The Experts below are selected from a list of 10995 Experts worldwide ranked by ideXlab platform
Gary K Smith - One of the best experts on this subject based on the ideXlab platform.
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antibody directed enzyme prodrug therapy with the t268g mutant of human Carboxypeptidase A1 in vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors gw1031 and gw1843
Bioconjugate Chemistry, 1999Co-Authors: Lawrence A Wolfe, Todd Andrew Blumenkopf, Michael Cory, Joan E Humphreys, Ronald M Laethem, John F Miller, Robert J Mullin, B R Keith, Gary K SmithAbstract:Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human Carboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme−antibody conjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mous...
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toward antibody directed enzyme prodrug therapy with the t268g mutant of human Carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate
Journal of Biological Chemistry, 1997Co-Authors: Gary K Smith, Sheila D Banks, Todd Andrew Blumenkopf, Michael Cory, Joan E Humphreys, Ronald M Laethem, John F Miller, Cary P Moxham, Robert J Mullin, Paul H RayAbstract:Abstract Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stablein vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme Carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine Carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human Carboxypeptidases A. Two of these analogs are MTX-α-3-cyclobutylphenylalanine and MTX-α-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human Carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-α-phenylalanine). Thus, thek cat/K m value for the wild type hCPA1 with MTX-α-phenylalanine is 0.44 μm −1 s−1, andk cat/K m values for hCPA1-T268G with MTX-α-3-cyclobutylphenylalanine and MTX-α-3-cyclopentyltyrosine are 1.8 and 0.16 μm −1 s−1, respectively. The cytotoxic efficiency of hCPA1–268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with thein vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.
Joan E Humphreys - One of the best experts on this subject based on the ideXlab platform.
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antibody directed enzyme prodrug therapy with the t268g mutant of human Carboxypeptidase A1 in vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors gw1031 and gw1843
Bioconjugate Chemistry, 1999Co-Authors: Lawrence A Wolfe, Todd Andrew Blumenkopf, Michael Cory, Joan E Humphreys, Ronald M Laethem, John F Miller, Robert J Mullin, B R Keith, Gary K SmithAbstract:Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human Carboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme−antibody conjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mous...
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toward antibody directed enzyme prodrug therapy with the t268g mutant of human Carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate
Journal of Biological Chemistry, 1997Co-Authors: Gary K Smith, Sheila D Banks, Todd Andrew Blumenkopf, Michael Cory, Joan E Humphreys, Ronald M Laethem, John F Miller, Cary P Moxham, Robert J Mullin, Paul H RayAbstract:Abstract Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stablein vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme Carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine Carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human Carboxypeptidases A. Two of these analogs are MTX-α-3-cyclobutylphenylalanine and MTX-α-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human Carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-α-phenylalanine). Thus, thek cat/K m value for the wild type hCPA1 with MTX-α-phenylalanine is 0.44 μm −1 s−1, andk cat/K m values for hCPA1-T268G with MTX-α-3-cyclobutylphenylalanine and MTX-α-3-cyclopentyltyrosine are 1.8 and 0.16 μm −1 s−1, respectively. The cytotoxic efficiency of hCPA1–268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with thein vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.
Robert J Mullin - One of the best experts on this subject based on the ideXlab platform.
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antibody directed enzyme prodrug therapy with the t268g mutant of human Carboxypeptidase A1 in vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors gw1031 and gw1843
Bioconjugate Chemistry, 1999Co-Authors: Lawrence A Wolfe, Todd Andrew Blumenkopf, Michael Cory, Joan E Humphreys, Ronald M Laethem, John F Miller, Robert J Mullin, B R Keith, Gary K SmithAbstract:Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human Carboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme−antibody conjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mous...
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toward antibody directed enzyme prodrug therapy with the t268g mutant of human Carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate
Journal of Biological Chemistry, 1997Co-Authors: Gary K Smith, Sheila D Banks, Todd Andrew Blumenkopf, Michael Cory, Joan E Humphreys, Ronald M Laethem, John F Miller, Cary P Moxham, Robert J Mullin, Paul H RayAbstract:Abstract Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stablein vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme Carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine Carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human Carboxypeptidases A. Two of these analogs are MTX-α-3-cyclobutylphenylalanine and MTX-α-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human Carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-α-phenylalanine). Thus, thek cat/K m value for the wild type hCPA1 with MTX-α-phenylalanine is 0.44 μm −1 s−1, andk cat/K m values for hCPA1-T268G with MTX-α-3-cyclobutylphenylalanine and MTX-α-3-cyclopentyltyrosine are 1.8 and 0.16 μm −1 s−1, respectively. The cytotoxic efficiency of hCPA1–268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with thein vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.
Todd Andrew Blumenkopf - One of the best experts on this subject based on the ideXlab platform.
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antibody directed enzyme prodrug therapy with the t268g mutant of human Carboxypeptidase A1 in vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors gw1031 and gw1843
Bioconjugate Chemistry, 1999Co-Authors: Lawrence A Wolfe, Todd Andrew Blumenkopf, Michael Cory, Joan E Humphreys, Ronald M Laethem, John F Miller, Robert J Mullin, B R Keith, Gary K SmithAbstract:Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human Carboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme−antibody conjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mous...
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toward antibody directed enzyme prodrug therapy with the t268g mutant of human Carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate
Journal of Biological Chemistry, 1997Co-Authors: Gary K Smith, Sheila D Banks, Todd Andrew Blumenkopf, Michael Cory, Joan E Humphreys, Ronald M Laethem, John F Miller, Cary P Moxham, Robert J Mullin, Paul H RayAbstract:Abstract Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stablein vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme Carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine Carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human Carboxypeptidases A. Two of these analogs are MTX-α-3-cyclobutylphenylalanine and MTX-α-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human Carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-α-phenylalanine). Thus, thek cat/K m value for the wild type hCPA1 with MTX-α-phenylalanine is 0.44 μm −1 s−1, andk cat/K m values for hCPA1-T268G with MTX-α-3-cyclobutylphenylalanine and MTX-α-3-cyclopentyltyrosine are 1.8 and 0.16 μm −1 s−1, respectively. The cytotoxic efficiency of hCPA1–268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with thein vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.
John F Miller - One of the best experts on this subject based on the ideXlab platform.
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antibody directed enzyme prodrug therapy with the t268g mutant of human Carboxypeptidase A1 in vitro and in vivo studies with prodrugs of methotrexate and the thymidylate synthase inhibitors gw1031 and gw1843
Bioconjugate Chemistry, 1999Co-Authors: Lawrence A Wolfe, Todd Andrew Blumenkopf, Michael Cory, Joan E Humphreys, Ronald M Laethem, John F Miller, Robert J Mullin, B R Keith, Gary K SmithAbstract:Antibody-directed enzyme prodrug therapy (ADEPT) is a technique to increase antitumor selectivity in cancer chemotherapy. Our approach to this technology has been to design a mutant of human Carboxypeptidase A (hCPA1-T268G) which is capable of hydrolyzing in vivo stable prodrugs of MTX and targeting this enzyme to tumors on an Ep-CAM1-specific antibody, ING1. Through the use of this >99% human enzyme which is capable of catalyzing a completely nonhuman reaction, we hope to increase ADEPT selectivity while decreasing overall immunogenicity of the enzyme−antibody conjugate. In the current report, prodrugs of the thymidylate synthase inhibitors GW1031 and GW1843 and the dihydrofolate reductase inhibitor methotrexate were studied for their wild-type and mutant hCPA enzyme hydrolysis, their in vivo stability, and their use in therapy. Prodrugs with high kcat/Km ratios for mutated versus wild-type hCPA1 were examined in vitro for their stability in human pancreatic juice, and in vivo for their stability in mous...
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toward antibody directed enzyme prodrug therapy with the t268g mutant of human Carboxypeptidase A1 and novel in vivo stable prodrugs of methotrexate
Journal of Biological Chemistry, 1997Co-Authors: Gary K Smith, Sheila D Banks, Todd Andrew Blumenkopf, Michael Cory, Joan E Humphreys, Ronald M Laethem, John F Miller, Cary P Moxham, Robert J Mullin, Paul H RayAbstract:Abstract Antibody-directed enzyme prodrug therapy (ADEPT) has the potential of greatly enhancing antitumor selectivity of cancer therapy by synthesizing chemotherapeutic agents selectively at tumor sites. This therapy is based upon targeting a prodrug-activating enzyme to a tumor by attaching the enzyme to a tumor-selective antibody and dosing the enzyme-antibody conjugate systemically. After the enzyme-antibody conjugate is localized to the tumor, the prodrug is then also dosed systemically, and the previously targeted enzyme converts it to the active drug selectively at the tumor. Unfortunately, most enzymes capable of this specific, tumor site generation of drugs are foreign to the human body and as such are expected to raise an immune response when injected, which will limit their repeated administration. We reasoned that with the power of crystallography, molecular modeling and site-directed mutagenesis, this problem could be addressed through the development of a human enzyme that is capable of catalyzing a reaction that is otherwise not carried out in the human body. This would then allow use of prodrugs that are otherwise stablein vivo but that are substrates for a tumor-targeted mutant human enzyme. We report here the first test of this concept using the human enzyme Carboxypeptidase A1 (hCPA1) and prodrugs of methotrexate (MTX). Based upon a computer model of the human enzyme built from the well known crystal structure of bovine Carboxypeptidase A, we have designed and synthesized novel bulky phenylalanine- and tyrosine-based prodrugs of MTX that are metabolically stable in vivo and are not substrates for wild type human Carboxypeptidases A. Two of these analogs are MTX-α-3-cyclobutylphenylalanine and MTX-α-3-cyclopentyltyrosine. Also based upon the computer model, we have designed and produced a mutant of human Carboxypeptidase A1, changed at position 268 from the wild type threonine to a glycine (hCPA1-T268G). This novel enzyme is capable of using the in vivo stable prodrugs, which are not substrates for the wild type hCPA1, as efficiently as the wild type hCPA1 uses its best substrates (i.e. MTX-α-phenylalanine). Thus, thek cat/K m value for the wild type hCPA1 with MTX-α-phenylalanine is 0.44 μm −1 s−1, andk cat/K m values for hCPA1-T268G with MTX-α-3-cyclobutylphenylalanine and MTX-α-3-cyclopentyltyrosine are 1.8 and 0.16 μm −1 s−1, respectively. The cytotoxic efficiency of hCPA1–268G was tested in an in vitro ADEPT model. For this experiment, hCPA1-T268G was chemically conjugated to ING-1, an antibody that binds to the tumor antigen Ep-Cam, or to Campath-1H, an antibody that binds to the T and B cell antigen CDw52. These conjugates were then incubated with HT-29 human colon adenocarcinoma cells (which express Ep-Cam but not the Campath 1H antigen) followed by incubation of the cells with thein vivo stable prodrugs. The results showed that the targeted ING-1:hCPA1-T268G conjugate produced excellent activation of the MTX prodrugs to kill HT-29 cells as efficiently as MTX itself. By contrast, the enzyme-Campath 1H conjugate was without effect. These data strongly support the feasibility of ADEPT using a mutated human enzyme with a single amino acid change.