Isolation and comparative characterization of Ki-67 equivalent antibodies from the HuCAL® phage display library

Abstract

It has been shown that a repetitive motif with the sequence FKEL(F) within the Ki-67 antigen (pKi-67) serves as an epitope for the Ki-67 antibody and equivalent clones. However, no direct correlation between reactivity towards Ki-67 epitopes and reactivity in formalin-fixed paraffin-embedded (FFPE) tissue could be found. In this study our aim was the isolation and characterization of new monoclonal Ki-67 equivalent antibodies in an in vitro approach. To select pKi-67 reactive phage antibodies, we used a large naive Fab-phage library (Human Combinatorial Antibody Library; HuCAL). We implemented a panning strategy against two different overlapping peptides, both containing the 'FKELF' epitope. ELISA screening of randomly picked phage antibody clones after the third selection round yielded six highly reactive clones against the 'FKELF' epitope, of which five were found to be reactive in FFPE tissue, showing a Ki-67 equivalent staining pattern. Substitutional epitope analysis on peptide arrays of the new recombinant pKi-67 binders and of the established murine clones Ki-67, Mib-1 and Mib-5 were carried out to compare their fine specificities. The results suggest that the lysine residue in the epitope is critical for recognition of Ki-67 antigen in FFPE tissue.

Selection and in vitro characterization of human CD44v6‐binding antibody fragments

“…In addition, the linker regions that connect the synthetic target peptide to the carrier proteins were different for the two carriers to avoid selection of linker‐specific binders. All selections were performed at 20°C and in three successive rounds, essentially as described in Jarutat et al and Prassler et al .…”

Affinity maturation by targeted diversification of the CDR-H2 loop of a monoclonal Fab derived from a synthetic naïve human antibody library and directed against the internal trimeric coiled-coil of gp41 yields a set of Fabs with improved HIV-1 neutralization potency and breadth

“…Specific binding to N CCG -gp41 was confirmed by ELISA and the 10 antibodies with the highest signal on N CCG -gp41 were selected for further characterization. These Fabs were also subcloned and expressed in the bivalent Fab-dHLX-MH format (Jarutat et al, 2006; Gustchina et al, 2007). Composition and purity of antibodies were confirmed by SDS-PAGE, and Western blot analysis against the panel of gp41 ectodomain-derived constructs was carried out as described previously (Gustchina et al, 2007).…”

Natural and man-made V-gene repertoires for antibody discovery

“…The CDR cassettes were again based on trinucleotide technology and were built to accurately reflect the canonical structures and diversity found in the families upon which each of the consensus frameworks were built, consciously including structures known to be preferred in the recognition of peptides. The finalized library was found to be of very high practical utility, routinely generating antibody specificities and affinities in the single digit nM range that were useful for both therapeutic and reagent purposes (Jarutat et al, 2006, 2007; Ohara et al, 2006; Prassler et al, 2009). This library design had, however, made a critical concession to optimize its function in E. coli expression and phage display technology: the FRs were codon optimized specifically to maximize for E. coli periplasmic expression rate.…”