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B Cell-Based Seamless Engineering of Antibody Fc Domains.

ABSTRACT: Engineering of monoclonal antibodies (mAbs) enables us to obtain mAbs with additional functions. In particular, modifications in antibody's Fc (fragment, crystallizable) region can provide multiple benefits such as added toxicity by drug conjugation, higher affinity to Fc receptors on immunocytes, or the addition of functional modules. However, the generation of recombinant antibodies requires multiple laborious bioengineering steps. We previously developed a technology that enables rapid in vitro screening and isolation of specific mAb-expressing cells from the libraries constructed with chicken B-cell line DT40 (referred to as the 'ADLib system'). To upgrade this ADLib system with the ability to generate customized mAbs, we developed a novel and rapid engineering technology that enables seamless exchanges of mAbs' Fc domains after initial selections of mAb-producing clones by the ADLib system, using a gene-replacement unit for recombinase-mediated cassette exchange (RMCE). In this system, Cre-recombinase recognition sites were inserted into the Fc region of the active DT40 IgM allele, allowing the replacement of the Fc domain by the sequences of interest upon co-transfection of a Cre recombinase and a donor DNA, enabling the rapid exchange of Fc regions. Combining this method with the ADLib system, we demonstrate rapid Fc engineering to generate fluorescent antibodies and to enhance affinity to Fc receptors.

SUBMITTER: Hashimoto K

PROVIDER: S-EPMC5131995 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Rationale: Fc engineering has become the focus of antibody drug development. The current mutagenesis and in silico protein design methods are confined by the limited throughput and high cost, while the high-throughput phage display and yeast display technologies are not suitable for screening glycosylated Fc variants. Here we developed a mammalian cell display-based Fc engineering platform. Methods: By using mammalian cell display and next generation sequencing, we screened millions of Fc variants for optimized affinity and specificity for FcγRIIIa or FcγRIIb. The identified Fc variants with improved binding to FcγRIIIa were substituted into trastuzumab and rituximab and the effector function of antibodies were examined in the PBMC-based assay. On the other hand, the identified Fc variants with selectively enhanced FcγRIIb binding were applied to CD40 agonist antibody and the activities of the antibodies were measured on different cell assays. The immunostimulatory activity of CD40 antibodies was also evaluated by OVA-specific CD8+ T cell response model in FcγR/CD40-humanized mice. Results: Using this approach, we screened millions of Fc variant and successfully identified several novel Fc variants with enhanced FcγRIIIa or FcγRIIb binding. These identified Fc variants displayed a dramatic increase in antibody-dependent cellular cytotoxicity in PBMC-based assay. Novel variants with selectively enhanced FcγRIIb binding were also identified. CD40 agonist antibodies substituted with these Fc variants displayed activity more potent than the parental antibody in the in vitro and in vivo models. Conclusions: This approach increased the throughput of Fc variant screening from thousands to millions magnitude, enabled screening variants containing multiple mutations and could be integrated with glycoengineering technology, represents an ideal platform for Fc engineering. The initial efforts demonstrated the capability of the platform and the novel Fc variants could be substituted into nearly any antibody for the next generation of antibody therapeutics.

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B Cell-Based Seamless Engineering of Antibody Fc Domains.

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