Simultaneous and Independent Dual Site-Specific Self-Labeling of Recombinant Antibodies
Antibody-based diagnostic and therapeutic reagents armed with effector molecules such as dyes and drugs offer hope in the battle against cancer. Several site-specific conjugation methods have been developed to equip antibodies with such effector molecules, but they tend to be expensive and involve m...
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| Veröffentlicht in: | Bioconjugate chemistry 2018-11, Vol.29 (11), p.3586-3594 |
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| creator | Wollschlaeger, Carolin Meinhold-Heerlein, Ivo Cong, Xiaojing Bräutigam, Karen Di Fiore, Stefano Zeppernick, Felix Klockenbring, Torsten Stickeler, Elmar Barth, Stefan Hussain, Ahmad Fawzi |
| description | Antibody-based diagnostic and therapeutic reagents armed with effector molecules such as dyes and drugs offer hope in the battle against cancer. Several site-specific conjugation methods have been developed to equip antibodies with such effector molecules, but they tend to be expensive and involve multiple reaction steps. The conjugation of two different effector molecules to a single antibody also remains a major challenge. Here we describe a simple, controlled, and robust method for the dual site-specific conjugation of an antibody with two effector molecules in a single-pot reaction using the self-labeling SNAP and CLIP protein tags. We verified the principle of the method by labeling an epidermal growth factor receptor (EGFR)-specific single-chain antibody fragment (scFv-425) simultaneously with IRDye700 and Alexa-Fluor647. This dual-labeled antibody bound to EGFR+ ovarian cancer cell lines and tissue samples with high specificity, and its phototherapeutic efficacy was confirmed by the selective killing of EGFR+ cells in vitro. |
| doi_str_mv | 10.1021/acs.bioconjchem.8b00545 |
| format | Article |
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Several site-specific conjugation methods have been developed to equip antibodies with such effector molecules, but they tend to be expensive and involve multiple reaction steps. The conjugation of two different effector molecules to a single antibody also remains a major challenge. Here we describe a simple, controlled, and robust method for the dual site-specific conjugation of an antibody with two effector molecules in a single-pot reaction using the self-labeling SNAP and CLIP protein tags. We verified the principle of the method by labeling an epidermal growth factor receptor (EGFR)-specific single-chain antibody fragment (scFv-425) simultaneously with IRDye700 and Alexa-Fluor647. 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Several site-specific conjugation methods have been developed to equip antibodies with such effector molecules, but they tend to be expensive and involve multiple reaction steps. The conjugation of two different effector molecules to a single antibody also remains a major challenge. Here we describe a simple, controlled, and robust method for the dual site-specific conjugation of an antibody with two effector molecules in a single-pot reaction using the self-labeling SNAP and CLIP protein tags. We verified the principle of the method by labeling an epidermal growth factor receptor (EGFR)-specific single-chain antibody fragment (scFv-425) simultaneously with IRDye700 and Alexa-Fluor647. This dual-labeled antibody bound to EGFR+ ovarian cancer cell lines and tissue samples with high specificity, and its phototherapeutic efficacy was confirmed by the selective killing of EGFR+ cells in vitro.</description><subject>Antibodies</subject><subject>Cancer</subject><subject>Cell Line, Tumor</subject><subject>Cells</subject><subject>Coloring Agents - chemistry</subject><subject>Conjugation</subject><subject>Diagnostic systems</subject><subject>Diagnostics</subject><subject>Drug therapy</subject><subject>Epidermal growth factor</subject><subject>Epidermal growth factor receptors</subject><subject>ErbB Receptors - immunology</subject><subject>ErbB Receptors - metabolism</subject><subject>Female</subject><subject>Flow Cytometry</subject><subject>Growth factors</subject><subject>Humans</subject><subject>Immunoconjugates - chemistry</subject><subject>Immunoglobulins</subject><subject>Labeling</subject><subject>Microscopy, Confocal</subject><subject>Ovarian cancer</subject><subject>Ovarian Neoplasms - pathology</subject><subject>Protein Binding</subject><subject>Protein Conformation</subject><subject>Proteins</subject><subject>Reagents</subject><subject>Recombinant Fusion Proteins - chemistry</subject><subject>Recombinant Fusion Proteins - immunology</subject><subject>Robust control</subject><subject>Single-Chain Antibodies - chemistry</subject><subject>Single-Chain Antibodies - immunology</subject><subject>Tumor cell lines</subject><issn>1043-1802</issn><issn>1520-4812</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2018</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNqFkEtP3DAURi1UBFPgL9BI3bDJcP1I4lmOeBVppEoExDLy47r1KLGncbLov6_RDKjqppvruzjfZ_sQ8oXCkgKj18qkpfbRxLA1P3FYSg1QieqILGjFoBSSsk95B8FLKoGdks8pbQFgRSU7IaccmFwxwRbktfXD3E8qYJxToYItHoPFHeYRpuJ2Vn3R-gnLdofGO2-KFntXbpTG3ocfRXTFE5o4aB9U5tdh8jpaj-mcHDvVJ7w4nGfk5f7u-eZbufn-8Hiz3pSKy2oqtWNCo1lpy1QNVoETjguUKyqYhQYbtNJWaLgBp-tGgUSuWVNXDlyF0vAzcrXv3Y3x14xp6gafDPb9_kcdo7SWFeNUZPTrP-g2zmPIr8tUTbMRWkOmmj1lxpjSiK7bjX5Q4--OQvfmvsvuu7_cdwf3OXl56J_1gPYj9y47A3wPvDV83P2_2j8r4pZ1</recordid><startdate>20181121</startdate><enddate>20181121</enddate><creator>Wollschlaeger, Carolin</creator><creator>Meinhold-Heerlein, Ivo</creator><creator>Cong, Xiaojing</creator><creator>Bräutigam, Karen</creator><creator>Di Fiore, Stefano</creator><creator>Zeppernick, Felix</creator><creator>Klockenbring, Torsten</creator><creator>Stickeler, Elmar</creator><creator>Barth, Stefan</creator><creator>Hussain, Ahmad Fawzi</creator><general>American Chemical Society</general><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>7QO</scope><scope>7TM</scope><scope>8FD</scope><scope>FR3</scope><scope>P64</scope><scope>7X8</scope><orcidid>https://orcid.org/0000-0002-5051-2392</orcidid><orcidid>https://orcid.org/0000-0002-1053-6939</orcidid></search><sort><creationdate>20181121</creationdate><title>Simultaneous and Independent Dual Site-Specific Self-Labeling of Recombinant Antibodies</title><author>Wollschlaeger, Carolin ; Meinhold-Heerlein, Ivo ; Cong, Xiaojing ; Bräutigam, Karen ; Di Fiore, Stefano ; Zeppernick, Felix ; Klockenbring, Torsten ; Stickeler, Elmar ; Barth, Stefan ; Hussain, Ahmad Fawzi</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-a385t-bf24bec9bd2a60da0f4f34e89142d07e7ed8d5ec3c0fb67a08e3b2765f0f5e8c3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2018</creationdate><topic>Antibodies</topic><topic>Cancer</topic><topic>Cell Line, Tumor</topic><topic>Cells</topic><topic>Coloring Agents - chemistry</topic><topic>Conjugation</topic><topic>Diagnostic systems</topic><topic>Diagnostics</topic><topic>Drug therapy</topic><topic>Epidermal growth factor</topic><topic>Epidermal growth factor receptors</topic><topic>ErbB Receptors - immunology</topic><topic>ErbB Receptors - metabolism</topic><topic>Female</topic><topic>Flow Cytometry</topic><topic>Growth factors</topic><topic>Humans</topic><topic>Immunoconjugates - chemistry</topic><topic>Immunoglobulins</topic><topic>Labeling</topic><topic>Microscopy, Confocal</topic><topic>Ovarian cancer</topic><topic>Ovarian Neoplasms - pathology</topic><topic>Protein Binding</topic><topic>Protein Conformation</topic><topic>Proteins</topic><topic>Reagents</topic><topic>Recombinant Fusion Proteins - chemistry</topic><topic>Recombinant Fusion Proteins - immunology</topic><topic>Robust control</topic><topic>Single-Chain Antibodies - chemistry</topic><topic>Single-Chain Antibodies - immunology</topic><topic>Tumor cell lines</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Wollschlaeger, Carolin</creatorcontrib><creatorcontrib>Meinhold-Heerlein, Ivo</creatorcontrib><creatorcontrib>Cong, Xiaojing</creatorcontrib><creatorcontrib>Bräutigam, Karen</creatorcontrib><creatorcontrib>Di Fiore, Stefano</creatorcontrib><creatorcontrib>Zeppernick, Felix</creatorcontrib><creatorcontrib>Klockenbring, Torsten</creatorcontrib><creatorcontrib>Stickeler, Elmar</creatorcontrib><creatorcontrib>Barth, Stefan</creatorcontrib><creatorcontrib>Hussain, Ahmad Fawzi</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>Biotechnology Research Abstracts</collection><collection>Nucleic Acids Abstracts</collection><collection>Technology Research Database</collection><collection>Engineering Research Database</collection><collection>Biotechnology and BioEngineering Abstracts</collection><collection>MEDLINE - Academic</collection><jtitle>Bioconjugate chemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Wollschlaeger, Carolin</au><au>Meinhold-Heerlein, Ivo</au><au>Cong, Xiaojing</au><au>Bräutigam, Karen</au><au>Di Fiore, Stefano</au><au>Zeppernick, Felix</au><au>Klockenbring, Torsten</au><au>Stickeler, Elmar</au><au>Barth, Stefan</au><au>Hussain, Ahmad Fawzi</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>Simultaneous and Independent Dual Site-Specific Self-Labeling of Recombinant Antibodies</atitle><jtitle>Bioconjugate chemistry</jtitle><addtitle>Bioconjugate Chem</addtitle><date>2018-11-21</date><risdate>2018</risdate><volume>29</volume><issue>11</issue><spage>3586</spage><epage>3594</epage><pages>3586-3594</pages><issn>1043-1802</issn><eissn>1520-4812</eissn><abstract>Antibody-based diagnostic and therapeutic reagents armed with effector molecules such as dyes and drugs offer hope in the battle against cancer. Several site-specific conjugation methods have been developed to equip antibodies with such effector molecules, but they tend to be expensive and involve multiple reaction steps. The conjugation of two different effector molecules to a single antibody also remains a major challenge. Here we describe a simple, controlled, and robust method for the dual site-specific conjugation of an antibody with two effector molecules in a single-pot reaction using the self-labeling SNAP and CLIP protein tags. We verified the principle of the method by labeling an epidermal growth factor receptor (EGFR)-specific single-chain antibody fragment (scFv-425) simultaneously with IRDye700 and Alexa-Fluor647. 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| ispartof | Bioconjugate chemistry, 2018-11, Vol.29 (11), p.3586-3594 |
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| subjects | Antibodies Cancer Cell Line, Tumor Cells Coloring Agents - chemistry Conjugation Diagnostic systems Diagnostics Drug therapy Epidermal growth factor Epidermal growth factor receptors ErbB Receptors - immunology ErbB Receptors - metabolism Female Flow Cytometry Growth factors Humans Immunoconjugates - chemistry Immunoglobulins Labeling Microscopy, Confocal Ovarian cancer Ovarian Neoplasms - pathology Protein Binding Protein Conformation Proteins Reagents Recombinant Fusion Proteins - chemistry Recombinant Fusion Proteins - immunology Robust control Single-Chain Antibodies - chemistry Single-Chain Antibodies - immunology Tumor cell lines |
| title | Simultaneous and Independent Dual Site-Specific Self-Labeling of Recombinant Antibodies |
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