Attainment of 15-Fold Higher Affinity of a Fusarium-specific Single-Chain Antibody by Directed Molecular Evolution Coupled to Phage Display

Abstract

Fusarium head blight (FHB) caused by Fusarium graminearum infection is a devastating disease of wheat, maize, and other cereals. A previously isolated chicken single-chain Fv antibody (scFv), CWP2, that conferred durable resistance in planta was subjected to directed evolution by error-prone PCR and DNA shuffling, generating a mutated library. Panning of the mutated library against cell wall-bound proteins (CWPs) from F. graminearum by phage display enriched phage clones that were used for a further round of DNA shuffling to construct a combinatorial library comprising 3 × 10(6) variants. Screening of this library by phage display for variants reactive against the CWPs led to the identification of a number of clones. Comparative enzyme-linked immunosorbent assay analyses revealed eight clones exhibiting a higher reactivity than the parent, CWP2, and containing four different single-chain antibody sequences. Surface plasmon resonance measurements confirmed that three mutated scFvs, CWPa, CWPb, and CWPd, displayed 15-fold, 11-fold, and 7-fold higher affinities, respectively, compared with CWP2. Three-dimension modeling of CWPa illustrates a conformational change bringing all six complementary domain regions on the antibody surface in one direction. These results provide promising unique resistance molecules for effective control of FHB and its associated mycotoxins in food/feed chains.

Antibody discovery and engineering by enhanced CRISPR-Cas9 integration of variable gene cassette libraries in mammalian cells

“…Deep sequencing of our libraries allowed us to more thoroughly interrogate library diversity, selection and enrichment and uncover mutational preferences, which all suggested that PnP screening was a robust approach for antibody engineering. It is reasonable to expect that additional mutagenesis libraries, such as extension of EP-PCR to the V L , variable region shuffling, 49,50 and combining the beneficial mutations of different clones could also be successfully integrated for PnP screening. Importantly, compared to the most common strategies for affinity maturation using phage and yeast display to express antibody fragments, our PnP system presents the fundamental advantage of selection in the native mammalian cell IgG context, which may further aid in the selection of candidates with favorable drug development parameters.…”

Generation of a Highly Reactive Chicken-Derived Single-Chain Variable Fragment against Fusarium verticillioides by Phage Display

“…FvCA2 had a higher binding value in phage ELISA than FvCA3 but a lower binding was seen in soluble antibody ELISA than for FvCA3. This discrepancy may be due to the alteration of antibody configuration when fused with pIII protein of phage particle, as observed by others [ 40 ].…”

“…Previous studies have also confirmed that the length and heterogeneity of the CDRH3 sequence were associated to antibody binding affinity, specificity, paratope shape, and activity (such as neutralization potency) [ 52 – 56 ]. Properties of the selected scFvs such as FvCA1, FvCA2 or FvCA3 might be further improved by CDR shuffling [ 57 – 59 ], chain shuffling [ 60 ], or DNA shuffling technology [ 39 , 40 ].…”

“…ScFv antibodies apparently have some advantages over polyclonal antisera or mAbs and can be isolated together with their coding sequences by phage display [ 37 ]. These antibodies carry only variable fragments and their relatively small size allows for easy genetic manipulation and molecular evolution to further improve their affinity [ 38 – 40 ]. In addition, fungus-specific scFvs, either alone or as a fusion with other molecules, have been shown to confer resistance to pathogens in transgenic plants [ 32 – 34 ].…”

Characterization of single-domain antibodies against Foot and Mouth Disease Virus (FMDV) serotype O from a camelid and imaging of FMDV in baby hamster kidney-21 cells with single-domain antibody-quantum dots probes

“…The sdAbs were diluted at concentrations of 25, 50, 75, 100, and 150 nM with HBS-EP buffer and then added onto the sensor chips at a flow rate of 30 μL/min. The binding kinetics were recorded, and the kinetic rate constants (ka and kd) were used to calculate the equilibrium dissociation constant (KD) [ 27 ]. Sensor chips were regenerated by 20 mM glycinate hydrochloride (pH 3.0) after each measurement.…”