Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein
MFE-CP is a recombinant antibody-enzyme fusion protein used for antibody-mediated delivery of an enzyme to cancer deposits. After clearance from normal tissues, the tumor-targeted enzyme is used to activate a subsequently administered prodrug to give a potent cytotoxic in the tumor. MFE-CP localizes...
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| Veröffentlicht in: | Glycobiology (Oxford) 2004-01, Vol.14 (1), p.27-37 |
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| creator | Medzihradszky, Katalin F. Spencer, Daniel I.R. Sharma, Surinder K. Bhatia, Jeetendra Pedley, R. Barbara Read, David A. Begent, Richard H.J. Chester, Kerry A. |
| description | MFE-CP is a recombinant antibody-enzyme fusion protein used for antibody-mediated delivery of an enzyme to cancer deposits. After clearance from normal tissues, the tumor-targeted enzyme is used to activate a subsequently administered prodrug to give a potent cytotoxic in the tumor. MFE-CP localizes to cancer deposits in vivo, but we propose that its therapeutic potential could be improved by N-glycosylation, obtained by expression in Pichia pastoris. Glycosylation could enhance clearance from healthy tissue and result in better tumor:normal tissue ratios. To test this, glycosylated MFE-CP was expressed and purified from P. pastoris. The resultant MFE-CP fusion protein was enzymatically active and showed enhanced clearance from normal tissues in vivo. Furthermore, it showed effective tumor localization. This favorable glycosylation pattern was analyzed by tandem mass spectrometry. High-resolution, high-detection sensitivity collision-induced dissociation experiments proved essential for this task. Results showed that of the three potential N-glycosylation sites only two were consistently occupied with oligomannose structures. Asn-442 appeared the most heterogeneously populated with oligomannose carbohydrates extending from 5 to 13 units in length. Asn-484 was found only in its nonglycosylated form. There was less heterogeneity at Asn-492, which was glycosylated with oligosaccharide structures ranging from 8 to 10 mannose units. Nonglycosylated forms of Asn-442 and Asn-492 were not observed. |
| doi_str_mv | 10.1093/glycob/cwh001 |
| format | Article |
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The resultant MFE-CP fusion protein was enzymatically active and showed enhanced clearance from normal tissues in vivo. Furthermore, it showed effective tumor localization. This favorable glycosylation pattern was analyzed by tandem mass spectrometry. High-resolution, high-detection sensitivity collision-induced dissociation experiments proved essential for this task. Results showed that of the three potential N-glycosylation sites only two were consistently occupied with oligomannose structures. Asn-442 appeared the most heterogeneously populated with oligomannose carbohydrates extending from 5 to 13 units in length. Asn-484 was found only in its nonglycosylated form. There was less heterogeneity at Asn-492, which was glycosylated with oligosaccharide structures ranging from 8 to 10 mannose units. Nonglycosylated forms of Asn-442 and Asn-492 were not observed.</description><identifier>ISSN: 0959-6658</identifier><identifier>ISSN: 1460-2423</identifier><identifier>EISSN: 1460-2423</identifier><identifier>DOI: 10.1093/glycob/cwh001</identifier><identifier>PMID: 14514711</identifier><language>eng</language><publisher>England: Oxford University Press</publisher><subject>ADEPT ; Amino Acid Sequence ; Antibodies - therapeutic use ; antibody directed enzyme prodrug therapy ; Antineoplastic Agents - chemical synthesis ; Antineoplastic Agents - therapeutic use ; Carbohydrate Conformation ; Carbohydrate Sequence ; carboxypeptidase enzyme G2 ; carcinoembryonic antigen ; CEA ; CID ; collision-induced dissociation ; CPG2 ; electrospray ionization mass spectrometry ; Enzymes - therapeutic use ; ESI-MS ; Glycopeptides - chemical synthesis ; Glycopeptides - therapeutic use ; high-performance liquid chromatography ; HPLC ; liquid chromatography ; MALDI ; mass spectrometry ; matrix-assisted laser desorption ionization ; methotrexate ; Molecular Sequence Data ; MTX ; N-glycosylation ; Oligosaccharides - chemistry ; Peptide Fragments - chemistry ; Pichia - genetics ; Pichia - immunology ; Pichia pastoris ; postsource decay ; PSD ; Recombinant Fusion Proteins - therapeutic use ; Spectrometry, Mass, Electrospray Ionization - methods ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods ; TFA ; trifluoracetic acid</subject><ispartof>Glycobiology (Oxford), 2004-01, Vol.14 (1), p.27-37</ispartof><rights>Copyright Oxford University Press(England) Jan 2004</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c424t-ba1f12fbc5124d4b49a60fdd3b0872e09afc0df81abdfcd145ebf72e0b6dbcbb3</citedby></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><link.rule.ids>314,776,780,27903,27904</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/14514711$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Medzihradszky, Katalin F.</creatorcontrib><creatorcontrib>Spencer, Daniel I.R.</creatorcontrib><creatorcontrib>Sharma, Surinder K.</creatorcontrib><creatorcontrib>Bhatia, Jeetendra</creatorcontrib><creatorcontrib>Pedley, R. Barbara</creatorcontrib><creatorcontrib>Read, David A.</creatorcontrib><creatorcontrib>Begent, Richard H.J.</creatorcontrib><creatorcontrib>Chester, Kerry A.</creatorcontrib><title>Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein</title><title>Glycobiology (Oxford)</title><addtitle>Glycobiology</addtitle><description>MFE-CP is a recombinant antibody-enzyme fusion protein used for antibody-mediated delivery of an enzyme to cancer deposits. After clearance from normal tissues, the tumor-targeted enzyme is used to activate a subsequently administered prodrug to give a potent cytotoxic in the tumor. MFE-CP localizes to cancer deposits in vivo, but we propose that its therapeutic potential could be improved by N-glycosylation, obtained by expression in Pichia pastoris. Glycosylation could enhance clearance from healthy tissue and result in better tumor:normal tissue ratios. To test this, glycosylated MFE-CP was expressed and purified from P. pastoris. The resultant MFE-CP fusion protein was enzymatically active and showed enhanced clearance from normal tissues in vivo. Furthermore, it showed effective tumor localization. This favorable glycosylation pattern was analyzed by tandem mass spectrometry. High-resolution, high-detection sensitivity collision-induced dissociation experiments proved essential for this task. Results showed that of the three potential N-glycosylation sites only two were consistently occupied with oligomannose structures. Asn-442 appeared the most heterogeneously populated with oligomannose carbohydrates extending from 5 to 13 units in length. Asn-484 was found only in its nonglycosylated form. There was less heterogeneity at Asn-492, which was glycosylated with oligosaccharide structures ranging from 8 to 10 mannose units. Nonglycosylated forms of Asn-442 and Asn-492 were not observed.</description><subject>ADEPT</subject><subject>Amino Acid Sequence</subject><subject>Antibodies - therapeutic use</subject><subject>antibody directed enzyme prodrug therapy</subject><subject>Antineoplastic Agents - chemical synthesis</subject><subject>Antineoplastic Agents - therapeutic use</subject><subject>Carbohydrate Conformation</subject><subject>Carbohydrate Sequence</subject><subject>carboxypeptidase enzyme G2</subject><subject>carcinoembryonic antigen</subject><subject>CEA</subject><subject>CID</subject><subject>collision-induced dissociation</subject><subject>CPG2</subject><subject>electrospray ionization mass spectrometry</subject><subject>Enzymes - therapeutic use</subject><subject>ESI-MS</subject><subject>Glycopeptides - chemical synthesis</subject><subject>Glycopeptides - therapeutic use</subject><subject>high-performance liquid chromatography</subject><subject>HPLC</subject><subject>liquid chromatography</subject><subject>MALDI</subject><subject>mass spectrometry</subject><subject>matrix-assisted laser desorption ionization</subject><subject>methotrexate</subject><subject>Molecular Sequence Data</subject><subject>MTX</subject><subject>N-glycosylation</subject><subject>Oligosaccharides - chemistry</subject><subject>Peptide Fragments - chemistry</subject><subject>Pichia - genetics</subject><subject>Pichia - immunology</subject><subject>Pichia pastoris</subject><subject>postsource decay</subject><subject>PSD</subject><subject>Recombinant Fusion Proteins - therapeutic use</subject><subject>Spectrometry, Mass, Electrospray Ionization - methods</subject><subject>Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods</subject><subject>TFA</subject><subject>trifluoracetic acid</subject><issn>0959-6658</issn><issn>1460-2423</issn><issn>1460-2423</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2004</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkU1v1DAQhi0EokvhyBVZHLiF2onjbI6woh-iAg5FQlwsf4y3LokdbAea_gH-Nl52RSUuHEZzmGee0ehF6Dklrynpm5PtsOigTvTPa0LoA7SijJOqZnXzEK1I3_YV5-36CD1J6aYAnK7bx-iIspayjtIV-nW227chjgkHlaXzYLBaMNxOEVJywWPn8Senr53Ek0w5RJewG6cYfgDW0muIOMu4hez8Fk8hg89ODjhYLHEEHUblvPQZl3IqmKUCf7eMgO38x15EGZx_ih5ZOSR4dujH6PPpu6vNeXX58exi8-ay0qxmuVKSWlpbpVtaM8MU6yUn1phGkXVXA-ml1cTYNZXKWG3Kn6DsbqC4UVqp5hi92nvL3e8zpCxGlzQMg_QQ5iQ62rY9I-S_YE0Y73pOC_jyH_AmzNGXJ0RNScNY1_QFqvaQjiGlCFZM0Y0yLoISsctR7HMU-xwL_-IgndUI5p4-BHcvdCnD7d-5jN8E75quFedfvoq3_OoDPX2_EbT5DVaNrs0</recordid><startdate>20040101</startdate><enddate>20040101</enddate><creator>Medzihradszky, Katalin F.</creator><creator>Spencer, Daniel I.R.</creator><creator>Sharma, Surinder K.</creator><creator>Bhatia, Jeetendra</creator><creator>Pedley, R. Barbara</creator><creator>Read, David A.</creator><creator>Begent, Richard H.J.</creator><creator>Chester, Kerry A.</creator><general>Oxford University Press</general><general>Oxford Publishing Limited (England)</general><scope>BSCLL</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7QL</scope><scope>7QO</scope><scope>7TK</scope><scope>7U9</scope><scope>8FD</scope><scope>C1K</scope><scope>FR3</scope><scope>H94</scope><scope>K9.</scope><scope>M7N</scope><scope>P64</scope><scope>RC3</scope><scope>7X8</scope></search><sort><creationdate>20040101</creationdate><title>Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein</title><author>Medzihradszky, Katalin F. ; Spencer, Daniel I.R. ; Sharma, Surinder K. ; Bhatia, Jeetendra ; Pedley, R. 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Barbara</au><au>Read, David A.</au><au>Begent, Richard H.J.</au><au>Chester, Kerry A.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein</atitle><jtitle>Glycobiology (Oxford)</jtitle><addtitle>Glycobiology</addtitle><date>2004-01-01</date><risdate>2004</risdate><volume>14</volume><issue>1</issue><spage>27</spage><epage>37</epage><pages>27-37</pages><issn>0959-6658</issn><issn>1460-2423</issn><eissn>1460-2423</eissn><abstract>MFE-CP is a recombinant antibody-enzyme fusion protein used for antibody-mediated delivery of an enzyme to cancer deposits. After clearance from normal tissues, the tumor-targeted enzyme is used to activate a subsequently administered prodrug to give a potent cytotoxic in the tumor. MFE-CP localizes to cancer deposits in vivo, but we propose that its therapeutic potential could be improved by N-glycosylation, obtained by expression in Pichia pastoris. Glycosylation could enhance clearance from healthy tissue and result in better tumor:normal tissue ratios. To test this, glycosylated MFE-CP was expressed and purified from P. pastoris. The resultant MFE-CP fusion protein was enzymatically active and showed enhanced clearance from normal tissues in vivo. Furthermore, it showed effective tumor localization. This favorable glycosylation pattern was analyzed by tandem mass spectrometry. High-resolution, high-detection sensitivity collision-induced dissociation experiments proved essential for this task. Results showed that of the three potential N-glycosylation sites only two were consistently occupied with oligomannose structures. Asn-442 appeared the most heterogeneously populated with oligomannose carbohydrates extending from 5 to 13 units in length. Asn-484 was found only in its nonglycosylated form. There was less heterogeneity at Asn-492, which was glycosylated with oligosaccharide structures ranging from 8 to 10 mannose units. Nonglycosylated forms of Asn-442 and Asn-492 were not observed.</abstract><cop>England</cop><pub>Oxford University Press</pub><pmid>14514711</pmid><doi>10.1093/glycob/cwh001</doi><tpages>11</tpages><oa>free_for_read</oa></addata></record> |
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| ispartof | Glycobiology (Oxford), 2004-01, Vol.14 (1), p.27-37 |
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| source | Oxford University Press Journals All Titles (1996-Current); MEDLINE; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; Alma/SFX Local Collection |
| subjects | ADEPT Amino Acid Sequence Antibodies - therapeutic use antibody directed enzyme prodrug therapy Antineoplastic Agents - chemical synthesis Antineoplastic Agents - therapeutic use Carbohydrate Conformation Carbohydrate Sequence carboxypeptidase enzyme G2 carcinoembryonic antigen CEA CID collision-induced dissociation CPG2 electrospray ionization mass spectrometry Enzymes - therapeutic use ESI-MS Glycopeptides - chemical synthesis Glycopeptides - therapeutic use high-performance liquid chromatography HPLC liquid chromatography MALDI mass spectrometry matrix-assisted laser desorption ionization methotrexate Molecular Sequence Data MTX N-glycosylation Oligosaccharides - chemistry Peptide Fragments - chemistry Pichia - genetics Pichia - immunology Pichia pastoris postsource decay PSD Recombinant Fusion Proteins - therapeutic use Spectrometry, Mass, Electrospray Ionization - methods Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - methods TFA trifluoracetic acid |
| title | Glycoforms obtained by expression in Pichia pastoris improve cancer targeting potential of a recombinant antibody-enzyme fusion protein |
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