title: Engineering of Immunoglobulin Fc Heterodimers Using Yeast Surface-Displayed Combinatorial Fc Library Screening.
abstract: Immunoglobulin Fc heterodimers, which are useful scaffolds for the generation of bispecific antibodies, have been mostly generated through structure-based rational design methods that introduce asymmetric mutations into the CH3 homodimeric interface to favor heterodimeric Fc formation. Here, we report an approach to generate heterodimeric Fc variants through directed evolution combined with yeast surface display. We developed a combinatorial heterodimeric Fc library display system by mating two haploid yeast cell lines, one haploid cell line displayed an Fc chain library (displayed FcCH3A) with mutations in one CH3 domain (CH3A) on the yeast cell surface, and the other cell line secreted an Fc chain library (secreted FcCH3B) with mutations in the other CH3 domain (CH3B). In the mated cells, secreted FcCH3B is displayed on the cell surface through heterodimerization with the displayed FcCH3A, the detection of which enabled us to screen the library for heterodimeric Fc variants. We constructed combinatorial heterodimeric Fc libraries with simultaneous mutations in the homodimer-favoring electrostatic interaction pairs K370-E357/S364 or D399-K392/K409 at the CH3 domain interface. High-throughput screening of the libraries using flow cytometry yielded heterodimeric Fc variants with heterodimer-favoring CH3 domain interface mutation pairs, some of them showed high heterodimerization yields (~80-90%) with previously unidentified CH3 domain interface mutation pairs, such as hydrogen bonds and cation-π interactions. Our study provides a new approach for engineering Fc heterodimers that could be used to engineer other heterodimeric protein-protein interactions through directed evolution combined with yeast surface display.
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea.
authors.forename: Hye-Ji
authors.initials: HJ
authors.lastname: Choi
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea.
authors.forename: Ye-Jin
authors.initials: YJ
authors.lastname: Kim
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea.
authors.forename: Dong-Ki
authors.initials: DK
authors.lastname: Choi
authors.affiliation: Department of Molecular Science and Technology, Ajou University, Suwon 16499, Korea.
authors.forename: Yong-Sung
authors.initials: YS
authors.lastname: Kim
mesh_terms: D000595:Amino Acid Sequence; D002462:Cell Membrane / Q000378:metabolism*; D015723:Gene Library; D005820:Genetic Testing / Q000379:methods; D006801:Humans; D007141:Immunoglobulin Fc Fragments / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007074:Immunoglobulin G / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D015202:Protein Engineering / Q000379:methods*; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism
chemical_list: D007141:Immunoglobulin Fc Fragments; D007074:Immunoglobulin G