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W2102970512 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. 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. A current major challenge to cancer therapy is to increase antitumor selectivity. One approach to realizing this goal is to use the exquisite selectivity of the antibody:antigen reaction to target therapeutic entities specifically to tumors. While absolutely tumor-specific antibodies are not known, many antibodies are available that can deliver tumor-selective targeting (for review, see Ref.1Sell S. Reisfeld R.A. Monoclonal Antibodies in Cancer. Humana Press, Clifton, NJ1985Crossref Google Scholar).One investigational therapy that makes use of this principle of antibody targeting is antibody-directed enzyme prodrug therapy (ADEPT). 1The abbreviations used are: ADEPT, antibody-directed enzyme prodrug therapy; CPA, carboxypeptidase; hCPA, human CPA; bCPA, bovine CPA; MTX, methotrexate; HPLC, high pressure liquid chromatography; WT, wild type; Sulfo-SMCC, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. 1The abbreviations used are: ADEPT, antibody-directed enzyme prodrug therapy; CPA, carboxypeptidase; hCPA, human CPA; bCPA, bovine CPA; MTX, methotrexate; HPLC, high pressure liquid chromatography; WT, wild type; Sulfo-SMCC, sulfosuccinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate. ADEPT is a powerful strategy with the potential for tumor-specific long-term delivery of chemotherapy (2Bagshawe K.D. Br. J. Cancer. 1989; 60: 275-281Crossref PubMed Scopus (118) Google Scholar, 3Senter P.D. FASEB. 1990; 4: 188-193Crossref PubMed Scopus (124) Google Scholar, 4Senter P.D. Wallace P.M. Svensson H.P. Vrudhula V.M. Kerr D.E. Hellstrom I. Hellstrom K.E. Bioconjugate Chem. 1993; 4: 3-9Crossref PubMed Scopus (82) Google Scholar, 5Melton R.G. Sherwood R.F. J. Natl. Cancer Inst. 1996; 88: 153-165Crossref PubMed Scopus (102) Google Scholar, 6Huennekens F.M. Trends Biotechnol. 1994; 12: 234-239Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 7Jungheim L.N. Shepherd T.A. Chem. Rev. 1994; 94: 1553-1566Crossref Scopus (96) Google Scholar). The premise of ADEPT is to target an enzyme of interest specifically to tumor cells by coupling it to a tumor-specific antibody. This conjugate is delivered to the patient systemically and then allowed to bind to the antigen-expressing target cells. Unbound conjugate is allowed to clear from circulation, and when the circulating levels of conjugate are sufficiently low, a prodrug is administered, also systemically, that can be converted to a toxic chemotherapeutic drug by the targeted enzyme-antibody conjugate. The action of the enzyme-antibody conjugate on the prodrug then ideally generates lethal levels of drug specifically at the tumor site. For this therapy to be selective, however, nonspecific activation of the prodrug at sites distant to the tumor must be minimized. This is generally accomplished by using a conjugate enzyme with an activity not endogenous to the host or at least not accessible to the prodrug (2Bagshawe K.D. Br. J. Cancer. 1989; 60: 275-281Crossref PubMed Scopus (118) Google Scholar, 3Senter P.D. FASEB. 1990; 4: 188-193Crossref PubMed Scopus (124) Google Scholar, 4Senter P.D. Wallace P.M. Svensson H.P. Vrudhula V.M. Kerr D.E. Hellstrom I. Hellstrom K.E. Bioconjugate Chem. 1993; 4: 3-9Crossref PubMed Scopus (82) Google Scholar, 5Melton R.G. Sherwood R.F. J. Natl. Cancer Inst. 1996; 88: 153-165Crossref PubMed Scopus (102) Google Scholar, 6Huennekens F.M. Trends Biotechnol. 1994; 12: 234-239Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 7Jungheim L.N. Shepherd T.A. Chem. Rev. 1994; 94: 1553-1566Crossref Scopus (96) Google Scholar, 8Bignami G.S. Senter P.D. Grothaus P.G. Fischer K.J. Humphreys T. Wallace P.M. Cancer Res. 1992; 52: 5759-5764PubMed Google Scholar, 9Vrudhula V.M. Senter P.D. Fischer K.J. Wallace P.M. J. Med. Chem. 1993; 36: 919-923Crossref PubMed Scopus (63) Google Scholar, 10Wallace P.M. Senter P.D. Bioconjugate Chem. 1991; 2: 349-352Crossref PubMed Scopus (66) Google Scholar, 11Senter P.D. Schreiber G.J. Hirschberg D.L Ashe S.A. Hellstrom K.E. Hellstrom I. Cancer Res. 1989; 49: 5789-5792PubMed Google Scholar, 12Svensson H.P. Vrudhula V.M. Emswiler J.E. MacMaster J.F. Cosand W.L. Senter P.D. Wallace P.M. Cancer Res. 1995; 55: 2357-2365PubMed Google Scholar, 13Kerr D.E. Schreiber G.J. Vrudhula V.M. Svensson H.P. Hellstrom I. Hellstrom K.E. Senter P.D. Cancer Res. 1995; 55: 3558-3563PubMed Google Scholar, 14Bagshawe K.D. Springer C.J. Searle F. Antoniw P. Sharma S.K. Melton R.G. Sherwood R.F. Br. J. Cancer. 1988; 58: 700-703Crossref PubMed Scopus (257) Google Scholar, 15Eccles S.A. Court W.J. Box G.A. Dean C.J. Melton R.G. Springer C.J. Cancer Res. 1994; 54: 5171-5177PubMed Google Scholar, 16Blakey D.C. Burke P.J. Davies D.H. Dowell R.I. East S.J. Eckersley K.P. Fitton J.E. McDaid J. Melton R.G. Niculescu-Duvaz I.A. Pinder P.E. Sharma S.K. Wright A.F. Springer C.J. Cancer Res. 1996; 56: 3287-3292PubMed Google Scholar, 17Meyer D.L. Jungheim L.N. Law K.L Mikolajczyk S.D. Shepherd T.A. Mackensen D.G. Briggs S.L. Starling J.J. Cancer Res. 1993; 53: 3956-3963PubMed Google Scholar, 18Meyer D.L. Law K.L. Payne J.K. Mikolajczyk S.D. Zarrinmayen H. Jungheim L.N. Kling J.K. Shepherd T.A. Starling J.J. Bioconjugate Chem. 1995; 6: 440-446Crossref PubMed Scopus (24) Google Scholar, 19Kuefner U. Lohrmann U. Montejano Y.D. Vitols K.S. Huennekens F.M. Biochemistry. 1989; 28: 2288-2297Crossref PubMed Scopus (60) Google Scholar, 20Haenseler E. Esswein A. Vitols K.S. Montejano Y. Mueller B.M. Reisfeld R.A. Huennekens F.M. Biochemistry. 1992; 31: 891-897Crossref PubMed Scopus (49) Google Scholar, 21Vitols K.S. Haag-Zeino B. Baer T. Montejano Y.D. Huennekens F.M. Cancer Res. 1995; 55: 478-481PubMed Google Scholar, 22Haisma H.J. Boven E. van Muijen M. de Jong J. van der Vigh W.J.F. Pinedo H.M. Br. J. Cancer. 1992; 66: 474-478Crossref PubMed Scopus (100) Google Scholar, 23Wang S-M. Chern J-W. Yeh M-Y. Ng J.C. Tung E. Roffler S.R. Cancer Res. 1992; 52: 4484-4491PubMed Google Scholar, 24Bosslet K. Czeh J. Hoffmann D. Cancer Res. 1994; 54: 2151-2159PubMed Google Scholar, 25Senter P.D. Su P.C. Katsuragi T. Sakai T. Cosand W.L. Hellstrom I. Hellstrom K.E. Bioconjugate Chem. 1991; 2: 447-451Crossref PubMed Scopus (81) Google Scholar, 26Kerr D.E. Garrigues U.S. Wallace P.M. Hellstrom K.E. Hellstrom I. Senter P.D. Bioconjugate Chem. 1993; 4: 353-357Crossref PubMed Scopus (29) Google Scholar, 27Ledermann J.A. Begent R.H.J. Massof C. Kelly A.M.B. Adam T. Bagshawe K.D. Int. J. Cancer. 1991; 47: 659-664Crossref PubMed Scopus (39) Google Scholar, 28Sharma S.K. Bagshawe K.D. Melton R.G. Sherwood R.F. Cell Biophys. 1992; 21: 109-120Crossref PubMed Scopus (59) Google Scholar, 29Springer C.J. Poon G.K. Sharma S.K. Bagshawe K.D. Cell Biophys. 1993; 22: 9-26Crossref PubMed Scopus (23) Google Scholar).A number of ADEPT strategies have been reported (2Bagshawe K.D. Br. J. Cancer. 1989; 60: 275-281Crossref PubMed Scopus (118) Google Scholar, 3Senter P.D. FASEB. 1990; 4: 188-193Crossref PubMed Scopus (124) Google Scholar, 4Senter P.D. Wallace P.M. Svensson H.P. Vrudhula V.M. Kerr D.E. Hellstrom I. Hellstrom K.E. Bioconjugate Chem. 1993; 4: 3-9Crossref PubMed Scopus (82) Google Scholar, 5Melton R.G. Sherwood R.F. J. Natl. Cancer Inst. 1996; 88: 153-165Crossref PubMed Scopus (102) Google Scholar, 6Huennekens F.M. Trends Biotechnol. 1994; 12: 234-239Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 7Jungheim L.N. Shepherd T.A. Chem. Rev. 1994; 94: 1553-1566Crossref Scopus (96) Google Scholar, 8Bignami G.S. Senter P.D. Grothaus P.G. Fischer K.J. Humphreys T. Wallace P.M. Cancer Res. 1992; 52: 5759-5764PubMed Google Scholar, 9Vrudhula V.M. Senter P.D. Fischer K.J. Wallace P.M. J. Med. Chem. 1993; 36: 919-923Crossref PubMed Scopus (63) Google Scholar, 10Wallace P.M. Senter P.D. Bioconjugate Chem. 1991; 2: 349-352Crossref PubMed Scopus (66) Google Scholar, 11Senter P.D. Schreiber G.J. Hirschberg D.L Ashe S.A. Hellstrom K.E. Hellstrom I. Cancer Res. 1989; 49: 5789-5792PubMed Google Scholar, 12Svensson H.P. Vrudhula V.M. Emswiler J.E. MacMaster J.F. Cosand W.L. Senter P.D. Wallace P.M. Cancer Res. 1995; 55: 2357-2365PubMed Google Scholar, 13Kerr D.E. Schreiber G.J. Vrudhula V.M. Svensson H.P. Hellstrom I. Hellstrom K.E. Senter P.D. Cancer Res. 1995; 55: 3558-3563PubMed Google Scholar, 14Bagshawe K.D. Springer C.J. Searle F. Antoniw P. Sharma S.K. Melton R.G. Sherwood R.F. Br. J. Cancer. 1988; 58: 700-703Crossref PubMed Scopus (257) Google Scholar, 15Eccles S.A. Court W.J. Box G.A. Dean C.J. Melton R.G. Springer C.J. Cancer Res. 1994; 54: 5171-5177PubMed Google Scholar, 16Blakey D.C. Burke P.J. Davies D.H. Dowell R.I. East S.J. Eckersley K.P. Fitton J.E. McDaid J. Melton R.G. Niculescu-Duvaz I.A. Pinder P.E. Sharma S.K. Wright A.F. Springer C.J. Cancer Res. 1996; 56: 3287-3292PubMed Google Scholar, 17Meyer D.L. Jungheim L.N. Law K.L Mikolajczyk S.D. Shepherd T.A. Mackensen D.G. Briggs S.L. Starling J.J. Cancer Res. 1993; 53: 3956-3963PubMed Google Scholar, 18Meyer D.L. Law K.L. Payne J.K. Mikolajczyk S.D. Zarrinmayen H. Jungheim L.N. Kling J.K. Shepherd T.A. Starling J.J. Bioconjugate Chem. 1995; 6: 440-446Crossref PubMed Scopus (24) Google Scholar, 19Kuefner U. Lohrmann U. Montejano Y.D. Vitols K.S. Huennekens F.M. Biochemistry. 1989; 28: 2288-2297Crossref PubMed Scopus (60) Google Scholar, 20Haenseler E. Esswein A. Vitols K.S. Montejano Y. Mueller B.M. Reisfeld R.A. Huennekens F.M. Biochemistry. 1992; 31: 891-897Crossref PubMed Scopus (49) Google Scholar, 21Vitols K.S. Haag-Zeino B. Baer T. Montejano Y.D. Huennekens F.M. Cancer Res. 1995; 55: 478-481PubMed Google Scholar, 22Haisma H.J. Boven E. van Muijen M. de Jong J. van der Vigh W.J.F. Pinedo H.M. Br. J. Cancer. 1992; 66: 474-478Crossref PubMed Scopus (100) Google Scholar, 23Wang S-M. Chern J-W. Yeh M-Y. Ng J.C. Tung E. Roffler S.R. Cancer Res. 1992; 52: 4484-4491PubMed Google Scholar, 24Bosslet K. Czeh J. Hoffmann D. Cancer Res. 1994; 54: 2151-2159PubMed Google Scholar, 25Senter P.D. Su P.C. Katsuragi T. Sakai T. Cosand W.L. Hellstrom I. Hellstrom K.E. Bioconjugate Chem. 1991; 2: 447-451Crossref PubMed Scopus (81) Google Scholar, 26Kerr D.E. Garrigues U.S. Wallace P.M. Hellstrom K.E. Hellstrom I. Senter P.D. Bioconjugate Chem. 1993; 4: 353-357Crossref PubMed Scopus (29) Google Scholar, 27Ledermann J.A. Begent R.H.J. Massof C. Kelly A.M.B. Adam T. Bagshawe K.D. Int. J. Cancer. 1991; 47: 659-664Crossref PubMed Scopus (39) Google Scholar, 28Sharma S.K. Bagshawe K.D. Melton R.G. Sherwood R.F. Cell Biophys. 1992; 21: 109-120Crossref PubMed Scopus (59) Google Scholar, 29Springer C.J. Poon G.K. Sharma S.K. Bagshawe K.D. Cell Biophys. 1993; 22: 9-26Crossref PubMed Scopus (23) Google Scholar). The concept has been shown to be effective both in in vitro and in vivo models, and at least one ADEPT strategy is currently undergoing clinical evaluation (27Ledermann J.A. Begent R.H.J. Massof C. Kelly A.M.B. Adam T. Bagshawe K.D. Int. J. Cancer. 1991; 47: 659-664Crossref PubMed Scopus (39) Google Scholar, 28Sharma S.K. Bagshawe K.D. Melton R.G. Sherwood R.F. Cell Biophys. 1992; 21: 109-120Crossref PubMed Scopus (59) Google Scholar, 29Springer C.J. Poon G.K. Sharma S.K. Bagshawe K.D. Cell Biophys. 1993; 22: 9-26Crossref PubMed Scopus (23) Google Scholar). ADEPT in vitroefficacy has been demonstrated with enzyme-antibody conjugates of (a) carboxypeptidase G2 along with several nitrogen mustards (14Bagshawe K.D. Springer C.J. Searle F. Antoniw P. Sharma S.K. Melton R.G. Sherwood R.F. Br. J. Cancer. 1988; 58: 700-703Crossref PubMed Scopus (257) Google Scholar, 15Eccles S.A. Court W.J. Box G.A. Dean C.J. Melton R.G. Springer C.J. Cancer Res. 1994; 54: 5171-5177PubMed Google Scholar, 16Blakey D.C. Burke P.J. Davies D.H. Dowell R.I. East S.J. Eckersley K.P. Fitton J.E. McDaid J. Melton R.G. Niculescu-Duvaz I.A. Pinder P.E. Sharma S.K. Wright A.F. Springer C.J. Cancer Res. 1996; 56: 3287-3292PubMed Google Scholar), (b) alkaline phosphatase with phosphorylated prodrugs of mitomycin, a phenol mustard, and etoposide (3Senter P.D. FASEB. 1990; 4: 188-193Crossref PubMed Scopus (124) Google Scholar, 10Wallace P.M. Senter P.D. Bioconjugate Chem. 1991; 2: 349-352Crossref PubMed Scopus (66) Google Scholar, 11Senter P.D. Schreiber G.J. Hirschberg D.L Ashe S.A. Hellstrom K.E. Hellstrom I. Cancer Res. 1989; 49: 5789-5792PubMed Google Scholar), (c) β-lactamase with lactam prodrugs of doxorubicin, vinca alkaloid analogs, and a nitrogen mustard (7Jungheim L.N. Shepherd T.A. Chem. Rev. 1994; 94: 1553-1566Crossref Scopus (96) Google Scholar, 12Svensson H.P. Vrudhula V.M. Emswiler J.E. MacMaster J.F. Cosand W.L. Senter P.D. Wallace P.M. Cancer Res. 1995; 55: 2357-2365PubMed Google Scholar, 13Kerr D.E. Schreiber G.J. Vrudhula V.M. Svensson H.P. Hellstrom I. Hellstrom K.E. Senter P.D. Cancer Res. 1995; 55: 3558-3563PubMed Google Scholar, 17Meyer D.L. Jungheim L.N. Law K.L Mikolajczyk S.D. Shepherd T.A. Mackensen D.G. Briggs S.L. Starling J.J. Cancer Res. 1993; 53: 3956-3963PubMed Google Scholar, 18Meyer D.L. Law K.L. Payne J.K. Mikolajczyk S.D. Zarrinmayen H. Jungheim L.N. Kling J.K. Shepherd T.A. Starling J.J. Bioconjugate Chem. 1995; 6: 440-446Crossref PubMed Scopus (24) Google Scholar), (d) penicillin-G amidase with prodrugs of palytoxin, doxorubicin and melphalan (8Bignami G.S. Senter P.D. Grothaus P.G. Fischer K.J. Humphreys T. Wallace P.M. Cancer Res. 1992; 52: 5759-5764PubMed Google Scholar, 9Vrudhula V.M. Senter P.D. Fischer K.J. Wallace P.M. J. Med. Chem. 1993; 36: 919-923Crossref PubMed Scopus (63) Google Scholar), (e) penicillin-V amidase with a prodrug of doxorubicin (2Bagshawe K.D. Br. J. Cancer. 1989; 60: 275-281Crossref PubMed Scopus (118) Google Scholar, 7Jungheim L.N. Shepherd T.A. Chem. Rev. 1994; 94: 1553-1566Crossref Scopus (96) Google Scholar), (f) human orEscherichia coli β-glucuronidase with glucuronide prodrugs of epirubicin, doxorubicin and a nitrogen mustard (22Haisma H.J. Boven E. van Muijen M. de Jong J. van der Vigh W.J.F. Pinedo H.M. Br. J. Cancer. 1992; 66: 474-478Crossref PubMed Scopus (100) Google Scholar, 23Wang S-M. Chern J-W. Yeh M-Y. Ng J.C. Tung E. Roffler S.R. Cancer Res. 1992; 52: 4484-4491PubMed Google Scholar, 24Bosslet K. Czeh J. Hoffmann D. Cancer Res. 1994; 54: 2151-2159PubMed Google Scholar), (g) cytosine deaminase and 5-fluorocytosine (25Senter P.D. Su P.C. Katsuragi T. Sakai T. Cosand W.L. Hellstrom I. Hellstrom K.E. Bioconjugate Chem. 1991; 2: 447-451Crossref PubMed Scopus (81) Google Scholar, 26Kerr D.E. Garrigues U.S. Wallace P.M. Hellstrom K.E. Hellstrom I. Senter P.D. Bioconjugate Chem. 1993; 4: 353-357Crossref PubMed Scopus (29) Google Scholar), and (h) bovine carboxypeptidase A and α-amino acid prodrugs of MTX (19Kuefner U. Lohrmann U. Montejano Y.D. Vitols K.S. Huennekens F.M. Biochemistry. 1989; 28: 2288-2297Crossref PubMed Scopus (60) Google Scholar, 20Haenseler E. Esswein A. Vitols K.S. Montejano Y. Mueller B.M. Reisfeld R.A. Huennekens F.M. Biochemistry. 1992; 31: 891-897Crossref PubMed Scopus (49) Google Scholar, 21Vitols K.S. Haag-Zeino B. Baer T. Montejano Y.D. Huennekens F.M. Cancer Res. 1995; 55: 478-481PubMed Google Scholar). Further, in vivo antitumor efficacy has been shown in a number of systems using the enzymes alkaline phosphatase, carboxypeptidase G2, β-lactamase, and β-glucuronidase (2Bagshawe K.D. Br. J. Cancer. 1989; 60: 275-281Crossref PubMed Scopus (118) Google Scholar, 3Senter P.D. FASEB. 1990; 4: 188-193Crossref PubMed Scopus (124) Google Scholar, 7Jungheim L.N. Shepherd T.A. Chem. Rev. 1994; 94: 1553-1566Crossref Scopus (96) Google Scholar,10Wallace P.M. Senter P.D. Bioconjugate Chem. 1991; 2: 349-352Crossref PubMed Scopus (66) Google Scholar, 11Senter P.D. Schreiber G.J. Hirschberg D.L Ashe S.A. Hellstrom K.E. Hellstrom I. Cancer Res. 1989; 49: 5789-5792PubMed Google Scholar, 12Svensson H.P. Vrudhula V.M. Emswiler J.E. MacMaster J.F. Cosand W.L. Senter P.D. Wallace P.M. Cancer Res. 1995; 55: 2357-2365PubMed Google Scholar, 13Kerr D.E. Schreiber G.J. Vrudhula V.M. Svensson H.P. Hellstrom I. Hellstrom K.E. Senter P.D. Cancer Res. 1995; 55: 3558-3563PubMed Google Scholar, 15Eccles S.A. Court W.J. Box G.A. Dean C.J. Melton R.G. Springer C.J. Cancer Res. 1994; 54: 5171-5177PubMed Google Scholar, 16Blakey D.C. Burke P.J. Davies D.H. Dowell R.I. East S.J. Eckersley K.P. Fitton J.E. McDaid J. Melton R.G. Niculescu-Duvaz I.A. Pinder P.E. Sharma S.K. Wright A.F. Springer C.J. Cancer Res. 1996; 56: 3287-3292PubMed Google Scholar, 17Meyer D.L. Jungheim L.N. Law K.L Mikolajczyk S.D. Shepherd T.A. Mackensen D.G. Briggs S.L. Starling J.J. Cancer Res. 1993; 53: 3956-3963PubMed Google Scholar, 18Meyer D.L. Law K.L. Payne J.K. Mikolajczyk S.D. Zarrinmayen H. Jungheim L.N. Kling J.K. Shepherd T.A. Starling J.J. Bioconjugate Chem. 1995; 6: 440-446Crossref PubMed Scopus (24) Google Scholar, 24Bosslet K. Czeh J. Hoffmann D. Cancer Res. 1994; 54: 2151-2159PubMed Google Scholar).An inherent problem with antibody-targeted therapies is the immune response mounted by the host to the foreign proteins and other antigens used in the therapy (1Sell S. Reisfeld R.A. Monoclonal Antibodies in Cancer. Humana Press, Clifton, NJ1985Crossref Google Scholar, 28Sharma S.K. Bagshawe K.D. Melton R.G. Sherwood R.F. Cell Biophys. 1992; 21: 109-120Crossref PubMed Scopus (59) Google Scholar). For example, monoclonal antibodies used in antibody targeting-based therapies are in general rodent in origin and as such recognized by the immune system (1Sell S. Reisfeld R.A. Monoclonal Antibodies in Cancer. Humana Press, Clifton, NJ1985Crossref Google Scholar, 28Sharma S.K. Bagshawe K.D. Melton R.G. Sherwood R.F. Cell Biophys. 1992; 21: 109-120Crossref PubMed Scopus (59) Google Scholar). ADEPT has the additional problem that the enzyme used to generate the site-specific drug synthesis can also be immunogenic, especially when a foreign enzyme is used. The immunogenicity associated with these foreign antibody or enzyme proteins decreases the utility of the antibody-targeting strategies by decreasing the ability of the physician to perform multiple dosing regimens.Attempts are being made to overcome the immune response to the rodent antibodies through “humanization” of the antibodies (30Reichmann L. Clark M. Waldmann H. Winter G. Nature. 1988; 332: 323-327Crossref PubMed Scopus (1307) Google Scholar). In this strategy, much of the sequence of the mouse monoclonal antibody is replaced with corresponding human antibody sequence. Only selected residues at the antigen combining site are left intact, leaving relatively few “rodent residues” remaining in the antibody.We reasoned that the imunogenicity associated with the use of enzymes of nonhuman origin might be circumvented through a similar strategy. However, we chose not to precisely follow the strategy of antibody humanization, which commences the process with the binding site of a foreign protein. Rather, our approach to generate a composite human/nonhuman enzyme was to start with a fully human enzyme and change the “active site” at one or two residues to produce a >99.5% human enzyme capable of efficiently performing a non-human reaction. This human enzyme with non-human specificity, along with humanized antibodies, should then facilitate the production of enzyme:antibody conjugates having lower immunogenicity and benefit the development of multiple dosing regimen ADEPT strategies.Our initial target to generate a human enzyme with non-human specificity was human pancreatic carboxypeptidase A, recently cloned and expressed in our laboratory (31Laethem R.M. Blumenkopf T.A. Cory M. Elwell L. Moxham C.P. Ray P.H. Walton L.M. Smith G.K. Arch. Biochem. Biophys. 1996; 332: 8-18Crossref PubMed Scopus (23) Google Scholar). Pancreatic carboxypeptidase A is a zinc-containing exopeptidase released into the small intestine from the pancreas as a zymogen (32Christianson D.W. Lipscomb W.N. Acc. Chem. Res. 1989; 22: 62-69Crossref Scopus (645) Google Scholar, 33Peterson L.M. Sokolovsky M. Vallee B.L. Biochemistry. 1976; 15: 2501-2508Crossref PubMed Scopus (49) Google Scholar). The pancreatic CPA has two further subclasses, CPA1 and CPA2, in both humans (31Laethem R.M. Blumenkopf T.A. Cory M. Elwell L. Moxham C.P. Ray P.H. Walton L.M. Smith G.K. Arch. Biochem. Biophys. 1996; 332: 8-18Crossref PubMed Scopus (23) Google Scholar, 34Catasús L. Villegas V. Pascual R. Aviles F.X. Wicker-Planquart C. Puigserver A. Biochem. J. 1992; 287: 299-303Crossref PubMed Scopus (10) Google Scholar, 35Catasús L Vendrell J. Aviles F.X. Carreira S. Puigserver A. Billeter M. J. Biol. Chem. 1995; 270: 6651-6657Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) and rats (36Quinto C. Quiroga M. Swain W.F. Nikovitis Jr., W.C. Standring D.N. Pictet R.L. Valenzuela P. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 31-35Crossref PubMed Scopus (67) Google Scholar,37Gardell S.J. Craik C.S. Clauser E. Goldsmith E.J. Stewart C-B. Graf M. Rutter W.J. J. Biol. Chem. 1988; 263: 17828-17836Abstract Full Text PDF PubMed Google Scholar). While the amino acid sequences of CPA1 and CPA2 active sites are similar and both enzymes prefer aromatic C-terminal amino acids, CPA2 enzyme prefers bulkier aromatic C-terminal amino acids (31Laethem R.M. Blumenkopf T.A. Cory M. Elwell L. Moxham C.P. Ray P.H. Walton L.M. Smith G.K. Arch. Biochem. Biophys. 1996; 332: 8-18Crossref PubMed Scopus (23) Google Scholar, 37Gardell S.J. Craik C.S. Clauser E. Goldsmith E.J. Stewart C-B. Graf M. Rutter W.J. J. Biol. Chem. 1988; 263: 17828-17836Abstract Full Text PDF PubMed Google Scholar). This was shown for the rat enzyme with di- and tripeptide substrates and for the human with amino acid prodrugs of MTX (31Laethem R.M. Blumenkopf T.A. Cory M. Elwell L. Moxham C.P. Ray P.H. Walton L.M. Smith G.K. Arch. Biochem. Biophys. 1996; 332: 8-18Crossref PubMed Scopus (23) Google Scholar, 37Gardell S.J. Craik C.S. Clauser E. Goldsmith E.J. Stewart C-B. Graf M. Rutter W.J. J. Biol. Chem. 1988; 263: 17828-17836Abstract Full Text PDF PubMed Google Scholar). High resolution crystal structures for bCPA have been determined (32Christianson D.W. Lipscomb W.N. Acc. Chem. Res. 1989; 22: 62-69Crossref Scopus (645) Google Scholar). Of the nine active site residues within 4.5 Å of the bound substrate, only three vary among bovine CPA (32Christianson D.W. Lipscomb W.N. Acc. Chem. Res. 1989; 22: 62-69Crossref Scopus (645) Google Scholar), rat CPA1 (36Quinto C. Quiroga M. Swain W.F. Nikovitis Jr., W.C. Standring D.N. Pictet R.L. Valenzuela P. Rutter W.J. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 31-35Crossref PubMed Scopus (67) Google Scholar), rCPA2 (37Gardell S.J. Craik C.S. Clauser E. Goldsmith E.J. Stewart C-B. Graf M. Rutter W.J. J. Biol. Chem. 1988; 263: 17828-17836Abstract Full Text PDF PubMed Google Scholar), hCPA1 (31Laethem R.M. Blumenkopf T.A. Cory M. Elwell L. Moxham C.P. Ray P.H. Walton L.M. Smith G.K. Arch. Biochem. Biophys. 1996; 332: 8-18Crossref PubMed Scopus (23) Google Scholar, 34Catasús L. Villegas V. Pascual R. Aviles F.X. Wicker-Planquart C. Puigserver A. Biochem. J. 1992; 287: 299-303Crossref PubMed Scopus (10) Google Scholar), and hCPA2 (31Laethem R.M. Blumenkopf T.A. Cory M. Elwell L. Moxham C.P. Ray P.H. Walton L.M. Smith G.K. Arch. Biochem. Biophys. 1996; 332: 8-18Crossref PubMed Scopus (23) Google Scholar, 35Catasús L Vendrell J. Aviles F.X. Carreira S. Puigserver A. Billeter M. J. Biol. Chem. 1995; 270: 6651-6657Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These changes, at residues" @default.
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W2102970512 title "Toward Antibody-directed Enzyme Prodrug Therapy with the T268G Mutant of Human Carboxypeptidase A1 and Novel in VivoStable Prodrugs of Methotrexate" @default.
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