Abstract
"Antibody-breeding" approach potentially generates therapeutic/diagnostic antibody mutants with greater performance than native antibodies. Therein, antibody fragments (e.g., single-chain Fv fragments; scFvs) with a variety of mutations are displayed on bacteriophage to generate diverse phage-antibody libraries. Rare clones with improved functions are then selected via panning against immobilized or tagged target antigens. However, this selection process often ended unsuccessful, mainly due to the biased propagation of phage-antibody clones and the competition with a large excess of undesirable clones with weaker affinities. To break radically from such panning-inherent problems, we developed a novel method, clonal array profiling of scFv-displaying phages (CAP), in which colonies of the initial bacterial libraries are examined one-by-one in microwells. Progenies of scFv-displaying phages generated are, if show sufficient affinity to target antigen, captured in the microwell via pre-coated antigen and detected using a luciferase-fused anti-phage scFv. The advantage of CAP was evidenced by its application with a small error-prone-PCR-based library (~ 105 colonies) of anti-cortisol scFvs. Only two operations, each surveying only ~ 3% of the library (9,400 colonies), provided five mutants showing 32–63-fold improved Ka values (> 1010 M−1), compared with the wild-type scFv (Ka = 3.8 × 108 M−1), none of which could be recovered via conventional panning procedures operated for the entire library.
Highlights
"Antibody-breeding" approach potentially generates therapeutic/diagnostic antibody mutants with greater performance than native antibodies
In our clonal array profiling of scFv-displaying phages (CAP), we examined the colonies of the initial bacterial library grown on agar plates one-by-one in different microwells, so as not to disturb their original diversity or compete with the different clones (Fig. 1C)
The colonies grown on agar were transferred individually into microwells in 96-well plates pre-coated with the target antigen and filled with liquid medium containing the KM13 helper phage[17]
Summary
"Antibody-breeding" approach potentially generates therapeutic/diagnostic antibody mutants with greater performance than native antibodies. We devised “clonal array profiling of scFv-displaying phages (CAP),” a reliable and robust system for recovering rare affinity-matured scFv phages that does not require sophisticated machinery or intelligent technology This system, schematically illustrated, directly (i.e., without any enrichment step) provided a set of scFv mutants showing 32–63-fold improved and 1010-order Ka values against a small biomarker cortisol (hydrocortisone; Fig. 2C and Supplementary Fig. S1), contained in a small (~ 105-order) scFv library generated via a simple mutagenesis based on error-prone polymerase chain reaction (PCR). None of such improved species could be recovered via a multiple operation of panning including three different protocols. We applied CAP to a related but different scFv library, and again succeeded in isolating affinity-matured mutants with a minimum experimental effort
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