De Novo Isolation of an Antigen-Binding Antibody by Targeted CDR-H3 Diversification of a Germline-Derived Scaffold in a Site-Specifically Integrated Mammalian Display Platform: A Proof-of-Principle Study

Source abstract

Background/Objectives: Mammalian display enables direct screening of full-length IgG antibodies, but genetically controlled library construction remains technically demanding. Here, we developed a mammalian display platform in which a target-naïve ΔDH antibody scaffold is first established at a defined human artificial chromosome (HAC) acceptor site, and CDR-H3 diversity is then introduced directly into that fixed genomic background by CRISPR-Cas9-mediated homology-directed repair (HDR). Methods: A site-specifically integrated display cell line carrying the ΔDH scaffold on an HAC was generated by Cre-mediated recombination. Randomized CDR-H3 libraries were introduced into this defined genomic background by HDR and screened by magnetic- and fluorescence-activated cell sorting together with orthogonal antigen-detection formats. Binding of isolated clones was characterized by flow cytometry and enzyme-linked immunosorbent assay. Results: Although early selection enriched clones reactive with a fluorescent reagent rather than the intended antigen, refinement of the selection scheme enabled isolation of AFFVVF. This selected antibody variant recognized multiple SARS-CoV-2 receptor-binding domain (RBD) variants by flow cytometry, with no detectable binding to unrelated antigens. As a recombinant protein, AFFVVF bound the JN.1 and XEC spike trimers by ELISA, but not monomeric RBD, including the XEC RBD used for selection, indicating that the form of antigen recognized differed between the cell-based and plate-based assays. Exploratory selections against epidermal growth factor receptor (EGFR) and glycoprotein A33 (GPA33) also produced antigen-dependent population shifts and enrichment of discrete sequence variants, although individual candidates were not independently validated as recombinant antibodies. Conclusions: These results provide proof of principle that targeted CDR-H3 diversification within a pre-established, genetically defined, target-naïve full-length IgG scaffold can yield a new antigen-binding antibody without starting from a pre-existing antigen-specific antibody or repertoire.