{"id":{"repo_id":"mit","oai_identifier":"oai:dspace.mit.edu:1721.1/61232"},"canonical_url":"https://search.dev.ndltd.org/etd/mit/oai:dspace.mit.edu:1721.1/61232","repository":{"repo_id":"mit","name":"MIT","base_url":"https://dspace.mit.edu/oai/request"},"display":{"title":"Engineering aglycosylated antibody variants with immune effector functions","abstract":"Monoclonal antibodies have emerged as a promising class of therapeutics for the treatment of human disease, and in particular human cancer. While multiple mechanisms contribute to antibody efficacy, the engagement and activation of immune effector cells - mediated by the interaction of the conserved Fc regions of the antibody with the Fc gamma receptors (Fc[gamma]Rs) on immune cells - is critical to the efficacy of several. This thesis describes the engineering of antibody Fc domain interactions with Fc[gamma]Rs, using the' yeast S. cerevisiae. In an initial step, a microbial system for the production of full-length antibodies in S. cerevisiae in milligram per liter titers has been developed, which serves as a platform for the engineering of antibody Fc domains with defined properties. The presence of a single N-linked glycan on each chain of the antibody Fc, as well as the specific composition of the glycoforms comprising it, are critical to the binding of the Fc to Fc[gamma]Rs, and have largely limited the production of therapeutic antibodies to mammalian expression systems. Using a display system that tethers full-length antibodies on the surface of yeast, we identify and characterize aglycosylated antibody variants that bind a subset of the human low-affinity Fc[gamma]Rs, Fc[gamma]RIIA and Fc'yRIIB, with approximately wildtype binding affinity and activate immune effector functions in vivo. In a separate approach, we identify aglycosylated variants that weakly bind a third low-affinity receptor, Fc[gamma]RIIIA, and through subsequent engineering generate variants that bind all of the low-affinity Fc[gamma]Rs with approximately wild-type binding affinity. By decoupling the function of the antibody from its post-translational processing, these variants have the potential to open up therapeutic antibody production to a far wider array of expression systems than currently available. Finally, in parallel work, we use a similar system to screen for glycosylated Fc variants with improved affinity and specificity for the activating receptor Fc[gamma]RIIIA compared to the inhibitory receptor Fc[gamma]RIIB, properties which have been hypothesized to lead to more potent antibody therapeutics.","abstract_html":"Monoclonal antibodies have emerged as a promising class of therapeutics for the treatment of human disease, and in particular human cancer. While multiple mechanisms contribute to antibody efficacy, the engagement and activation of immune effector cells - mediated by the interaction of the conserved Fc regions of the antibody with the Fc gamma receptors (Fc[gamma]Rs) on immune cells - is critical to the efficacy of several. This thesis describes the engineering of antibody Fc domain interactions with Fc[gamma]Rs, using the' yeast S. cerevisiae. In an initial step, a microbial system for the production of full-length antibodies in S. cerevisiae in milligram per liter titers has been developed, which serves as a platform for the engineering of antibody Fc domains with defined properties. The presence of a single N-linked glycan on each chain of the antibody Fc, as well as the specific composition of the glycoforms comprising it, are critical to the binding of the Fc to Fc[gamma]Rs, and have largely limited the production of therapeutic antibodies to mammalian expression systems. Using a display system that tethers full-length antibodies on the surface of yeast, we identify and characterize aglycosylated antibody variants that bind a subset of the human low-affinity Fc[gamma]Rs, Fc[gamma]RIIA and Fc'yRIIB, with approximately wildtype binding affinity and activate immune effector functions in vivo. In a separate approach, we identify aglycosylated variants that weakly bind a third low-affinity receptor, Fc[gamma]RIIIA, and through subsequent engineering generate variants that bind all of the low-affinity Fc[gamma]Rs with approximately wild-type binding affinity. By decoupling the function of the antibody from its post-translational processing, these variants have the potential to open up therapeutic antibody production to a far wider array of expression systems than currently available. Finally, in parallel work, we use a similar system to screen for glycosylated Fc variants with improved affinity and specificity for the activating receptor Fc[gamma]RIIIA compared to the inhibitory receptor Fc[gamma]RIIB, properties which have been hypothesized to lead to more potent antibody therapeutics.","abstract_has_math":false,"creators":["Sazinsky, Stephen L. (Stephen Lael)"],"institution":"Massachusetts Institute of Technology","degree_name":null,"degree_level":null,"degree_discipline":null,"degree_department":"Massachusetts Institute of Technology. Dept. of Biological Engineering.","school":null,"contributors":[],"advisors":["K. Dane Wittrup."],"committee_chairs":[],"committee_members":[],"year":2009,"date_issued":"2009","date_published":"2009","updated_at":"2026-07-22T22:21:58Z","subjects":["Biological Engineering."],"languages":["eng"],"rights":["MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."],"rights_urls":["http://dspace.mit.edu/handle/1721.1/7582"],"identifier_entries":[]},"links":{"outbound_url":"http://hdl.handle.net/1721.1/61232","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.advisor","label":"Advisor","values":["K. Dane Wittrup."]},{"key":"dc:contributor.department","label":"Department","values":["Massachusetts Institute of Technology. Dept. of Biological Engineering."]},{"key":"dc:contributor.other","label":"Dc Contributor Other","values":["Massachusetts Institute of Technology. Dept. of Biological Engineering."]},{"key":"dc:creator","label":"Author","values":["Sazinsky, Stephen L. 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They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://dspace.mit.edu/handle/1721.1/7582"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/1721.1/61232"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description","label":"Description","values":["Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biological Engineering, February 2009.","\"February 2008.\" Cataloged from PDF version of thesis.","Includes bibliographical references (p. 110-114)."]},{"key":"dc:description.abstract","label":"Abstract","values":["Monoclonal antibodies have emerged as a promising class of therapeutics for the treatment of human disease, and in particular human cancer. While multiple mechanisms contribute to antibody efficacy, the engagement and activation of immune effector cells - mediated by the interaction of the conserved Fc regions of the antibody with the Fc gamma receptors (Fc[gamma]Rs) on immune cells - is critical to the efficacy of several. This thesis describes the engineering of antibody Fc domain interactions with Fc[gamma]Rs, using the' yeast S. cerevisiae. In an initial step, a microbial system for the production of full-length antibodies in S. cerevisiae in milligram per liter titers has been developed, which serves as a platform for the engineering of antibody Fc domains with defined properties. The presence of a single N-linked glycan on each chain of the antibody Fc, as well as the specific composition of the glycoforms comprising it, are critical to the binding of the Fc to Fc[gamma]Rs, and have largely limited the production of therapeutic antibodies to mammalian expression systems. Using a display system that tethers full-length antibodies on the surface of yeast, we identify and characterize aglycosylated antibody variants that bind a subset of the human low-affinity Fc[gamma]Rs, Fc[gamma]RIIA and Fc'yRIIB, with approximately wildtype binding affinity and activate immune effector functions in vivo. In a separate approach, we identify aglycosylated variants that weakly bind a third low-affinity receptor, Fc[gamma]RIIIA, and through subsequent engineering generate variants that bind all of the low-affinity Fc[gamma]Rs with approximately wild-type binding affinity. By decoupling the function of the antibody from its post-translational processing, these variants have the potential to open up therapeutic antibody production to a far wider array of expression systems than currently available. Finally, in parallel work, we use a similar system to screen for glycosylated Fc variants with improved affinity and specificity for the activating receptor Fc[gamma]RIIIA compared to the inhibitory receptor Fc[gamma]RIIB, properties which have been hypothesized to lead to more potent antibody therapeutics."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Ph.D."]},{"key":"dc:title","label":"Title","values":["Engineering aglycosylated antibody variants with immune effector functions"]}]}],"canonical_facts":{"dc:contributor.advisor":["K. Dane Wittrup."],"dc:contributor.department":["Massachusetts Institute of Technology. Dept. of Biological Engineering."],"dc:contributor.other":["Massachusetts Institute of Technology. Dept. of Biological Engineering."],"dc:creator":["Sazinsky, Stephen L. (Stephen Lael)"],"dc:date.accessioned":["2011-02-23T14:33:36Z"],"dc:date.available":["2011-02-23T14:33:36Z"],"dc:date.issued":["2009"],"dc:description":["Thesis (Ph. 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In an initial step, a microbial system for the production of full-length antibodies in S. cerevisiae in milligram per liter titers has been developed, which serves as a platform for the engineering of antibody Fc domains with defined properties. The presence of a single N-linked glycan on each chain of the antibody Fc, as well as the specific composition of the glycoforms comprising it, are critical to the binding of the Fc to Fc[gamma]Rs, and have largely limited the production of therapeutic antibodies to mammalian expression systems. Using a display system that tethers full-length antibodies on the surface of yeast, we identify and characterize aglycosylated antibody variants that bind a subset of the human low-affinity Fc[gamma]Rs, Fc[gamma]RIIA and Fc'yRIIB, with approximately wildtype binding affinity and activate immune effector functions in vivo. In a separate approach, we identify aglycosylated variants that weakly bind a third low-affinity receptor, Fc[gamma]RIIIA, and through subsequent engineering generate variants that bind all of the low-affinity Fc[gamma]Rs with approximately wild-type binding affinity. By decoupling the function of the antibody from its post-translational processing, these variants have the potential to open up therapeutic antibody production to a far wider array of expression systems than currently available. Finally, in parallel work, we use a similar system to screen for glycosylated Fc variants with improved affinity and specificity for the activating receptor Fc[gamma]RIIIA compared to the inhibitory receptor Fc[gamma]RIIB, properties which have been hypothesized to lead to more potent antibody therapeutics."],"dc:description.degree":["Ph.D."],"dc:identifier.uri":["http://hdl.handle.net/1721.1/61232"],"dc:language.iso":["eng"],"dc:publisher":["Massachusetts Institute of Technology"],"dc:rights":["MIT theses are protected by copyright. They may be viewed, downloaded, or printed from this source but further reproduction or distribution in any format is prohibited without written permission."],"dc:rights.uri":["http://dspace.mit.edu/handle/1721.1/7582"],"dc:subject":["Biological Engineering."],"dc:title":["Engineering aglycosylated antibody variants with immune effector functions"],"dc:type":["Thesis"]},"updated_at":"2026-07-22T22:21:58Z"}