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.
Highlights
Immunoglobulin G (IgG) is a monospecific, bivalent antigen-binding antibody consisting of two identical heavy chains and two identical light chains
If the two fragment crystallizable (Fc) variants favor heterodimerization over homodimerization, during secretion, the secreted FcCH3B will be anchored on the cell surface through its heterodimeric interaction with the displayed FcCH3A, such that the secreted FcCH3B can be detected on the yeast cell surface by immunofluorescence
Semi-quantitative comparisons of the mean fluorescence intensity (MFI) showed a close correlation between Flag MFI and the previously reported heterodimerization yields of Fc variants, with higher MFI values for Fc variants with higher heterodimerization yields (Fig 1c). These results suggested that the strength of Fc heterodimerization is directly correlated with the amount of cell surface-displayed secreted FcCH3B, allowing semi-quantitative monitoring of Fc heterodimerization on the yeast cell surface
Summary
Immunoglobulin G (IgG) is a monospecific, bivalent antigen-binding antibody consisting of two identical heavy chains and two identical light chains. Its assembly is driven by homodimerization of the fragment crystallizable (Fc) regions of the heavy chains and disulfide linkages between each heavy chain and each light chain [1]. Fc homodimerization of the heavy chains is initially driven by noncovalent inter-CH3 domain interactions and subsequently by disulfide. Bispecific antibodies simultaneously bind to two different target antigens within a single molecule. Such bispecific antibodies have potential clinical benefits for the treatment of complicated diseases, such as tumors and immune disorders [5, 6], and more than 50 different bispecific antibodies have been reported [3, 4]. The heterodimeric Fc-based IgG-like format is attractive because it can be designed as close as possible to the natural IgG architecture such that it possesses desirable physicochemical properties, such as high stability, large-scale manufacturing capability, and low immunogenicity, in addition to the natural IgG-like properties of a long serum half-life and immune cell-recruiting effector functions [3, 4, 6]
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