Engineering aglycosylated antibody variants with immune effector functions
Author(s)Sazinsky, Stephen L. (Stephen Lael)
Massachusetts Institute of Technology. Dept. of Biological Engineering.
K. Dane Wittrup.
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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.
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).
DepartmentMassachusetts Institute of Technology. Dept. of Biological Engineering.
Massachusetts Institute of Technology