., GENBANK™ Accession No. NM_152295) is occupied by the same amino acid residue as the corresponding position in the other sequence (e.g., a mutant form of TARS), then the molecules have identity at that position. The percent identity or percent similarity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity or % similarity=number of identical positions/total number of positions×100). Such an alignment can be performed using any one of a number of well-known computer algorithms designed and used in the art for such a purpose.
An isolated nucleic acid molecule encoding a TARS polypeptide having a percent identity or similarity to the protein of GENBANK™ Accession No.: NM_152295 can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of GENBANK™ Accession No.: NM_152295 such that one or more amino acid substitutions, additions or deletions are introduced into the encoded polypeptide. Mutations can be introduced into the sequence set forth as GENBANK™ Accession No. NM_152295 by standard techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis. In some embodiments of the invention conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues. A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in TARS may be replaced with another amino acid residue from the same side chain family. Alternatively, in another embodiment, mutations can be introduced randomly along all or part of a TARS coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for TARS activity to identify mutants that retain TARS activity. Following mutagenesis of a sequence such as that set forth as GENBANK™ Accession No. NM_152295, the encoded protein can be expressed recombinantly and the TARS activity of the polypeptide can be determined, for example using an assay described herein or other suitable assay.
As used herein, the terms “protein” and “polypeptide” are used interchangeably and thus the term polypeptide may be used to refer to a full-length protein and may also be used to refer to a fragment of a full-length protein. As used herein with respect to polypeptides, proteins, or fragments thereof, and nucleic acids that encode such polypeptides the term “exogenous” means the compound is administered to a cell or subject and was not naturally present in the cell or subject. It will be understood that an exogenous TARS polypeptide or TARS polypeptide-encoding nucleic acid may be identical to an endogenous TARS polypeptide or TARS polypeptide-encoding nucleic acid, respectively, in terms of its sequence, but was administered to the cell or subject.
According to some aspects of the invention, full-length TARS polypeptides or fragments of full-length TARS polypeptide may be administered in methods of the invention. Fragments of the invention may be fragments that retain a distinct functional capability of the polypeptide. Functional capabilities that can be retained in a fragment include interaction with antibodies, and interaction with other polypeptides or fragments thereof. Polypeptide fragments may be natural fragments or may be synthesized using art-known methods, and tested for function using the methods exemplified herein. Full-length TARS and functional and non-functional fragments of TARS that are useful in methods and compositions of the invention may be recombinant polypeptides.
A fragment of a full-length TARS polypeptide may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, or 50 amino acids fewer of the contiguous amino acids of TARS polypeptide having a consecutive sequence found in a wild-type TARS polypeptide or in a modified TARS polypeptide sequence as described herein. Such TARS polypeptides that are fragments of full-length TARS polypeptide may be useful for a variety of purposes, including for administration as TARS-modulating compounds and for preparing TARS-modulating compounds such as antibodies that bind specifically to synthetic and natural TARS polypeptides.
A “modified” wild-type or mutant full-length TARS polypeptide or polypeptide that is a fragment thereof may include deletions, point mutations, truncations, amino acid substitutions and/or additions of amino acids or non-amino acid moieties. Modifications of a polypeptide of the invention may be made by modification of the nucleic acid that encodes the polypeptide or alternatively, modifications may be made directly to the polypeptide, such as by cleavage, addition of a linker molecule, addition of a detectable moiety, such as a fluorescent label, and the like. Modifications also embrace fusion proteins comprising all or part of the polypeptide's amino acid sequence.
In some embodiments of the invention modified polypeptides (e.g. modified TARS wild-type or mutant polypeptides) may include polypeptides that are modified specifically to alter a feature of the polypeptide related or unrelated to its physiological activity. TARS polypeptides can be synthesized with modifications and/or modifications can be made in a TARS polypeptide by selecting and introducing an amino acid substitution, deletion, or addition. Modified polypeptides then can be tested for one or more activities (e.g., modulating TARS-polypeptide activity in a cell or subject, treatment of an angiogenic or immune system disease or condition, etc., to determine which modification provides a modified polypeptide with the desired properties.
The skilled artisan will also realize that conservative amino acid substitutions may be made in a polypeptide to provide functionally equivalent polypeptides, i.e., a modified TARS polypeptide that retains a functional capability of an un-modified TARS polypeptide in a treatment method of the invention. As used herein, a “conservative amino acid substitution” refers to an amino acid substitution that does not alter the relative charge or size characteristics of the polypeptide in which the amino acid substitution is made. Modified TARS polypeptides can be prepared according to methods for altering polypeptide sequence and known to one of ordinary skill in the art such. Exemplary functionally equivalent TARS polypeptides include conservative amino acid substitutions of a TARS polypeptide, or fragments thereof, such as a modified TARS polypeptide. Conservative amino-acid substitutions in a TARS polypeptide typically are made by alteration of a nucleic acid encoding the polypeptide. Such substitutions can be made by a variety of methods known to one of ordinary skill in the art. For example, amino acid substitutions may be made by PCR-directed mutation, site-directed mutagenesis, or by chemical synthesis of a gene encoding the TARS polypeptide. Where amino acid substitutions are made to a small fragment of a polypeptide, the substitutions can be made by directly synthesizing the polypeptide. The activity of functionally equivalent fragments of TARS polypeptides, or non-functional fragments of TARS polypeptides, can be tested by cloning the gene encoding the altered polypeptide into a bacterial or mammalian expression vector, introducing the vector into an appropriate host cell, expressing the altered polypeptide, and testing for a functional capability of the polypeptide as disclosed herein.
In some embodiments of the invention, a level or function of a TARS polypeptide may be modulated by genetically introducing a TARS polypeptide into a cell, tissue, and/or subject and reagents and methods are provided for genetically targeted expression of TARS polypeptides. Genetic targeting can be used to deliver TARS or other therapeutic polypeptides to specific cell types, to specific cell subtypes, to specific spatial regions within an organism, and to sub-cellular regions within a cell. Genetic targeting also relates to the control of the amount of a TARS polypeptide expressed, and the timing of the expression. Some embodiments of the invention include a reagent for genetically targeted expression of a TARS polypeptide, wherein the reagent comprises a vector that contains a nucleic acid that encodes a functional or non-functional TARS polypeptide or encodes a functional or non-functional fragment of a TARS polypeptide.
As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting between different genetic environments another nucleic acid to which it has been operatively linked. The term “vector” also refers to a virus or organism that is capable of transporting the nucleic acid molecule. One type of vector is an episome, i.e., a nucleic acid molecule capable of extra-chromosomal replication. Some useful vectors are those capable of autonomous replication and/or expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors”. Other useful vectors, include, but are not limited to viruses such as lentiviruses, retroviruses, adenoviruses, and phages. Vectors useful in some methods of the invention can genetically insert TARS polypeptides into dividing and non-dividing cells and can insert TARS polypeptides to cells that are in vivo, in vitro, or ex vivo cells.
Vectors useful in methods of the invention may include additional sequences including, but not limited to one or more signal sequences and/or promoter sequences, or a combination thereof. Expression vectors and methods of their use are well known in the art. In certain embodiments of the invention, a vector may be a lentivirus comprising a nucleic acid or gene that encodes a TARS polypeptide of the invention or a variant thereof. A lentivirus is a non-limiting example of a vector that may be used to create stable cell line. The term “cell line” as used herein is an established cell culture that will continue to proliferate given the appropriate medium.
Promoters that may be used in methods and vectors of the invention include, but are not limited to, cell-specific promoters or general promoters. Methods for selecting and using cell-specific promoters and general promoters are well known in the art. A non-limiting example of a general purpose promoter that allows expression of an TARS polypeptide in a wide variety of cell types—thus a promoter for a gene that is widely expressed in a variety of cell types, for example a “housekeeping gene” can be used to express a TARS polypeptide in a variety of cell types. Non-limiting examples of general promoters are provided elsewhere herein and suitable alternative promoters are well known in the art. In certain embodiments of the invention, a promoter may be an inducible promoter, examples of which include, but are not limited to tetracycline-on or tetracycline-off, etc.
Certain aspects of the invention include methods of administering antibodies or antigen-binding fragments thereof which specifically bind to a TARS polypeptide to treat an angiogenic or immune system disease or condition characterized by abnormal TARS polypeptide activity. In some embodiments of the invention such antibodies or antigen-binding fragments thereof may be administered to a cell and/or subject to inhibit TARS polypeptide activity in the cell and/or subject. The term “antigen-binding fragment” of an antibody as used herein, refers to one or more portions of an antibody that retain the ability to specifically bind to an antigen (e.g., a TARS polypeptide). One may prepare and test an antigen-binding fragment of a TARS-modulating antibody for use in methods of the invention using art-known methods and routine procedures. In some embodiments of the invention, the antibodies are recombinant antibodies, polyclonal antibodies, monoclonal antibodies, humanized antibodies or chimeric antibodies, or a mixture of these. Antibodies for use in methods of the invention may be produced and tested using art-known methods in conjunction with the disclosure herein.
Additional TARS-Activity-Inhibiting Compounds
Additional compounds that may be administered in treatment methods of the invention include small molecules or chemicals that inhibit TARS polypeptide activity. Methods of identifying and testing such small molecules and chemicals may include use of art-known library screening and testing procedures in conjunction with the teaching provided herein.
It will be understood that additional TARS-modulating compounds can be identified and used in methods of the invention. For example, candidate compounds can be can be tested for their ability to decrease TARS polypeptide activity (level and/or function) and their ability to treat an angiogenic or immune system disease or condition using assays and methods presented herein.
TARS-Activity-Inhibiting Compound Administration
TARS polypeptide modulating compounds of the invention may be administered singly or in combination with one or more additional compounds. In some embodiments, a compound of the invention may act in a synergistic manner with one or more other therapeutic agents or treatments and increase the effectiveness of the one or more therapeutic agents or activities, thus a TARS inhibitor compound may act synergistically to increase the effectiveness of one or more agents or treatments that can be administered to treat an angiogenic or immune system disease or condition.
Compositions, compounds, and methods of the invention may be enhanced by utilization in combination with other procedures for treating a angiogenic or immune system disease or condition. In some instances a treatment procedure may involve administration of another therapeutic agent or treatment such a medicament and/or a behavioral treatment, surgery, etc. Thus, in some embodiments of the invention, administration of a compound of the invention (e.g., administration of an anti-TARS antibody or functional fragment thereof, a TARS polypeptide-encoding nucleic acid, TARS polypeptide (e.g., non-functional version), or a small molecule TARS inhibitor) may be performed in conjunction with therapies for treating the angiogenic or immune system disease or condition such as surgery, etc. Treatment methods of the invention that include administration of a TARS-modulating compound can be used at any stages of pre-angiogenic or pre-immune system disease or condition or when the angiogenic or immune system disease or condition is at a later stage, including but not limited to early-stage, mid-stage, and late-stage of the angiogenic or immune system disease or condition, including all times before and after any of these stages. Methods of the invention may also be used for subjects who have previously been treated with one or more other medicaments or therapy methods that were not successful, were minimally successful, and/or are no longer successful at slowing or stopping progression of the angiogenic or immune system disease or disorder in the subject.
TARS-modulating compounds of the invention (such as compounds comprising a non-functional TARS molecule, an anti-TARS polypeptide antibody or functional fragment thereof, a small molecule TARS inhibitor, etc.) described herein can be used alone or in conjugates with other molecules such as targeting agents, labeling agents, and/or cytotoxic agents in treatment methods of the invention.
Targeting agents useful according to the methods of the invention are those that direct a compound of the invention to a specific cell type including but not limited to Human umbilical vein epithelial cells (HUVEC); prostate cancer cells; ovarian cancer cells. A targeting compound of choice will depend upon the nature of the angiogenesis-associated disease or condition. In some instances it may be desirable to target the agent to skeletal muscle, cardiac muscle, kidney, liver, brain, etc. Those of ordinary skill in the art will be aware of and able to select and use suitable targeting agents for use in methods of the invention.
Labeling agents may be used in methods of the invention to determine the location of TARS polypeptides and treatment compounds, etc.) in cells and tissues and also, may be used to assess the cell, tissue, or organelle location of treatment compounds that have been administered. Procedures for attaching and utilizing labeling agents such as enzymatic labels, dyes, radiolabels, etc. are well known in the art.
Effective Amounts for Treatments
TARS-modulating compounds of the invention, (e.g., an anti-TARS antibody or functional fragment thereof, a TARS polypeptide-encoding nucleic acid, TARS polypeptide, or a small molecule TARS inhibitor, etc.) are administered to the subject in an effective amount for treating the angiogenic or immune system disease or condition. An “effective amount for treating an angiogenic or immune system disease or condition is an amount necessary or sufficient to realize a desired biologic effect. For example, an effective amount of a compound of the invention could be that amount necessary to (i) slow or halt progression of the disease or condition; or (ii) reverse one or more symptoms of the angiogenic or immune system disease or condition. According to some aspects of the invention, an effective amount is that amount of a compound of the invention alone or in combination with another medicament or treatment, which when combined or co-administered or administered alone, results in a therapeutic response in the angiogenic or immune system disease or condition, either in the prevention or the treatment of the angiogenic or immune system disease or condition. The biological effect may be the amelioration and or absolute elimination of symptoms resulting from the angiogenesis-associated disease or condition. In another embodiment, the biological effect is the complete abrogation of the angiogenic or immune system disease or condition, as evidenced for example, by a diagnostic test that indicates the subject is free of the disease or condition.
Typically an effective amount of a compound or drug to decrease the function or level of a TARS polypeptide will be determined in clinical trials, establishing an effective dose for a test population versus a control population in a blind study. In some embodiments, an effective amount will be that results in a desired response, e.g., an amount that diminishes one or more symptoms of an angiogenic or immune system disease or condition in cells or tissues in a subject with the angiogenic or immune system disease or condition. Thus, an effective amount to treat an angiogenic or immune system disease or condition characterized by an increased TARS polypeptide activity may be the amount that when administered decreases the amount of TARS polypeptide activity in the subject to an amount that that is below the amount that would occur in the subject or tissue without the administration of the composition. In some embodiments of the invention, an effective amount of a compound or drug to decrease the function or level of a TARS polypeptide and to treat an angiogenic or immune system disease or condition is an amount that decreases the TARS polypeptide activity in a cell, tissue, or subject, and is an amount of the compound that is lower than an amount that results in an amino acid starvation response in the cell, tissue, or subject, respectively. Thus, an effective amount of a treatment compound of the invention may be an amount that does not result in an amino acid starvation response, but that reduces TARS polypeptide activity sufficiently to treat the angiogenic or immune system disease or condition associated with elevated TARS polypeptide activity.
In the case of treating an angiogenic or immune system disease or condition the desired response may be reducing or eliminating one or more symptoms of the disease or condition in the cell, tissue, and/or subject. The reduction or elimination may be temporary or may be permanent. The status of the disease or condition can be monitored using methods of determining TARS polypeptide activity or levels of nucleic acids that encode a TARS polypeptide, etc. In some aspects of the invention, a desired response to treatment of the angiogenic or immune system disease or condition also can be delaying the onset or even preventing the onset of the angiogenic or immune system disease or condition.
An effective amount of a compound that modulates (e.g., decreases) TARS polypeptide activity (also referred to herein as a pharmaceutical compound) may also be determined by assessing physiological effects of administration on a cell or subject, such as a decrease of an angiogenic or immune system disease or condition following administration. Assays suitable to determine efficacy of a pharmaceutical compound of the invention will be known to those skilled in the art and can be employed for measuring the level of the response to a treatment and an amount of a pharmaceutical compound administered to a subject can be modified based, at least in part, on such measurements. The amount of a treatment may be varied for example by increasing or decreasing the amount of a therapeutic composition, by changing the therapeutic composition administered, by changing the route of administration, by changing the dosage timing and so on. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated; the severity of the condition, the duration of the treatment, the nature of the concurrent therapy (if any), the specific route of administration, and additional factors within the knowledge and expertise of the health practitioner. For example, an effective amount may depend upon the degree to which an individual has abnormally high levels of TARS polypeptide activity.
The effective amount of a compound of the invention in the treatment of an angiogenic or immune system disease or condition or in the reduction of the risk of developing an angiogenic or immune system disease or condition may vary depending upon the specific compound used, the mode of delivery of the compound, and whether it is used alone or in combination. The effective amount for any particular application can also vary depending on such factors as the angiogenic or immune system disease or condition being treated, the particular compound being administered, the size of the subject, or the severity of the disease or condition. A skilled artisan can empirically determine the effective amount of a particular compound of the invention without necessitating undue experimentation. Combined with the teachings provided herein, by choosing among the various active compounds and weighing factors such as potency, relative bioavailability, patient body weight, severity of adverse side-effects and preferred mode of administration, an effective prophylactic or therapeutic treatment regimen can be planned which does not cause substantial toxicity and yet is entirely effective to treat the particular subject.
A pharmaceutical compound dosage may be adjusted by an individual health care provider or veterinarian, particularly in the event of any complication. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, from about 0.1 mg/kg to about 200 mg/kg, or from about 0.2 mg/kg to about 20 mg/kg, in one or more dose administrations daily, for one or more days. In some aspects of the invention, a pharmaceutical compound dosage used in treatment methods of the invention may be a dose that is below a dose that if administered to a cell, tissue, or subject, would result in an amino acid starvation response in the cell, tissue or subject, respectively. For example, in some aspects of the invention an effective dose of a treatment compound to administer to a cell, tissue, or subject may include an amount of the compound that is less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% of an amount of the compound that results in an amino acid starvation response in the treated cell, tissue, or subject.
The absolute amount will depend upon a variety of factors including a concurrent treatment, the number of doses and the individual subject parameters including age, physical condition, size and weight. These are factors well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. In some embodiments, a maximum dose can be used, that is, the highest safe dose according to sound medical judgment.
Multiple doses of compounds of the invention are also contemplated. In some instances, a compound of the invention, (e.g., an anti-TARS antibody or functional fragment thereof, a TARS polypeptide-encoding nucleic acid, TARS polypeptide, or a small molecule TARS inhibitor, etc.) can be administered at least daily, every other day, weekly, every other week, monthly, etc. Doses may be administered once per day or more than once per day, for example, 2, 3, 4, 5, or more times in one 24 hour period.
Pharmaceutical compounds of the invention may be administered alone, in combination with each other, and/or in combination with other drug therapies, or other treatment regimens that are administered to subjects with an angiogenic or immune system disease or condition. Pharmaceutical compositions used in the foregoing methods preferably are sterile and contain an effective amount of a therapeutic compound that will modulate a TARS polypeptide activity to a level sufficient to produce the desired response in a unit of weight or volume suitable for administration to a subject.
The doses of a composition to modulate the TARS polypeptide activity that is administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.
Administration Methods
A variety of administration routes for a TARS-modulating compound are available. The particular delivery mode selected will depend, of course, upon the particular condition being treated and the dosage required for therapeutic efficacy. Methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of protection without causing clinically unacceptable adverse effects. In some embodiments of the invention, a compound of the invention may be administered via an oral, enteral, mucosal, percutaneous, and/or parenteral route. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intraperitoneal, and intrasternal injection, or infusion techniques. Other routes include but are not limited to nasal (e.g., via a gastro-nasal tube), dermal, vaginal, rectal, and sublingual. Delivery routes of the invention may include intrathecal, intraventricular, or intracranial. In some embodiments of the invention, a compound of the invention may be placed within a slow release matrix and administered by placement of the matrix in the subject. In some aspects of the invention, a compound (such as an anti-TARS antibody or functional fragment thereof, a TARS polypeptide-encoding nucleic acid, TARS polypeptide, or a small molecule TARS or inhibitor, etc.) may be delivered to a subject cell using nanoparticles coated with a delivery agent that targets a specific cell or tissue. In some aspects of the invention a delivery agent may be a microbead that targets a desired cell or tissue. Examples of delivery agents are well known in the art.
Compounds of the invention may be administered in formulations, which may be administered in pharmaceutically acceptable solutions, which may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, adjuvants, and optionally other therapeutic ingredients. According to methods of the invention, the compound may be administered in a pharmaceutical composition. In general, a pharmaceutical composition comprises the compound of the invention and a pharmaceutically-acceptable carrier. Pharmaceutically-acceptable carriers are well-known to those of ordinary skill in the art. As used herein, a pharmaceutically-acceptable carrier means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients, e.g., the ability of the compound such as an anti-TARS antibody or functional fragment thereof, a TARS polypeptide-encoding nucleic acid, TARS polypeptide, or a small molecule TARS inhibitor, etc. to treat the angiogenic or immune system disease or condition.
Pharmaceutically acceptable carriers include diluents, fillers, salts, buffers, stabilizers, solubilizers and other materials that are well-known in the art. Exemplary pharmaceutically acceptable carriers are described in U.S. Pat. No. 5,211,657 and others are known by those skilled in the art. Such preparations may routinely contain salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.
Compounds of the invention may be administered directly to a tissue. In some embodiments, the tissue to which the compound is administered is a tissue in which the angiogenic or immune system disease or condition is likely to arise. Direct tissue administration may be achieved by direct injection. Compounds may be administered once, or alternatively they may be administered in a plurality of administrations. If administered multiple times, the compounds may be administered via different routes. For example, the first (or the first few) administrations may be made directly into the affected tissue while later administrations may be systemic.
The compounds, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with or without an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like. Lower doses will result from other forms of administration, such as intravenous administration. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. Multiple doses per day may be used as needed to achieve appropriate systemic or local levels of compounds.
In yet other embodiments, a delivery vehicle is a biocompatible microparticle or implant that is suitable for implantation into the mammalian recipient. Exemplary bioerodible implants that are useful in accordance with this method are described in PCT Publication No. WO 95/24929 (incorporated by reference herein), which describes a biocompatible, biodegradable polymeric matrix for containing a biological macromolecule. Such delivery means are well known in the art and can be used to achieve sustained release of a compound of the invention in a subject, and may be selected not to degrade, but rather, to release by diffusion over an extended period of time.
Both non-biodegradable and biodegradable polymeric matrices can be used to deliver the compounds of the invention to the subject. In some embodiments, a matrix may be biodegradable. Matrix polymers may be natural or synthetic polymers. A polymer can be selected based on the period of time over which release is desired, generally in the order of a few hours to a year or longer. Typically, release over a period ranging from between a few hours and three to twelve months can be used. The polymer optionally is in the form of a hydrogel that can absorb up to about 90% of its weight in water and further, optionally is cross-linked with multivalent ions or other polymers.
In general, compounds of the invention may be delivered using the bioerodible implant by way of diffusion, or by degradation of the polymeric matrix. Exemplary synthetic polymers for such use are well known in the art. Biodegradable polymers and non-biodegradable polymers can be used for delivery of compounds of the invention using art-known methods. Bioadhesive polymers such as bioerodible hydrogels (see H. S. Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26, 581-587, the teachings of which are incorporated herein) may also be used to deliver compounds of the invention for treatment. Additional suitable delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the compound, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. (See for example: U.S. Pat. Nos. 5,075,109; 4,452,775; 4,675,189; 5,736,152; 3,854,480; 5,133,974; and 5,407,686 (the teaching of each of which is incorporated herein by reference). In addition, pump-based hardware delivery systems can be used, some of which are adapted for implantation.
Use of a long-term sustained release implant may be particularly suitable for prophylactic treatment of subjects and for subjects at risk of developing a recurrent angiogenic or immune system disease or condition. Long-term release, as used herein, means that the implant is constructed and arranged to delivery therapeutic levels of the active ingredient for at least 30 days, 60 days, 90 days or longer. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.
Therapeutic formulations of compounds of the invention may be prepared for storage by mixing the compound having the desired degree of purity with optional pharmaceutically acceptable carriers, excipients or stabilizers [Remington's Pharmaceutical Sciences 21st edition, (2006)], in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counter-ions such as sodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionic surfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).
In Vivo Imaging Techniques
A molecule used in a treatment method of the invention, (e.g., a TARS polypeptide or fragment thereof, a small molecule that binds to TARS or to TARS in association with another polypeptide, etc.—(also referred to herein as “a therapeutic molecule of the invention”) may also be used for imaging purposes, for example, to detect tumor metastasis. Suitable labels that may be attached to a treatment molecule and used in methods of the invention include, but are not limited to, radioisotopes, iodine (125I, 121I), carbon (14C), sulphur (35S), tritium (3H), indium (112In), and technetium (99mTc), fluorescent labels, such as fluorescein and rhodamine, and biotin, and nano- or micro-particles.
In some embodiments a therapeutic molecule (e.g., a TARS-binding polypeptide, functional fragment thereof etc.) used for a treatment method of the invention may be labelled, or otherwise modified, to permit detection. Such labeled treatment molecules can be used for real-time in vivo imaging using sample that remains within (e.g., is not removed from) a subject or for in vitro imaging using a sample that is removed from a subject. Detectable labels that can be used in conjunction with a therapeutic molecule of the invention may be any that do not substantially interfere with the therapeutic molecule function to treat the angiogenesis-associated disease or condition, but that allow external detection. Examples of detectable labels and methods suitable for use in in vitro treatment methods of the invention are described in detail elsewhere herein. Suitable in vivo detectable labels may include those that may be detected by X-radiography, NMR or MRI. For X-radiographic techniques, suitable detectable labels include any radioisotope that emits detectable radiation but that is not overtly harmful to the patient, such as barium or cesium, for example. Suitable detectable labels for NMR and MRI generally include those with a detectable characteristic spin, such as deuterium, which may be incorporated into the antibody by suitable labeling of nutrients for the relevant hybridoma, in the case of an antibody, for example.
The size of the subject, and the imaging system used, will determine the quantity of imaging moiety needed to produce in vivo images. In the case of a radioisotope moiety, for a human subject, the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of technetium-99m. The labeled therapeutic molecule (for example, labeled antibody or antibody fragment thereof or other TARS molecule) will then preferentially accumulate at the location of sample cells that contain TARS. The labeled therapeutic molecule can then be detected using known techniques.
Assessing Treatments of TARS-Associated Diseases and Conditions
In some aspects of the invention methods are provided that include comparing a level of TARS determined or measured a sample obtained from a subject to a control value for determining the efficacy of a treatment of the invention. In addition, the effectiveness of a treatment of the invention can be assessed by measuring TARS levels in samples obtained from a subject. Thus, methods of the invention, in some aspects include, assessing the onset, progression, or regression of an angiogenic or immune system disease or condition that is characterized by increased TARS activity, by measuring TARS levels in samples obtained from or tested in the subject at two, three, four, five, or more different times, e.g., before, during and after a treatment regimen of the invention. Thus, for example, in a method that utilizes two or more samples obtained from a subject at different times, values obtained from a sample obtained at one time can be compared to values obtained at other times as a measure of the efficacy of a treatment of the invention. For example, a first level obtained from the subject may serve as a baseline level or control level for that subject, thus allowing comparison of the TARS level and the determination of change or stability of the TARS level over time and across a treatment regimen. Thus, in some aspects of the invention TARS levels may be measured after a specific course of treatment against cancer or other diseases has been initiated, with the intent of determining the efficacy of that treatment or the onset of relapse as a consequence of resistance to the treatment.
The status of the angiogenic or immune system disease or condition can be monitored using methods of determining TARS polypeptide activity or levels of nucleic acids that encode a TARS polypeptide, etc. In some aspects of the invention, a desired response to treatment of the angiogenic or immune system disease or condition also can be delaying the onset or even preventing the onset of the angiogenic or immune system disease or condition.
The invention, in some aspects, includes methods and assays (e.g. binding assays, gel electrophoresis; mass spectrometry; NMR; etc.) to determine changes in TARS level and/or activity in a subject or cell sample (e.g., cell culture) over time. This allows monitoring of TARS levels and/or activity in a subject who is to undergo a treatment for an angiogenic or immune system disease or condition and also enables to monitoring in a subject who is currently undergoing therapy for the angiogenic or immune system disease or condition. Thus, methods of the invention may be used to treat an angiogenic or immune system disease or condition (e.g., a cancer or proliferative disease or condition) in a subject and may also be used to assess the efficacy of a therapeutic treatment of the disease or condition and for assessment of the activity or level of a TARS molecule in a subject at various time points. For example, a subject's TARS level and/or activity can be determined prior to the start of a therapeutic regimen (either prophylactic or as a treatment of an angiogenic or immune system disease or condition), during the treatment regimen and/or after a treatment regimen, thus providing information on the status of the angiogenic or immune system disease or condition in the subject.
Methods of Detection
Thus, in addition to the treatment methods of the invention, in some aspects, the invention includes detection methods useful for assessing treatment methods and for identifying candidate compounds for use in treatment methods of the invention. Detection methods may permit detecting the presence of a TARS molecule in a biological sample. The methods may comprise contacting the biological sample with an agent capable of detecting TARS polypeptide or nucleic acid molecules (e.g., TARS mRNA or DNA, etc.) such that the presence of TARS is detected in the biological sample. An agent for detecting TARS mRNA using methods of the invention may be a labeled or labelable nucleic acid probe capable of hybridizing to TARS mRNA. The nucleic acid probe may be, for example, the full-length TARS cDNA of GENBANK™ Accession No. NM_152295 or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to TARS mRNA.
Detection methods useful in methods of the invention, including but not limited to those described above herein, can be used to monitor efficacy of a treatment of the invention that is administered to a subject. In some embodiments of the invention, methods include contacting a biological sample obtained from the subject (or isolate of the sample) who has undergone a treatment of the invention, with an agent capable of detecting TARS polypeptide or nucleic acid such that the presence and/or level of TARS polypeptide or nucleic acid is detected in the biological sample or isolate, thereby permitting the practitioner to assess the efficacy of the treatment. In certain embodiments, methods of the present invention may include comparing the level of TARs polypeptide or nucleic acid in a sample or isolate with the level of TARS polypeptide or nucleic acid in a control sample. A control sample may be a sample from the subject prior to, or at an earlier stage of the subject's treatment and a difference in the TARS activity in the subsequent sample can indicate whether there has been a change in the status of the disease or condition in response to the treatment of the invention. For example, if a treatment of the invention results in a decrease in TARS activity that is detected in a follow up assessment, it is an indication of the efficacy of the treatment to treat the angiogenic or immune system disease or condition. Such a determination of the efficacy of the treatment can also include a step of determining an appropriate treatment for a subject by a health-care provided based at least in part on the determination of the TARS level in a sample from the subject and the efficacy of the treatment the subject has received.
In some embodiments of the invention, a control level of TARS activity is a level determined from cells that do not have the disease or condition associated with altered TARS activity that is being tested for in the subject's sample. For example, in some embodiments, a control level of TARS is a level determined in normal cells that do not have a cancer or angiogenic or immune disease or condition that is suspected to be in the biological sample obtained from the subject. In such a case, the efficacy of the treatment of a compound or treatment of the invention can be determined based on a decrease in the TARS activity in the subject's sample as compared to the control that is free of the disease or condition.
Thus, some aspects of the invention include methods of assisting a health care provided to select a treatment to inhibit angiogenesis or the immune system in a subject in need of such treatment. In some embodiments of the invention, such methods may include obtaining a cell sample from a subject having or at risk of having an angiogenic or immune system disease or condition, determining the threonyl-tRNA synthetase (TARS)/von Hippel Lindau factor (VHL) interaction in the cell sample; comparing the determined TARS/VHL interaction to a control TARS/VHL interaction, and selecting a treatment for the angiogenic or immune system disease or condition in the subject based at least in part on the difference between the determined TARS/VHL interaction and the control TARS/VHL interaction, wherein if the determined TARS/VHL interaction is greater than the control interaction, the selected treatment is a treatment that inhibits the TARS/VHL interaction in the subject and inhibits angiogenesis and/or the immune system disease or condition in the subject. The interaction of TARS with VHL may include the formation, maintenance, or activity of a TARS/VHL complex, and decreasing the interaction of the TARS/VHL complex may include: 1) decreasing the formation of a TARS/VHL complex in the subject; 2) decreasing the activity of a TARS/VHL complex in the subject; 3) decreasing the maintenance of a TARS/VHL complex in the subject. In some embodiments decreasing the maintenance of the TARS/VHL complex activity comprises increasing disassociation of the TARS/VHL complex. A control value for a TARS/VHL interaction may be a predetermined standard TARS/VHL, which may be a normal control or a disease control. In certain embodiments of the invention, methods of selecting a treatment may include determining the level of the TARS/VHL complex in a tissue sample from a subject, comparing the level of the TARS/VHL complex with a control level of TARS/VHL complex and basing the selection at least in part on the comparison; comparing the level of HIF-1α in the subject to a control level of HIF-1α and basing the selection at least in part on the comparison; comparing the level of ubiquitination of HIF-1α in the subject to a control level of ubiquitination and basing the selection at least in part on the comparison; or comparing the level of vascular endothelial growth factor (VEGF) in the subject to a control level of VEGF and basing the selection at least in part on the comparison. A treatment of the angiogenic or immune system disease or condition may include administering to the subject an effective amount of a TARS-activity-inhibiting compound to decrease angiogenesis and/or the immune system response in the subject.
Identifying Candidate Compounds
The invention, in some aspects also includes methods to identify candidate compounds that decrease TARS activity when administered to a cell, tissue, or subject, and methods to assess the efficacy of candidate TARS-modulating compounds to decrease expression of TARS polypeptide-encoding nucleic acid or a TARS polypeptide in a cell or tissue. Such methods may be carried out in vivo in human or animal subjects; or using in vitro assays of the invention such as in cells from culture—e.g., as screening assays to assess candidate TARS-modulating compounds to modulate TARS polypeptide activity. TARS-modulating compounds that alter TARS polypeptide activity in a cell, tissue, or subject may be used in the treatment of an angiogenic or immune system disease or condition or as a pretreatment for an angiogenic or immune system disease or condition (e.g., to prepare a cell or subject for subsequent treatment).
It will be understood that a therapeutic regimen may be either prophylactic or a treatment of an angiogenic or immune system disease or condition in a subject. The invention in some aspects provides methods that may be used to monitor a subject's response to prophylactic therapy and/or treatment for an angiogenic or immune system disease or condition provided to a subject. Methods of the invention (e.g. binding assays, gel electrophoresis; mass spectrometry; NMR; etc.) may also be useful to monitor the efficacy of a treatment of the invention. TARS polypeptide levels and/or activity or TARS-encoding nucleic acid levels may be determined in two, three, four, or more biological samples obtained from a subject at separate times. The TARS polypeptide levels and/or activity or the TARS-encoding nucleic acid levels determined in the samples may be compared and changes in the levels and/or activity over time may be used to assess the status and stage of an angiogenic or immune system disease or condition in the subject (or in a cell or tissue sample) and/or the effect of a treatment strategy on the angiogenic or immune system disease or condition in a subject (or a cell or tissue sample). Some embodiments of methods of the invention can be used to assess treatments for an angiogenic or immune system disease and can be used to select a therapy for the subject, for example, to select a drug therapy, behavioral therapy, surgical therapy, etc.
Assays for assessing TARS levels in embodiments of the invention may include determining one or more TARS levels and/or activities, including but not limited to determining levels of nucleic acids that encode TARS polypeptides and/determining levels of TARS polypeptides in cells, tissues, and subjects. Levels of TARS polypeptide-encoding nucleic acids and TARS polypeptides can be determined in a number of ways when carrying out the various methods of the invention. In some embodiments of the invention, a level of a TARS polypeptide-encoding nucleic acid or TARS polypeptide is measured in relation to a control level of TARS polypeptide-encoding nucleic acid or TARS polypeptide, respectively, in a cell, tissue, or subject. One possible measurement of the level of TARS polypeptide-encoding nucleic acid or polypeptide is a measurement of an absolute level of TARS polypeptide-encoding nucleic acid or TARS polypeptide. This could be expressed, for example, in the level of TARS polypeptide-encoding nucleic acid or polypeptide per unit of cells or tissue. Another measurement of a level of TARS polypeptide-encoding nucleic acid or TARS polypeptide is a measurement of the change in the level of the TARS polypeptide-encoding nucleic acid or TARS polypeptide over time. This may be expressed in an absolute amount or may be expressed in terms of a percentage increase or decrease over time. Antibodies or antigen-binding fragments or other compounds that specifically bind a TARS polypeptide or a nucleic acid that encodes a TARS polypeptide may be used in embodiments of methods of the invention to assess TARS polypeptide and TARS polypeptide-encoding nucleic acid molecules to assess the status of an angiogenic or immune system disease or condition and/or the efficacy of treatments for an angiogenic or immune system disease or condition.
In some aspects, the invention includes methods that provide information on the efficacy of a method of the invention used to treat an angiogenic or immune system disease or condition. In certain aspects, the invention includes to assess activity and efficacy of compounds administered in treatment methods of the invention. Information about the stage or status of a disease or condition and the efficacy of a compound or treatment of the invention can be used to assist a health-care provided to select a treatment for administration to the subject or can be used by a health-care professional to adjust (e.g., increase, decrease, or stop) a treatment that is being provided to the subject.
As used herein a “subject” refers to any warm-blooded animal, such as, but not limited to a human, a non-human primate, a rodent, a dog, cat, or other animal. Thus, in addition to human medical application, some aspects of the invention include veterinary application of methods described herein. A subject may be known to have a disease or condition characterized by an altered TARS activity as compared to a control level, and thus may be a subject diagnosed with the disease or condition. In some embodiments, a subject may not have been previously or currently diagnosed with such a disease or condition, but may be considered to be at risk of for having the disease or condition, for example, a subject who may be free of a detectable disease or condition in which TARS activity is altered. In some embodiments of the invention, a subject may have previously been diagnosed with a disease, for example diagnosed with a cancer or other angiogenic disease or condition, or immune system disease or condition but the subject may be in remission at the time a treatment is performed using methods of the invention.
In some embodiments of the invention, a biological sample comprises a cell or tissue or extracellular material from a subject. In some embodiments a sample is a tumor sample. A tissue sample or tumor sample may comprise tissue or a suspension of cells. A tissue section, for example, a freeze-dried, paraffin embedded, or fresh frozen section of tissue removed from a subject, or a section of a tumor biopsy can be used as the biological sample. Moreover, a biological sample may be a biological fluid obtained from a subject (e.g., blood, Aqueous humour and vitreous humour, bile, blood, serum, breast milk, cerebrospinal fluid, lymph, female or male ejaculate, gastric fluid, mucus, peritoneal fluid, plural fluid, saliva, sebum, semen, sweat, tears, vaginal secretion, urine, ascites, spinal fluid, etc.).
In some aspects of the invention, a biological sample includes one or more pre-vascular cells, angioblasts, vascular cells, immune cells, including T cells; fibroblasts; neuronal cells, glial cells, cells of the lymphatic system, tumor cells, stem cells, progenitor cells, and inflammatory cells. In certain embodiments of the invention, a biological sample that includes vascular cells includes endothelial cells, adventitial cells, pericytes and/or smooth muscle cells. Biological samples for use in methods of the invention (e.g., for assessing a treatment, etc.) may be obtained from any number of sources. A sample obtained directly from a cancer or tumor, such as the stroma or cytosol, etc.
In some embodiments of the invention, a biological sample in which a TARS molecule is to be detected is a prostate or ovarian tissue and/or tumor sample. In certain embodiments of the invention, a biological sample in which TARS mRNA or TARS polypeptide is to be detected is, for example, a prostate, colon, cervical, ovarian, or other tumor. In some aspects of the invention, a biological sample may comprise TARS that has been secreted from the cell in which it was produced. In certain aspects of the invention, a biological sample may comprise a TARS molecule that is a non-secreted molecule, which as used herein, is a TARS molecule that was produced in a cell but not secreted by that cell into the extracellular environment.
As used herein, the term “isolated”, when used in the context of a biological sample, is intended to indicate that the biological sample has been removed from a subject. In some embodiments of the invention, a biological sample comprises a sample that has been isolated from a subject and is subjected to a method of the present invention without further processing or manipulation subsequent to its isolation. In some embodiments of the invention, a biological sample can be processed or manipulated subsequent to being isolated and prior to being subjected to a method of the invention. For example, a sample can be refrigerated (e.g., stored at 4° C.), frozen (e.g., stored at −20° C., stored at −135° C., frozen in liquid nitrogen, or cryopreserved using any one of many standard cryopreservation techniques known in the art). Furthermore, a sample can be purified subsequent to isolation from a subject and prior to subjecting it to a method of the present invention.
As used herein, the term “purified” when used in the context of a biological sample, is intended to indicate that at least one component of the isolated biological sample has been removed from the biological sample such that fewer components, and consequently, purer components, remain following purification. For example, a serum sample can be separated into one or more components using centrifugation techniques known in the art to obtain partially-purified sample preparation. Furthermore, it is possible to purify a biological sample such that substantially only one component remains. For example, a tissue or tumor sample can be purified such that substantially only the polypeptide or mRNA component of the biological sample remains.
Furthermore, it may be desirable to amplify a component of a biological sample such that detection of the component is facilitated. For example, the mRNA component of a biological sample can be amplified (e.g., by RT-PCR) such that detection of TARS mRNA is facilitated. As used herein, the term “RT-PCR” (an abbreviation for reverse transcriptase-polymerase chain reaction) involves subjecting mRNA to the reverse transcriptase enzyme results in the production of cDNA which is complementary to the base sequences of the mRNA. Large amounts of selected cDNA can then be produced by means of the polymerase chain reaction which relies on the action of heat-stable DNA polymerase for its amplification action. Alternative amplification methods include: self-sustained sequence replication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA 87: 1874-1878), transcriptional amplification system (Kwoh, D. Y. et al., 1989, Proc. Natl. Acad. Sci. USA 86: 1173-1177), Q-Beta Replicase (Lizardi, P. M. et all, 1988, Bio/Technology 6: 1197), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
TARS Detection Techniques
Some aspects of the invention include detection methods useful to assess treatments of the invention. In addition, certain methods of the invention may include detection methods to identify a candidate therapeutic compound and/or may to assess the efficacy of a therapeutic compound. For example, the efficacy of compounds and treatments of the invention to decrease TARS activity can be assessed by detecting and measuring TARS molecules (for example, TARS polypeptides and nucleic acids that encode TARS polypeptides). For such methods of the invention TARS molecules can be detected and measured using any suitable means known in the art. In some embodiments of the invention, a detection or measurement means for TARS molecules includes an immunological assay, nucleotide determination (mRNA or DNA), mass spectrometry assessment, TARS aminoacylation, GTPase, or Ap4A synthesis assay, TARS active site determination assay, or a TARS binding assay that may include a TARS-binding reporter molecule. Examples of immunological assays suitable for use to assess treatment methods of the invention may include, but are not limited to ELISA assays, assays that utilize an anti-TARS antibody (or FV derivative) to which is conjugated a detectable label, (examples of which include but are not limited to a radiolabel, non-limiting examples of which are technicium and indium). In some aspects of the invention, TARS levels may be measured in complex mixtures using an amino acid (threonine) activation assay, aminoacylation assay, or binding of a threonine specific tRNA, or a nucleic acid aptamer designed and selected to bind to threonyl-tRNA synthetase.
In some embodiments of the invention, levels of a TARS polypeptide may be detected in complex protein mixtures using mass spectrometry methods, which may include a TARS-specific peptide as an internal standard to allow quantitation. Methods of measuring levels of nucleic acids encoding TARS (i.e. TARS mRNA) may include, but are not limited to, real-time polymerase chain reaction (qRT-PCR), DNA array, and next generation sequencing methods.
The present invention features agents that are capable of detecting and/or quantitating a TARS polypeptide or a TARS-encoding nucleic acid such that the presence and/or level of TARS are determined. As defined herein, an “agent” refers to a substance that is capable of identifying or detecting TARS in a biological sample (e.g., identifies or detects TARS mRNA, TARS DNA, TARS polypeptide, TARS activity, etc.). In some embodiments of the invention, the agent is a labeled or a labelable antibody or molecule (e.g., a binding partner) that specifically binds to a TARS polypeptide. It will be understood that as used herein, the term “polypeptide” is used in reference to an amino acid sequence of a full-length TARS protein or a portion of a TARS protein. As used herein, the terms “labeled” or “labelable” refers to the attaching or including of a label (e.g., a marker or indicator) or ability to attach or include a label (e.g., a marker or indicator). Markers or indicators useful in methods of the invention may include, but are not limited to, for example, radioactive molecules, colorimetric molecules, and enzymatic molecules that produce detectable changes in a substrate.
In some embodiments of the invention, an agent is an antibody that specifically binds to all or a portion of a TARS polypeptide. As used herein, the phrase “specifically binds” refers to binding of, for example, an antibody to an epitope or antigen or antigenic determinant in such a manner that binding can be displaced or competed with a second preparation of identical or similar epitope, antigen or antigenic determinant. In an exemplary embodiment, the agent is an antibody that specifically binds to all or a portion of the human TARS polypeptide. In some embodiments of the invention, an ELISA is used in conjunction with the antibody to determine the presence and/or level of TARS polypeptide in a biological sample. Methods of the invention for detecting the presence and/or quantity of a TARS molecule may also include procedures such as an immunological assay, a polymerase chain reaction, real-time polymerase chain reaction (qRT-PCR), mass spectrometry, a TARS aminoacylation assay, TARS active site determination assay, or a TARS binding assay comprising a TARS-binding reporter molecule. In addition, embodiments of the invention include may include nucleic “aptamers”, i.e. nucleic acids (DNA, RNA or peptide nucleic acids [PNAs]) that possess high affinity for TARS derived polypeptides and can be readily labeled for high throughput binding assays. Aptamers can be produced by standard molecular biological techniques by those skilled in the art by repeated rounds of binding, selection, and affinity, and amplification (Hamaguchi, et al. Anal. Biochem. (2001) 294; pt 2, pages 126-131).
In some embodiments of the invention an agent is a labeled or labelable nucleic acid probe capable of hybridizing to a TARS nucleic acid, (e.g., a TARS RNA or DNA). For example, the agent can be an oligonucleotide primer for the polymerase chain reaction that flanks or lies within the nucleotide sequence encoding human TARS. In some embodiments of the invention, the biological sample being tested is an isolate, for example, RNA. In yet another embodiment, the isolate (e.g., the RNA) is subjected to an amplification process that results in amplification of TARS nucleic acid. As defined herein, an “amplification process” is designed to strengthen, increase, or augment a molecule within the isolate. For example, where the isolate is mRNA, an amplification process such as RT-PCR can be utilized to amplify the mRNA, such that a signal is detectable or detection is enhanced. Such an amplification process is beneficial particularly when the biological, tissue, or tumor sample is of a small size or volume.
TARS Nucleic Acid Binding Agents
Types of binding agents that can be used in treatments of the invention include, but are not limited to cDNA, riboprobes, RNAi compounds, and synthetic oligonucleotides, etc. The type of binding agent used in a treatment of the invention or to assess a treatment or treatment compound of the invention will generally be dictated by the particular situation, such as riboprobes for in situ hybridization, and cDNA for Northern blotting, antisense probe for binding, for example. Methods of the invention, in some embodiments, include identifying a candidate therapeutic compound and/or include assessing the efficacy of a therapeutic compound. In some embodiments of the invention a binding agent or probe can be directed to nucleotide regions unique to the polypeptide. Detection of the TARS-encoding gene, per se, may be useful for treatment methods of the invention and for assessing treatment methods and treatment compounds of the invention. Other forms of assays to detect TARS activity to determine efficacy of compounds of the invention that reduce activity of TARS transcripts and other expression products—will generally be useful as well. An RNA binding agent that is useful in a treatment of the invention may be as short as is required to differentially recognize TARS mRNA transcripts, and may be as short as, for example, 15 bases; however, agents of at least 17 bases, 18 bases, 19, bases, 20 bases, or more may be used.
An RNA or cDNA binding agent useful in methods of the invention may be reverse-engineered by one skilled in the art, for example using the amino acid sequence of GENBANK™ Accession No.: NM_152295. However use of such agents may be more limited than the native DNA sequence, as it will be appreciated that any one given reverse-engineered sequence will not necessarily hybridize well, or at all, with any given complementary sequence reverse-engineered from the same peptide, owing to the degeneracy of the genetic code. This is a factor common in the calculations of those skilled in the art, and the degeneracy of any given sequence is frequently so broad as to yield a large number of probes for any one sequence.
The form of labeling of a binding agent or probe used in an embodiment of the invention may be any that is appropriate, such as the use of radioisotopes, for example, 32P and 35S, etc. Labeling with radioisotopes may be achieved, whether the agent or probe is synthesized chemically or biologically, by the use of suitably labeled bases using methods well known in the art.
TARS RNA Detection Techniques
To identify a candidate therapeutic compound or to assess the efficacy of a therapeutic compound for treatment of an angiogenic or immune system disease or condition, RNA transcripts may be detected using art known methods. For example, Northern blotting, can be performed in which a preparation of RNA is run on a denaturing agarose gel, and transferred to a suitable support, such as activated cellulose, nitrocellulose or glass or nylon membranes. Radiolabeled cDNA or RNA is then hybridized to the preparation, washed and analyzed by autoradiography.
Detection of RNA transcripts can further be accomplished using known amplification methods. For example, it is within the scope of the present invention to reverse transcribe mRNA into cDNA followed by real-time polymerase chain reaction (RT-PCR); or, to use a single enzyme for both steps as described in U.S. Pat. No. 5,322,770, or reverse transcribe mRNA into cDNA followed by symmetric gap ligase chain reaction (RT-AGLCR). Each of these methods is well known and routinely used in the art. Other known amplification methods can also be utilized in methods of the invention, including, but not limited to:
In situ hybridization visualization may also be employed, wherein a radioactively labeled antisense RNA probe is hybridized with a thin section of a biopsy sample, washed, cleaved with RNase and exposed to a sensitive emulsion for autoradiography. Biological samples may be stained with haematoxylin to demonstrate the histological composition of the sample, and dark field imaging with a suitable light filter shows the developed emulsion. Non-radioactive labels such as digoxigenin, etc. may also be used.
TARS Antibodies and Additional Binding Agents
It will be appreciated that antibodies for use in accordance with treatment methods of the present invention, or to assess candidate compounds for use in treatment methods, or to assess efficacy of a treatment of the invention, may be monoclonal or polyclonal as appropriate. Antibody equivalents of these may comprise: the Fab′ fragments of the antibodies, such as Fab, Fab′, F(ab′)2 and Fv; idiotopes; or the results of allotope grafting (where the recognition region of an animal antibody is grafted into the appropriate region of a human antibody to avoid an immune response in the patient), for example. Single chain antibodies may also be used. Other suitable modifications and/or agents will be apparent to those skilled in the art. Chimeric and humanized antibodies are also within the scope of the invention and a variety of approaches for making chimeric antibodies are known in the art. Additionally, a chimeric antibody can be further “humanized” such that parts of the variable regions, especially the conserved framework regions of the antigen-binding domain, are of human origin and only the hypervariable regions are of non-human origin. Such altered immunoglobulin molecules may be made by any of several techniques known in the art.
In addition to using antibodies to bind to a TARS molecule in a treatment method of the invention, antibodies that bind to TARS polypeptide may also be used to assess efficacy of a TARS treatment of the invention, e.g., to quantify TARS in a treated subject, etc. Treatment methods of the invention may include administration of other molecules or compounds that bind to a TARS molecule to reduce the TARS activity in a subject. For example, it may be possible to identify antagonists, compounds, and/or molecules such as polypeptides, chemicals, or other small molecules that specifically bind to a TARS molecule and reduce its activity. In addition, it may also be possible to use an antibody or other compound or molecule that binds to, and permits detection of a TARS molecule in a biological sample to assess a treatment of the invention and to identify candidate compounds useful in a treatment of the invention. In some embodiments a TARS molecule will detected as part of a complex with one or more additional polypeptides. One non-limiting example of a binding molecule that may be useful in treatment methods of the invention is a von Hippel Lindau (VHL) polypeptide. VHL polypeptides bind to and form a complex with TARS, and in some embodiments of the invention may be used to detect the presence and/or to quantify a TARS molecule in a biological sample to assess a treatment of the invention or to identify a candidate therapeutic agent.
An isolated TARS polypeptide, or fragment thereof, can be used as an immunogen to generate antibodies that bind TARS (e.g., candidate compounds for use in a treatment of the invention) using standard techniques for polyclonal and monoclonal antibody preparation. The full-length TARS polypeptide can be used or, alternatively, the invention provides antigenic peptide fragments of TARS for use as immunogens. The antigenic peptide of TARS may comprise at least 8 amino acid residues of the amino acid sequence shown in GENBANK™ Accession No.: NM_152295 and encompasses an epitope of TARS such that an antibody raised against the peptide forms a specific immune complex with TARS. Polypeptides that may be used as immunogens include but are not limited to the sequence set forth as SEQ ID NO:7 RAELNPWPEYIYTRLEMYNILKAEHDSILAEKAEKDSKPIKVTLPDGKQVDAESW KTTPYQIACGISQGLADNTVIAKVNNVVWDLDRPLEEDCTLELLK, which is a portion of the sequence set forth in GENBANK™ Accession No.: NM_152295. In some aspects of the invention, an antigenic peptide may comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more residues. Antigenic polypeptides comprising at least 50, 100, 150, 200 or 250 amino acid residues are also within the scope of the present invention. Preferred epitopes encompassed by the antigenic peptide are regions of TARS that are located on the surface of the polypeptide, e.g., hydrophilic regions. Antibodies that bind to TARS can be tested using routine methods to assess whether they are candidate compounds that may be administered to a subject to reduce TARS activity in a cell, tissue, or subject in a treatment method of the invention.
A TARS immunogen typically may be used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen. An appropriate immunogenic preparation can contain, for examples, recombinantly expressed TARS polypeptide or a chemically synthesized TARS polypeptide. The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic TARS preparation induces a polyclonal anti-TARS antibody response. The immunogen may further include a portion of non-TARS polypeptide, for example, a polypeptide useful to facilitate purification.
Accordingly, another aspect of the invention pertains to the use of anti-TARS antibodies to administer to a subject in an amount suitable to reduce the level and/or activity of TARS polypeptides and to treat an angiogenic or immune system disease or condition. The term “antibody” as used herein refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as TARS. The invention may include use of polyclonal and monoclonal antibodies that bind TARS. The term “monoclonal antibody” or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of TARS. A monoclonal antibody composition thus typically displays a single binding affinity for a particular TARS polypeptide with which it immunoreacts.
Polyclonal antibodies generated by the above or another technique may be used directly, or suitable antibody producing cells may be isolated from the animal and used to form a hybridoma by known means [Kohler and Milstein, Nature 256:795. (1975)]. Selection of an appropriate hybridoma will also be apparent to those skilled in the art, and the resulting antibody may be used in a suitable assay to identify and/or quantify a TARS molecule.
TARS Protein Detection Techniques
Methods of the invention may include the use of TARS binding molecules (e.g., antibodies, antibody equivalents, small molecules, etc.) to detect TARS polypeptides to permit identification of candidate therapeutic compounds and/or for assessment of the efficacy of a therapeutic compound of the invention. Thus, TARS binding molecules can be used to measure whether the activity of a TARS molecule in a subject or cell is altered by contact with a candidate compound or by a treatment regimen of the invention. Methods for the detection of polypeptides are well known to those skilled in the art, and include ELISA (enzyme linked immunosorbent assay), RIA (radioimmunoassay), Western blotting, and immunohistochemistry. Methods for immunoassays are routinely used and are well known in the art.
ELISA and RIA procedures may be conducted such that a TARS standard is labeled (with a radioisotope such as 125I or 35S, or an assayable enzyme, such as horseradish peroxidase or alkaline phosphatase), and, together with the unlabelled sample, brought into contact with the corresponding antibody, whereon a second antibody is used to bind the first, and radioactivity or the immobilized enzyme assayed (competitive assay). Alternatively, TARS in the sample is allowed to react with the corresponding immobilized antibody, radioisotope- or enzyme-labeled anti-TARS antibody is allowed to react with the system, and radioactivity or the enzyme assayed (ELISA-sandwich assay). Other conventional methods may also be employed as suitable.
The above techniques may be conducted essentially as a “one-step” or “two-step” assay. A “one-step” assay may involve contacting antigen with immobilized antibody and, without washing, contacting the mixture with labeled antibody. A “two-step” assay may involve washing before contacting the mixture with labeled antibody. Other conventional methods may also be employed as suitable.
Enzymatic and radiolabeling of a detection agent (e.g., antibodies, binding molecules, etc.) may be carried out by conventional means. Such means will generally include covalent linking of the enzyme to the detection agent, such as by glutaraldehyde, specifically so as not to adversely affect the activity of the enzyme, by which is meant that the enzyme must still be capable of interacting with its substrate, although it is not necessary for all of the enzyme to be active, provided that enough remains active to permit the assay to be effected. Indeed, some techniques for binding enzyme are non-specific (such as using formaldehyde), and will only yield a proportion of active enzyme.
It is usually desirable to immobilize one component of an assay system on a support, thereby allowing other components of the system to be brought into contact with the component and readily removed without laborious and time-consuming labor. It is possible for a second phase to be immobilized away from the first, but one phase may be sufficient.
Enzymes employable for labeling are not particularly limited, but may be selected from the members of the oxidase group, for example. These catalyze production of hydrogen peroxide by reaction with their substrates, and glucose oxidase is often used for its good stability, ease of availability and cheapness, as well as the ready availability of its substrate (glucose). Activity of the oxidase may be assayed by measuring the concentration of hydrogen peroxide formed after reaction of the enzyme-labeled detection agent with the substrate under controlled conditions well-known in the art.
Other techniques may be used to detect TARS molecules according to a practitioner's preference based upon the present disclosure. One such technique is Western blotting (Towbin et at., Proc. Nat. Acad. Sci. 76:4350 (1979)), wherein a suitably treated sample is run on an SDS-PAGE gel before being transferred to a solid support, such as a nitrocellulose filter. Anti-TARS antibodies (unlabeled) are then brought into contact with the support and assayed by a secondary immunological reagent, such as labeled protein A or anti-immunoglobulin (suitable labels including but not limited to 125I, horseradish peroxidase and alkaline phosphatase). Chromatographic detection may also be used.
Immunohistochemistry may be used to detect expression of human TARS in a biopsy sample. A suitable antibody is brought into contact with, for example, a thin layer of cells, washed, and then contacted with a second, labeled antibody. Labeling may be by fluorescent markers, enzymes, such as peroxidase, avidin, or radiolabelling. The assay may be scored visually, using microscopy, or using any other suitable methods. Using detection methods one can determine efficacy of a treatment of the invention and can identify additional candidate agents for use in treatments of the invention.
The following examples are provided to illustrate specific instances of the practice of the present invention and are not intended to limit the scope of the invention. As will be apparent to one of ordinary skill in the art, the present invention will find application in a variety of compositions and methods.
Threonyl tRNA Synthetase (TARS) is an Angiogenic Chemokine Secreted by Endothelial Cells in Response to VEGF
Cell Culture, Reagents and Antibodies—
Human umbilical vein endothelial cells (HUVEC) (a gift from C. Holmes, University of Vermont) were grown in Clonetics® EGM®-2 complete media (Lonza, Annandale, N.J.). Borrelidin analog BC194 was a gift from Dr. Barrie Wilkinson, (Biotica). Purified basic-fibroblast growth factor (bFGF) was a gift from J. Spees, Univ. of Vermont. Retinoic acid and cycloheximide were purchased from Sigma-Aldrich, and VEGF and TNF-α were purchased from Cell Signaling Technology, Danvers, Mass. and Calbiochem, San Diego, Calif., respectively.
Western Blot—
After treatments, cells were harvested into sample buffer containing: 0.2 M Tris-HCL, 4% SDS, 4% β-mercaptoethanol, 40% glycerol, 4 μM pyronin Y. Extracts were sheared through a 24-gauge syringe. Samples were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane and probed with specific antibody as described (Lounsbury, Beddow et al. 1994). Primary antibodies are as follows: Rabbit monoclonal anti-P-eIF2α (1:1000; Cell Signaling Technology, Danvers, Mass.), rabbit monoclonal anti-Cleaved Caspase-3 (1:1000; Cell Signaling Technology, Danvers, Mass.), rabbit polyclonal anti-TARS (1:500; Santa Cruz Biotechnology, Santa Cruz, Calif.). Loading control antibodies were rabbit monoclonal anti-β-actin and anti-β-tubulin (1:1000; Cell Signaling Technology, Danvers, Mass.). Secondary antibodies were HRP-goat-anti-mouse and HRP-goat-anti-rabbit (1:5,000; Jackson Laboratories, Bar Harbor, Me.).
In Vitro Tube Formation Assay—
Tube formation assays were performed as described (Arnaoutova and Kleinman 2010; Cassavaugh, Hale et al. 2011). Human Umbilical Vein Endothelial Cells (HUVECs) were seeded in 48-well plates (1.5×104 cells/well) coated with 100 μl of Matrigel™ Basement Membrane Matrix Growth Factor Reduced (BD Biosciences, San Jose, Calif.) and incubated in Clonetics® EGM®-2 complete media (Lonza, Annandale, N.J.) or EGM®-2 with reduced serum (0.2% fetal bovine serum). Cells were incubated at 37° C. for 6 h then fixed in 10% formalin. Fixed samples were imaged by phase-contrast microscopy or stained with Oregon Green 488 Phalloidin (Molecular Probes, Eugene, Oreg.) then imaged with fluorescence microscopy (2× objective). Number of tubes and tube lengths (in pixels) were quantified using the Simple Neurite Tracer (Longair, Baker et al. 2011) plug-in on ImageJ software (NIH). Statistical analysis of one-way ANOVA was performed with GraphPad Software. Multiple comparisons were performed using the Tukey Test.
Cell Viability—
Cell viability was measured by counting cells in a hemacytometer with Trypan Blue exclusion (Sigma-Aldrich, St. Louis, Mo.) according to manufacturer's instructions. Measurements were normalized to untreated cells.
Nascent Protein Synthesis Assay—
Nascent protein synthesis was measured using Invitrogen Click-iT® metabolic labeling reagents [Dieterich, D. C., et al., Nat Protoc 2, 532-40 (2007)]. HUVEC cultures were pre-incubated in methionine-free Dulbecco's Modified Eagle Medium (D-MEM) high glucose (Invitrogen, Life Technologies, Grand Island, N.Y.) supplemented with 10% dialyzed fetal bovine serum (Invitrogen) containing the control or test compounds. Cycloheximide (50 μM) was used as a positive control. After 45 minutes, 25 μM Click-iT® AHA (L-azidohomoalanine) (Invitrogen) was added and cultures were incubated for 3 h. Cells were lysed with 1% SDS in 50 mM Tris-HCl with protease and phosphatase inhibitors: 1 mM phenyl-methylsulfonamide, 20 mg/ml aprotinin, and 4 mg/ml leupeptin. Extracts were sonicated and protein concentration was determined by Bradford assay. Protein samples were labeled with biotin alkyne (PEG4 carboxamide-propargyl biotin) (Invitrogen) using the Click-iT® Protein Reaction Buffer Kit (Invitrogen) according to manufacturer's instructions. Equal concentrations of protein were run on a 10% SDS-PAGE and transferred to nitrocellulose membrane, incubated with streptavidin-HRP reagent (Pierce Thermo Scientific, Rockford, Ill.) followed by reaction with ECL reagent (Pierce) and exposed on film.
Expression and Purification of Human Aminoacyl tRNA Synthetases—
N-terminal His6-tagged human TARS (ThRS) was expressed and purified from E. coli Rossetta™ 2(DE3)pLysS competent cells (EMD Millipore, Billerica, Mass.) transformed with derivatives of plasmid pET28a hctThrRS. Transformant cultures were grown in terrific broth supplemented with 100 mg/ml kanamycin and 100 mg/ml chloramphenicol at 37° C. to a cell density of A600=0.6. Expression of TARS was induced with 1 mM isopropyl 1-thio-β-D-galactoside overnight at 15° C. The bacterial pellet was lysed by sonication in buffer A (20 mM potassium phosphate buffer pH 8.0, 100 mM KCl, 35 mM imidazole, and 5 mM β-mercaptoethanol) and cleared by centrifugation at 17050×g for 30 minutes. Nucleic acids were precipitated by the addition of protamine sulfate to a final concentration of 0.3% followed be centrifugation. The supernatant was loaded onto a HisTrap™ FF column (GE Healthcare, Pittsburgh, Pa.) in buffer A and eluted by an imidazole gradient of 35-250 mM in buffer A over 20 column volumes. TARS containing fractions were identified by SDS-PAGE and GelCode™ Blue (Thermo Scientific, Rockford, Ill.), pooled, and dialyzed into buffer B (100 mM potassium phosphate buffer pH 6.8 and 5 mM β-mercaptoethanol). The sample was loaded onto a CHT-Tricorn Hydroxyapatite column and eluted over 20 column volumes by using a gradient of buffer B to buffer C (500 mM potassium phosphate pH 8.0 and 5 mM β-mercaptoethanol). TARS-containing fractions were determined by SDS-PAGE. Buffer B (10 mM HEPES pH 8.0, 100 mM KCl, 2.5 mM β-mercaptoethanol, and 40% glycerol), and stored at −20° C. TARS-containing fractions were pooled and dialyzed into buffer D (10 mM HEPES pH 8.0, 100 mM KCl, 2.5 mM β-mercaptoethanol, and 40% glycerol), and stored at −20° C. Protein concentration was determined by Abs260. The protein purity and stability were evaluated by Coomassie stain following SDS-PAGE, and concentration of active sites was determined using a steady state aminoacylation assay (
The L567V mutant was derived from the wildtype TARS plasmid using Quikchange II Site-Directed Mutagenesis (Stratagene, Cedar Creek, Tex. [Agilent Technologies Inc. Santa Clara, Calif.]) with the forward primer 5′ cac cag tgt gca acc atc cag ctg gat ttc cag gtg ccc atc aga ttt aat c 3′ (SEQ ID NO:8) and its reverse compliment [5′-gat taa atc tga tgg gcc act gga aat cca get gga tgg ttg cac act ggt g-3′ (SEQ ID NO:9)] and transformed into XL1-Blue cells. Colonies positive for the mutation were isolated, grown in LB media, and the plasmid purified via Qiagen miniprep kit. The plasmid was then transformed into Rosetta II cells for use in protein expression using the same protocol as for wildtype TARS.
N-terminal His6-tagged human leucyl tRNA synthetase (LARS) was expressed using the plasmid pPROEX hTb-LARS and purified using a similar purification scheme to the TARS purification with minor modifications described in (Francklyn, First et al. 2008). The protein purity and stability were confirmed using SDS-PAGE and Coomassie stain (
Steady State Aminoacylation Assay—
The aminoacylation activities of the TARS constructs were determined using modifications to established procedures (Francklyn et al 2008). Briefly, reaction mixtures consisted of 20 mM Tris-HCl pH 8.0, 100 mM KCl, 10 mM MgCl2, 1 mM dithiothreitol, 2 mM ATP, 2.5 U pyrophosphatase (Roche), 80 μM threonine, 20 μM [14C]-threonine, and 5 μM of E. coli or human tRNAThr. Reactions were initiated with the addition of 0.25-0.75 μM TARS and run at 37° C. Aliquots were taken at varying time points and spotted onto Whatmann 3 MM paper filters pre-soaked in 5% trichloroacetic acid (TCA). Upon completion, the filters were washed 3 times in excess TCA, once in 95% ethanol, and dried under a heating lamp. The formation of Thr-tRNAThr was detected by scintillation counter and the activity determined by linear regression of threonyl-tRNAThr formed per active site per unit time.
LARS steady state ATPase activity was determined using the same procedure as for TARS aminoacylation with the following modifications. The reaction mixture did not include labeled threonine or tRNAThr and 1 nM [α-32P] ATP (PerkinElmer, Waltham, Mass.) was added. Reactions were incubated for 3 minutes at 37° C. and initiated with the addition of 1 μM human LARS. At various time points 5 μl aliquots were quenched in 45 μl of 500 mM sodium acetate and 0.1% sodium dodecyl sulfate. For each sample, 1 μl was spotted onto a CCM cellulose PEI F plates (EMD) and resolved via thin-layer chromatography in 0.75 M potassium phosphate buffer mobile phase. Radioactive signals were detected via phosphorimaging and AMP production overtime was quantified using Quantity One v 4.6.6 software (Bio-Rad, Hercules, Calif.).
Chick Chorioallantoic Membrane Assay—
Fertilized chicken eggs (Sunrise Farms, Catskill, N.Y.) day 1-2 post-laying were incubated in a humidified incubator at 37° C. for 72 h. Cleaned eggs were cracked and plated in a sterile 10 cm2 tissue culture-treated dish and incubated at 37° C. for another 7 days. On developmental day 10, 1 mm3 sterile gelatin sponge pieces (Surgifoam®; Johnson & Johnson Wound Management, Somerville, N.J.) were placed within the outer one-third of the membrane between large vessels. 40 μg/ml human bFGF and 2 μg/ml human VEGF were used as pro-angiogenic control compounds; 100 μg/mL retinoic acid (Sigma-Aldrich, St. Louis, Mo.) diluted in phosphate-buffered saline (PBS) was used as an angiostatic control. All compounds were applied in 10 μl to the CAM every 24 h for 72 h. Images were taken using a Leica MZ6 stereomicroscope every 24 h. Compounds were scored according to a modified version of Intensity Scoring as previously described (Ribatti, Nico et al. 2006). Briefly, each experimental condition was given a blinded score from 0-5 based on the change in the extent of vessel convergence and formation in proximity to the sponge from day 0 to day 3. Total score is averaged individual experimental condition scores from at least 15 replicates.
ELISA and Lactate Dehydrogenase Assays—
Confluent HUVEC cultures (passage 4) were incubated at 37° C. in Clonetics® EGM®-2 modified with 0.2% fetal bovine serum with the addition of 50 ng/ml human VEGF or 50 ng/ml human TNF-α for 6 h as indicated. Culture media supernatants were tested for levels of secreted TARS protein using the Threonyl tRNA Synthetase (TARS) ELISA Kit (USCN Life Science, Wuhan, Hubei, PRC) according to manufacturer's instructions. Cell membrane integrity was confirmed using the lactate dehydrogenase assay CytoTox-ONE™ Homogeneous Membrane Integrity Assay (Promega, Madison, Wis.) according to manufacturer's instructions and reported as percent cytotoxicity relative to a lysis control. Levels of secreted VEGF were measured using the Human VEGF ELISA kit (Thermo Scientific) per manufacturer's instructions.
Quantitative RT-PCR—
Total RNA was extracted from cells using the RNeasy column protocol and cDNA was generated using an Omniscript reverse transcriptase assay according to the manufacturer's instructions (Qiagen, Frederick, Md.). Primers and probes for TARS and β-microglobulin were Assays-on-Demand (Applied Biosystems, [Life Technologies, Carlsbad, Calif.]). RT-qPCR was performed using an ABI prism 7700 Sequence Detection System (Applied Biosystems). The relative quantity of mRNA level was determined using the comparative CT (ΔΔCT) method using β-microglobulin to normalize mRNA level (Cassavaugh, Hale et al. 2011).
Endothelial Cell Proliferation Assay—
The MTT-based alamarBlue® (Invitrogen) reagent was used to assess cell proliferation (Ahmed, Gogal et al. 1994). HUVECs were seeded in a 96-well dish (1×103 cells/well) and grown for 48 h in EGM®-2 media. Cells were incubated in 0.2% FBS EGM®-2 media or EGM®-2 complete media as indicated; VEGF (50 ng/ml) and media alone served as controls. After 48 h, 72 h, or 96 h in culture, 10 μl well premixed alamarBlue® (Invitrogen) was added and after 3 h at 37° C. the amount of reduced alamarBlue® was quantified by fluorescence (excitation at 530 nm, emission at 590 nm) on a microplate reader (Synergy™ HT, BioTek, Winooski, Vt.).
Transwell Migration Assay—
Migration was assessed using transwell inserts (Svensson, Kucharzewska et al. 2011). HUVEC cultures were serum-depleted overnight in Clonetics® EGM®-2 modified with 0.2% fetal bovine serum then 5×104 cells were plated in 90% EBM®-2, 10% EGM®-2 in the upper chamber of 0.2% gelatin-coated 24-well 8 μm Transwell® inserts (Corning, Tewksbury, Mass.) with 90% EBM®-2, 10% EGM®-2 media plus 50 ng/ml VEGF, 1-100 nM TARS protein, or 10 nM BC194 in the lower chamber. Cultures were incubated for 4 h, fixed in 10% formalin, and stained with 10 μg/ml DAPI solution (Roche) following removal of cells from the top layer of the chamber with a cotton swab. Migrated cells were imaged using a 4× objective on the Olympus IX70 Inverted microscope (Olympus). DAPI-stained nuclei were counted using ImageJ software.
Statistical Analysis—
Data are presented as mean±SEM, and p<0.05 is considered significant. Except where indicated, one-way ANOVA for multiple comparisons was performed on all data. A Kruskal-Wallis adjustment was used where necessary. All pairwise comparisons were assessed using the Student's t-test.
Concentration-Dependent Effects of a TARS Inhibitor Reveal a Specific Angiogenic Function for TARS.
Inhibition of TARS by BC194 has been shown previously to reduce in vitro endothelial tube formation (Wilkinson, Gregory et al. 2006); however, because TARS is a component of the protein synthesis machinery, this effect could be explained by cell toxicity through the unfolded protein response or apoptosis pathways. By using a range of BC194 concentrations, the sensitivity of HUVECs to the anti-angiogenic versus cell stress effects of BC194 was compared. As shown in
Exogenously Added TARS Stimulates Angiogenesis.
In light of the potential role for TARS in angiogenic signaling and the secreted activity of select other aminoacyl tRNA synthetases (Wakasugi and Schimmel 1999; Greenberg, King et al. 2008), the ability of purified TARS to stimulate angiogenesis was tested using the in vitro tube formation assay. Human His-tagged TARS was expressed in E. coli and purified by nickel chromatography followed by sequential column chromatography to produce an active and pure preparation (
To confirm and expand these results, a chorioallantoic membrane (CAM) assay was used to examine a role for TARS in an in vivo angiogenesis environment. Daily application of BC194 to a gel sponge on the CAM over 4 days inhibited vessel formation at both the basal level and after stimulation with either bFGF or VEGF (
TARS is Secreted in Response to VEGF and TNF-α.
Although TARS exerts significant pro-angiogenic effects, there was no prior evidence that TARS is physiologically present in the extracellular space except as a result of cell lysis. To explore the possibility that TARS is actively secreted, endothelial cells were treated with VEGF or TNF-α followed by measurement of TARS in the media using ELISA. As shown in
TARS Stimulates Endothelial Cell Migration.
An increase in angiogenesis by TARS signaling to endothelial cells could have resulted through either an increase in cell proliferation or an increase in cell migration. Unlike VEGF, TARS did not exert a significant effect on cell proliferation, and BC194 did not significantly reduce the VEGF proliferative response (
TARS may be playing a substantial role in normal and pathogenic angiogenesis as a proangiogenic chemokine activated by endothelial cells in response to VEGF or TNF-α stimulation (
Database Assessment of TARS in Disease
Database analysis was used to assess TARS expression in cancers including Cancer Gene Anatomy Project (CGAP)(Strausberg 2001), GEO database, and Human Protein Atlas (Uhlen, Oksvold et al.). Using the CGAP database, TARS mRNA was found to be over-expressed in cells derived from prostate carcinoma, colon adenocarcinoma, ovarian carcinoma, and in certain stem cell lines. Furthermore, whereas other synthetases were found to be relatively unchanged, TARS protein was found to be selectively upregulated in ovarian tumors from tissue arrays displayed in the Human Protein Atlas. (proteinatlas.org/ENSG00000113407).
A preliminary investigation using GEO data from a prostate cancer progression study by Tomlins et al. (Tomlins, Mehra et al. 2007) revealed that mRNA levels of TARS exhibited a 2.9 fold increase in prostate carcinoma versus normal (p<0.0001). To expand on these findings, GEO dataset GSE6919, 171 sample CEL files (scanned chip image files) were downloaded. GSE6919 is a GEO SuperSeries that includes GSE6604 (normal prostate tissue from 18 patients), GSE6605 (metastatic prostate tumor included 25 samples from 4 patients and 9 sites, some paired), and paired sets GSE6606 (primary prostate tumor from 65 patients), and GSE6608 (normal prostate tissue adjacent to tumor from 63 of those patients). Probe-level intensities were background-corrected, normalized, and summarized, and Robust Multichip Average (RMA) statistics are calculated for each probe set and sample as is implemented in Partek Genomic Suites, version 6.6 Beta (Copyright 2009, Partek Inc., St. Louis, Mo., USA). Sample quality was assessed based on the 3′:5′ ratio, relative log expression (RLE), and normalized unscaled standard error (NUSE). Principal Component Analysis (PCA) was also used to look for outlier samples that would potentially introduce latent variation into the analysis of differential expression across sample groups. Based on these analyses, 13 samples were eliminated from further analysis. Additional analysis included assessment of GEO datasets. Results of the analysis, which included a comparison of samples of normal and metastatic prostate cancer tumors, indicated that TARS mRNA was found to be significantly elevated. These findings were selective for TARS in that other aminoacyl tRNA synthetases were not elevated in prostate cancer.
Analysis of TARS Expression in Prostate Cancer Patients
Patient Selection for IHC Studies—
Using an IRB protocol (CHRMS #:08-218) approved by UVM Committee on Human Subjects, FAHC patient registries were searched to identify patients with high grade PCa from 2008-2010 for whom archived tissue samples were available. The search was confined to those patients for whom a clinical record was available, and who had all undergone prostatectomies, and for whom there were clinical samples available. An initial set of 54 cases with PC surgeries from 10/08 to 3/10 was collected in this way. A second group of 79 patients with high-grade disease was identified with surgeries over the interval 12/99-8/02.
Immunohistochemistry—
The immunohistochemistry procedures were conducted essentially as described (Conant, Penz, et al., 2011). Slide mounted 5 μm tissue sections cut from formalin-fixed, paraffin-embedded (FFPE) prostate carcinoma specimens were dewaxed by 3× 5 mins washes in xylene followed by rehydration through graded ethanol washes (100%, 95%, 70% and 50%; 2× 3 mins in each). After rinses in Milli-Q ultra-pure water (EMD Millipore, Billerica, Mass.), heat induced epitope retrieval (HIER) was performed by immersing the slides in Target Retrieval solution pH 6.0 (Dako North America Inc., Carpenteria, Calif.) and heating at 100° C. for 15 mins in a Decloaking Chamber™ Pro pressure cooker (Biocare Medical, Concord, Calif.). Slides were then allowed to cool in the pressure cooker unit for another 20 minutes. After 3× 5 minute rinses in TBST (25 mM Tris, 0.15M NaCl, 0.05% Tween 20), slides were immersed in 3% H2O2/TBST for 15 mins as to inactivate any endogenous peroxidase in the tissues. After 3× 5 min washes in TBST slides were immersed in protein block, serum-free ((Dako North America Inc., Carpenteria, Calif.) for 15 minutes to block non-specific protein binding sites in the tissues. Primary antibody (anti-TARS, mouse monoclonal clone 1A9, Abnova, Walnut, Calif.) at a 1:200 dilution was then applied for 30 min at room temperature. As a negative control test, IHC was also performed substituting primary antibody with a mouse monoclonal (mAb) IgG1 antibody to Aspergillus niger glucose oxidase (Dako North America Inc., Carpenteria, Calif.). After TBST washes, secondary detection was performed by incubating the slides for 30 mins RT with EnVison+ Dual Link polymer HRP (horseradish peroxidase) reagent (Dako North America Inc., Carpenteria, Calif.). Following a further series of TBTS washes, slides were incubated for ˜6 minutes with DAB+ chromogen substrate (Dako North America Inc., Carpenteria, Calif.) and then rinsed with tap water. Tissues were then counterstained with hematoxylin for ˜7 minutes, rinsed with TBST and water and the dehydrated through 50%, 70%, 95% and 100% ethanol. Finally, slides were cover-slipped with Cytoseal mountant (ThermoFisher Scientific, Waltham, Mass.) for viewing by bright-field microscopy.
Imaging Details—
Images of IHC were captured at the Micrscopy Imaging Center at the UVM College of Medicine using an Olympus BX50 light microscope, QImaging Retiga 2000R camera, and QCapture Pro Software. The final images used for standards in grading are seen in
Immunohistochemistry Scoring—
The stained slides were initially scored by a primary pathologist, and then a secondary review was provided two other expert pathologists. In the scoring procedure, the pathological grade and the intensity of TARS immunochemical staining were evaluated independently. In the pathological grading, each slide was evaluated by assigning different region of the slide to benign (non-tumor) and tumor. The total tumor area was further subdivided into Gleason primary pattern 3, 4, or 5, and a of tumor region estimated for each. (There are no Gleason scores below 2, because such patients were never subjected to prostatectomies.) The AJCC criteria were used to make these pathological assessments. The pathological assessment also included the recording and grading of HGPIN (a precursor lesion to PCa) and several benign controls, including BPH and atrophy (characterized by small, hyperchromatic nuclei with no prominent nucleoli). The experimental analysis also noted tissue staining pattern (diffuse, focal, or scattered), and any additional notes (such as tertiary Gleason grade, lymph node metastasis, or extraprostatic extension (EPE). In cases where there were uncertainties and/or ambiguities, the original H&E stained slides corresponding to each case were referenced. The TARS IHC staining intensity was graded independently, using a semiqualitative scale from 0-3 (0=negative, 1=mild, 2=moderate, and 3=strong). A set of reference slides that served to calibrate the scoring procedure is shown in
Statistical Analysis.
Univariate statistics were used to determine the significance between TARS staining and tumor type. Secondary t-tests were done to correlate TARS intensity with progression from Gleason 3 to 4 and to correlate TARS intensity with PSA and biochemical failure.
ELISA Assays. TARS ELISA was perform on neat serum samples, according the manufacturer's (CUSABIO Biotech, Wuhan, P.R. China) instructions. The serum samples from four age matched male non-cancer subjects was used as the control group.
Immunohistochemical Analysis of TARS Expression in Prostatectomy Sections.
To assess the relationship between TARS expression and prostate cancer progression, patient tumor samples were analyzed by immunohistochemistry and scored by intensity as shown in
TARS expression was also compared with 10-year outcome. When TARS staining of the various anatomical grades was examined, there was a strong relationship between Gleason 5 staining and elevated PSA at 10-years. Specifically, a one unit increase in TARS staining on the Gleason 5 portion of the slide increases the odds by a factor of 2.211 that subject will experience biochemical failure. Taken together these results suggest that TARS expression correlates with diagnosis of prostate cancer, progression of disease and likelihood of biochemical failure.
Analysis of Circulating TARS Levels in Prostate Cancer Patients.
The essential features of a useful human biomarker are that it be present in medium that can be readily obtainable in a non-invasive fashion (e.g. serum or urine), that it be readily quantifiable using a robust and repeatable assay, and that it provide useful information that reflects on subject disease state. As part of the very initial process of TARS biomarker discovery, serum samples were collected from 10 consenting subjects of the Fletcher Allan Urology Clinic. This small set included patients at various points along the prostate cancer diagnosis/treatment continuum, including immediately after diagnosis prior to treatment; under active surveillance; and under androgen deprivation therapy following prior radiation or prostatectomy surgery. Serum samples from four age and gender matched control subjects were also analyzed. All samples were measured in duplicate, and the values reported in
The mean value of the control samples was 105±19.5 pg/mL. Two patients (TARS 0012 and TARS 0013) had values higher than the controls, and the other eight patients all exhibited values lower than the controls. In three cases (TARS 0014, TARS 0016, and TARS0018) the levels of circulating TARS were undetectable, and significantly decreased levels were seen in three others (TARS0011, TARS0014, and TARS 0017). In three of the six cases where TARS levels were significantly decreased or not detectable, the patients were on androgen deprivation therapy. In one patient under androgen deprivation therapy, TARS levels were increased 50% relative to the controls. Notably, the two patients with TARS levels closest to the controls had either received no treatment or were under active surveillance. These data allow several important conclusions regarding the potential utility of TARS as a prostate cancer biomarker to be drawn. First, the TARS enzyme can be readily detectable in human serum samples by a conventional and commercially ELISA kit without any extensive modification or adaptation. Secondly, the variation in levels among different subjects is within the dynamic detection range of the kit. Thirdly, the values seen in untreated or active surveillance patients were closer to the values seen in the controls than samples derived from patients who had undergone past surgery/radiation treatments and were currently under androgen deprivation therapy. This provides initial support for the hypothesis that circulating TARS levels change in prostate cancer patients in response to treatment. It is noteworthy that the significant drop in TARS levels seen with patients under androgen deprivation suggests that TARS expression is at least partially under the control by the androgen receptor.
TARS Interacts with VHL and its Inhibition Interferes with the Ovarian Cancer Cell Response to Hypoxia.
Co-Immunoprecipitation—
Plasmids expressing biotinylatable TARS (pTARS) and myc-tagged VHL (pVHL) were transfected into HEK293 cells, and then extracts were prepared. Biotin-TARS was precipitated using streptavidin-coupled beads. Myc-VHL was precipitated using anti-myc antibodies. Precipitates were separated by SDS-PAGE, transferred and blots probed with anti-TARS antibody or anti-myc (VHL) antibody.
Western Blot—
After treatments, cells were harvested into sample buffer containing 0.2 M Tris-HCL, 4% SDS, 4% β-mercaptoethanol, 40% glycerol, 4 μM pyronin Y. Extracts were sheared through a 24-gauge syringe. Samples were separated by 10% SDS-PAGE and transferred to nitrocellulose membrane and probed with rabbit polyclonal anti-TARS (1:500; Santa Cruz Biotechnology, Dallas, Tex.) or monoclonal anti-HIF-1α (BD Transduction Laboratories, [BD Biosciences, San Jose, Calif.]) (Lounsbury, Beddow et al. 1994). Secondary antibodies were HRP-goat-anti-mouse and HRP-goat-anti-rabbit (1:5,000; Jackson Laboratories, Bar Harbor, Me.).
Mass Spectrometry.
Culture dishes were seeded with 2×106 human embryonic kidney cells (HEK293) and maintained in DMEM (Mediatech, Manassas, Va.) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, Calif.), penicillin/streptomycin (Gibco), and L-glutamine (Gibco) at 37° C. and 5% CO2 in a humidified incubator. Cells were transfected by polyethylenimine with plasmids encoding a TARS construct with C-terminal HA tag and BirA biotinylation site, BirA, and C-terminally myc-tagged VHL. Control experiments substituted an empty vector plasmid for the TARS construct. Following a 48 hour incubation, cells were lysed with 1% Triton X, 0.5% NP-40, 140 mM NaCl, 25 mM Tris-HCl pH 7.6, and 1 Complete Mini protease inhibitor tablet (Roche) per 10 ml. TARS was then “pulled-down” with streptavidin immobilized on magnetic beads (Invitrogen, Dynabeads MyOne Streptavidin) and unbound proteins were washed away with three exchanges of lysis buffer. The bound proteins were eluted from the beads through boiling and resolved on a reducing, SDS-PAGE gel. Major bands and their empty vector counterparts were detected using SilverSNAP Stain Kit II (Pierce) and excised. Fragments were trypically digested using the in gel procedure for ProteaseMAX Surfactant (Promega, Madison, Wis.) according to the manufacturers specifications. Briefly, free cysteines were alkylated by incubation with 55 mM iodoacetamide:50 mM NH4HCO3 followed by trypsin digestion (2 ng/μl) in 0.01% ProteaseMAX surfactant:50 mM NH4HCO3. Peptides were analyzed by electospray ionization (ESI) liquid chromatography mass spectrometry (LC-MS). Samples were resolved over a fused-silica microcapillary MagicC18 LC column (12 cm×100 μm i.d.) using a 5-50% acetonitrile gradient in 0.1% formic acid. Spectra were obtained using collision-induced dissociation with an LTQ linear quadrupole ion trap-Orbitrap mass spectrometer (Thermo Electro, San Jose, Calif.) and analyzed using SEQUEST (Bioworks software package, version 3.3.1; Thermo Electron, San Jose, Calif.). Acquired TARS data were compared to empty vector equivalents in order to identify non-specific interactions.
An interaction between TARS and VHL may affect hypoxia signaling. VHL is the E3 ubiquitin ligase for Hypoxia inducible factor-1α (HIF-1α), thus if TARS interferes with VHL activity, it may influence the induction of HIF-1α by hypoxia. Shown in
A large-scale study examining protein-protein interactions in human cells featured the immunoprecipitation of flag tagged bait proteins, followed by the LC-ESI/MS analysis of interacting proteins. Using the Von Hippel Lindau tumor suppressor as bait, TARS was identified as a potential binding partner. This result was confirmed in two independent approaches. In the first of these experiments, HEK cells were transfected with expression plasmids for TARS-[hemagglutinin tag]-[biotinylation recognition] and VHL-[myc-tag]. The TARS construct possessed an appended peptide tail that served as recognition site for the E. coli biotin ligase, whose gene was also transfected into cells. As shown in
In order to provide additional validation of the proposed VHL-TARS interaction, and perform the converse experiment of the original Ewing et al experiment, the proteins that associate with TARS in vivo were identified by precipitating biotinylated TARS, resolving all proteins by SDS polyacrylamide gel electrophoresis, and then subjecting isolated bands to mass spectrometry analysis. The resulting TARS binding partners that were identified are shown in
Angiogenesis Related Secondary Functions of Human Threonyl-tRNA Synthetase
TARS Preparation and Nucleotide Assays.
TARS purification and active site determination were performed as previously described (Williams, Mirando et al., 2013). TARS non-canonical catalytic activities were characterized using modifications from published methods (Guo, Chong et al. 2009). For Ap4A reactions 5 μM of wildtype TARS or R442A TARS were incubated for 10 minutes on ice with 2 mM threonine, and 10 μM BC194 or borrelidin as indicated in
Ap4G and GTPase assays were performed as above with the following exceptions: Ap4G reactions always included 2 mM ATP and 2 mM threonine and 10 μM BC194 where indicated in
CAM Assays.
Fertilized chicken embryos were cultured ex-ova and, starting at developmental day 10, agents were applied daily to gelfoam sponges on the CAM. Images were recorded daily over 72 h and scored blindly according to a modified version Intensity Scoring as previously described (Ribatti, Nico et al. 2006). Additional details regarding methods are provided in the Methods and Materials section of Example 1.
An important scientific question is the extent to which the pro-angiogenic functions of TARS are directly linked to aminoacylation function. Alternatively, stimulation of angiogenesis might be linked to alternate catalytic functions, employing substrates and products that are distinct from those aminoacylation. To directly test whether aminoacylation is required for stimulation of angiogenesis, a mutant version of hTARS was produced in which an essential arginine in the active site (Arg442) was substituted with alanine. As shown in
In light of the previous observation that aminoacylation activity is not required to stimulation of angiogenesis, the hypothesis that alternative catalytic function might be involved was explored. One such alternative is the production of diadenosine tetraphosphate (Ap4A), which is produced by human lysyl-tRNA synthetase in immune cells that become activated by antigen. The Ap4A produced by LysRS binds to Hint, liberating the associated microphthalmia transcription factor (MITF) to execute a complicated program of gene expression (Lee, Nechushtan et al. 2004; Ofir-Birin, Fang et al. 2013). This confirms the role of Ap4A as an intracellular signaling molecule. There is also data to suggest that Ap4A can function as an extracellular signaling molecule (McLennan 2000) (Delicado, Miras-Portugal et al. 2006). Significantly, Ap4A is released extracellularly from platelets, and is capable interacting with the P2Y and P2X receptors. These interactions have the potential of modulating angiogenesis in endothelial cells (Chang, Yanachkov et al. 2010; Roedersheimer, Nijmeh et al. 2011).
As shown in
Another reaction in which aminoacyl-tRNA synthetases can potentially modulate signaling is GTP hydrolysis. Recent published work that LeuRS may contribute amino acid sensing properties to the Mammalian Target of Rapamycin complex (mTOR) by virtue of interactions with the Rag GTPase a mediator of amino acid signaling to mTORC1 (Bonfils, Jaquenoud et al. 2012; Han, Jeong et al. 2012). In contrast to the dinucleotide synthesis, TARS GTPase activity differs greatly in response to similar treatments. As shown in
TARS is Overexpressed in Ovarian Cancer
Ovarian Cancer Study Group—
The ovarian cancer studies were approved by the University of Vermont's institutional review board (CHRMS 01-026 and M12-004). The study group consisted of 58 patients diagnosed with epithelial ovarian cancer at Fletcher Allen Health Care/University of Vermont between 1999 and 2001. The control group consisted of 16 women who underwent oophorectomies for gynecologic reasons (other than ovarian cancer) and the final pathology demonstrated normal ovarian tissue. Serum and paraffin embedded samples from both the study and control group were obtained after adequate portions of the samples were evaluated for pathologic diagnosis. Histological subtype was based according to the WHO criteria and grading of tumors. Formalin-fixed, paraffin embedded tissue samples from each patient were retrieved. Three serial sections (6 μm) from each specimen were cut and transferred to slides, then analyzed using immunohistochemistry to measure the expression of VEGF and TARS as previously described (Wong, Wellman et al. 2003). After deparaffinization and rehydration, slides were incubated at 97° C. for 15 min with DAKO target retrieval solution, containing 100 mM Tris base, pH 10.5 and 0.1% Triton X-100. Immunoperoxidase staining was performed using the mouse ImmunoCruz staining system (Santa Cruz Biotechnology, Santa Cruz, Calif.) according to the manufacturer's protocol. Antibodies were mouse monoclonal anti-VEGF (1:100, Santa Cruz Biotechnology) and mouse monoclonal anti-TARS (1:100, Clone 1A9 Abnova, Taipei City, Taiwan). Normal mouse IgG was used as a negative control. After immunoperoxidase staining, cells were lightly stained with Mayers' hematoxylin and eosin. Slides were dehydrated through xylenes then mounted with coverslips using Cytoseal 60 (Richard-Allan Scientific, Kalamazoo, Mich.). Images were obtained using an Olympus BX50 light microscope coupled to a CCD camera and Metamorph image capture software. Slides were scored for the expression of VEGF and TARS on a scale of 1-4 where 1=no staining and 4=intense staining. TARS ELISA was performed on undiluted serum following the manufacturer's instructions (Cusabio Biotech). Statistical significance between groups was determined using Kruskal-Wallis test. Correlation between TARS and VEGF expression was evaluated using multiple regression correlation coefficient.
It has previously been shown that VEGF is overexpressed in ovarian cancer and correlates with progression of disease (Wong, C. et al. (2003) Gynecol Oncol 91 (3), 513-517). To determine whether TARS is dysregulated in human ovarian cancer, immunohistochemical staining for TARS was performed on patient tumor sections and correlated with staining of VEGF and serum levels of TARS. In the samples analyzed, TARS staining colocalized with VEGF and was selectively overexpressed in the tumor cells (
Identifying a Clinical Relationship between Cancer and TARS Protein Level and Activity in Tissue and Serum
Results of these experiments better define the newly discovered pathway whereby cells regulate angiogenesis through unconventional signaling by an aminoacyl tRNA synthetase. These experiments also determine the anti-angiogenic activity of TARS inhibitors, which suggest their development for and use as a therapeutic in ovarian and other angiogenesis-dependent cancers. Furthermore, these experiments determine if TARS secretion can be used as a means of detecting angiogenic ovarian cancer to assist in earlier diagnosis and improved treatment regimens for ovarian cancer patients.
Studies are performed that include measuring TARS expression in tumors and serum obtained from cancer patients, including but not limited to ovarian cancer patients, prostate cancer patients, etc. In the studies, angiogenesis markers such as PECAM are compared with control levels and the cancer's status, stage, and progression and the subject's prognosis is determined. Additional types of cancers tested are metastatic carcinoma of the cervix; sarcoma of the kidney; renal cell carcinoma; prostate cancer; androgen independent prostate cancer; Kaposi's sarcoma; colorectal cancer, hepatobilliary cancer, gastric cancer, epithelial ovarian cancer; lung cancer, and/or mesothelioma.
Experiments are performed and TARS activity and/or expression is measured in samples from one or more subjects that have or are suspected of being at risk of having additional diseases and conditions such as angiogenesis-associated disorder in which the level of TARS is increased as compared to a normal control level. A TARS level is determined to assess a cancer, a tumor, a hemangioma, a vascular overgrowth, a venous malformation, an arterial malformation, overweight, macular degeneration, an inflammatory disease, psoriasis, diabetes, or rheumatoid arthritis.
Additional studies are performed and TARS activity and/or expression is determined in samples from one or more subjects that have or are suspected of being at risk of having additional diseases and conditions such as angiogenesis-associated disorder in which the level of TARS is decreased as compared to a normal control level. A TARS level is determined for a tissue implant, an organ implant, ischemia, cardiac infarction, tissue trauma, cartilage to bone transformation, stroke, surgery, pregnancy, macular degeneration, and/or vascular occlusion.
The stage and prognosis for one or more of the diseases and conditions listed above elsewhere herein are assessed by determining the level of TARS in a sample and comparing the level to a level in a control sample.
Although several embodiments of the present invention have been described and illustrated herein, those of ordinary skill in the art will readily envision a variety of other means and/or structures for performing the functions and/or obtaining the results and/or one or more of the advantages described herein, and each of such variations and/or modifications is deemed to be within the scope of the present invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. It is, therefore, to be understood that the foregoing embodiments are presented by way of example only and that, within the scope of the appended claims and equivalents thereto; the invention may be practiced otherwise than as specifically described and claimed. The present invention is directed to each individual feature, system, article, material, and/or method described herein. In addition, any combination of two or more such features, systems, articles, materials, and/or methods, if such features, systems, articles, materials, and/or methods are not mutually inconsistent, is included within the scope of the present invention.
All definitions, as defined and used herein, should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and/or ordinary meanings of the defined terms.
The indefinite articles “a” and “an,” as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one.”
The phrase “and/or,” as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified, unless clearly indicated to the contrary.
All references, patents and patent applications and publications that are cited or referred to in this application are incorporated in their entirety herein by reference.
This application is a National Stage Filing under U.S.C. § 371 of PCT International Application PCT/US2013/51808, filed Jul. 24, 2013 which was published under PCT Article 21(2) in English, which claims benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/675,661, filed Jul. 25, 2012 and the content of each of which is incorporated by reference herein in its entirety.
This invention was made with government support under RO1 GM54899 and by training grant T32 ES007122-23 both awarded by the National Institutes of Health. The government has certain rights in the invention.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2013/051808 | 7/24/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/018620 | 1/3/2014 | WO | A |
| Number | Name | Date | Kind |
|---|---|---|---|
| 5795757 | Hodgson et al. | Aug 1998 | A |
| 8986681 | Greene | Mar 2015 | B2 |
| 20120058133 | Whitman et al. | Mar 2012 | A1 |
| 20130129704 | Greene et al. | May 2013 | A1 |
| 20150166976 | Francklyn et al. | Jun 2015 | A1 |
| 20150177243 | Francklyn et al. | Jun 2015 | A1 |
| Number | Date | Country |
|---|---|---|
| 1576160 | May 2007 | EP |
| 2011097031 | Aug 2011 | WO |
| 2011139801 | Nov 2011 | WO |
| WO2011139801 | Nov 2011 | WO |
| WO2011139801 | Nov 2011 | WO |
| Entry |
|---|
| Ruan et al, JBC, 280:571-577, 2005. |
| Funahashi, Y. et al., “Establishment of a quantitative mouse dorsal air sac model and its application to evaluate a new angiogenesis inhibitor”, Oncol. Res., 1999, vol. 11, pp. 319-329. |
| Moss, S.J. et al., “Biosynthesis of the angiogenesis inhibitor borrelidin:directed biosynthesis of novel analogues”, Chem. Communications, Jun. 14, 2006, vol. 22, pp. 2341-2343. |
| Tsuchiya, E. et al., “Borrelidin inhibits a Cyclin-dependent kinase (CDK), Cdc28/Cln2 of Saccharomyces cerevisiae”, J. of Antibiotics, Jan. 2011, vol. 54. pp. 84-90. |
| Wakabayashi, T. et al., “Borreldin is an angiogenesis inhibitor; disruption of angiogenic capillary vessels in rat aorta matrix culture model” J. antibiotics, Aug. 1997, vol. 50, pp. 671-676. |
| Gantt, J.S. et al., “Increased levels of threonyl-tRNA synthetase in a borrelidin resistant Chinese hamster ovary cell line” PNAS, Sep. 1981, vol. 78, pp. 5367-5370. |
| Herzog, W. et al., “Genetic evidence for a non-canonical function of seryl-tRNA synthetase in vascular development”, Circulation Research, Jun. 5, 2009, vol. 104, pp. 1260-1266. |
| Kontis, K. and S. Arfin, “Isolation of a cDNA clone for human threonyl-tRNA synthetase: amplification of the structural gene in borrelidin-resistant cell lines”, Molecular and Cellular Biology, May 1989, vol. 9, pp. 1832-1838. |
| Nass, G. and K. Poralla, “Genetics of borrelidin resistant mutants of Saccharomyces cerivisiae and properties of their threonyl-tRNA-synthetase”, Mol. Gen. Genet., Aug. 1976, vol. 10, pp. 39-43. |
| Seibold, M. et al., “Homoserine and threonine pools of borrelidin resistant Saccharomyces cerevisiae mutants with an altered aspartokinase”, Arch Microbiol., Jul. 1981, vol. 129, pp. 368-370. |
| Habibi, D. et al., “Borrelidin, a small molecule nitrile-containing macrolide inhibitor of threonyl-tRNA synthetase, is a potent inducer of apoptosis in acute lymphoblastic leukemia”, Springer, Jun. 17, 2011, pp. 1-10. |
| Nagamitsu, T. et al., “Total Synthesis of Borrelidin”, J. Org. Chem., 2007, vol. 72, pp. 2744-2756. |
| Vong, B.G. et al., “Stereoselective Total Syntheis of (−)-Borrelidin”, Angew. Chem. Int. Ed., 2004, vol. 43, pp. 3947-3951. |
| Woolard, J. et al., “Borrelidin modulates the alternative splicing of VEGF in favour of anti-angiogenic isoforms”, Chemical Science, 2011, vol. 2, pp. 273-278. |
| Chandran, U.R. et al., “Gene expression profiles of prostate cancer reveal involvement of multiple molecular pathways in the metastatic process”, BMC Cancer, Apr. 12, 2007, vol. 7, pp. 1-21. |
| Fox, P.L. et al., “Noncanonical Functions of Aminoacyl-tRNA Synthetases in Translational Control”, Translational Control in Biology and Medicine, 2007, vol. 29, pp. 829-854. |
| Ghanipour, A. et al., “The Prognostic Significance of Tryptophanyl-tRNA Synthetase in Colorectal Cancer”, Cancer Epidemiol Biomarkers, Nov. 2009, vol. 18, pp. 2949-2956. |
| Harisi, R. et al., “Differential Inhibition of Single and Cluster Type Tumor Cell Migration”, AntiCancer Research, 2009, vol. 29, pages. |
| Kim, S. et al., “Aminoacyl-tRNA synthetases and tumorigenesis: more than housekeeping”, Nature, Oct. 2011, vol. 11, pp. 708-719. |
| Mathews, M.B. et al., “Anti-Threonyl-tRNA Synthetase, A Second Myositis-Related Autoantibody”, J. ExP. Med, Aug. 1984, vol. 160, pp. 420-434, Downloaded from jem.rupress.org on Jan. 29, 2013. |
| Simirnova, E.V. et al., “Noncanonical Functions of AminoacyltRNA Synthetases”, Biochemisty (Moscow), 2012, vol. 77, pp. 15-25. |
| Vazquez-Mena, O. et al., “Amplified Genes May Be Overexpressed, Unchanged, or Downregulated in Cervical Cancer Cell Lines”, PLoS ONE, Mar. 2012, vol. 7, pp. 1-17. |
| Vellaichamy, A. et al., “Proteomic Interrogation of Androgen Action in Prostate Cancer Cells Reveals Roles of Aminoacyl tRNA Synthetases”, PLos ONE, Sep. 2009, vol. 4, pp. 1-12. |
| Zampieri, S. et al., “Polymyositis, dermatomyositis and malignancy: A further intriguing link”, Autoimmunity Reviews, 2010, vol. 9, pp. 449-453. |
| Guo, M. et al., “New functions of aminoacyl-tRNA synthetases beyond translation”, Nature Reviews, Sep. 2010, vol. 11, pp. 668-674. |
| Ko, Y.G. et al., “Glutamine-dependent Antiapoptotic Interaction of Human Glutaminyl-tRNA Synthetase with Apoptosis Signal-regulating Kinase 1”, The Journal of Biological Chemistry, Feb. 2001, vol. 276, pp. 6030-6036. |
| Even-Zohar, N. et al., “Nutrition-induced catch-up growth increases hypoxia inducible factor 1 α RNA levels in the growth plate”, Bone, 2008, vol. 42, pp. 505-515. |
| Greenberg, Y. et al., “The novel fragment of tyrosyl tRNA synthetase, mini-TyrRS, is secreted to induce an angiogenic response in endothelial cells”, The FASEB Journal, May 2008, vol. 22, pp. 1597-1605. |
| Howard, O.M. Z. et al., “Histidyl-tRNA Synthetase and Asparaginyl-tRNA Synthetase, Autoantigens in Myositis, Activate Chemokine Receptors on T Lymphocytes and Immature Dendritic Cells”, The Journal of Experimental Medicine, Sep. 16, 2002, vol. 196, pp. 781-791. |
| Kawamura, T. et al., “Anti-angiogenesis Effects of Borrelidin are Mediated through Distinct Pathways: Threonyl-tRNA Synthetase and Caspases are Independently Involved in Suppression of Proliferation and Induction of Apoptosis in Endothelial Cells”, The Journal of Antibiotics, Aug. 2003, vol. 56, pp. 709-715. |
| NCBI, GenBank accession No. NM-152295.4, Jun. 27, 2012. |
| Olano,C. et al., “Biosynthesis of the Angiogenesis Inhibitor Borrelidin by Streptomyces parvulus Tü4055: Cluster Analysis and Assignment of Functions”, Chemistry & Biology, Jan. 2004, vol. 11, pp. 87-97. |
| Park, S. et al., “Aminoacyl tRNA systhetases and their connections to disease”, PNAS, Aug. 12, 2008, vol. 105, pp. 11043-11049. |
| Ruan, B. et al., “A Unique Hydrophobic Cluster Near the Active Site Contributes to Differences in Borrelidin Inhibition among Threonyl-tRNA Sythetases”, The Journal of Biological Chemistry, Jan. 7, 2005, vol. 280, pp. 571-577. |
| Williams, T. et al., “Secreted Threonyl-tRNA synthetase stimulates endothelial cell migration and angiogenesis”, Scientific Reports, Feb. 21, 2013, vol. 3:1317, pp. 1-7. |
| International Search Report and the Written Opinion of the International Searching Authority dated Oct. 10, 2013 for the International Patent Application No. PCT/US2013/051806, 15 pages. |
| International Preliminary Report on Patentability dated Jan. 27, 2015 for the International Patent Application No. PCT/US2013/051806, 5 pages. |
| International Search Report and the Written Opinion of the International Searching Authority dated Oct. 24, 2013 for International Patent Application No. PCT/US2013/051808, 17 pages. |
| International Preliminary Report on Patentability dated Feb. 5, 2015 for the International Patent Application No. PCT/US2013/051808, 10 pages. |
| International Search Report and the Written Opinion of the International Searching Authority dated Oct. 22, 2013 for International Patent Application No. PCT/US2013/051807, 15 pages. |
| International Preliminary Report on Patentability dated Feb. 5, 2015 for the International Patent Application No. PCT/US2013/051807, 12 pages. |
| Ahmed, S. A., et al., “A new rapid and simple non-radioactive assay to monitor and determine the proliferation of lymphocytes: an alternative to [3H]thymidine incorporation assay.” J Immunol Methods, 1994, vol. 170, pp. 211-224. |
| Altundag, K., et al., “CA125 Nadir values as a prognostic factor in epithelial ovarian cancer.” J Clin Oncol, 2005, vol. 23, pp. 2435-2436. |
| Arnaoutova, I. and H. K. Kleinman, “In vitro angiogenesis: endothelial cell tube formation on gelled basement membrane extract.” Nat Protoc, 2010, vol. 5, pp. 628-635. |
| Cassavaugh, J. M., et al., “Negative regulation of HIF-1alpha by an FBW7-mediated degradation pathway during hypoxia.” J Cell Biochem, 2011, vol. 112, pp. 3882-3890. |
| Ewing, R. M., et al., “Large-scale mapping of human protein-protein interactions by mass spectrometry.” Mol Syst Biol, 2007, vol. 3, p. 89. |
| Francklyn, C. S., et al., “Methods for kinetic and thermodynamic analysis of aminoacyl-tRNA synthetases.” Methods, 2008, vol. 44, pp. 100-118. |
| Longair, M. H., et al., “Simple Neurite Tracer: open source software for reconstruction, visualization and analysis of neuronal processes.” Bioinformatics, 2011, vol. 27, pp. 2453-2454. |
| Lounsbury, K. M., et al., “A family of proteins that stabilize the Ran/TC4 GTPase in its GTP-bound conformation.” J Biol Chem, 1994, vol. 269, pp. 11285-11290. |
| Mor, G., et al., “Serum protein markers for early detection of ovarian cancer.” Proc Natl Acad Sci U S A, 2005, vol. 102, pp. 7677-7682. |
| Ribatti, D., et al., “The gelatin sponge-chorioallantoic membrane assay.” Nat Protoc, 2006, vol. 1, pp. 85-91. |
| Strausberg, R. L. “The Cancer Genome Anatomy Project: new resources for reading the molecular signatures of cancer” J Pathol, 2001, vol. 195, pp. 31-40. |
| Svensson, K. J., et al., “Hypoxia triggers a proangiogenic pathway involving cancer cell microvesicles and PAR-2-mediated heparin-binding EGF signaling in endothelial cells.” Proc Natl Acad Sci U S A, 2011, vol. 108, pp. 13147-13152. |
| Tomlins, S. A., et al., “Integrative molecular concept modeling of prostate cancer progression.” Nat Genet, 2007, vol. 39, pp. 41-51. |
| Uhlen, M., et al., “Towards a knowledge-based Human Protein Atlas.” Nat Biotechnol, 2010, vol. 28, pp. 1248-1250. |
| Wakasugi, K. and P. Sshimmel, “Two distinct cytokines released from a human aminoacyl-tRNA synthetase.” Science, 1999, vol. 284, pp. 147-151. |
| Wilkinson, B., et al., “Separation of anti-angiogenic and cytotoxic activities of borrelidin by modification at the C17 side chain.” Bioorg Med Chem Lett, 2006, vol. 16, pp. 5814-5817. |
| Wong, C., et al., “VEGF and HIF-1alpha expression are increased in advanced stages of epithelial ovarian cancer.” Gynecol Oncol, 2003, vol. 91, pp. 513-517. |
| Bikfalvi, A. and Bicknell, R., “Recent advances in angiogenesis, anti-angiogenesis and vascular targeting”, Trends in Pharmacological Sciences, Dec. 2002, vol. 23, pp. 576-582. |
| Bonfils, G.M. et al., “Leucyl-tRNA synthetase controls TORC1 via the EGO complex”, Molecular Cell, 2012, vol. 46, pp. 105-110. |
| Brown, M.V. et al., “Mammalian aminoacyl-tRNA synthetases: cell signaling functions of the protein translation machinery”, Vascul Pharmacol, 2010, vol. 52, pp. 21-26. |
| Chang, H., et al., “Agonist and antagonist effects of diadenosine tetraphosphate, a platelet dense granule constituent, on platelet P2Y1, P2Y12 and P2X1 receptors”, Thromb Res, 2010, vol. 125, pp. 159-165. |
| Conant, JL. et al., “Sarcomatiod renal cell carcinoma is an examples fo epithelial-mesenchymal transition”, J. Clin. Pat., 2011, vol. 64, pp. 1088-1092. |
| Delicado, E.G. et al., “Dinucleoside polyphosphates and their interaction with other nucleotide signaling pathways”, Pfluger Arch, 2006, vol. 452, pp. 563-572. |
| Dieterich, D.C. et al., “Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid tagging”, Nat Protoc, 2007, vol. 2, pp. 532-540. |
| Folkman, J., “Angiogenesis-dependent diseases”, Semin Oncol, Dec. 2001, vol. 28, pp. 536-452. |
| Guo, R.T. et al., “Crystal structures and biochemical analyses suggest a unique mechanism and role for human glycyl-tRNA synthetase in Ap4A homeostasis” J Biol Chem, 2009, vol. 284, pp. 28968-28976. |
| Han, J.M. et al., “Leucyl-tRNA synthetase is an intracellular leucine sensor for the mTORC1-signaling pathway”, Cell, 2012, vol. 149, pp. 410.-424. |
| Lee, Y.N. et al., “The function of lysyl-tRNA synthetase and Ap4A as signaling regulators of MITF activity in FcepsilonRI-activated mast cells”, Immunity, 2004, vol. 20, pp. 145-151. |
| Liao, D. and Johnson, Rs., “Hypoxia a key regulator of angiogenesis in cancer”, Cancer and Metastasis Reviews, 2007, vol. 26, pp. 281-290. |
| McLennan, A.G. “Dinucleoside ployphosphates-friend or foe?” Pharmacol Ther, 2000, vol. 87, pp. 73-89. |
| Ofir-Birin, Y. et al., “Structural Switch of Lysyl-tRNA Synthetase between Translation and Transcription”, Mol Cell, 2013, vol. 49, pp. 30-42. |
| Olsson, A.K. et al., “VEGF receptor signalling—in control of vascular function”, Nat Rev Mol Cell Biol, 2006, vol. 7, pp. 359-371. |
| Roedersheimer, M. et al., “Complementary effects of extracellular nucleotides and platelet-derived extracts on angiogenesis of vasa vasorum endothelial cells in vitro and subcutaneous Matrigel plugs in vivo”, Vasc Cell, 2011, vol. 3, p. 4. |
| Mehta, R. et al “Proteosomal Regulation of the Hypoxic Response modulates aging in C. elegans” Science, May 2009, vol. 324, pp. 1196-1198. |
| Eastwood, EL. and SE. Schaus, “Borrelidin induces the transcription of amino acid biosynthetic enzymes via a GCN4-dependent pathway”, Bioorg. Med. Chem. Lett., Jul. 2003, vol. 13, pp. 2235-2237. |
| Cura, et al., (2000) Eur. J. Biochem 267, pp. 379-393. |
| Sankaranarayanan et al., (1999) Cell, vol. 97, pp. 371-381. |
| Yampolsky et al., (2005) Genetics 170, pp. 1459-1472. |
| Number | Date | Country | |
|---|---|---|---|
| 20150210997 A1 | Jul 2015 | US |
| Number | Date | Country | |
|---|---|---|---|
| 61675661 | Jul 2012 | US |
