Escherichia coli inner membrane display system for high-throughput screening of dimeric proteins.

Multimer formation is indispensable to the intrinsicbiologicalfunctions of many natural proteins. For example, the human immunoglobulin G (IgG) antibody has two variable regions (heavy chain variable domain [VH] and light chain variable domain [VL]) that must be assembled for specific antigen binding, and homodimerization of the antibody's Fc domain is essential for eliciting therapeutic effector functions. For the more efficient high-throughput directed evolution of multimeric proteins with ease of cultivation and handling, here we report a membrane protein drift and assembly (MPDA) system, in which a multimeric protein is displayed on a bacterial inner membrane by drifting and auto-assembling membrane-anchored subunit polypeptides. This system enabled the auto-assembly of membrane-tethered Fv domains (VH and VL) or the monomeric Fc domain into a functional hetero- or homodimeric protein complex on the bacterial inner membrane. This system could also be used to enrich a desired engineered Fc variant from a mixture containing a million-fold excess of wild-type Fc domain, indicating the applicability of the MPDA system for the high-throughput directed evolution of a variety of multimeric proteins, such as cytokines, enzymes, or structural proteins.

Optimal combination of beneficial mutations for improved ADCC effector function of aglycosylated antibodies.

The Fc region of IgG antibodies is crucial for binding to Fc receptors expressed on the surfaces of various immune leukocytes and eliciting therapeutic effector functions such as clearance of antibody-opsonized tumor cells. Despite abrogated Fc gamma receptor (FcγR) binding and therapeutic effector function in the absence of N-linked glycosylation at Asn297, the aglycosylated Fc region of IgG antibodies has bioprocessing advantages such as the absence of glycan heterogeneity and simple bacterial antibody production. Therefore, these antibodies have been comprehensively engineered as effector functional units for human therapy. In this work, we constructed a huge library of Fc variants with combinations of 25 beneficial mutations that were previously identified to improve binding of glycosylated or aglycosylated Fc regions to human FcγRs in previous studies. High-throughput screening of the resulting library led to the identification of an aglycosylated Fc variant that exhibited almost double the antibody-dependent cell-mediated cytotoxicity than wild-type glycosylated Fc. All mutations in this aglycosylated Fc variant were derived from previously identified beneficial mutations for engineered aglycosylated Fc variants as opposed to glycosylated variants, suggesting that significantly different sets of beneficial mutations are necessary to improve the effector function of aglycosylated Fc.

The Highly Evolvable Antibody Fc Domain.

The Fc region of the IgG antibody recruits immune leukocytes or serum complement molecules, which in turn triggers the clearance of defective cells such as tumor cells or infected cells. In addition, the Fc region is crucial for the prolonged serum persistence of circulating IgG antibodies through an intracellular trafficking and recycling mechanism. Recently, the utility of antibody Fc has been further expanded to include a new class of antigen-binding scaffolds. This review presents the recent progress in the field of antibody Fc engineering and highlights new biomolecular, cellular, and evolutionary approaches to overcome the limitations of conventional monoclonal therapeutic antibodies by engineering the antibody Fc domain.